CN109283282A - A kind of amino acid analysis method detected in a low voltage state using electric conductivity detector - Google Patents
A kind of amino acid analysis method detected in a low voltage state using electric conductivity detector Download PDFInfo
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- CN109283282A CN109283282A CN201810486862.8A CN201810486862A CN109283282A CN 109283282 A CN109283282 A CN 109283282A CN 201810486862 A CN201810486862 A CN 201810486862A CN 109283282 A CN109283282 A CN 109283282A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
- G01N2030/645—Electrical detectors electrical conductivity detectors
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Abstract
A kind of amino acid analysis method detected in a low voltage state using electric conductivity detector, its processing step include: 1. by eluent low-lift pump input chromatography column, electric signal is converted by electric conductivity detector when the eluent exported from chromatography column flows through conductance flow cell, is surveyed and drawn to form baseline；2. the tested sample containing amino acid enters chromatography column under the promotion of eluent generates ion exchange, the hydrogen ion under positively charged amino acid replacement on chromatography column is simultaneously retained on chromatography column；3. eluent continuously to be inputted to chromatography column elution amino acid ion, electric signal is converted by electric conductivity detector when the solution of the acid ion containing amino exported from chromatography column flows through conductance flow cell, is surveyed and drawn to form spectrogram.Such method not only can be carried out good analysis to amino acid, and simple process, analysis cost are low.
Description
Technical field
The invention belongs to the analysis methods of amino acid, in particular to a kind of to be carried out in a low voltage state using electric conductivity detector
The amino acid analysis method of detection.
Background technique
Analysis in relation to amino acid starts from twentieth century three, the forties.The forties later period, Moore and Stein will be from
Sub-exchange resin in amino acid analysis, and had developed in 1958 in the world First using ion-exchange chromatography and
The automatic dedicated amino-acid analyzer of the derivative detection method of column deutomerite triketone, since then, the above method and necessary instrument are always in amino
Occupy an leading position in acid analysis: since the seventies, high performance liquid chromatography is especially bonded reverse-phase chromatography and column front derivation technology
Development, new method is provided for amino acid analysis, and develop a series of derivating agents in addition to saving triketone.Though above method
It so solves the quantitative analysis of amino acid, but requires just to can be carried out detection using derivatization reaction, and wrap in its processing step
Gradient elution is included, is operated very loaded down with trivial details: in addition, all need to complete to detect in 2000~4000psi high pressing operation.
Summary of the invention
The present invention is examined using electric conductivity detector in a low voltage state in view of the shortcomings of the prior art, providing one kind
The new method of the amino acid analysis method of survey, such method both can guarantee analytical effect, and can simplification of flowsheet and reduction point
Analyse cost.
The technical scheme is that having the both sexes ionization characteristics according to base again according to amino existing in amino acid molecular, dividing
The property of sub- side-chain radical and the pH range of isoelectric point, using cation-exchange chromatography design technology step, processing step according to
Secondary includes baseline mapping, sample introduction and ion exchange, elution analysis.
1, baseline is surveyed and drawn
Eluent is conveyed into chromatography column with the low-lift pump that operating pressure is 30~40Psi and reaches balance, from
Electric signal is converted by electric conductivity detector when the eluent of chromatography column output flows through conductance flow cell, to form baseline quilt
Mapping.
Tested sample containing amino acid enters chromatography column under the promotion of eluent and generates ion exchange, positively charged
Hydrogen ion under the amino acid replacement of lotus on chromatography column is simultaneously retained on chromatography column.
3, elution analysis
Chromatography column is continuously conveyed in eluent constantly various amino acid are unfolded on chromatography column, various ammonia
Base acid ion generates differential migration and is separated from each other, and is eluted in different retention times, and what is exported from chromatography column contains ammonia
Electric signal is converted by electric conductivity detector when the solution of base acid ion flows through conductance flow cell, is surveyed and drawn to form spectrogram.
Eluent is selected according to the acid-base property of amino acid, when analysis is acid and neutral amino acid, eluent use concentration for
The HN03 solution of 0.5X10-3mol/L~2.0Xl0-3mo1/L, analyze basic amino acid when, eluent use concentration for
The lemon of 6X10-3mol/L~10X10-3mol/L is according to sour potassium solution, to keep tested amino acid positively charged, with cationic shape
There are a for formula
The filler of chromatography column is the surface ion exchanger resin of low ion exchange capacity, preferably selection styrene _ divinyl
Base benzene high molecular polymer simultaneously controls exchange capacity in 0.01~0.04mmo1/g.The filler particle size of chromatography column is generally
10~30 μm, the diameter of section of chromatography column is generally 3~6mM, column length are generally 40~200mm.
The invention has the following advantages: 1, a variety of common amino acids can be carried out with good separation, the coefficient of variation 1
~5%, detection is limited up to 10-6g1mol, and in the concentration range of 10-3~10-5mol/L, peak height is in good line to concentration
Sexual intercourse.
2, due to being detected using electric conductivity detector, it is not necessary to carry out gradient elution to tested sample, thus simplify
Process flow.
3, when analyzing, sample is directly injected into chromatography column and spectrogram can be obtained through electric conductivity detector detection, is removed
Before column in existing analysis method or post-column derivation step, keep the process flow greatly simplified.
4, it is conveyed using low-lift pump, the cost of required mating analysis instrument is greatly lowered, and technique simplifies, and has both saved reagent
It is asked when can shorten analysis again, thus analysis cost is low, it is easy to popularize.
Detailed description of the invention
Fig. 1 is common acid, the neutral amino acid chromatogram that the method provided according to the present invention obtains；
Fig. 2 is the common alkali amino acid chromatogram that the method provided according to the present invention obtains；
Fig. 3 is surveyed amino acid His, Ala, Phe, Pro of the method acquisition provided according to the present invention in 10-3~10-
The relational graph of concentration and peak height in the concentration range of 4mo1/L；
Fig. 4 is surveyed amino acid Met, Pro, Glu of the method acquisition provided according to the present invention in 10-4~10-5mol/L
Concentration range in concentration and peak height relational graph.
Specific embodiment
Embodiment 1:
The present embodiment is analyzed common acid, neutral, basic amino acid, and sample used is the amino of known concentration
Acidity scale sample.
1) acid, neutral amino acid analysis
The instrument used: by method provided by the invention design amino-acid analyzer, operating pressure be 30~
40psi。
Eluent: 1.0X10-3mol/L nitric acid solution, flow velocity 1.2mol/min.
Chromatography divides Gao Zhu: filler is one divinylbenzene high molecular polymer of styrene, and exchange capacity 0.02mmol/g is filled out
25~301'm of material granularity, column section diameter 5mm, column length 200mm.
Analysis step is wanted:
A, baseline is surveyed and drawn
Amino-acid analyzer is operated, eluent is conveyed into chromatography column and reaches balance, it is defeated from chromatography column
Electric signal is converted by electric conductivity detector when eluent out flows through conductance flow cell, is surveyed and drawn to form baseline.
B, spectrogram is surveyed and drawn
By in the liquid flow path of the tested sample injection amino-acid analyzer containing amino acid, amino-acid analyzer is operated,
Make eluent that tested sample be pushed to enter chromatography column and generates ion exchange, chromatographic isolation under positively charged amino acid replacement
Hydrogen ion on column is simultaneously retained on chromatography column；
Chromatography column is continuously conveyed in eluent constantly various amino acid are unfolded on chromatography column, various ammonia
Base acid ion generates differential migration and is separated from each other, and asks and is eluted in different reservations, and what is exported from chromatography column contains ammonia
Electric signal is converted by electric conductivity detector when the solution of base acid ion flows through conductance flow cell, is surveyed and drawn to form spectrogram.Institute
The spectrogram of mapping is shown in Fig. 1.
2) analysis of basic amino acid
The instrument used: by method provided by the invention design amino-acid analyzer, operating pressure be 30~
40psl。
Eluent: 9.0X10-3mol/L lemon Specifications acid potassium solution, flow velocity 1.3m1/min.
Chromatography column: filler is one divinylbenzene high molecular polymer of styrene, and exchange capacity 0.02mmol/g is filled out
20~25 μm of material granularity, column section diameter 5mm, column length 40mm.
Analysis step ring is identical as Suan Xing, the analysis step ring of neutral amino acid.The spectrogram surveyed and drawn is shown in Fig. 2.
Embodiment 2:
The present embodiment is analyzed the acidity in protein hydrolysis injection, neutrality, basic amino acid.
1) acid, neutral amino acid analysis
The instrument used: by method provided by the invention design amino-acid analyzer, operating pressure be 30~
40Psi。
Eluent: 1.5X10-3mol/L nitric acid solution, flow velocity is l.2mol/min.
Chromatography divides Gao Zhu: filler be one divinylbenzene high molecular polymer of styrene, exchange capacity 0.015mmol/g,
25~30l'm of filler particle size, column section diameter 6mm, column length 150mm.
Analysis step ring is same as Example 1.
2) analysis of basic amino acid
The instrument used: by method provided by the invention design amino-acid analyzer, operating pressure be 30~
40psi。
Eluent: 8.0X10-3mol/L lemon Specifications acid potassium solution, flow velocity 1.3m/min.
Chromatography column: filler be one divinylbenzene high molecular polymer of styrene, exchange capacity 0.015mmol/g,
20~251'm of filler particle size, column section diameter 6mm, column length 50mm.
Analytical procedure is identical as Suan Xing, the analysis step ring of neutral amino acid.
It is following (mol/L) to analyze result:
Asp7.29X10-4Pro1.16X10-3Ya9.75x10-4phe3.12x10-3
His2.20X10-3Lys1.88x10-4Arg8.06x10-4
Embodiment 3:
The present embodiment is analyzed the acidity in amino acids oral-liquor, neutrality, basic amino acid.
1) acid, neutral amino acid analysis
The instrument used: by method provided by the invention design amino-acid analyzer, operating pressure be 30~
40psl。
Eluent: 0.8X10-3mo1/L nitric acid solution, flow velocity 1.2mol/min.
Chromatography column: filler is one divinylbenzene high molecular polymer of styrene, and exchange capacity 0.03mmol/g is filled out
Material granularity 25~30l, m, column section diameter 5mm, column length 180mm.
Analysis step ring is same as Example 1.
2) analysis of basic amino acid
The instrument used: by method provided by the invention design amino-acid analyzer, operating pressure be 30~
40Psi。
Eluent: 8.5X10-3mo1/L lemon Specifications acid potassium solution, flow velocity is l.3murnin.
Chromatography column: filler is one divinylbenzene high molecular polymer of styrene, and exchange capacity 0.03mmol/g is filled out
20~25 μm of material granularity, column section diameter 5mm, column length 45mm.
Analysis step ring is identical as Suan Xing, the analytical procedure of neutral amino acid.
It is following (mol/L) to analyze result:
Asp8.33X10-4Glu5.48Xl0-4Ya5.00xl0-4Met3.50x10-4
Phe6.60Xl0-3His3.48X10-3Lys2.50X10-4Arg7.64X10-4
The concentration for the partial amino-acid surveyed in above-described embodiment and the relationship of peak height are shown in Fig. 3, Fig. 4.
Claims (6)
1. a kind of amino acid analysis method detected in a low voltage state using electric conductivity detector, it is characterised in that technique step
Suddenly successively include:
1) baseline is surveyed and drawn
Eluent is conveyed into chromatography column with the low-lift pump that operating pressure is 30~40Psi and reaches balance, from chromatography
Electric signal is converted by electric conductivity detector when the eluent of splitter output flows through conductance flow cell, so that it is tested to form baseline
It draws,
2) sample introduction and ion exchange
Tested sample containing amino acid enters chromatography column under the promotion of eluent and generates ion exchange, positively charged
Hydrogen ion under amino acid replacement on chromatography column is simultaneously retained on the high column of chromatography point,
3) elution analysis
Chromatography column is continuously conveyed in eluent constantly various amino acid are unfolded on chromatography column, various amino acid
Ion generates differential migration and is separated from each other, and asks and is eluted in different reservations, contains amino acid from what the high column of chromatography point exported
Electric signal is converted by electric conductivity detector when the solution of ion flows through conductance flow cell, is surveyed and drawn to form spectrogram.
2. the analysis method of amino acid according to claim 1, it is characterised in that:
1) when analysis acidity is with neutral amino acid, eluent uses concentration for 0.5X10-3mol/L~2.0x10-3mol/L's
HN03 solution, when analyzing basic amino acid, eluent uses concentration for the potassium citrate of 6x10-3mo1/L~10x10-3mol/L
Solution,
2) filler of chromatography column is the surface ion exchanger resin of low ion exchange capacity.
3. the analysis method of amino acid according to claim 2, it is characterised in that the surface ion of low ion exchange capacity exchanges
Resin is one divinylbenzene high molecular polymer of styrene.
4. the analysis method of amino acid according to claim 2 or 3, it is characterised in that the exchange of the filler of chromatography column
Capacity is 0.01~0.04mmo1/g.
5. the analysis method of amino acid according to claim 1 or 2 or 3, it is characterised in that the filler grain of chromatography column
Degree is 10~30 μm, and the diameter of section of chromatography column is 3~6mm, and column length is 40~200mm.
6. the analysis method of amino acid according to claim 4, it is characterised in that the filler particle size of chromatography column is 10
~30 μm, chromatography divides the diameter of section of Gao Zhu to be 3~6mm, and column length is 40~200mm.
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| CN201810486862.8A CN109283282A (en) | 2018-05-18 | 2018-05-18 | A kind of amino acid analysis method detected in a low voltage state using electric conductivity detector |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86100091A (en) * | 1986-01-11 | 1987-09-02 | 成都科技大学 | The chromatography of ions of analysis of anions and instrument |
| CN1038522A (en) * | 1988-06-09 | 1990-01-03 | 成都科技大学 | Low-voltage ion chromatograph for analyzing cation |
| CN2119659U (en) * | 1991-12-04 | 1992-10-21 | 成都科技大学 | Low-pressure ion chromatograph for analysis of transition metal |
| CN2585231Y (en) * | 2002-12-03 | 2003-11-05 | 四川大学 | Low-pressure ion chromatograph for analysing amino acid |
| CN1172189C (en) * | 2002-12-03 | 2004-10-20 | 四川大学 | Analysis method of amino acid |
-
2018
- 2018-05-18 CN CN201810486862.8A patent/CN109283282A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86100091A (en) * | 1986-01-11 | 1987-09-02 | 成都科技大学 | The chromatography of ions of analysis of anions and instrument |
| CN1038522A (en) * | 1988-06-09 | 1990-01-03 | 成都科技大学 | Low-voltage ion chromatograph for analyzing cation |
| CN2119659U (en) * | 1991-12-04 | 1992-10-21 | 成都科技大学 | Low-pressure ion chromatograph for analysis of transition metal |
| CN2585231Y (en) * | 2002-12-03 | 2003-11-05 | 四川大学 | Low-pressure ion chromatograph for analysing amino acid |
| CN1172189C (en) * | 2002-12-03 | 2004-10-20 | 四川大学 | Analysis method of amino acid |
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Application publication date: 20190129 |