JP6393964B2 - Time-controlled sample injection device and liquid chromatography provided with the same - Google Patents

Description

  The present invention relates to a sample injection apparatus capable of easily introducing a small amount of sample into a column in liquid chromatography or the like.

  In “flow analysis equipment” that performs analysis by introducing a sample into a liquid flow such as liquid chromatography or flow injection, it is common to contribute to improvement of analysis accuracy by keeping the sample injection amount constant. . In such a flow analyzer, various detectors such as photodetection represented by an ultraviolet-visible detector and a fluorescence detector, an electrical conductivity detector based on conductivity, and an electrochemical detector based on oxidation-reduction potential Is used depending on the purpose. Since these detectors change the signal intensity in proportion to the concentration, the volume of the sample introduced into the “flow analyzer” is required to be kept constant with high accuracy.

  In liquid chromatography, which is representative of a “flow analyzer”, a sample is introduced into the eluent stream, separated into individual components with an analytical column, and changes in signal intensity are continuously monitored with a detector. The elution time, peak height and area are calculated, and identification calculation and quantitative calculation are performed.

  Liquid chromatography may be classified according to analytical pressure, such as low-pressure chromatography with an analytical pressure of several hundred kPa, high-pressure chromatography with an analytical pressure of about 10 MPa, and ultra-high pressure chromatography with an analytical pressure of 30 MPa or more. In many cases, the same structure and injection method are used.

  In general, there are two ports for connecting a sample holding loop for holding a certain amount of sample, a sample introduction port, a sample discharge port, an eluent inlet port, an eluent outlet port (analysis column). A first state in which the sample can be introduced and held in the sample holding loop (Load), and the sample in the sample holding loop can be pushed out by the flow of the eluent and introduced into the column. The structure can be switched between the second state (Injection) (see FIG. 1).

  The following three types of sample injection methods are roughly classified into proposed products.

(1) Loop total volume injection method (see Fig. 2)
The structure of the basic injection mechanism is the same as described above. First, in the second state, the sampling channel is washed (step 1) and then switched to the first state, and the sample holding loop is completely filled with the sample by the sample suction mechanism (steps 2 and 3). Next, it switches to a 2nd state and introduce | transduces a sample into a column (steps 4, 5). The sample injection amount is determined by the volume of the sample holding loop. In this method, even if the amount of the sample sucked into the sample holding loop varies to some extent, the sample injection amount actually introduced into the column is constant, so that the reproducibility of the injection amount is very good. However, since the injection volume is determined by the volume of the sample holding loop, to change the injection volume, it is necessary to replace the sample holding loop attached to the injection valve with a tool suitable for the volume using a tool or the like. There is a drawback that takes time and effort. Moreover, since an extra amount is used rather than the sample volume actually injected into the column, there is a disadvantage that the sample is wasted.

(2) Loop variable injection method (see Fig. 3)
The structure of the basic injection mechanism is the same as described above. The injection operation is started from the second state. First, in the second state, the sampling channel is washed (step 1), and then the sample is sucked by the sample suction mechanism until it comes out from the port f of the injection valve (step 2). Next, the state is switched to the first state, and the target injection amount is accurately sucked by the sample suction mechanism (step 3). Next, the state is switched to the second state, and the weighed sample is introduced into the column (steps 4, 5). The sample injection amount is determined by the volume sucked by the sample suction mechanism (the sample holding loop volume or less). This method has an advantage that the sample injection amount can be varied within a certain range, although the injection reproducibility is slightly lowered as compared with the method (1). In addition, although not as in the case of (1), since an extra amount is used rather than the sample volume actually injected into the column, there is a disadvantage that the sample is wasted.

(3) Direct injection method (see Fig. 4)
The structure of the injection mechanism is slightly different from the above (1) and (2). In this method, the sample weighed by the sample metering mechanism is directly introduced from the port e of the injection valve.

  First, in the second state, the sampling channel is washed (step 1), and then the sample is directly sucked by the sample weighing mechanism (step 2). Next, switching to the first state, a sample weighing mechanism containing the weighed sample is connected to the port e of the injection valve, and the sample is accurately pushed into the sample holding loop (steps 3 and 4).

  Next, switching to the second state, the weighed sample is introduced into the column (steps 5, 6). The amount of sample injection is determined by the volume aspirated by the sample metering mechanism (below the sample holding loop volume). This method has an advantage that the sample injection amount can be varied within a certain range, although the injection reproducibility is slightly lowered as compared with the method (1). In addition, since an extra amount of sample as in (1) and (2) is hardly required, there is an advantage that the sample is not wasted. However, it has the disadvantage that the mechanism is complicated.

  FIG. 5 shows an external view of a general two-position switching six-way valve used in the above (1) to (3). Generally, a hard part called a stator (15) has two ports (11a, 11d) connected to a sample holding loop for holding a certain amount of sample, a port (11e) for introducing a sample, and a sample discharge port. (11f), 6 ports of an eluent inlet port (11b) and an eluent outlet port (analytical column inlet) (11c), each connected to a sample holding loop (4) or a pipe (13) Have There is a rotor seal (17) having three grooves (18) for switching the flow path inside the valve, and the flow path is switched by rotating the rotor seal (FIG. 7). FIG. 5a is a view showing a state where the valve body (14) and FIG. 5b are each pipe (13) or the sample holding loop (4). The piping used is 1/16 inch in outer diameter, 0.1 to 1.0 mm in inner diameter, the material is SUS, PEEK resin, PTFE resin, etc., and the piping etc. is valved by Osine (nut) (12), ferrule It is connected to the main body and sealed.

  As can be seen from FIG. 5b, the stator surface of the valve is in a state where six pipes / ocine (nuts) (12) are densely packed.

  The sample holding loop (4) has the same shape as the piping used in liquid chromatography. The outer shape is 1/16 inch, the inner diameter is 0.25 to 1.0 mm, the material is SUS, PEEK resin, PTFE resin, etc., and the structure is connected and sealed to the valve body with osine (nut) (12) and ferrule. Take.

  In the case of the loop total amount injection method of (1) described above, the sample amount introduced into the column is the sum of the sample holding loop capacity, the capacity of the stator flow path, and the groove capacity of the rotor seal.

Since the capacity of the stator passage and the groove capacity of the rotor seal are usually as small as several microliters, the capacity can be almost ignored when the injection amount is large. However, when the injection amount is as small as several tens of microliters, the above-mentioned capacity needs to be taken into consideration.
V = S + R + L * 3.14 * (D / 2) ²
Injection amount: V
Piping inner diameter: D
Pipe length: L
Rotor seal groove capacity: R
Stator channel capacity: S
In consideration of the fact that the sample holding loop (4) has the osine (12) / ferrule (16) at both ends, the relationship between the physical arrangement and the workability of attachment / detachment, the pipe length needs to be approximately 10 cm or more (see FIG. 6). Table 1 shows the calculated values of the inner diameter and length of the pipes when producing sample holding loops of 1, 2, 5, 10, 20 microliters. The () part in the table indicates that the length of the pipe is 10 cm or less, indicating that the production is impossible or difficult.

このように、２０マイクロリッタの試料保持ループを作製する場合は、内径０．４ｍｍ、長さ１６ｃｍ、または、内径０．２５ｍｍ、長さ４１ｃｍ程度の配管を使用すればよく、作製は可能である。ところが、５マイクロリッタの試料保持ループを作製する場合は、内径０．４ｍｍを使用すると長さは４ｃｍとなり作製できない。内径０．２５ｍｍを使用すると長さは１０ｃｍとなる。これは作製可能な長さの下限に近い。ループ容量は計算上５マイクロリッタであるが、実際には前述のとおり、ロータシールの溝容量、ステータの流路容量が加算されるため、注入量は５マイクロリッタ＋α（数マイクロリッタ）となってしまう。注入量を正確に５マイクロリッタにする場合は、αの容量を考慮する必要があり、ループの配管長は１０ｃｍ以下となり、作製は困難となる。

Thus, when producing a sample holding loop of 20 microliters, a pipe having an inner diameter of 0.4 mm and a length of 16 cm, or an inner diameter of about 0.25 mm and a length of 41 cm can be used. . However, when a sample holding loop of 5 microliters is produced, if an inner diameter of 0.4 mm is used, the length becomes 4 cm and cannot be produced. If an inner diameter of 0.25 mm is used, the length is 10 cm. This is close to the lower limit of the length that can be produced. The loop capacity is calculated to be 5 microliters. However, as described above, since the groove capacity of the rotor seal and the flow path capacity of the stator are added, the injection amount is 5 microliters + α (several microliters). End up. When the injection amount is precisely 5 microliters, it is necessary to consider the capacity of α, and the pipe length of the loop is 10 cm or less, which makes it difficult to manufacture.

  Even in the variable injection method of loop (2) and the direct injection method of (3), since the measured sample is put into the sample holding loop and introduced into the column, the groove capacity of the rotor seal and the flow path of the stator Capacities below the sum of the capacities cannot be injected, and it is difficult to inject low capacities with high accuracy.

  From the above, it is quite difficult to use a general six-way switching valve when accurately injecting a volume of 5 microliters or less. In order to make up for this, valves with extremely reduced rotor seal groove capacity and stator flow path capacity and pipes used for sample holding loops with ultra-thin tubes have been commercialized. Clogging is likely to occur due to a slight amount of insoluble matter contained in the material, making it difficult to use (Non-Patent Document 1).

  In addition, in order to inject a low-volume sample, a valve with a sample holding loop connected directly to the rotor seal and arranged inside the valve, or a groove or hole in the rotor seal that is used as a holding part for sample weighing is also proposed. / Commercialized (see FIG. 8). In any case, injection of a minute volume is possible, but there is a difficulty in that the structure is complicated or expensive (Non-patent Document 2).

IDEX Health & Science, Manual Sample Injectors Rheodyne 7520, [Search July 18, 2013], Internet <URL: http://www.idex-hs.com/products/4139/Manual-Sample-Injectors.aspx? ProductTypeID = 153 & ProductFamilyID = 2> VICI Valco Instruments, Inc. CHEMINERT INJECTORS / Model C74H, [Search July 18, 2013], Internet <URL: http://www.vici.com/cval/c74_16-250-10k.php>

  It is an object of the present invention to provide an injection apparatus that can easily introduce a small amount of sample less than the capacity of a sample holding loop to be mounted even if a general injection valve that does not reduce the void capacity of a rotor seal or a stator is used. Objective.

The present invention has been made for the purpose of solving the above problems, and is specifically as follows.
at least,
(1) a sample injection unit for injecting a sample;
(2) means for carrying the sample into the sample injection portion;
(3) Sample suction / cleaning means for sucking or cleaning the sample into the sample injection portion;
(4) A sample injection apparatus including an injection condition input unit for setting injection conditions, and (5) a sample injection control unit for controlling (1), (2), and (3) according to the conditions of (4). ,
The sample injection part of (1) is
Two ports connected to both ends of a sample holding loop for holding a sample, a sample introduction port, a sample discharge port, a fluid inlet port, a fluid outlet port, and 6 ports,
At least a first state in which the fluid inlet port and the outlet port are directly conducted without going through the sample holding loop, and the sample entering from the sample introduction port goes out to the sample discharge port through the sample holding loop And the fluid that has entered from the fluid inlet port flows to the fluid outlet port via the sample holding loop, and the sample introduction port and the sample discharge port directly conduct without passing through the sample holding loop. It is a valve that can take the state of
By the sample injection control unit (5),
After all or part of the sample holding loop is filled with the sample in the first state, the valve is switched to the second state, and specified by the injection condition input unit in (4) above, based on the switching time. The sample injection device is characterized in that a part of the sample in the sample holding loop is transferred to the fluid flow port and injected into the fluid outlet port by switching the valve to the first state at a later time. is there.

  Further, in the above-described sample injection device, the product of the flow rate of the fluid and the time for switching from the time for switching to the second state to the time for switching to the first state becomes the sample injection amount.

  Furthermore, the sample injection apparatus according to any one of the above, wherein the sample injection amount is 0.3 to 10 microliters.

  A liquid chromatography is provided with any of the sample injection devices described above.

  As a result, a part of the sample in the sample holding loop is quantitatively placed on the fluid flow and transferred to the fluid outlet port. Can also be loaded.

  Hereinafter, the present invention will be described in detail. In “flow analysis” represented by liquid chromatography, the flow rate of the fluid is constant, so the amount of sample injection can be changed by controlling the time to switch the valve from the second state to the first state. Is possible. FIG. 9 is a schematic diagram showing the operation of the injection device based on time control according to the present invention, and FIG. 10 is a diagram showing an example of the system configuration.

(Step 1)
The sample injection valve starts from the second state (Injection). In this state, the cleaning line including the sample suction / cleaning means-injection valve port f-port e is cleaned. Note that step 1 can be omitted.
(Step 2)
The sample injection valve is switched to the first state (Load).
(Step 3)
While the sample injection valve is in the first state (Load), the sample is sucked in the sample holding loop by the sample suction / washing means in whole or in part.
(Step 4)
The sample injection valve is switched to the second state (Injection).
(Step 5)
The sample sucked into the sample holding loop is pushed out by the pump and introduced into the column.
(Step 6)
Before all the sample sucked into the sample holding loop in step 3 is introduced into the column, the sample injection valve is switched to the first state (Load). That is, the time for switching the sample injection valve to the first state (Load) is set so that the sample introduced into the column is less than the sample sucked into the sample holding loop.

  Thus, the product of the difference between the time when the sample injection valve is switched to the second state (Injection) in Step 4 and the time when the sample injection valve is switched to the first state (Load) in Step 6 and the flow rate of the pump is The amount of sample introduced into the column. When performing the analysis, the flow rate of the pump is used at a constant value. Therefore, by changing the time difference, for example, by changing the time for switching to the first state (Load) in Step 6, the injection amount is changed. be able to. The time difference corresponds to the injection time.

  Next, a general-purpose column (inner diameter: 4.6 mm), semi-micro column (inner diameter: 2.0 mm), micro column (inner diameter: 1.0 mm), and capillary column (inner diameter: 0.5 mm), which are currently used in analysis, are taken as examples. The injection amount will be described.

  The injection volume varies greatly depending on the sample characteristics, concentration, and type of column used, but it is 100 to 10 microliters for general purpose columns, 20 to 2 microliters for semi-microcolumns, 5 to 0.5 microliters for microcolumns, capillary The column is often used in the range of 1 to 0.1 microliter. In the general-purpose column region, the injection amount is 10 microliters or more, and this region can be sufficiently handled by a general injection method / apparatus. In the semi-microcolumn to capillary column region, the injection amount is often 10 microliters or less, and this is a region that is difficult to use with high accuracy by a general injection method / apparatus.

  An example in which the time-controlled injection method according to the present invention is applied to the semi-microcolumn to capillary column region is shown below (all assume that a sample holding loop larger than the target injection amount is provided). When a semi-micro column is fed at 200 microliters per minute and the injection time is 3 seconds, 10.0 microliters are injected into the semi-microcolumn. When the liquid is fed at 50 microliters per minute using a microcolumn and the injection time is 3 seconds, 2.5 microliters are injected into the microcolumn. In addition, when the liquid is fed at 12 microliters per minute in the capillary column and the injection time is set to 3 seconds, 0.6 microliter is injected into the capillary column. Thus, by adjusting the injection time, it is possible to load the column even in an amount of 10 microliters or less, particularly 0.3 to 10 microliters.

  FIG. 11a is a view schematically showing the conventional injection method, and FIG. 11b is a view schematically showing the injection operation of the injection device by the time control of the present invention. The shaded portion indicates the total amount of the volume of the sample holding loop and the void volume. In the conventional method (FIG. 11a), all of the total amount is introduced into the column, whereas the injection method according to the present invention ( In FIG. 11b) less than the total amount is injected into the column.

  Thus, by switching the injection valve from the second state (Injection) to the first state (Load) before all the samples in the sample holding loop are pushed out, a smaller amount of sample than the capacity of the sample holding loop Can be injected quantitatively. In particular, even when the shortest sample holding loop that can be physically manufactured is used, it is possible to inject a very small amount of sample smaller than the capacity.

It is the figure which showed the flow-path system of the general liquid chromatograph apparatus. It is the figure which showed the injection principle of the general loop whole quantity injection method. It is the figure which showed the injection principle of the general loop variable injection method. It is the figure which showed the injection principle of the general direct injection method. It is the figure which showed the external appearance of the general sample injection valve. a shows a state before piping and b shows piping. It is the figure which showed the external appearance of the sample holding loop used for a sample injection valve. a is an external appearance, b is an enlarged view. It is the figure which showed the external appearance of the rotor seal | sticker of a general sample injection valve. It is the figure which showed an example of the structure of the slide-type valve used for a micro injection. It is the figure which showed the injection | pouring principle of the injection apparatus by the time control of this invention. It is the figure which showed an example of the system configuration | structure of the injection apparatus by the time control of this invention. It is the figure which showed injection | pouring operation | movement typically. a shows the conventional injection | pouring method, b shows the injection | pouring method by the time control of this invention. 1 is a flow chart of a system used in Example 1. FIG. a indicates the first state (Load), and b indicates the second state (Injection). 2 is a chromatogram showing a change in detector signal when the length of the sample holding loop is changed in Example 1. FIG. FIG. 3 is a diagram showing the relationship between the capacity of the sample holding loop and the peak area in Example 1. 2 is a chromatogram showing a change in detector signal when the sample injection time is changed in Example 1. FIG. It is the figure which showed the relationship between sample injection time and the peak height or area in Example 1. It is the figure in Example 1 which showed the precision of the injection apparatus by the time control of this invention. a is a chromatogram, b is a diagram showing reproducibility of peak height and area. FIG. 6 is a flow chart of the system used in Example 2. a shows the eluent A flow / sample injection valve in the first state (Load), and b shows the eluent A flow / sample injection valve in the second state (Injection). c shows the first state (Load) of the eluent B flow / sample injection valve. It is the chromatogram which showed the change of the detector signal at the time of changing sample injection time in Example 2. FIG. FIG. 20 is an enlarged view of the chromatogram when the sample holding loop having an inner diameter of 0.5 mm and a length of 10 cm in FIG. 19 is used and the injection time is 0.03 minutes. It is the figure which showed the relationship between sample injection time and total area in Example 2. FIG. The black circle in the legend indicates a case where a sample holding loop having an inner diameter of 0.25 mm and a length of 10 cm and the legend white triangle has an inner diameter of 0.5 mm and a length of 10 cm is used.

  The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[Example 1]
In order to verify the effect of the present invention, the following was carried out.

  FIG. 12 shows a flow chart of the used liquid chromatograph. It consisted of a pump (2) for feeding the eluent, a 2-position 6-port injection valve (20) for injecting the sample, a resistance tube (35), and a UV-visible detector (7).

  The pump (2) used was DP-8020 manufactured by Tosoh Corporation, and the detector (7) used UV-8020 manufactured by Tosoh Corporation at a wavelength of 415 nm. The resistance tube (35) used as a substitute for the analytical column was a spirally wound SUS pipe having an inner diameter of 0.2 mm and a length of 2 m. As a 2-position 6-port injection valve (20) for injection, a solenoid-driven 2-position 6-port valve (MTV-6SL-N32UF-1) manufactured by Takasago Electric was used. The sample holding loop (4) attached to the valve was obtained by cutting PEEK piping having an outer diameter of 1/16 inch, an inner diameter of 0.25 mm, 0.5 mm, and 0.75 mm to a specified length. As eluent (1), pure water was fed at a rate of 1 milliliter per minute, and whole blood was diluted to 1/400 as a sample.

  First, in order to calculate the channel capacity of the rotor seal groove and stator of the valve used for verification, the length of the sample holding loop was changed from 10 to 50 cm, and the change in peak area was confirmed (inner diameter 0.25 mm). , PEEK piping). FIG. 13 shows the change of the detector signal, and FIG. 14 shows the relationship of the peak area to the sample holding loop capacity. In addition, the capacity at each pipe length is as shown in Table 2 in the calculation.

図１４から分かるように、保持ループ容量とピーク面積は良好な直線関係が見られる。しかしながらその近似直線は原点を通らず、一定の切片を有している。つまり、試料保持ループの長さをゼロにしても、計算上はピーク面積が存在することになる。これは、切り替えバルブのロータシール溝やステータ部の流路容量が存在することを意味している。この条件下での近似式は下記の通りとなる。
Ａｒｅａ＝７５．６２７＊Ｖ＋４５６．８９
Ａｒｅａ：ピーク面積
Ｖ：試料保持ループ容量（計算値）
ピーク面積（Ａｒｅａ） にゼロを代入して得られたＶが切り替えバルブのロータシール溝やステータ部の流路等のボイド容量となる。上記近似式から、今回使用した注入バルブの総ボイド容量は約６．０マイクロリッタと計算される。
０＝７５．６２７＊Ｖ＋４５６．８９
Ｖ＝−４５６．８９／７５．６２６＝−６．０μＬ
このことから、この注入バルブでは理論上６．０マイクロリッタ以下は注入することはできない。実際には物理的配置から１０ｃｍ以下の試料保持ループは作製できないことから、内径０．２５ｍｍの配管を使用した場合は、１０ｃｍのときのループ配管容量（４．９マイクロリッタ）と総ボイド容量（６．０マイクロリッタ）の合計値、１０．９マイクロリッタ以下の注入はできないことになる。

As can be seen from FIG. 14, there is a good linear relationship between the holding loop capacity and the peak area. However, the approximate line does not pass through the origin and has a certain intercept. In other words, even if the length of the sample holding loop is zero, there is a peak area in the calculation. This means that the rotor seal groove of the switching valve and the flow path capacity of the stator portion exist. The approximate expression under this condition is as follows.
Area = 75.627 * V + 456.89
Area: Peak area V: Sample holding loop capacity (calculated value)
V obtained by substituting zero for the peak area (Area) is the void capacity of the rotor seal groove of the switching valve, the flow path of the stator portion, and the like. From the above approximate expression, the total void volume of the injection valve used this time is calculated to be about 6.0 microliters.
0 = 75.627 * V + 456.89
V = −456.89 / 75.626 = −6.0 μL
For this reason, it is theoretically impossible to inject below 6.0 microliters with this injection valve. Actually, a sample holding loop of 10 cm or less cannot be produced from the physical arrangement. Therefore, when a pipe having an inner diameter of 0.25 mm is used, the loop pipe capacity (4.9 microliter) and the total void capacity ( The total value of 6.0 microliters) cannot be injected below 10.9 microliters.

Next, the effect of the injection device by time control of the present invention was verified. As the sample holding loop, PEEK piping having an outer diameter of 1/16 inch, an inner diameter of 0.25 mm, and a length of 10 cm was used (loop capacity: 4.9 μL). As the eluent, pure water was fed at 100 microliters per minute, and whole blood was diluted to 1/400 as a sample. Other device configurations and basic conditions are the same as described above.
In calculation, it takes 0.1 minute to completely push the sample in the loop with the eluent by switching the injection valve from the first state (Load) to the second state (Injection).
(Loop volume 4.9 μL + Void volume 6.0 μL) / (100 μL / min) = 0.1 min The injection valve is switched from the first state (Load) to the second state (Injection), and after a certain time, the first It returned to the state and it verified whether the injection | pouring of the sample below a sample holding | maintenance loop capacity could be performed quantitatively. For comparison, data was also acquired for a time sufficient for the sample in the loop to be pushed out (0.2 minutes) and held in the second state (corresponding to loop full volume injection). Table 3 shows the injection time and the calculated injection amount.

図１５はクロマトグラム、図１６は注入時間に対するピークの面積、高さの変化を示した図である。計算上、注入時間が０．２分と０．１分では同量となることから、ピーク高さ、面積とも近い値を示している。注入時間を０．１分から短くするに連れて、ピーク強度が減少して行くことが見てとれる。このことから、注入時間を試料が完全に押し出される時間より短い時間に設定することで、（試料保持ループ＋バルブのボイド容量）以下の微量を定量的に注入できることが分かる。

FIG. 15 is a chromatogram, and FIG. 16 is a graph showing changes in peak area and height with respect to injection time. In calculation, since the injection amount is the same at 0.2 minutes and 0.1 minutes, the peak height and the area are close to each other. It can be seen that the peak intensity decreases as the injection time is reduced from 0.1 minutes. From this, it can be seen that by setting the injection time to a time shorter than the time when the sample is completely pushed out, it is possible to quantitatively inject a trace amount less than (sample holding loop + valve volume of valve).

  Next, the reproducibility of the injection device by time control was verified. The injection time was 0.02 minutes (injection volume 2 μL), and n = 10, 3 sets. The first set of chromatograms is shown in FIG. 17a and the area and height Cv% are shown in FIG. 17b. Despite the small injection volume of 2 μL, reproducibility of about 4% in both area and height is obtained.

[Example 2]
In order to verify the effect of microinjection by the time control of the present invention, hemoglobin A1c was measured as follows.

  A mechanism capable of a two-liquid step gradient was added to the system used in Example 1, and a microanalysis column (33) was mounted instead of the resistance tube (35) (see FIG. 18).

  Between the feed pump A (27) of the eluent A (29) and the injection valve (20), a 2-position 6-port flow path switching valve (31) is arranged, and the eluent holding loop (32) is provided. Step gradient was performed by filling the eluent B and switching the valve after a certain time from the sample injection.

  The eluent holding loop (32) was a PEEK pipe having an outer diameter of 1/16 inch, an inner diameter of 0.75 mm, and a length of 30 cm (capacity: 132 microliters).

  A micro analytical column (33) was prepared by filling a column tube having an inner diameter of 1.0 mm and a length of 10 mm with TSKgelBorate-5PW (10 μm) in a column oven (34) temperature-controlled at 40 ° C.

  As eluents A and B (29, 30), the eluent for affinity of Tosoh Corporation automatic glycohemoglobin analyzer GHbVIII was used. The eluent A was sent at 200 microliters per minute, and whole blood was diluted to 1/200 as a sample. The step gradient was performed under conditions where the eluent A flows from 0 to 0.65 minutes and the eluent B flows from 0.65 to 1.05 minutes after sample injection.

  When the injection valve (20) is in the first state (Load), the sample holding loop is filled with the sample, switched to the second state (Injection), and the sample held in the void portion of the sample holding loop and the injection valve is microanalyzed. The sample was extruded into a column and returned to the first state before the entire amount of the retained sample was extruded, and a microinjection was performed. FIG. 19 is a chromatogram when the time for returning from the second state to the first state (injection time) is changed from 0.2 minutes to 0.01 minutes, and FIG. 19a is outside the sample holding loop (4). When a diameter of 1/16 inch, an inner diameter of 0.25 mm, and a length of 10 cm (capacity: 4.9 microliter) are attached, FIG. 19b shows an outer diameter of 1/16 inch, an inner diameter of 0.5 mm, and a sample holding loop (4). A chromatogram with a 10 cm length (capacity: 19.6 microliters) attached. FIG. 20 shows a chromatogram with an injection time of 0.03 minutes using a sample holding loop with an inner diameter of 0.5 mm and a length of 10 cm. FIG. 21 is an enlarged view showing a change in the total area of the hemoglobin A1c peak with respect to the injection time.

  As shown in Table 4, the sample injection time was changed from 0.2 minutes to 0.01 minutes.

図２１から分かるように、注入時間が０．０１分から０．０５分の場合、内径０．２５ｍｍと、内径０．５ｍｍの試料保持ループのどちらを搭載しても、得られる総面積はほぼ同一の値を示す。注入時間とピークの総面積はほぼ原点を通る１次式で近似でき、注入時間を制御することで注入量を正確に制御できることを表している。

As can be seen from FIG. 21, when the injection time is 0.01 minute to 0.05 minute, the total area obtained is almost the same regardless of whether the sample holding loop having the inner diameter of 0.25 mm or the inner diameter of 0.5 mm is mounted. Indicates the value of. The injection time and the total area of the peak can be approximated by a linear expression that almost passes through the origin, indicating that the injection amount can be accurately controlled by controlling the injection time.

  When a sample holding loop with an inner diameter of 0.25 mm is mounted, the total area of the peak hardly changes from the injection time of 0.2 minutes to 0.1 minutes. The injection amount at a calculation injection time of 0.2 minutes is 40 microliters (0.2 min × 200 μL / min), which greatly exceeds (loop capacity + injection valve void capacity). This is the loop total injection method exemplified in.

  When the injection time is shortened from 0.05 minutes, the total area decreases accordingly, and the injection time and the total area can be approximated by a linear expression passing through the origin. In other words, a minute amount of sample below the void volume of the sample holding loop and the valve is injected into the column, suggesting that the injection amount can be changed by controlling the injection time.

  The same tendency as described above is observed when a sample holding loop having an inner diameter of 0.5 mm is mounted. Since the capacity of the sample holding loop is quadrupled, the range of linearity of injection time and peak total area is expanded to 0.1 minutes.

  In general, when a small amount of sample is injected regardless of a method such as loop total injection, variable loop injection, or direct injection, the accuracy of injection is improved by suppressing the volume of the sample holding loop as much as possible. For this reason, a sample holding loop is produced using a tube having a small inner diameter, but the risk increases such as an increase in the suction resistance of the sample and an increase in the frequency of clogging in the tube.

  In the injection device with time control according to the present invention, the injection amount is determined by the flow rate and the injection time, and therefore, in the low volume region, it is hardly affected by the form and volume of the sample holding loop. That is, it is not necessary to prepare the sample holding loop with a tube having an inner diameter smaller than necessary, and the risk can be avoided.

  The accuracy of the injection amount in the injection device according to the time control of the present invention depends on the accuracy of the valve switching time and the accuracy of the flow rate of the eluent. Therefore, the injection accuracy is slightly lower than that of the general loop total injection method, loop variable injection method, and direct injection method, but there are many advantages such that the injection amount can be easily changed or a minute injection is possible. As exemplified in Example 2, it is very effective in a measuring instrument that performs quantitative calculation from the area ratio of each component, such as glycohemoglobin measurement.

1. 1. Eluent Pump 3. 3. Sample injection valve 4. Sample holding loop Sample 6 6. analytical column Detector 8. 8. Cleaning solution Sample suction mechanism 10. 10. Switching valve Port 12. Osine (nut)
13. Piping 14. Valve body 15. Stator 16. Ferrule 17. Rotor seal 18. Groove 19. Syringe 20. Injection valve 21. Stator A
22. Stator B
23. Suction needle 24. Surada 25. Sample holding hole 26. Flow path 27. Liquid feed pump A
28. Liquid feed pump B
29. Eluent A
30. Eluent B
31. Flow path switching valve 32. Eluent retention loop 33. Micro analysis column 34. Column oven 35. Resistance tube 36. Sample carrying means 37. Sample suction / cleaning means 38. Sample injection control unit 39. Injection condition input section

Claims (3)

at least,
(1) a sample injection unit for injecting a sample;
(2) means for carrying the sample into the sample injection portion;
(3) Sample suction / cleaning means for sucking or cleaning the sample into the sample injection portion;
(4) A sample injection apparatus including an injection condition input unit for setting injection conditions, and (5) a sample injection control unit for controlling (1), (2), and (3) according to the conditions of (4). ,
The sample injection part of (1) is
Two ports connected to both ends of a sample holding loop for holding a sample, a sample introduction port, a sample discharge port, a fluid inlet port, a fluid outlet port, and 6 ports,
At least a first state in which the fluid inlet port and the outlet port are directly conducted without going through the sample holding loop, and the sample entering from the sample introduction port goes out to the sample discharge port through the sample holding loop And the fluid that has entered from the fluid inlet port flows to the fluid outlet port via the sample holding loop, and the sample introduction port and the sample discharge port directly conduct without passing through the sample holding loop. It is a valve that can take the state of
By the sample injection control unit (5),
After all or part of the sample holding loop is filled with the sample in the first state, the valve is switched to the second state, and specified by the injection condition input unit in (4) above, based on the switching time. By switching the valve to the first state at a predetermined time, a part of the sample in the sample holding loop is transferred to the fluid outlet port by being put on the fluid flow, and the sample injection amount is 0.3 to 10 A sample injection device characterized by being a microliter . The sample injection apparatus according to claim 1, wherein a product of a flow rate of the fluid and a time for switching from the time for switching to the second state to the time for switching to the first state is a sample injection amount. Liquid chromatography comprising the sample injection device according to claim 1 or 2.

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