JP6348258B2 - Separation analysis method - Google Patents

Description

  The present invention relates to a method for separating and analyzing components in a sample.

  In various fields, various separation analysis methods are known as methods for quantifying or qualifying each of a plurality of components contained in a sample. For example, high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), etc. are known, and the separation and analysis methods employed particularly in the biochemical field include gels. Electrophoresis is known.

  In recent years, capillary electrophoresis has been developed to enable high-speed analysis with a small amount of sample. A capillary is a thin glass tube that stores a solvent containing a substance to be measured. Capillary electrophoresis is a method of applying a potential to the solvent in the capillary to cause the substance to be measured to undergo electrophoresis. In electrophoresis in narrow gaps in capillaries, convection is greatly suppressed even if the viscosity of the electrophoresis medium is low, so there is no need to fill the capillaries with gels. Polyacrylamide, polyethylene glycol, dextran If filled with a solution of a synthetic polymer such as pullulan or a natural polymer, they sufficiently function as a molecular sieving medium. In capillary electrophoresis, in order to detect a substance to be measured contained in a solvent in a capillary, for example, light (for example, ultraviolet light) is applied to a part of the capillary (referred to as a light detection unit) and the transmitted light is transmitted. Measure the spectrum of.

  Regarding capillary electrophoresis, for example, Non-Patent Document 1 (Nadako Tadakai, Shinobu Endo, Hiroto Akagawa, Yutaka Itabashi, fatty acid composition analysis by indirect ultraviolet detection capillary electrophoresis, BUNSEKI KAGAKU, Vol. 52, No. 9, pp. 847-850, 2003) includes a plurality of fatty acids [stearic acid (18: 0) (the carbon number is shown on the left side and the number of unsaturated bonds on the right side) contained in vegetable oil or the like using a capillary electrophoresis apparatus. The same applies hereinafter), oleic acid (18: 1), linoleic acid (18: 2), linolenic acid (18: 3), palmitic acid (16: 0), myristic acid (14: 0), etc.] Examination and quantitative results are disclosed for the separation analysis method.

  Non-Patent Document 2 (F. Haddadian, SA Shamsi, And I. M. Warner, Separation of Saturated and Unsaturated Free Fatty Acids Using Capillary Electrophoresis with Indirect Photometric Detection, Journal of Chromatographic Science, Vol. 37, pp. .103-107, April 1999) is a reference document of Non-Patent Document 1, and here, regarding separation of saturated fatty acids and unsaturated fatty acids corresponding to C12-C31, differences in electrophoretic liquid composition and changes in capillary temperature (20 (40 ° C).

  Patent Document 1 (Japanese Patent Laid-Open No. 5-317699) describes a method for producing a silver-supporting silica gel used for chromatography such as HPLC. The separation of linoleic acid (18: 2) and linolenic acid (18: 3) by chromatography using silver-supported silica gel is described. However, the separation of other fatty acids has not been studied.

  Non-Patent Document 3 (Yoshio Murata, Application of Two-Dimensional NMR Method (NOESY) to Elucidation of Micelle Structure of Potassium Oleate in Aqueous Solution, Journal of Science, Fukuoka University, 40, 2, pp.203-216, 2010) Discloses a result of analyzing the state change of potassium oleate according to temperature and concentration using a two-dimensional NMR method. FIG. 14 shows that potassium oleate takes M (micelle region) and A to E (different association states and liquid crystal states, respectively), and further, 20 ° C. from B region to C region, and from C region to D region. It is described that a state change of 50 ° C. is predicted. Also, FIG. 17 shows the result of analysis of the temperature dependence of the rotational correlation time (τc) in the carbon chain of potassium oleate (18: 1), also using the two-dimensional NMR method. Here, it is shown that the structure of the carbon chain of potassium oleate (18: 1), particularly in the vicinity of the double bond, changes greatly in a temperature range of about 30 ° C. to 40 ° C. or more. Thus, Non-Patent Document 3 shows detailed research results on the state change of potassium oleate (18: 1). However, there is no mention of how to use these contents.

Japanese Patent Laid-Open No. 5-317699

Atsuko Tadaumi, Shinobu Endo, Hiroto Akagawa, Yutaka Itabashi, Fatty acid composition analysis by indirect UV detection capillary electrophoresis, BUNSEKI KAGAKU, Vol.52, No.9, pp.847-850, 2003 F. Haddadian, S.H. A. Shamsi, And I. M. Warner, Separation of Saturated and Unsaturated Free Fatty Acids Using Capillary Electrophoresis with Indirect Photometric Detection, Journal of Chromatographic Science, Vol.37, pp.103-107, April 1999 Yoshio Murata, Application of two-dimensional NMR (NOESY) to elucidation of micelle structure of potassium oleate in aqueous solution, Journal of Fukuoka University, 40, 2, pp.203-216, 2010

  Non-Patent Document 1 p. In the right column, line 848, “18: 3 (linolenic acid) and 16: 0 (palmitic acid) were eluted before the difference in mobility occurred, and good separation was not obtained”. . As described above, the separation and analysis method described in Non-Patent Document 1 has a problem that a highly accurate quantitative result cannot be obtained for a component whose mobility is close to that of linolenic acid or palmitic acid.

  In Non-Patent Document 2, the separation of components such as linolenic acid and palmitic acid which are difficult to separate is not studied. Moreover, in patent document 1, isolation | separation of fatty acids other than linoleic acid and linolenic acid is not examined.

  In view of these problems, the present invention provides a separation and analysis method capable of obtaining a highly accurate measurement result that enables separation between components having similar mobility characteristics such as mobility in the separation and analysis of components in a sample. The purpose is to provide.

  The inventors of the present invention have improved resolution by changing the states of components such as linolenic acid (18: 3) and palmitic acid (16: 0) that are close in peak detection time. The headline, the present invention has been reached.

That is, the present invention is a method of separating and analyzing the components by moving a plurality of components contained in a sample and utilizing a difference in the movement characteristics of the plurality of components.
A separation analysis method including a step of causing a state change of a plurality of the components.

  The state change of the component is preferably a three-dimensional state change. Moreover, it is preferable to include the process of heating the said sample. The heating temperature is preferably 30 ° C to 80 ° C. In the separation analysis method of the present invention, it is preferable to use capillary electrophoresis.

Further, the present invention is a method for separating and analyzing the components by moving a plurality of components contained in a sample and utilizing a difference in movement characteristics of the plurality of components,
The present invention also relates to a separation and analysis method including a step of heating the sample so that the difference in the movement characteristics increases.

  The heating temperature is preferably 30 ° C to 80 ° C.

  According to the separation and analysis method of the present invention, it is possible to separate components with close mobility in a sample, and a highly accurate measurement result can be obtained. In particular, in separation analysis of fatty acids in a sample, components having close mobility such as linolenic acid and palmitic acid can be separated, and highly accurate measurement results can be obtained.

It is the schematic which shows an example of a capillary electrophoresis apparatus. 2 is an electropherogram at 20 ° C. of a standard sample of Example 1 (2 ppm each). 2 is an electropherogram at 25 ° C. of a standard sample of Example 1 (2 ppm each). 2 is an electropherogram at 30 ° C. of the standard sample of Example 1 (2 ppm each). 2 is an electropherogram of a standard sample of Example 1 (2 ppm each) at 40 ° C. FIG. 2 is an electropherogram of a standard sample of Example 1 (2 ppm each) at 50 ° C. FIG. 2 is an electropherogram at 55 ° C. of a standard sample of Example 1 (2 ppm each). 2 is an electropherogram of a standard sample of Example 1 (2 ppm each) at 60 ° C. FIG. 2 is an electropherogram of canola oil of Example 2 (5-fold dilution) at 60 ° C. FIG. 2 is an electropherogram of olive oil of Example 2 (5-fold dilution) at 60 ° C. FIG. It is an electropherogram at 60 degreeC of the sesame oil of Example 2 (100 times dilution). FIG. 2 is an electropherogram at 60 ° C. of the oil of Example 2 (100-fold dilution). It is the electropherogram of the butter (20 g / L) of Example 2 at 60 ° C.

  The separation analysis method of the present invention is a method for separating and analyzing (quantitative / qualitative) when a plurality of components (analysis target) are contained in a sample. However, not only when there are a plurality of components (measurement components) in the sample to be measured directly, but one type of measurement component and other components (contaminants) whose movement characteristics in separation analysis are similar to this component The separation analysis method of the present invention can also be applied to the case where it is necessary to separate and analyze the measurement component and the contaminants.

  The separation analysis method of the present invention is a method for separating and analyzing components by moving a plurality of components contained in a sample and utilizing the difference in movement characteristics of the plurality of components (hereinafter referred to as “flow separation analysis method”). And can be applied to various known separation analysis methods.

  The component in the sample is not particularly limited as long as it is a compound that causes a difference in transfer characteristics between components so that it can be separated by flow separation analysis. As this component, an organic compound is mentioned, for example, A fatty acid is mentioned as an organic compound. Note that the present invention is particularly useful when the measurement sample contains a plurality of components whose movement characteristics in the flow separation analysis method approximate. Examples of the plurality of components having similar mobility characteristics in the flow separation analysis method include linolenic acid and palmitic acid among fatty acids.

  Specific examples of the separation analysis method include high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), gel electrophoresis, and capillary electrophoresis. Among these, it is particularly preferable to use capillary electrophoresis. That is, it is preferable to use a capillary electrophoresis apparatus as the analyzer.

  FIG. 1 is a schematic diagram illustrating an example of a capillary electrophoresis apparatus. The capillary electrophoresis apparatus shown in FIG. 1 includes a capillary 1. The inner diameter of the capillary 1 is, for example, 100 μm or less. Many portions of the capillary 1 are accommodated in the chamber 11, and the inside of the chamber 11 can be heated by the heating means 12. The heating means 12 is, for example, a temperature control device that can circulate in the chamber 11 using warmed air or liquid as a medium.

  Both ends of the capillary 1 are in communication with liquids including measurement samples, electrophoresis liquid, washing liquid and the like in the vials 21 and 22, and the capillary 1 is also filled with the same liquid. The vials 21 and 22 are set in an auto sampler (not shown) or the like, and are sequentially replaced with vials containing a measurement sample and the like.

  Moreover, the power supply 4 is provided and an electric field is applied between both ends of a capillary using this at the time of a measurement. Thereby, the component in the sample in the capillary 1 moves, and the component can be separated according to the difference in mobility of each component.

  Further, as an optical system for detecting separated components, a light source 31 (D2 lamp, etc.) and a detector 32 (diode array, photodiode, etc.) for measuring the absorbance of light emitted from the light source 31 and the like. ). For example, the light source 31 irradiates a part of the capillary 1 with predetermined light, and the light transmitted through the capillary 1 is detected by the detector 32. Data obtained by the irradiated detector 32 (such as an electropherogram indicating time-series absorbance) is sent to the data arithmetic processing unit 5 (such as a personal computer) for analysis of the measurement results. Note that the data arithmetic processing device 5 may include an instruction function for performing hardware operations. The detector of the capillary electrophoresis apparatus is not limited to an optical system, and a mass spectrometer, a non-contact type conductivity detector, a fluorescence detector, and the like can also be used.

  The “movement characteristic” of a component is, for example, the movement speed of the component in various chromatographic methods or the mobility of the component in capillary electrophoresis. In capillary electrophoresis, separation of components in a sample is basically performed using the principle of electrophoresis. In this case, the mobility Ue of the sample component can be expressed as follows.

Ue = q / 6πηr
(Ue: mobility, q: charge, η: viscosity of solvent, r: Stokes radius)
In order to improve the resolution, it is necessary to increase the difference in Ue between the measurement components. From the above equation, the difference in q (charge) or r (Stokes radius) between the measurement components increases. It can be seen that when the measurement is performed under various electrophoresis conditions, the difference in Ue increases and the resolution improves.

  However, for example, when the measurement sample is a mixture of a plurality of types of fatty acids, the functional group having the possibility of dissociating and having a charge (q) is usually only one carboxyl group in the fatty acids, Since there are many similar fatty acids with respect to the number of carbon chains related to the Stokes radius (r), it is often difficult to separate fatty acids having a slight difference in r. In the present invention, even for a plurality of components that have such a small difference in r that cannot be separated as they are, the structural state of the component itself is changed, and the difference in mobility between components (r It is considered that separation and analysis of these components becomes possible by increasing the difference.

  The separation analysis method of the present invention is characterized in that components are separated by utilizing a change in the state of the components contained in the sample. Here, the “component state change” is preferably a three-dimensional state change. The “three-dimensional state change” is specifically a change in the three-dimensional structure of the measurement component. One index of this three-dimensional state change is the rotational correlation time (τc) disclosed in Non-Patent Document 3. That is, when the rotation correlation time (τc) for a certain component changes, it is considered that a three-dimensional state change has occurred. In addition, it is preferable that the state change in this invention is a state change which changes the Stokes radius (r) of the measurement component mentioned later, and enlarges the difference of the mobility (Ue) between each component.

  As a suitable means for causing the above “change in the state of components”, heating a sample containing each component may be mentioned. Therefore, the separation analysis method of the present invention preferably includes a step of heating the sample. In this step, the state of the measurement component contained in the sample is changed by heating the sample. For example, after the sample is heated, if the rotational correlation time (τc) for a certain component is longer than that at room temperature, the component state change (three-dimensional state change) occurs due to heating. Can be judged. In addition, the method of causing the change in the state of the component is not limited to the method of heating. For example, in the case of a separation analysis method in a liquid phase (capillary electrophoresis, HPLC, etc.), the pH of the liquid phase is adjusted. It is also possible to use a method, a method of adding any compound in the liquid phase, and the like.

  The specific method of heating is not particularly limited as long as the sample (measurement component in the sample) reaches a predetermined temperature. For example, when a capillary electrophoresis apparatus is used, the heating is performed using a capillary. A method of heating an ambient atmosphere (such as air in a chamber in which a capillary is accommodated) to a predetermined temperature can be used. In addition, heating may be performed during measurement (for example, in capillary electrophoresis, while a charge is applied to the capillary), may be performed before measurement, or performed before and during measurement. Also good.

  The heating temperature is preferably 30 to 80 ° C, more preferably 40 to 70 ° C, and most preferably 55 to 65 ° C. In the range of 30 to 80 ° C., separation and analysis of organic compounds (for example, linolenic acid and palmitic acid among fatty acids) having similar mobility characteristics in the flow separation analysis method are possible, and in the range of 55 to 65 ° C. Enables more accurate separation and analysis of organic compounds with similar mobility characteristics in flow separation analysis. The heating temperature here is the temperature of the sample during heating, and is usually the temperature when a certain steady state is reached. However, in practice, for example, the temperature of the sample can be adjusted by adjusting the temperature of the atmosphere around the capillary of the capillary electrophoresis apparatus (such as air in the chamber containing the capillary) or the temperature of the atmosphere around the HPLC column. Is adjusted.

  By heating the sample, for example, in the case of capillary electrophoresis, the temperature of the electrophoresis solution filled in the capillary and the temperature of the sample are increased, and the viscosity thereof is reduced, thereby shortening the analysis time. The advantage that is possible is also obtained.

  In addition, when the separation and analysis method of the present invention is applied to capillary electrophoresis, there is an advantage that laborious pretreatment such as derivatization of sample components, which is necessary in conventional HPLC and GC analysis methods, is unnecessary. can get.

Example 1
In this example, using a capillary electrophoresis apparatus (Agilent 7100 capillary electrophoresis system) as shown in FIG. 1 described above, six types of fatty acids (stearic acid (18: 0), oleic acid (18: 1), About standard samples (manufactured by Tokyo Chemical Industry Co., Ltd.) each containing 2 ppm of linoleic acid (18: 2), linolenic acid (18: 3), palmitic acid (16: 0), myristic acid (14: 0)) Separation analysis was performed by changing the temperature conditions.

(Method of operation)
In this example, the following operations (1) to (6) were performed in this order.
(1) Set the capillary.
(2) Set a vial containing the electrophoresis solution.
(3) Set a vial containing the standard sample.
(4) Turn on the capillary electrophoresis device and the personal computer (data processing unit having an instruction function).
(5) Start up the PC software and program the following analysis conditions into the dedicated software.

1. Capillary temperature control temperature: 20, 25, 30, 40, 50, 55 or 60 ° C
2. Filling capillary with electrophoresis solution: Flash, 300 sec
3. Sample injection into the capillary: 50 mbar, 4 sec
4). Detection wavelength: 254 nm (reference wavelength: 400 nm)
5. Applied voltage: 25 kV, measurement time: 20 min
(6) Start the measurement by executing dedicated software.

  As the capillary, a capillary made of fused silica (inner diameter 50 μm, total length 64.5 cm, effective length 56 cm) was used. The total length is the entire length of the capillary, and the effective length is the length from the sample inlet to the detection portion.

  As the electrophoresis solution, N-methylformamide / dioxane / distilled water (4: 6: 1, v / v / v), 2.5 mM adenosine-1-phosphate, 50 mM Tris (2-amino-2- (hydroxymethyl) ) -1,3-propanediol) was used.

  As a sample solvent, N-methylformamide / dioxane (4: 1, v / v) was used, and a sample diluted and dissolved with the solvent was filled in a vial.

  In the detection system, a D2 lamp was used as the light source, and a diode array was used as the detector. The detection wavelength was set to 254 nm as described above. In addition, assuming that the reference wavelength is 400 nm, the absorbance at 400 nm is subtracted from the absorbance at 254 nm, which is the maximum absorption wavelength of adenosine-1-phosphate, so that the electropherogram peak is displayed as a positive peak. .

  The conditions for the electrophoresis solution used for the analysis, the solvent of the sample, the detection wavelength, the applied voltage and the like were set in substantially the same manner as in Non-Patent Document 1. Further, separation analysis was performed for each fatty acid in the standard sample by changing the temperature of the capillary (sample) heating within a range of 20 to 60 ° C. The measurement results at each temperature are shown in FIGS.

  In this example, when the temperature was 20 ° C. and 25 ° C. (FIGS. 2 and 3), the separation of linolenic acid and palmitic acid was insufficient, as in the results of Non-Patent Document 1, and all fatty acids The species could not be separated and analyzed simultaneously. On the other hand, when the temperature was 30 ° C. or higher (FIGS. 4 to 8), the peaks of linolenic acid and palmitic acid were separable, and all fatty acids could be separated and analyzed simultaneously. Furthermore, when the temperature is 55 ° C. and 60 ° C. (FIGS. 7 and 8), the peaks of linolenic acid and palmitic acid are almost completely separated, and therefore, more accurate analysis of these fatty acids is possible. it is conceivable that. In addition, the analysis time can be shortened compared to Non-Patent Document 1.

(Example 2)
Measurement of fatty acids (separation) for five types of samples (canola oil, olive oil, sesame oil, lard oil, butter) in the same manner as in Example 1 except that the temperature of the capillary (sample) was fixed at 60 ° C. Analysis was carried out. The sample used was diluted or dissolved with a solvent in the same manner as in Example 1. The measurement results for each sample are shown in FIG. 9: canola oil (5 times dilution), FIG. 10: olive oil (5 times dilution), FIG. 11: sesame oil (10 times dilution), FIG. 12: rar oil (100 times dilution), and FIG. 13: Shown in butter (20 g / L).

  From the results shown in FIGS. 9 to 13 (particularly FIG. 9), it is understood that separation and analysis are possible even for components that are difficult to separate, such as linolenic acid and palmitic acid, by the separation analysis method of the present invention.

  Table 1 summarizes the types of fatty acids (qualitative results) detected for each sample. From the results shown in Table 1, the fatty acids detected for each sample are listed in the “Fifth Amendment Japanese Food Standard Composition Table Fatty Acid Composition Table” published on the Ministry of Education, Culture, Sports, Science and Technology website (URL: http: //www.mext .go.jp / b_menu / shingi / gijyutu / gijyutu3 / houkoku / 1299179.htm), it was confirmed that it was consistent with the main components in each sample.

  It should be understood that the embodiments and examples disclosed herein are illustrative and non-restrictive in every respect. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

  1 Capillary, 11 Chamber, 12 Heating means, 21, 22 Vials, 31 Light source, 32 Detector, 4 Power supply, 5 Data processing unit.

Claims (6)

By moving a plurality of components (excluding DNA) contained in a sample by capillary electrophoresis using a capillary (however, the electrophoresis polymer is not included in the capillary), the difference in the movement characteristics of the plurality of components A method for separating and analyzing the components using
Heating the capillary to warm the sample;
A separation analysis method comprising a step of causing a state change of a plurality of the components.   The separation analysis method according to claim 1, wherein the component is an organic compound (excluding DNA).   The separation analysis method according to claim 1 or 2, wherein the state change of the component is a three-dimensional state change. The separation analysis method according to any one of claims 1 to 3 , wherein the heating temperature is 30 ° C to 80 ° C. By moving a plurality of components (excluding DNA) contained in a sample by capillary electrophoresis using a capillary (however, the electrophoresis polymer is not included in the capillary), the difference in the movement characteristics of the plurality of components A method for separating and analyzing the components using
A separation analysis method comprising a step of heating the capillary and heating the sample so that the difference in movement characteristics increases. The separation analysis method according to claim 5 , wherein the heating temperature is 30 ° C. to 80 ° C.