CN101762665B - Method for measuring tyrosine, phenylalanine and tryptophane in wort, fermentation liquid and beer - Google Patents
Method for measuring tyrosine, phenylalanine and tryptophane in wort, fermentation liquid and beer Download PDFInfo
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- CN101762665B CN101762665B CN2008101875965A CN200810187596A CN101762665B CN 101762665 B CN101762665 B CN 101762665B CN 2008101875965 A CN2008101875965 A CN 2008101875965A CN 200810187596 A CN200810187596 A CN 200810187596A CN 101762665 B CN101762665 B CN 101762665B
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- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 title claims abstract description 42
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 title claims abstract description 42
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 title claims abstract description 41
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 239000006035 Tryptophane Substances 0.000 title claims abstract description 41
- 229960004799 tryptophan Drugs 0.000 title claims abstract description 41
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 title claims abstract description 40
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 239000007788 liquid Substances 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 31
- 235000013405 beer Nutrition 0.000 title claims abstract description 27
- 238000000855 fermentation Methods 0.000 title claims abstract description 19
- 230000004151 fermentation Effects 0.000 title claims abstract description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 239000003643 water by type Substances 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 16
- 241000209140 Triticum Species 0.000 claims description 15
- 235000021307 Triticum Nutrition 0.000 claims description 15
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 abstract description 8
- 239000000337 buffer salt Substances 0.000 abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 5
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 238000009795 derivation Methods 0.000 abstract description 2
- 238000004811 liquid chromatography Methods 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract description 2
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 abstract 1
- 238000002372 labelling Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 32
- 150000001413 amino acids Chemical class 0.000 description 11
- 239000000243 solution Substances 0.000 description 8
- 230000003595 spectral effect Effects 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000001212 derivatisation Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- BDKLKNJTMLIAFE-UHFFFAOYSA-N 2-(3-fluorophenyl)-1,3-oxazole-4-carbaldehyde Chemical compound FC1=CC=CC(C=2OC=C(C=O)N=2)=C1 BDKLKNJTMLIAFE-UHFFFAOYSA-N 0.000 description 2
- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-hydroxyphenyl)ethanol Chemical compound OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 238000004886 process control Methods 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 229940087562 sodium acetate trihydrate Drugs 0.000 description 2
- MBBOMCVGYCRMEA-UHFFFAOYSA-N tryptophol Chemical compound C1=CC=C2C(CCO)=CNC2=C1 MBBOMCVGYCRMEA-UHFFFAOYSA-N 0.000 description 2
- DBLDQZASZZMNSL-QMMMGPOBSA-N L-tyrosinol Natural products OC[C@@H](N)CC1=CC=C(O)C=C1 DBLDQZASZZMNSL-QMMMGPOBSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 235000004330 tyrosol Nutrition 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method utilizing HPLC (high performance liquid chromatography) to measure tyrosine, phenylalanine and tryptophane in wort, fermentation liquid and beer. The method comprises the following procedures: the preparation of a standard sample; the procedure for pre-treating the beer to be measured; the procedure for gradient elution; and the procedure for the comparison detecting of the liquid to be measured and a spectrogram of the standard sample, wherein a mobile phase A and a mobile phase D in the procedure for gradient elution are respectively trifluoroacetic acid water solution (0.01%) and acetonitrile; the procedure for gradient elution carried out on a Waters AtlantisC18 (250mm*46mm, id) chromatographic column includes seven steps, the flowing speed of each step is 1.0ml/min, and the proportion conversion of the mobile phase is carried out in isogradient and linear manners. Tyrosine and tryptophane are detected at 280nm, and phenylalanine and tryptophane are detected at 210nm. The invention can directly carrying out measuring without derivation, which not only avoids an expensive as well as unstable derivating agent, but also avoids buffer salts that greatly damage a liquid chromatography system; as the pH value of the mobile phase does not need to be adjusted, the operation is simple; moreover, the labeling recovery rate of the method is within 99.5 to 100.5%, the reproducibility % RSD (n=6) is lower than or equal to 1.8%, and the minimal detection limit is 0.1mg/L.
Description
Technical field
The present invention relates to the beer technical field, and the method for tyrosine, phenylalanine and tryptophane in high-performance liquid chromatogram determination wheat juice, fermentation liquor and the beer is adopted in definite saying so.
Background technology
Tyrosine, phenylalanine and tryptophane all belong to amino acid, derive from wheat juice at first, are the important nutriments of yeast.Yeast forms corresponding higher alcohol to the metabolism meeting of this three seed amino acid, and the same respectively tyrosol of tyrosine, phenylalanine and tryptophane, bata-phenethyl alcohol are corresponding with tryptophol.These three kinds of alcohol are the important flavor substances of beer, and the content that detects this three seed amino acid to the generation of control beer flavoring substances highly significant.And the content of tyrosine, phenylalanine and tryptophane can reflect the dissolution degree of Fructus Hordei Germinatus, helps making the control that becomes more meticulous of wheat process.In addition, the variation of its content before and after fermentation can also reflect that yeast to amino acid whose assimilation situation, embodies the fermentation activity of yeast.It is thus clear that the content of this three seed amino acid is significant for the process control of Beer Production.
Most amino acid detects mostly need derive, and not only cost is very high, detection time is very long, and requires also very high to each item of system.
Because tyrosine, phenylalanine and tryptophane have ultraviolet absorption characteristic, can directly use the liquid-phase chromatography method sample detection in theory.To the detection of this three seed amino acid, have method adopt pH be 2.80 0.1M potassium dihydrogen phosphate aqueous solution as moving phase, detect tyrosine, phenylalanine and tryptophane in the amino acids oral-liquor.Because potassium dihydrogen phosphate is a kind of buffer salt, heavier to the liquid chromatographic system damage, be prone to cause system jams, the carrying out of impact analysis.Application for method is very unfavorable.Also need accurately to regulate the pH value of moving phase, operate comparatively loaded down with trivial details.
And in the beer this three seed amino acid detection all be to adopt before the post or the derivatization method behind the post, do not have the report of direct determination method.The accuracy of derivatization method receives the influence of derivating agent stability.And all to use the sodium acetate trihydrate solution that accurately to regulate the pH value with the post-column derivation method before these posts and make moving phase.Sodium acetate trihydrate solution equally also is a kind of buffer salt, and system is had damage.In addition, because these derivatization methods also will use acetonitrile as organic phase, if buffer salt and acetonitrile use simultaneously, system receives just polluting and results in blockage more easily.Therefore, these method costs are higher, complex operation, and it is very long to expend time in, and detects the time of a sample and wants mostly about 1 hour.
Summary of the invention
For fear of the amino acid derived reagent, the buffer salt that adopt various costlinesses; Thereby reduce buffer salt to the infringement of chromatographic system and practice thrift analysis time; The invention provides a kind of liquid direct injected to be measured, quick and precisely detect high performance liquid chromatography (the High Performance Liquid Chromatography of tyrosine, phenylalanine and tryptophane in wheat juice, fermentation liquor and the beer; Be called for short HPLC) method, to the process control of Beer Production highly significant.
The method of tyrosine, phenylalanine and tryptophane comprises in mensuration wheat juice of the present invention, fermentation liquor and the beer:
(1) standard specimen preparation procedure: configuration tyrosine, phenylalanine and tryptophane standard solution;
(2) liquid pre-treatment program to be measured: beer or fermentation liquor filter after exhaust, sample introduction after the wheat juice direct filtration;
(3) respectively to the gradient elution program of standard specimen and liquid to be measured: mobile phase A: 0.01% trifluoroacetic acid aqueous solution; Moving phase D: acetonitrile;
(4) liquid to be measured and standard specimen spectrogram comparison and detection: liquid to be measured and the contrast of standard specimen spectrogram, qualitative with retention time, chromatographic peak area is quantitative.
In more detail, in (1) standard specimen configurator, tyrosine, phenylalanine and tryptophane standard reserving solution are respectively 158.76mg/L, 225.7mg/L and 143.28mg/L.
(2) liquid pre-treatment program to be measured: 0.22um syringe-type filter film is adopted in described filtration.
(3) respectively to the gradient elution program of standard specimen and liquid to be measured: (250mm*46mm, id) chromatographic column, the temperature of chromatographic column are set in 30 ℃, detect wavelength and fix on 280nm to adopt Waters Atlantis C18.
When carrying out gradient elution, at 0-6 minute, the scale dimension of mobile phase A was held in 95%, and the scale dimension of moving phase D is held in 5%; 6.0-6.5 minute, the ratio of mobile phase A is by 95% to 93%, and the ratio of moving phase D is changed to 7% by 5%, and this ratio remained to 22.0 minutes always; At 22.0-22.5 minute, the ratio of mobile phase A reduced to 40% by 93%, and the ratio of moving phase D rises to 60% by 7% variation, and this ratio lasted till 25.5 minutes with thorough cleaning chromatographic column since 22.5 minutes; At 25.5-26.0 minute, the ratio of mobile phase A was increased to 95% by 40%, and the ratio of moving phase D reduces to 5% by 60%; At 26.0-35.0 minute; The moving phase ratio remains on 95% A and 5% D always, when finishing in the 35th minute, accomplishes the whole gradient operation of a liquid to be measured; Each step flow velocity all is 1.0ml/ minute, and the conversion of moving phase ratio is carried out with linear mode such as degree such as grade, and promptly the rate of change of the rate of change of A and D equates, and always with the constant rate variation through calculating.
(4) liquid to be measured and standard specimen spectrogram comparison and detection.Liquid to be measured and the contrast of standard specimen spectrogram, qualitative with retention time, chromatographic peak area is quantitative.Computing formula is following:
In the formula: C
Appearance---the content of tyrosine, phenylalanine and tryptophane in the liquid to be measured, unit: mg/L;
C
Mark---the content of tyrosine, phenylalanine and tryptophane in the standard specimen, unit: mg/L;
A
Appearance---the chromatographic peak area of tyrosine, phenylalanine and tryptophane in the beer to be measured;
A
Mark---the chromatographic peak area of tyrosine, phenylalanine and tryptophane in the standard specimen.
The present invention adopts direct determination method, need not use expensive derivative reagent, and pH value that also need not regulator solution and there is the buffer salt of damage in system is simple to operate.Adopt the purpose of gradient elution to be to shorten analysis time in the liquid chromatography, improve separating effect.Contain thousands of kinds of chemical constitutions in the beer, form very complicatedly, the polarity difference between the component is very big.In order to practice thrift analysis time, improve separation efficiency and clean chromatographic column effectively each the analysis when finishing, make chromatographic column remain optimum condition, be necessary very much to carry out gradient elution.The gradient elution program that the present invention adopts can successfully be separated tyrosine, phenylalanine and the tryptophane in the beer at 20 minutes, and in 35 minutes, accomplishes whole gradient operation, and analysis fast, accurately.
Through above-described method; The method recovery of standard addition of making is 99.5%-100.5%; Reappearance %RSD (n=6)≤1.8%; Minimum detects and is limited to 0.1mg/L, thereby measures wheat juice accurately, reliably, tyrosine, phenylalanine and tryptophane in fermentation liquor and the beer, has very high sensitivity.
Description of drawings
Fig. 1 is that standard specimen separates spectrogram with liquid to be measured under 280nm.
The meaning of capitalization English letter representative wherein:
The A-fermentation liquor separates spectrogram
B-wheat juice separates spectrogram
C-beer separates spectrogram
D-standard specimen spectrogram
Fig. 2 is that standard specimen separates spectrogram with liquid to be measured under 210nm.
The meaning of capitalization English letter representative wherein:
The A-fermentation liquor separates spectrogram
B-wheat juice separates spectrogram
C-beer separates spectrogram
D-standard specimen spectrogram
Embodiment
(1) standard specimen preparation
Standard reserving solution: accurately take by weighing tyrosine 0.0162g respectively with balance; Phenylalanine 0.0228g; Tryptophane 0.0144g with the aqueous hydrochloric acid solution dissolving of 0.1M, and is settled to 100ml; The concentration that obtains tyrosine, phenylalanine and tryptophane standard reserving solution is 158.76mg/L, 225.7mg/L and 143.28mg/L.
Standard operation liquid: respectively get tyrosine, phenylalanine and the tryptophane standard reserving solution of 0.4ml, mixing obtains comprising the hybrid standard working fluid of this three seed amino acid.
(2) liquid pre-treatment to be measured
Beer or fermentation liquor are after exhaust, and 0.22um syringe-type filter film filters, and wheat juice is sample introduction after 0.22um syringe-type filter film filters directly.
(3) gradient elution program
3.1 selection chromatographic column:
Adopting the production of Waters company, brand is the C18 chromatographic column of Atlantis
(250mm*46mm，id)。It is the analytical approach of master's moving phase that this chromatographic column is highly suitable for adopting water, and on common C18 chromatographic column, keeping very weak component by force for polarity has good reservation and stalling characteristic.The temperature of chromatographic column is set in 30 ℃ during detection, and sample size is 10ul.The characteristic absorption wavelength of tyrosine is (220,274.5), because the interference of adjacent impurity peaks in the liquid to be measured under 220nm fixes on 280nm so it detects wavelength.The characteristic absorption wavelength of phenylalanine is (210), detects wavelength 210nm.The characteristic absorption wavelength of tryptophane is (219.0,279.2), can detect with 280nm 210.Use 2996 PDADs of Waters company during detection.
3.2 the preparation of moving phase
Mobile phase A: get the trifluoroacetic acid of 100ul with liquid-transfering gun, add ultrapure water and be settled to 1L, obtain 0.01% trifluoroacetic acid aqueous solution.
Moving phase D: acetonitrile
All moving phases all need through 0.45 μ m filter membrane suction filtration, and use on the machine after 5 minutes with the ultrapure helium degassing.
3.3 gradient elution
See table 1.Waters 2695 high performance liquid chromatographs that adopt U.S. Waters company to provide.When carrying out gradient elution, at 0-6 minute, the scale dimension of mobile phase A was held in 95%, and the scale dimension of moving phase D is held in 5%; 6.0-6.5 minute, the ratio of mobile phase A is by 95% to 93%, and the ratio of moving phase D is changed to 7% by 5%, and this ratio remained to 22.0 minutes always; At 22.0-22.5 minute, the ratio of mobile phase A reduced to 40% by 93%, and the ratio of moving phase D rises to 60% by 7% variation, and this ratio lasted till 25.5 minutes with thorough cleaning chromatographic column since 22.5 minutes; At 25.5-26.0 minute, the ratio of mobile phase A was increased to 95% by 40%, and the ratio of moving phase D reduces to 5% by 60%; At 26.0-35.0 minute; The moving phase ratio remains on 95% A and 5% D always, when finishing in the 35th minute, accomplishes the whole gradient operation of a testing sample; Each step flow velocity all is 1.0ml/ minute, and the conversion of moving phase ratio is carried out with linear mode such as degree such as grade.Be that the rate of change of A and the rate of change of D equate, and always with the constant rate variation through calculating, i.e. curve 6 in the form.Curve 6 is the data processing software Empower of Waters company a kind of parameter settings to the moving phase commutation.Total 1-11 bar curve is available.And curve 6 is wherein the most frequently used a kind of forms.
Table 1: the gradient elution program of moving phase
(4) liquid to be measured and standard specimen spectrogram comparison and detection
Fig. 1: will detect with the standard specimen direct injected, and obtain the separation spectrogram under 280nm.Wherein spectral line A is a fermentation liquor to be measured, and spectral line B is a wheat juice to be measured, and spectral line C is a beer to be measured, and spectral line D is the hybrid standard of tyrosine, phenylalanine and tryptophane.Tyr on the spectrogram is the english abbreviation of tyrosine, and Phe is the english abbreviation of phenylalanine, and Trp is the english abbreviation of tryptophane.
Fig. 2: liquid to be measured and standard specimen direct injected are detected, obtain the separation spectrogram under 210nm.Wherein spectral line A is a fermentation liquor to be measured, and spectral line B is a wheat juice to be measured, and spectral line C is a beer to be measured, and spectral line D is the hybrid standard of tyrosine, phenylalanine and tryptophane.Tyr on the spectrogram is the english abbreviation of tyrosine, and Phe is the english abbreviation of phenylalanine, and Trp is the english abbreviation of tryptophane.
Liquid to be measured and the contrast of standard specimen spectrogram, qualitative with retention time, chromatographic peak area is quantitative.Computing formula is following:
In the formula: C
Appearance---the content of tyrosine, phenylalanine and tryptophane in the liquid to be measured, unit: mg/L;
C
Mark---the content of tyrosine, phenylalanine and tryptophane in the standard specimen, unit: mg/L;
A
Appearance---the chromatographic peak area of tyrosine, phenylalanine and tryptophane in the beer to be measured;
A
Mark---the chromatographic peak area of tyrosine, phenylalanine and tryptophane in the standard specimen;
The reagent that is adopted in the above-mentioned steps is following:
The trifluoroacetic acid of reagent: DIKMA (enlightening horse) company (analyze pure), the tyrosine (98% of Aldrich (Sigma) company; Chromatographically pure), phenylalanine (99%; Chromatographically pure) and tryptophane (99.5%, chromatographically pure), the acetonitrile (chromatographically pure) of Burdick & Jackson company; The hydrochloric acid of Nanjing chemical reagents corporation (37%, analyze pure).
Claims (3)
1. method that adopts tyrosine, phenylalanine and tryptophane in high-performance liquid chromatogram determination wheat juice, fermentation liquor and the beer; Comprise standard specimen preparation, liquid pre-treatment program to be measured, gradient elution program and liquid to be measured and standard specimen spectrogram comparison and detection program, it is characterized in that being formulated as of gradient elution program moving phase: mobile phase A: 0.01% trifluoroacetic acid aqueous solution; Moving phase D: acetonitrile; Said liquid pre-treatment program to be measured is: after 0.22um syringe-type filter film filters, wheat juice is sample introduction after 0.22um syringe-type filter film filters directly through exhaust for beer or fermentation liquor; The gradient elution program is: at 0-6 minute, the scale dimension of mobile phase A was held in 95%, and the scale dimension of moving phase D is held in 5%; 6.0-6.5 minute, the ratio of mobile phase A is by 95% to 93%, and the ratio of moving phase D is changed to 7% by 5%, and this ratio remained to 22.0 minutes always; At 22.0-22.5 minute, the ratio of mobile phase A reduced to 40% by 93%, and the ratio of moving phase D rises to 60% by 7% variation, and this ratio lasted till 25.5 minutes with thorough cleaning chromatographic column since 22.5 minutes; At 25.5-26.0 minute, the ratio of mobile phase A was increased to 95% by 40%, and the ratio of moving phase D reduces to 5% by 60%; At 26.0-35.0 minute; The moving phase ratio remains on 95% A and 5% D always, when finishing in the 35th minute, accomplishes the whole gradient operation of a testing sample; Each step flow velocity all is 1.0ml/ minute, and the conversion of moving phase ratio is carried out with linear mode such as degree such as grade.
2. the method for tyrosine, phenylalanine and tryptophane in high-performance liquid chromatogram determination wheat juice according to claim 1, fermentation liquor and the beer; It is characterized in that said gradient elution program adopts the Atlantis C18 chromatographic column of Waters company; The temperature of chromatographic column is set in 30 ℃, detects wavelength and fixes on 280nm.
3. according to the method for tyrosine, phenylalanine and tryptophane in the described high-performance liquid chromatogram determination wheat of claim 1 juice, fermentation liquor and the beer, it is characterized in that liquid to be measured and standard specimen spectrogram comparison and detection computing formula are following:
In the formula: C
Appearance---the content of tyrosine, phenylalanine and tryptophane in the liquid to be measured, unit: mg/L;
C
Mark---the content of tyrosine, phenylalanine and tryptophane in the standard specimen, unit: mg/L;
A
Appearance---the chromatographic peak area of tyrosine, phenylalanine and tryptophane in the beer to be measured;
A
Mark---the chromatographic peak area of tyrosine, phenylalanine and tryptophane in the standard specimen.
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| CN108896679A (en) * | 2018-07-18 | 2018-11-27 | 精晶药业股份有限公司 | A kind of efficient liquid phase detection method of l-tyrosine |
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| CN1825111A (en) * | 2006-03-21 | 2006-08-30 | 扬州大学 | P-acetamido benzene sulfonyl fluorine column front derivation of amino-acid in beer and capillary electrophoresis quantitative detecting method |
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| CN1825111A (en) * | 2006-03-21 | 2006-08-30 | 扬州大学 | P-acetamido benzene sulfonyl fluorine column front derivation of amino-acid in beer and capillary electrophoresis quantitative detecting method |
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