JP7547586B2 - Method for detecting amino acids and chromatograph - Google Patents

Description

The present invention relates to liquid chromatographs such as high performance liquid chromatographs (HPLC) and ultra high performance liquid chromatographs (UHPLC), separation columns used therein, and packing materials packed in the separation columns.

Ion exchange chromatography is one of the separation modes of HPLC. In order to achieve both high separation and high speed analysis when analyzing ions such as amino acids by ion exchange chromatography, a packing material for a separation column is known in which a core made of a polymer or the like is coated with a polymer shell, and the core has lower permeability than the shell (see, for example, Patent Document 1).

Here, ion exchange chromatography generally uses a column packed with ion exchange resin. Ion exchange resin is a polymer packing material, and its mechanical strength is inferior to that of silica gel. In other words, if too much pressure is applied to the packed layer, the polymer packing material will be significantly deformed, the packing material arrangement will be disturbed, and a certain separation performance such as the theoretical plate number will not be obtained and it will not return to its original state. There is also an image of gaps being formed in the packed layer. This state where the yield point, which is the critical point in terms of mechanical strength, is exceeded is called compaction. If pressure is continued to be applied beyond the yield point, not only will separation performance not be obtained, but a significant pressure increase will occur and the column will no longer function. There is also an image of the packing material being mechanically destroyed if too much pressure is applied. In addition, ion exclusion chromatography and ligand exchange chromatography are also available as separation modes of HPLC, but both use ion exchange resin. Organic acids and sugars are representative analytical species.
The components separated using these ion exchange resins are organic compounds with polar functional groups such as carboxyl, amino, or hydroxyl groups, which may become charged, i.e., ions, in aqueous solution. Organic compounds that do not become ions may still have polarity. For example, analytes including nucleotides, nucleosides, biogenic amines, sugars, oligosaccharides, or biopolymers such as proteins, polypeptides, antibodies, and nucleic acids can be separated.

Re-table No. 2012-026569

However, even with packing materials in which the polymer core is coated with a polymer shell as described above, it was not easy to significantly increase the speed of separation and analysis of ions or polar organic compounds by increasing the eluent pressure.

In view of the above, the present invention aims to facilitate faster analysis of the separation of ions or polar organic compounds.

In order to achieve the above object, the present invention provides
A method for detecting an amino acid, comprising the steps of:
a separation column comprising a column body filled with a packing material in which a carrier made of an inorganic material is modified with an ion exchange group and a functional group that contributes to hydrophobic interaction;
A pump capable of pumping an eluent at a pressure of 20 MPa or more;
Using
The pump delivers the eluent to the separation column, and amino acids are separated and detected by two separation modes, ion exchange and hydrophobic interaction.

As a result, carriers made of inorganic materials are less likely to compact, making it easier to analyze ions quickly and with high separation rates.

The present invention makes it easy to perform faster analysis of amino acids, organic acids, sugars, and the like.

FIG. 2 is a diagram showing the flow path configuration of a liquid chromatograph system. 1 is a graph showing the relationship between particle diameter, linear velocity, and height equivalent to a theoretical plate.

The following describes in detail an embodiment of the present invention with reference to the drawings.

(Schematic configuration of chromatograph 100)
In a chromatograph 100 for analyzing ions such as amino acids, for example, as shown in FIG. 1, first to fourth buffer solutions 111 to 114 and a column regenerating solution 115 are selected as eluents or the like by a series of solenoid valves 116A to 116E, and are sent to an autosampler 119 via an ammonia filter column 118 by a buffer pump 117 at a pressure of preferably 20 MPa or more, more preferably 40 MPa or more. An amino acid sample is introduced into the autosampler 119, and is sent to a separation column 120 for separation. The separated amino acids are mixed in a mixer 123 with a ninhydrin reagent 121 sent by a ninhydrin pump 122, and the amino acids (Ruhemann's purple) colored by a reaction in a heated reaction column 124 are continuously detected by a detector 125, and are output as a chromatogram and data by a data processing device 200, which are recorded and stored.

(Separation Column 120)
The separation column 120 is a cylindrical column body made of stainless steel or the like, filled with packing material using an inorganic carrier, and is capable of high-speed, high-separation analysis without compaction even when high-pressure liquid is delivered as described above. More specifically, the packing material is, for example, a carrier made of an inorganic material modified with an ion exchange group and a functional group that contributes to hydrophobic interaction, and has a particle size of 3 μm or less, preferably 2 μm or less.

More specifically, for example, the inorganic material may be at least one of silica, titania, alumina, zirconia, and organic-inorganic composite silica materials. In addition, the cation exchange group may be a sulfonic acid group or a carboxymethyl (CM) group. As the anion exchange group, a quaternary amine, a diethylaminoethyl (DEAE) group, or a tertiary amine may be introduced. Furthermore, as the functional group that contributes to the hydrophobic interaction, at least one of C18, C8, C1, C30, C4, PFP (pentafluorophenyl), and a phenyl group (numbered L01, L07, L13, L62, L26, L43, and L11 according to the USP regulations) may be used.

By using a carrier made of an inorganic material as described above, consolidation does not occur, which would cause an irreversible and sudden loss of pressure when the critical pressure is reached, as occurs when, for example, a resin material is used, so the pump pressure can be set to 40 MPa or more as described above, and high speed, high separation performance can be easily obtained. Note that the upper limit of the pressure can actually be set based on the pressure resistance of each part of the device, but since consolidation is unlikely to occur as described above, there is no particular restriction and it is thought that it can be set to 60 MPa or more.

Here, the pressure loss ΔP (MPa) follows the Kozeny Carman formula:

ΔP=(η×u0×L)/Kv
η (mPa·s): Viscosity u0 (mm/s): Linear velocity L (mm): Column length Kv (m^2): Column permeability (for example, proportional to the square of the particle diameter dp)
So, for example,
Viscosity η is 0.5 (mPa·s)
Linear velocity u0 is 1.0 or 2.0 (mm/s)
Column length L is 60 (mm)
Column permeability Kv is 3 or 1.5 (m^2)
Then, the pressure loss ΔP is as shown below (Table 1).

As shown in the van Deemter plot in Figure 2, by setting the particle size of the packing material to 3 μm or less, preferably 2 μm or less, a smaller theoretical plate height H can be obtained, and a relatively stable theoretical plate height H can be obtained regardless of the linear velocity. More specifically, in the linear velocity range of 0.8 to 1.2 mm/s, which corresponds to a practical flow rate of 0.4 to 0.6 mL/min, it is desirable to set the particle size to 3 μm or less, preferably 2 μm or less, since this results in a small theoretical plate height H.

In addition, by introducing a functional group that contributes to hydrophobic interactions, such as a phenyl or biphenyl group, even when an inorganic carrier is used, a separation mode of amino acids that contributes to hydrophobic interactions similar to those of resin-derived carriers can be obtained in addition to ion exchange chromatography.

The present invention overcomes the shortcomings of ninhydrin ion exchange chromatography, which was previously thought to require the use of resin. By making the packing material inorganic and hard, and by further miniaturizing it from the perspective of the van Deemter plot mentioned above, and by allowing high pressure, high speed and high separation can be achieved. In other words, the higher the pressure that can be allowed, the higher the flow rate, which contributes to higher speeds. It is also possible to extend the length of the column, improving the number of theoretical plates, i.e., separation performance.

As described above, by using a packing material that uses an inorganic carrier, high-speed, high-separation analysis can be performed without compaction. Moreover, even when a packing material that uses an inorganic carrier is used, by applying post-column derivatization ion exchange chromatography using ninhydrin, quantitative analysis with higher reproducibility in terms of daily variation can be performed than the pre-column derivatization method of reversed-phase chromatography, because the ninhydrin reagent is stable and the reaction itself is also stable. In addition, because two separation modes, ion exchange and hydrophobic interaction, can be used, separation of any amino acid component can be easily performed.

An example of a preferred form of inorganic material is an organic-inorganic hybrid silica material.

(Other matters)
When ion exchange chromatography is to be performed at 20 MPa or more, preferably 60 MPa or more, in order to achieve UHPLC, it is possible to avoid compaction and to switch the polymer to, for example, silica gel. Silica gel particles can be relatively easily refined to 3 μm or less. In other words, by chemically modifying silica gel with ion exchange groups such as sulfonic acid groups, a packing material for ion exchange chromatography for UHPLC can theoretically be created. However, when the intention is to achieve UHPLC by replacing the packing material, it is assumed that the hydrophobic interaction properties that the ion exchange resin originally possesses will not be obtained. In other words, since the resin matrix such as polystyrene has hydrophobic interaction properties, these properties may be lost by replacing it with silica gel.
Therefore, it is possible to add a functional group exhibiting hydrophobic interactions to the ion exchange group for chemical modification. Usually, alkyl groups such as C18 are used to introduce hydrophobic interactions, but polystyrene and other materials have double bonds between carbon atoms, i.e., interactions specific to π electrons, and it may be desirable to introduce benzene rings such as phenyl groups and biphenyl groups.

As a result, carriers made of organic-inorganic composite silica-based inorganic materials are less likely to compact, and in addition, the introduction of hydrophobic interaction functional groups can replace the inherent chemical properties of the resin, making it easy to convert ion exchange chromatography into UHPLC, i.e., to high-speed, high-separation analysis.

As described above, it is possible to easily perform faster analysis of not only amino acids, but also organic acids and sugars. In other words, it is possible to separate and analyze analytes that could not be analyzed by UHPLC using ion exchange resins used in ion exchange chromatography, ion exclusion chromatography, or ligand exchange chromatography because they have a pressure of 20 MPa or more, preferably 60 MPa or more, and a particle size of 3 μm or less. For example, analytes include nucleotides, nucleosides, biogenic amines, sugars, oligosaccharides, or biopolymers such as proteins, polypeptides, antibodies, and nucleic acids.

100 Chromatograph 111-114 1st to 4th buffer solutions 115 Column regenerant solution 116A-116E Solenoid valve series 117 Buffer pump 118 Ammonia filter column 119 Autosampler 120 Separation column 121 Ninhydrin reagent 122 Ninhydrin pump 123 Mixer 124 Reaction column 125 Detector 200 Data processing device

Claims (8)

a separation column comprising a column body filled with a packing material in which a carrier made of an inorganic material is modified with an ion exchange group and a functional group that contributes to hydrophobic interaction;
A pump capable of pumping an eluent at a pressure of 20 MPa or more;
Using
A method for detecting amino acids, comprising the steps of: delivering the eluent to the separation column by the pump; and separating and detecting amino acids by two separation modes, ion exchange and hydrophobic interaction . The method for detecting an amino acid according to claim 1,
The method for detecting an amino acid, wherein the inorganic material is at least one of silica, titania, alumina, zirconia, and an organic-inorganic composite silica material. A method for detecting an amino acid according to any one of claims 1 to 2, comprising:
The method for detecting an amino acid, wherein the ion exchange group is at least one of sulfonic acid, quaternary amine, carboxymethyl, and diethylaminoethyl. A method for detecting an amino acid according to any one of claims 1 to 3, comprising:
A method for detecting an amino acid , wherein the functional group that contributes to the hydrophobic interaction is at least one of a C18, C8, C1, C30, C4, PFP, phenyl, and biphenyl group. A method for detecting an amino acid according to any one of claims 1 to 4, comprising:
A method for detecting an amino acid, wherein the particle size of the filler is 3 μm or less. A method for detecting an amino acid according to any one of claims 1 to 5, comprising:
A method for detecting an amino acid, wherein the eluent delivered to the column is selected from a plurality of buffer solutions. 7. The method for detecting an amino acid according to claim 6,
Further comprising a ninhydrin pump for pumping a ninhydrin reagent;
A method for detecting an amino acid, comprising mixing the amino acid separated in the separation column with the ninhydrin reagent to detect the amino acid. a separation column comprising a column body filled with a packing material in which a carrier made of an inorganic material is modified with an ion exchange group and a functional group that contributes to hydrophobic interaction;
A pump capable of pumping an eluent at a pressure of 20 MPa or more;
A detector,
A chromatograph characterized in that the pump sends the eluent to the separation column, and the amino acids separated by two separation modes, ion exchange and hydrophobic interaction, can be detected by the detector.

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