JP2010112960A - Liquid chromatography apparatus and analysis program - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To reduce an analysis cycle time. <P>SOLUTION: In a liquid chromatograph analysis, the cycle time from current sample injection to next sample injection is fixed. A sample is injected, before a column is equilibrated by an eluting solution. Since the timing, when the eluting solution is switched in each injection/analysis cycle and the timing, when the sample is injected are synchronized, the properties for comparatively and reproducibly solving the hold time of each component peak is applied, even though the column does not necessarily become equilibriated. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a liquid chromatographic analysis method.

  As an example of the liquid chromatograph apparatus, a high-speed amino acid analyzer will be described. Amino acid analyzers can be broadly classified into standard analysis methods that target about 20 amino acids of protein hydrolysates and biological fluid analysis methods that target about 40 or more components of biological fluid amino acids. it can.

  Examples of biological fluid analysis methods include JP-A-53-60291, JP-A-59-10849, JP-A-4-194570, JP-A-9-80037, JP-A-2002-71660. There is. The report includes Journal of Chromatography, 224; 315-321 (1981) “Resolution of 52 ninhydrin-positive compounds with a High-speed amino acid analyzer”, Clinical Chemistry 43; 8, 1421-1428 (1997) “Amino” Acid determination in biological fluids by automated ion-exchange chromatography: performance of Hitachi L-8500A ”.

JP-A-53-60291 JP 59-10849 A JP-A-4-194570 Japanese Patent Laid-Open No. 9-80037 JP 2002-71660 A

Journal of Chromatography, 224; 315-321 (1981) “Resolution of 52 ninhydrin-positive compounds with a High-speed amino acid analyzer” Clinical Chemistry 43; 8, 1421-1428 (1997) “Amino Acid determination in biological fluids by automated ion-exchange chromatography: performance of Hitachi L-8500A”

  In liquid chromatographic analysis, high speed analysis is an important issue. In general, in liquid chromatography, a column is sufficiently equilibrated with a first eluent, and then a sample is injected to obtain a chromatogram. This is because when the column is not sufficiently equilibrated, the retention time of each component becomes unstable, and as a result, good quantitative reproducibility cannot be obtained.

  The present invention relates to shortening of analysis cycle time, fast connection of two different analysis method programs, and the like.

  The present invention relates to injecting a sample before equilibrating a column with an eluent in a liquid chromatographic analysis, with a constant cycle time from sample injection to the next sample injection. By synchronizing the eluent switching timing and sample injection timing in each injection analysis cycle, we applied the property that the retention time of each component peak is eluted with relatively reproducibility even if the column does not necessarily reach equilibrium. Is.

  Preferably, in order to prevent the retention time from becoming unstable, the eluent switching timing of the stepwise elution method or gradient elution method is set to a constant interval, and the sample injection interval is synchronized with the eluent switching timing of the elution method. Let

  Preferably, in the connection of two different analysis method programs, the end of the program to be executed before and the leading end element of the program to be executed later are taken up to create the fastest connection intermediate program. The intermediate program for the fastest connection ends the previous execution program when the previous execution program elutes the final peak to be analyzed. Immediately, the tail part of the backward program is cut out and sent. The intermediate program for fastest connection is inserted between the end of interruption of the front program and the cut-out program at the end of the rear program.

  Reducing the analysis cycle time, even if the column is not sufficiently replaced with the first eluent, the retention time of each component is reproduced by repeating the degree of eluent replacement by making the switching time constant. Good properties can be obtained. If good retention time reproducibility is obtained, as a result, the eluent switching and the injection timing synchronization have an effect of obtaining good quantitative reproducibility.

  For the connection of two different analysis method programs, the fastest analysis method transition can be realized by efficiently terminating the forward program and starting the end of the backward program.

FIG. Chromatogram obtained by the PFS-94 method. Chromatogram obtained by the PFS-75 method. Analysis program A. Analysis program B. Example of using the fastest connection intermediate program. Fastest connection intermediate program.

  The above and other novel features and effects of the present invention will be described below with reference to the drawings.

  FIG. 1 is an apparatus configuration and flow path explanatory diagram of the amino acid analyzer of this example. 1 to 4 are first to fourth buffer solutions, and 5 is a column regeneration solution. Among these, a buffer solution is selected by the solenoid valve series 6, and the amino acid sample introduced by the ammonia filter column 8 and the autosampler 9 is separated by the separation column 10 by the buffer solution pump 7. The separated amino acids are mixed with the ninhydrin reagent 11 sent by the ninhydrin pump 12 by the mixer 13 and reacted with the heated reaction column 14. The amino acid (Luemann purple) colored by the reaction is continuously detected by the detector 15 and is output as a chromatogram and data by the data processor 16 and recorded and stored.

(Reducing analysis cycle time)
In the conventional amino acid analysis using ion exchange chromatography, an ion exchange column is washed with about 0.1 mol / L of sodium hydroxide or lithium hydroxide regeneration solution, and then sodium citrate or lithium citrate eluent. For a sufficient period of time to equilibrate the column. The so-called column equilibration liquid amount from the regenerating solution to the eluent before the sample is injected is 9.5 ml (flow rate: 0.56 ml / min, flow rate) in the conventional PF-94 method as shown in Table 1. Time: 17 minutes). The column has an inner diameter of 5.4 mm and a length of 50 mm, and has a volume of 1.1 ml. When the equilibration liquid amount is normalized by the column volume, it becomes a conversion in which 8.4 times the column volume is fed. In the case of an ion exchange resin, since the eluent penetrates into the packing material, the porosity in the column is not considered. When the regenerant in the column container is replaced by feeding the eluent, in the model in which the sent eluent is quickly uniform in the column container, the regenerant is in proportion to the power of the natural logarithm base e. Remains. In other words, in the above-described example, if 8.4 times the column volume is fed, it is calculated that only e ^−8.4 = 0.0000225 = 0.0225% of the regenerated solution remains in the column.

  In this embodiment, it is important how much the column equilibration liquid can be reduced by allowing the fluctuation and variation of the retention time and peak area to the limit as compared with the conventional column volume of 7 times. .

  The PFS-75 method, which is an amino acid analysis method of this example, is compared with the PFS-94 method, which is an amino acid analysis method equipped with a conventional technique. In the PFS-94 method and the PFS-75 method, the analysis cycle time is 94 minutes and 75 minutes, respectively, and only the column regeneration solution feeding time and the equilibration time by the first buffer solution are different. Regarding the above analysis method and the conventional protein hydrolyzate analysis method (hereinafter referred to as PH) and biological fluid analysis method (hereinafter referred to as PF), the remaining ratio of the regenerated solution when the liquid in the column is replaced with the first buffer solution. Is shown in Table 1. When the volume in the column is V (mL) and the volume of the first buffer solution sent from the column regeneration to the next sample injection is VRI (mL), the remaining ratio R of the regeneration solution in the column R ( %) Is expressed by Equation 1. Table 2 shows the lithium ion concentration, pH, and the like of the buffer solution used in the PFS-75 method and the PFS-94 method.

  From Table 1, the regenerated liquid remaining rate R in the analytical method of this example is 600 times the regenerated liquid remaining rate R in the conventional PF method, and the regenerated liquid remaining rate R of the analytical method of this example has so far been This is far greater than any other analysis method. In other words, conventionally, the liquid in the column is sufficiently replaced with the first buffer, and the sample is injected after the column is equilibrated. On the other hand, in this embodiment, a method of injecting the sample in a state where the liquid replacement is insufficient and the regenerated solution of 1% order remains in the column is adopted. In other words, in the conventional method, it is considered that good holding time reproducibility was obtained by injecting the sample after sufficient column equilibration. However, in this example, it can be said that priority was given to high-speed analysis even if the reproducibility was slightly reduced, and the sample injection method was adopted despite the state where the column equilibration was slightly insufficient. It can be seen from Equation 1 that when R is 1%, the VRI / V ratio is 4.6.

  PFS-94 has the same analysis program from the first buffer solution to the fourth buffer solution, but the program for the portion that sends the column regeneration solution and the first buffer solution for equilibration is different. PFS-94 and PFS-75 are analysis methods in which the required time per analysis is 94 minutes and 75 minutes, respectively.

  FIGS. 2 and 3 compare the chromatograms of the first injection analysis and the second injection analysis for the PFS-94 method and the PFS-75 method, respectively, when analysis is started without RG start using each analysis method. It is a thing. In the PFS-75 method of FIG. 3, the retention times of the components do not match in the chromatograms of the first injection analysis and the second injection analysis, but in the PFS-94 method of FIG. 2, the retention times match.

  Conventionally, the column has been sufficiently equilibrated so that peaks can be identified in the same time window, that is, the retention times of each component are matched as much as possible in the first injection analysis and the second injection analysis. In the PFS-94 method, regarding the peak identification during the cycle, the VRI / V ratio was 8.4 and the regenerated solution remaining rate was 0.0225%, which was sufficiently equilibrated as before and the retention times were matched. This time, another method of matching the retention time was examined without being bound by the conventional method of sufficiently equilibrating the column with the first buffer after the first injection analysis.

  This time, regardless of the column equilibration time, even if the residual ratio of the regenerated solution is 1% or more, that is, the VRI / V ratio is 4.6 or less, the injection interval cycle is made constant. I found a way to match the retention time. Since the injection interval can be shortened without sufficiently feeding the first buffer solution, it is possible to speed up the analysis. If the data in the case of RG start in which the analysis is started first from the column regeneration (RG) step is always used, the retention time of the second injection chromatogram in FIG. 3 can be obtained. The analysis cycle can be speeded up by using RG start or using data from the second and subsequent injection analyses. In this example, although the PFS-75 method had a VRI / V ratio of 2.5 (see Table 1), the retention times in the first injection analysis and the second injection analysis could be matched. .

  Also in the protein hydrolyzate (PH) method, the VRI / V ratio is as large as 6.8 (see Table 1) as before.

(Regarding the connection of two different analytical method programs)
The analysis program consists of an elution step that separates the components adsorbed on the column, a column regeneration step that regenerates the column with a strong alkali regeneration solution, and a column equilibration step that equilibrates the column with a first buffer solution for subsequent analysis. Consists of The analysis program also specifies the pump flow rate, column oven temperature, etc. along the time axis.

  Here, consider the case of continuous analysis using two types of analysis programs. As an example of two types of analysis programs, an analysis program A (FIG. 4) and an analysis program B (FIG. 5) are taken as examples. RG start is effective for efficiently performing continuous component analysis in a sample using the same analysis program. The RG start is a method for eliminating the difference between the retention time of the component peak in the chromatogram obtained by the first analysis and the retention time of the component peak in the chromatogram obtained after the second analysis.

  However, when analyzing the components in the sample using the analysis program B after the analysis program A, if the analysis program B is executed subsequently to the analysis program A, the chromatogram obtained in the first analysis of the analysis program B The reproducibility of the retention time of the component peak and the retention time of the component peak in the chromatogram obtained after the second analysis is lowered. By interrupting the program immediately after elution of arginine (Arg), which is the final component of analysis program A, and inserting the RG start dedicated to analysis program B without carrying out the regenerative liquid feeding step by analysis program A This phenomenon can be avoided. However, the following problems occur at the joint between the RG start for the analysis program A and the analysis program B.

(1) When the set value of the pump flow rate is not equal between the two analysis programs, pressure fluctuation occurs at the joint of the analysis programs. This has a negative effect on the column life. (2) If the temperature setting value of the column oven does not match at the joint of the analysis program, the reproducibility of the amount of enthalpy received by the column from the outside of the column decreases, so the reproducibility of the component peak retention time decreases.

  In order to solve these problems, as shown in FIG. 6, a connection program that smoothly connects both programs is inserted at the joint of the programs. FIG. 7 shows the fastest connection intermediate program corresponding to the analysis program A and the analysis program B shown in FIGS. In the intermediate program for fastest connection shown in FIG. 7, the column oven temperature value was set to be the first column oven temperature in the RG step of the analysis program B. Regarding the flow rate setting value of the pump, the setting value of the lowest stage of the analysis program A and the setting of the first stage of the RG process of the analysis program B are smoothly connected. The flow rate setting value of the pump 2 was connected from 0.400 mL / min to 0.350 mL / min by a linear gradient to 0.400 mL / min. By inserting this connection program, before the RG of the analysis program B is executed, the pump flow rate and the column oven temperature are changed to the pump flow rate setting value and the column oven temperature setting value at the first stage of the RG process of the analysis program B. By linearly connecting using a linear gradient program and RG-starting the analysis program B in a normal state, continuous analysis can be performed with good reproducibility.

  DESCRIPTION OF SYMBOLS 1 ... 1st buffer solution, 2 ... 2nd buffer solution, 3 ... 3rd buffer solution, 4 ... 4th buffer solution, 5 ... Column regeneration liquid, 6 ... Solenoid valve series, 7 ... Buffer solution pump, 8 ... Ammonia filter Column: 9 ... Autosampler, 10 ... Amino acid sample is separation column, 11 ... Ninhydrin reagent, 12 ... Ninhydrin pump, 13 ... Mixer, 14 ... Reaction column, 15 ... Detector, 16 ... Data processing device.

Claims (2)

A liquid chromatograph apparatus comprising:
Gradient elution program for connection including the components of the first gradient elution program and second gradient elution program is included in the connection gradient elution program inserted during the transition from the first gradient elution program to the second gradient elution program. A liquid chromatograph apparatus characterized by that. An analysis program for use in a liquid chromatograph device,
A gradient elution program for connection to be inserted during the transition from the first gradient elution program to the second gradient elution program;
A program for analysis including a gradient elution program for connection including components of each of the first gradient elution program and the second gradient elution program.

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