DE2200882A1 - Chromatographic process - Google Patents

Description

Durrum Chemical Corporation Palo Alto, Calif. (V.St.A.)

Chromatographic process.

For this application, priority is derived from the corresponding U.S. Registration Ser. No 105 257 of January 11, 1971 claimed.

Samples made from mixtures of amino acids of the protein hydrolyzate type (such as the 20 α-amino acids) or consist of physiological fluids (such as urine) can be chromatographically mediated Separate different'Verfahren in order to enable the subsequent quantitative determination of the individual components. One particularly effective method is to apply a sample of a mixture of amino acids to an ion exchange resin column supplied, which consist of a fixed matrix (stationary phase) of crosslinked with diviny! benzene! Polystyrene, to which a negative functional group, generally sulfonic acid, is attached. The SuIfongruppe attracts the positively charged amino acids, each of which has a characteristic affinity for it. Then the individual amino acid components are eluted one after the other, i.e. washed out and thus in reverse Order of the degree of resin affinity of each component under the action of a pressurized feed

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Alkali metal citrate buffer solution separated from the column. A common method of quantitative determination is in reacting the amino acids with ninhydrin reagent and to form a compound which absorbs light in the wavelength range of 570 nm, the mixture is passed through a flow cuvette past a colorimeter detector (e.g. a photocell), which is quantitatively responsive to the wavelength in question. Certain "imino acids" (such as proline) are involved in the formation Ninhydrin is a compound which absorbs in the wavelength region of 440 nm, and this absorption can be mediated - determine a second monitoring device. The detectors are connected to a recording device, with which the response behavior is recorded on a strip of paper, for example. The quantitative mood is done by integrating the individual peak values (i.e. the peaks) in the recording over time, since each peak value has a characteristic location and shape that depends on the type of the respective one Amino acid dependent.

When developing the chromatographic procedure described above, a sodium citrate buffer solution was used constant composition at the top of a relatively long column (e.g. 150 cm length) fed to the sample from an amino acid mixture to elute. However, this method requires very long elution times, namely about 48 hours

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for protein hydrolyzates and 90 hours for physiological fluids.

To reduce the time required for chromatographic separation, a two-column system was developed in which a first, long column (150 cm long) and a second, shorter column (15 cm long) are used, as in detail in one Article by Spackman DC, Stein WH and Moore S. "Automatic Recording Apparatus for Use in the Chromatography of Amino Acids", 30 Anal. Chem. 1190 (July 1997). In this system (which will be referred to as the Stein-Moore method in the following) the sample is split into two fractions, which are each fed to one of the two columns: two discrete buffer solutions with pH values of 3.2 and 4.5 are introduced sequentially into the long column to elute the more easily removable amino acids of the fraction, and a third buffer solution with a pH of 7 is retained to elute the more strongly Ten amino acids from the second, shorter column are used. With this two-column system with three separate buffers, the separation time is significantly shorter than with the individual column as described above. For example, Spackman, Stein, and Moore were able to analyze protein hydrolyzates in about 22 hours and physiological fluids in about HH hours. Technological improvement

Ments and the development of synthetic resins with higher resolving power allow a shortening nowadays this analysis times to 4 hours for hydrolysates and 11 hours for physiological fluids.

A quick separation in the two-pillar system is based on a compromise between a known separation force, namely the pH of the buffer solution, and the desired one Degree of resolution. However, increasing the pH of the buffer solution will reduce the analysis time at the same time the resolution is also reduced. The more easily removable amino acids are used along with the Lower pH buffer solutions eluted in the long column because of a relatively small elution separation force is required. The more strongly retained amino acids can be eluted in that a solution with higher pH is used in the short column.

The Stein-Moore method is used nowadays in the It is stated that a sodium citrate buffer solution consisting of three equal fractions is used, in which the first portion has a pH of about 3.25 with O, 2N sodium citrate, the second portion has a pH of about 4.25 with the same normality and the third proportion (for the short column) has a pH of 5> 35 with a normality of 0.35.

This two-pillar approach suffers from several serious disadvantages. Imagine once error very quickly when the original sample

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is not divided into two exactly equal fractions. In addition, a larger amount of possibly very valuable samples is required, as one is required for the second column additional fraction is needed. Furthermore, the operation of the two-column system is complicated, including the higher cost of an automatic sample loader come.

One attempt to overcome some of the disadvantages described above is to use a single column with a variable buffer system similar to that used in the two-column system. Such a system is described in an article by Hamilton PB "Ion Exchange Chromatography of Amino Acids" 35 Anal. Chem. 2056 (December 1963). This system also requires extremely long separation times (of around 2k hours, for example).

A serious problem with both of the aforementioned one- and two-column systems is baseline drift or drift in the course of the quantitative determination. This can cause larger errors, and there are particular problems when using small amounts of sample (for example, less than 100 nanomoles), since the errors become very large. Initially, ammonia impurities in the buffers were assumed to be the source of the baseline migration. It was assumed that when the

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pH of the solution would be eluted from the column in increasing amounts. Ammonia contamination as the only one The cause of the baseline shift was questioned on page 2060 of the aforementioned article by Hamilton, however, no new theory was put forward. In practice, however, these assumptions are refuted by that even with buffer solutions from which the ammonia impurities have previously been filtered out, baseline shifts occur especially when small samples (e.g., less than 50 nanomoles) are being analyzed will.

The invention is based on the object of a method for the chromatographic separation of one from one Amino acid mixture using existing sample to provide an ion exchange resin column which does not suffer from the aforementioned disadvantages of the known methods is, the separation in a short time in a single ion exchange column and in particular with a buffer system allows which baseline migrations during the port run of the analysis are essentially eliminated will.

The chromatographic method proposed for this purpose for separating the components of a sample contained, for example, in a protein hydrolyzate or in a physiological fluid from a mixture of amino acids using a column with a cation exchange resin is characterized according to the invention.

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indicates that the sample is fed into the column in such a way that the amino acid components are distributed in the resin and retained by it, and a sufficiently large amount of an aqueous buffer solution is passed through the column and the components are washed out one after the other and thereby separated, wherein the buffer solution used is an alkali metal salt of an acid with a pK value less than 5, which does not react in a harmful manner with the resin and the sample, and the pH value of the buffer solution during the elution is less than about 4 * 0 and the pH -Value fluctuation is less than 30 % .

For eluting a typical sample of amino acid components, for example of the protein hydrolyzate type within an acceptably short time (e.g. less than 5 hours), a buffer system is preferably used with a low initial "separation force" is used and this separation force is used according to the port course of the eluted amino acids from those of lower to those of higher resin affinity increased. The separation force is defined than the rate at which certain amino acids are eluted from the column by means of a buffer system. The invention is based on the discovery that the conventional measures to increase the separation force for the Elution of the strongly retained amino acids by increasing the pH of the buffer solution to a substantial more than 4.0 and through

Increase in the total concentration of the buffer a shift the baseline. It has been found that this baseline migration is essentially a result of this can turn off the fact that the pH value within relatively small fluctuation ranges to less than about 4.0 is held. The separation force is increased by increasing the cation concentration. Suitable Buffers consist of alkali metal salt citrates, acetates, formats, phosphates or other anions with the properties described below.

In the case of a protein hydrolyzate sample, the buffer solution is generally preferably at a pH of kept about 3 to 4.0 during the final stage of elution, although it is also possible under certain circumstances is to increase the pH up to 4.2. An exception for this rule, phosphate is formed during elution

preferably has a pH value between 2.0 and 3 _s 0 so that it corresponds more to the pK value 2.1 in the manner described below. The total variation in pH during the process (defined below) is less than 30 % and preferably less than 20ί. The alkali metal ion concentration is preferably at least 500 % greater at the end than at the beginning of the elution, so that an adequate increase in the separation force is obtained for the amino acids which are more difficult to remove.

The invention is explained in detail below.

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Fig * 1 is a drawing with no baseline migration as they are when using the Buffer system is obtained.

Fig. 2 is a record showing the relocation the baseline can be seen with a conventional buffer system.

According to the invention, a sample from an amino acid mixture of the protein hydrolyzate type or from a physiological fluid (such as urine) is separated in a column with a cation exchange resin. The following description is directed to a Proteinhydrolysatprobe containing 18 amino acid components, as shown in Figures 1 and 2. FIG.

The amino acid sample will be the top end of a Ion exchange resin (e.g. sulfone type) in the manner supplied so that the amino acid components are distributed in the resin and retained by this. Then it will be an aqueous buffer solution under pressure through the top the column introduced to carry the amino acid components through successive washing out (elution) of the same to separate the outlet end of the column.

The expression "pH value fluctuation" or "pH value fluctuation range" used here is defined by the following equation:
(1) pH value fluctuation = [(pH ₂ - PH ₁ ) Z (PH ₂ + PH ₁ ) Za] X100?

in what pH _? the pH value of the 3 buffer after elution of the last amino acid and PH ₁ the pH value of the buffer

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Elution of the first amino acid is.

The cation exchange resin is typically made of relatively small particles (e.g. from 10 to 20 μm in diameter) made of polystyrene with a smaller Share (of e.g. $ 8) Diviny! Benzene is linked to the several negatively charged groups (e.g. from sulfonic acid) are attached. For a single column, it is convenient to use two a resin depth of 50 to 60 cm is used.

A protein hydrolyzate type amino acid sample is generally made from an unknown protein sample prepared by mixing it with an aqueous solution of a strong acid, and by heating at At elevated temperature, the protein is hydrolyzed into the separate amino acid components. Part of the sample is then inserted into the top of the ion exchange column, in which the individual amino acid groups and through electrostatic bonding with the functional groups of the synthetic resin are retained. The resin affinity of each ingredient is a differentiator, which serves as the basis for the chromatographic separation. The alkali metal of the buffer solution is displaced one after the other the individual amino acid components of the resin in reverse order to the resin affinity of the individual components. As from the records 1 and 2, forms aspartic acid in the protein hydrolyzate group used for example the most weakly retained amino acid, while

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Arginine is the most retained acid.

A known method for the quantitative determination of the column elution is to bring the amino acid components to reaction with ninhydrin reagent to form a compound that absorbs light in the wavelength range of 570 nm and then to pass the mixture through a flow cell to a flow colorimeter, the quantitative is responsive to this wavelength. As stated above, a second monitoring device responsive to kkO nm can be used for imino acids. These detectors are connected to a recorder which graphically records the results on a strip of paper. The quantitative determination is carried out by integrating the individual curve peaks over time, each of which has a characteristic position and shape in the recording.

According to the invention, the buffer system used for the chromatographic separation has particular ones Properties that allow high speed separation in a single column and essentially without moving the baseline. The system can also be used for several columns and prevents a migration of the baseline even when relatively small sample amounts (e.g. 50 nanomoles or less).

The buffer solution is added to the column in discrete

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Amounts totaling, for example, three or four are added, the composition of each amount being different is. Instead, a so-called "gradient buffer solution" can also be used, in which the buffer continuously fed and its composition is constantly changing. With every buffer solution system used however, the change is predetermined in such a way that the separation force of the solution increases with the rate of elution is increased so that the more retained amino acids are washed out within a reasonable time can. If the separation force is too high at the beginning, the force required to separate the individual components may be Degree of resolution get lost. The one to dissolve The relatively low separation force required for the first amino acids, on the other hand, can lead to excessively long elution times for the more retained amino acids. As a compromise, a relatively small separation force is used Beginning of the process and a relatively high separation force used at the end of the elution.

Controlling the pH during the elution process is an important feature in preventing an Baseline hike. It has been found that for pH values greater than 4.0 a strong increase in baseline migration can be observed. This effect occurs higher analytical sensitivity, such as a reduction in the amount of sample, even more noticeable. A fluctuation of the pH value within a large range

wisely more than $ 20 or $ 25) during the procedure also a baseline migration that increases with the absolute pH value. An optimal condition would be the use of essentially constant pH buffers throughout the process. Small fluctuations in pH of the buffer are allowed, however the amount of fluctuation depends on the absolute sensitivity of the respective Analysis is dependent. Higher sensitivities (i.e. smaller amounts of sample) require that the pH - Fluctuations during the analysis reduced to a minimum will.

The buffer solutions used in the process according to the invention do not significantly impair the development of color when the amino acids react with the ninhydrin reagent. In addition, the acids forming the buffer anions according to the invention have a pK value (−10 g _{10 of} the dissociation constant) less than 5. The pK value preferably does not differ significantly from the pH value of the buffer during elution, so that the buffer has a retains adequate effectiveness. The pH value preferably deviates from the pK value of the acid by no more than plus or minus 30%. There are many acids available which satisfy these conditions and which can be used in accordance with the invention, since, in contrast to known processes, the pH value is kept within a relatively small fluctuation range (of 30/5). Suitable acids and their corresponding ones

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pK values are acetic acid (4.75K formic acid (3.25), phosphoric acid (2 _{} 1} 1) and citric acid (3.1)

Suitable buffer solutions are alkali metal salts for example sodium, potassium and lithium. Citrate, acetate, format, phosphate and use.

With the exception of the phosphate buffer, a particularly suitable pH range for the buffer solution is during the first part of the elution between 3.1 to 3.4 or 3.4 to 3.7 for protein hydrolyzate samples. When using a buffer solution in a range from 3j3 to 3.4 Difficulty dissolving due to movement of the movable Cysteine component in the area of the glycine and alanine components and superposition of the same result. With a The pH range of about 3.1 to 3.3 will turn the cysteine into one free area in front of glycine and alanine and with a range of 3.4 to 3.7 in a free area eluted after them.

A phosphate buffer has been shown to be effective for an initial pH of 2.6, which is closer is due to the pK value of phosphoric acid (2.1). It is assumed that other buffer solutions with pK values use below 3.0 with a corresponding, lower pH value. The separation force is through use a relatively high initial cation concentration (e.g. 0.5 N or higher).

In the aforementioned Stein-Moore method, the third buffer solution has a pH value of 5.35,

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is significantly higher than the maximum value according to the invention of 4.0. For pH values greater than 4.0, there is a significant baseline shift, especially when used relatively small samples (e.g. 50 nanomoles Or less). In the known method, this high pH value is required in order to elute the more strongly retained amino acid constituents within a reasonable period of time required separation force to create.

The separation force required for a chromatographic Separation in a single column in four hours or less can be achieved by increasing the alkali metal ion concentration of the citrate buffer, keeping the pH and the anion value maintained relatively constant values. The elution of the more strongly retained Amino acids made change in the separation force by adding an inorganic alkali metal salt of a Halogen, a phosphate, sulfate or the like. Take place. The baseline is obviously up against large increases insensitive to the alkali metal ion concentration. At the beginning of the elution, therefore, the alkali metal ion content from an initial value of about 0.10 to 0.35 moles / liter to a final value of about 0.25 to 3.0 or less moles / liter can be changed without shifting the baseline.

In accordance with the above principles, a particularly effective three-stage sodium citrate

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Buffer system for a protein hydrolyzate in the beginning the sodium concentration between 0.1 to 0.35 »and the pH fluctuates from about 3.1 to 3 _S 3 or from 3> 4 to 3.7 * The second and third buffers have a pH -Value of 3 to 4.4 and a sodium ion concentration of 0.2 to 3 moles / liter.

The theoretical explanation for the sensitivity of the baseline to pH is insufficient clear. The shift in the baseline is apparently caused by certain external, colored interfering media, which are eluted from the resin column and have a coloration that matches the color of the ninhydrin reaction product is similar. It can therefore be hypothesized that the interfering media consist of constituents of the resin themselves exist, which are washed out of the resin in different amounts depending on the pH value. This hypothesis can be partially confirmed when a resin column is filled with an aqueous solution and the pH is changed. This shows that the solution has a certain characteristic at higher pH values Takes on color, but does not appear at lower pH values. It is very possible that this Component indicated by the detector and thereby the baseline shift is caused. After this In theory, the disorder occurs when the pH is increased to values of 4.0 or higher by erratic and intermittent Emission from the resin, whereby the considerable

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borrowed deviations from the baseline are caused. This theory also explains why large pH fluctuations also shift below a pH value of 4.0 have as a consequence. The baseline changes as the pH ^ value of the buffer increases the amount of interfering medium emitted influenced in a corresponding manner.

In order to better illustrate the method according to the invention, a specific exemplary embodiment is described below, which, however, is intended only for explanation and not as a limitation of the invention.
example 1

A pot of a calibrated mixture of about 50 nanomoles of the 18 amino acids shown in FIG consisted of a polystyrene base which was crosslinked with about 8% divinylbenzoi and had a diameter of 15 to 20 μm (DG-IA from Durrum Chemical Corp., PaIo Alto, Calif., V.St # A.).

Three discrete aqueous sodium citrate buffers (the labeled as A, B and C) were taken one after the other a pump pressure of 19.6 atü in a constant throughput of 70 ml / h of the column and fed in this way the sample pushed through the column. The composition of the fuffer was as follows:

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buffer PH value Citration sodium chloride "(Mol / 1) (Moles / 1) (Moles / 1) A. 3.25 0.067 0.2 - - B. 3.50 0.067 0.7 0.5 C. 3.65 0.067 1.6 1.4

Before performing the experiment, buffers A and B were filtered to remove ammonia impurities from them.

The buffer sequence during the experiment was as follows: buffer A for 85 minutes, buffer B for 35 minutes and buffer C. for 120 minutes with a total analysis time of 240 minutes. . The temperature of the column was 45 ^° C. during the first 90 minutes and then 65 ^° C. and was controlled by means of a liquid circulation jacket through which a heat transfer liquid (warm water) was circulated.

After washing out, the completely separated amino acids were mixed in a mixing tube with ninhydrin reagent, which was pumped into the tube under sufficiently high pressure to maintain a throughput of 35 ml / h. The mixture was ^{fed to a reaction coil kept at about 100 °} C., and the reaction products formed typically absorbed the light either in the wavelength range of 570 nm (amino acids) or in the range of 440 nm (Iiairto * acids). These colored products were passed through a flow cell which was irradiated with light of 570 nm. The transmitted light was from one

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Silicon diodes photometer collected _s and whose output voltage is supplied to a recording device. The absorption of the light by the colored product caused a voltage loss in the detector, and this voltage loss was recorded by the recorder. 1 shows a graph of the absorption as a function of time. As can be seen from this record, about 45 minutes pass before the first amino acid is washed out. The recording result shows an essentially steady baseline throughout the experiment. Example 2

For example 2, a sample of the same nature as that of example 1 was used, with the Exception that the amino acid concentrations were doubled to 100 nanomoles / 1. The same analysis as in Example 1 was used, with the exception that a conventional buffer system was used as follows:

buffer PH value Citration Sodium ion Chloride ion (Minor) (Minor) (Minor) A. 3.25 0.067 0.2 - B. 4.40 0.067 0.2 - C. 6.35 0.20 1.6 1.0

The results of Example 2 shown in the plot corresponding to Fig. 2 are shown compared to example 1 a considerable change in the baseline, especially between 100 and 200 minutes

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the elution process. This is particularly evident from the fact that Example 1 has twice the sensitivity (the
half the sample concentration) compared to Example 2
was executed. The only apparent reason for the lack of any baseline shift in Example 1 is the use of a buffer system according to the invention in place of a conventional buffer system.

- claims

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Claims (8)

-21 - Patent claims: Chromatographic method for separating the components of a sample contained, for example, in a protein hydrolyzate or in a physiological fluid from a mixture of amino acids using a column with a cation exchange resin, characterized in that the sample is fed into the column in such a way that the amino acid components are in are distributed and retained by the resin, and a sufficiently large amount of an aqueous buffer solution is passed through the column and the components are washed out one after the other and thereby separated, an alkali metal salt of an acid with a pK value less than 5 being used as the buffer solution, which does not react deleteriously with the resin and sample, and the pH of the buffer solution during elution is less than about 4.0 and the pH fluctuation is less than 30 % . 2. The method according to claim 1, characterized in that the alkali metal salt is selected from Group consisting of sodium, lithium and potassium. 3. The method according to claim 2, characterized in that the pH of the buffer solution at the beginning of Elution to about 3.1-3.3 or 3.4-3.7 and over the elution is made about 3.0 to 4.0. 209829/075/4 4. The method according to claim 2, characterized in that the anion of the buffer solution is selected from Group consisting of citrate, acetate, format and phosphate. 5. The method according to claim 4, characterized in that citrate is used as the anion. 6. The method according to claim 2, characterized in that the concentration of the alkali metal ions at the beginning of the elution is made about 0.10 to 0.35 mol / l, and at the end of the elution about 0.25 to ₃ O mol / l and the alkali metal ion concentration at the end of the elution is at least 500 / S higher than that at the start of the same. 7. The method according to claim 2, characterized in that the aqueous buffer solution of the column in separate, discrete fractions. 8. The method according to claim 2, characterized in that the aqueous buffer solution of the column in one Stream is supplied with a continuously increasing alkali metal ion content. 9/075 ^ Blank page