JPH01503805A - How to determine the composition of substances in microcolumn liquid chromatography - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] How to determine the composition of substances in microcolumn liquid chromatography Technical field The present invention is useful in analytical chemistry, particularly in methods for the analysis of complex mixtures of substances dissolved in fluid media. Regarding, and in more detail, substances by microcolumn liquid chromatography Concerning a method for determining the composition of

Conventional technology An application for determining the composition of substances in microcolumn liquid chromatography. According to methods known in the industry, a sample of the substance is chromatographed along with an eluent. is pumped through the rough microcolumn, while at its outlet the eluent and Both quantitative and qualitative analyzes of the solution containing the components of the test sample are performed; A control solution is analyzed; the results of both analyzes are compared, and the results of these comparative analyses. The composition of the test substance is determined from Analysis of solutions containing eluent and test sample components transfer it to a detector equipped at the outlet from the microcolumn. ect-flow) through a measurement cuvette. Analysis of control solution Place it in the detector's comparison kit similar to the measurement cuvette at the same flow rate as the test solution. This is done by passing it through a tube. Pure eluent is used as control solution . The signals from both cuvettes are compared and the signal obtained from the measurement cuvette is From the cuvette, the signal from the comparison cuvette is subtracted, and the resulting signal is recorded and used for comparison of sample compositions (J, 'Kirkland, M odd state of 1iquid chroa+atograpby + 1974, Moscow, MIRPublishers + 82 pages 84) 4 copies The conventional method of and has low sensitivity due to incompatibility of the mobile phase in the measurement cuvent. measurement key In the tube, the mobile phase contains residues of the preceding test sample, traces of dissolved and adsorbed background signals in comparison cuvettes, as well as sensitive detectors. backgrounds that may differ substantially in their level and may vary. contains other impurities that determine the level and nature of the change in signal. Furthermore, this The conventional method requires twice the amount of eluent to perform the analysis and a second pressure source. Due to the presence of , it is characterized by high analytical costs.

In addition, microcolumn liquid chromatography, which is known in the industry, A method for determining the composition of a substance in which a sample of the test compound is The components of the sample are pumped through the chromatographic column at its outlet. Quantitative and qualitative analysis of solutions containing eluent and quantitative analysis of control solutions and Perform a qualitative analysis; compare the results of the former and latter analyses, and Evaluate the composition of the test substance from the results. of the solution containing the eluent and test sample components. For analysis, at the outlet from the chromatographic column, this solution is transferred to the detector for measurement. Direct flow - done by passing through a cuvette. Analysis of control solution by passing it through the detector's comparison direct-flow cuvette. It will be done. This solution contains the solvent solution and the components of the comparison sample used for comparison purposes. Has (U, S, , A, 38475550).

This method also has low sensitivity since it is impossible to use absolutely identical columns. It is characterized by The conditions for separation of similar samples will always be different on the two columns. cormorant. Furthermore, since different samples passed through them, they have different histories. do. For sensitive detectors, due to the different histories of the working and comparison columns, Differences in the composition of the mobile phases emanating from them will be recorded.

In addition, methods for determining the composition of substances in microcolumn liquid chromatography A method is known in which a sample of a substance is chromatinized along with a stream of eluent. The solution is pumped through the matoographic microcolumn and then at its outlet. Both quantitative and qualitative analysis of solutions containing syneresis and test sample components This is done by passing the sample through the detector comparison cuvette. Furthermore, the control solution By passing the sample through the comparison cuvette of the detector, both quantitative and qualitative analysis can be performed. It is applied to the person. The results of the former and latter analyzes are compared, and the results of the comparison are tested. Used for comparative evaluation of test substances. In this method, comparative cuvettes The effluent from the measurement cuvette was used as a control solution and 10% of the sample ．． 000-100.000 times more in volume and filled with pure eluent The mixture is passed through a dilution container. The outlet from this dilution vessel is connected to the inlet of the comparison cuvette. (US, A, 3981179).

The solvent volume required to perform one analysis is 1-2% of the dilution vessel volume. The result As a result, the eluate flowing out of the column has a non-significant effect on the composition of the liquid in the dilution vessel. Even will have some effect, however, the compositional substitution will be very gradual. The eluate generated at Not representative of the true composition of the eluate flowing out of the tograph column. passed through the column The composition of the eluate may differ considerably from that of the eluate that did not pass through the column.

The eluate is washed away as a result of the dissolution of the adsorbent or left after the preceding analysis. If the substance contains substances introduced into it as a residue of impurities that are difficult to There is. Furthermore, due to stratification in the container, gas oxidation, and changes in ambient temperature, the eluate itself may change its properties over time. Each feature uses a high-sensitivity detector. In a specific analysis, the background line of the measured signal is may be on different levels of the scale of the location. Fluctuations in composition and properties of solvent solutions If the compensation is done with insufficient precision, different modes of measurement may cause incorrect A signal may be generated, which can lead to an analytical result for a mixture of substances. Distorts the pattern of the distribution of romatograph peaks.

This prior art method, like those previously described, is characterized by low sensitivity. Na Because in every particular analysis that has that particular number for comparison, If the control solution corresponding to the analysis has a number substantially different than the one performed at This is because it is used.

The purpose of the present invention is to determine the composition of substances in microcolumn liquid chromatography. The goal is to improve the accuracy and certainty of decisions.

Disclosure of invention The present invention is useful for determining the composition of substances in microcolumn liquid chromatography. This method consists of a method in which the test sample is transferred to a chromatographic column along with a stream of eluent. pumped through the system and at its outlet containing the eluent and the components of the test sample. Quantitative and qualitative analysis of the solution, and quantitative and qualitative analysis of the control sample. , compare the results of the former analysis and the latter analysis, and are analyzed from the results of the comparison. comprising determining the composition of the substance, which according to the invention serves as a control solution. Just before introducing the test sample into the chromatographic microcolumn, Use a solvent solution that is pumped by

For determining the composition of substances in the microcolumn liquid chromatography of the present invention The method for Improving the accuracy and certainty of determining the composition of substances based on the substantial increase in detection 5 degrees and make it possible.

Brief description of the drawing The invention will now be described by way of a description of particular embodiments thereof, with reference to the accompanying drawings, in which: In the figure, substances in microcolumn liquid chromatography of the present invention are shown. An apparatus for carrying out the composition determination method is shown.

BEST MODE FOR CARRYING OUT THE INVENTION Determination of the composition of substances in microcolumn liquid chromatography according to the invention The method consists of the following points.

A sample of a substance is pumped into a chromatographic column with a stream of eluent; At the outlet, quantitative analysis and qualitative analysis of the solution containing the eluent and the components of the test sample Both analyzes are performed together with quantitative and qualitative analyzes of control solutions; The results of the comparison are compared, and the results of the comparison are used to determine the composition of the analyzed substance. used. Introducing the test sample into the chromatographic microcolumn as a control solution An eluent is used which is pumped onto the column immediately before the injection.

Composition of substances in microcolumn liquid chromatography shown in the attached drawings The device for performing the method for determining It has a pressure source 1 with a cavity communicating via a dispenser 2.

A cuvette unit 4 is provided at the outlet from the microcolumn 3, and this This is a measurement direct-fl flow connected directly to the outlet of microcolumn 3. ow) Cuvette 5, a comparison cuvette with a similar arrangement adjacent through a common side wall. It has a bet of 6. The outlet of the measuring cuvette 5 is connected to a drainage container by a switch tap 7. 8 (this connection is shown as a solid line in the figure) or the inlet of the comparison cuvette 6 (this connection is shown as a solid line in the figure). can be connected to either one of the two (indicated by a dotted line in the figure). cuvette unit A laser light source 9 is placed on the opposite side to 4, and two parallel beams emitted therefrom are The system 10 passes through the corresponding cuvettes 5 and 6, respectively.

And it hits the optical sensor 11 after the cuvette unit. Output of optical sensor 11 The leads are compared by input leads (each input lead to its corresponding output lead). unit 12, this comparison unit has at its output a recording unit 13. connected to.

The device shown in the figures operates as follows according to the invention.

A pressure source 1 presses the adsorbent in the dispenser 2 and the chromatographic microcolumn 3. Ensure flow of eluent through. A sample of the mixture of substances to be analyzed is dispensed. This flow of eluent is introduced at the inlet to the microcolumn 3 by means of a sensor 2. entered. The eluent stream is pumped through the microcolumn 3 along with the sample. then through the measuring cuvette 5 of the transparent cuvette unit 4, and then through the switch. It passes through the chitap 7 and enters the large capacity reservoir 8. Two laser beams from light source 9 The beams 10 each pass through a measurement cuvette 5 and a comparison cuvette 6 and into a light sensor. It is accepted at the entrance of Service 11. The two optical signals are converted into electrical signals, which and is compared by the comparison unit 12. The resulting signal is sent to the recording unit 13. applied and recorded here.

After completion of the analysis, the measurement cuvette 5 and the comparison cuvette 6 are connected in series. A switch tap is located at the position connected to the reservoir 8 (as shown by the solid line in the figure). 7 is placed. Chromatograph switching on chromatograph microcolumn 3 Eluent is pumped through the liquid path of the graph. Background image from detector When the sound signal (recording level in the recording unit 13) reaches a predetermined level. After ensuring that the switch tap 7 has stabilized in its initial position, The column 3 is now connected to the reservoir 8 via the measuring cuvette 5 (in this case If so, the comparison cuvette 6 is closed at both ends). In addition to the mixture of substances to be analyzed sample is introduced into the eluent stream and the analysis is performed again. After its completion, the comparison key The tube is washed with an eluent whose composition corresponds to the time point of the next analysis. Ru. The background signal is the amount of sorbent in the chromatographic column at a given time. It will be shadowed by the actual situation. Is the signal obtained from the measurement cuvette? This can be corrected in the measurement by subtracting the signal obtained from the comparison cuvette. will be taken into consideration.

The method of determining the composition of substances according to the invention ensures a higher accuracy of measurements. because, Cleaning based on adsorbent impurities, residues of analyzed substances, and fluctuations in eluent composition over time. Changes in the composition of the eluent actually coming out of the romatograph microcolumn 3 can be identified. This is because it is considered after one analysis and before the next analysis.

For a better understanding of this invention, some specific examples are provided to illustrate specific aspects thereof. Described below: To Seto Differential-type refractometer detector for detection purposes is used.

Separation of polystyrene and proteins with molecular weights between 2X103 and 2X106 was carried out. It will be done.

Fluoroplasty with an internal diameter of 0.5 mm and a length of 300 m for the analysis of polystyrene. Microcolumns made by Microcolumns are used. Matrix with different bore sizes as filling A mixture of black porous glasses is used. The average particle size of the adsorbent is 6±1- be. At the end of the microcolumn 3, a porous titanium film with a thickness of about 0.2 mm is placed. A filter is provided.

Methyl ethyl ketone (chemical purity grade) is used as eluent. elution The liquid pumping speed is 4-6 m/min. The sample volume is approximately 0.5 μ/cm The concentration of tyrene is 0.03j1! not exceed. Five calibration methods for polystyrene The separated solution is separated with a separation factor of 0.8. Column efficiency is 10,000 min It is a theory plate.

After each analysis, microcolumn 3 is regenerated to a stable buffer by washing with eluent. to obtain a background signal. In doing so, cuvettes 5 and 6 of the detector (measuring cuvette and comparison cuvette) are connected to the outlet of column 3 and drain container 8. are connected in series between them.

After this, the inlet and outlet of the comparison cuvette 6 are closed by the switch tap 7, The column then interacts with the analyzer in the comparison path of the detector. is removed from the liquid path of the chromatograph. Place measuring cuvette 5 in drain container 8. The same switch tap 7 is used for the connection.

The optical background at a given time point obtained during the operation of the chromatograph preparation for analysis The specific level of the round signal is determined by the chroma, pure due to the composition of the eluent, and then the next sample is introduced into this column for analysis. Ru.

The level thus obtained of background signal of all comparison (difference) measurements made, it is closest to the next separation condition.

The method of the invention makes it possible to take full advantage of the high sensitivity of laser differential refractive detectors. do.

1 piece Protein from a mixture of myoglobin, egg albumin, serum albumin and ferridine Separate the white matter. Trifluoroacetic acid containing 5% water by volume was used as the eluent. Ta.

A microphone of the same size as in Example 1 above, filled with macroporous glass for separation. Use Locolumn 3. The average particle size of the adsorbent is 5-6μ.

A mixture of proteins with a concentration of 1 mg/ml was used for analysis, and the sample volume was O , OM. Each peak of the mixture components has a scale range value of approximately 0.8. have The zero point of light after each analysis changed significantly and microcolumn 3 as well as Washing of cuvettes 5 and 6 with pure eluent was necessary.

The zero point set on the measurement scale most accurately corresponded to the conditions for the next analysis. 0 Transfer a portion of the washing solution after setting to comparison cuvette 6 just before starting the next analysis. was retained and used as a control solution.

In the above example of implementation of the method of the invention the following data were obtained.

1. Typical analysis of polymers with molecular weights ranging from 2X103 to 2X10b Duration: Approximately 7 minutes.

2. Of solvents for typical analysis! ...0.1d.

3. Minimum detectable amount of substance: 2 ng.

4. Lower limit of detection: 0.5 ng.

5. Root mean square deviation of output signal: Deviation of peak height...2% or less ;Difference in retention time: 1% or less.

6. Effective capacity of cuvette...0.1#.

7. Working pressure at column inlet: 120 atm.

Known methods for detecting the composition of substances in liquid microcolumn chromatography Compared to the method of By considering the potential, it is possible to increase the detection sensitivity by nearly two orders of magnitude.

Industrial applicability The method of the invention for determining the composition of substances is particularly useful in biotechnology, medicine, agriculture, chemistry. For research in the analysis of complex mixtures of substances that can be dissolved in liquids. It can be used at any time.

Claims (1)

[Claims] 1. A method for determining the composition of substances in microkeram liquid chromatography. A sample of a substance is passed through a chromatographic microcolumn with a stream of eluent. Pumping and quantifying a solution containing the eluent and the components of the sample to be analyzed Analytical and qualitative analyzes are performed at the outlet of the chromatographic microcolumn, and a control Perform quantitative analysis and qualitative analysis of the solution, and compare and differentiate the results of the first and second analysis. It consists of evaluating the composition of the substance to be analyzed and, as a control solution, Immediately before introducing the sample to be processed into the chromatographic microcolumn, A method characterized in that the eluent is pumped by a pump.