CN112858522A - Method for separating and measuring hybrid amino acids in high-purity valine by high performance liquid chromatography - Google Patents
Method for separating and measuring hybrid amino acids in high-purity valine by high performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for separating and determining hybrid amino acid in high-purity valine by high performance liquid chromatography, in particular to the technical field of separating amino acid by liquid chromatography, which comprises the following steps: the method comprises the following steps: preparing instruments and reagents; step two: preparing a sample; step three: and (4) performing chromatographic analysis. The method directly adopts an ultraviolet detector for detection at 204nm, adopts a C8 reversed phase bonding chromatographic column with weaker hydrophobicity, takes a mixed solution of a phosphate buffer solution and acetonitrile as a mobile phase, and adds a sodium pentane sulfonate solution with proper concentration, has the characteristics of high accuracy, wide linear range and good reproducibility, does not need derivatization operation and complex sample pretreatment, has better separation between valine and each impurity amino acid, has quicker peak-out time and short detection time, has higher sensitivity, can better realize the detection of the quality of the high-purity valine, and is favorable for meeting the requirement of the quality control of the high-purity valine product in the production process.
Description
Technical Field
The embodiment of the invention relates to the technical field of amino acid separation by liquid chromatography, in particular to a method for separating and determining high-purity valine and hybrid amino acid by high performance liquid chromatography.
Background
Valine, one of the 20 amino acids constituting proteins, has the chemical name of 2-amino-3-methylbutyric acid, belongs to a branched chain amino acid, and is also an essential 8 amino acids and a glycogenic amino acid for the human body, and works together with the other two branched chain amino acids (isoleucine and leucine) to promote the normal growth of the body, repair tissues, regulate blood sugar, and supply required energy.
Valine is usually produced by fermentation, and is limited by process conditions, and industrial grade valine extracted and produced from fermentation liquor often contains amino acids such as alanine, leucine, isoleucine, phenylalanine and the like.
Valine is a starting material for preparing crude drugs such as ketovaline calcium and the like, has high requirements on the quality of valine raw materials, and particularly has strict limit standards on the content of the heteroamino acids. Such highly pure valine is usually produced by a special purification process. In the series of processes for producing and applying high-purity valine, the quality of products, particularly the detection of the hybrid amino acids, is often required.
Amino acid analysis can be accomplished using a dedicated amino acid analyzer. The amino acid analyzer is an apparatus for separating amino acids by cation exchange chromatography and performing qualitative and quantitative analysis. Separating amino acids in the sample by ion exchange chromatographic column, reacting the separated single amino acid component with ninhydrin reagent to obtain purple compound, measuring absorbance at 570nm with visible light detector, and comparing with absorbance of standard solution to calculate amino acid content in the sample. The special amino acid analyzer is expensive, single in use, complex in operation and long in analysis time.
Amino acid analysis can also be accomplished using high performance liquid chromatography. Usually, reversed phase column separation and ultraviolet detection method before or after column derivation are adopted. However, these methods require derivatization, are complicated to operate, require long analysis time, and generally require 1 hour for one sample. Gradient elution is also needed, and the requirements on a high performance liquid chromatograph and reagents for preparing a mobile phase are high.
The hetero amino acids in the highly pure valine mainly refer to alanine, isoleucine, leucine and phenylalanine, and other amino acids are not generally present. As long as the several kinds of hetero amino acids and valine can be separated from each other, it is possible to find a simple liquid chromatography separation method for separating the several kinds of hetero amino acids. However, the content of the main component valine is up to more than 99%, while the content of the hetero amino acid is about 0.01-0.5%, and the difference between the content of the main component valine and the content of the hetero amino acid can reach 10000 times, so that the accurate determination of the content of the low-concentration hetero amino acid in the high-concentration background is still difficult.
Therefore, it is necessary to establish an analytical method for simply, rapidly and accurately determining the content of the impurity amino acid in the high-purity valine so as to meet the requirement of quality control in the production process of the high-purity valine.
Disclosure of Invention
Therefore, the embodiment of the invention provides a method for separating and determining the hybrid amino acid in the high-purity valine by using a high performance liquid chromatography, which avoids the derivative reaction of sample components by directly adopting an ultraviolet detector to detect at 204nm, simplifies the operation steps, shortens the analysis time and obtains higher detection sensitivity; by adopting the C8 reversed phase bonding column with weaker hydrophobicity, the separation degree of the amino acid components is improved, the mixed solution of phosphate buffer solution and acetonitrile is taken as a mobile phase, and sodium pentanesulfonate solution with proper concentration is added, the anion of the pentanesulfonate and the amino acid component with positive charge form an ion pair compound, thereby reducing the possibility that the amino acid component is absorbed by silicon hydroxyl possibly existing on a chromatographic column carrier, reducing the tailing of a chromatographic peak, realizing the separation between valine and various impurity amino acids by adopting the liquid chromatography condition provided by the invention, solves the problems that in the prior art, as the special amino acid analyzer is expensive and has single use, the method for detecting amino acid by deriving ultraviolet light before or after the column has fussy operation and long analysis time, high requirements on a high performance liquid chromatograph and reagents for preparing a mobile phase, and low accuracy of separating and detecting the hybrid amino acid.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions: a method for separating and measuring hybrid amino acids in high-purity valine by high performance liquid chromatography comprises the following specific steps:
the method comprises the following steps: preparation of instruments and reagents: performing high performance liquid chromatography analysis under the following chromatographic conditions to determine the content of the hetero amino acid in the valine:
a chromatographic column: (C8, 5 μm,150 × 4.6 mm i.d.) reverse phase bonded chromatography column;
mobile phase: a mixed solution of acetonitrile/sodium pentanesulfonate-phosphate buffer solution in a volume ratio of 0:100-20: 80;
flow rate: 0.6-1.0 mL/min;
column temperature: 25-35 ℃;
detection wavelength: 204 nm;
step two: preparing a sample: weighing a proper amount of high-purity valine and industrial grade valine samples, adding water for dissolving, fixing the volume to 100mL by using water, filtering by using a 0.45um microporous filter, and taking the filtrate as an analysis solution for sample injection analysis;
step three: and (3) chromatographic analysis: and injecting the prepared 20uL of analysis solution into a liquid chromatograph by adopting a six-way sampling valve, recording a chromatogram, and carrying out chromatographic analysis.
Further, an ultraviolet detector was used for detection at 204nm when the chromatogram was recorded in step three.
Further, the reverse phase bonding column in the first step is a C8 reverse phase column.
Further, the method for preparing the mobile phase in the step one comprises the following steps: taking 0.17-0.85g of sodium pentane sulfonate and 0.78-2.72g of monopotassium phosphate, adding 900mL of water for dissolving, dropwise adding 5% diluted phosphoric acid until the pH value is 2.0-4.7, then supplementing water to 1000mL, wherein the solution is a sodium pentane sulfonate-phosphate buffer solution, mixing acetonitrile and the sodium pentane sulfonate-phosphate buffer solution according to the volume ratio of 0:100-20:80, filtering through a 0.45-micrometer microporous filter membrane, and performing ultrasonic degassing to obtain a mobile phase solution, wherein the concentration of the sodium pentane sulfonate in the mobile phase solution is 1-5 mmol/L, the concentration of the phosphate buffer solution is 5-20mmol/L, and the pH value is 2.0-4.7.
Furthermore, the weighing amount of the high-purity valine and the industrial grade valine sample in the second step is 1.00g, and the sample injection volume of the second step is 20 uL.
The embodiment of the invention has the following advantages:
1. according to the invention, the ultraviolet detector is directly adopted for detection at 204nm, compared with the prior art, the derivatization reaction of sample components is avoided, the operation steps are simplified, the analysis time is shortened, and higher detection sensitivity is obtained;
2. according to the invention, the C8 reversed phase bonding column with weak hydrophobicity is adopted, compared with the prior art, the separation degree of amino acid components is improved, the mixed solution of phosphate buffer solution and acetonitrile is taken as a mobile phase, a sodium pentane sulfonate solution with proper concentration is added, an ion pair compound is formed by pentane sulfonate anions and amino acid components with positive charges, the possibility that the amino acid components are adsorbed by free silicon hydroxyl possibly existing on a silica gel carrier of the reversed phase column is reduced, the tailing of a chromatographic peak is reduced, and the separation between valine and various impurity amino acids is realized by adopting the liquid chromatography condition provided by the invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a chromatogram of a standard;
FIG. 2 is a chromatographic separation of technical grade valine;
FIG. 3 is a diagram showing the chromatographic separation of highly pure valine.
In the figure: 1 alanine, 2 valine, 3 isoleucine, 4 leucine, 5 phenylalanine.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the invention provides a method for separating and measuring hybrid amino acids in high-purity valine by using high performance liquid chromatography, which comprises the following specific steps:
the method comprises the following steps: preparation of instruments and reagents: performing high performance liquid chromatography analysis under the following chromatographic conditions to determine the content of the hetero amino acid in the valine:
a chromatographic column: (C8, 5 μm,150 × 4.6 mm i.d.) reverse phase bonded chromatography column;
mobile phase: dissolving 0.17g of sodium pentanesulfonate and 0.78g of monopotassium phosphate in 900mL of water, dropwise adding 5% diluted phosphoric acid until the pH value is 2.0, supplementing water to 1000mL, wherein the solution is a sodium pentanesulfonate-phosphate buffer solution, and mixing acetonitrile and the phosphate buffer solution in a ratio of 5: 95 (volume ratio), filtering by a 0.45um microporous filter membrane, and performing ultrasonic degassing to obtain the mixture with the volume ratio of acetonitrile/sodium pentanesulfonate-phosphate of 5: 95, the concentration of the sodium pentane sulfonate is 1 mmol/L, the concentration of the phosphate buffer solution is 5mmol/L, and the pH value is 2.0;
flow rate: 0.6 mL/min;
column temperature: 25 ℃;
detection wavelength: 204 nm;
step two: preparing a sample: weighing a high-purity valine and industrial grade valine sample with the weight of 1.00g, adding water for dissolving, fixing the volume to 100mL by using water, filtering by using a 0.45um microporous filter, and taking the filtrate as an analysis solution for sample injection analysis;
step three: and (3) chromatographic analysis: and injecting the prepared 20uL of analysis solution into a liquid chromatograph by adopting a six-way sampling valve, detecting and recording a chromatogram by adopting an ultraviolet detector at 204nm, and carrying out chromatographic analysis.
Example 2:
the invention provides a method for separating and measuring hybrid amino acids in high-purity valine by using high performance liquid chromatography, which comprises the following specific steps:
the method comprises the following steps: preparation of instruments and reagents: performing high performance liquid chromatography analysis under the following chromatographic conditions to determine the content of the hetero amino acid in the valine:
a chromatographic column: (C8, 5 μm,150 × 4.6 mm i.d.) reverse phase bonded chromatography column;
mobile phase: dissolving 0.35g of sodium pentanesulfonate and 1.36g of monopotassium phosphate in 900mL of water, dropwise adding 5% diluted phosphoric acid until the pH value is 3.0, supplementing water to 1000mL, wherein the solution is a sodium pentanesulfonate-phosphate buffer solution, and mixing acetonitrile and the phosphate buffer solution in a ratio of 10: 90 (volume ratio), filtering through a 0.45um microporous filter membrane, and performing ultrasonic degassing to obtain a mixture with an acetonitrile/sodium pentanesulfonate-phosphate volume ratio of 10: 90, the concentration of the sodium pentane sulfonate is 2 mmol/L, the concentration of the phosphate buffer solution is 10mmol/L, and the pH value is 3.0;
flow rate: 0.7 mL/min;
column temperature: 27 ℃;
detection wavelength: 204 nm;
step two: preparing a sample: weighing a high-purity valine and industrial grade valine sample with the weight of 1.00g, adding water for dissolving, fixing the volume to 100mL by using water, filtering by using a 0.45um microporous filter, and taking the filtrate as an analysis solution for sample injection analysis;
step three: and (3) chromatographic analysis: and injecting the prepared 20uL of analysis solution into a liquid chromatograph by adopting a six-way sampling valve, detecting and recording a chromatogram by adopting an ultraviolet detector at 204nm, and carrying out chromatographic analysis.
Example 3:
the invention provides a method for separating and measuring hybrid amino acids in high-purity valine by using high performance liquid chromatography, which comprises the following specific steps:
the method comprises the following steps: preparation of instruments and reagents: performing high performance liquid chromatography analysis under the following chromatographic conditions to determine the content of the hetero amino acid in the valine:
a chromatographic column: (C8, 5 μm,150 × 4.6 mm i.d.) reverse phase bonded chromatography column;
mobile phase: dissolving 0.70g of sodium pentanesulfonate and 1.36g of monopotassium phosphate in 900mL of water, dropwise adding 5% of dilute phosphoric acid until the pH value is 4.0, supplementing water to 1000mL, wherein the solution is a sodium pentanesulfonate-phosphate buffer solution, and mixing acetonitrile and the phosphate buffer solution in a ratio of 5: 95 (volume ratio), filtering by a 0.45um microporous filter membrane, and performing ultrasonic degassing to obtain the mixture with the volume ratio of acetonitrile/sodium pentanesulfonate-phosphate of 5: 95, the concentration of the sodium pentane sulfonate is 4 mmol/L, the concentration of the phosphate buffer solution is 10mmol/L, and the pH value is 4.0;
flow rate: 0.8 mL/min;
column temperature: 30 ℃;
detection wavelength: 204 nm;
step two: preparing a sample: weighing a high-purity valine and industrial grade valine sample with the weight of 1.00g, adding water for dissolving, fixing the volume to 100mL by using water, filtering by using a 0.45um microporous filter, and taking the filtrate as an analysis solution for sample injection analysis;
step three: and (3) chromatographic analysis: and injecting the prepared 20uL of analysis solution into a liquid chromatograph by adopting a six-way sampling valve, detecting and recording a chromatogram by adopting an ultraviolet detector at 204nm, and carrying out chromatographic analysis.
Example 4:
the invention provides a method for separating and measuring hybrid amino acids in high-purity valine by using high performance liquid chromatography, which comprises the following specific steps:
the method comprises the following steps: preparation of instruments and reagents: performing high performance liquid chromatography analysis under the following chromatographic conditions to determine the content of the hetero amino acid in the valine:
a chromatographic column: (C8, 5 μm,150 × 4.6 mm i.d.) reverse phase bonded chromatography column;
mobile phase: dissolving 0.17g of sodium pentanesulfonate and 1.36g of monopotassium phosphate in 900mL of water, dropwise adding 5% diluted phosphoric acid until the pH value is 4.0, supplementing water to 1000mL, wherein the solution is a sodium pentanesulfonate-phosphate buffer solution, and mixing acetonitrile and the phosphate buffer solution in a ratio of 20:80 (volume ratio), filtering by a 0.45um microporous filter membrane, and ultrasonically degassing to obtain a mobile phase solution with the volume ratio of acetonitrile/sodium pentane sulfonate-phosphate being 20:80, the concentration of the sodium pentane sulfonate being 1 mmol/L, the concentration of a phosphate buffer solution being 10mmol/L, and the pH value being 4.0;
flow rate: 1.0 mL/min;
column temperature: 35 ℃;
detection wavelength: 204 nm;
step two: preparing a sample: weighing a high-purity valine and industrial grade valine sample with the weight of 1.00g, adding water for dissolving, fixing the volume to 100mL by using water, filtering by using a 0.45um microporous filter, and taking the filtrate as an analysis solution for sample injection analysis;
step three: and (3) chromatographic analysis: and injecting the prepared 20uL of analysis solution into a liquid chromatograph by adopting a six-way sampling valve, detecting and recording a chromatogram by adopting an ultraviolet detector at 204nm, and carrying out chromatographic analysis.
Example 5:
the following data were obtained from examples 1 to 4:
as can be seen from fig. 1, under the optimal chromatographic conditions, there were significant differences in the retention of valine and the respective impurity amino acids (i.e., alanine, isoleucine, leucine, phenylalanine), and the separation of these five amino acids was achieved (fig. 1).
In the case of valine as a main component, the chromatographic peak thereof drags a long 'big tail', which often affects the accurate quantification of the following amino acid, so that the chromatographic peak of valine and the following isoleucine must have a considerable distance. The process of the present invention meets this need.
And deducting a blank mobile phase peak from a sample spectrogram, and calculating the content of each amino acid component by using an area normalization method.
The detection limit (S/N) of the method was determined as S/N =3 by actual measurement series of amino acid standard samples at low concentrations.
The recovery of the process was determined by standard addition methods.
The repeatability test was performed by repeating the injection 6 times.
TABLE 1 detection limits, Linear Range, correlation coefficients, repeatability and recovery for various amino acid components
Taking the filtrate obtained in the step two as an analysis solution for sample injection analysis; chromatograms were recorded, see fig. 2 and 3. The analytical results are shown in Table 2.
TABLE 2 analysis results of technical grade valine and highly pure valine (%)
It can be known from the above data that examples 1-4 all have the characteristics of high accuracy, wide linear range and good reproducibility compared with the prior art, but example 4 has the greatest improvement degree, under the separation condition provided by the present invention, the ultraviolet detector is directly adopted for detection at 204nm, the derivatization operation and complex sample pretreatment are not required, the operation steps are simplified, the analysis time is shortened, the C8 reversed phase bonding chromatographic column with weak hydrophobicity is adopted, the mixed solution of phosphate buffer solution and acetonitrile is used as the mobile phase, and the sodium pentanesulfonate solution with proper concentration is added, so that the separation between valine and each impurity amino acid is good, the peak emergence time is fast, the detection time is short, and the sensitivity is high. Therefore, the method provided by the invention can better realize the detection of the quality of the high-purity valine, and is favorable for meeting the requirement of quality control of the high-purity valine product in the production process.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (5)
1. A method for separating and measuring hybrid amino acids in high-purity valine by high performance liquid chromatography is characterized by comprising the following steps: the method comprises the following specific steps:
the method comprises the following steps: preparation of instruments and reagents: performing high performance liquid chromatography analysis under the following chromatographic conditions to determine the content of the hetero amino acid in the valine:
a chromatographic column: (C8, 5 μm,150 × 4.6 mm i.d.) reverse phase bonded chromatography column;
mobile phase: a mixed solution of acetonitrile/sodium pentanesulfonate-phosphate buffer solution in a volume ratio of 0:100-20: 80;
flow rate: 0.6-1.0 mL/min;
column temperature: 25-35 ℃;
detection wavelength: 204 nm;
step two: preparing a sample: weighing a proper amount of high-purity valine and industrial grade valine samples, adding water for dissolving, fixing the volume to 100mL by using water, filtering by using a 0.45um microporous filter, and taking the filtrate as an analysis solution for sample injection analysis;
step three: and (3) chromatographic analysis: and injecting the prepared 20uL of analysis solution into a liquid chromatograph by adopting a six-way sampling valve, recording a chromatogram, and carrying out chromatographic analysis.
2. The method for separating and determining the high-purity valine medium-impurity amino acid by using the high performance liquid chromatography as claimed in claim 1, which is characterized in that: and detecting at 204nm by using an ultraviolet detector when recording the chromatogram in the third step.
3. The method for separating and determining the high-purity valine medium-impurity amino acid by using the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the reverse phase binding column in step one is a less hydrophobic C8 column.
4. The method for separating and determining the high-purity valine medium-impurity amino acid by using the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the preparation method of the mobile phase in the first step comprises the following steps: taking 0.17-0.85g of sodium pentane sulfonate and 0.78-2.72g of monopotassium phosphate, adding 900mL of water for dissolving, dropwise adding 5% diluted phosphoric acid until the pH value is 2.0-4.7, then supplementing water to 1000mL, wherein the solution is a sodium pentane sulfonate-phosphate buffer solution, mixing acetonitrile and the sodium pentane sulfonate-phosphate buffer solution according to the volume ratio of 0:100-20:80, filtering through a 0.45-micrometer microporous filter membrane, and performing ultrasonic degassing to obtain a mobile phase solution, wherein the concentration of the sodium pentane sulfonate in the mobile phase solution is 1-5 mmol/L, the concentration of the phosphate buffer solution is 5-20mmol/L, and the pH value is 2.0-4.7.
5. The method for separating and determining the high-purity valine medium-impurity amino acid by using the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the weighing amount of the high-purity valine and the industrial grade valine samples in the second step is 1.00g, and the sample injection volume of the second step is 20 uL.
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