JP2015215320A - Simultaneous analytical method of sample containing compounds having different polarities - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simultaneous analytical method of a hydrophilic compound and a hydrophobic compound.SOLUTION: A analytical method uses a chromatograph that comprises: a moving phase liquid feeding part; a separation column arranged in an analysis channel of the moving phase liquid feeding part; a sample injection part injecting a sample to the analysis channel between the moving phase liquid feeding part and the separation column; a detector detecting a sample component eluted from the separation column; and a back-pressure valve arranged downstream of the separation column with respect to flow of the moving phase of the analysis channel. The method includes the steps of: changing the compositions of supercritical fluid and a modifier in the moving phase sent from the moving phase liquid feeding part over a range from a supercritical fluid-excess state where the moving phase comes into a supercritical state to a modifier-excess state where the moving phase comes into a liquid state, so as to temporally change a ratio of the modifier to the supercritical fluid in the moving phase; injecting the sample by the sample injection part; and detecting the sample component eluted from the separation column with the detector.

Description

  The present invention relates to supercritical fluid chromatography (SFC) using a supercritical fluid as a mobile phase, and more specifically, chromatography including a wider mobile phase composition range than supercritical fluid chromatography, so-called unification. It relates to chromatography (Unified Chromatograpy).

  In chromatography, a single-phase mobile phase such as liquid, gas, or supercritical fluid is usually used, and any mobile phase is selected according to the purpose. However, if the target compound chemistry is extensive, multiple chromatographies with different mobile phases are combined.

Supercritical fluid chromatography is a complementary separation technique for gas chromatography (GC) and liquid chromatography (LC), and uses supercritical carbon dioxide (SCCO ₂ ) or subcritical carbon dioxide as the mobile phase. Supercritical is a state where the critical temperature and critical pressure are exceeded, and subcritical is a state in the vicinity of a region lower than the critical point.

Supercritical carbon dioxide, which is a fluid composed of carbon dioxide (CO ₂ ) near or beyond the critical temperature and / or critical pressure, has a critical pressure of 7.38 MPa and a critical temperature of 31.1 ° C. It is most commonly used in supercritical fluid chromatography because it is relatively close to room temperature, has no flammability or chemical reactivity, and has high purity at low cost. Supercritical carbon dioxide has favorable properties as chromatography with low viscosity and high diffusivity, and supercritical carbon dioxide chromatography can obtain faster and better separation than liquid chromatography.

  Supercritical fluid carbon dioxide, the main fluid of the mobile phase of supercritical fluid chromatography, is nonpolar and similar to n-hexane, so supercritical fluid chromatography is normal phase chromatography and nonpolar It has been considered suitable for the analysis of compounds. However, since nonpolar supercritical fluid carbon dioxide is compatible with polar organic solvents such as methanol and ethanol, adding these polar organic solvents as modifiers makes the mobile phase polar. Things have been done. The modifier is added gradually over time and is generally increased to 30-40%.

  In supercritical fluid chromatography, not only non-polar compounds but also polar compounds may be analyzed by adding modifiers. There is a report (see Non-Patent Documents 1 and 2) that supercritical fluid chromatography is used for simultaneous analysis of polar compounds and nonpolar compounds in that way, but the number is very low.

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  The present invention is directed to a method for simultaneous analysis of samples containing compounds having different polarities, such as simultaneous analysis of polar compounds and nonpolar compounds, or simultaneous analysis of hydrophilic compounds and hydrophobic compounds. In the embodiments described below, vitamins are taken as an example of such compounds. Vitamins are essential nutrients for humans and animals and are necessary for normal growth, body maintenance and functioning. Also, when vitamins are deficient or taken too much, it becomes a specific disease. Therefore, intensive research has been conducted to measure vitamins in food or in the body. Vitamins are divided into two groups according to their solubility. That is, fat-soluble vitamins (FSV) and water-soluble vitamins (WSV). There are significant differences between the two groups with respect to water solubility. As shown in Table 1, the difference ranges from −2.11 to 10.12.

  In Table 1, Nos. 1-8 are fat-soluble vitamins and Nos. 9-17 are water-soluble vitamins. Since fat-soluble vitamins and water-soluble vitamins also behave differently as acid bases, it is difficult to analyze them simultaneously by conventional chromatography.

  An object of the present invention is to provide a method for analyzing samples containing compounds having different polarities simultaneously by developing supercritical fluid chromatography.

The present invention injects a sample into a mobile phase liquid feeding section, a separation column disposed in an analysis flow path from the mobile phase liquid feeding section, and an analysis flow path between the mobile phase liquid feeding section and the separation column. A chromatograph provided with a sample injection part, a detector for detecting a sample component eluted from the separation column, and a back pressure valve arranged downstream of the separation column with respect to the flow of the mobile phase in the analysis flow path was used. This analysis method includes the following steps (A) to (C).
(A) The composition of the supercritical fluid and the modifier in the mobile phase fed from the mobile phase feeding section is changed from the supercritical fluid excess state in which the mobile phase is in the supercritical state to the liquid phase in the mobile phase. Varying the ratio of modifier to supercritical fluid in the mobile phase over time, up to the excess state;
(B) A step of injecting a sample from the sample injection unit, and (C) a step of detecting a sample component eluted from the separation column by the detector.

  Chromatographic techniques are generally classified as gas chromatography if the physical state of the mobile phase is gas, supercritical fluid chromatography if it is a supercritical fluid, and liquid chromatography if it is a liquid. It is. For this reason, the method of the present invention is not exactly called supercritical fluid chromatography from the viewpoint of a general classification method. This is because the state of the mobile phase observed through the method of the present invention is not limited to the supercritical state.

  A separation technique that uses two or more mobile phase states is called unified chromatography. Since the method of the present invention uses three states, ie, a supercritical state, a subcritical state, and a liquid state, regarding the phase state of carbon dioxide, it belongs to unified chromatography, and by changing the mobile phase and analysis conditions with time. In one chromatography, both supercritical fluid chromatography and liquid chromatography are realized. Although the detailed method will be described in the embodiment, as an example, the state of the mobile phase is continuously changed from supercritical to subcritical to liquid. For this purpose, a gradient method in which the modifier is added to carbon dioxide as the mobile phase is adopted, and the analysis is started in a supercritical state, for example, supercritical carbon dioxide near 100%. Gradually increase the modifier ratio to near 100% until it reaches a state.

  An important point in the unified chromatography of the present invention is that no discontinuous transition is observed in the mobile phase even when the phase state of carbon dioxide changes. By controlling the temperature or pressure, the mobile phase can be continuously changed from gas to supercritical or from supercritical to liquid. In so doing, carbon dioxide is always or almost always present through a series of analyses. At the start of analysis, the phase state of carbon dioxide is supercritical, but it is thought that it changes from subcritical to liquid as the proportion of modifiers increases. But that is not all. In the present invention, since the composition of the mobile phase is changed until the carbon dioxide is replaced by the modifier until it is in a liquid state, the method of the present invention is further modified in comparison with supercritical fluid chromatography and liquid chromatography. The solvating power of the mobile phase can be increased as compared with the conventional unified chromatography in which the proportion of a is increased only to 30-40%. That is, the possibility of analyzing various compounds in a wide polar range can be provided. It also eliminates the boundary between supercritical fluid chromatography and liquid chromatography, and integrates the two chromatographic techniques.

  There are important things to achieve the unified chromatography of the present invention. It is to select an appropriate column. This increases the possibility of simultaneous analysis of various compounds. In addition to increasing the polarity diversity of the mobile phase by replacing the carbon dioxide in the mobile phase with a modifier, it is important in practical terms to select a column so that various compounds can be separated. is there.

  Therefore, in a preferred embodiment of the present invention, as a separation column, a packing material in which octadecyl carbon chains are bonded to silica is used as a stationary phase, a packing material in which silica is bonded to a propylfluorophenyl group is used as a stationary phase, or silica. In this case, a solid phase (BEH: Ethylene Bridged Hybrid) having a filler bonded with octadecylsilyl group is used, and methanol or a solution containing water in methanol is used as a modifier. By so selecting column characteristics and mobile phase solvent, unified chromatography becomes useful for the analysis of a variety of compounds.

  Since supercritical fluid carbon dioxide is compatible with acids, bases, and water, the range of compounds that can be analyzed simultaneously can be further expanded by adding these substances as additives in addition to the modifier. . Therefore, in still another embodiment of the present invention, the mobile phase may further contain an additive containing ammonium ions in addition to the modifier.

  In yet another embodiment, in step (A), the ratio of modifier to supercritical fluid in the mobile phase is set in step (A) so that the mobile phase is in an excessive modifier state in which the mobile phase is in a liquid state. It may be increased to 70% or more, preferably 85% or more, and may further be increased to 100%.

  In a preferred embodiment, a mixed solution containing a fat-soluble vitamin and a water-soluble vitamin having different polarities is separated and analyzed as a sample. This does not limit the method of the present invention to the analysis of specific compounds, but includes fat-soluble vitamins and water-soluble vitamins to see how far the range of compounds can be expanded with a single analysis of unified chromatography. This is because simultaneous analysis of vitamins can be a practical and direct indicator.

  According to the unified chromatography of the present invention, the composition of the supercritical fluid and the modifier in the mobile phase is changed from the supercritical fluid excess state in which the mobile phase is in the supercritical state to the modifier excess state in which the mobile phase is in the liquid state. Since the time range is changed over time, the polar range of the mobile phase can be expanded by one analysis. For example, as shown in the Examples, the logP containing fat-soluble vitamin and water-soluble vitamin is 2.11. Allows simultaneous analysis of 17 different vitamins in a wide range from 1 to 10.12. In addition, the analysis can be performed in a shorter time than conventional supercritical fluid chromatography.

It is a flow chart which shows an example of a chromatograph in which unified chromatography of the present invention is carried out. It is a chromatogram which shows the influence which it has on a peak shape when a base is added as an additive to a mobile phase in Example 1. 2 is a chromatogram showing the peak shapes of thiamine and pyridoxine when ammonium formate is added to the mobile phase in Example 1. FIG. It is a chromatogram which shows the influence which it has on the peak shape which varied the length of the column in Example 1. FIG. 2 is a chromatogram showing a peak shape when formic acid is added to a mobile phase in Example 1. It is a chromatogram which shows the peak shape when ammonium formate is added to a mobile phase in Example 1 and the concentration is changed. 2 is a chromatogram showing the peak shape obtained in Example 2. FIG.

  An example of a chromatograph in which the method of the present invention is carried out is shown in FIG. A liquid feed flow path 6 for feeding the supercritical fluid stored in the cylinder 2 by the SFC pump 4 and a modifier liquid feed flow path 12 for feeding the modifier solution 8 by the modifier pump 10 are connected to the mixer 14. ing. A flow path including the pumps 4 and 10 and the mixer 14 is a mobile phase liquid feeding unit. Supercritical carbon dioxide is used as the supercritical fluid.

  The flow path through which the mobile phase is sent from the mixer 14 is the analysis flow path 16. On the analysis channel 16, a sample injection unit 18, a separation column 20, a back pressure valve 22, and a detector 24 for injecting a sample into the analysis channel 16 are arranged from the upstream side with respect to the flow of the mobile phase. The separation column 20 is accommodated in a column oven 21 and kept at a constant temperature. The sample injection unit 18 is, for example, an autosampler. The back pressure valve 22 is for maintaining the analysis channel 16 at a predetermined pressure in order to maintain the state of the mobile phase in the analysis channel 16.

  The detector 24 is not particularly limited, but a mass spectrometer such as a tandem quadrupole mass spectrometer is used in this embodiment. The mass spectrometer as the detector 24 includes an ESI (electrospray ionization) source. In the analysis flow path 16 upstream of the back pressure valve 22, the mobile phase is in a supercritical state, a subcritical state, or a liquid state. However, since the mobile phase is released under atmospheric pressure on the downstream side of the back pressure valve 22, the column The sample component separated and eluted at 20 is released in the form of a mist together with the mobile phase on the downstream side of the back pressure valve 22. By applying a voltage (electrospray voltage) between the mobile phase outlet and the ionization chamber of the mass spectrometer, the eluted sample components are ionized and analyzed by the mass spectrometer.

  When a mass spectrometer is used as the detector 24, an ionization accelerator such as formic acid or ammonia may be added to the mobile phase in order to promote ionization of sample components in the ionization chamber of the mass spectrometer. . Further, a makeup solution serving as an ionization aid may be supplied to the analysis flow path between the column 20 and the back pressure valve 22 by a pump. As the makeup solution, for example, a solution containing an ionization accelerator such as formic acid or ammonia in an organic solvent such as methanol or water can be used.

  In this chromatograph, the pressure of the back pressure valve 22, the ratio of supercritical carbon dioxide and modifier in the mobile phase, the temperature of the separation column 20, and the sample injection from the sample injection unit 18 are managed by a control unit (not shown). ing. When the analysis operation is started, the supercritical fluid and the modifier solution are sent by the pumps 4 and 10, mixed by the mixer 14, and introduced into the analysis flow path 16 as a mobile phase. In order to perform the gradient analysis, the flow rates of the pumps 4 and 10 are adjusted according to the program held in the control unit so that the ratio of the supercritical fluid and methanol changes with time. The analysis channel 16 is controlled to have a predetermined internal pressure by the back pressure valve 22, and the phase of the mobile phase introduced into the analysis channel 16 changes from supercritical to subcritical and liquid. The sample injected by the sample injection unit 18 is conveyed to the separation column 20 by the mobile phase and separated for each component, and is introduced from the separation column 20 through the back pressure valve 22 to the mass spectrometer of the detector 24 and detected.

Example 1
In Example 1, a gradient method in which the proportion of the modifier as a mobile phase was increased to 60% was carried out, the optimum combination of the modifier and the column was examined, and the effectiveness of the additive was further examined. The gradient conditions are as shown in Table 2. The pressure of the back pressure valve was 1800 psi.

(Mobile phase) Supercritical carbon dioxide and modifier The following four types were examined as modifiers.
(1) 2-propanol (IPA),
(2) Acetonitrile (ACN),
(3) methanol (MeOH),
(4) Methanol / water (95/5, v / v) (95% MeOH).
The mobile phase contains 0.1% (v / v) formic acid as an ionization accelerator during mass spectrometry.

Mobile phase flow rate: 1.0 mL / min Sample injection volume: 1 μL
Column temperature: 35 ° C
Sample temperature: 6 ° C
Back pressure (ABPR): 14.2 MPa

(column)
The following six types of columns were examined.
(1) ACQUITY UPC ² BEH (BEH)
(2) ACQUITY UPC ² 2-Ethylpyridine (2-EP)
(3) ACQUITY UPC ² CSH Fluoro-Phenyl (FP)
(4) ACQUITY UPC ² HSS C18 SB (C18SB)
(5) ACQUITY HSS Cyano (Cyano)
(6) ACQUITY CSH Phenyl-Hexyl (PH)
(All are Waters products, the size is 3.0 mm inside diameter, 100 mm length, and the filler size is 2 μm)

Instrument parameters for mass spectrometry:
Capillary voltage: 0.8KV
Desolvation temperature: 600 ° C
Conical gas flow rate: 50 L / hour Desolvation gas flow rate: 1200 L / hour

  17 kinds of vitamins were detected by a mass spectrometer, and MRM (Multiple Reaction Monitoring) parameters for detecting a chromatogram peak at that time were set as shown in Table 3.

  For the ionization, an electrospray ionization method which is one of the atmospheric pressure ionization methods was adopted. A high voltage (capillary voltage) is applied between the capillary for supplying the sample solution from the supercritical fluid chromatograph and the counter electrode, and the atomized gas is sprayed. As a result, a conical liquid cone (Taylor cone) is formed at the capillary tip, and highly charged droplets are generated at the tip. Further, the charged droplets are vaporized by spraying the desolvation gas through the heat block and the desolvation temperature in the desolvation tube, and finally the positive ions by proton addition (Selected Ion in Table 3). [M + H]) and negative ions are generated by proton elimination. The generated ions are “Precursor Ion” in Table 3. Thereafter, the ions were cleaved into specific ions by collision activated dissociation (CID) using Ar gas (Collision Energy in Table 3) inside the mass spectrometer, and selected ions among the generated fragment ions (in Table 3) Product Ion) is measured with a detector. The MRM chromatograms shown in FIGS. 2 to 7 show the detection signal intensity of ions (Product Ion) selected for each vitamin with respect to time. In Table 3, “Span” is the width of m / z monitored by MRM, “Cone Voltage” is the voltage value of the sampling cone, and is the voltage applied to introduce ions generated at the interface into the mass spectrometer. In addition, said apparatus parameter has shown the general-purpose numerical value as an example in an electrospray ionization mass spectrometer, and is not limited to such an apparatus parameter.

(Sample preparation)
Each vitamin was dissolved in a diluent to prepare a standard solution. Diluents are as follows.
Diluent for fat-soluble vitamins: chloroform: methanol = 50: 50
Diluent for water-soluble vitamins: methanol: water = 50:50
Diluent for vitamin VC: 100% water
These standard solutions were further diluted with methanol to obtain a final mixed standard solution.

Column considerations:
Six columns of BEH, 2-EP, FP, C18SB, Cyano and PH described above, and four types of modifiers of 2-propanol, acetonitrile, methanol, methanol / water (95/5 (v / v)) described above. The combination of a was examined. The modifier contains 0.1% (v / v) formic acid to facilitate ionization upon detection by the mass spectrometer.
The results are shown in Table 4.

The code | symbol AF of Table 4 is a fat-soluble vitamin, and code | symbol GN is a water-soluble vitamin (WSV). Based on the results of Table 4, the following three were selected as preferred combinations of columns and modifiers depending on the peak shape and the number of detected peaks.
(1) Combination of C18SB column and methanol (2) Combination of C18SB column and methanol / water (95/5 (v / v)) (3) Combination of FP column and methanol

  The FP column uses a packing material in which a propyl fluorophenyl group as a ligand is bonded to silica as a stationary phase. When methanol is used as a modifier, all vitamins are eluted. However, the FP column has a lower retention time for fat-soluble vitamins (FSV) and a shorter retention time than the C18SB column.

  The C18SB column uses a filler in which octadecyl carbon chain as a ligand is bound to silica as a stationary phase, and in the studied column, there is a tendency to have a hydrophobic interaction with the compound. Strongest. This is why the C18SB column retains fat-soluble vitamins very well. On the other hand, C18SB columns can also retain hydrophilic compounds due to hydrophilic interactions due to residual silanol groups on the non-endcapping stationary phase. It is thought that the opposite properties of the octadecyl carbon chain and the silanol group in the C18SB column give important retention behavior to both fat-soluble and water-soluble vitamins. That is, the octadecyl carbon chain contributes to retention of the water-soluble vitamin, and the silanol group on the stationary phase contributes to retention of the water-soluble vitamin. As a result, even a single column can be applied to a wide range of polar compounds.

For the C18SB column, the modifier methanol and methanol / water were not significantly different with respect to the number of peaks detected. However, the observed peak shape was slightly better with methanol as the modifier than with methanol / water.
Based on the above examination results, the following analysis uses a C18SB column as the column.

(Examination of additives)
If a C18SB column is used as the column and methanol is used as the modifier, both fat-soluble vitamins and water-soluble vitamins can be retained, but there is room for improvement with respect to the chromatogram. One is that the peak of thiamine (symbol L) is missing in the results of Table 4, and the other is that the peak shape of pyridoxine (symbol I) and vitamin VC (symbol N) has tailing. .

  The data in Table 4 was obtained when the measurement time was set to 10 minutes. When the measurement time was further increased, a thiamine peak was observed, but the problem was that the thiamine retention time was too long.

  Peak tailing was observed at the pyridoxine and vitamin VC peaks. With respect to pyridoxine, pyridoxine has a heterocyclic amine ring, and the interaction with the silanol group in the column is considered to cause tailing. Regarding tailing, there is a report that the peak shape of a basic compound is improved by adding a basic additive to the mobile phase (see Non-Patent Document 3). There is also a report that the peak shape of a basic compound is improved when ammonium hydroxide is present in the mobile phase of a supercritical fluid chromatograph (see Non-Patent Document 4).

  Therefore, FIG. 2 shows an MRM chromatogram as a result of examining the effect of a base as an additive on the peak tailing of pyridoxine. The code | symbol 1-13 in FIG. 2 has shown the following 13 types of vitamins. 1 is A acetate and A palmitate, 2 is α-Tocopherol, 3 is K2, 4 is K1, 5 is β-Carotene, 5 is Nicotinamide, 7 is Nicotinic acid, 8 is Pyridoxine, 9 is d-Pantothenic acid, 10 is Biotin, 11 is Thiamine, 12 is Riboflavin, and 13 is VC. A to C indicate the type and concentration of the base added to the mobile phase, A is 0.1% (v / v) formic acid, B is 0.2% (v / v) formic acid, and C is 0.1. % (V / v) ammonium hydroxide.

  According to the results of FIG. 2, the peak shape of pyridoxine (symbol 8) is often improved when ammonium hydroxide is added to the mobile phase, but is not improved even when formic acid is added.

  Furthermore, the peak shapes of thiamine and pyridoxine when ammonium formate is added to the mobile phase are shown in FIG. A is when 0.1% (w / v) ammonium formate is added, and B is when 0.1% (v / v) formic acid and 0.1% (v / v) ammonium hydroxide are added. Similarly, tailing is improved. From this result, it is the ammonium ion, not the hydroxide ion, that leads to improved peak shape, and the ammonium ion peaks by cleaving the interaction between the silanol residue and the amine in pyridoxine on the C18SB column. It is thought that it acts to shorten tailing.

  Next, the point that the retention time of thiamine is too long and the problem of peak tailing of vitamin VC will be examined. Prior to further study of the additive, it was considered to shorten the column length. That is, when the column length is shortened, it is considered that the problem of retention time and peak tailing may be solved by reducing silanol groups. The result is shown in FIG. A is when the column length is 5 cm, and B is when the column length is 10 cm. 1-12 represents the type of vitamin, 1 is A acetate and A palmitate, 2 is α-Tocopherol, 3 is K2, 4 is K1, 5 is β-Carotene, 6 is Nicotinamide, 7 is Nicotinic acid, 8 is Pyridoxine, 9 is D-Pantothenic acid, 10 is Biotin, 11 is Thiamine, and 12 is VC. When the column length is shortened from 10 cm to 5 cm, the thiamine is eluted within 7 minutes with a shorter retention time, and the peak shape is improved. Pyridoxine also has a better peak shape. However, the peak shape of vitamin VC has not been improved.

  When the sample contains a basic compound and an acidic compound, there is a report that the peak shape is improved by adding not only an acid but also a base as an additive to be added to the mobile phase together with the modifier (see Non-Patent Document 5). .) In supercritical fluid chromatography, the addition of an acid stronger than the solute is effective for improving the peak shape of the acidic solute, and for the peak tailing of the carboxylic acid, an acid is added to suppress the ionization of the solute. Is also very important (see Non-Patent Documents 3 and 6). Although the use of a strong acid can improve the peak shape of vitamin VC, the use of a strong acid such as trifluoroacetic acid has the problem of causing background noise when using a mass spectrometer as a detector.

  Therefore, in order to see whether or not improvement of the peak shape of vitamin VC can be achieved using weak acid, formic acid was added and the effect was examined. The resulting MRM chromatogram is shown in FIG. A is vitamin B12, B is vitamin VC, C is pyridoxine, and D is thiamine. Reference numeral 1-4 represents the weak acid added. Reference numeral 1 is 0.1% (w / v) ammonium formate only, reference numeral 2 is 0.1% (w / v) ammonium formate and 0.2% formic acid, reference numeral 3 is 0.1% (w / v) formic acid. Ammonium and 0.4% formic acid, code 4 is 0.1% (w / v) ammonium formate and 0.6% formic acid. The modifier is methanol.

  As shown in FIG. 5, it is understood that the peak shape of vitamin VC (symbol B) is improved when the formic acid concentration is increased. In pyridoxine (symbol C), the peak shape is distorted. In addition, the sensitivity of vitamin B12 (symbol A) decreases, which indicates that the addition of formic acid prevents the ionization of vitamin B12 (see Non-Patent Document 7).

  Next, the results when the ammonium formate concentration is changed are shown in FIG. A is for 0.2% (w / v) ammonium formate and B is for 0.1% (w / v) ammonium formate. The modifier is methanol / water (95/5 (v / v)). The code | symbol 1-15 represents the kind of vitamin. 1 is A acetate, 2 is A palmitate, 3 is α-Tocopherol, 4 is K2, 5 is K1, 6 is β-Carotene, 7 is Nicotinamide, 8 is Nicotinic acid, 9 is Pyridoxine, 10 is d-Pantothenic acid, 11 is Biotin, 12 is Thiamine, 13 is Riboflavin, 14 is VC, and 15 is B12. Vitamin VC (symbol 14) is improved so that the peak width becomes narrower when the ammonium formate concentration increases from 0.1% to 0.2%. The peak shape of the other vitamins has not deteriorated with increasing ammonium formate concentration.

  The results of FIGS. 5 and 6 show that the additive concentration is another important factor in improving the peak shape of vitamin VC in the unified chromatographic separation. In the analysis for obtaining the result of FIG. 6, water was added to methanol in order to suppress precipitation of ammonium formate by unified chromatography as much as possible, but this did not cause any adverse effects.

  Regarding vitamin B12, the peak shape of reference numeral 1 in FIG. 5 (the modifier is methanol and 0.1% (w / v) ammonium formate added thereto) and the reference numeral 15 in FIG. 6 (the modifier is methanol) Of water / water (95/5 (v / v) with 0.1% (w / v) ammonium formate added), tailing is improved by adding water .

(Example 2)
Study of modifier composition:
In the unified chromatographic separation, it was found that the peak shape can be improved to some extent by adding additives. However, it is not enough. In particular, there is still room for improvement regarding vitamin VC and vitamin B12. As a rule of thumb, a preferred analyte in supercritical fluid chromatography is one having a solubility of at least 1 mg / mL in methanol (see Non-Patent Document 8). In fact, vitamin VC has a solubility in methanol lower than the standard value of 1 mg / mL, so it cannot be said that it is a suitable compound for measurement by unified chromatography on the extended line of supercritical fluid chromatography. With respect to vitamin B12, vitamin B12 has a solubility of 1 mg / mL in methanol, but vitamin B12 has a phosphate group, which provides hydrophilicity. Therefore, it is considered that the solvating power of the mobile phase is not strong enough to elute vitamin VC and vitamin B12. Based on this idea, an attempt was made to further increase the polarity of the mobile phase in order to elute the compound with a better peak shape.

  The simplest way to make the modifier polar is to add water. In Example 1, the case where 5% of water was added was already examined. If water is further added, the system pressure may become unstable due to miscibility with carbon dioxide.

  Therefore, as another method, we investigated further increasing the composition of the modifier in the gradient. In conventional unified chromatography, the modifier composition is usually increased to 30-40%, and in Example 1 it was increased to 60%. In this example, it was further increased to 70% or 100%.

In this example, ACQUITY UPC ² BEH (BEH) (product of Waters, size 3.0 mm, length 50 mm, packing size 1.7 μm) was used as the column. This column uses a packing material in which octadecylsilyl groups are bonded to silica as a stationary phase. As the mobile phase, the following solvent A and solvent B were used. When the modifier composition was increased to 100%, the composition was changed with time based on the gradient conditions in Table 5. Solvent A is carbon dioxide, and solvent B is methanol / water (95/5 (v / v)) as a modifier (containing 0.2% ammonium formate). The column temperature was 40 ° C.

Table 6 shows the pressure gradient conditions of the back pressure valve.

  The analysis was performed for 10 minutes under the above conditions. The result is shown in FIG. FIG. 7 is an MRM chromatogram of 17 types of vitamins. Reference numerals 1 to 17 indicate the types of vitamins. 1 is A acetate, 2 is A palmitate, 3 is D2, 4 is α-Tocopherol, 5 is K2, 6 is K1, 7 is α-Tocopherol acetate, 8 is β-Carotene, 9 is Nicotinamide, 10 is Nicotinic acid, 11 is Pyridoxine, 12 is d-Pantothenic acid, 13 is Biotin, 14 is Thiamine, 15 is Riboflavin, 16 is B12, and 17 is VC. The horizontal axis is the retention time in unified chromatography. According to the results of FIG. 7, the final composition of the modifier was increased to 100%, but the elution of all vitamins was completed when the composition of the modifier was 85%. As a result, the peak width was reduced and the peak shape was improved. For example, looking at the peak shapes of vitamin VC (symbol 17) and vitamin B12 (symbol 16), the peak width of vitamin VC is from 0.23 minutes to 0.11 compared to the case where the final composition of the modifier is up to 70%. In Vitamin B12, it was reduced from 0.25 minutes to 0.10 minutes.

  Then, since the peak width is reduced and the separation between the peaks of each vitamin is improved, the analysis can be completed in a short time. As shown in FIG. Separation was possible in 4 minutes. This separation time is shorter than the separation time of supercritical fluid chromatography that has been reported so far (see Non-Patent Documents 9-13).

  It was also confirmed that each vitamin was detected with good reproducibility by repeated measurement. The results are shown in Table 5.

  Table 7 shows an average value (Ave.) and a relative standard deviation (% RSD) (= (standard deviation / average value) × 100) of peak vitamin retention times and peak area measurements. . According to the result, the reproducibility of the retention time is 0-0.51% RSD, and the reproducibility of the peak area is 1.24-5.63% RSD, indicating a good reproducibility.

  The present invention enables simultaneous analysis of various compounds using unified chromatography ranging from supercritical fluid chromatography to liquid chromatography, as specifically shown in the Examples. In the examples, analysis examples using vitamins are shown, but the present invention is not limited to vitamin analysis. If only supercritical fluid chromatography is used, the solvating power of the mobile phase is limited. However, the unified chromatography of the present invention moves the mobile phase by largely replacing carbon dioxide in the mobile phase with methanol. Diversity of phase polarity can be expanded. As a result, when applied to the analysis of vitamins, fat-soluble vitamins and water-soluble vitamins were simultaneously applied in 4 minutes by the gradient method of the example in which the ratio of methanol to carbon dioxide in the mobile phase was changed from 2% to 85%. I was able to analyze. The method of Example 2 has a shorter analysis time than conventional supercritical fluid chromatography.

  Unified chromatography cannot be achieved without carbon dioxide, so it can be said that it is a technique developed from supercritical fluid chromatography. The present invention provides a new approach to unified chromatography. That is, unified chromatography can increase the solvating power in the mobile phase by controlling the physical state of the mobile phase and largely replacing carbon dioxide with methanol. As a result, it is apparent that the present invention can be applied as a method for analyzing compounds having various chemical properties, not limited to vitamins, and can be a separation means for various compounds having a wide range of polarities.

4 SFC pump 10 modifier pump 14 mixer 16 analysis flow path 18 sample injection part 20 separation column 22 back pressure valve 24 detector

Claims (6)

A mobile phase feeding unit, a separation column arranged in an analysis channel from the mobile phase feeding unit, a sample injection unit for injecting a sample into an analysis channel between the mobile phase feeding unit and the separation column, An analysis method using a chromatograph provided with a detector for detecting a sample component eluted from the separation column and a back pressure valve disposed downstream of the separation column with respect to the flow of the mobile phase in the analysis flow path. And
An analysis method comprising the following steps (A) to (C):
(A) The composition of the supercritical fluid and the modifier in the mobile phase fed from the mobile phase feeding section is changed from the supercritical fluid excess state in which the mobile phase is in the supercritical state to the liquid phase in the mobile phase. Varying the ratio of modifier to supercritical fluid in the mobile phase over time, up to the excess state;
(B) A step of injecting a sample from the sample injection unit, and (C) a step of detecting a sample component eluted from the separation column by the detector.   The analysis method according to claim 1, wherein the vitamin is separated and analyzed by injecting a mixed solution containing a fat-soluble vitamin and a water-soluble vitamin from the sample injection portion. As the separation column, a filler having octadecyl carbon chain bonded to silica as a stationary phase, a filler having a propylfluorophenyl group bonded to silica as a stationary phase, or a filler having octadecylsilyl group bonded to silica. Is a stationary phase,
The analysis method according to claim 2, wherein the modifier is methanol or a solution containing water in methanol.   The analysis method according to claim 2 or 3, wherein the mobile phase further contains an additive containing ammonium ions.   The step (A) increases the ratio of the modifier to the supercritical fluid in the mobile phase to 85% or more in order to obtain a modifier-excess state in which the mobile phase is in a liquid state in the step (A). 5. The analysis method according to any one of items 1 to 4.   The analysis method according to claim 5, wherein in step (A), the ratio of the modifier to the supercritical fluid in the mobile phase is increased to 100.