JPS6270755A - Method for examining concentration of solvent for liquid chromatography - Google Patents

Abstract

PURPOSE:To accurately calculate the concn. of a solvent to be examined, by calculating a calibration curve of solvent concn. and a holding time with respect to a solvent having the same component as the solvent to be examined but known concn. different from that of said solvent to be examined and comparing the same with the holding time of the solvent to be examined. CONSTITUTION:For example, a water-methanol mixture is used as a solvent to be examined. At first, solvents of which the methanol concns. are different over several stages are prepared and used as the mobile phase of liquid chromatography and a sample is injected therein to measure the hold times of said solvents. Then, the calibration curves between solvent concns. and teh holding times are calculated. Further, the sample is injected in the water- methanol solvent mixture being the solvent to be examined and the holding time of said solvent is measured by the liquid chromatography and the holding time of the solvent to be examined is compared with the calibration curves to calculate concn. Because the concn. is calculated from the holding time of the solvent to be examined by the calibration curve of each solvent having known concn., the concn. can be rapidly calculated with high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for testing the concentration of a solvent for liquid chromatography, and more particularly to a method for accurately, quickly and easily testing the concentration of a solvent used as a moving layer in liquid chromatography.

[Prior Art] Liquid chromatography (hereinafter sometimes abbreviated as rLCJ) is used for the purpose of separating and purifying soluble substances, and is used for separation and purification on an industrial scale in fields such as pharmaceuticals, foods, and fine chemistry. Applied to refining. Separation by LC
The purification method involves introducing the sample, developer, and eluent from one end of a column filled with an adsorbent, developing and separating the target component from other components or impurities, and detecting a portion of the effluent from the other end of the column. At the same time, the target component and other components or impurities are sequentially recovered.

In LC, alcohol solutions such as methanol and ethanol are available as solvents used as developing solutions or eluents, that is, as moving layers in LC, but LC solvents have a tendency to retain the sample +I range 11!1 depending on the solvent concentration.
＋ Iζ -phase KO1-1- 目 ＾h 正” けへ し φ h knee くんね - The time it takes for the brain to become conceivable is greatly affected. For this reason, in separation and purification by LC, if the concentration of the solvent used is not accurately known in advance, an error will occur in the retention time of the sample, making it impossible to perform separation laboratory purification with a high recovery rate.

Conventionally, methods for verifying the concentration of solvents generally include gas chromatography, specific gravity measurement, viscosity measurement, or U.S.
In addition to the V measurement method, the Karl Fischer method has been proposed for measuring the concentration of water.

[Problems to be Solved by the Invention] However, in the gas chromatography method, (1) It takes about half a day to age the column, so it takes time to prepare for analysis.

@ When creating a calibration curve using samples with known concentrations, the peak area or peak height is used, so the accuracy is affected by the error in the sample loading amount, resulting in a lack of accuracy.

There are drawbacks such as. In addition, specific gravity measurement, viscosity measurement or U
The method using VK11l has a problem in terms of accuracy.

Furthermore, since the Karl Fischer method is a method for measuring the absolute amount of water, it is prone to errors due to sample preparation and loading amount, making it difficult to determine Δ11 with high accuracy, and the analysis time is long. be.

As described above, all of the conventional solvent concentration verification methods have problems in analysis time, analysis operations, accuracy, etc., but in industrial-level separation and purification using LC, especially large amounts of solvent are handled, and Since precise fine-tuning of the concentration is required, there has been a strong desire for a method that can accurately, quickly, and easily detect the concentration of an LC solvent.

[Means for Solving the Problems] The present invention has been made to solve the above-mentioned conventional problems and to accurately, quickly and easily measure the concentration of a solvent for LC. In the method of verifying concentration, ■ A sample is loaded into a liquid chromatography system using a moving phase of each solvent of known different concentration, which has the same components as the test solvent, and its retention time is measured, thereby determining the solvent concentration. The measurement in step [2] includes a step of calculating a calibration curve with the retention time, and a step of injecting the sample into liquid chromatography using the test solvent as a moving phase and measuring the retention time. The gist of the present invention is a method for testing the concentration of a solvent for liquid chromatography, which is characterized in that the concentration of the solvent to be tested is determined from the results and the calibration curve of step [1].

The present invention will be explained in detail below.

First, the step [1] of the present invention will be explained.

Generally, as a solvent for LC, hexane, benzene, chloroform, ether, alcohol, water, or a mixed solvent thereof, such as a water-methanol mixed solvent, is used. Prepare multiple types of solvents (hereinafter sometimes referred to as "standard solvents") that have the same components as the test solvent but have different concentrations. It is preferable to prepare a wide variety of standard solvents at electrification intervals, for example, at intervals of about 5% by weight.

Next, an LC using each of the prepared standard solvents as a moving phase is created. In this case, there are no particular restrictions on the column shape, and there are no particular restrictions on the LC stationary phase, which may be selected appropriately depending on the sample, solvent, etc. used, but examples include alumina, silica, magnesia, clay, cellulose, carbon, etc. , porous polymer, etc.

Next, input the samples into each of these LCs.
There are no particular restrictions on the sample to be used, but one should be one that is compatible with the test solvent and allows for accurate measurement of retention time, that is, one in which the peak of the sample in the LC effluent appears sharp and clear. is preferable.

Then, from the measured retention time and the concentration of the standard solvent,
A calibration curve is created on coordinates consisting of two axes: solvent concentration and retention time.

Next, step (2) will be explained.

Create an LC. In this case, the model of LC, type of stationary phase, and size should be the same as the LC prepared in step ①.Next, load the same sample used in step ① into this LC, and hold it for a long time. Measure.

By comparing the measured value of this retention time with the calibration curve obtained in step (2), the concentration of the test solvent can be determined.

[Effect] Generally, in LC, a slight change in the solvent concentration of the moving phase has a significant effect on the retention time of the sample. In other words, the binding between the sample and the stationary phase in LC is mostly due to hydrophobic interactions. The higher the concentration, that is, the higher the concentration of the hydrophobic component of the LC solvent, the easier the sample will be washed out by the solvent, and the retention time will be shorter.

For example, when using a column packed with C6 as the stationary phase and loading biphenyl as a sample onto the column with a mobile phase of water-methanol solvent, if the concentration of methanol in the solvent is high, the retention of biphenyl will decrease. becomes shorter, and at lower methanol concentrations, the retention time of biphenyl becomes longer.

The present invention makes use of the phenomenon that the retention time of a sample varies depending on the concentration of the solvent, selects an LC and sample suitable for the solvent to be tested, performs LC analysis, and calculates the retention time of the sample and the solvent. A calibration curve is created between the concentration of the test solvent and the concentration corresponding to the retention time measured by the test solvent is determined from this calibration curve, and the concentration of the test solvent can be determined very easily.

[Example] Examples will be described below.

Example 1 An experiment was conducted to verify the concentration of a two-part water-methanol mixed solvent.

The liquid chromatography device used is - Shimadzu LC4A (with autosampler) analytical column (
6 mm in diameter and 50 mm in length) and filled with Ca filler as a stationary phase. In addition, Tokyo Rika Kikai ■ can be used as a constant temperature bath.
UA-100 was used. Furthermore, methanol aqueous solutions with methanol concentrations of 67.5% by weight, 70% by weight, and 72.5% by weight were used as standard solvents, and samples in which uracil and biphenyl were dissolved in methanol were used.

<Creating a calibration curve> First, at a constant temperature of 35°C, after purging the column with a 67.5% methanol aqueous solution, the sample was injected and developed using this aqueous solution as the moving phase, and the retention time of the sample was determined. I asked for it. In this case, uracil is not retained at C8, so if uracil elution time 11D is to and biphenyl elution time t and 3, then biphenyl retention time t
R is determined by t R = t B t U .

Similar operation was carried out with 70% methanol aqueous solution and 72.5%
It was also carried out for a methanol aqueous solution of 1% by weight.
L h M 3-t (F Iv 7J
1+Zl-/'-1-Il, s-sorghum 1ta was determined.

The relationship between the measured tR and methanol concentration was plotted to obtain a calibration curve as shown in FIG.

<Concentration test of detected solvent> A methanol aqueous solution with a concentration of 69.4% by weight was prepared from high purity methanol and water.

Using this aqueous solution as a mobile phase at the same temperature as when creating the calibration curve, an LC column was created in the same way, a sample was loaded and developed, and the retention time tR of biphenyl was determined; tll = 1. It was 8 minutes.

Using the calibration curve in Figure 1, we interpolated the solvent concentration corresponding to a retention time of 1.8 minutes, and found that the methanol concentration was 6 from the intersection point.
It was determined that the content was 9.4%.

As a result, it is clear that according to the method of the present invention, the concentration of the test solvent can be determined accurately, quickly, and easily.

[Effect of the invention] As detailed above, the uninvented LC...9F of Kasuri b9! ! According to the hemp assay method, the concentration of the LC solvent can be determined accurately, quickly, and easily.

Therefore, by employing the method of the present invention, the measurement time can be shortened, and the measurement operation can be simplified because high technique and precision are not required for sample implantation, etc. Furthermore, LC such as gas chromatography equipment
Since no equipment other than the device is required, it is possible to reduce equipment costs and is extremely advantageous in terms of cost.

[Brief explanation of drawings]

FIG. 1 is a graph showing the calibration curve obtained in Example 1. Agent Patent Attorney Tsuyoshi Shigeno Figure 1 Methanol concentration (!% by weight) Procedural amendment November 25, 1985 To the Commissioner of the Japan Patent Office Vba1, l
Display of 415 items 1985 #Permit No. 211760 2 Name of IJJ Concentration verification method of solvent for liquid chromatography 3 Person making amendment 4 Relationship with item Patent applicant name Name (106) Kurita Water Industries, Ltd. 4 Agent address: 4-8-19 Akasaka, Minato-ku, Tokyo 107
Akasaka Omotemachi Building No. 502 No. 6 Claims column and Detailed Description of the Invention column of the specification subject to amendment. 7. Contents of amendment (1) The scope of claims is corrected as shown in the attached sheet. (2) Lines 3 and 17 of page 2 of the specification, line 4 of page 4, line 4 of page 5, line 3 and bottom of page 6 of the specification, line 10 of page 7 of the specification , line 15 and bottom line, page 9, number 11
line and page 10, line 8 of the same page, "mobile phase" is corrected to read "r mobile phase" and "1, respectively." The above attached claims (]) Method for assaying the concentration of a solvent for liquid chromatography In this case, ■ Inject the sample into a liquid chromatography system using L&J as each solvent with the same component as the test solvent and at a known different concentration, measure the retention time, and use this to create a calibration curve between solvent concentration and retention time. (1) Injecting the sample into a liquid chromatography system using the solvent to be tested and measuring its retention time; A method for testing the concentration of a liquid chromatography solvent, characterized by determining the concentration of the solvent to be tested from a calibration curve.

Claims (1)

[Claims] (1) In a method for verifying the concentration of a liquid chromatography solvent, [1] A sample is introduced into a liquid chromatography system in which each of the solvents having the same components as the test solvent and having different known concentrations is used as a moving phase. A step of measuring the retention time and thereby obtaining a calibration curve between solvent concentration and retention time; [2] A step of injecting the sample into liquid chromatography using the test solvent as a moving phase and measuring the retention time. , A method for testing the concentration of a solvent for liquid chromatography, characterized in that the concentration of the solvent to be tested is determined from the measurement results of step [2] and the calibration curve of step [1].