JP6754225B2 - Sample introduction device and sample introduction method - Google Patents

Description

The present invention relates to liquid chromatography, and more particularly to high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC), an operation method under conditions that give high performance and ultra high performance, particularly. The present invention relates to a method for introducing a sample, and further relates to a sample introduction apparatus required for these methods.

Here, "high performance liquid chromatography (HPLC)" means liquid chromatography performed under a pressure of up to 40 MPa. Further, "ultra-high performance liquid chromatography (UHPLC)" means liquid chromatography performed under a pressure of more than 40 MPa and up to 140 MPa.

In HPLC or UHPLC, it is necessary to use a column packed with a small filler in order to speed up the separation, that is, to generate a large number of theoretical plates in a short time and shorten the separation time. However, a small column packing material requires a high pressure for liquid feeding because it reduces the gap between the packing materials (the flow path through which the mobile phase passes). Therefore, a short column is used to reduce the pressure.

Further, when the liquid is sent to the gap between the small fillers at high speed, heat is generated due to the friction between the liquid and the particle wall. The generated heat is dissipated from the column tube wall to the outside of the column, but the temperature in the center of the column becomes high, and as a result of the temperature distribution in the column, the solution transfer resistance based on the viscosity of the mobile phase solvent and the distance between the mobile phase and the stationary phase Since the partition coefficient of the solute is non-uniform, the moving speed of the same solute is non-uniform depending on the position in the column. This widens the bandwidth of the solute and reduces the column separation performance. Therefore, in order to quickly achieve the temperature equilibrium in the column and maintain the separation performance of the column, it is effective to make the column thinner.

For the above reasons, a small filler is packed in a short and thin column in order to exhibit the high performance of the column. The solute band eluted from a small high performance column has a very small volume and dispersion (Non-Patent Document 8).

Further, as the solvent for preparing the sample by dissolving the solute, a solvent having a strong dissolution output, a solvent having a large dissolving power, or a solvent having the same composition as the mobile phase is often used. Then, when the sample is injected into the column, the solute advances in the column due to the solvent itself in the sample, so that the sample bandwidth is widened and the number of theoretical plates is reduced. Therefore, it is very important to reduce the volume of the sample to be injected, but it is generally used when using a column with an inner diameter of 1.0 to 4.6 mm, based on the ease of handling the sample and the sensitivity of the detector. 0.1 to 20 μL of sample is injected. Then, when the sample becomes a laminar flow (in the form of a parabolic flow profile that is fast at the center of the pipe and slow at the wall) while flowing through the flow path from the sample injection device such as an injector to the column, for example, the connecting pipe, , The presence of unswept volume (effect of retention) widens the sample bandwidth.

The peaks of each sample component (solute) separated by the column appearing on the chromatogram are approximated by a Gaussian distribution. The variance of the Gaussian distribution (σ ² : standard deviation σ squared) is additive, and the variance of the solute bands separated using a certain HPLC device and column (dispersion of the entire solute band, σ _total ² ) is , As shown in Equation 1, it is expressed as the sum of the dispersion σ _col ^{2 of the} solute band brought about by the column body and the dispersion σ _extra ² of the solute band in the flow path outside the column (Non-Patent Document 1).

(Equation 1)
σ _total ² = σ _col ² + σ _extra ²

Dispersion of the solute band outside the column σ _extra ² is, as shown in Equation 2, (1) dispersion of the solute band (σ _inj ) in the sample injection device (inj) and the pipe (tube 1) from the sample injection device to the column inlet. ² + σ _tube1 ² ), (2) Dispersion of solute band in the pipe (tube2) from the column outlet to the detector and the detector cell (det) (σ _tube2 ² + σ _det ² ) and (3) from the detector It consists of the variance of the solute band σ _data ^{2 provided} by the signal averaging and the time interval during which the data processor captures the signal.

(Equation 2)
σ _extra ² = σ _inj ² + σ _tube1 ² + σ _tube2 ² + σ _det ² + σ _data ²

The peak dispersion (dispersion of the entire solute band, σ _total ² ) actually obtained in the HPLC apparatus or UHPLC apparatus is obtained by adding the dispersion of the solute band in the column (σ _col ² ) to the right side of Equation 2 in Equation 3. It is shown as.

(Equation 3)
σ _total ² = σ _inj ² + σ _tube1 ² + σ _col ² + σ _tube2 ² + σ _det ² + σ _data ²

Number of theoretical plates of the column (N), as shown in Equation 4, solute retention times (t _R) (or holding volume (V _R)) and is calculated using the dispersion of the solute band. Here, σ _total-t ² is the variance calculated as the observed time, and σ _total-v ² is the variance calculated as the volume. Hereinafter, the number of theoretical plates was calculated assuming that the peak width at half maximum (peak width at 1/2 of the peak height) = 2.35σ.

(Equation 4) N = (t _R ² / σ _total-t ² ) = (V _R ² / σ _total-v ² )

Compared with the theoretical plate number (N _col ) that the column should show under ideal conditions without σ _extra ² shown in Equation 5, the column is actually connected to the HPLC apparatus or UHPLC apparatus shown in Equation 4. The number of theoretical plates (N) to be obtained is generally small. This is based on the contribution of the dispersion σ _extra ² of the solute band outside the column shown in Equations 1 and 2.

(Equation 5) N _col = (t _R ² / σ _col-t ² ) = (V _R ² / σ _col-v ² )

Here, the reason for including σ _data ² in (3) above in σ _extra ² will be described below. The chromatogram plots the detector signal over time. When the chromatogram is displayed on the data processor, the slow output of the detector and the slow data capture of the data processor also provide time-scale peak spread (variance). When the mobile phase liquid is flowing at a constant flow rate, the dispersion of the solute band on the time scale can be converted into the dispersion on the volume scale using the flow velocity and expressed.

In order to obtain high performance in HPLC separation and UHPLC separation using a small column, it is necessary to minimize the dispersion σ _extra ² of the solute band outside the column. Among the σ _extra ² , the σ _data ^{2 in} (3) above is minimized by shortening the response time of the detector and shortening the data acquisition interval of the data processing device.

Further, σ _tube ^{2 2} + σ _det ^{2 in} (2) above is minimized by reducing the inner diameter and length of the connecting pipe connecting the column outlet and the detector cell, and reducing the inner diameter and capacity of the detector cell flow path. Be transformed.

When injecting a liquid sample into an HPLC device or UHPLC device, a sample band flows from a sample injection device such as an autosampler or an injector to a separation column through a pipe, and a laminar flow (parabolic flow profile) and It diffuses due to the effect of retention due to unswept volume brought about by the corners of the connecting parts. _{Σ inj} ² + σ tube1 ² above (1), reduced pipe inside diameter from the sample injector to the column, reduced and reducing the length, the internal diameter of the sample loop and sample needle sample injection device, a sample It is minimized by reducing the orifice of the valve through which the sample is passed, but the sample loop, sample needle, and valve orifice of the sample injection device need to pass the liquid sample reproducibly under attractive pressure or atmospheric pressure. A practical inner diameter that does not easily become a resistance is required.

As described above, the dispersion σ _extra ² of the solute band outside the column makes a relatively large contribution as the column size becomes smaller, and the smaller the column, the more difficult it is for the column to exhibit its original performance. In a commonly used HPLC apparatus or UHPLC apparatus, when the capacity of the column tube is less than 200 μL (for example, a column having an inner diameter of 2.1 mm and a length of 5 cm (column tube capacity of about 173 μL), an inner diameter of 1.0 mm, and a length of 25 cm (for example). In the case of a column having a column tube capacity of about 196 μL) or a column having a smaller capacity), this tendency becomes remarkable.

Specifically, in UHPLC equipment, columns with an inner diameter of 2.1 mm and a length of 5 cm are often used, and a sample of 0.1 to 2.0 μL is often injected. Then, in many UHPLC devices, the solute that gives the retention coefficient k = about 5 by the dispersion σ _extra ² of the solute band outside the column is about 30% in the column having an inner diameter of 2.1 mm and a length of 5 cm, and the inner diameter is 1. It has been reported that the number of theoretical plates is reduced by about 70% or more in a column having a length of 0.0 mm and a length of 5 cm (column tube capacity of about 39 μL) (Non-Patent Document 2).

Further, the smaller the column, the larger the ratio of the dispersion σ _extra ² of the solute band outside the column to the dispersion σ _total ² of the entire solute band, and this tendency is seen on the chromatogram. It becomes even more remarkable when the retention coefficient of is small. In the UHPLC apparatus, it has been reported that the number of theoretical plates of a solute giving a retention coefficient k = about 1 is reduced by about 50% even in a column having an inner diameter of 2.1 mm and a length of 5 cm (Non-Patent Document 3). For columns with an inner diameter of 1.0 mm and a length of 5 cm, σ _extra ² brings about a further deterioration in performance (Non-Patent Document 8).

Further, in order to reduce the dispersion σ _extra ² of the solute band outside the column of the UHPLC device, the flow path piping from the sample injection device to the column was originally provided in the device (inner diameter 0.127 mm, length 55 cm). By changing to a shorter pipe (inner diameter 0.127 mm, length 25 cm), we confirmed the performance improvement of the column with inner diameter 2.1 mm and length 5 cm, and the column with inner diameter 2.1 mm and length 10 cm. An example of this has been reported (Non-Patent Document 5).

The sample injection device of the HPLC device or UHPLC device is installed between the liquid feed pump and the column, and usually has an injection port for storing the sample and a sample loop for temporarily storing the sample, and is input from the injection port. The sample is incorporated into the flow path between the liquid feed pump and the column and introduced into the column. Then, since the sample incorporated in the flow path moves while diffusing the flow path to the column inlet, the capacity of the flow path greatly affects the dispersion of the sample band. The autosampler, which is a type of sample injection device, is equipped with a flow path switching valve to which a sample loop with a capacity of 5.0 to 50 μL is connected, and the connection from the outlet of this flow path switching valve to the column inlet has an outer diameter. Piping with a diameter of 1.6 mm, an inner diameter of 0.1 to 0.25 mm, and a length of 20 to 30 cm is often used. Among stainless steel tubes with an outer diameter of 1.6 mm, which have a pressure resistance and are not easily damaged even if columns are replaced repeatedly, an inner diameter of 0.1 mm is the smallest. In addition, when the autosampler and the column oven are separate units, a fixed length of piping is required from the flow path switching valve to the column inlet.

The dispersion σ _extra ² of the solute band outside the column makes a relatively large contribution as the column size becomes smaller, and lowers the column performance. It is necessary to minimize σ _extra ^{2 in} order to obtain the performance (theoretical plate number) that the column originally has by using a column with a small inner diameter and a short length. Among them, a sample injection device and a sample Since the piping from the injection device to the column inlet has the above-mentioned actual restrictions on minimization, the sample band that diffuses in the flow path from the sample injection device to the column inlet has been so far. An effective solution other than the above method or the method of adsorbing the sample as a narrow band on the stationary phase at the inlet of the column and concentrating the sample using a solvent having a small elution output (Non-Patent Documents 6 and 7) is available. Not found.

On the other hand, in a column for capillary gas chromatography or a column for capillary liquid chromatography in which the amount of filler (stationary phase) is small, the volume of the sample or the weight of the solute contained in the sample may overload the column. By split injection, the volume of the sample or the weight of the solute contained in the sample is reduced to the extent that the column can be processed, and the column performance is maintained (Patent Document 1, Non-Patent Document 4).

This split injection is performed by taking a portion of the sample that is too large for the column, i.e., taking in a portion of the flowing sample band from the column inlet and discharging most of it for disposal. In this split injection method, a large amount of sample is inevitably discarded. Further, this split injection method is performed when the volume of the sample or the weight of the solute contained in the sample is excessive with respect to the column, and is not performed when the amount of the sample is appropriate for the column. ..

Further, in the HPLC apparatus and the UHPLC apparatus, this split injection is performed in the form of branching the flow path in the middle of the piping from the sample injection apparatus to the column inlet, so that the dispersion of the solute band cannot be reduced.

On the other hand, in the HPLC apparatus, a method of providing a second liquid feed port at the column inlet separately from the first liquid feed port to distinguish between liquid feed to the center of the column and liquid feed to the periphery of the column is a column. It has been proposed for the purpose of controlling the laminar flow (flow profile that draws a parabolic shape) that occurs within (Non-Patent Documents 9 and 10).

U.S. Pat. No. 3,513,636

A. Prus, C. Kempter, J. Gysler, T. Jira, Journal of Chromatography A, 1016, 129-141 (2003). Extracolumn band broadening in capillary liquid chromatography. N. Wu, A. C. Bradley, Journal of Chromatography A, 1261, 113-120 (2012). Effect of column dimension on observed column efficiency in very high pressure liquid chromatography F. Gritti, C. A. Sanchez, T. Farkas, G. Guiochon, Journal of Chromatography A, 1217, 3000-3012 (2010). Achieving the full performance of highly efficient columns by optimizing conventional benchmark high-performance liquid chromatography instruments. N. Wu, J. A. Lippert, M. L Lee, Journal of Chromatography A, 911, 1-12 (2001). Practical aspects of ultrahigh pressure capillary liquid chromatography. F. Gritti, CA Sanchez, T. Farkas, G. Guiochon, Journal of Chromatography A, 1217, 7677-7689 (2010). On the extra-column band-broadening contributions of modern, very high pressure liquid chromatographs using 2.1mmI. D. columns packed with sub-2 μm particles. J. P. C. Vissers, A. H. de Ru, M. Ursem, J.-P. Chervet, Journal of Chromatography A, 746, 1-7 (1996). Optimised injection techniques for micro and capillary liquid chromatography. A. C. Sanchez, J. A. Anspach, T. Farkas, Journal of Chromatography A, 1228, 338-348 (2012) Performance optimizing injection sequence for minimizing injection band broadening contributions in high efficiency liquid chromatographic separations. Y. Ma, AW Chassy, S. Miyazaki, M. Motokawa, K. Morisato, H. Uzu, M. Ohira, M. Furuno, K. Nakanishi, H. Minakuchi, K. Mriziq, T. Farkas, O. Fiehn , N. Tanaka, Journal of Chromatography A, 1383, 47-57 (2015). Efficiency of short, small-diameter columns for reversed-phase liquid chromatography under practical operating conditions. T. J. N. Webber, E. H. McKerrell, Journal of Chromatography, 122, 243-258 (1976). Optimisation of liquid chromatographic performance on columns packed with microparticulate silicas. R. A. Shalliker, M. Camenzuli, L. Pereira, H. J. Ritchie, Journal of Chromatography A, 1262, 64-69 (2012). Parallel segmented flow chromatography columns: Conventional analytical scale column formats presenting as a ‘virtual’ narrow bore column.

As described above, the sample injected into the HPLC apparatus or UHPLC apparatus causes a large dispersion in the piping from the sample injection apparatus to the column. When using a small column (small inner diameter and short length), it is necessary to minimize the dispersion of the solute band outside the column in order to obtain the performance (theoretical plate number) that the column originally has. For the sample band that diffuses in the flow path from the sample injection device to the column inlet while having an appropriate volume and solute weight for the sample band, the above-mentioned prior art, particularly a solvent having a small elution output, has been used so far at the column inlet. No effective solution was found other than the method of adsorbing the sample as a narrow band on the stationary phase of the sample and concentrating it.

Further, a method is also used in which a valve containing a concentration column is provided between the sample injection device and the column, the sample is once adsorbed on the concentration column as a narrow band, and then the sample is introduced into the column. It is necessary to concentrate according to the solute of the sample, and it cannot be applied under conditions where it is difficult to concentrate.

The split injection described in Non-Patent Document 4 is performed when the volume of the sample or the weight of the solute in the sample is excessive with respect to the column. It is not done when the sample is already in the proper amount for the column. Further, since the flow path is branched in the middle of the piping from the sample injection device to the column inlet, the dispersion of the solute band cannot be reduced.

Further, there is a method in which the valve attached to the sample injection device is repeatedly switched to divide the sample and cut it into a narrow band, but after that, the sample band causes a large dispersion in the piping leading to the column.

Further, in the examples of Non-Patent Document 9 and Non-Patent Document 10, a second liquid feeding port is provided at the column inlet separately from the first liquid feeding port, and the liquid is fed to the center of the column and to the peripheral portion of the inner wall of the column tube. The purpose is to distinguish the liquids, keep the speed ratio between the liquid feed ports constant, and reduce the effect of the speed difference of the mobile phase solvent between the center of the column and the periphery of the inner wall of the column tube over the entire length of the column. It was not a technique to use the liquid sent from the two liquid feeding ports for dividing the sample band introduced from the first liquid feeding port and reducing the dispersion of the solute outside the column.

Therefore, the present invention exhibits even higher column performance not only when the volume of the sample and the solute weight in the sample are excessive with respect to the column, but also with respect to the sample which is considered to be an appropriate amount for the column. The purpose is to reduce the dispersion of solute bands outside the column.

In the present invention as a means for solving the above problems, when a sample is introduced into a separation column in liquid chromatography, a sample band is cut and divided in a flow path between the sample injection device and the column. It is an invention that improves the separation performance of a column by reducing the dispersion of sample bands that enter the column at one time.

When a liquid sample is injected into a chromatography device, especially a liquid chromatography device, the sample band diffuses in the process from the sample injection device (autosampler, injector, etc.) through the flow path to the column, and the sample band spreads. Wake up. The sample band that extends to the column inlet reduces column performance. The effect that this band spreading outside the column (dispersion of solute bands outside the column) reduces the column performance (outside column effect) makes a relatively large contribution as the column size decreases, and high performance liquid chromatography is performed on small columns. When used in a (HPLC) device or an ultra-high pressure liquid chromatography (or ultra-high performance liquid chromatography, UHPLC) device, the column cannot exhibit its original performance. In the present invention, the sample band injected by the sample injection device and diffused in the pipe is cut at the column inlet in a direction intersecting the axial direction (flow direction) of the sample band, divided, and introduced into the column at once. By reducing the volume and dispersion of the sample band, the separation capacity (theoretical plate number) of the column is improved, and it is important to reduce the dispersion of the solute band outside the column in the HPLC apparatus and the UHPLC apparatus. It solves the problem.

More specifically, it is a device provided between the sample injection device of the sample introduction device and the column, and is a sample band dividing device for dividing the sample band.

Further, the sample band dividing device, which is a device provided between the sample injection device of the sample introduction device for introducing a sample into a column and the column in a liquid chromatography device, for sending the sample band to the column. It is a sample band dividing device for dividing a sample band, which includes a first liquid feeding port and a dividing port for dividing the sample band sent from the first liquid feeding port.

Further, in the sample band dividing device, the sample band dividing device is a sample band dividing device for dividing a sample band in a direction intersecting in the axial direction.

Further, in the sample band dividing device, the dividing port is configured to include a second liquid feeding port for sending the mobile phase to the column and / and a first discharging port for discharging a part of the sample band. It is a sample band dividing device characterized by.

Further, in the sample band dividing device, the capacity of the flow path from the flow path switching point between the first liquid supply port and the second liquid supply port or the first discharge port to the column inlet is 1 of the column void capacity. It is a sample band dividing device characterized by being ／ 13.9 or less.

Further, in the sample band dividing device, the capacity of the flow path from the flow path switching point between the first liquid supply port and the second liquid supply port or the first discharge port to the column inlet is 1 of the column void capacity. It is a sample band dividing device characterized by being / 21.1 or less.

Further, in the sample band dividing device, the capacity of the flow path from the flow path switching point between the first liquid supply port and the second liquid supply port or the first discharge port to the column inlet is 1 of the column void capacity. It is a sample band dividing device characterized by being / 21.9 or less.

Further, in the sample band dividing device, the capacity of the flow path from the flow path switching point between the first liquid supply port and the second liquid supply port or the first discharge port to the column inlet is 1 of the column void capacity. It is a sample band dividing device characterized by being / 259 or less.

Further, the sample band dividing device is provided with a flow path switching valve.

Further, in the sample band dividing device, the flow path switching valve is a sample band dividing device characterized by being a three-way valve, a four-way valve, a six-way valve, an eight-way valve, or a ten-way valve.

Further, the sample band dividing device is characterized in that it is provided with a three-way or four-way joint.

Further, the sample introduction device is provided with the sample band dividing device.

Further, the sample introduction device is characterized in that the sample introduction device is provided with a second discharge port for discharging a part of the sample band upstream of the sample injection device.

Further, it is a liquid chromatography apparatus provided with the sample introduction apparatus.

Further, the liquid chromatography apparatus is a liquid chromatography apparatus including a column having an inner diameter of 4.6 mm or less.

Further, the liquid chromatography apparatus is characterized in that the peak elution volume is 10.2 mL or less.

Further, in liquid chromatography, sample introduction is characterized in that a sample band is divided in a flow path in front of the column from a sample injection device by switching the flow path, and at least a part of the divided sample band is introduced into the column. The method.

Further, in the above sample introduction method, when the sample band straddles the column inlet or the flow path switching point closest to the column inlet, the flow path is switched and the sample band is divided. The method.

Further, in the sample introduction method, the sample introduction method is characterized in that the volume of a part of the sample bands to be introduced into the column at one time among the divided sample bands is 1/13.9 or less of the column void volume. Is.

Further, the sample introduction method is characterized in that the sample band is divided in a direction intersecting the axial direction of the sample band.

Further, in the above sample introduction method, the sample introduction method is characterized in that all of each portion of the divided sample band is introduced into the column.

The sample introduction method is characterized in that a part of the divided sample band is introduced into the column.

Further, in the above sample introduction method, the sample introduction method is characterized in that the front portion of the divided sample band is introduced into the column.

Further, in the sample introduction method, the sample introduction method is characterized in that an intermediate portion of the divided sample band is introduced into the column.

Further, in the above sample introduction method, the sample introduction method is characterized in that the rear portion of the divided sample band is introduced into the column.

Further, in the above sample introduction method, the sample introduction method is characterized in that a portion of the divided sample band other than the portion introduced into the column is discharged.

Further, in the above sample introduction method, the rear portion including the intermediate portion of the sample band was placed in the flow path without being introduced into the column, and the front portion of the divided sample band was introduced into the column or discharged from the flow path. After that, the sample introduction method is characterized in that the rear portion of the sample band placed in the flow path is divided and the intermediate portion of the sample band is introduced into the column.

Further, in the above sample introduction method, a chromatograph device configured by connecting a pump, a sample injection device, a column, and a detector in this order is used, and the front part of the sample band injected from the sample injection device enters the column to enter the sample. Stop the feed from the first feed port that feeds the sample band to the column when the band straddles the column inlet or when the sample band straddles the flow path switching point closest to the column inlet on the flow path. , Start the liquid feeding from the second liquid feeding port provided near the column inlet, divide the sample band, introduce the front part of the sample band into the column, and the rear part of the sample band from the sample injection device. It is a sample introduction method characterized by being fastened in the connecting pipe of.

Further, in the above sample introduction method, a chromatograph device configured by connecting a pump, a sample injection device, a column, and a detector in this order is used, and the front part of the sample band injected from the sample injection device enters the column to enter the sample. Stop the feed from the first feed port that feeds the sample band to the column when the band straddles the column inlet or when the sample band straddles the flow path switching point closest to the column inlet on the flow path. Alternatively, the liquid feeding is maintained, the liquid feeding is started from the second liquid feeding port provided near the column inlet, the sample band is divided, the front part of the sample band is introduced into the column, and the vent valve. Is opened, the liquid feeding from the first liquid feeding port is restarted, or the liquid feeding is maintained, and the rear part of the sample band is discharged from the first discharge port provided in the flow path near the column inlet. This is a sample introduction method.

Further, in the above sample introduction method, a chromatograph device configured by connecting a pump, a sample injection device, a column, and a detector in this order is used, and a first discharge port is created between the sample injection device and the column to inject the sample. When the front part of the sample band injected from the apparatus enters the column and the sample band straddles the column inlet, or when the sample band straddles the flow path switching point closest to the column inlet on the flow path, the above. This is a sample introduction method characterized by opening the first discharge port and discharging the rear portion of the sample band.

Further, in the above sample introduction method, the injected sample band is divided by the liquid feed from the second liquid feed port, and the liquid is discharged between the pump and the sample injection device via a joint and a valve. A second discharge port is provided so that liquid can be discharged at any time by opening and closing the valve. After the sample band is divided, the rear part of the sample band placed in the first liquid feed port is fed from the second liquid feed port. This is a sample introduction method, characterized in that the sample is pushed back through the sample injection device and discharged from a second discharge port provided upstream of the sample injection device.

Further, in the above sample introduction method, the injected sample band is divided by the liquid feeding from the second liquid feeding port, the rear part of the sample band is placed in the first liquid feeding port, and the sample band introduced into the column. After the sample component contained in the front part is eluted from the column, the rear part of the sample band placed in the first liquid feeding port is introduced into the column by restarting the liquid feeding from the first liquid feeding port. This is a characteristic sample introduction method.

Further, in the above sample introduction method, the rear part of the sample band placed in the first liquid feeding port is further divided, and at least a part thereof is introduced into the column by restarting the liquid feeding from the first liquid feeding port. It is a sample introduction method characterized by.

Further, in the above sample introduction method, the range of the ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) from the time of sample injection to the sample band division shall be 10/0 to 1/5. It is a sample introduction method characterized by.

According to the present invention, in liquid chromatography, a sample band extended in the flow path from the sample injection device to the separation column is divided at the column inlet in a direction intersecting the axial direction of the sample band and introduced into the column. By reducing the dispersion of the sample band, it became possible to improve the separation performance of the column, that is, the number of theoretical plates.

Schematic diagram of the sample introduction device Sample band division method 1 Example process diagram The figure which shows the embodiment of the sample band dividing apparatus The figure which shows the embodiment of the sample band dividing apparatus and the dividing process. The figure which shows the Example of the sample band dividing apparatus The figure which shows the Example of the sample band dividing apparatus The figure which shows the Example of the sample band dividing apparatus The figure which shows the Example of the sample band dividing apparatus The figure which shows the Example of the sample band dividing apparatus The figure which shows the Example of the sample introduction apparatus which has a vent structure. The figure which shows the Example of the sample band dividing apparatus which has a vent structure. The figure which shows the Example of the sample band dividing apparatus which has a vent structure. The figure which shows the Example of the sample band dividing apparatus which has a vent structure. The figure which shows the Example of the sample band dividing apparatus which has a vent structure. The figure which shows the Example of the sample band dividing apparatus which has a vent structure. The figure which shows the Example of the sample band dividing apparatus which has a vent structure. The figure which shows the Example of the sample band dividing apparatus which has a vent structure. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Chromatogram of analysis results using the sample band division method of the present invention and the injection method of the comparative example. Graph diagram showing the correlation between the liquid feed capacity from the first liquid feed port and the introduction rate to the column until the sample band is divided.

The present invention includes the sample injection device and the sample injection device to the column inlet in the sample band spread outside the column that occurs for the sample band injected from the sample injection device in liquid chromatography including HPLC and UHPLC. The sample band spread in the flow path including the piping of the sample band is cut in the direction intersecting the axial direction of the sample band in front of the column entrance, specifically, in the sample band dividing device provided between the sample injection device of the sample introduction device and the column. By dividing and introducing a part of the sample band into the column, the dispersion of the solute band in the sample injection device (inj) and the pipe (tube1) from the sample injection device to the column inlet, that is, (σ _inj ) of the formula 2 ² + σ _tube1 ² ) is reduced to reduce the dispersion of the solute band outside the column (σ _extra ² ), and (σ _total ² ) in equations 1 and 4, that is, the dispersion of the solute band throughout the separation system. The sample band dividing device, the sample band introducing device, and the sample band are divided and introduced into the column to increase the column performance (theoretical number of stages N, equation 4), that is, to improve the separation ability actually provided by the column. This is a sample band introduction method.

Liquid chromatography includes analysis and preparative use. The amount of the sample to be injected from the sample injection device is not particularly limited, but in the case of analytical HPLC, it is appropriate for a sample having a minute volume of 0.01 to 5 μL, and in the case of preparative HPLC, it is appropriate depending on the size of the device and column. Can be injected in large amounts. Further, the chromatography device using the method of the present invention or installing the sample introduction device of the present invention is a known device configured by connecting a pump, a sample injection device, a column, and a detector in this order.

Also in the present invention, when the sample band reaches the column inlet, more specifically, when the sample band is injected and then the front part of the expanded sample band enters the column, that is, the sample band straddles the column inlet. When the sample band is straddling the flow path switching point closest to the column inlet, the sample band is cut by switching the flow path, and a part of the sample band, for example, the front part and the rear part (tail). Separation of the column by reducing the volume and dispersion of the sample band entering the column by dividing it into parts) and allowing only the front part of the sample band to enter the column without allowing the rear part to enter the column at least temporarily. A method and device for improving performance. Of the portions where the sample band is cut and divided, the portion forward in the traveling direction is referred to as a front portion, the portion rearward in the traveling direction is referred to as a rear portion, and the portion other than the front portion and the rear portion is referred to as an intermediate portion.

The sample injection device includes, for example, an injector using a 6-way switching valve or a similar valve, an auto-injector, and an auto sampler, and a known one generally used in HPLC or UHPLC can be used.

The method of cutting the sample band near the column inlet to divide the sample band and introducing the divided sample band into the column, the sample band dividing device for dividing the sample band and introducing it into the column, and the sample introducing device are shown below. Methods and devices can be adopted.

(1) Division of sample band-Sample introduction method and equipment with rear partial placement When a sample is entering the column inlet from the pipe (first liquid feed port) from the sample injection device, the joint between the pipe and the column (that is, that is) At the column inlet), the sample band is divided into two or three or more by starting the supply of the mobile phase solvent from a route (second liquid feeding port) different from the first liquid feeding port (FIGS. 1 and 2). .. At this time, if the supply of the mobile phase solvent from the first liquid feed port is stopped, only the front part of the sample band enters the column, and the rear part of the sample band is kept in the first liquid feed port (FIG. 2 (4)). ~ (6)). After that, the components in the front part of the sample band introduced into the column can be separated by the liquid feeding from the second liquid feeding port. FIG. 1 shows an outline of a sample introduction device provided with a sample band dividing device having a second liquid feeding port, and FIGS. 3, 4, and 5 show embodiments and examples of the sample band dividing device. .. As will be described later, when the separation analysis of the rear part of the sample band is not necessary, a discharge port (vent) may be provided at an appropriate position upstream or downstream of the sample injection device to discharge the rear part. Yes (Fig. 6 and Fig. 7).

(2) Division of sample band-Sample introduction method and equipment with rear partial discharge At the connection between the piping from the sample injection device and the column inlet, the second liquid feed port that supplies the mobile phase solvent by another route, and A third port (first discharge port or second discharge port) is provided to allow liquid to be discharged from the flow path by operating a valve, and the sample band is fed from the second liquid feeding port when the front part is entering the column. By starting the liquid and starting the discharge from the third port, the sample band can be cut, the rear part of the sample band can be discharged from the flow path, and only the front part of the sample band can be introduced into the column (Fig.). 6 and FIG. 7).

(3) Sample introduction method and equipment that involves sample division by partial discharge from the sample band At the connection between the piping from the sample injection device and the column inlet, a port that allows liquid to be discharged from the flow path by valve operation (first discharge) A port) is provided, and when the front part of the sample band enters the column and the rear part (tail part) of the sample band remains in the pipe, the sample band is discharged from the first discharge port to the rear of the sample band. The portion can be discharged from the flow path and only the front portion of the sample band can be introduced into the column (FIG. 8).

Further, in these methods, at the sample band dividing portion of the sample band dividing device, a second liquid feeding port and / and a dividing port composed of a first discharging port for discharging a part of the sample band are used. The sample band injected from the sample injection device is divided in the direction intersecting the axial direction (traveling direction) of the sample band, the front part of the sample band is introduced into the column, and the rear part of the sample band is discharged. Discharge and introduce only the rear part of the sample band into the column, discharge the front and rear parts of the sample band and introduce only the middle part into the column, or part or all of the sample band continuously. It can be divided and the divided portions can be continuously introduced into the column at predetermined intervals. That is, the sample band can be divided into two or more, one or more divided portions can be introduced into the column, and the other portions can be discharged.

By the above method, a part of the sample band, for example, the rear part (tail part) is discharged, and only the other part of the sample band, for example, the front part is introduced into the column to reduce the dispersion of the sample band and improve the column performance. It is possible to improve. By reducing the dispersion of the sample band entering the column, it is possible to reduce the peak width of the chromatogram.

Next, a sample introduction method involving division of the sample band and post-partial placement will be described in detail. In carrying out this method, as shown in FIG. 1, a sample is a liquid chromatograph device provided with a sample band dividing device 10 having a sample band dividing portion 11 for dividing the sample band in the vicinity of the column 1 inlet. A liquid chromatograph device equipped with an introduction device 91 is used. The sample introduction device 91 is configured to include a sample band dividing device 10 and a column 1 from the liquid feeding pump 21 through the sample injection device 22 and the connecting pipe 23, and the sample band dividing device 10 includes the first liquid feeding port 20 and others. In addition, a second liquid feeding port 29 capable of separately supplying the mobile phase solvent and a flow path 28 to the inlet of the column 1 can be provided. Further, the sample band dividing device may be provided in the sample introduction device together with the second discharge port 601 as shown in FIG. 6, or may be provided in the first discharge port 60 as shown in FIG. ..

As shown in FIG. 1, the sample band dividing portion 11 includes a flow path switching point 12 for switching the flow paths of the first liquid feeding port and the second liquid feeding port, and the first liquid feeding port near the flow path switching point 12. The tip portion of 20 is provided, the tip portion of the second liquid feeding port 29, and the flow path 28 to the inlet of the column 1 are provided. The sample band is cut by switching the liquid feed at the flow path switching point 12 which is the outlet of the first liquid feed port. In FIG. 1, a mobile phase is supplied to the second liquid feeding port 29 by a route different from that of the first liquid feeding port 20 or from another liquid feeding pump (not shown). A filter 25 may be installed at the tip of the column 1.

As shown in FIG. 1, the sample is supplied from the sample injection device 22 to the column 1 through the first liquid feeding port 20. The first liquid feeding port 20 may be arranged between the connection pipe 23 connected to the sample injection device 22 and the column 1, or may be arranged between the sample injection device 22 and the column 1. The structure is such that the mobile phase solvent and the sample are sent to the column 1 only through the connecting pipe 23 and the first liquid feeding port 20 or the first liquid feeding port 20.

FIG. 2 shows the progress of the sample band 3 in the first liquid feeding port 20 from the sample injection device 22 to the column 1 and the operating status of the pump (+ = on, 0 =) in the sample introduction device 91 and the sample band dividing device 10. It is a liquid transfer process diagram which shows off, A shows the liquid feed pump 21 for sending liquid from the first liquid feed port 20, and B shows the liquid feed pump 291) for sending liquid from the second liquid feed port 29. As shown in FIGS. 2 (1) to 2 (3), the operation of the liquid feeding pump 21 causes the sample band 3 to advance in the first liquid feeding port 20, and as shown in FIG. 2 (4), the sample band 3 travels. When 3 straddles the flow path switching point 12 of the sample band dividing portion 11, the liquid feeding pump 21 is stopped to stop the liquid feeding from the first liquid feeding port 20, and at the same time, the liquid feeding pump 291 is operated. When the liquid feeding is started from the second liquid feeding port 29, the sample band 3 is divided by the sample band dividing portion 11 as shown in FIGS. 2 (5) to 2 (6), and the front portion 31 of the divided sample band 3 is divided. It is pumped into the column 1 from the column inlet, and the rear portion 32 of the sample band 3 is retained in the first liquid feed port 20.

Instead of exchanging the liquid feeding states of the two pumps, the liquid feeding pump 21 and the liquid feeding pump 291, a flow path switching valve is installed at the outlet of the liquid feeding pump 21, and the flow path is continued while the pump is feeding liquid. By switching, it is also possible to stop the liquid feeding from the first liquid feeding port 20 and start the liquid feeding from the second liquid feeding port 29 at the same time.

In the division of the sample band 3, when the front part of the sample band 3 injected from the sample injection device enters the column, the liquid feeding from the first liquid feeding port for sending the sample band 3 to the column 1 is stopped, and the column is divided. The liquid feeding from the second liquid feeding port provided near the inlet may be started. Further, the liquid transfer from the second liquid supply port 29 is continuously performed in advance at the same time as the liquid transfer from the first liquid supply port 20 before the sample is injected, and the liquid supply from the first liquid supply port 20 is performed when the sample band is divided. In the method of stopping and continuing the liquid feeding from the second liquid feeding port 29, the liquid feeding from the second liquid feeding port 29 is performed in advance at the same time as the liquid feeding from the first liquid feeding port 20 before the sample is injected. It is also possible to adopt a method in which the liquid feeding from the first liquid feeding port 20 is stopped at the time of dividing the sample band to increase the liquid feeding amount (flow velocity) from the second liquid feeding port 29.

After that, when the liquid feeding from the second liquid feeding port 29 is continued, the front portion 31 of the sample band 3 introduced into the column 1 is separated and eluted through a normal chromatographic separation process. According to this introduction method, the sample band is divided into a front part and a rear part (tail part) near the entrance of the column 1 by dividing the sample band 3 in an intersecting direction including a direction orthogonal to the axial direction (flow direction). , Only the front portion can be introduced into the column 1 to make the band spread (dispersion) of the sample entering the column 1 very small.

After the separation and elution of the sample band front portion 31 introduced into the column 1 is completed, if the liquid feeding from the first liquid feeding port 20 is restarted by restarting the liquid feeding pump 21, the sample band is retained in the first liquid feeding port 20. The rear portion 32 of the sample band is introduced into column 1 and a second chromatographic separation is performed. By repeating this operation, the entire sample band 3 injected at one time can be introduced into the column 1 twice or more, and each portion can be separated and eluted or separated and analyzed.

After introducing the sample band front portion 31 into the column 1, if separation analysis is not required for the sample band rear portion 32, as shown in FIG. 6, a second is provided between the liquid feed pump 21 and the sample injection device 22. A sample band is provided by providing a discharge port (vent) 601 for discharging, or as shown in FIG. 7, providing a first discharge port 60 between the sample injection device 22 and the column 1 for discharging. After introducing the front portion of the sample band into the column 1, the rear portion of the sample band can be discharged from the discharge port before the start of separation and elution of the front part of the sample band, during separation and elution, or after the end of separation and elution. These structures and operation methods will be described later.

As an example of the sample band dividing device for carrying out the method of dividing the sample band by supplying liquid from the second liquid feeding port and introducing the sample band into the column, as shown in FIG. 3, a T-shaped connector (three-way joint) ) 41 and the double tube 42 are used to form a chromatograph device having a sample band dividing device 10 having the inner tube 421 of the double tube 42 as the first liquid feeding port and the outer tube 422 as the second liquid feeding port. Can be used. The structure of this sample band dividing device makes it possible to divide the sample band of a commonly used column immediately before the solvent filter (frit) at the column inlet. By configuring the first liquid feeding port and the second liquid feeding port using a double pipe, the sample of the present invention is introduced into a column having a general column end joint (column end joint having one connecting pipe port). The method can be performed, and there is an advantage that a column end joint having a special structure having a plurality of liquid feeding channels is not required.

In FIG. 3, the T-shaped connector 41 can be connected to a general tube having an outer diameter of 1/16 inch (outer diameter 1.6 mm). The inner pipe 421 of the double pipe, that is, the first liquid feeding port 20, is a stainless steel pipe (length 10 to 25 cm) having an inner diameter of 0.05 to 0.25 mm and an outer diameter of 0.1 to 0.4 mm. The outer pipe 422 and the second liquid feeding port 29 of the double pipe connected to the inlet of the column 1 are stainless steel pipes having an inner diameter of 0.4 to 1.0 mm and an outer diameter of 1.6 mm. The flow path switching point 12 for switching the flow paths of the first liquid supply port 20 and the second liquid supply port 29 is configured in the outer pipe 422 of the double pipe. The branch pipe for feeding liquid to the second liquid feeding port is a stainless steel pipe having an inner diameter of 0.1 to 0.5 mm and an outer diameter of 1.6 mm. The size of the connecting tube is appropriately adjusted depending on the scale of the liquid chromatograph device and the flow rate range, regardless of the above range. Connections between pipes and with T-connectors and column end joints are made using appropriate ferrules and cines or nuts.

As shown in FIG. 3A, the ends of the inner pipe 421 and the outer pipe 422 of the double pipe have a structure on the same plane as the orifice inlet of the column end joint 17. Further, the inner tube may be longer than the outer tube (FIGS. 3B and 3C) or slightly shorter than the outer tube as long as the inner tube of the double tube does not damage the filter 25 and the filler layer of the column 1. .. Further, the sample can be directly introduced into the filler layer 18 through the filter 25 in the column (FIG. 3 (D)).

As shown in FIG. 3, when a double-tube inner tube 421 is used as the first liquid delivery port 20 and a double-tube outer tube 422 is used as the second liquid delivery port 29, a T-shaped connector is used to use the inner tube. The outer pipe 422 penetrates the T-shaped connector, and the outer pipe 422 is located outside one of the inner pipes 421 and is connected to one port of the T-shaped connector. When the sample band is moving from the liquid port to the column, the sample band is divided by stopping the liquid feeding from the first liquid feeding port and switching to the liquid feeding from the second liquid feeding port. A sample introduction method can be adopted in which the rear portion is kept in the first liquid feeding port and only the front portion (tip portion) of the sample band is introduced into the column.

Further, as shown in FIG. 4, a flow path switching valve for switching the liquid feed flow path from the pump, for example, a three-way, four-way, six-way, eight-way or ten-way switching valve, is used with the sample injection device 22 and the column 1. A second liquid feeding port 52 is created on the flow path near the inlet of the column 1 between the two, and the sample band is cut at the flow path switching point 50 using the second liquid feeding port 52, the sample band is divided into a front part and a rear part, and a loop is formed. Even with a structure having a function of indwelling in 53 or discharging from the loop cleaning pipes 57 and 58 using a separate liquid feed pump, the sample introduction method including division of the sample band and partial placement of the sample can be carried out.

As shown in these examples, by dividing the sample band at the flow path switching point near the column 1 inlet, the sample injection device and the sample diffused in the piping from the sample injection device to the column inlet are extremely dispersed. Can be made smaller. If the capacity of the flow path from the flow path switching point to the column inlet is large, the divided sample band will diffuse before being introduced into the column, so the capacity of the flow path from the flow path switching point to the column inlet The smaller the value, and if other factors affecting the dispersion of the solute are constant, the smaller the ratio of the capacity of the flow path from the flow path switching point to the column inlet / the void capacity of the column, the sample of the present invention. The effectiveness of the introduction method is high.

Below, three examples of the volume / column void volume ratio from the flow path switching point to the column inlet are shown from the examples in which the effectiveness of the sample introduction method of the present invention was confirmed.

In Examples 1 to 9, 12 and 13 (FIGS. 10 to 18, 21 and 22), since the same sample dividing device is used, the capacitance from the flow path switching point to the column inlet is the same. This volume is determined from the elution volume of naphthalene in the second chromatogram of Example 4 (E) of 0.65 μL (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.013 min). The zero dead volume union (ZDU, capacity 0.02 μL), the capacity from the ZDU outlet to the detector cell inlet (about 0.55 μL), and the detector cell capacity 6 nL are subtracted to calculate 0.07 μL. Non-Patent Document 2 is referred to as a method for measuring the capacity of the device flow path based on the measured value. Regarding the porosity of the column, the porosity of InertStain C18 (filler diameter 2 μm, inner diameter 1.0 mm, length 5 cm, column tube capacity about 39.3 μL) was measured for uracil in Example 3 and FIG. 12 (A). The value (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.40 min) and the measured value of naphthalene in Example 4 and FIG. 13 (B) (measured value at a flow rate of 0.05 mL / min). , 0.05 mL / min × 0.037 min), calculated as 20.0-1.85 = 18.15 μL, and further, the volume of ZDU used in Example 4 and FIG. 13 (B) is 0.02 μL. Is calculated to be 18.13 μL, and the porosity is about 46.1%.

In these calculations, the response of the detector is 0.01 seconds without the column, the peak top value of the chromatogram at the data acquisition interval of 5 mSec, and the response of the detector with the column is 0. At 05 seconds, the data acquisition interval uses the peak top value of the chromatogram at 50 mSec, and it seems that the contribution to the original peak shape and retention time is small, so the response of the detector and the data acquisition interval are calculated. Not put in. From these values, the capacity from the flow path switching point to the column inlet / the void capacity of the column = 1/259. As will be described later, in Example 3, 1.2 to 2.8 times as many theoretical plates are generated by the sample introduction method of the present invention as compared with the conventional injection method. The rate of increase in the number of theoretical plates is higher for peaks with a smaller retention capacity, and in particular, the number of theoretical plates for non-retention (k = 0) uracil peaks is 2.8 times higher. Since the uracil peak passes through the column, the volume required for the uracil peak to elute only from the column is the void volume (18.13 μL) of the column.

On the other hand, in Examples 14 and 15 (FIGS. 23 and 24), the sample dividing device using the micro 6-way flow path switching valve shown in FIG. 4 is used, and the column inlet from the micro 6-way flow path switching valve is used. Since the pipe up to is 0.13 mm in inner diameter and 6.5 cm in length, the capacity from the flow path switching point to the column inlet is at least 0.86 μL or more, and the flow path inside the micro 6-way flow path switching valve The value is the sum of the capacitance (because the valve has a flow path diameter of 0.25 mm, the length of the groove of the rotor seal is 5 mm and is about 0.25 μL). The average value of the elution volume of uracil obtained from the second to sixth chromatograms of Example 15 and FIG. 24 (F) is 2.45 μL (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min ×). It is 0.049 min), and is calculated as 1.304 μL by subtracting the ZDU (capacity 0.02 μL), the capacity from the ZDU outlet to the detector cell inlet (about 1.12 μL), and the detector cell capacity 6 nL. Non-Patent Document 2 is referred to as a method for measuring the capacity of the device flow path based on the measured value. Regarding the porosity of the column, the porosity of InertStain C18 (filler diameter 2 μm, inner diameter 1.0 mm, length 5 cm, column tube capacity about 39.3 μL) was measured by uracil in Example 14 and FIG. 23 (D). The value (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.53 min) and the measured value of uracil in Examples 15 and 24 (D) (measured value at a flow rate of 0.05 mL / min). , 0.05 mL / min × 0.167 min), calculated as 26.5-8.35 = 18.15 μL, and further, the volume of ZDU used in Example 15 and FIG. 24 (D) is 0.02 μL. Is calculated to be 18.13 μL, and the porosity is about 46.1%.

In these calculations, the response of the detector is 0.01 seconds without the column, the peak top value of the chromatogram at the data acquisition interval of 5 mSec, and the response of the detector with the column is 0. At 05 seconds, the data acquisition interval uses the peak top value of the chromatogram at 50 mSec, and it seems that the contribution to the original peak shape and retention time is small, so the response of the detector and the data acquisition interval are calculated. Not put in. From these values, the capacity from the flow path switching point to the column inlet / the void capacity of the column = 1 / 13.9. Further, at least 0.86 μL / 18.15 μL = 1 / 21.1 or more. As will be described later, comparing Example 14, FIGS. 23 (D) and (E), the number of theoretical plates generated by the sample introduction method of the present invention is 1.03 to 3.06 times that of the conventional injection method. doing. The rate of increase in the number of theoretical plates is higher for peaks with a smaller holding capacity.

On the other hand, in Examples 16 and 17 (FIGS. 25 and 26), the sample band dividing device using the cloth shown in FIG. 7B is used, and the inner diameter of the pipe from the cloth to the column inlet is 0. Since 03 mm and 6 cm in length are used, the capacitance from the flow path switching point to the column inlet is at least 0.042 μL or more. Regarding the porosity of the column, the porosity of MonoCap C18 (silica monolith column, inner diameter 0.1 mm, length 15 cm, column tube capacity of about 1.18 μL) was measured for thiourea in Example 16 and FIG. 25 (A). The value (measured value at a flow rate of 0.002 mL / min, 0.002 mL / min × 0.77 min) and the measured value of naphthalen in Examples 17 and 26 (A) (measured value at a flow rate of 0.003 mL / min). , 0.003 mL / min × 0.18 min), calculated as 1.54-0.54 = 1.00 μL, and further, of the fused silica capillary tube used in Example 17, FIG. 26 (A). By subtracting the capacitance of 0.042 μL and the capacitance of the zero dead volume union for connecting the fused silica capillary tube inlet / outlet (0.02 μL × 2), it is calculated as 0.918 μL, and the porosity is about 77.8%. It becomes.

In these calculations, the response of the detector is 0.01 seconds without the column, the peak top value of the chromatogram at the data acquisition interval of 5 mSec, and the response of the detector with the column is 0. At 05 seconds, the data acquisition interval uses the peak top value of the chromatogram at 50 mSec, and it seems that the contribution to the original peak shape and retention time is small, so the response of the detector and the data acquisition interval are calculated. Not put in. From these values, the capacity from the flow path switching point to the column inlet / the void capacity of the column = 1 / 21.9. As will be described later, in Example 16, the number of theoretical plates is 2.5 to 5.0 times that of the conventional injection method, depending on the sample introduction method of the present invention. The rate of increase in the number of theoretical plates is higher for peaks with a smaller holding capacity.

As described above, among the ratios of the volume from the flow path switching point to the column inlet / the void volume of the column in which the effectiveness of the sample introduction method of the present invention was confirmed, the largest value was 1 / 13.9. there were. At a ratio of values smaller than this, the effectiveness of the sample introduction method of the present invention will be exhibited.

As an example of the sample band dividing device, the sample band introducing device, and the sample band dividing and introducing method, FIG. 4 shows a sample band dividing device 10 and a sample band introduction using the micro 6-way flow path switching valve 5 as the flow path switching valve piping. The structure of the apparatus 93, the division of the sample band using the apparatus 93, and the process of introduction into the column are shown. FIG. 4A shows a state in which the sample band 3 begins to enter the flow path of the flow path switching valve 5 from the sample injection device 22, and FIG. 4B shows a state in which the sample band 3 reaches the entrance of the column 1. It shows a state of straddling the adjacent flow path switching point 50 (liquid feeding port switching point). FIG. 4C shows a state in which the sample band 3 is divided into two by switching the flow path from FIG. 4B, and from this state, as shown in FIG. 4D, from the second liquid feeding port 52. The sample band front portion 31 enters the column 1 by the liquid feeding, and the components contained in the sample band front portion 31 are separated and eluted. In the state of FIG. 4D, it is possible to discharge the sample band rear portion 32 indwelled in the first liquid feeding port 51 and the loop 53, but after the elution of the sample band front portion 31 is completed, FIG. As shown in (E), by switching to the liquid feeding from the first liquid feeding port 51, the indwelled sample band rear portion 32 can be introduced into the column 1 and separated and eluted.

Further, when the sample band rear portion 32 is large in FIG. 4D, after the elution of the sample band front portion 31 is completed, the flow path is switched and the sample band rear portion is fed from the first liquid feeding port 51. Can be introduced into the column (FIG. 4 (E)), returned to FIG. 4 (D), and eluted by the liquid feed from the second liquid feed port 52. After that, by repeating the flow path switching of FIGS. 4 (E) to 4 (D), the sample injected at one time from the sample injection device can be introduced into the column in a large number of times and separated and eluted. ..

In the sample introduction method of the present invention, by controlling the liquid transfer rate (flow velocity) from the first liquid supply port and the time from sample injection to division of the sample band and introduction into the column, the entire sample band can be used. The ratio of the portion introduced into the column, that is, the sample introduction rate can be adjusted. The timing of dividing the sample band and introducing it into the column is from when the tip of the sample band reaches the column inlet or the flow path switching point to when the rear end of the sample band reaches the column inlet or the flow path switching point, that is, While the sample band straddles the column inlet or flow path switching point. The sample introduction rate can be 96 to 4%, preferably 93 to 10%.

Further, in the sample introduction method of the present invention, at the time of sample injection, not only the liquid is fed only from the first liquid feed port, but also the mobile phase solvent is partially fed from the second liquid feed port. The introduction rate of the sample band to the column can also be controlled by stopping the liquid transfer from the first liquid supply port and increasing the liquid transfer rate from the second liquid supply port when the sample is divided and introduced into the column. Is possible. The range of the ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) from the time of sample injection to the sample division can be 10/0 to 1/9.

Further, at the time of sample injection, the mobile phase solvent is partially fed from the second liquid feed port, and the ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) is adjusted. , The sample introduction rate to the column can be easily adjusted. In addition, the ratio of (first liquid feed port flow rate) / (second liquid feed port flow rate) from sample injection to sample division, which maximizes column performance, and the sample to the column without causing deterioration of column performance. You can choose the introduction rate. The ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) from the time of sample injection to the sample division is preferably 10/0 to 3/7.

As an example of the sample band dividing device, even when the column is directly connected to the injector, the first liquid feeding port is provided, and as shown in FIG. 5A, the second liquid feeding using the T-shaped connector and the double tube is used. A port 52 is provided, or as shown in FIG. 5B, a column is directly connected to the connection port of the sample injection device (injector), and a second liquid feeding port 52 is provided at the column inlet, or as shown in FIG. 5C. As shown, by arranging a T-shaped connector between the sample injection device and the column 1 and providing the second liquid feeding port 52 with a branch pipe, the present invention can be performed by the same principle and operation as in the above description and FIG. It is possible to carry out the method of sample introduction with the division of the sample band-post-partial placement of the invention.

As an example of the sample band dividing device, as shown in FIG. 5D, the sample band can be divided at the entrance of the column 1 and introduced into the column by forming the second liquid feeding port 52 in the column end joint 19. However, in that case, a column having a specially structured end joint having a second liquid feeding port 52 is required.

As an example of the sample introduction device, when the analysis of the rear portion 32 of the sample band 3 after the division of the sample band is unnecessary, a second discharge port 601 is provided upstream of the sample injection device 22 as shown in FIG. By the operation of the vent valve 62, the liquid is fed from the second liquid feed port 29 before, during, or after the separation and elution of the front portion 31 of the sample band 3 through the resistance tube 63 or without the resistance tube. Allows the rear portion 32 of the sample band 3 to be ejected.

Next, a sample introduction method involving division of the sample band and partial discharge of the sample band will be described in detail. In carrying out this method, in a liquid chromatograph device, as shown in FIG. 6, in addition to the first liquid feed port 20, a second liquid feed port 29 capable of separately supplying a mobile phase solvent, and a column 1 A sample band dividing device 10 at the inlet of column 1 having a flow path 28 to the inlet and cutting and dividing the sample band, and a sample injection device 22 and a first liquid feeding port 20 from a liquid feeding pump 21 in a normal liquid chromatograph. A sample introduction device 95 provided with a connection pipe 23 connected to the sample introduction device 95 can be used. The confluence of the first liquid supply port 20 and the second liquid supply port 29 is the flow path switching point 12. In FIG. 6, the mobile phase is supplied to the second liquid feeding port 29 by a route different from that of the first liquid feeding port 20 or from another liquid feeding device (not shown). Further, as described above, the second discharge port 601 is provided upstream of the sample injection device 22.

Then, as shown in FIG. 7A, when the sample band injected from the sample injection device (not shown) is entering the column 1 inlet (or straddling the flow path switching point 121), the first feed is performed. Stop the liquid feed from the liquid port 20 or maintain the liquid feed, cut the sample band by the liquid feed from the second liquid feed port 29, divide the sample band, and divide the sample band, and sample band from the first discharge port 60. A sample band dividing device 101 having a structure having a sample band dividing portion for discharging (venting) the rear portion (tail portion) can be used.

In FIG. 7B, a four-way joint 7 is arranged between the sample injection device (not shown) and the column 1, and in addition to the first liquid feeding port 20 and the connection port to the column 1, the second liquid feeding port is shown. It is a sample band dividing device 101 having a structure incorporating 29 and a first discharge port (vent port) 60. In the case of this configuration, the connection portion of the first liquid feed port 20, the second liquid feed port 29, and the first discharge port 60 serves as the flow path switching point 121.

When the device and method having such a configuration are used, when the sample band is passing through the central portion where the flow paths of the four-way joint (cross) 7 intersect, that is, the flow path switching point 121, in other words, straddling the flow paths. Alternatively, when the sample band straddles the column inlet, the sample band is stopped by stopping the liquid feeding from the first liquid feeding port 20 or by maintaining the liquid feeding and starting the liquid feeding from the second liquid feeding port 29. The sample band front part is introduced into column 1 by cutting and dividing, and the vent valve is opened before the start of separation and elution of the sample band, during separation and elution, or after the end of separation and elution, and the sample band is opened from the first liquid feed port. It is possible to improve the column performance by restarting or maintaining the liquid feeding and discharging (venting) the rear part of the sample band from the first discharge port 60 to reduce the dispersion of the sample band entering the column. .. A storage pipe (not shown) may be provided in the first discharge port 60, and the rear portion of the sample band sent to the first discharge port 60 may be placed in the storage pipe instead of being discharged.

In particular, in the case of a sample band that extends forward and backward (front part and rear part), the front part of the sample band is introduced into the column or discharged to the outside of the system, and then placed in the connecting pipe from the sample injection device to the column. The rear part of the sample band is divided and placed, or the operation is repeated to divide the rear part of the sample band, and only the middle part of the sample band is introduced into the column. As a result, the dispersion of the sample band entering the column can be reduced, and the separation performance of the column can be improved.

In this way, the first discharge port 60 is provided together with the second liquid feed port 29 between the sample injection device and the column 1, and the start of the liquid feed from the second liquid feed port 29 and the discharge of the rear part of the sample band are performed. As an example of the case where the sample band is divided and the front part of the sample band is introduced into the column, the injector and the column 1 are connected to the 4-way joint 7, and the other two ports are the second liquid feed port 29 and the second. 1 Used as a discharge port 60. In this case, when the sample band straddles the center of the four-way joint, the liquid feeding from the second liquid feeding port 29 is started, the sample band is divided, and the sample band is introduced into the column in front of the sample band. The rear portion of the sample band can be discharged from the first discharge port before the start of separation and elution of the solute contained in the front portion of the sample band, during the separation and elution, or after the end of the separation and elution. By installing the second liquid feed port 29 and the first discharge port 60 in another positional relationship between the sample injection device and the column, the liquid feed from the second liquid feed port can be started and the first discharge can be started in the same manner as described above. It is possible to discharge the rear part of the sample band from the port to divide the sample band and introduce the front part of the sample band into the column. Further, in either case, by arranging a storage pipe (not shown) between the first discharge port and the vent valve 62, the rear part of the sample band discharged from the flow path connecting the sample injection device and the column is temporarily separated. It is also possible to have a structure for storing the sample.

In addition, using the first liquid feeding port and the second liquid feeding port, even when the sample band straddles the center of the four-way joint, the vent valve is opened without stopping the liquid feeding from the first liquid feeding port. By starting the liquid feeding from the second liquid feeding port, the sample band may be divided and the rear portion of the sample band may be discharged. Further, the discharge of the rear portion of the sample band can be discharged from the discharge port upstream of the sample injection device as shown in FIG. In this case, not only the 4-way joint but also the sample band dividing portion using the T-shaped connector and the double tube as shown in FIG. 3 can be used.

FIG. 5E is an example of a sample band dividing device in which the first liquid feeding port 51, the second liquid feeding port 52, and the first discharging port 60 are connected to the flow path switching point provided in the column end joint. It can be used in the same manner as the sample band dividing device shown in FIGS. 7 (A) and 7 (B).

Next, an example of a sample introduction method involving sample division by discharging the rear portion (tail portion) from the sample band will be described in detail. In carrying out this method, in a liquid chromatograph device, as shown in FIG. 8, when the sample band is entering the column 1 inlet, it is opened and closed by operating a valve 62 provided immediately before the column 1 inlet. A liquid chromatograph device provided with a sample band dividing device 101 having a structure having a sample band dividing portion 11 for discharging (venting) the rear portion (tail portion) of the sample band from the first discharge port 60 can be used. You can. The sample band dividing device 101 discharges the rear portion (tail portion) of the sample band from the first discharge port 60 at the flow path switching point 13 which is a connection portion between the first liquid feeding port 20 and the first discharge port 60. , Divide the sample band.

FIG. 8A shows a sample band dividing device 101 having a structure in which the column 1 is connected to the column port 77 that communicates with the first liquid feeding port 20 and the first discharging port 60 is provided. The sample band dividing device 101 is configured to include a first liquid feeding port 20 and a first discharging port 60 without providing a second liquid feeding port. FIG. 8B shows a structure in which the outlet of the sample injection device (not shown), the sample band dividing portion 11 and the column 1 inlet are integrated by using the T-shaped connector 41 and the double tube 42, and FIG. 8C shows the first structure. A first discharge port 60 is provided on the outside of the liquid feed port 20 with a T-shaped connector 41 and a double pipe 42, and the sample band dividing portion 11 is arranged at a position very close to the column inlet. In FIG. 8D, a T-shaped connector 41 is arranged between the sample injection device and the column 1, and the branch pipe is used as the discharge port 60.

In principle, in any of FIGS. 8A to 8D, a T-shaped connector (three-way joint) is arranged between the sample injection device and the column 1, and the first liquid feeding port 20 and the connection port to the column 1 are provided. In addition, the structure incorporates the first discharge port (vent port) 60. Further, as shown in FIG. 8E, the sample band dividing device 101 is provided with a sample dividing portion 11 in the column end joint 28, that is, the tip portions of the first liquid feeding port 20 and the first discharging port 60 are connected to the column end joint. It may be provided in 28 and configured.

In the case of the configurations of FIGS. 8A to 8E, the front portion of the sample band passes through the connection portion of the first discharge port 60, that is, the flow path switching point 13, and the rear portion (tail portion) of the sample band is the flow path switching point 13. At a later time, the vent valve 62 is opened and the rear portion of the sample band is discharged from the first discharge port 60 to cut and divide the sample band so that the rear portion of the sample band is not introduced into the column. , It is possible to improve the column performance by reducing the dispersion of the sample band entering the column.

When the injected sample band enters the column inlet, or when the sample band straddles the flow path switching point 13, the liquid feeding rate (flow velocity) to the column 1 is temporarily reduced, or By not changing the flow velocity or increasing the flow velocity and opening the vent valve 62 to start the flow to the first discharge port 60, the rear portion of the sample band is separated from the first discharge port 60 and the vent valve 62. It can be placed or discharged in a storage pipe (not shown) provided between the two. When the rear portion of the indwelled sample band is introduced into the column, a liquid feed pump or the like capable of feeding liquid from the first discharge port 60 toward the column is separately required.

In this way, as an example of the case where the sample band is divided and introduced into the column by providing the first discharge port 60 between the sample injection device and the column and discharging the rear portion of the sample band, the injector And the column are connected to the three-way joint, and the branch pipe is used as the discharge port 60. In the case of FIGS. 8A and 8D, the sample band is divided by operating the vent valve 62 when the sample band straddles the flow path branch point 13, and in the case of FIGS. 8B, 8C and 8E, the sample band is divided. The sample band is divided by operating the vent valve 62 while straddling the inlet of the column 1, the rear part of the sample band is discharged from the first discharge port 60, and then the vent valve is closed and the liquid is sent to the column. By restarting, it is possible to introduce the sample band in front of the column, separate and elute, and analyze. In either case, by arranging a storage pipe (not shown) between the first discharge port 60 and the vent valve 62, the rear part of the sample band discharged from the flow path connecting the sample injection device and the column is temporarily separated. It is also possible to have a storage structure.

As shown in FIG. 9, when the T-shaped connector 73 or the 4-way joint 74 is used for the sample band dividing portion 11, FIGS. 9 (A) and 9 (B) corresponding to FIGS. 7 (B) and 8 (D). ), The sample band is divided and introduced into the column in order to prevent the diffusion of the front part of the sample band to be analyzed and the rear part of the discharged sample band from re-entering the flow path to the column 1 due to diffusion. It is preferable to have a structure in which the gap between the pipes on the flow path connecting the sample injection device and the column, which has entered the inside of the joint for performing the above, is narrowed to 0.5 mm or less. The same structure is possible in the connection form of the other sample band dividing portion (FIG. 5C).

Next, the ratio of the timing of starting liquid feeding from the second liquid feeding port and (first liquid feeding port flow velocity) / (second liquid feeding port flow velocity) will be described. In the method of dividing the sample band with the liquid feeding from the second liquid feeding port, the sample band is divided by stopping the liquid feeding from the first liquid feeding port and starting the liquid feeding from the second liquid feeding port at the same time. It can be divided and introduced into the column, but before the sample is injected, the liquid flow from the second liquid feed port is in the range of 1 to 90% of the flow velocity from the first liquid feed port. You can start it. Preliminary feeding from the second feeding port at the time of sample injection facilitates the division of the sample band and the acceleration of the second feeding port liquid feeding pump at the time of introduction into the column, thus maximizing the column performance. It is effective for enabling the introduction of the sample to be expressed in the sample and for adjusting the introduction rate of the sample band into the column.

As described above, the present invention differs from the split injection method conventionally used in capillary liquid chromatography and gas chromatography in the following points.
1. The present invention further divides a sample of sample volume (0.01-5 μL), which is standard in conventional HPLC or UHPLC and is considered to be sufficiently small by modern state of the art.
2. In the present invention, instead of venting (releasing) most of the flow of a large amount of sample solution and taking a part into the column, a standard amount of sample is divided in an axially intersecting direction. At the same time that the part (front part) is sent to the column for separation elution and analysis, a part (rear part) of the divided sample is placed in the flow path between the sample injection device and the column, or discharged to the outside of the system. ..
3. In the present invention, the rear portion of the sample placed in the flow path between the sample injection device and the column is separated and eluted after the separation and elution of the front portion of the sample is completed, or is discharged from the discharge port before and after the separation and elution. Will be done.
4. In the split injection used in conventional gas chromatography, the difference in behavior between solutes (discrimination) becomes a problem, but in the method of the present invention, does the method of the present invention cause a difference in behavior between solutes due to the division of the sample band? Since it is possible to continuously analyze the front part and the rear part of the divided sample band to see if it does not occur, it is possible to investigate by injecting a single sample.
5. In the conventional sample injection method, even the solute band provided by the minimum sample volume that gives the best performance increases the band spread when flowing from the sample injection device to the column inlet, but the present invention increases this at the column inlet. , Can be further divided and introduced into the column.

Hereinafter, a method for embodying the apparatus and the operation method of the present invention will be described as an example together with a comparative example. The following examples describe the sample band division-sample introduction method with posterior partial placement, sample band division-sample introduction method with posterior partial discharge, and sample introduction method with sample division by posterior partial discharge from the sample band. Each example is shown. In each method, these examples include column size, sample injection capacity, detector cell capacity, and (first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio from sample injection to sample division. In some combinations of the above, the sample introduction method of the present invention shows the effect of increasing the number of theoretical stages of the column as compared with the conventional sample injection method, and the example without the column shows the sample injection capacity and detection. How the injected sample band expands in some combinations of vessel cell capacity, (first liquid delivery port flow velocity) / (second liquid supply port flow velocity) ratio from sample injection to sample division, the present invention. It directly visualizes how the sample is divided and introduced into the column by the sample introduction method of. In the following example, the conventional sample injection method (incorporating the sample band in the sample loop into the flow path of the mobile phase by operating the sample injection valve) is referred to as "conventional sample injection method" or "conventional sample injection". Expressed as "law" or "injection".

For the sample introduction method with sample band division-posterior partial placement, the sample injection capacity is 0.1 to 5.0 μL, the column inner diameter is 1.0 to 4.6 mm, the column length is 5 to 15 cm, and the detector cell capacity is 1 μL. Examples include 6 nL and some combinations of ((first feed port flow velocity) / (second feed port flow velocity) ratios from sample injection to sample splitting. On the chromatograms of FIGS. 10-28. The numerical value attached to the peak of is the peak half price width (unit: min), and in the case of Gaussian distribution, it corresponds to 2.35σ (σ: standard deviation) of the peak. From this numerical value and the holding time (t _R ). It can be directly understood that the theoretical number of columns can be calculated (Equation 4), and that the bandwidth, that is, the peak dispersion (σ ² ) can be reduced by the sample introduction method based on the present invention. Table 1 shows the method of dividing the sample band in each Example and Comparative Example, the column size, the detector cell capacity, the sample injection capacity, and the relationship between each Example.

In order to confirm the improvement of column performance by the sample introduction method involving division of the sample band and post-partial placement, the conventional sample injection method (FIG. 10 (A)) (comparative example) and the sample introduction method of the present invention (FIG. 10 (B)) )) The column performance was compared.

Among the sample introduction methods of the present invention, in the sample introduction method involving division of the sample band and placement of the rear portion, the sample band injected with 0.4 μL is divided into two and introduced into the column, and the first from the front portion of the sample band. This is an example showing an improvement in the number of theoretical plates in the chromatogram and a second chromatogram provided by the rear part of the sample band fastened to the first liquid feed port.

A UHPLC device LC800 (GL Sciences, Inc.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection device, and a column acetonitrile C18 (filler diameter 2 μm, inner diameter 1.0 mm) was used. , 5 cm in length, GL Sciences, Inc.) was used to deliver the mobile phase (acetonitrile / water = 60/40) at 0.05 mL / min. The temperature condition was room temperature. The room temperature is 24.5 ± 0.5 ° C. (the same applies hereinafter). For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. As the connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. Samples include sample I: uracil (U, 0.13 mg / mL), methyl benzoate (MB, 1.8 mg / mL), toluene (T, 2.6 mg / mL), naphthalene (N, 0.7 mg / mL). mL) A mixture of acetonitrile / water = 60/40 was used.

The sample band dividing portion of the sample band dividing device used in this example has the structure shown in FIG. 3 (A), uses a T-shaped connector U-428 (inner diameter 0.5 mm, IDEX), and has a double tube. , A stainless steel pipe (capacity: about 0.92 μL) with an outer diameter of 0.236 mm, an inner diameter of 0.108 mm, and a length of 10 cm for the connecting pipe from the sample injection valve to the column inlet (inner pipe for liquid feeding at the first liquid feeding port). The outer pipe for liquid feeding at the second liquid feeding port was composed of a stainless steel pipe having an outer diameter of 1.6 mm, an inner diameter of 0.5 mm, and a length of 5 cm. A stainless steel pipe having an outer diameter of 1.6 mm and an inner diameter of 0.25 mm was used as the connecting pipe (branch pipe) for supplying liquid to the second liquid feeding port of the T-shaped connector.

FIG. 10 (A) is a chromatogram obtained by injecting 0.4 μL of sample I and separating the sample I using a conventional sample injection method. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at a flow rate of 0.05 mL / min.

In FIGS. 10 to 28, the numerical value on each peak indicates the bandwidth (half width, unit: min) at the 50% peak height. This full width at half maximum corresponds to 2.35σ (σ: standard deviation) in the case of Gaussian distribution, and the variance of the peak and the theoretical plate number of the column (N, equation 4) can be calculated from this value. When the variance of the peak (σ ² , Equation 1) is expressed in the volume dimension, the flow velocity (mL / min) is used to convert σ into the volume dimension.

In FIG. 10 (B), 0.4 μL of sample I was injected under the same mobile phase conditions as in FIG. 10 (A), and 0.03 minutes after the injection, the first liquid feed port was stopped and the second liquid feed port was stopped. 4. Start the liquid transfer (acetonitrile / water = 60/40, 0.05 mL / min) to divide the sample band, introduce the front part of the divided sample band into the column for elution separation, and perform elution separation. After 0 minutes, stop the liquid feeding from the 2nd liquid feeding port, return to the liquid feeding from the 1st liquid feeding port (acetonitrile / water = 60/40, 0.05 mL / min), and retain it in the 1st liquid feeding port. This is a chromatogram obtained by introducing the rear part of the sample band into a column and separating and elution.

In FIG. 10 (A) (comparative example), the number of theoretical plates 450, 2540, 4400, and 5110 are obtained for U, MB, T, and N, respectively. In contrast, about 60% of the injected sample was left in the first liquid delivery port by splitting the sample band, and about 40% was introduced into the column in the first half chromatogram of FIG. 10 (B) (first). In the chromatogram), the bandwidth of each peak is narrowed by dividing the sample band and introducing the front part of the sample band into the column, and the theoretical plates 960, 4640, 6120 and 6740 are used for U, MB, T and N, respectively. Is obtained, and it is possible to generate about 1.2 to 2.1 times as many theoretical plates as in FIG. 10 (A).

As shown in the chromatogram (second chromatogram) in the latter half of FIG. 10B, the liquid was returned from the first liquid feed port 4.0 minutes after the sample injection, so that 0.03 from the sample injection. The rear part of the sample band, which had been left in the first liquid feed port tube since the division of the sample band after minutes, was introduced into the column and separated and eluted. The peaks all show a smaller bandwidth and dispersion than the elution peak in FIG. 10 (A) (Comparative Example), which was eluted without dividing the sample band. That is, all of the sample bands introduced into the column divided into two in the direction intersecting the axial direction are separated with high performance as compared with the case where the sample bands are injected without being divided.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, as compared with the conventional sample injection method (FIG. 11 (A)) (comparative example), the division of the sample band of the present invention-after The reduction in sample bandwidth by the sample introduction method with partial placement (FIG. 11B) was demonstrated without a column.

Among the sample introduction methods of the present invention, in the sample introduction method involving division of the sample band and placement of the rear portion, the sample band injected with 0.4 μL is divided into two and introduced into the zero dead volume union, and the sample band is introduced from the front portion of the sample band. The first chromatogram of the sample band and the second chromatogram provided by the rear part of the sample band held in the first liquid feeding port are shown in this example. It should be noted that each peak on the chromatogram includes band spread in the connecting tube and the detection cell. 11 (A) and 11 (B) can be considered to represent the state of the sample band at the column entrance in FIGS. 10 (A) and 10 (B), respectively.

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as the sample injection device, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU) was used. The same sample band dividing portion as in Example 1 having the structure shown in FIG. 3 (A) was used. The mobile phase (acetonitrile / water = 60/40) was sent at 0.05 mL / min, and the temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The response of the detector was 0.01 seconds, and the data acquisition interval was 5 mSec. As the connecting tube from the ZDU outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

In FIG. 11 (A), 0.4 μL of sample naphthalene is injected using the conventional sample injection method, and the liquid is fed from the first liquid feed port (acetonitrile / water = 60/40, 0.05 mL / min). It is an eluted chromatogram. FIG. 11B is a chromatogram in which 0.4 μL of sample naphthalene was divided into two parts using the sample introduction method of the present invention, introduced into ZDU, and eluted. 0.03 minutes after sample injection, the liquid feed from the first liquid feed port (acetonitrile / water = 60/40, 0.05 mL / min) is stopped, and the liquid feed from the second liquid feed port (acetonitrile / water =). 60/40, 0.05 mL / min) is started to divide the sample band, the front part is introduced into ZDU and eluted (first chromatogram), and 0.2 minutes later, the sample band is fed to the second liquid feed port. Stop the liquid, return it to the liquid (acetonitrile / water = 60/40, 0.05 mL / min) from the first liquid feed port, and return to the ZDU at the rear part of the sample band that was retained in the first liquid feed port. Is introduced and eluted (second chromatogram).

In the second chromatogram of FIG. 11 (B), the sample left in the first liquid feed port when the sample band was divided 0.03 minutes after the sample injection was eluted, and the sample band was eluted near the ZDU inlet. Is divided into two in the direction of intersecting the axial direction, and it is directly shown that each of them is introduced into the ZDU. Compared with the peak of FIG. 11A (Comparative Example), each peak of the two chromatograms of FIG. 11B has a reduced bandwidth.

In both Examples 1 and 2, after the sample was injected, each peak passed through the cell (capacity 1 μL) of the UV detector regardless of whether or not the sample band was divided. Therefore, on the chromatograms shown in FIGS. 10 and 11. Each peak contains a connecting tube from the column outlet or ZDU outlet to the detector cell, and the dispersion of the solute band in the detector cell.

In order to confirm the improvement of column performance by the sample introduction method involving division of the sample band and post-partial placement, the conventional sample injection method (FIGS. 12A and 12B) (comparative example) and the sample introduction method of the present invention (comparative example) The column performances in FIGS. 12 (C), 12 (D), and (E)) were compared.

Among the sample introduction methods of the present invention, the sample introduction method involving division of the sample band and placement of the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band and keeps the sample in the first liquid feeding port. This is an example showing the second chromatogram given by the posterior part of the sample band.

A UHPLC device LC800 (GL Sciences, Inc.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection device, and a column acetonitrile C18 (filler diameter 2 μm, inner diameter 1.0 mm) was used. , 5 cm in length, GL Sciences, Inc.) was used to deliver the mobile phase (acetonitrile / water = 60/40) at 0.05 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. For the connecting pipe from the column outlet to the detector cell entrance, a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm is connected to the ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected and used. As the sample band dividing device, the same sample band dividing portion as in Examples 1 and 2 having the structure shown in FIG. 3 (A) was used. Samples include sample I: uracil (U, 0.13 mg / mL), methyl benzoate (MB, 1.8 mg / mL), toluene (T, 2.6 mg / mL), naphthalene (N, 0.7 mg / mL). mL) A mixture of acetonitrile / water = 60/40 was used.

12 (A) and 12 (B) are chromatograms obtained by injecting 0.2 μL of sample I and 0.1 μL of (B), respectively, and separating them using a conventional sample injection method. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at a flow rate of 0.05 mL / min.

12 (C), (D), and (E) show the first liquid feed port (acetonitrile / water) 0.03 minutes after the injection of 0.4 μL, 0.2 μL, and 0.1 μL of sample I, respectively. = 60/40, flow velocity 0.05 mL / min) is stopped, the second liquid delivery port feed (acetonitrile / water = 60/40, flow velocity 0.05 mL / min) is started to divide the sample band, and the sample band is divided. The front part was introduced into the column for separation and elution, and 4.0 minutes after sample injection, the first liquid feed port feed (acetonitrile / water = 60/40, flow velocity 0.05 mL / min) was restarted, and the second Liquid feeding port This is a chromatogram obtained by dividing sample I into two parts by stopping the liquid feeding, introducing the sample I into a column, and separating the samples.

In FIG. 12A (comparative example), theoretical plates of 1240, 4870, 6370, and 6900 are obtained for U, MB, T, and N, respectively.

On the other hand, 0.4 μL of sample I was injected, and about 40% of the structure shown in FIG. 3 (A) was introduced into the column using the same sample band dividing portion as in Examples 1 and 2. In the 12 (C) first chromatogram, the theoretical plates 3330, 6840, 7480 and 7800 are obtained for U, MB, T and N, respectively. In the first chromatogram of FIG. 12 (D), where 0.2 μL of sample I was injected and about 47% was introduced into the column using the same sample band dividers as in Examples 1 and 2, U, MB, T. For N and N, the number of theoretical plates 3530, 8560, 8140 and 8410 are obtained, respectively. In the first chromatogram of FIG. 12 (E), 0.1 μL of sample I was injected and about 48% was introduced into the column using the same sample band dividers as in Examples 1 and 2, U, MB, T. The number of theoretical plates 3330, 8720, 8160 and 8610 are obtained for and N, respectively.

In Example 3, as compared with the conventional injection method, the peak width of the chromatogram was narrowed due to the division of the sample band in the direction intersecting the axial direction and the introduction into the column, and as shown in Table 2, 1.2. It is possible to generate about 2.8 times the number of theoretical plates. Since the sample bandwidth is also small when the injection capacity is small, the proportion of the sample left in the first liquid feeding port is reduced in the division of the sample band the same time after the sample injection. However, as seen in FIGS. 12 (D) and 12 (E), 0.2 μL or 0.1 μL at a slow flow rate (0.05 mL / min), which is a condition in which diffusion of the sample in the flow path is unlikely to occur. When the injection volume is reduced to some extent as in the above, the sample bandwidth after diffusion does not change, so that the ratio of the front portion of the divided sample band introduced into the column does not change.

When a capillary UV cell (6 nL) having a small capacity was used in Example 3, a large number of theoretical plates was obtained as compared with the case where a 1 μL cell was used in Example 1. Further, especially for solutes (U, MB) having a small retention, a large increase in the number of theoretical plates was observed as compared with the conventional injection method by dividing the sample band and introducing the solute into the column. These results show the capacity of the pipe from the column outlet to the detector cell inlet (about 0.48 μL in Example 1, about 0.55 μL in Example 3) and the detector cell capacity (1 μL in Example 1, carried out). In Example 3, 6 nL), a larger number of theoretical plates can be obtained under the condition that each solute peak eluted from the column is difficult to disperse, and the number of theoretical plates is greatly increased by the sample introduction method of the present invention. Is shown. In addition, the effect of solute dispersion outside the column was reduced by the sample introduction method of the present invention, and the performance (theoretical plate number) close to the original performance of the column was obtained even for the peak of the solute with a small retention that appeared at the beginning of the chromatogram. It is shown that.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, as compared with the conventional sample injection methods (FIGS. 13 (A), (B), (C)) (comparative example), this The reduction of the sample bandwidth by the sample introduction method (FIGS. 13 (D), (E), (F)) with the division of the sample band of the present invention and the rear partial placement was demonstrated without a column.

Among the sample introduction methods of the present invention, in the sample introduction method involving division of the sample band-post-partial placement, the sample bands injected with 0.4 μL, 0.2 μL, and 0.1 μL are divided and introduced into the ZDU, and the sample is introduced. This is an example showing a first chromatogram from the front part of the band and a second chromatogram given by the rear part of the sample band fastened to the first liquid feed port. 13 (B) and (C), 13 (D), (E) and (F) are FIGS. 12 (A) and 12 (B) (conventional sample injection method) and FIGS. 12 (C), respectively. It can be considered to represent the state of the sample band at the column entrance in (D) and (E) (the sample introduction method of the present invention). However, these chromatograms also include a connecting tube from the ZDU outlet to the detector cell and dispersion of the solute band in the detector cell.

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as the sample injection device, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU) was used. The mobile phase (acetonitrile / water = 60/40) was sent at 0.05 mL / min, and the temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.01 seconds, and the data acquisition interval was 5 mSec. The connecting tube from the ZDU outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm, and another ZDU (capacity: 0.02 μL). ), And further connected to a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. As the sample band dividing device, the same sample band dividing portion as in Examples 1 to 3 having the structure shown in FIG. 3 (A) was used. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

13 (A), (B), and (C) show that 0.4 μL, 0.2 μL, and 0.1 μL of sample naphthalene were injected using a conventional sample injection method using a valve, and acetonitrile / water was used as the mobile phase. = 60/40 is a chromatogram eluted by feeding liquid at 0.05 mL / min from the first liquid feeding port.

In FIGS. 13 (D), (E), and (F), 0.4 μL, 0.2 μL, and 0.1 μL of sample naphthalene were injected, respectively, and the sample band was divided into ZDU by the sample introduction method of the present invention. It is a chromatogram introduced and eluted. 0.03 minutes after sample injection, the liquid feed from the first liquid feed port (acetonitrile / water = 60/40, 0.05 mL / min) is stopped, and the liquid feed from the second liquid feed port (acetonitrile / water =). 60/40, 0.05 mL / min) was started to divide the sample band, the front part was introduced into ZDU and eluted (first chromatogram), 0.2 minutes after sample injection, and the second liquid delivery Stop the liquid feeding at the port, return to the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.05 mL / min), and introduce and elute the rear part of the divided sample band into the ZDU. We are doing (second chromatogram).

In FIGS. 13 (A), (B) and (C) (comparative example), the peak of naphthalene shows a large tailing, whereas in FIGS. 13 (D), (E) and (F), The peak of the first chromatogram has a narrow width, and has a shape in which the tailing portion is cut off from the peaks of FIGS. 13 (A), (B), and (C). Even a device using a small-capacity pipe with an inner diameter of 0.108 mm and a length of 10 cm (capacity: about 0.92 μL) for the pipe from the sample injection device to the column inlet reduced the injection volume by the conventional sample injection method. It has been shown that a narrow sample band, which cannot be obtained by itself, was obtained by splitting the sample band in the axially intersecting direction near the column (in this case, ZDU) inlet according to the present invention. The half width (50% peak height) of naphthalene in the first chromatograms of FIGS. 13 (D), (E), and (F) is the peak of naphthalene in FIGS. 13 (A), (B), and (C), respectively. Compared to the width, it is reduced to about 1/2. Further, as a result of using the sample band dividing device having the sample band dividing portion having the structure shown in FIG. 3 (A), even in the sample injected with a small amount of 0.1 μL (100 nL), the sample band is formed near the column inlet. It is shown that the effect of solute dispersion outside the column can be reduced by dividing and introducing.

The measured value of the elution volume of naphthalene in Example 4 and FIG. 13 (B) (measured value at a flow rate of 0.05 mL / min, 0.05 mL / min × 0.037 min) was 1.85 μL. When the ZDU (capacity 0.02 μL), the capacitance from the ZDU outlet to the detector cell inlet (about 0.55 μL), and the detector cell capacitance 6 nL were subtracted from this value, it became 1.274 μL. Therefore, the timing for dividing the sample at a flow rate of 0.05 mL / min was set 0.03 min after sample injection (0.05 mL / min × 0.03 min = after 1.5 μL liquid delivery). As a result, in FIGS. 13 (A), (B) and (C) (Comparative Example), the peak heights of the peaks of naphthalene injected and eluted at 0.4 μL, 0.2 μL and 0.1 μL, respectively, were 50%. The symmetry coefficients in the above were 2.5, 2.4 and 2.1, but in FIGS. 13 (D), (E) and (F), 0.4 μL, 0.2 μL and 0.1 μL, respectively. At a peak height of 50% of the peak of naphthalene that was injected, divided by the sample introduction method of the present invention, introduced into ZDU (sample introduction rates were about 29%, about 41% and about 40%, respectively) and eluted. The symmetry coefficients were 0.99, 0.93 and 1.04, which were peaks with good symmetry.

In FIGS. 13 (A), (B) and (C) (comparative example), the eluted peaks are tailing, and the peak with a larger injection volume has the entire peak on the back side (the one with a longer holding time). It has spread to. Therefore, when the sample is divided after a certain period of time, 0.03 min after the sample injection, the ratio of the most divided sample (100% -29%) when 0.4 μL is injected (FIG. 13 (D)). = 71%) has increased.

Figure 13 (A), (B) in and (C) (Comparative Example), the variance of the peak of naphthalene, the moment method (BEM), 0.34MyuL ^2, and 0.42MyuL ² and 0.41MyuL ² calculated. On the other hand, in FIGS. 13 (D), (E) and (F), 0.04 μL ² , 0.05 μL ² and 0.07 μL ² were calculated, and 0.3 to 0.3 to 0.07 according to the sample introduction method of the present invention. As much as 0.4 μL ^2, the dispersion of the solute was reduced (about 80% or more). By using a 6 nL capillary cell with a small capacity for UV detection and reducing the capacity of the flow path from the ZDU to the detector cell, under the condition that the element in which the solute is dispersed outside the column (outside the ZDU) is small. It was shown that the sample introduction method of the present invention has a very large effect of reducing the dispersion of the sample band by 80% or more as compared with the conventional sample injection method. The fact that these results were obtained under the same conditions as in Example 3, FIGS. 12 (C), (D) and (E) except for the column is shown in FIGS. 12 (C), (D) and (E). It is considered that the factor that made it possible to exhibit the performance close to the original performance of the small column having an inner diameter of 1.0 mm and a length of 5 cm is the effect of reducing the dispersion of the sample band near the column inlet. When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used. The reason why the variance of the peak is calculated by the moment method is that the variance that more reflects the peak shape can be calculated for the peak having poor symmetry. For peaks with good symmetry, the half price width (unit: min) is multiplied by the flow velocity and converted to the unit of volume, then σ is calculated with this value as 2.35σ, and the square (σ ² ) can be used as the variance. , The variance calculated by the moment method and the value are close.

Further, in Example 3 and FIG. 12 (A), 0.2 μL of the sample was injected by the conventional sample injection method, and the dispersions of the solutes U, MB, T and N separated and eluted by the column peaked, respectively. half-value width (unit: min) from, 0.31μL ^2, 0.93μL ^2, were calculated to 2.04MyuL ² and 2.96MyuL ² (half-value width (unit: min) to multiply the flow rate 0.05 mL / min After changing to the unit of volume, σ was calculated with this value as 2.35σ, and the square (σ ² ) was used as the variance). For these, in the FIG. 12 (D) first chromatogram, 0.11μL ^2, 0.55μL ^2, calculated to 1.66MyuL ² and 2.53MyuL ^2, the sample introduction method of the present invention, the solute peak The dispersion was reduced by 0.3 to 0.4 μL ² , and the theoretical number of solute peaks could be increased by 1.2 to 2.8 times as compared with the conventional sample injection method.

Among the sample introduction methods of the present invention, the sample introduction method involving division of the sample band and placement of the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band and keeps the sample in the first liquid feed port. This is an example showing the second chromatogram given by the rear part of the sample band (Fig. 14 (B)). Further, an example of improving the number of theoretical plates in the first chromatogram from the front part of the sample band and discharging the rear part of the sample band held in the first liquid feeding port from the discharge port shown in FIG. 6 without introducing it into the column. (Fig. 14 (C)). Further, the sample band is divided, the rear part of the sample band is placed in the first liquid feeding port, the front part of the sample band is separated and eluted, and then the first liquid feeding port liquid feeding and the second liquid feeding port liquid feeding are alternately performed. By repeating this, the rear part of the sample band was introduced into the column at regular intervals in 5 steps, separated and eluted, and the improvement of the separation performance in each chromatogram was shown (FIG. 14 (D)). Further, for comparison of column performance, an example by a conventional sample injection method using a valve is also shown (FIG. 14 (A)) (comparative example).

Using a UHPLC device LC800 (GL Sciences, Inc.) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) as a sample injection device, a column acetonitrile C18 (filler diameter 2 μm, inner diameter 1.0 mm) was used. , Length 5 cm, GL Sciences), mobile phase (acetonitrile / water = 60/40) from both the first and second feed ports, first feed port flow velocity (0.09 mL / min), The liquid was fed at the second liquid feed port flow velocity (0.01 mL / min), totaling 0.1 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. For the connecting pipe from the column outlet to the detector cell entrance, a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm is connected to the ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected and used. As the sample band dividing device, the same sample band dividing portion as in Examples 1 to 4 having the structure shown in FIG. 3 (A) was used. Further, as shown in FIG. 6, a sample introduction device having a vent discharge function provided with a second discharge port 601 upstream of the sample injection device 22 was used. Samples include Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0.3 mg / mL), naphthalene (N, 0.3 mg / mL). , Hexanophenone (HxPh, 0.44 mg / mL), Heptanophenone (HpPh, 0.46 mg / mL), Octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 14A (Comparative Example) is a chromatogram separated by injecting 1 μL of Sample II using a conventional sample injection method using a valve. The mobile phase (acetonitrile / water = 60/40) is from both the first and second liquid feed ports, the first liquid feed port flow velocity (0.09 mL / min), and the second liquid feed port flow velocity (0.01 mL / min). , A total of 0.1 mL / min was sent.

In FIG. 14 (B), 1 μL of sample II was injected under the same mobile phase conditions as in FIG. 14 (A), and 0.03 minutes later, the first liquid feed port was fed (acetonitrile / water = 60/40, 0. 09 mL / min) was stopped, and the sample band was divided by increasing the flow rate of the second liquid feed port (acetonitrile / water = 60/40, 0.01 mL / min) to 0.1 mL / min, and the front part was divided. It was introduced into a column, separated and eluted, and 6.0 minutes after sample injection, the flow velocity of the first liquid delivery port (0.09 mL / min) and the flow velocity of the second liquid feed port (0.01 mL / min), totaling 0.1 mL / min. It is a chromatogram which was returned to the liquid feed of min, the rear part of the sample band was introduced into the column, and separated and eluted.

In FIG. 14 (C), 1 μL of sample II was injected under the same mobile phase conditions as in FIG. 14 (A), and 0.03 minutes later, the first liquid feed port was fed (acetoyl / water = 60/40, 0. 09 mL / min) was stopped, and the sample band was divided by feeding the liquid by increasing the flow velocity of the second liquid feed port feed (acetoyl / water = 60/40, 0.01 mL / min) to 0.187 mL / min. , The front part is introduced into the column to start separation and elution, and the rear part of the sample band is discharged from the second discharge port 601 by opening the vent valve 62 in FIG. 6, and the vent valve 62 is discharged 1.0 minute after the sample injection. Closed and returned to the first liquid feed port flow velocity (0.09 mL / min) and the second liquid feed port flow velocity (0.01 mL / min), totaling 0.1 mL / min, and separated the front part of the sample band. It is an eluted chromatogram.

Regarding the details of the partial discharge after the sample band, a second discharge port 601 is provided upstream of the sample injection device 22 of the sample band dividing device shown in FIG. 6 for 0.03 to 1.0 minutes after the sample injection. The vent valve 62 installed was opened, and the rear part of the sample band was pushed back to the upstream of the sample injection device and discharged through the flow path resistance of the resistance tube 63 (fused silica tube having an inner diameter of 0.03 mm and a length of 30 cm). Emission). From 0.03 to 1.0 minutes after sample injection, the flow rate required for separation and elution of the sample band front part from the column is added to the flow rate required for discharge of the sample band rear part from the discharge port. The flow rate of the second liquid feed port liquid feed was 0.187 mL / min. The fused silica tube with an inner diameter of 0.03 mm and a length of 30 cm functions as a liquid feed resistor and prevents a sudden pressure drop due to opening the vent valve. Further, by adjusting the flow velocity of the second liquid feed port, it is possible to obtain a separation chromatogram equivalent to that of the first chromatogram when there is no discharge operation (FIG. 14 (B)). In addition, by dividing the sample band with discharge from the discharge port and introducing it into the column, separation and elution can be performed in the same time as the conventional valve injection method (FIG. 14 (A)), and the column performance. Was shown to be able to improve.

FIG. 14 (D) shows the same mobile phase conditions as in FIGS. 14 (A), (B), and (C), that is, the mobile phase (acetoyl / water = 60/40) is applied to both the first and second liquid feed ports. Then, 3 μL of sample II was injected while feeding at a total of 0.1 mL / min, that is, the flow velocity of the first liquid feed port (0.09 mL / min) and the flow velocity of the second liquid feed port (0.01 mL / min). .02 minutes later, stop the first liquid feed port feed (acetoyl / water = 60/40, 0.09 mL / min), and stop the second feed port feed (acetoyl / water = 60/40, 0.01 mL / min). ) Was increased to 0.1 mL / min, the sample band was divided, introduced into the column, separated and eluted (Fig. 14 (D) first chromatogram), and 6.0 minutes to 0.01 minutes after sample injection. Between (between 6.00 and 6.01 minutes), 1st liquid feed port flow velocity (0.09 mL / min), 2nd liquid feed port flow velocity (0.01 mL / min), total 0.1 mL / min feed By making it a liquid, only 0.01 minutes of liquid feed from the sample band held in the first liquid feed port is introduced into the column, and between 6.01 minutes and 12.00 minutes after sample injection. , The first liquid feed port liquid feed was stopped, the flow velocity of the second liquid feed port liquid feed was set to 0.1 mL / min, and the sample introduced into the column was separated and eluted (FIG. 14 (D) second chromatogram). ).

From 12.00 minutes to 12.01 minutes after sample injection, the first liquid feed port flow velocity (0.09 mL / min) and the second liquid feed port flow velocity (0.01 mL / min), totaling 0.1 mL / min. After min feeding, only 0.01 minutes of liquid feeding from the sample band held in the first liquid feeding port is introduced into the column, and 12.01 minutes to 18.00 minutes after sample injection. During that time, the feed from the first feed port was stopped, the flow velocity of the feed from the second feed port was 0.1 mL / min, and the sample introduced into the column was separated and eluted (FIG. 14 (D), third chromatogram). In the same operation, the fourth sample band was introduced between 18.00 minutes and 18.01 minutes after the sample injection, and between 18.01 minutes and 24.00 minutes after the sample injection. The sample introduced into the column was separated and eluted (Fig. 14 (D) 4th chromatogram), and the fifth sample band was introduced between 24.00 minutes and 24.01 minutes later. The sample introduced into the column was separated and eluted from 01 minutes to 30.00 minutes (Fig. 14 (D) 5th chromatogram), and after 30.00 minutes, the flow rate of the first liquid feed port (0). .09 mL / min), 2nd liquid delivery port flow velocity (0.01 mL / min), total 0.1 mL / min, and the entire amount of sample band remaining in the 1st liquid supply port was introduced into the column. Separation and elution were performed (FIG. 14 (D) 6th chromatogram).

In this way, after the elution of the front part of the sample band injected into the column was completed, the liquid feeding from the first liquid feeding port was restarted by restarting the liquid feeding pump, and the liquid was left in the first liquid feeding port 20. The middle part and the rear part 32 of the sample band are introduced into the column 1, the next chromatographic separation and elution are performed, and this operation is repeated, every 6 minutes from the sample band retained in the first liquid feeding port. By repeatedly dividing the sample band in the direction intersecting the axial direction and introducing it into the column to separate and elute, a large amount of sample is injected, and as a large tail, in the first liquid feed port and in the loop of the Rheodyne 8125 valve. The possibility of continuous analysis was shown for the posterior part of the fastened sample band.

Further, the result of FIG. 14D shows that the number of theoretical plates of the column can be increased as compared with the conventional sample injection method by discharging the front part and the rear part of the sample band and introducing only the middle part of the sample band into the column. It shows that it is possible to increase. For example, in FIG. 13, the sample band injected by the conventional sample injection method is tailing, but the sample band divided and introduced into the ZDU has a small peak width in both the front part and the rear part. Further, in FIG. 10B, the peak widths of the first and second chromatograms in which the front portion and the rear portion of the sample band divided and introduced into the column are separated from each other are shown in FIG. Since the peak width derived from the same solute is smaller in (A), the front part of the sample band is discharged without being introduced into the column by using the sample introduction method of the present invention, and only the rear part of the sample band is discharged. It is self-evident that the column performance can be improved by reducing the dispersion of the entire sample band by introducing the above into the column.

If a sample dividing portion for arranging a T-shaped connector with a discharge port or a four-way joint in front of the column inlet as shown in FIG. 8 is used, the front part or the rear part of the sample band is discharged, and only the intermediate part is discharged. It can also be introduced into the column.

In FIG. 14A (Comparative Example), 1 μL of sample II was injected by the conventional sample injection method, and the number of theoretical plates for TU, N, and OccPh separated and eluted (for the 1st, 4th, and 7th peaks), respectively. 500, 6690 and 7990 stages have been obtained. On the other hand, in the first chromatogram of FIG. 14 (B), 1 μL of the injected sample II was divided, the front portion (about 65% of the total injected sample) was introduced into the column, and separated and eluted. Theoretical plates 1910, 9240, and 8620 are obtained for TU, N, and OccPh, respectively. Further, in the first chromatogram of FIG. 14 (C) in which 1 μL of sample II was divided, the front portion was introduced into the column, and the sample II was separated and eluted, the theoretical plates 2130 and 9140 and the number of theoretical plates 2130 and 9140 were obtained for TU, N and OccPh, respectively. 8870 steps were obtained. Regardless of whether or not the rear part of the sample band is ejected, the number of theoretical plates is about 1.1 to 4 times that of the conventional sample injection method by dividing the sample band in the direction intersecting the axial direction and introducing it into the column. Is possible.

Further, even under the condition that the liquid is fed from both the first liquid feeding port and the second liquid feeding port at the time of sample injection, the liquid feeding from the first liquid feeding port is stopped and the liquid feeding speed from the second liquid feeding port is increased. By increasing the sample band, the sample band can be divided and introduced into the column, and the flow velocity ratio between the first liquid feed port and the second liquid feed port (first liquid feed port flow velocity / second liquid feed port flow velocity). It was shown that the column performance can be improved by the sample introduction method of the present invention without impairing the column performance even if the value is changed from 9/1 to 0/10 and then to 9/1 again. In FIG. 14B, the splitting of the sample band and the introduction rate into the column at the time when the liquid feeding capacity from the first liquid feeding port was 2.7 μL from the time of sample injection was about 65%.

In order to confirm the performance improvement by the sample introduction method involving the division of the sample band and the rear partial placement, the conventional sample injection method using a valve (FIG. 15 (A)) (comparative example) and the sample introduction method of the present invention (FIG. 15) The column performances in (B) and (C)) were compared.

Among the sample introduction methods of the present invention, the sample introduction method involving division of the sample band and placement of the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band and keeps the sample in the first liquid feeding port. This is an example in which the second chromatogram given by the rear part of the sample band is shown (FIGS. 15 (B) and 15 (C)).

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as the sample injection device, and the column Kinex C18 (filler diameter 2.6 μm, inner diameter 2. A mobile phase (acetonitrile / water = 60/40) was delivered from both the first and second delivery ports at a total of 0.2 mL / min using a 1 mm length, 5 cm length, Phenomenex). The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. As the connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. As the sample band dividing device, the same sample band dividing portion as in Examples 1 to 5 having the structure shown in FIG. 3 (A) was used. Samples include Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0.3 mg / mL), naphthalene (N, 0.3 mg / mL). , Hexanophenone (HxPh, 0.44 mg / mL), Heptanophenone (HpPh, 0.46 mg / mL), Octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 15A is a chromatogram obtained by injecting 1 μL of sample II by a conventional sample injection method for separation. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.2 mL / min.

In FIG. 15B, the mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.2 mL / min, and 1 μL of sample II was injected 0.02 minutes after the first feed. Stop the liquid transfer port, start the second liquid transfer port (acetonitrile / water = 60/40, 0.2 mL / min), divide the sample band, introduce the front part into the column, and separate and elute. After 6.0 minutes from the sample injection, the liquid feeding from the second liquid feeding port was stopped, and the liquid was returned to the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.2 mL / min). This is a chromatogram in which the rear part of the sample band was introduced into the column and separated.

FIG. 15C shows the mobile phase (acetoyl / water = 60/40) from both the first and second liquid feed ports, the first liquid feed port flow velocity (0.199 mL / min), and the second liquid feed port flow velocity. (0.001 mL / min), feed at a total of 0.2 mL / min, inject 1 μL of sample II, stop the first feed port feed after 0.02 minutes, and flow rate of the second feed port feed. The sample band is divided by increasing (0.2 mL / min), the front part is introduced into the column for separation and elution, and 6.0 minutes after sample injection, the sample band is fed from both the first and second liquid feed ports. It is a chromatogram that was returned to the liquid and separated and eluted at the first liquid feed port flow velocity (0.199 mL / min) and the second liquid feed port flow velocity (0.001 mL / min), for a total of 0.2 mL / min.

In FIG. 15A (comparative example), the theory is given for the first, third, and fourth peaks, TU (non-retention), BuPh (retention coefficient k = 1.8), and N (k = 2.8), respectively. The number of stages 3030, 8520 and 10200 stages has been obtained. On the other hand, in the first chromatogram of FIG. 15 (B), the theoretical plates of 3760, 9340 and 10960 were obtained for TU, BuPh and N, respectively. The sample introduction method of the present invention, which divides the sample in the direction intersecting the axial direction and introduces the sample into the column, makes it possible to generate about 1.07 to 1.25 times the number of theoretical plates as compared with the conventional sample injection method. .. Great effect on peaks eluted early in the chromatogram.

At the time of sample injection, liquid is fed from both the first and second liquid feeding ports at a ratio of 1st liquid feeding port flow velocity / 2nd liquid feeding port flow velocity = 199/1, and 0.02 as in FIG. 15 (B). In the first chromatogram of FIG. 15C, in which the sample band was split after minutes and the front portion was introduced into the column, TU (non-retention), BuPh (retention coefficient k = 1.8) and N (k = 2). For 0.8), the theoretical plates 3830, 9070 and 10970 are obtained, respectively, and the sample introduction method of the present invention, which is divided in the direction intersecting the axial direction and introduced into the column, is compared with the conventional sample injection method. It is possible to generate about 1.07 to 1.26 times the number of theoretical plates. Transferring the liquid from the second liquid feeding port at a flow velocity in an appropriate range between the time of sample injection and the division of the sample band is one form in which the effect of the sample introduction method of the present invention can be obtained. Therefore, when the sample band is divided and the front portion is introduced into the column, there is an effect of facilitating an increase in the liquid feeding speed of the second liquid feeding port.

In order to confirm the improvement of column performance by the sample introduction method involving division of the sample band and post-partial placement, the conventional sample injection method using a valve (FIG. 16 (A)) (comparative example) and the sample introduction method of the present invention (FIG. The column performance in 16 (B)) was compared.

Among the sample introduction methods of the present invention, the sample introduction method involving division of the sample band and placement of the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band and keeps the sample in the first liquid feeding port. It is an example showing the second chromatogram given by the rear part of the sample band (FIG. 16 (B)).

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection device, and a column Kinex C18 (filler diameter 2.6 μm, inner diameter 2. A mobile phase (acetonitrile / water = 60/40) was delivered at 0.3 mL / min using a 1 mm length, 5 cm length, Phenomenex). The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. For the connecting pipe from the column outlet to the detector cell entrance, a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm is connected to the ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected and used. As the sample band dividing device, the same sample band dividing portion as in Examples 1 to 6 having the structure shown in FIG. 3 (A) was used. Samples include Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0.3 mg / mL), naphthalene (N, 0.3 mg / mL). , Hexanophenone (HxPh, 0.44 mg / mL), Heptanophenone (HpPh, 0.46 mg / mL), Octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 16A is a chromatogram obtained by injecting 1 μL of sample II by a conventional sample injection method for separation. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.3 mL / min.

FIG. 16B shows the first solution 0.01 minutes after the mobile phase (acetonitrile / water = 60/40) was supplied from the first solution port at 0.3 mL / min and 1 μL of sample II was injected. Stop the liquid feed port feed, start the second liquid feed port feed (acetonitrile / water = 60/40, 0.3 mL / min) to divide the sample band, introduce the front part into the column and separate and elute. , 5.0 minutes after sample injection, stop the liquid feeding from the 2nd liquid feeding port and return to the liquid feeding from the 1st liquid feeding port (acetonitrile / water = 60/40, 0.3 mL / min). This is a chromatogram in which the rear part of the sample band was introduced into the column and separated and eluted.

In FIG. 16A (comparative example), the first, third, fourth, and seventh peaks are TU (non-retention), BuPh (retention coefficient k = 1.8), N (k = 2.8), and For OccPh (k = 10.9), the number of theoretical plates 3330, 9490, 11220 and 10890 are obtained, respectively. On the other hand, in the first chromatogram of FIG. 16 (B) in which the sample band was divided in the axially intersecting direction and the front portion was introduced into the column, TU (non-retention) and BuPh (retention coefficient k =). For 1.8), N (k = 2.8) and OccPh (k = 10.9), the theoretical plates 6130, 11530, 12320 and 11570 were obtained, respectively. The sample introduction method of the present invention makes it possible to generate about 1.06 to 1.84 times the number of theoretical plates as compared with the conventional sample injection method. Great effect on peaks eluted early in the chromatogram.

Here, with respect to FIG. 16B, Table 3 shows the results of the sample introduction rate for which the peak area of the first chromatogram / (the peak area of the first + second chromatogram) was calculated. For each peak (TU, AcPh, BuPh, N, HxPh, HpPh, OccPh in order from the left of the first chromatogram of FIG. 16 (B)), the ratio of the divided sample bands introduced into the column was 65.2 ± 0. Within the range of 0.8%, it is considered that no particular solute has been sorted by the sample introduction method of the present invention and introduced into the column more than other solutes (difference in behavior between solutes). , Discrimination is not considered to have occurred).

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, the sample band of the present invention is compared with the conventional sample injection methods (FIGS. 17A and 17B) (comparative example). The reduction of the sample bandwidth by the sample introduction method (FIG. 17 (C)) with the division-posterior partial placement was demonstrated without a column.

Among the sample introduction methods of the present invention, the sample introduction method involving division of the sample band and placement of the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band and keeps the sample in the first liquid feed port. This is an example showing the second chromatogram given by the rear part of the sample band (Fig. 17 (C)). 17 (A) and 17 (C) show the state of the sample band at the column inlet in FIGS. 16 (A) (conventional sample injection method) and FIG. 16 (B) (sample introduction method of the present invention), respectively. ..

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as the sample injection device, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU) was used. The mobile phase (acetonitrile / water = 60/40) was sent at 0.3 mL / min, and the temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.01 seconds, and the data acquisition interval was 5 mSec. The connecting tube from the ZDU outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm, and another ZDU (capacity: 0.02 μL). ), And further connected to a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. As the sample band dividing device, the same sample band dividing portion as in Examples 1 to 7 having the structure shown in FIG. 3 (A) was used. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

In FIGS. 17A and 17B, 1.0 μL and 0.5 μL of sample naphthalene are injected, respectively, using the conventional sample injection method, and acetonitrile / water = 60/40 is injected as the mobile phase from the first liquid feed port. It is a chromatogram eluted by sending the solution at 0.3 mL / min.

FIG. 17C is a chromatogram in which 1.0 μL of sample naphthalene was injected, the sample band was divided into two, introduced into ZDU, and eluted. 0.01 minutes after the sample injection, the liquid feed from the first liquid feed port (acetonitrile / water = 60/40, 0.3 mL / min) is stopped, and the liquid feed from the second liquid feed port (acetonitrile / water =). 60/40, 0.3 mL / min) was started to divide the sample band, the front part was introduced into ZDU and eluted (first chromatogram), 0.2 minutes after sample injection, and the second liquid delivery The liquid feeding at the port was stopped, and the liquid was returned from the first liquid feeding port (acetonitrile / water = 60/40, 0.3 mL / min) to elute the rear part of the divided sample band (No. 1). 2 chromatograms).

In FIGS. 17 (A) and 17 (B) (comparative example), the peak of naphthalene shows a large tailing, whereas in FIG. 17 (C), the peak width of the first chromatogram is small. The tailing portion is scraped off from the peak in FIG. 17 (A). The direction in which a narrow sample band, which cannot be obtained only by reducing the injection volume by the conventional sample injection method (FIG. 17 (B)), intersects the axial direction of the sample near the inlet of the column (ZDU in this case) according to the present invention. It is shown that it was obtained by the division at, and the introduction to ZDU. In FIG. 17 (C), about 69% of the sample injected with 1 μL is introduced into the ZDU and appears in the first chromatogram, and in FIG. 17 (B), the sample of 0.5 μL by the conventional sample injection method. Although the sample volume introduced into the ZDU is larger than the peak that appears as a result of the injection, it gives a narrow peak. FIG. 17 (D) is an overlay of the first chromatograms of FIGS. 17 (A), (B) and 17 (C), and the peak width of the first chromatogram of FIG. 17 (C) is the narrowest. You can clearly see that.

In FIG. 17 (A), (B) ( Comparative Example), the variance of the peak of naphthalene, the moment method, 1.44MyuL ^2, was calculated to 1.33μL ^2. On the other hand, in FIG. 17C, it was calculated to be 0.43 μL ^2, and the dispersion was reduced by about 70% by the sample introduction method of the present invention as compared with the conventional sample injection method. The fact that this result was obtained under the same conditions as in Example 7 and FIG. 16 (B) except for the column means that a small column having an inner diameter of 2.1 mm and a length of 5 cm originally has in FIG. 16 (B). It is considered that the factor that made it possible to develop the performance close to the performance is the effect of reducing the dispersion of the sample band near the column inlet. When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used.

In order to confirm the improvement of column performance by the sample band division-sample introduction method with post-partial placement, the conventional sample injection method using a valve (FIGS. 18 (A), (G)) (comparative example) and the sample of the present invention The column performances in the introduction methods (FIGS. 18 (B), (C), (D), (E), (F), (H), (I)) were compared. Further, with respect to FIGS. 18B and 18C, the peak area of the first chromatogram / (peak area of the first + second chromatogram) was calculated, and the reproducibility was confirmed. In addition, the effect of the timing of discharge of the rear part of the sample band and the process from sample injection to division of the sample band applied to the sample introduction method of the present invention (first liquid feed port liquid feed rate) / (second liquid feed port). It was confirmed that the effect of the liquid feed port liquid feed rate) ratio and the liquid feed time from sample injection to the division of the sample band on the column performance.

18 (B) and 18 (C) show that, among the sample introduction methods of the present invention, the number of theoretical plates in the first chromatogram from the front part of the sample band is improved by the sample introduction method involving division of the sample band and placement of the rear part. And, the second chromatogram given by the posterior part of the sample band held in the first liquid feed port is shown. Reproducibility of the sample introduction method of the present invention at a ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) = 0.6 / 1.2 mL / min from the time of sample injection to the time of sample band division. I was able to confirm the sex.

In FIGS. 18 (D), (E), and (F), among the sample introduction methods of the present invention, in the sample introduction method involving division of the sample band and placement of the rear part, the discharge time of the rear part of the sample band and the sample band are shown. It was confirmed that the (first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio and the liquid feed time after the discharge of the rear part had an effect on the column performance. The (first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio from the time of sample injection to the time of sample band division was set to 0.6 / 1.2 mL / min. In FIG. 18 (G), the effect of the (first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio from the time of sample injection to the end of separation and elution by the conventional sample injection method using a valve (FIG. Comparison with 18 (A)) was confirmed. The ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) from the time of sample injection to the end of separation and elution was set to 0.3 / 1.5 mL / min.

In FIGS. 18 (H) and 18 (I), among the sample introduction methods of the present invention, in the sample introduction method involving sample band division-posterior partial placement, from the time of sample injection to the sample band division (first feed). The effect of the liquid port flow velocity) / (second liquid port flow velocity) ratio (comparison with FIGS. 18B and 18C) was confirmed. The (first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio from the time of sample injection to the time of sample band division was set to 0.3 / 1.5 mL / min.

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection device, and a column acetonitrile C18 (filler diameter 2 μm, inner diameter 4.6 mm) was used. , 5 cm in length, GL Sciences, Inc.), the mobile phase (acetonitrile / water = 60/40) was fed from both the first and second feed ports at a total of 1.8 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. As the connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. As the sample band dividing device, the same sample band dividing portion as in Examples 1 to 8 having the structure shown in FIG. 3 (A) was used. Further, as shown in FIG. 6, a sample introduction device having a vent discharge function provided with a second discharge port 601 upstream of the sample injection device 22 was used. Samples include Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0.3 mg / mL), naphthalene (N, 0.3 mg / mL). , Hexanophenone (HxPh, 0.44 mg / mL), Heptanophenone (HpPh, 0.46 mg / mL), Octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 18A is a chromatogram obtained by injecting 2 μL of sample II by a conventional sample injection method and separating and eluting the sample II, which is a comparative example. The mobile phase (acetonitrile / water = 60/40) was fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio = 0.6 / 1). .2 mL / min, total 1.8 mL / min). In FIGS. 18B and 18C, the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) / (second feed). Liquid port flow velocity) ratio = 0.6 / 1.2 mL / min, total 1.8 mL / min), 2 μL of sample II was injected, and 0.01 minutes after sample injection, the first liquid feed port feed (acetonitrile / water). = 60/40, 0.6 mL / min) was stopped, and the flow rate of the second liquid feed port (acetonitrile / water = 60/40, 1.2 mL / min) was increased to 1.8 mL / min for the sample. The band was split, the front part was introduced into the column, and separated and eluted. 9.0 minutes after sample injection, liquid feed from both the first and second feed ports ((first feed port flow velocity) / (second feed port flow velocity) ratio = 0.6 / 1.2 mL / It is a chromatogram in which the posterior portion of the sample band retained in the first liquid feeding port is separated and eluted by returning to min (1.8 mL / min in total).

In FIG. 18A (Comparative Example), the theoretical plates 4540, 9510, 10070, and 11290 were obtained for the first, second, third, and fourth peaks TU, AcPh, BuPh, and N, respectively. In comparison with this, in FIGS. 18B and 18C, the average values of the theoretical plates of 5980, 10930, 11480, and 11490 were obtained for TU, AcPh, BuPh, and N, respectively. The sample introduction method of the present invention, in which the sample is divided in the direction intersecting the axial direction and introduced into the column, is 1.02 to 1.30 for the peak that elutes at the initial stage of the chromatogram as compared with the conventional sample injection method. It is possible to generate about twice the number of theoretical plates.

Table 4 shows the results of the sample introduction rate for which the peak area of the first chromatogram / (peak area of the first + second chromatogram) was calculated for FIGS. 18 (B) and 18 (C). For each peak (TU, AcPh, BuPh, N, HxPh, HpPh, OccPh in order from the left of the first chromatogram in FIG. 18 (B)), the ratio of the divided sample bands introduced into the column was 90.0 ±. It was in the range of 0.8%, demonstrating the reproducibility of the sample introduction method of the present invention. In addition, a specific solute has not been selected by the sample introduction method of the present invention and introduced into a column more than other solutes (discrimination has not occurred).

In FIGS. 18 (D), (E), and (F), the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) /. (Second liquid feed port flow velocity) Ratio = 0.6 / 1.2 mL / min, total 1.8 mL / min), inject 2 μL of sample II, and 0.01 minutes after sample injection, liquid feed to the first liquid feed port. Stop (acetonitrile / water = 60/40, 0.6 mL / min) and increase the flow rate of the second liquid feed port feed (acetonitrile / water = 60/40, 1.2 mL / min) to 1.9 mL / min. By doing so, the sample band is divided, the front part of the sample band is introduced into the column to start separation and elution, and the rear part of the sample band is opened from the vent valve of the discharge port shown in FIG. From 01 minutes to 1.0 minutes, the sample was discharged from the upstream of the sample injection device (back discharge) through the resistance of a fused silica capillary tube having an inner diameter of 0.03 mm and a length of 30 cm. The operations of the first liquid feed port liquid feed, the second liquid feed port liquid feed, and the vent valve of the discharge port from 1.0 minute after the sample injection are shown in FIGS. 18 (D) and 18 (E) and as shown below. Each is different in (F). The reason why the flow velocity from the second liquid feed port was set to 1.9 mL / min between 0.01 minutes and 1.0 minutes after the sample injection was to separate and elute the front part of the sample band from the column. This is because the flow rate required for discharging the rear part of the sample band from the discharge port is added to the required flow rate.

In FIGS. 18 (D), (E), and (F), from 1.0 minute to 9.0 minutes after sample injection, the first liquid feed port liquid feed, the second liquid feed port liquid feed, and the discharge port Regarding the operation of the vent valve, the following operations are performed. In FIG. 18D, the ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) = 0 / 1.9 mL / min was maintained, and the vent valve was left open. In FIG. 18 (E), the ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) was 0.6 / 1.2 mL / min, and the vent valve was closed. In FIG. 18F, the ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) was 1.8 / 0 mL / min, and the vent valve was closed.

Presence or absence of the sample introduction method of the present invention, from the time of sample injection to the sample band division, and after the sample is divided and the front portion is introduced into the column (first liquid feed port flow velocity) / (second liquid feed). Table 5 shows the effect of the port flow velocity ratio and the presence or absence of the discharge operation on the theoretical plate number of columns.

In all of FIGS. 18 (D), (E), and (F), the first, third, and fourth peaks TU, BuPh, and N are shown in FIG. 18 (A) by the conventional sample injection method. A 1-20% larger number of theoretical plates was obtained as compared with the number of theoretical plates obtained. These results show that the sample introduction method of the present invention for a column having an inner diameter of 4.6 mm, in which the sample is divided in the direction intersecting the axial direction and introduced into the column, is effective in increasing the number of column theoretical plates. After the divided sample band front part is introduced into the column and the sample band rear part is discharged, the (first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio is the total flow through the column. It is shown that if the flow velocities are the same, the column performance is not significantly affected (the difference in the number of column theoretical plates is 4.2% or less).

The fused silica capillary tube with an inner diameter of 0.03 mm and a length of 30 cm also functions as a liquid feed resistance in this example, preventing a sudden pressure drop due to the discharge operation of opening the vent valve, and at the same time, the flow velocity of the second liquid feed port. The adjustment made it possible to obtain a separation chromatogram equivalent to that without the discharge operation. Further, by performing the sample introduction method of the present invention accompanied by the use of the discharge port, the column performance can be improved as compared with the conventional sample injection method, and separation and elution can be performed in the same time as the analysis by the conventional sample injection method. Was shown.

FIG. 18 (G) is a comparative example, which is a chromatogram obtained by injecting 2 μL of sample II by a conventional sample injection method and separating and eluting. The mobile phase (acetonitrile / water = 60/40) was fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio = 0.3 / 1). .5 mL / min, total 1.8 mL / min).

In FIGS. 18 (H) and 18 (I), the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) / (second feed). Liquid port flow velocity) ratio = 0.3 / 1.5 mL / min, total 1.8 mL / min), 2 μL of sample II was injected by the conventional sample injection method, and 0.02 minutes after sample injection, the first liquid delivery port Stop the liquid feed (acetonitrile / water = 60/40, 0.3 mL / min), and change the flow velocity of the liquid feed to the second liquid feed port (acetonitrile / water = 60/40, 1.5 mL / min) to 1.8 mL / min. The sample band was divided by increasing to min, the front part was introduced into the column, and the sample band was separated and eluted. 9.0 minutes after sample injection, liquid feed from both the first and second feed ports ((first feed port flow velocity) / (second feed port flow velocity) ratio = 0.3 / 1.5 mL / It is a chromatogram in which the posterior portion of the sample band retained in the first liquid feeding port is separated and eluted by returning to min (1.8 mL / min in total).

In FIG. 18 (G) (Comparative Example), the theoretical plates 1850, 6300, 8690, and 9140 were obtained for the first, second, third, and fourth peaks TU, AcPh, BuPh, and N, respectively. In comparison, in the first chromatogram of FIG. 18 (H) and the first chromatogram of FIG. 18 (I), the average number of theoretical plates of TU, AcPh, BuPh, and N is 2840, 7700, 9450, and 9760. Obtained. In the sample introduction method of the present invention, in which the sample is divided in the direction intersecting the axial direction and introduced into the column, the peak elution at the initial stage of the chromatogram is 1.02 to 1.50 as compared with the conventional sample injection method. It is possible to generate about twice the number of theoretical plates. However, as shown in Table 5, the mobile phase liquid feed conditions ((first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio = 0.6 / 1) in FIGS. 18 (B) and 18 (C). A lower theoretical plate number was obtained than in the case of 2 mL / min, 1.8 mL / min in total), and from this result, the flow rate from the time of sample injection to the time of sample band division (first flow velocity at the feed port) / (second feed feed). It has been shown that proper selection of the port flow velocity) ratio results in a higher number of theoretical plates.

Table 6 shows the results of the sample introduction rate for which the peak area of the first chromatogram / (peak area of the first + second chromatogram) was calculated for FIGS. 18 (H) and 18 (I). For each peak (TU, AcPh, BuPh, N, HxPh, HpPh, OccPh in order from the left of the first chromatogram in FIG. 18 (H)), the ratio of the divided sample bands introduced into the column was 92.4 ±. It was within the range of 0.8%, demonstrating the reproducibility of the sample introduction method of the present invention. In addition, a specific solute has not been selected by the sample introduction method of the present invention and introduced into a column more than other solutes (discrimination has not occurred).

When the flow rate of the first liquid feed port was 0.3 mL / min, the sample band was divided at 0.02 minutes and introduced into the column, and about 92% of the injected sample was analyzed as the first chromatogram. Reproducibility was observed in the ratio of the peak areas of the first chromatogram and the second chromatogram. Further, as shown in Table 4, in FIGS. 18B and 18C, when the flow velocity of the first liquid feed port is 0.6 mL / min, the sample band is divided in 0.01 minutes and introduced into the column. , About 90% of the injected sample was analyzed as the first chromatogram. Reproducibility was observed in the ratio of the peak areas of the first chromatogram and the second chromatogram. The results of these examples are compared with the case where the sample introduction method of the present invention uses the conventional sample injection method even for a large column having an inner diameter of 4.6 mm and a length of 5 cm, especially for the peak at the initial stage of the chromatogram. As a result, it is shown that a large number of theoretical plates up to about 50% can be obtained, that there is no difference in behavior between solutes (discrimination), and that the sample introduction method of the present invention is reproducible.

The results of Examples 1 to 9 are
1. The division of the sample band and the volume of the liquid transfer from the first liquid supply port until the introduction of the sample front part into the column, that is, the flow velocity of the first liquid supply port, the division of the sample band from the sample injection, and the sample front part. The ratio (sample introduction rate) of the front part of the sample band used for the first separation analysis to the whole sample can be controlled based on the time until the introduction into the column.
2. Even if a large flow rate is required for a large column, if the flow rate of the first liquid supply port is reduced and the liquid flow from the second liquid supply port is partially used, the flow velocity of the whole and the first liquid supply port can be adjusted. By considering the timing, the sample band can be divided and the front part of the sample can be properly introduced into the column.
3. If the (first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio from the time of sample injection to the sample band division becomes too small, the number of theoretical plates of the column will decrease, so a better number of theoretical plates (higher). It is appropriate to choose a flow velocity ratio that gives performance).
And
4. The effect of the (first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio on the column performance is limited to the time of sample injection until the sample band is divided, and the sample band is divided and the front part of the sample band is divided. After the introduction to the column and the rear part of the sample band being discharged from the discharge port, the ratio of (first liquid feed port flow velocity) / (second liquid feed port flow velocity) = 100/0 to 0/100. The flow velocity ratio in the range gives the same result.
Etc. are shown.

Among the sample introduction methods of the present invention, the conventional sample injection method using a valve (FIG. 19 (A)) and the present invention are used to confirm the improvement of column performance by the sample introduction method involving division of the sample band and post-partial placement. The column performance in the sample introduction method (FIG. 19B) was compared. Among the sample introduction methods of the present invention, it was confirmed that the number of theoretical plates in the first chromatogram given by the front part of the sample band was improved by the sample introduction method involving the division of the sample band and the placement of the rear part.

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as a sample injection device, and a column Chromolith RP-18 column (C18 modified monolith type silica gel, inner diameter) was used. 4.6 mm, length 10 cm, using Merck KGaA, Darmstadt, Germany), mobile phase (acetonitrile / water = 60/40) from both the first and second liquid delivery ports at a total of 1.0 mL / min. The liquid was sent. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. As the connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. For the sample band dividing device, the sample band dividing portion having the structure shown in FIG. 3 (A) is used, and the connection pipe between the injection valve and the column inlet (the inner pipe of the double pipe for the first liquid feeding port). Was a stainless steel tube having an outer diameter of 0.363 mm, an inner diameter of 0.185 mm, and a length of 10 cm (capacity: about 2.7 μL). Further, as shown in FIG. 6, a sample introduction device having a vent discharge function provided with a second discharge port 601 upstream of the sample injection device 22 was used. Samples include Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0.3 mg / mL), naphthalene (N, 0.3 mg / mL). , Hexanophenone (HxPh, 0.44 mg / mL), Heptanophenone (HpPh, 0.46 mg / mL), Octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 19A (Comparative Example) is a chromatogram obtained by injecting 5 μL of Sample II by a conventional sample injection method and separating and eluting. The mobile phase (acetonitrile / water = 60/40) was fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio = 0.3 / 0. .7 mL / min, 1.0 mL / min in total).

In FIG. 19B, the mobile phase (acetoyl / water = 60/40) is fed from both the first and second liquid feed ports ((first liquid feed port flow rate) / (second liquid feed port flow rate)). Ratio = 0.3 / 0.7 mL / min, 1.0 mL / min in total), inject 5 μL of sample II by the conventional sample injection method, and 0.03 minutes after sample injection, the first liquid feed port feed (acetojet). Stop (/ water = 60/40, 0.3 mL / min) and increase the flow rate of the second liquid feed port (acetoyl / water = 60/40, 0.7 mL / min) to 1.0 mL / min. The sample band was divided by, the front part of the sample band was introduced into the column for separation and elution, and 12 minutes after the sample injection, the vent valve was opened while keeping the flow rate of the second liquid feed port at 1.0 mL / min. It was opened and the rear part of the sample band left in the first liquid feeding port was discharged.

In FIG. 19A (comparative example), the first, third, fourth, and seventh peaks are TU (non-retention), BuPh (retention coefficient k = 1.1), N (k = 1.7), and OccPh. For (k = 6.5), the theoretical plates 7850, 12450, 13280 and 11930 were obtained, respectively. In comparison with this, in FIG. 19 (B), TU (non-retention), BuPh (retention coefficient k = 1.1), N (k = 1.7) and OccPh (k = 6.5), respectively. The theoretical plates 9550, 13900, 13620 and 11940 were obtained. In the sample introduction method of the present invention, in which the sample is divided in the direction intersecting the axial direction and introduced into the column, the TU (non-retention) and BuPh (retention) eluted at the initial stage of the chromatogram are compared with the conventional sample injection method. It is possible to generate 1.03 to 1.22 times the number of theoretical plates for the three peaks of the coefficient k = 1.1) and N (k = 1.7).

In this example, the mobile phase was fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio = 0.3 / 0.7 mL / min. , Total 1.0 mL / min). Transferring the liquid from the second liquid feeding port at a flow rate within an appropriate range between the sample injection and the division of the sample band facilitates the adjustment of the introduction rate of the sample band to the column, and also makes it easy to adjust the introduction rate of the sample band. It has the effect of facilitating an increase in the flow velocity of the second liquid feed port during division and introduction into the column.

Further, in FIGS. 19A and 19B, the elution volume of OccPh at the 7th peak was 10.2 mL, which was among the elution volumes of the peaks for which the effect of the sample introduction method of the present invention was confirmed in Examples. Was the largest value.

The present invention relates to the spread and shape of the sample band in the conventional sample injection method using a valve and the sample introduction method of the present invention, as compared with the conventional sample injection methods (FIGS. 20A and 20B) (comparative example). The reduction of the sample bandwidth by the sample introduction method (FIGS. 20C and 20D) with the division of the sample band and the rear partial placement of the sample band was demonstrated without a column.

Among the sample introduction methods of the present invention, the sample introduction method involving division of the sample band and placement of the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band and keeps the sample in the first liquid feed port. This is an example in which the second chromatogram given by the rear part of the sample band is shown (FIGS. 20 (C) and 20 (D)). 20 (B) and (D) show the state of the sample band at the column inlet in FIGS. 19 (A) (conventional sample injection method) and FIG. 19 (B) (sample introduction method of the present invention), respectively. It can be thought of as representing.

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as the sample injection device, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU) was used. The mobile phase (acetonitrile / water = 60/40) was fed from both the first and second liquid feed ports at a total of 1.0 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 1 μL and a detection wavelength of 254 nm was used. The response of the detector was 0.01 seconds, and the data acquisition interval was 5 mSec. As the connecting tube from the column outlet to the detector cell inlet, a PEEK tube (capacity: about 0.48 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.0635 mm, and a length of 15 cm was used. For the sample band dividing device, the sample band dividing portion having the structure shown in FIG. 3 (A) is used, and the connection pipe between the injection valve and the column inlet (the inner pipe of the double pipe for the first liquid feeding port). Was a stainless steel tube having an outer diameter of 0.363 mm, an inner diameter of 0.185 mm, and a length of 10 cm (capacity: about 2.7 μL). Further, as shown in FIG. 6, a device having a vent discharge function provided with a second discharge port 601 upstream of the sample injection device 22 was used. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

20 (A) and 20 (B) are chromatograms obtained by injecting 2 μL and 5 μL of Sample II by a conventional sample injection method, respectively, and separating and eluting. The mobile phase (acetonitrile / water = 60/40) was fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) / (second liquid feed port flow velocity) ratio = 0.3 / 0. .7 mL / min, 1.0 mL / min in total). In FIGS. 20C and 20D, the mobile phase (acetonitrile / water = 60/40) is fed from both the first and second liquid feed ports ((first liquid feed port flow velocity) / (second feed). Liquid port flow velocity) ratio = 0.3 / 0.7 mL / min, total 1.0 mL / min), 2 μL and 5 μL of sample II were injected, respectively, and 0.02 minutes after sample injection, the liquid was sent to the first liquid supply port. Stop (acetonitrile / water = 60/40, 0.3 mL / min) and increase the flow rate of the second liquid feed port (acetonitrile / water = 60/40, 0.7 mL / min) to 1.0 mL / min. By doing so, the sample band is divided, the front part of the sample band is introduced into ZDU to elute, and 0.2 minutes after the sample injection, (first liquid feed port flow velocity) / (second liquid feed port flow velocity). The ratio was returned to 0.3 / 0.7 mL / min, for a total of 1.0 mL / min. 20 (E) is an overlay of FIGS. 20 (A) and 20 (C), and FIG. 20 (F) is an overlay of FIGS. 20 (B) and 20 (D).

In FIGS. 20 (A) and 20 (B) (comparative example), the peak of naphthalene shows large tailing, whereas in FIGS. 20 (C) and 20 (D), the peak width of the first chromatogram is small. The tailing portion is cut off from the peaks in FIGS. 20 (A) and 20 (B), respectively. It is shown that the rear part (tail part) of the sample band was removed by the division in the direction intersecting the axial direction of the sample band immediately before the column inlet and the introduction into the ZDU.

In FIGS. 20 (D) and 19 (B), the liquid feeding capacities from the first liquid feeding port from the injection of the sample to the division of the sample band are different between 9 μL and 6 μL. However, this point and the fact that this result was obtained under the same conditions except for the column means that in the chromatogram of FIG. 19 (B), even with a large column having an inner diameter of 4.6 mm and a length of 10 cm, especially in the early stage of the chromatogram. It is considered that the factor that made it possible to develop the performance close to the original performance of the column is the effect of reducing the dispersion of the sample band near the column inlet.

20 In (A), (B) (Comparative Example), the variance of the peak of naphthalene, the moment method, 22.6MyuL ^2, was calculated to 60.1μL ^2. On the other hand, in the first chromatograms of FIGS. 20 (C) and 20 (D), it was calculated as 14.7 μL ² and 23.1 μL ^2, and in the first chromatogram of FIG. 20 (D), FIG. 20 (B). ), The dispersion of the solute band was reduced by 60% or more. On the other hand, in the first chromatogram of FIG. 20 (C), the decrease of the band dispersion of the solute was only about 35% as compared with FIG. 20 (A). These results are shown in FIG. 20 under the condition that the volume of the mobile phase fed from the first liquid feeding port from sample injection to sample division is 0.3 mL / min × 0.02 min = 0.006 mL = 6 μL. In C), the sample injection capacity was 2 μL, and a small part was placed in the first liquid feeding port when the sample band was divided, whereas in FIG. 20 (D), the sample injection capacity was 5 μL. It is based on the fact that more parts were placed in the first liquid delivery port when the sample band was split. When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, as compared with the conventional sample injection method (FIG. 21 (A)) (comparative example), the division of the sample band of the present invention-after The reduction in sample bandwidth by the sample introduction method with partial placement (FIGS. 21B, 21C) was demonstrated without a column.

Among the sample introduction methods of the present invention, in the sample introduction method involving division of the sample band and partial placement after the sample, the sample band injected with 1 μL is divided into two at different timings and introduced into the ZDU and eluted. An example shown as a first chromatogram given by the front part of the band and a second chromatogram given by the rear part of the sample band held in the first liquid feeding port (FIGS. 21 (B) and 21 (C)), and a sample. This is an example (FIG. 21 (D)) in which the sample band does not reach the ZDU inlet and the first chromatogram cannot be obtained at the timing of division and introduction into ZDU. 21 (A) and 21 (C) are Examples 13, 22 (A) (conventional sample injection method using a valve) and FIG. 22 (B) (sample introduction method of the present invention), which will be described later, respectively. It is considered to represent the state of the sample band at the column entrance in.

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as the sample injection device, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU) was used. The mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.01 seconds, and the data acquisition interval was 5 mSec. The connecting tube from the ZDU outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm, and another ZDU (capacity: 0.02 μL). ), And further connected to a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. As the sample band dividing device, the same sample band dividing portion as in Examples 1 to 9 having the structure shown in FIG. 3 (A) was used. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

FIG. 21 (A) is a chromatogram in which 1 μL of sample naphthalene was injected and eluted by a conventional sample injection method. The mobile phase (acetonitrile / water = 60/40) was delivered at 0.1 mL / min from the first feed port. In FIGS. 21 (B), (C), and (D), the mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feed port at 0.1 mL / min, and 1 μL of sample naphthalene was injected. After 0.03 minutes, 0.02 minutes, and 0.01 minutes from the sample injection, the first liquid feed port feed was stopped, and the second feed port feed (acetonitrile / water = 60/40, 0. Start 1 mL / min) to divide the sample, introduce it into ZDU, and elute it. 0.2 minutes after injecting the sample, stop the liquid transfer from the second liquid supply port and feed it from the first liquid supply port. It is a chromatogram which was returned to the liquid (acetonitrile / water = 60/40, 0.1 mL / min) and separated.

In FIGS. 21 (A) (comparative example), the peak of naphthalene shows large tailing, whereas in FIGS. 21 (B) and 21 (C), the peak of the first chromatogram is shown in FIG. 21 (A). The tailing part is scraped off from the peak of the sample band, and it is shown that the rear part of the sample band was placed in the first liquid feeding port by dividing the sample band and introducing it into the ZDU.

In FIG. 21 (A) (Comparative Example), the variance of the naphthalene peak was calculated to be 0.95 μL ² by the moment method. On the other hand, in the first chromatograms of FIGS. 21 (B) and 21 (C), it was calculated as 0.27 μL ² and 0.17 μL ^2, and the sample of the present invention was divided in the direction intersecting the axial direction. It was reduced by 0.7 to 0.8 μL ² by the sample introduction method introduced into the column (ZDU in this case). By using a 6 nL capillary cell with a small capacity for UV detection and reducing the capacity of the flow path from the ZDU to the detector cell, under the condition that the element in which the solute is dispersed outside the column (outside the ZDU) is small. It was shown that the sample introduction method of the present invention has a very large effect of reducing the dispersion of the sample band by 80% as compared with the conventional sample injection method. When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used.

The measured value of the elution volume of naphthalene in Example 12 and FIG. 21 (A) (measured value at a flow rate of 0.1 mL / min, 0.1 mL / min × 0.025 min) was 2.5 μL. Subtracting the ZDU (capacity 0.02 μL), the capacity from the ZDU outlet to the detector cell inlet (about 0.55 μL), and the detector cell capacity of 6 nL, the value was 1.924 μL. In FIG. 21 (D), the sample is divided 0.01 minutes after the sample injection (the volume of the liquid fed from the first liquid feeding port is 0.1 mL / min × 0.01 min = 0.001 mL = 1 μL). When attempting to introduce into ZDU, the tip of the sample band did not reach the outlet of the first liquid feed port (inner pipe of the double tube), that is, the flow path switching point, and the entire sample band was the first liquid feed. No peak appeared in the first chromatogram because it was anchored in the loop of the port and Rheodyne 8125 valve (injector). It is shown that the entire sample band was eluted as the peak of the second chromatogram with the resumption of liquid feeding from the first liquid feeding port.

In FIGS. 21B and 21C, 0.1 mL / min × 0.03 min = 0.003 mL = 3 μL and 0.1 mL / min × 0.02 min = 0.002 mL = 2 μL are sent from the sample injection, respectively. When the sample is divided and introduced into ZDU at the time of liquidization, about 78% of the sample in FIG. 21 (B) and about 40% of the sample in FIG. 21 (C) are introduced into ZDU as the peak of the first chromatogram. Eluted. The symmetry coefficient of the peak of FIG. 21 (A) at the 50% peak height is 2.0, 1.32 for the peak of the first chromatogram of FIG. 21 (B), and that of the first chromatogram of FIG. 21 (C). The peak was 1.0. From these results, by controlling the liquid feeding capacity from the first liquid feeding port from the sample injection to the division of the sample band, the sample band having a different volume from the fixed volume sample band injected by the conventional method Was shown to be able to be introduced into the column.

In order to confirm the improvement of column performance by the sample introduction method involving division of the sample band and post-partial placement, the conventional sample injection method using a valve (FIG. 22 (A)) (comparative example) and the sample introduction method of the present invention (FIG.) The column performance in 22 (B)) was compared.

Among the sample introduction methods of the present invention, the sample introduction method involving division of the sample band and placement of the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band and keeps the sample in the first liquid feed port. It is an example showing the second chromatogram given by the rear part of the sample band (FIG. 22 (B)).

Using a UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) as a sample injection device, column Monobis C18 (C18 modified monolith type silica gel, inner diameter 1.0 mm). , Length 15 cm, Kyoto Monotech Co., Ltd.), the mobile phase (acetonitrile / water = 60/40) was sent at 0.1 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. For the connecting pipe from the column outlet to the detector cell entrance, a fused silica capillary tube (capacity: about 0.29 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 15 cm is connected to the ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected and used. As the sample band dividing device, the same sample band dividing portion as in Examples 1 to 8 and 12 having the structure shown in FIG. 3 (A) was used. Samples include Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0.3 mg / mL), naphthalene (N, 0.3 mg / mL). , Hexanophenone (HxPh, 0.44 mg / mL), Heptanophenone (HpPh, 0.46 mg / mL), Octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 22 (A) is a chromatogram obtained by injecting 1 μL of Sample II by a conventional sample injection method for separation. The mobile phase (acetonitrile / water = 60/40) was delivered at 0.1 mL / min from the first feed port.

In FIG. 22 (B), the mobile phase (acetonitrile / water = 60/40) is fed from the first feed port at 0.1 mL / min, and 1 μL of sample II is injected, and 0.02 minutes after the first feed. Stop the liquid transfer port, start the second liquid transfer port (acetonitrile / water = 60/40, 0.1 mL / min), divide the sample band, introduce the front part into the column, and separate and elute. , 10 minutes after sample injection, stop the liquid feeding from the second liquid feeding port, return to the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min), and return to the sample band. It is a chromatogram in which the rear part was introduced into the column and separated and eluted.

In FIG. 22 (A) (comparative example), the first, third, fourth, and seventh peaks are TU (non-retention), BuPh (retention coefficient k = 1.4), N (k = 2.3), and For OccPh (k = 9.4), the number of theoretical plates 6060, 1970, 15360 and 15960 are obtained, respectively. On the other hand, in the first chromatogram of FIG. 22 (B) in which the sample band was divided and the front portion was introduced into the column, TU (non-retention), BuPh (retention coefficient k = 1.4), N ( For k = 2.3) and OccPh (k = 9.4), the theoretical plates 13330, 17120, 18890, and 17350 were obtained, respectively. The sample introduction method of the present invention, in which a sample is divided in a direction intersecting the axial direction and introduced into a column, can generate about 1.09 to 2.20 times the number of theoretical plates as compared with the conventional sample injection method. It is said.

In order to confirm the improvement of column performance by the sample introduction method involving division of the sample band and the rear partial placement, the conventional sample injection method using a valve (FIG. 23 (A)) (comparative example) and the sample introduction method of the present invention (FIG.) The column performances in 23 (B) and (C)) were compared. Further, as compared with the conventional sample injection method (FIG. 23 (D)), the sample bandwidth according to the example (FIG. 23 (E)) in which the sample introduction method involving the division of the sample band and the rear partial placement of the present invention is continuously performed. Confirmed a decrease in.

Among the sample introduction methods of the present invention, the method of dividing the sample band and indwelling the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band, and the valve for switching between the inside of the first liquid feed port and the micro 6-way. It is an example showing the improvement of the number of theoretical plates in the second chromatogram given by the rear part of the sample band fixed in the loop of (FIGS. 23 (B) and 23 (C)). For comparison of column performance with these examples, examples by the conventional sample injection method are also shown (FIG. 23 (A)).

Further, the sample band is divided, the rear part of the sample band is placed in the first liquid feed port and the loop of the micro 6-way flow path switching valve, and the front part of the sample band is separated and eluted, and then the liquid is fed to the first liquid feed port. In this example, the rear part of the sample band is introduced into the column at regular intervals, separated and eluted by alternately repeating the second liquid feeding port and the second liquid feeding port, and the separation performance is improved in each chromatogram. There is (Fig. 23 (E)). For comparison of column performance with this example, an example by the conventional sample injection method is also shown (FIG. 23 (D)) (comparative example).

UHPLC device LC800 (GL Sciences, Inc.) equipped with a Rheodyne 8125 valve (2 μL loop, inner diameter 0.13 mm, length 15.0 cm and 5 μL loop, inner diameter 0.20 mm, length 15.9 cm were used properly) as a sample injection device. The mobile phase (acetonitrile / water = 60/40) was 0.1 mL / min and 0. using the column IntertStain C18 (filler diameter 2 μm, inner diameter 1.0 mm, length 5 cm, GL Sciences). The solution was sent at 05 mL / min. The temperature condition was 40 ° C. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 240 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. For the connecting pipe from the column outlet to the detector cell entrance, a stainless steel pipe (capacity: about 0.86 μL) with an outer diameter of 1.6 mm, an inner diameter of 0.13 mm, and a length of 6.5 cm is connected to the ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected and used. For the sample band dividing device, a sample band dividing portion having the structure shown in FIG. 4 was used.

Between the Rheodyne8125 valve and the column, a micro 6-way flow path switching valve (C72X-1696EH, VALCO) for switching the flow path is shown in FIG. 4 in the flow path configuration (first liquid supply port 51, second liquid supply port). 52, the flow path switching point 50, the loop 53, and the column 1) were incorporated, and this valve was operated by LC800 to divide the sample band and introduce it into the column.

A stainless steel pipe (capacity: about 0.79 μL) with an outer diameter of 1.6 mm, an inner diameter of 0.1 mm, and a length of 10 cm is used for the piping from the Rheodyne 8125 valve to the C72X-1696EH valve, and the capacity of the loop 53 is 2 μL (Fig. 23 (A), (B), (C)) and 5 μL (used in FIGS. 23 (D), (E)). A stainless steel pipe (capacity: about 0.86 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.13 mm, and a length of 6.5 cm was used for the piping from the flow path switching point 50 to the column inlet. The other pipes connected to the C72X-1696EH valve were stainless steel pipes with an outer diameter of 1.6 mm and an inner diameter of 0.1 mm.
Samples include, in FIGS. 23 (A), (B) and (C), Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0. 3 mg / mL), naphthalene (N, 0.3 mg / mL), hexanophenone (HxPh, 0.44 mg / mL), heptanophenone (HpPh, 0.46 mg / mL), octanophenone (OcPh, 0. A 60/40 solution of acetonitrile / water of the mixture (6 mg / mL) was used.

Further, in FIGS. 23 (D) and 23 (E), Sample III: uracil (0.005 mg / mL), acetoanilide (AcAn, 0.05 mg / mL), acetophenone (AcPh, 0.05 mg / mL), propiophenone. (PrPh, 0.05 mg / mL), butyrophenone (BuPh, 0.05 mg / mL), benzophenone (BePh, 0.05 mg / mL), valerophenone (VaPh, 0.05 mg / mL), hexanophenone (HxPh, 0) A mixture of 0.05 mg / mL), heptanophenone (HpPh, 0.05 mg / mL) and octanophenone (OcPh, 0.05 mg / mL) in acetonitrile / water = 65/35 was used.

In FIGS. 23 (A), (B) and (C), the loop capacitance of the Rheodyne8125 valve and the loop capacitance of the C72X-1696EH valve were set to 2 μL. In FIGS. 23 (D) and 23 (E), the loop capacitance of the Rheodyne 8125 valve and the loop capacitance of the C72X-1696EH valve were set to 5 μL. The numerical value attached to each peak on the chromatogram indicates the peak half width.

FIG. 23 (A) is a chromatogram obtained by injecting 0.2 μL of Sample II by a conventional sample injection method for separation. The mobile phase (acetonitrile / water = 60/40) was delivered at 0.1 mL / min from the first feed port.

FIG. 23 (B) shows 0.04 minutes after the mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.1 mL / min and 0.2 μL of sample II was injected. Operate the micro 6-way flow path switching valve (C72X-1696EH valve) shown in FIG. 4 to switch, stop the first liquid feed port feed, and the second feed port feed (acetonitrile / water = 60/40, 0). .1 mL / min) is started to divide the sample band, the front part is introduced into the column for separation and elution, and 5.0 minutes after sample injection, the micro 6-way flow path switching valve is operated to perform sample injection. By switching to the flow path of, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) is returned to the first. This is a chromatogram in which the rear part of the sample band, which was placed in the liquid feed port and in the loop of the micro 6-way flow path switching valve, was introduced into the column and separated and eluted.

FIG. 23 (C) shows 0.05 minutes after the mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.1 mL / min and 0.2 μL of sample II was injected. Operate the micro 6-way flow path switching valve (C72X-1696EH valve) shown in FIG. 4 to switch, stop the first liquid feed port feed, and the second feed port feed (acetonitrile / water = 60/40, 0). .1 mL / min) is started to divide the sample band, the front part is introduced into the column for separation and elution, and 5.0 minutes after sample injection, the micro 6-way flow path switching valve is operated to perform sample injection. By switching to the flow path of, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) is returned to the first. This is a chromatogram in which the rear part of the sample band, which was placed in the liquid feed port and in the loop of the micro 6-way flow path switching valve, was introduced into the column and separated and eluted.

FIG. 23 (D) is a chromatogram obtained by injecting 0.5 μL of Sample III by a conventional sample injection method and separating the sample III. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.05 mL / min.

In FIG. 23 (E), the mobile phase (acetoyl / water = 60/40) is fed from the first liquid feed port at a flow rate of 0.05 mL / min, 5 μL of sample III is injected, and 0.09 minutes later, micro 6 By operating the direction flow path switching valve, the first liquid feed port feed is stopped, and the sample band is split by switching to the second feed port feed (acetoyl / water = 60/40, 0.05 mL / min). , Introduced into a column, separated and eluted (Fig. 23 (E) first chromatogram), 10.00 minutes to 0.02 minutes (10.00 to 10.02 minutes) after sample injection, micro By operating the 6-way flow path switching valve, the 2nd liquid feed port feed is stopped, the liquid is fed from the 1st liquid feed port (acetoyl / water = 60/40, 0.05 mL / min), and the 1st feed is performed. 0.02 minutes of liquid feed (1 μL capacity) was introduced into the column from the sample band placed in the liquid port and in the loop of the micro 6-way flow path switching valve, and 10.02 minutes after sample injection, micro By operating the 6-way flow path switching valve, the first liquid feed port liquid feed was stopped, and the second liquid feed port liquid feed (acetoyl / water = 60/40, 0.05 mL / min) was switched to separate elution. (FIG. 23 (E) second chromatogram). Further, every 10 minutes thereafter, that is, 0.01 minutes each from 20.00, 30.00, 40.00, and 50.00 minutes after sample injection, the capacity is increased by operating the micro 6-way flow path switching valve as described above. A sample band of 0.5 μL was introduced into the column, separated and eluted to obtain the third to sixth chromatograms of FIG. 23 (E). Finally, 60.00 minutes after the sample injection, the micro 6-way flow path switching valve was operated to switch to the liquid feed (acetonitrile / water = 60/40, 0.05 mL / min) from the first liquid feed port. The sample band remaining in the first liquid feed port and the loop of the micro 6-way flow path switching valve was introduced into the column and separated and eluted to obtain the 7th chromatogram of FIG. 23 (E).

In FIG. 23A (Comparative Example), the theoretical plates 440, 3520, 5220 and 7880 were obtained for the first, third, fourth and seventh peaks TU, BuPh, N and OccPh, respectively. On the other hand, in the first chromatogram of FIG. 23 (B), in which the sample band was divided by switching the liquid feeding port using a valve and the front portion was introduced into the column, TU, BuPh, N and OccPh were shown, respectively. , 900, 5660, 7140 and 8540 theoretical plates were obtained. The sample introduction method of the present invention, in which a sample is divided in a direction intersecting the axial direction and introduced into a column, can generate about 1.08 to 2.00 times the number of theoretical plates as compared with the conventional sample injection method. It is said. Great effect on the peak at the beginning of the chromatogram.

In the first chromatogram of FIG. 23 (C), the sample band was divided by switching the liquid feed port using the micro 6-way flow path switching valve and the front part was introduced into the column, for TU, BuPh, N and OccPh. The number of theoretical plates 680, 4900, 6410 and 8640 were obtained, respectively. The sample introduction method of the present invention makes it possible to generate about 1.07 to 1.50 times the number of theoretical plates as compared with the conventional sample injection method. In FIG. 23 (B), a sample introduction rate of 41% was obtained by dividing the sample 0.04 minutes after the sample injection and introducing the sample into the column, whereas in FIG. 23 (C), the sample was injected. A sample introduction rate of 73% was obtained by dividing the sample after 0.05 minutes and introducing it into the column. When the sample introduction rate is high, the sample band introduced into the column contains a larger rear portion (tail portion), and therefore, in the first chromatogram of FIG. 23 (B) and the first chromatogram of FIG. 23 (C). , FIG. 23 (B), the first chromatogram has a higher number of theoretical plates.

In FIGS. 23 (B) and 23 (C), the number of theoretical plates of the peak of the second chromatogram given by the rear portion of the divided sample band is also larger than that in FIG. 23 (A). In FIG. 23 (A), theoretical plates of 440, 3520, 5220 and 7880 were obtained for TU, BuPh, N and OccPh, respectively, whereas in the second chromatogram of FIG. 23 (B), TU, Theoretical plates 640, 4790, 6320 and 8330 were obtained for BuPh, N and OccPh, respectively. Further, in the second chromatogram of FIG. 23C, theoretical plates of 750, 5280, 6770 and 8460 were obtained for TU, BuPh, N and OccPh, respectively. The sample introduction method of the present invention, in which a sample is divided in a direction intersecting the axial direction and introduced into a column, enables generation of a higher number of theoretical plates as compared with a conventional sample injection method.

In FIG. 23 (D) (Comparative Example), the theoretical plates 930, 3910, 5110, and 6640 were obtained for the 2, 5, 7, and 9th peaks, AcAn, BuPh, VaPh, and HpPh, respectively. On the other hand, in the third to sixth chromatograms of FIG. 23 (E), average theoretical plates of 2840, 6950, 7270, and 6860 were obtained for AcAn, BuPh, VaPh, and HpPh, respectively. The sample introduction method of the present invention, in which a sample is divided in a direction intersecting the axial direction and introduced into a column, can generate 1.03 to 3.06 times as many theoretical plates as a conventional sample injection method. It is said. Comparing the bandwidth (half width) at 50% height of the peak, it is 1.7 to 43% smaller in the 3rd to 6th chromatograms in FIG. 23 (E) than in FIG. 23 (D).

A small part of the sample band intermediate portion existing in the rotor channel of the valve immediately before the flow path switching point 50 of the first liquid feeding port shown in FIG. 4B is a micro 6-way flow path switching valve. With the switching, the sample band moves along with the front part or the rear part depending on the rotation direction of the rotor. Further, as shown in FIG. 4D, after the sample band is divided by switching the flow path, the loop cleaning pipe 58 of the switching valve is used in the loop during or after the separation and elution of the front part of the sample band. It is possible to eject the rear part of the indwelling sample band.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, as compared with the conventional sample injection method (FIG. 24 (A)) (comparative example), the division of the sample band of the present invention-after The reduction of the sample bandwidth by the sample introduction method with partial placement (FIGS. 24 (B) and 24 (C)) was demonstrated without a column. Further, as compared with the conventional sample injection method (FIGS. 24 (D) and 24 (E)), an example in which the sample introduction method involving the division of the sample band of the present invention and the rear partial placement is continuously performed (FIG. 24 (F)). The reduction in sample bandwidth due to the above was demonstrated without a column.

Among the sample introduction methods of the present invention, the sample introduction method involving division of the sample band and placement of the rear part improves the number of theoretical plates in the first chromatogram from the front part of the sample band, and switches between the inside of the first liquid feed port and the switching valve. This is an example showing an improvement in the number of theoretical plates in the second chromatogram given by the rear part of the sample band fastened in the loop of (FIGS. 24 (B) and 24 (C)). For comparison with these examples, examples by the conventional sample injection method are also shown (FIG. 24 (A)). Further, the sample band is divided, the rear part of the sample band is placed in the first liquid feed port and the loop of the micro 6-way flow path switching valve, and the front part of the sample band is separated and eluted, and then the liquid is fed to the first liquid feed port. This is an example in which the rear part of the sample band is introduced into the ZDU in 6 batches at regular intervals and eluted by alternately repeating the second liquid feeding port and the second liquid feeding port, and the separation performance in each chromatogram is improved. (FIG. 24 (F)). For comparison with this example, an example by the conventional sample injection method is also shown (FIGS. 24 (D) and (E)). 24 (A), (B) and (C) are Examples 14, 23 (A) (conventional sample injection method) and 23 (B) and (C) (sample introduction method of the present invention, respectively). ) Represents the state of the sample band at the column entrance. Further, FIGS. 24 (E) and 24 (F) are shown at the column inlets in FIGS. 14, 23 (D) (conventional sample injection method) and 23 (E) (sample introduction method of the present invention), respectively. Represents the state of the sample band.

UHPLC device LC800 (GL Sciences, Inc.) equipped with a Rheodyne 8125 valve (2 μL loop, inner diameter 0.13 mm, length 15.0 cm and 5 μL loop, inner diameter 0.20 mm, length 15.9 cm were used properly) as a sample injection device. A zero dead volume union (U-435, inner diameter 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU) was used instead of the column. The mobile phase (acetonitrile / water = 60/40) was fed at 0.1 mL / min and 0.05 mL / min. The temperature condition was 40 ° C. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 240 nm was used. The response of the detector was 0.01 seconds, and the data acquisition interval was 5 mSec. The connecting pipe from the ZDU outlet to the detector cell entrance is a stainless steel pipe (capacity: about 0.86 μL) with an outer diameter of 1.6 mm, an inner diameter of 0.13 mm, and a length of 6.5 cm, and another ZDU (capacity: 0.02 μL). ), And further connected to a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. For the sample band dividing device, a sample band dividing portion having the structure shown in FIG. 4 was used.

Between the Rheodyne8125 valve and the column, a micro 6-way flow path switching valve (C72X-1696EH, VALCO) for switching the flow path is shown in FIG. 4 in the flow path configuration (first liquid supply port 51, second liquid supply port). 52, the flow path switching point 50, the loop 53, and the column 1) were incorporated, and this valve was operated by LC800 to divide the sample band and introduce it into the ZDU. A stainless steel pipe (capacity: about 0.79 μL) with an outer diameter of 1.6 mm, an inner diameter of 0.1 mm, and a length of 10 cm is used for the piping from the Rheodyne8125 valve to the C72X-1696EH valve, and the capacity of the loop 53 is 2 μL (Fig. 24 (A), (B), (C)) and 5 μL (used in FIGS. 24 (D), (E), (F)). A stainless steel pipe (capacity: about 0.86 μL) having an outer diameter of 1.6 mm, an inner diameter of 0.13 mm, and a length of 6.5 cm was used for the pipe from the flow path switching point 50 to the ZDU inlet. The other pipes connected to the C72X-1696EH valve were stainless steel pipes with an outer diameter of 1.6 mm and an inner diameter of 0.1 mm. As a sample, a solution of sample uracil: uracil (0.07 mg / mL) in acetonitrile / water = 60/40 was used. The numerical values attached to each peak on the chromatogram indicate tR: retention time and tW: half width of peak.

FIG. 24 (A) is a chromatogram in which 0.2 μL of sample uracil was injected and eluted by a conventional sample injection method. The mobile phase (acetonitrile / water = 60/40) was delivered at 0.1 mL / min from the first feed port.

FIG. 24 (B) shows 0.04 minutes after the mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feed port at 0.1 mL / min and 0.2 μL of sample uracil was injected. Operate the micro 6-way flow path switching valve (C72X-1696EH valve) shown in FIG. 4 to switch, stop the first liquid feed port feed, and the second feed port feed (acetonitrile / water = 60/40, 0). .1 mL / min) is started to divide the sample band, the front part is introduced into ZDU to separate and elute, and 0.5 minutes after sample injection, the micro 6-way flow path switching valve is operated to perform sample injection. By switching to the flow path of, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) is returned to the first. This is a chromatogram in which the rear part of the sample band, which was indwelled in the liquid feed port and the loop of the flow path switching valve, was introduced into ZDU and separated and eluted.

FIG. 24 (C) shows 0.05 minutes after the mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feed port at 0.1 mL / min and 0.2 μL of sample uracil was injected. Operate the micro 6-way flow path switching valve (C72X-1696EH valve) shown in FIG. 4 to switch, stop the first liquid feed port feed, and the second feed port feed (acetonitrile / water = 60/40, 0). .1 mL / min) is started to divide the sample band, the front part is introduced into ZDU to separate and elute, and 0.5 minutes after sample injection, the micro 6-way flow path switching valve is operated to perform sample injection. By switching to the flow path of, the liquid feeding from the second liquid feeding port is stopped, and the liquid feeding from the first liquid feeding port (acetonitrile / water = 60/40, 0.1 mL / min) is returned to the first. This is a chromatogram in which the rear part of the sample band, which was placed in the liquid feed port and in the loop of the micro 6-way flow path switching valve, was introduced into ZDU and separated and eluted.

FIG. 24D is a chromatogram obtained by injecting 0.5 μL of sample uracil by a conventional sample injection method for separation. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.05 mL / min. FIG. 24 (E) is a chromatogram obtained by injecting 5.0 μL of sample uracil by a conventional sample injection method for separation. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.05 mL / min.

In FIG. 24 (F), the mobile phase (acetoyl / water = 60/40) was fed from the first liquid feed port at a flow rate of 0.05 mL / min, 5 μL of sample uracil was injected, and 0.09 minutes later, micro 6 By operating the direction flow path switching valve, the liquid feed at the first liquid feed port is stopped, and the sample band is divided by switching to the liquid feed at the second liquid feed port (acetoyl / water = 60/40, 0.05 mL / min). , Introduced into ZDU, separated and eluted (Fig. 24 (F) 1st chromatogram), between 0.50 minutes and 0.02 minutes (0.50 minutes to 0.52 minutes) after sample injection. ), By operating the micro 6-way flow path switching valve, the liquid feed from the 2nd liquid feed port is stopped and returned to the liquid feed from the 1st liquid feed port (acetoyl / water = 60/40, 0.05 mL / min). 0.02 minutes of liquid feed (capacity 1 μL) was introduced into the ZDU from the sample band placed in the first liquid feed port and in the loop of the micro 6-way flow path switching valve, and 0.52 from the sample injection. After a minute, by operating the micro 6-way flow path switching valve, the first liquid feed port liquid feed is stopped and switched to the second liquid feed port liquid feed (acetoyl / water = 60/40, 0.05 mL / min). (Fig. 24 (F) second chromatogram). After that, every 0.5 minutes, that is, 1.0, 1.5, 2.0, and 2.5 minutes after sample injection, 0.01 minutes each, operate the micro 6-way flow path switching valve as described above. A sample band having a volume of 0.5 μL was introduced into ZDU and eluted to obtain the third to sixth chromatograms of FIG. 24 (F). Finally, 3.0 minutes after the sample injection, the micro 6-way flow path switching valve is operated to switch to the liquid feed (acetonitrile / water = 60/40, 0.05 mL / min) from the first liquid feed port. The sample band remaining in the first liquid feed port and the loop of the micro 6-way flow path switching valve was eluted to obtain the 7th chromatogram of FIG. 24 (F).

As shown in FIG. 24 (B), when the liquid feed capacity from the first liquid feed port from sample injection to division in the direction intersecting the axial direction of the sample band and introduction into ZDU is 4 μL, About 41% of the total injected sample was introduced and appeared as the peak of the first chromatogram. At the peak of FIG. 24 (A) (comparative example), the variance of the sample band calculated by the moment method was 1.68 μL ² , whereas at the peak of the first chromatogram of FIG. 24 (B), the sample band was The variance was 0.68 μL ² , a decrease of about 60% from the peak in FIG. 24 (A). When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used.

As shown in FIG. 24 (C), when the liquid feed capacity from the first liquid feed port from sample injection to division in the direction intersecting the axial direction of the sample band and introduction into ZDU is 5 μL, About 73% of the total injected sample was introduced and appeared as the peak of the first chromatogram. At the peak of the first chromatogram of FIG. 24 (C), the variance of the sample band calculated by the moment method was 0.97 μL ² , which was about 40% less than the peak of FIG. 24 (A). In both cases of FIGS. 24 (B) and 24 (C), a decrease in the sample bandwidth was observed at the peak of the second chromatogram as compared with the peak of FIG. 24 (A), and the magnitude of the width was determined. It is similar to the tendency that the peak width at the peak of the second chromatogram of FIGS. 23 (B) and 23 (C) decreases as compared with the peak of FIG. 23 (A). When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used.

In FIGS. 24 (B) and 24 (C), the elution volume of the peak of the second chromatogram was 0.1 mL / min × 0.035 min = 0.0035 mL = 3.5 μL. When the capacity of ZDU (0.02 μL), the capacity of the pipe from ZDU to the inlet of the detector (1.12 μL), and the capacity of the detector cell (6 nL) are subtracted from this value, it becomes 2.354 μL, which is the flow shown in FIG. The measured value of the volume from the road switching point 50 to the ZDU inlet at 0.1 mL / min was 2.354 μL. Further, in FIG. 24 (A), the elution volume of the peak was 0.1 mL / min × 0.068 min = 0.0068 mL = 6.8 μL. Subtracting the capacity of ZDU (0.02 μL), the capacity of the pipe from ZDU to the inlet of the detector (1.12 μL), and the capacity of the detector cell (6 nL) gives 5.654 μL, and the above measured value 2 Subtracting .354 μL yielded 3.3 μL. Therefore, under the condition that the sample uracil having a volume of 0.2 μL is eluted at a flow rate of 0.1 mL / min, the volume from the injector to the flow path switching point is 3.3 μL, and the volume from the flow path switching point to the ZDU inlet is 2.354 μL. It became. Under this condition, when the liquid feed volumes from the first liquid feed port until the sample band division are 4 μL and 5 μL, respectively, in FIGS. 24 (B) and 24 (C), the sample introduction rates are 41% and 73, respectively. It became%. These sample introduction rates are in agreement with the sample introduction rates in FIGS. 23 (B) and 23 (C).

In FIG. 24 (D), the elution volume of the peak was 0.05 mL / min × 0.167 min = 0.00835 mL = 8.35 μL. Subtracting 0.02 μL of ZDU capacitance from this value gives 8.33 μL. In the device configuration using a 5 μL loop injector, when 0.5 μL sample is injected by the conventional sample injection method, it is outside the column calculated from the peak elution time (in this experiment, it is not outside the column but outside the ZDU). The volume of was 8.33 μL. On the other hand, in the third to sixth chromatograms of FIG. 24 (F), 0.5 μL of each sample band placed in the loop of the flow path switching valve of the sample band dividing device was introduced into the ZDU. Since the sample uracil was eluted on average 0.049 minutes after the introduction, the peak elution volume was 0.05 mL / min × 0.049 min = 0.00245 mL = 2.45 μL. Subtracting 0.02 μL of ZDU capacitance from this value gives 2.43 μL. It was confirmed that the volume outside the column (outside the ZDU in this experiment) was reduced by 5.9 μL as a measured value by the sample introduction method of the present invention. Further, when the capacity of the pipe from ZDU to the detector inlet (1.12 μL) and the capacity of the detector cell (6 nL) are subtracted from 2.43 μL, the result is 1.304 μL, which is obtained from the flow path switching point 50 shown in FIG. The measured value of the volume up to the ZDU inlet at 0.05 mL / min was 1.304 μL.

As disclosed in Non-Patent Document 3, in a normal UHPLC instrument, the extra-column volume measured in the same manner as in Non-Patent Document 2 is 12-18 μL at a flow rate of 0.1 to 2.0 mL / min. There is big. If the sample introduction method of the present invention is applied to these devices, the volume outside the column can be reduced and the dispersion of the solute can be made very small.

Figure 24 (D), (E) ( Comparative Example) each dispersion band peak uracil calculated by the moment method in, 1.00 L ^2, whereas was 1.16μL ^2, FIG. 24 (F The dispersion of the uracil peak band in the 3rd to 6th chromatograms of) was 0.26 μL ² on average. In both the 3rd to 6th chromatograms of FIGS. 24 (D) and 24 (F), a sample having a volume of 0.5 μL is injected or introduced into ZDU, but the volume is the same. The dispersion of eluted peaks is very different. This is the effect of the present invention, which is the division of the sample in the direction intersecting the axial direction and the introduction into the column (here, ZDU). When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used.

According to the examples shown in FIGS. 23 (E) and 24 (F) in Examples 14 and 15, the sample introduction method of the present invention can originally divide one sample band into a large number for analysis. Although it was confirmed, between the flow path switching point 50 of FIG. 4 and the column 1 used in this example, from the sample band dividing portion including the four-way joint shown in FIG. 7 and the T-shaped connector shown in FIG. If the sample band dividing portion is provided, the front portion of the sample band can be discharged without being introduced into the column, and the intermediate portion of the sample band can be quickly analyzed.

In Examples 14 and 15, the sample introduction method of the present invention using the micro 6-way flow path switching valve was shown, but the sample introduction method of the present invention using the 3-way flow path switching valve is separately used for the liquid feeding from the second liquid feeding port. The sample introduction method of the present invention can be carried out by using a liquid feed pump. Further, even if a 4-way flow path switching valve, an 8-way flow path switching valve, or a 10-way flow path switching valve is used, the sample introduction method of the present invention can be obtained by forming the same flow path configuration as the 6-way flow path switching valve. Can be carried out.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, as compared with the conventional sample injection method (FIG. 25 (A)) (comparative example), the division of the sample band of the present invention-after It was confirmed that the number of theoretical stages of the column was improved by the sample introduction method with partial placement (FIG. 25 (B)) and the sample introduction method by dividing the sample band of the present invention-rear partial discharge (FIG. 25 (C)).

FIG. 25B shows the sample introduction method of the present invention involving the division of the sample band and the placement of the rear part from the front part of the sample band by dividing the sample band and introducing the front part into the column. This is an example of improving the number of theoretical plates in the chromatogram and discharging the rear part of the sample band. A discharge port upstream of the sample injection device (injector) was used to discharge the rear part of the sample band as shown in FIG.

FIG. 25 (C) shows the sample introduction method of the present invention involving the division of the sample band and the discharge of the rear part from the front part of the sample band by dividing the sample band and introducing the front part into the column. This is an example of improving the number of theoretical plates in the chromatogram and discharging the rear part of the sample band. The discharge port connected to the cloth placed between the injector and the column as shown in FIG. 7B was used for discharging the rear part of the sample band.

Using a UHPLC device LC800 (GL Sciences) equipped with a Valco injector valve (VICI C4-0004-01, 10 nL internal loop) as a sample injection device, column Monocap C18 (C18 modified monolith type silica gel, inner diameter 0.1 mm, length) 15 cm, Kyoto Monotech Co., Ltd.) was used. The mobile phase (acetonitrile / water = 60/40) was fed at 0.002 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. The connecting tube from the column outlet to the detector cell inlet was used by connecting a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. As the sample band dividing device, a sample band dividing portion having the structure shown in FIG. 7 (B) was used. Further, as shown in FIG. 6, a discharge port was separately installed upstream of the sample injection device.

The details of the sample band dividing portion shown in FIG. 7B show that the Valco injector valve and the cloth (4-way joint, U-430, inner diameter 0.50 mm, internal capacity 0.72 μL, IDEX) have an outer diameter of 0. Fused silica capillary tube (capacity: about 0.035 μL) with a diameter of 375 mm, inner diameter of 0.03 mm, and length of 5 cm is connected, and the cloth and column inlet are fused silica with an outer diameter of 0.375 mm, inner diameter of 0.03 mm, and length of 6 cm. It was connected with a capillary tube (capacity: about 0.042 μL). As shown in FIG. 9 (A), the two fused silica capillary connecting pipes connected to the cloth were connected by being inserted into the inside of the cloth, and the gap between the two connecting pipes was set to 0.2 mm. In addition, a second liquid feed port and a discharge port (a vent valve and a resistance tube (inner diameter 0.03 mm, length 30 cm) are connected) are provided as separate ports on the cross.

Samples include Sample II: thiourea (TU, 0.1 mg / mL), acetophenone (AcPh, 0.16 mg / mL), butyrophenone (BuPh, 0.3 mg / mL), naphthalene (N, 0.3 mg / mL). , Hexanophenone (HxPh, 0.44 mg / mL), Heptanophenone (HpPh, 0.46 mg / mL), Octanophenone (OcPh, 0.6 mg / mL) in acetonitrile / water = 60/40 solution. used.

FIG. 25 (A) is a chromatogram obtained by injecting 0.01 μL of Sample II by a conventional sample injection method for separation. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.002 mL / min.

FIG. 25 (B) shows 0.15 minutes after the mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.002 mL / min and 0.01 μL of sample II was injected. Stop the first liquid feed port feed, start the second feed port feed (acetonitrile / water = 60/40, 0.02 mL / min) to divide the sample band, introduce the front part into the column and separate. After elution and 5.0 minutes after sample injection, stop the liquid feed from the second liquid feed port and switch to the liquid feed (acetonitrile / water = 60/40, 0.002 mL / min) from the first liquid feed port. This is the returned chromatogram.

From 0.15 minutes to 5.0 minutes after sample injection, the vent valve connected to the upstream discharge port of the sample injection device was opened as shown in FIG. 6, and the inner diameter was 0.03 mm and the length was 30 cm. The rear part of the sample band was discharged through the resistance of the fused silica capillary tube. From 0.15 minutes to 5.0 minutes after sample injection, the flow rate from the second liquid feed port was set to 0.02 mL / min, which is necessary for separating and eluting the front part of the sample band with a column. This is because the flow velocity required to discharge the rear part of the sample band from the discharge port is added to the normal flow velocity. The vent valve at the end of the discharge port connected to the cross, shown in FIG. 7B, was left closed.

FIG. 25 (C) shows 0.15 minutes after the mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.002 mL / min and 0.01 μL of sample II was injected. Stop the first liquid feed port feed, start the second feed port feed (acetonitrile / water = 60/40, 0.02 mL / min) to divide the sample band, introduce the front part into the column and separate. After elution and 5.0 minutes after sample injection, stop the liquid feed from the second liquid feed port and switch to the liquid feed (acetonitrile / water = 60/40, 0.002 mL / min) from the first liquid feed port. This is the returned chromatogram.

From 0.15 minutes to 5.0 minutes after sample injection, open the vent valve at the end of the discharge port connected to the cloth shown in FIG. 7 (B), and fuse with an inner diameter of 0.03 mm and a length of 30 cm. The rear part of the sample band was discharged through the resistance of the dosilica capillary tube. From 0.15 minutes to 5.0 minutes after sample injection, the flow rate from the second liquid feed port was set to 0.02 mL / min, which is necessary for separating and eluting the front part of the sample band with a column. This is because the flow velocity required to discharge the rear part of the sample band from the discharge port is added to the normal flow velocity. The vent valve at the end of the discharge port upstream of the sample injection device as shown in FIG. 6 was left closed.

As shown in FIG. 25 (A) (comparative example), when a very small capillary column (inner diameter 0.1 mm, length 15 cm) is used, the effect of dispersing the solute outside the column appears very large. For the 4th, 6th, and 7th peaks, N, HpPh, and OccPh, the theoretical plates 410, 1140, and 1960 were obtained, respectively. On the other hand, in FIG. 25 (B) from the front part of the sample band obtained by the sample introduction method of the present invention, the theoretical plates of 2190, 4360, and 5430 were obtained for N, HpPh, and OccPh, respectively. .. In FIG. 25C, theoretical plates of 2380, 4300, and 5440 are obtained for N, HpPh, and OccPh, respectively, and the sample of the present invention is divided in the direction intersecting the axial direction as compared with the conventional sample injection method. In the sample introduction method introduced into the column, the number of theoretical plates was 2.5 to 5.0 times.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, as compared with the conventional sample injection method (FIG. 26 (A)) (comparative example), the division of the sample band of the present invention-after The reduction of the sample bandwidth by the sample introduction method with partial placement (FIG. 26 (B)) and the sample introduction method with partial band division-posterior partial discharge of the present invention (FIG. 26 (C)) without a column. Demonstrated.

26 (A), (B) and (C) show the state of the sample band at the column entrance in FIGS. 25 (A), (B) and (C), respectively.

A UHPLC device LC800 (GL Sciences) equipped with a Valco injector valve (VICI C4-0004-01, 10 nL internal loop) was used as the sample injection device, and instead of the column, the outer diameter was 0.375 mm and the inner diameter was 0.03 mm. For a 6 cm long fused silica capillary tube (capacity approx. 0.042 μL) with a zero dead volume union (U-435, inner diameter 0.25 mm, internal capacity 0.02 μL, IDEX) connected to the inlet and outlet. There was. The mobile phase (acetonitrile / water = 60/40) was fed at 0.003 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.01 seconds, and the data acquisition interval was 5 mSec. Fused silica capillary tube instead of column The connecting tube from the outlet to the detector cell inlet connects a fused silica capillary tube (capacity: about 0.24 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. And used. As the sample band dividing device, a sample band dividing portion having the structure shown in FIG. 7 (B) was used. Further, as shown in FIG. 6, a discharge port was separately installed upstream of the sample injection device.

The details of the sample band dividing portion shown in FIG. 7 (B) are as follows: Valco injector valve and cross (4-way joint, U-430, inner diameter 0.50 mm, internal capacity 0.72 μL, IDEX), outer diameter 0.375 mm. Fused silica capillary tube with an inner diameter of 0.03 mm and a length of 5 cm (capacity: about 0.035 μL) is connected, and the fused silica capillary tube instead of the cloth and column has an outer diameter of 0.375 mm, an inner diameter of 0.03 mm, and a length. It was connected with a 6 cm fused silica capillary tube (capacity: about 0.042 μL). As shown in FIG. 9A, the two connecting pipes connected to the cloth were connected by being inserted into the inside of the cloth, and the gap between the two connecting pipes was set to 0.2 mm. In addition, a second liquid feed port and a discharge port (a vent valve and a resistance tube (inner diameter 0.03 mm, length 30 cm) are connected) are provided as separate ports on the cross. As a sample, a solution of sample naphthalene: naphthalene (N, 0.1 mg / mL) in acetonitrile / water = 60/40 was used.

FIG. 26 (A) is a chromatogram obtained by injecting 0.01 μL of sample naphthalene and eluting by the conventional sample injection method. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.003 mL / min.

FIG. 26 (B) shows 0.1 minutes after the mobile phase (acetonitrile / water = 60/40) was fed from the first liquid feed port at 0.003 mL / min and 0.01 μL of sample naphthalene was injected. Stop the first liquid feed port feed, start the second feed port feed (acetonitrile / water = 60/40, 0.004 mL / min) to divide the sample band, and fuse the front part instead of the column. It is introduced into a silica capillary tube and eluted, 1.0 minutes after sample injection, the liquid transfer from the second liquid supply port is stopped, and the liquid supply from the first liquid supply port (acetonitrile / water = 60/40, It is a chromatogram returned to 0.003 mL / min).

From 0.1 minutes to 1.0 minutes after sample injection, the vent valve connected to the upstream discharge port of the sample injection device was opened as shown in FIG. 6, and the inner diameter was 0.03 mm and the length was 30 cm. The rear part of the sample band was discharged through the resistance of the fused silica capillary tube. From 0.1 minutes to 1.0 minutes after sample injection, the flow rate from the second liquid feed port was set to 0.004 mL / min because the front part of the sample band was a fused silica capillary instead of a column. This is because the flow rate required for discharging the sample band from the discharge port is added to the flow rate required for elution from the tube. The vent valve at the end of the discharge port connected to the cross, shown in FIG. 7B, was left closed.

In FIG. 26 (C), the mobile phase (acetonitrile / water = 60/40) is fed from the first liquid feed port at 0.003 mL / min, and 0.01 μL of sample naphthalene is injected by the conventional sample injection method. After 0.1 minutes, stop the first liquid feed port feed, start the second feed port feed (acetonitrile / water = 60/40, 0.004 mL / min) and divide the sample band, the front part. Is introduced into a fused silica capillary tube instead of a column to elute, and 1.0 minutes after sample injection, the liquid transfer from the second liquid supply port is stopped, and the liquid transfer from the first liquid supply port (acetonitrile). / Water = 60/40, 0.003 mL / min).

From 0.1 minutes to 1.0 minutes after sample injection, open the vent valve at the end of the discharge port connected to the cloth shown in FIG. 7 (B), and fuse with an inner diameter of 0.03 mm and a length of 30 cm. The rear part of the sample band was discharged through the resistance of the dosilica capillary tube. From 0.1 minutes to 1.0 minutes after sample injection, the flow rate from the second liquid feed port was set to 0.004 mL / min because the front part of the sample band was a fused silica capillary instead of a column. This is because the flow rate required for discharging the sample band from the discharge port is added to the flow rate required for elution from the tube. The vent valve at the end of the discharge port upstream of the sample injection device as shown in FIG. 6 was left closed.

In FIGS. 26 (A) (comparative example), the peak of naphthalene shows large tailing, whereas in FIGS. 26 (B) and 26 (C), the peak width of the chromatogram is small, and FIG. 26 The tailing part is scraped off from the peak of (A). It was shown that the rear part (tail part) of the sample band was removed by dividing the sample band in the axially intersecting direction just before the column inlet and introducing it into the fused silica capillary tube instead of the column. There is.

In FIG. 26 (A) (Comparative Example), the variance of the naphthalene peak was calculated to be 0.092 μL ² by the moment method. In contrast, FIG. 26 (B), the variance of the peak of naphthalene (C), respectively, 0.023MyuL ^2, was calculated to 0.030μL ^2. According to the method of the present invention in which the sample is divided in the axially intersecting direction and the sample is introduced into the column (here, the fused silica capillary tube), 0.06-0.07 μL ² (about 60-70%) ) The variance became smaller. In FIGS. 26 (B) and 26 (C), these results were obtained under the same conditions as in FIGS. 25 (B) and 25 (C) except for the column and the flow velocity. Therefore, FIGS. 25 (B) and 25 (C) ), The reason why a column with an inner diameter of 0.1 mm and a length of 15 cm obtained a higher theoretical plate number than the chromatogram obtained by the conventional sample injection method (FIG. 25 (A)) is the column inlet. This is the effect of reducing the dispersion of the sample band in the vicinity. When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used.

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, the sample at the column inlet of the present invention is compared with the conventional sample injection method (FIG. 27 (A)) (comparative example). The column performance was compared by the sample introduction method (FIG. 27 (B)) involving sample division by discharging the rear part of the band.

Among the sample introduction methods of the present invention, improvement of column performance, that is, improvement of the number of theoretical plates by a sample introduction method involving sample division by discharging the rear part of the sample band using the sample band division portion shown in FIG. 8C. It is a chromatogram shown (FIG. 27 (B)).

Using a UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) as a sample injection device, column IntertStain C18 (filler diameter 2 μm, inner diameter 1.0 mm, A length of 5 cm (GL Sciences) was used. The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.05 seconds, and the data acquisition interval was 50 mSec. For the connecting pipe from the column outlet to the detector cell entrance, a fused silica capillary tube (capacity: about 0.88 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 45 cm is connected to the ZDU (capacity: 0.02 μL). Further, a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm was connected and used. As the sample band dividing device, a sample band dividing portion having the structure shown in FIG. 8C was used.

For the details of the sample band division shown in FIG. 8 (C), U-428 (inner diameter 0.5 mm, IDEX) is used for the T-shaped connector (three-way joint), and the double tube is from the sample injection valve to the column inlet. Stainless steel pipe (capacity: about 0.92 μL) with an outer diameter of 0.236 mm, an inner diameter of 0.108 mm, and a length of 10 cm for the connecting pipe (inner pipe for liquid feeding at the first liquid feeding port), and an outer diameter for the outer pipe. It was composed of a stainless steel tube having a diameter of 1.6 mm, an inner diameter of 0.5 mm, and a length of 5 cm. For the branch pipe of the T-shaped connector, a stainless steel pipe having an outer diameter of 1.6 mm and an inner diameter of 0.25 mm was used. A 6-way switching valve (Rheodyne7000) was used for the vent valve at the tip of the branch pipe. Samples include Sample IV: (thiourea (TU), acetanilide (AcAn), acetophenone (AcPh), propiophenone (PrPh), butyrophenone (BuPh), naphthalene (N), hexanophenone (HxPh), heptanophenone. An acetonitrile / water = 60/40 solution of a mixture of (HpPh) and octanophenone (OcPh) was used.

FIG. 27 (A) is a chromatogram obtained by injecting 0.2 μL of sample IV and separating by a conventional sample injection method. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.05 mL / min.

FIG. 27 (B) shows 0.03 minutes after the mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.05 mL / min and 0.2 μL of sample IV was injected. The vent valve was opened until about 0.04 minutes later, and the rear part of the sample band was discharged. The flow velocity of the first liquid feed port from 0.03 minutes to 0.04 minutes after the sample injection was 0.199 mL / min, and 0.05 mL / min after 0.04 minutes from the sample injection. From 0.03 minutes to 0.04 minutes after sample injection, the flow rate from the first liquid feed port was set to 0.199 mL / min, which is necessary to elute the front part of the sample band from the column. This is because the flow velocity required to discharge the rear part of the sample band from the discharge port is added to the flow velocity.

In FIG. 27 (A) (Comparative Example), the theoretical plates of 1090, 5800, 6490, and 6820 were obtained for the first, fifth, sixth, and ninth peaks, TU, BuPh, N, and OccPh, respectively. On the other hand, the number of theoretical plates in the chromatogram from the front part of the sample band obtained by the sample introduction method of the present invention in which the sample is divided in the direction intersecting the axial direction and introduced into the column is shown in FIG. 27 (B). ), BuPh, N, and OccPh were 4580, 8690, 8780, and 7310, respectively, showing a 1.07 to 4 times improvement in column performance. This is the result obtained by comparing the conventional sample injection method and the sample introduction method of the present invention using the same column in Example 3 (N in FIG. 12 (A) is 6900 steps, and FIG. 12 (D). The performance improvement is close to (N is 8410 steps).

Regarding the spread and shape of the sample band in the conventional sample injection method and the sample introduction method of the present invention, the sample at the column inlet of the present invention is compared with the conventional sample injection method (FIG. 28 (A)) (comparative example). The effect of reducing the sample bandwidth by the sample introduction method (FIG. 28 (B)) involving sample division by discharging the rear portion (tail portion) of the band was demonstrated without a column.

A UHPLC device LC800 (GL Sciences) equipped with a Rheodyne 8125 valve (5 μL loop, inner diameter 0.20 mm, length 15.9 cm) was used as the sample injection device, and a zero dead volume union (U-435, inner diameter) was used instead of the column. 0.25 mm, internal capacity 0.02 μL, IDEX, hereinafter referred to as ZDU) was used. The mobile phase (acetonitrile / water = 60/40) was fed at 0.05 mL / min. The temperature condition was room temperature. For detection, a UV detector (MU701, GL Sciences, Inc.) having a detection cell capacity of 6 nL and a detection wavelength of 254 nm was used. The response of the detector was 0.01 seconds, and the data acquisition interval was 5 mSec. The connecting tube from the column outlet to the detector cell inlet is a fused silica capillary tube (capacity: about 0.88 μL) with an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 45 cm, and another ZDU (capacity: 0.02 μL). ), And further connected to a fused silica capillary tube (capacity: about 0.24 μL) having an outer diameter of 0.375 mm, an inner diameter of 0.05 mm, and a length of 12 cm. As the sample band dividing device, the sample band dividing portion used in Example 18 having the structure shown in FIG. 8C was used. As a sample, a solution of sample uracil: uracil (U, 0.07 mg / mL) in acetonitrile / water = 60/40 was used.

FIG. 28 (A) is a chromatogram in which 0.2 μL of sample uracil was injected and eluted by a conventional sample injection method. The mobile phase (acetonitrile / water = 60/40) was fed from the first feed port at 0.05 mL / min.

In FIG. 28 (B), the mobile phase (acetonitrile / water = 60/40) is fed from the first liquid feed port at 0.05 mL / min, and 0.2 μL of sample uracil is injected by the conventional sample injection method. The vent valve was opened from 0.03 minutes to 0.04 minutes after that, and the rear part of the sample band was discharged. The flow velocity of the first liquid feed port from 0.03 minutes to 0.04 minutes after the sample injection was 0.199 mL / min, and 0.05 mL / min after 0.04 minutes from the sample injection. From 0.03 minutes to 0.04 minutes after sample injection, the flow rate from the first liquid feed port was set to 0.199 mL / min, which is necessary to elute the front part of the sample band from ZDU. This is because the flow velocity required to discharge the rear part of the sample band from the discharge port is added to the flow velocity. 28 (A) and 28 (B) show the extent and shape of the sample band at the column entrance in FIGS. 27 (A) and 27 (B), respectively.

In FIG. 28 (A) (comparative example), the uracil band shows a large tailing, whereas as shown in FIG. 28 (B), the liquid feed capacity from the first liquid feed port is 1.5 μL ( Approximately 71% of the injected sample band was introduced into the ZDU due to the axially intersecting division of the sample at a flow rate of 0.05 mL / min x 0.03 min), and the uracil band showed large tailing. Not. In FIG. 28 (A), the dispersion of the sample band calculated by the moment method was 0.95 μL ² , whereas in FIG. 28 (B), it was 0.22 μL ² , and the dispersion of the sample band was 70% or more. I was able to reduce it. In FIG. 28 (B), the fact that this result was obtained under the same conditions as in FIG. 27 (B) except for the column means that in the chromatogram of FIG. 27 (B), the inner diameter was 1.0 mm and the length was 5 cm. The reason why the number of theoretical plates obtained on the column is higher than that of the chromatogram (FIG. 27 (A)) obtained by the conventional sample injection method is the effect of reducing the dispersion of the sample band near the column inlet. When calculating the variance of peaks by the moment method, the full width at half maximum attached to each peak is not used.

Conventional sample injection under the conditions examined in Examples 1, 3, 5, 6, 7, 9, 10, 13, 16 (FIGS. 10, 12, 14, 15, 16, 18, 19, 22, 25). Compared with the method, the relationship between the increase ratio of the number of column theoretical plates by the sample introduction method of the present invention, the column size, and the detector cell capacity is as shown in Table 7.

As the column size increases, the effect of increasing the number of theoretical plates by dividing and introducing the sample band decreases, but when a small column is used, the column has a high potential, especially for the peak at the beginning of the chromatogram. In order to realize the performance (large number of theoretical plates), it is effective to cut and divide the sample band in the direction intersecting the axial direction at the column entrance and introduce only the front part of the sample band into the column. It has been shown that the structure and operation of the sample band dividing device described in the present invention make it possible to increase the theoretical plate number of columns.

Table 8 shows the results of calculating the dispersion σ _extra ² of the solute band outside the column from the examples measured by connecting a ZDU or a fused silica capillary pipe instead of the column. Peaks with good symmetry were calculated from the half width, and peaks with poor symmetry were calculated by the moment method. It has been shown that the sample introduction method of the present invention can reduce σ _extra ² by 30 to 80% as compared with the conventional sample injection method. Although σ _extra ² increases as the volume of the injected sample increases, the method of the present invention makes it possible to introduce the sample into the column as a very narrow band.

The dispersion of the sample band is the smallest at a low flow rate and when the time to sample division is shortened (at a low sample introduction rate). From this, even for the peak that appears at the beginning of the chromatogram, where the dispersion of the solute band originally in the column (σ _col ² , equation 1) is small, by reducing σ _extra ² , it is compared with the conventional sample injection method. It has become possible to obtain 1 to 5 times the number of theoretical plates (Table 8).

Further, in order to make the most effective use of the effect of the sample introduction method of the present invention, dispersion of solute bands (σ _tube ² + σ _det ) in the pipe ( _tube ² ) from the column outlet to the detector and the detector cell (det) It is appropriate to use a pipe or detector cell with a small inner diameter and a small capacity that can keep ² ) low.

The results of Examples 1, 3, 5, 6, 7, 9, 10, 13, and 14 (FIGS. 10, 12, 14, 15, 16, 18, 19, 22, and 23) show that the sample band was divided from the time of sample injection. The ratio of (first liquid feed port flow velocity / second liquid feed port flow velocity) is not only 100/0, but also various values such as 90/10, 199/1, 1/2, 1/5, and 3/7. Even if it is a value, it is shown that the sample introduction method of the present invention improves the column performance as compared with the conventional sample injection method.

Further, the results of Example 5 (FIG. 14) and Example 9 (FIG. 18) are obtained from the flow path in the rear part of the sample band by opening the vent valve at the time of dividing the sample band and increasing the flow velocity of the second liquid feed port. It is shown that the discharge of the sample band and the separation and elution of the sample band in the column can be performed at the same time. In addition, after the rear part of the sample band is discharged from the flow path, the vent valve is closed to separate and elute the front part of the sample band on the column at various ratios (first liquid feed port flow velocity / second liquid feed port flow velocity). It shows that it can be continued.

Further, the result of Example 9 (FIG. 18) shows that the column exhibits high performance in the ratio of (first liquid feed port flow velocity / second liquid feed port flow velocity) from the time of sample injection to the sample band division. It shows that there is a ratio.

Further, as shown in Examples 2, 4, 8, 11 and 12 (FIGS. 11, 13, 17, 20 and 21), the sample introduction method of the present invention was carried out using ZDU instead of the column, and sample injection was performed. The sample band injected from the apparatus is clearly divided in the direction intersecting the axial direction (traveling direction) of the sample band, and only the front part of the sample band is introduced into the column, so that the high performance of the column can be exhibited. Further, if a sample band dividing device having a vent port between the sample injection device and the column is used, the front part of the sample band can be discharged from the flow path and only the rear part of the sample band can be introduced into the column, or an embodiment. As suggested by 5, 14 and 15 (FIGS. 14, 23 and 24), it is clear that the anterior and posterior parts of the sample band can be expelled from the flow path and only the intermediate part can be introduced into the column.

From the above examples, the present invention presents the present invention when the sample band injected from the sample injection device (injector or the like) is entering the column from the first liquid feeding port through the connecting tube, that is, the column inlet or the flow path switching point. When the sample band straddled the sample band, the sample band that entered the column was divided by the liquid transfer from the second liquid supply port connected to the column inlet, and the sample band in front of the sample band was separated and eluted at the same time. By retaining the rear part (tail part) of the remaining sample band in the first liquid feed port tube or pushing it back and discharging it from the flow path or discharging it from the flow path, the spread of the sample band introduced into the column is reduced. Therefore, it is clear that the column performance can be improved.

In particular, for a sample band provided by a sample injection capacity of 0.01 to 5 μL, which is usually suitable for liquid chromatography, as a method for improving column performance, the structure of the disclosed sample band dividing device and the operation of the liquid feed flow path It is clear that the method is useful. It is clear that the improvement of column performance (increase in the number of theoretical plates) by dividing the sample band and introducing it into the column is achieved in the range of the sample introduction rate of 93 to 29%.

From the results of Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 18, 19 (FIGS. 10-18, 21, 22, 27, 28), the column from the sample injection device When the volume to the inlet is 1.274 μL (measured in Example 4, FIG. 13 (B) at a flow rate of 0.05 mL / min and a sample volume of 0.2 μL), the sample injection volume is 0.01 to 5 μL. At this time, it is clear that 1.5 to 6 μL is appropriate for the liquid feed capacity of the first liquid feed port until the sample band is divided. Further, as shown in Table 8, in the example in which the ZDU or the fused silica capillary pipe was used instead of the column, the sample band introduced into the ZDU or the like was dispersed (outside the column) at a sample introduction rate of 29 to 93%. (It can be regarded as the dispersion of the solute band in) can be reduced by 30 to 80% as compared with the conventional sample injection method. Therefore, the sample introduction method of the present invention can be used in Examples 1, 3, 7, 10, and 13. , 14, 16, 18 (FIGS. 10, 12, 16, 19, 22, 23, 25, 27), the number of theoretical plates of the column could be improved to 1.1 to 5 times.

Further, it is clear that the present invention can improve the column performance (theoretical plate number) by introducing the injected sample into the column with a very small volume. Then, in the present invention, it is clear that a sample having a capacity of 0.01 to 0.1 μL (10 to 100 nL), which is considered to be sufficiently small at the current state of the art, can be further divided at the column inlet and introduced into the column. Is.

In FIG. 29, when the volume of the sample injected by the injector is 1 μL (Examples 5, 6, 7, 8, 12, 13 (FIGS. 14, 15, 16, 17, 21, 22), the sample is divided. The relationship between the volume of the mobile phase fed from the first liquid feeding port and the introduction rate of the front part of the sample band into the column or ZDU is shown. The horizontal axis is "the first until the sample band is divided. 1 Liquid feeding capacity from the liquid feeding port (μL) ”, the vertical axis is the“ sample introduction rate into the column ”. However, when the liquid feeding capacity until the division of the sample band is 6 μL, the capacity of the injected sample. Is 2 μL (Example 9, FIG. 18), and when the liquid feeding capacity until the division of the sample band is 1.5 μL, the volume of the injected sample is 0.4 μL (Examples 1, 2, 3, 4 (FIG. 10). , 11, 12, 13))) are plotted.

By creating the calibration curve illustrated in FIG. 29 for the sample band dividing device having a fixed configuration, the sample band in the selection of the liquid feeding capacity from the first liquid feeding port from the sample injection to the dividing of the sample band and the setting thereof. Can be predicted in the column.

Table 9 shows the dispersion of solute bands (σ _col ² , equation 5) in which a column having a length of 5 cm and a porosity of 50% can obtain 10000 theoretical plates, and some retention coefficients between k = 0 to 10. The value simulated for the peak of (k value) is shown. If the column size to be compared and the column void volume are the same, it can be determined by comparing with Table 9 whether or not the peak of the chromatogram collected at various flow velocities approaches the theoretical plate number of 10,000. In order to obtain 10000 theoretical plates on a column with a length of 5 cm, it is generally necessary to pack a filler of 2 μm or less, but in particular, InertStain C18 (filler diameter 2 μm, inner diameter 1.0 mm, length 5 cm, GL Science). When used in a conventional liquid chromatography device, the sample loop capacity of the auto sampler is 0.2 μL, and the connecting pipe from the sample injection valve of the auto sampler to the column inlet has an inner diameter of 0.025 mm and a length of 10 cm (capacity). Approximately 0.049 μL), the connecting pipe from the column outlet to the detector cell inlet has an inner diameter of 0.025 mm and a length of 10 cm (capacity of approximately 0.049 μL), and the detector cell has an inner diameter of approximately 0.15 mm and a length of 5 cm (capacity of approximately 0). Even if it is used at a flow velocity of 0.05 mL / min with a microcapillary HPLC apparatus (Exgent ExpressLC-Ultra, AB SCIEX), which has a very small volume of the flow path through which the sample passes (.09 μL), the theory is that the retention coefficient k = 0. The limit was to obtain 7130 when the number of stages was 1790 and k = 2.2, 8550 when k = 4.0, and 8720 when k = 9.0 (Non-Patent Document 8). The porosity of the column differs depending on the type of filler, the filling method, the type of a single substance in the column, etc., but Table 9 shows the values of general porosity.

Further, when the value at which the dispersion of the solute is reduced by the sample introduction method of the present invention as compared with the conventional injection method shown in Table 8 is compared with the dispersion value shown in Table 9, the sample introduction method of the present invention is obtained. The column size and peak retention coefficient that can be more effective can be confirmed. For example, in Example 4 of Table 8 and FIG. 13 (D), the variance is reduced by 0.3 μL ² . Even if the retention coefficient k is 8 in the column with an inner diameter of 1.0 mm and a length of 5 cm in Table 9, the dispersion is 3.11 μL ^2. Therefore, if the dispersion is reduced by about 10%, the column performance is greatly affected. Further, an inner diameter of 2.1 mm, so 2.99MyuL ² in retention factor k is 1, a column of length 5 cm, although larger impact on column performance When 0.3 [mu] L ² decrease, k is large, 6.73MyuL ² In 2 Therefore, it is expected that the effect on the column performance will not be so large even if the decrease is 0.3 μL ² .

For example, in the case of the column of Example 3 and FIG. 12 (A) having an inner diameter of 1.0 mm and a length of 5 cm, the peak variance calculated from the full width at half maximum for the solutes U, MB, T and N is 0.31 μL ² . It was 0.93 μL ² , 2.04 μL ² and 2.96 μL ² . In FIG. 12 (D) obtained by dividing the sample band and introducing it into the column from the same sample injection volume of 0.2 μL, the peak dispersion for the solutes U, MB, T and N was 0.11 μL ² . It was 0.55 μL ² , 1.66 μL ² and 2.53 μL ² . As shown in Example 4, FIGS. 13 (B) and 13 (E), the decrease in the dispersion of these peaks was reduced by 0.3 to 0.4 μL ² due to the division of the sample band and the introduction into the ZDU. The number of theoretical plates of each peak in FIG. 12 (D) (k (U) = 0 is 3530, k (MB) = 2.5 is 8560, and k (T) = 4.9 is 8140. , K (N) = 6.4, 8410) is the number of theoretical plates of each peak in FIG. 12 (A) (k (U) = 0, 1240, k (MB) = 2.5, 4870, k (T) = Compared with 6370 at 4.9 and 6900 at k (N) = 6.4), the result was 1.2 to 2.8 times higher (Table 2). In the peak variance (σ _col ² ) by the column (N = 10000) shown in Table 9, for example, in a column having an inner diameter of 1.0 mm and a length of 5 cm, the variance of the peak of k = 6 is 1.88 μL ² . For N (peak dispersion 2.53 μL ² at k = 6.4) of (D), an HPLC apparatus having a larger volume of the flow path through which the sample passes is used than the microcapillary HPLC apparatus by the sample introduction method of the present invention. However, the number of theoretical plates can be increased from 6900 to 8410, and the column performance close to the column performance obtained by the microcapillary HPLC apparatus (8550 theoretical plates at k = 4.0 and 8720 at k = 9.0) is demonstrated. It was shown that it was done. Further, for U (peak dispersion 0.11 μL ² at k = 0) and MB (peak dispersion 0.55 μL ² at k = 2.5) in FIG. 12 (D), a UHPLC apparatus was used according to the sample introduction method of the present invention. The number of theoretical plates can be increased from 1240 to 3530 and from 4870 to 8560, respectively, and the column performance obtained by the microcapillary HPLC apparatus (k = 0, theoretical plate number 1790, k = 2.2, 7130) can be increased. ), It was shown that the column performance exceeded.

That is, according to the sample introduction method of the present invention, the number of theoretical plates is expected to increase in a wide range of the retention coefficient k for a column having an inner diameter of 1.0 mm and a length of 5 cm. Even in a column having an inner diameter of 2.1 mm and a length of 5 cm, or a column having an inner diameter of 3.0 mm and a length of 5 cm, improvement in column performance is expected, especially for peaks appearing in the early stage of the chromatogram.

Table 10 shows a column with a porosity of 46% and an inner diameter of 1.0 mm and a length of 5 cm, a column with a porosity of 50% and an inner diameter of 2.1 mm and a length of 5 cm, and a column with a porosity of 50% and an inner diameter of 3.0 mm and a length. The half-value width (unit: min) of the solute band that can obtain 10000 theoretical plates on a 5 cm column is shown by simulating several retention coefficients (k values) between k = 0 to 10. The full width at half maximum is affected by the flow velocity, but if the column size to be compared, the column void volume, and the flow velocity are the same, whether the peak of the collected chromatogram approached the theoretical plate number of 10,000 stages (or improved performance). It can be judged by comparing with Table 10. A half width (unit: min) is given to each peak of the embodiment. Further, for peaks to which the half width is not given, the half width is shown in Tables 11 to 13.

Tables 11 to 13 show the full width at half maximum of each peak for each example.

According to the sample introduction method of the present invention, it is possible to reduce the dispersion of solutes outside the column in columns for liquid chromatography having various inner diameters, and columns having inner diameters of 0.1 mm, 1.0 mm, 2.1 mm and 4.6 mm. The effect of increasing the number of column theoretical plates was observed. INDUSTRIAL APPLICABILITY According to the present invention, it has become possible to improve column performance for thin, short, and small columns required for UHPLC, especially for peaks that elute at the beginning of the chromatogram. The operation of the pump, the flow path switching valve, etc. used in the embodiment is controlled in units of 1/100 minutes, but if the controllable time interval can be shortened to, for example, 1/1000 minutes to 1/10000 minutes. It is self-evident that more suitable control can be performed to improve column performance.

According to the present invention, in liquid chromatography, it is possible to improve the separation performance of a column, that is, the number of theoretical plates, so that the analysis and sorting efficiency of substances are improved, and it is used in research and applications in various fields. Is possible.

1 Column 10 Sample band dividing device 11 Sample band dividing part 12 Flow path switching point 20 1st liquid feeding port 21 Liquid feeding pump 22 Sample injection device 29 2nd liquid feeding port 3 Sample band 31 Front part of sample band 32 Sample band Rear part 5 6-way valve 50 Flow path switching point 51 1st liquid supply port 52 2nd liquid supply port 60 1st discharge port 7 4-way joint 91 Sample introduction device

Claims (26)

In a liquid chromatography device, a sample introduction device for introducing a sample into a column, which is provided between a sample injection device equipped with a flow path switching valve for the sample injection device and a column, and for sending a sample band to the column. A sample band dividing device for dividing a sample band, comprising a first liquid feeding port and a dividing port for dividing the sample band sent from the first liquid feeding port. The sample band dividing device according to claim 1, wherein the sample band dividing device is a device for dividing a sample band in a direction intersecting in an axial direction. 1. The split port is configured to include a second liquid feed port for feeding the mobile phase to the column and / and a first discharge port for discharging a part of the sample band. Or the sample band dividing device according to 2. The capacity of the flow path from the flow path switching point between the first liquid supply port and the second liquid supply port or the first discharge port to the column inlet is 1/13.9 or less of the column void capacity. The sample band dividing device according to claim 3. The sample band dividing device according to claim 3 or 4 , wherein the first liquid feeding port and the second liquid feeding port are configured by using a flow path switching valve for dividing a sample band. The sample band dividing device according to claim 5, wherein the sample band dividing flow path switching valve is a three-way valve, a four-way valve, a six-way valve, an eight-way valve, or a ten-way valve. The sample band dividing device according to any one of claims 1 to 4 , wherein the first liquid feeding port and the dividing port are connected by using a three-way joint or a four-way joint. A sample introduction device comprising the sample band dividing device according to any one of claims 1 to 7. The sample introduction device according to claim 8, further comprising a second discharge port for discharging a part of the sample band upstream of the sample injection device. A liquid chromatography apparatus comprising the sample introduction apparatus according to claim 8 or 9. The liquid chromatography apparatus according to claim 10, further comprising a column having an inner diameter of 4.6 mm or less. The liquid chromatography apparatus according to claim 10 or 11, wherein the elution volume of the peak is 10.2 mL or less. In liquid chromatography, a sample band is divided in a flow path in front of a column from a sample injection device provided with a flow path switching valve for a sample injection device by switching the flow path, and at least a part of the divided sample band is used as a column. A sample introduction method characterized by introduction. The sample introduction method according to claim 13, wherein the flow path is switched and the sample band is divided when the sample band straddles the column inlet or the flow path switching point closest to the column inlet. The sample introduction method according to any one of claims 13 to 14, wherein the sample band is divided in a direction intersecting the axial direction of the sample band. The sample introduction method according to any one of claims 13 to 15, wherein all of each portion of the divided sample band is introduced into the column. The sample introduction method according to any one of claims 13 to 15, wherein a part of the divided sample band is introduced into the column. The sample introduction method according to claim 17, wherein a portion of the divided sample band other than the portion introduced into the column is discharged. The rear part including the middle part of the sample band is placed in the flow path without being introduced into the column, and the front part of the divided sample band is introduced into the column or discharged from the flow path and then placed in the flow path. The sample introduction method according to claim 13, wherein the rear portion of the sample band is divided and the intermediate portion of the sample band is introduced into the column. Pump, sample injection device provided with a flow path switching valve for sample injection device, columns, coupled in the order of the detector using a chromatographic device configured, front part of the injected sample bands from the sample injection device column From the first liquid feed port that enters and feeds the sample band to the column when the sample band straddles the column inlet, or when the sample band straddles the flow path switching point closest to the column inlet on the flow path. The liquid transfer of the mobile phase is stopped, the liquid transfer of the mobile phase is started from the second liquid transfer port provided near the column inlet, the sample band is divided, and the front part of the sample band is introduced into the column. The sample introduction method according to any one of claims 13 to 15, wherein the rear portion of the sample band is fastened in a connecting pipe from the sample injection device. Pump, sample injection device provided with a flow path switching valve for sample injection device, columns, coupled in the order of the detector using a chromatographic device configured, front part of the injected sample bands from the sample injection device column From the first liquid feed port that enters and feeds the sample band to the column when the sample band straddles the column inlet, or when the sample band straddles the flow path switching point closest to the column inlet on the flow path. Stop the transfer of the mobile phase or maintain the transfer, start the transfer of the mobile phase from the second liquid transfer port provided near the column inlet, divide the sample band, and divide the sample band into the sample band. The front part is introduced into the column, the vent valve is opened, the mobile phase liquid feeding from the first liquid feeding port is restarted or the liquid feeding is maintained, and the rear part of the sample band is provided in the flow path near the column inlet. The sample introduction method according to any one of claims 13 to 15, wherein the sample is discharged from the first discharge port. Using a chromatograph device configured by connecting a pump, a sample injection device, a column, and a detector in this order, a first discharge port is created between the sample injection device and the column, and the sample band injected from the sample injection device When the front part enters the column and the sample band straddles the column inlet, or when the sample band straddles the flow path switching point closest to the column inlet on the flow path, the first discharge port is opened and the sample is sampled. The sample introduction method according to any one of claims 13 to 15, wherein the rear portion of the band is discharged. A mobile phase from the first liquid feed port that sends the sample band to the column using a chromatograph device that is configured by connecting a pump, a sample injection device equipped with a flow path switching valve for the sample injection device, a column, and a detector in this order. Stop the liquid transfer or maintain the liquid transfer, start the transfer of the mobile phase from the second liquid transfer port provided near the column inlet, and use the sample band injected from the sample injection device for the second. divided by feeding the mobile phase from the liquid supply port, between the pump and the sample injector, provided with the second discharge port for discharging the liquid through the joints and valves, at any time by opening and closing the valve a structure in which liquid can be discharged, after the division of the sample band, the indwelling sample bands after partial in the first feeding port, the feeding of the mobile phase from the second liquid supply port, pushed back through the sample injector The sample introduction method according to any one of claims 13 to 15, wherein the sample is discharged from a second discharge port provided upstream of the sample injection device. A mobile phase from the first liquid feed port that sends the sample band to the column using a chromatograph device that is configured by connecting a pump, a sample injection device equipped with a flow path switching valve for the sample injection device, a column, and a detector in this order. Stop the liquid transfer or maintain the liquid transfer, start the mobile phase transfer from the second liquid transfer port provided near the column inlet, and use the sample band injected from the sample injection device for the second. The sample was divided by the transfer of the mobile phase from the liquid feed port, the rear part of the sample band was placed in the first liquid feed port, and the sample components contained in the front part of the sample band introduced into the column were eluted from the column. Of claims 13 to 15, the rear portion of the sample band indwelled in the first liquid feeding port is later introduced into the column by restarting the liquid feeding of the mobile phase from the first liquid feeding port. The sample introduction method according to any one item. From claim 13, the rear portion of the sample band placed in the first liquid feeding port is further divided, and at least a part thereof is restarted from the first liquid feeding port and introduced into the column. The sample introduction method according to any one of 15. A mobile phase from the first liquid feed port that feeds the sample band to the column using a chromatograph device that is configured by connecting a pump, a sample injection device equipped with a flow path switching valve for the sample injection device, a column, and a detector in this order. The mobile phase liquid transfer from the second liquid supply port provided near the column inlet is started, and the sample band injected from the sample injection device is moved from the second liquid supply port. divided by feed phase solution, that the range of the ratio of the time of sample injection to the sample-band division (first feeding port flow velocity) / (second feeding port flow velocity) is 10 / 0-1 / 5 The sample introduction method according to any one of claims 13 to 15, wherein the sample is introduced.

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