CN102621239A - High performance liquid chromatography determination method for volatile phenolic compounds in white spirits - Google Patents

High performance liquid chromatography determination method for volatile phenolic compounds in white spirits Download PDF

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CN102621239A
CN102621239A CN2011103915723A CN201110391572A CN102621239A CN 102621239 A CN102621239 A CN 102621239A CN 2011103915723 A CN2011103915723 A CN 2011103915723A CN 201110391572 A CN201110391572 A CN 201110391572A CN 102621239 A CN102621239 A CN 102621239A
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schardinger dextrin
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杨敏
寻思颖
冯永渝
彭黔荣
董睿
耿平兰
孙棣
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GUIZHOU PROVINCE PRODUCT QUALITY SUPERVISION AND INSPECTION INSTITUTE
Guizhou University
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Guizhou University
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Abstract

The invention discloses a high performance liquid chromatography determination method for volatile phenolic compounds in white spirits. By adopting a high performance liquid chromatogram-fluorescence detector (HPLC-FLD) technique to analyze volatile phenolic compounds in white spirits, the method of the invention, when used in analysis of volatile phenolic materials, can determine the following 10 volatile phenolic compounds of phenol, guaiacol, p-cresol, m-cresol, o-cresol, 4-methylguaiacol, 4-vinylphenol, 4-ethylphenol, 4-vinylguaiacol and 4-ethylguaiacol in white spirits. And the method has the advantages of no need for derivatization, high accuracy, high sensitivity and good repeatability.

Description

The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor
Technical field
The present invention relates to the high-performance liquid chromatogram determination method of Volatile Phenols compounds in a kind of liquor, belong to liquor detection technique field.
Background technology
Usually contain some volatility phenols materials in the alcoholic drink.They have come to light and have been present in beer, and grape wine also has in the Spirit.The Volatile Phenols compounds is the important aroma constituent of alcoholic drink, because aspects such as this type component is fragrant for the news of wine, taste and stability all have important effect, has therefore caused many researchers' attention.
Food fermentation research institute of domestic Ministry of Light Industry was main research object with Maotai in 1980; Adopt chemical derivatization to combine GC-FID/GC-MS in the analysis and research that Volatile Phenols unit compound carries out in the liquor; Pre-treatment is more loaded down with trivial details; Need the derivatization step, and wherein the recovery of standard addition of 4-methyl guaiacol and 4,4-ethyl guaiacol has only about 40%.
Nineteen ninety University Of Qingdao, Chinese medical courses in general institute medicine grind and the diligent sweet-smelling grass in Shandong University land, Liu Chunsheng, Yan Changtai extract 6 kinds of volatile phenols (University Of Qingdao's journal, 1990,3 (1) in the liquor with solvent extraction; 102～105), use to simulate wine appearance 500mL, through adding the anti-oxidant sodium thiosulfite; Add 2M NaOH solution and regulate PH=12.5; Make phenol form phenates and get into water, ethanol is removed in distillation, uses CH 2Cl 2Alkalescence and neutral compound are removed in extraction, use 4M HCl and 6%NaHCO again 3The WS is regulated PH=8.0, uses CH at last 2Cl 2Extraction phenol carries out GC-FID and analyzes.It is thus clear that its pre-treatment step is loaded down with trivial details; And only test simulating wine appearance, 6 kinds of volatile phenol average recovery rates are 79%, the recovery of phenol is 65%; 6 kinds of average RSD values of volatile phenol are 6.1%; Therefore can not reach the detection method recovery is 80～120%, and the analyzing detecting method of Volatile Phenols in the liquor is not set up in the requirement of RSD value＜5%.
The Zhu Yan of bioengineering institute of Southern Yangtze University in 2010. the model document received. Xu Yan. use and immerse SPME (DI-SPME) and gas chromatography-mass spectrography (GC-MS) technology (free phenolic compound in DI-SPME and the GC-MS analysis liquor; Food and fermentation industries; 2010; 36 (10): 138-143) Volatile Phenols compounds in the liquor is analyzed, this method recovery reaches 85%～115%.But adopt DI-SPME and GC-MS technology, equipment is expensive, and operation is had relatively high expectations, and promote the wider application to be limited to, and this method has only been analyzed 7 kinds of Volatile Phenols compounds.
Summary of the invention
The objective of the invention is to; The high-performance liquid chromatogram determination method of Volatile Phenols compounds in a kind of liquor is provided; Promptly set up high performance liquid chromatography-fluorescence detector and analyze the method for volatility phenols material in the China white wine, be used for China's liquor volatility phenols species analysis, can measure 10 kinds of Volatile Phenols compounds in the liquor simultaneously; Do not need derivatization, accuracy height, sensitivity and good reproducibility.
Technical scheme of the present invention.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor, gets 50～100mL wine appearance, uses CH 2Cl 2Or CHCl 3Carry out liquid-liquid extraction, the consumption of each extractant is that the percent by volume of wine appearance is 20～30vol%, extracts 3 times; Merge organic phase and form extract, the extract of gained, the about 15min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions; Flow out to no longer including distillate; Concentrate is settled to 25 or 50mL with 1% glacial acetic acid, cross 0.45 μ m filter membrane after, supply the analysis of high-efficient liquid phase color spectrometer;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Moving phase is made up of mobile phase A and Mobile phase B, mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3-8.81 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate 0min: contain mobile phase A 80%, Mobile phase B 20%; 30min: mobile phase A 40%, Mobile phase B 60%; 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm;
Concentration with 10 kinds of phenol of external standard method quantitative Analysis.
The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, the beta-schardinger dextrin-concentration of the said beta-schardinger dextrin-WS is: 7.05 * 10 -3-8.81 * 10 -3Mol/L.
The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor; Said extract adding 1-3% glacial acetic acid solution or ultrapure water are as the restraining barrier; Rotary evaporation concentrates again, and the addition on restraining barrier is that the percent by volume of wine appearance is 10-20vol%.
The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, said restraining barrier are 1% glacial acetic acid solution.
The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, said wine appearance adds ultrapure water or absolute ethyl alcohol is adjusted into the 45-55 degree with its alcohol concentration.
The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, said wine appearance adds ultrapure water or absolute ethyl alcohol is adjusted into the 52-53 degree with its alcohol concentration.
The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, volatility phenols material is in the said liquor: phenol, guaiacol, p-Cresol ,-10 kinds of Volatile Phenols compounds such as cresols, neighbour-cresols, 4-methyl guaiacol and 4,4-vinylphenol, 4-ethyl-phenol, 4-vinyl guaiacol and 4-ethyl guaiacol.
The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, said liquor is: the distinctive a kind of Spirit of China.With bent type, distiller's yeast is saccharifying ferment, utilizes starchy material, through boiling, saccharification, fermentation, distillation, ageing with blend and all kinds of wine that brew forms.
The inventor has carried out a large amount of experiments in order to adopt the mensuration of high performance liquid chromatography-fluoroscopic examination (HPLC-FLD) method to volatility phenols material in the China white wine, and is specific as follows:
1.1 instrument and reagent
Agilent 1100 high performance liquid chromatographs; Agilent 1100 fluorescence detectors; Mttler AE200 electronic balance (sensibility reciprocal: 0.1mg); MILLIPORE Direct Q5 type ultrapure water system; Rotary Evaporators (the inferior flourish biochemical instrument in Shanghai), 250ml separating funnel (Tianjin Glass Instrument Factory).
Acetate (analyze pure, Kingsoft, Chengdu chemical reagent company limited), acetonitrile (chromatographically pure, MERCK company); Methylene chloride (analyze pure, Tianjin Fu Yu Fine Chemical Co., Ltd), and ethanol (95%, analyze pure; 1Guanghua Chemical Plant Co., Ltd., Guangdong), beta-schardinger dextrin-(biochemical reagents, Tianjin Kermel Chemical Reagent Co., Ltd.), guaiacol; The 4-vinyl guaiacol, 4-vinylphenol, 4-methyl guaiacol and 4,4-ethyl guaiacol (Aldrich company); 4-ethyl-phenol (Fluka company), adjacent, p-Cresol (Long Huagongshijichang of Chengdu section) ,-cresols (Shanghai Ke Feng chemical reagent company limited); Phenol (the luxuriant industry chemical reagent in Chongqing company limited), vinasse, Daqu (Guizhou Province Maotai Zhen bumpkin brewery), size bent (Guizhou Dong Jiu incorporated company); Vinasse (Renhuai, Guizhou Province Maotai Zhen honored guest brewery), liquor: Guizhou Province Maotai Zhen bumpkin brewery provides, commercially available liquor.), ultrapure water (from MILLIPORE Direct Q5 type ultrapure water system).
1.2 experimental implementation
1.2.1 pre-treating method
Get 50～100mL wine appearance, use CH 2Cl 2Or CHCl 3Carry out liquid-liquid extraction, the consumption of each extractant is that the percent by volume of wine appearance is 20～30vol%, extracts 3 times; Merge organic phase and form extract, gained extract, the about 15min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions; Or add wine appearance amount 10～20vol%1～3% glacial acetic acid solution or ultrapure water concentrates as the restraining barrier again, till no longer including distillate and flowing out, concentrate is settled to 25 or 50mL with 1% glacial acetic acid solution; Cross 0.45 μ m filter membrane, supply HPLC to analyze.
1.2.2HPLC chromatographic condition
Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS (1/100); Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS (1/40/60), beta-schardinger dextrin-concentration is: 7.05 * 10 -3-8.81 * 10 -3Mol/L; Gradient elution: 0min:A 80%, B 20%; 30min:A40%, B 60%; 40min:A30%, B 70%; Chromatographic column: Synergi 4u Hydro-RP 80A (150 * 4.6mm, phenomenex company); Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L; Fluorescence detector condition: Ex=270nm, Em=315nm.
With 10 kinds of phenolic compound standard items mixed solutions; Set up concentration range and be concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound standard items of 0.02～4.0 μ g/mL; Peak area with 10 kinds of phenol being surveyed in the HPLC sample introduction; Go out the concentration of 10 kinds of corresponding phenol with the external standard method quantitative Analysis, be converted into former concentration of getting 10 kinds of phenol in the wine appearance again.
2 results and discussion
2.1 detect the selection of wavelength and detecting device
The fluorescence principle: the fluoroscopic examination of organic compound has two important parameters: excitation wavelength and emission wavelength, and only within the certain wavelengths scope, organism just might produce fluorescence, and the required wavelength coverage of different compounds is also inequality.
If energy level from high to low 1,2,3.Energy is that the photon of E13 is energized into energy level 1 to particle from energy level 3; Be called and excite 1 this excited state relaxation a period of time of energy level (general 1 to 10 nanosecond), the change of fluorescence molecule meeting occurred conformation in this section relaxation time; And possibly interact with surrounding environment, make:
A. the part particle on the excited state, its energy is dissipated by part, and this part particle is transferred on the lower energy level 2.
B. because the part particle is transferred, make that the population on the energy level 1 reduces, still this part particle on energy level 1 is with spontaneous radiation resilience level 3.
C. that part of particle that is transferred on the energy level 2 is the photon of E23 with the spontaneous radiation energy, returns energy level 3, and this part energy is that the photon of E23 is called fluorescent emission, and its light frequency and corresponding photon energy are less than frequency that excites and energy.
The relation of fluorescence intensity and solution concentration: fluorescence is to be gone out by emission behind the material absorbing luminous energy, and therefore, the degree of fluorescence intensity of solution F and solution absorption luminous energy and the fluorescence frequency of material are relevant:
F∝(I o-I t)→F＝K′(I o-I t)
I t＝I o10 -ebc
K ' is a constant, depends on the quantum efficiency φ of fluorescent material, according to the L-B law:
F＝K′(I o-I o10 -ebc)＝K′I o(1-10 -ebc)＝K′I o(1-e -2.303ebc)
e x = 1 + x + x 2 2 ! + Λ + x n n ! + Λ
When 2.303ebc≤0.05 (concentration is very little, when solution is rarer), e -2.303ebc≈ 1-2.303ebc,
F＝K′I o2.303ebc
For the lean solution of a fluorescent material, work as I oAnd b one regularly, and promptly when low concentration (2.303ebc≤0.05), the concentration of fluorescence intensity of solution and fluorescence is linear proportional relation to F=Kc.
Influence the extraneous factor of fluorescence intensity:
Excitation source: generally select λ for use ExMaximum.
Temperature: most of molecules are when temperature raises, and the collision frequency between molecule and the molecule, between molecule and the solvent molecule raises, and non-radiative energy shifts and raises, and F reduces, and therefore, reduce temperature, help improving F.
PH value of solution: the fluorescence of fragrance group compound that has acidity or alkaline cyclic substituents is generally all relevant with the pH value, and some compound does not show fluorescence when ionic condition.For this reason, when carrying out quantitative measurement with fluorescence intensity, should strict control pH value of solution value.
The fluorescence exciting wavelength of phenolic compound and emission wavelength all have a maximal value, and under this wavelength condition, the fluorescence intensity of compound is maximum, and detection sensitivity is the highest.Great majority have the compound of fluorescence at the 230nm place absorption to be arranged, so be made as excitation wavelength 230nm and be made in the line sweep emission spectrum, set the emission wavelength when measuring then, carry out the scanning of excitation wavelength again.The optimum detection wavelength of each Volatile Phenols is seen table 2.1.
The optimum detection wavelength of table 2.114 kind of Volatile Phenols
Figure BDA00001148423800061
The result shows that most of Volatile Phenols are 270nm in excitation wavelength, and emission wavelength is that the 315nm place has optimal response, selects this wavelength for detecting wavelength.
Contrasted the sensitivity of fluorescence (FLD) in the experiment with diode array (DAD) detecting device; With concentration is above-mentioned 10 kinds of phenolic compound mixed standard solutions of 1 μ g/mL; Under same chromatographic separation condition, (Ex=270nm Em=315nm) measures to use PDAD (280nm) and fluorescence detector respectively; Find that it is 17.3～43.0 that PDAD is measured the detectability signal to noise ratio (S/N ratio) of 10 kinds of Volatile Phenols; The detectability signal to noise ratio (S/N ratio) that fluorescence detector is measured is 35.5～317.6, and both compare, and fluorescence response exceeds 1.33～8.80 times than the response of diode array ultraviolet.Explanation is in the mensuration to phenolic compound, and fluorescence detector is higher than PDAD sensitivity.Because the volatile phenol concentrations presented in the liquor is lower; High sensitivity helps accurate quantitative test, and considers complicated component in the liquor, and fluorescence detector has higher selectivity; Help getting rid of and disturb, for accurate qualitative the playing a key effect of target analytes.Take all factors into consideration, select fluorescence detector evaluating objects thing.
2.2 the selection of volatility phenols material
Having only food fermentation research institute of Ministry of Light Industry owing to the research of volatility phenols material in the liquor was research object with Maotai in 1980; The analysis of adopting chemical derivatization to combine GC-FID/GC-MS that Volatile Phenols compounds in the Maotai is carried out; From Maotai, detected 12 kinds of Volatile Phenols compounds: phenol, neighbour-cresols ,-cresols, p-Cresol, guaiacol, 4-methyl guaiacol and 4,4-ethyl guaiacol, 4-ethyl-phenol, 3-ethyl-phenol, 2-ethyl-phenol, 2,4-xylenol and a kind of isopropyl-phenol.After having consulted domestic and international pertinent literature report; The Volatile Phenols that will in alcoholic drink, detect is all listed range of choice in; In order to avoid omit; Have 14 kinds: phenol, guaiacol, p-Cresol ,-cresols, neighbour-cresols, 4-methyl guaiacol and 4,4-vinylphenol, 4-ethyl-phenol, 3-ethyl-phenol, 4-vinyl guaiacol, 2,4-xylenol, 4-ethyl guaiacol, 2-ethyl-phenol, eugenol.
Split and separate the 9th～13 peak through interpolation beta-schardinger dextrin-paracresol isomeride in moving phase, 5 easy overlap peaks are groped the chromatographic condition that 14 kinds of Volatile Phenols separate simultaneously.After the factors such as the concentration through changing the beta-schardinger dextrin-WS, the flow velocity of moving phase and gradient, 14 kinds of materials obtain 13 chromatographic peaks, see
Fig. 1--14 kinds of Volatile Phenols separate colors spectrograms.13 chromatographic peaks are among the figure:
1: phenol 2: guaiacol 3: p-Cresol 4 :-cresols 5: neighbour-cresols 6:4-methyl guaiacol and 4 7:4-vinylphenol 8:4-ethyl-phenol 9:3-ethyl-phenol 10:4-vinyl guaiacol 11:2,4-xylenol 12:4-guaethol and 2-ethyl-phenol 13: eugenol
Visible by figure, the fractionation of paracresol isomeride can be accomplished, but the peak overlap of 4-ethyl guaiacol and 2-ethyl-phenol.Continue to grope the method that both possibly separate.Li Wei etc. studied in 2009 pure in the presence of R, S-1, the cyclodextrin chiral recognition principle of 1 '-2-dinaphthol enantiomorph, mention in the literary composition: alcohol is organic modifier commonly used in liquid chromatography (especially reverse-phase chromatography) moving phase, also is the object solvent of polarity.Alcohol exists down; Different when envelope form when the chirality guest molecule forms inclusion complex with beta-schardinger dextrin-and nothing alcohol; Alcohol have the chiral selectivity of appreciable impact cyclodextrin to the mapping compound, and can change enthalpy or the not too favourable enantiomorph envelope of entropy into favourable envelope.Situation under (0.2% volume fraction), the existence of salt exist with alcohol is similar.Different alcohols can be to some extent by envelope in cyclodextrin cavity; Produce the inclusion complex of different stability, this influence depends primarily on hydrophobicity and lipophilicity effect, its influence degree and pure characteristic; The concentration of alcohol, the size of cyclodextrin cavity is relevant with the attribute of guest molecule.Increase along with contained C atom number in the alcohol; Alcohol is filled in the beta-schardinger dextrin-cavity more and more easily; And guest molecule is squeezed cavity, as far as containing the alcohol of identical C atom number, beta-schardinger dextrin-is more eager to excel in whatever one does than branched-chain alcoho to the envelope degree of branched-chain alcoho; Branched-chain alcoho more helps forming hydrogen bond with the terminal hydroxy group of cyclodextrin on spatial configuration, and alcohol is the essence of the two effect with the formation of beta-schardinger dextrin-intermolecular ydrogen bonding.
With reference to above content, attempt in the moving phase of having added beta-schardinger dextrin-, adding buffer salt and alcohol.Groped after different buffer salts, different alcohols and both different proportions in moving phase; Obtain that buffer salt biphosphate ammonium concentration is 40mmol/L in moving phase; Isopropanol content is 0.2% o'clock; The degree of separation of overlapping 4-ethyl guaiacol and 2-ethyl-phenol improves, but still can not separate fully, sees Fig. 2.14 chromatographic peaks are among the figure:
1: phenol 2: guaiacol 3: p-Cresol 4 :-cresols 5: neighbour-cresols 6:4-methyl guaiacol and 4 7:4-vinylphenol 8:4-ethyl-phenol 9:3-ethyl-phenol 10:4-vinyl guaiacol 11:2,4-xylenol 12:4-guaethol 13:2-ethyl-phenol 14: eugenol
Though 4-ethyl guaiacol can not separate with the 2-ethyl-phenol fully; Can't be accurately quantitative, but can qualitative analysis, concrete kind of time-like of Volatile Phenols in not knowing liquor; With this chromatographic condition wine appearance in enormous quantities is known the real situation; There are not following 4 kinds of Volatile Phenols in discovery: 2-ethyl-phenol, 3-ethyl-phenol, 2,4-xylenol and eugenol in the liquor wine appearance of having analyzed.So Volatile Phenols to be analyzed in the liquor is confirmed as following 10 kinds: phenol, guaiacol, p-Cresol ,-cresols, neighbour-cresols, 4-methyl guaiacol and 4,4-vinylphenol, 4-ethyl-phenol, 4-vinyl guaiacol, 4-ethyl guaiacol.With this in 10 Volatile Phenols set up new chromatographic condition and analyze.
2.3 the selection of pre-treating method
Before liquor is selected pre-treating method, attempt direct injected, chromatogram is seen Fig. 3, the chromatogram of certain liquor direct injected.
Visible by Fig. 3, there is certain interfering material in the chromatogram of direct injected, and because the content of Volatile Phenols in liquor is lower, it is carried out qualitative and quantitative have difficulties.Gu consider the extraction of wine appearance is concentrated sample introduction analysis more afterwards.Select which kind of organic solvent that wine appearance is extracted? We have carried out the selection of Extractant experiment; The slective extraction agent is methylene chloride, methenyl choloride, ether, tetrahydrofuran, ethyl acetate and ether respectively: n-pentane=1: 1, ethyl acetate: the mixed solvent of sherwood oil=3: 1 is made extractant, obtains the recovery and sees table 2.2.
Table 2.2 extractant is to the influence of 10 kinds of Volatile Phenols recovery of extraction (%)
Figure BDA00001148423800091
Annotate: not stratified after the jolting when " ... " expression joins in the liquor of 45～55 degree as extractant, therefore can not extract the Volatile Phenols in the liquor.
Visible by table 2.2, methylene chloride and methenyl choloride obviously are superior to ether to the recovery of extraction of 10 kinds of Volatile Phenols: n-pentane=1: 1 and ethyl acetate: the recovery of extraction of the mixed solvent of sherwood oil=3: 1 during as extractant.But when being to use ether, tetrahydrofuran and ethyl acetate to make extractant; In the separating funnel that 100mL 50～52 degree wine appearance are housed, add ether and tetrahydrofuran; Carry out extracting operation, leave standstill 30mim and do not see layering, adding 30mL water is not stratified; It is also not stratified to add 50mL water again, and it is still not stratified to add 10mL ether or tetrahydrofuran again; 50～52 degree wine appearance are added ultrapure water, and to reduce to alcoholic strength be 26 degree; It is still not stratified to add ether or tetrahydrofuran or ethyl acetate extraction again; So think the extraction solvent of aldehydes matter in the uncomfortable cooperation liquor of ether or tetrahydrofuran or ethyl acetate; So after analysis in, select methylene chloride or methenyl choloride as extractant.
2.4 the selection of column temperature and flow velocity
The composition of moving phase is very big to the retention time influence.P-Cresol and-the cresols structure is very close with character, in common moving phase, can't separate, need beta-schardinger dextrin-can to split it as the moving phase adjuvant.The phenolic hydroxyl group easily ionizable has dual retention mechanism on the stationary phase surface.Non-polar solvent is difficult to the separating effect that reaches desirable.Employing contains 1% acetic acid to be done moving phase the ionization of phenol is suppressed, and becomes the hydrophobic association thing of neutral molecule under the anti-phase condition, and separating effect and peak type are greatly improved.Under moving phase isocratic elution condition, measure component and can not separate fully; The mensuration component is reached fully separates.
Keep other conditions constant, change column temperature and experimentize, the result shows that column temperature is very little to the influence that separates, and selects 25 ℃ of normal temperature.Because analyte is more, under the 1mL/min flow velocity, the equal backward delay of each material appearance time for practicing thrift analysis time and analysis cost, is chosen under the 1.5mL/min flow velocity and analyzes.
2.5 influence the extraneous factor that HPLC analyzes
Retention time is drifted about sometimes when analyzing with HPLC, takes place sometimes to change fast.The drift that retention time takes place is because temperature control is bad, and solution is to adopt thermostat, keeps column temperature constant; Moving phase changes, solution be prevent that moving phase from evaporating, reaction etc.; The pillar lack of equilibrium is good, needs pillar is carried out the balance of longer time.The quick variation of retention time then is that solution is to reset flow velocity because flow velocity changes, and makes it to keep stable; Bubble is arranged in the pump, can bubble be driven out of through operations such as exhausts; Moving phase is improper, and solution is for changing moving phase or moving phase suitably being mixed in the pulpit.
2.6 chromatographic peak is qualitative in the liquor sample
Qualitative for chromatographic peak in the liquor sample, it is qualitative to adopt retention time and the fluorescence spectrum at contrast standard specimen and liquor sample chromatogram peak that phenols component is carried out, and Fig. 4 is 10 kinds of Volatile Phenols standard specimen chromatograms, and Fig. 5 is the chromatogram of Volatile Phenols in the liquor:
10 kinds of Volatile Phenols standard specimens are respectively among Fig. 4: 1: phenol 2: guaiacol 3: p-Cresol 4 :-cresols 5: neighbour-cresols 6:4-methyl guaiacol and 4 7:4-vinylphenol 8:4-ethyl-phenol 9:4-vinyl guaiacol 10:4-guaethol
Volatile Phenols is respectively among Fig. 5: 1: phenol 2: guaiacol 3: p-Cresol 6:4-methyl guaiacol and 4 7:4-vinylphenol 8:4-ethyl-phenol 10:4-guaethol
2.7 working curve and detectability
Precision take by weighing phenol, guaiacol, p-Cresol ,-cresols, neighbour-cresols, 4-methyl guaiacol and 4,4-vinylphenol, 4-ethyl-phenol, 4-vinyl guaiacol, each 25mg of 4-ethyl guaiacol reference substance; Be settled to 25mL (4-vinylphenol, 4-vinyl guaiacol are used methanol constant volume) with 1% glacial acetic acid solution, be made into the standard reserving solution of 1mg/ml.Carry out doubling dilution with 1% glacial acetic acid solution, be made into mass and size concentration and be followed successively by 0.02,0.2, the phenolic compound of 1.0,2.0 and 4.0 μ g/mL mixes reference substance solution.With peak area (Y) mass concentration (X, μ g/mL) is carried out linear regression, the phenolic compound regression equation is seen table 2.3.With signal to noise ratio (S/N ratio) is 3 definite detectabilities.
The regression equation and the detectability of table 2.3 Volatile Phenols compounds
Figure BDA00001148423800111
The result shows that the related coefficient of this analytical approach is between 0.99984 to 0.99990, and concentration and peak area are good linear relationship.
2.8 the beta-schardinger dextrin-addition to-cresols and p-Cresol isomeride split and the 4-ethyl-phenol separates with 4-vinyl guaiacol overlap peak influence
Cyclodextrin is in the application that splits isomeride and enantiomorph body: cyclodextrin (CDs) is one type and is made up of through α-(1,4)-glycosidic bond several D-pyranose unit, hydrophilic " cone barrel " cyclic oligosaccharide outside hydrophobic in the chamber, the chamber.The nonpolar cavity of cyclodextrin forms host-guest complex through the coupling wedging function with the hydrophobic effect of guest molecule and shape size, optionally inorganic, organic, neutrality of inclusion and ionic species molecule.Temperature, pH value that this process with the spatial structure of guest molecule, electrically reaches polarity of solvent, system etc. is relevant; The body feature of cyclodextrin molecular and chemical feature are determining the selectivity of recombination process; Thereby demonstrate good molecular recognition, therefore be widely used in separating of molecular recognition, selectivity catalytic reaction, chipal compounds and isomeride.
With beta-schardinger dextrin-as the separable cresols isomeride of the RPLC of moving phase adjuvant, this separation be based on beta-schardinger dextrin-for p-Cresol ,-the selectivity bag of cresols and neighbour-cresols isomeride connects effect.There is hydrophilic hydroxyl the cavity structure outside of beta-schardinger dextrin-; Inside is the hydrocarbon structure of lipophilic, the methyl substituted phenyl ring in the cresols structure since lipophilicity will embed in the cavity of beta-schardinger dextrin-, and phenolic hydroxyl group will with the hydroxyl generation hydrogen bond action outside the beta-schardinger dextrin-chamber; Because p-Cresol ,-methyl of cresols and neighbour-cresols is in different positions; The difference of its space structure, cause they with the beta-schardinger dextrin-cavity to agree with degree different, formed inclusion compound stability is different; Thereby obtain different retention times, therefore can the cresols isomeride be split.According to the eluting order of cresols isomeride on reversed-phase column, can infer that the cresols isomeride gets into the beta-schardinger dextrin-cavity, the host-guest complex stability order of formation be p-Cresol＞-cresols＞neighbour-cresols.The beta-schardinger dextrin-of this result and bibliographical information and p-Cresol ,-cresols conforms to the stability constant of neighbour-cresols inclusion complex.Fig. 6-9 is the influence of different beta-cyclodextrin concentration to the isomer separation degree.Wherein
Fig. 6 (a): beta-schardinger dextrin-concentration is: 3.53 * 10 -3Mol/L Fig. 7 (b): beta-schardinger dextrin-concentration is: 5.29 * 10 -3Mol/L
Fig. 8 (c): beta-schardinger dextrin-concentration is: 7.05 * 10 -3Mol/L Fig. 9 (d): beta-schardinger dextrin-concentration is: 8.81 * 10 -3Mol/L
Volatile Phenols among the figure is respectively: 1: phenol 2: guaiacol 3: p-Cresol 4 :-cresols 5: neighbour-cresols 6:4-methyl guaiacol and 4 7:4-vinylphenol 8:4-ethyl-phenol 9:4-vinyl guaiacol 10:4-guaethol
Can know by Fig. 6～9, when beta-schardinger dextrin-concentration is 3.53 * 10 -3During mol/L (4g/L), 3, No. 4 peaks among Fig. 6 (a) (p-Cresol ,-cresols) and 8, No. 9 peaks (4-ethyl-phenol, 4-vinyl guaiacol) begin to separate, but can not separate fully.When beta-schardinger dextrin-concentration is 5.29 * 10 -3During mol/L (6g/L), 3, No. 4 peaks further separate among Fig. 7 (b), and 8, No. 9 the peak can separate fully.Along with the increase of beta-schardinger dextrin-concentration, to ,-the cresols degree of separation increases gradually, when concentration is 7.05 * 10 -3During mol/L (8g/L), p-Cresol among Fig. 8 (c) ,-the cresols degree of separation is 1.52, reaches fully to separate.When concentration is 8.81 * 10 -3During mol/L (10g/L), p-Cresol among Fig. 9 (d) ,-the cresols degree of separation further improves, and is 1.81, the beta-schardinger dextrin-addition to-influence that cresols, p-Cresol isomers split sees table 2.4.
Table 2.4 beta-schardinger dextrin-addition to-influence that cresols, p-Cresol isomers split
Figure BDA00001148423800121
The result shows that the degree of separation of cresols isomeride increases along with the increase of beta-schardinger dextrin-concentration.This possibly be because the increase of beta-schardinger dextrin-concentration helps strengthening the interaction between it and the isomers, makes that the chromatographic behavior difference between the isomers enlarges, and obtains different retention times, thereby reaches the purpose of separation.But when beta-schardinger dextrin-concentration is too high,, in moving phase, can not dissolve fully, make the phase transformation of flowing muddy, and be prone to stop up chromatographic column, therefore through relatively selecting 7.05～8.81 * 10 owing to receive the restriction of solubleness -3Mol/L beta-schardinger dextrin-solution is as optimal flow phase adjuvant.
2.9 the recovery
2.9.1 extraction times and volume are to the influence of the recovery
Get 100mL mark-on simulation wine appearance (0.1 μ g/mL), use CH 2Cl 2To its carry out liquid-liquid extraction (20mL/ time, extract respectively 1,2,3,4 and 5 time, add the 20mL1% glacial acetic acid and do the restraining barrier; Under 35 ℃ of conditions, rotate evaporation and concentration, till no longer including distillate and flowing out, concentrate is settled to 50mL with 1% glacial acetic acid; Cross 0.45 μ m filter membrane, supply HPLC to analyze.Investigate its recovery.
Table 2.5 extraction times and volume are to the recovery influence (%) of Volatile Phenols compounds
Figure BDA00001148423800131
Annotate: 3 ' expression is used CH to 100mL wine appearance 2Cl 2Extraction 30mL/ time, extracts 3 times; 3 " expression is used CHCl to 100mL wine appearance 3Extraction 20mL/ time, extracts 3 times.
The result shows, extract 3 times after, each material recovery extracts 4 recovery and slightly improves between 84.1 to 101.6, considers the use amount and the extraction time of reagent, slective extraction 3 times,, also investigated the influence of extractant consumption, use CH 2Cl 2Extraction, 30mL/ time, extract 3 times, the recovery is seen 3 ' items in the table.It is thus clear that CH 2Cl 2Extraction, 30mL/ time, extract 3 times, the recovery of 10 kinds of phenol is better.Also investigated extractant by CH 2Cl 2Become CHCl 3To the influence of the recovery, see in the table 3 ", visible CHCl 3Extraction, 20mL/ time, extract 3 times, the recovery of 10 kinds of phenol is also fine, so extractant CH 2Cl 2And CHCl 3Consumption elect 20～30mL as.
2.9.2 revolve in the steaming process restraining barrier to the influence of the recovery
Get 100mL mark-on simulation wine appearance (0.1 μ g/mL), use CH 2Cl 2It is carried out liquid-liquid extraction (20mL/ time, extract 3 times), merge organic phase; Add the 20mL1% glacial acetic acid as the restraining barrier, the about 15min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions is till no longer including the distillate outflow; Concentrate is settled to 50mL with 1% glacial acetic acid; With 0.45 μ m filtering with microporous membrane, the sample introduction analysis, external standard method is quantitative.The recovery is seen table 2.6.
Table 2.6 revolves and adds the restraining barrier in the steaming process and do not add restraining barrier and restraining barrier volume to measuring the influence of the recovery
Figure BDA00001148423800141
Can find out through last table 2.7 contrast; Revolve before the steaming; When in extract, not adding the restraining barrier; The recovery of 10 kinds of Volatile Phenols reaches 1 phenol that has only more than 90%, and the recovery that also has 2 phenol (paracresol and 4-ethyl guaiacol) is that 74.2% and 69.3% not reach the detection method recovery be 80%～120% requirement; But revolve before the steaming; Along with in extract, adding 1%HAc 4ml, after 10mL and 20mL did the restraining barrier, the recovery of 10 kinds of Volatile Phenols reached be increased to 8 more than 90%; All the other 2 also is 87.7% and 88.8%; Explain and revolve before the steaming, in extract, add the restraining barrier, the recovery of 10 kinds of Volatile Phenols obviously is superior to not adding steaming is directly revolved on the restraining barrier to extract result.Reason possibly be in revolving the inspissation compression process, carries the part Volatile Phenols when contained a large amount of extractant methylene chloride form the steam volatilization in the extract and evaporates together, causes the loss of volatility phenols material.Revolve in extract, add the restraining barrier before steaming after; It is on the main organic phase greater than 1 methylene chloride that the restraining barrier is covered in proportion; All must pass through the restraining barrier when making the phenolic compound evaporation that in revolving the steaming process methylene chloride and part carried by methylene chloride; Because methylene chloride is not dissolved in the restraining barrier of water character, can be through the restraining barrier evaporation; And contain hydroxyl in the phenolic compound molecule; Through the restraining barrier time can with wherein hydrone and acetic acid molecule generation interaction of hydrogen bond; Therefore the purpose of capture, absorption and reservation Volatile Phenols compounds can be played in the restraining barrier; The recovery of mensuration is improved, and the accuracy of therefore measuring is improved, and analysis result is stable.
Investigated of the influence of different blocking layer consumption to the recovery; Can know by table 2.7, add 4mL, the recovery that 10mL and 20mL restraining barrier can obtain; When the restraining barrier of adding just forms thin overlayer on extract; Measure to such an extent that the volume of restraining barrier liquid is 4ml, measure in the recovery that calculates 9 phenol data greater than the recovery of 80%, 1 phenol less than 80%.Add 10mL and 20mL restraining barrier and can obtain the better recovery, confirm that therefore the addition on restraining barrier is 10～20ml, i.e. the 10-20vol% of wine appearance percent by volume.
The recovery when investigating variable concentrations glacial acetic acid aqueous solution, ultrapure water as the restraining barrier, the result sees table 2.7.
Table 2.7 revolves and adds the recovery that the different blocking layer is measured in the steaming process
Figure BDA00001148423800151
Can know that by table ultrapure water is consistent relatively as the measured recovery in restraining barrier with the variable concentrations glacial acetic acid aqueous solution,, select 1% glacial acetic acid, examine or check its recovery, see table 2.8 as the restraining barrier for keeping sample identical with moving phase.
Table 2.8 revolves and adds the 1% glacial acetic acid mensuration recovery and repeatability in the steaming process
The result shows, revolves to add 1% glacial acetic acid solution or ultrapure water in the steaming process as the restraining barrier, and the relative standard deviation of 10 kinds of Volatile Phenols recovery analyzing shows that the repeatability of this method is good between 0.9% to 2.5%.
2.10 the liquor alcoholic strength is to the influence of extraction
When the liquor alcoholic strength was higher than 55 °, because extractant methylene chloride and ethanol dissolve each other, the liquor alcohol content was too high, and extractant and sample solution can not layerings afterwards to cause extraction, and analysis can't be proceeded; And when the liquor alcoholic strength is lower than 45 °, though can fine layering after the extraction, add 1% glacial acetic acid aqueous solution as the restraining barrier after in the concentrated process of rotary evaporation, be prone to cause the waterfall phenomenon of boiling cause the loss of extract.Analyze reason,, also can dissolve each other with methylene chloride because ethanol can dissolve each other with the WS; In the process that liquor is extracted with methylene chloride; Ethanol also partly is extracted out simultaneously, when rotary evaporation concentrates, because 35 ℃ bath temperatures can make lower floor's methylene chloride volatilization and can not make the upper water solution evaporation; The ethanol of certain content can make the restraining barrier WS and extractant methylene chloride partial miscibility in lower floor's extractant methylene chloride, makes lower floor's heat radiation evenly; When ethanol content reduced, two alternate mutual solubilities reduced, and the heat that lower floor's methylene chloride gathers can not evenly be discharged, and caused the boil appearance of phenomenon of waterfall.
Solution is: when the liquor alcoholic strength is higher than 55 °, with ultrapure water wine appearance is carried out a certain proportion of dilution, when the liquor alcoholic strength is lower than 45 °; After extraction finishes; Before rotary evaporation concentrates, add a spot of ethanol, make to revolve and steam ethanol content increase in the liquid; Two alternate ability partial miscibilities can prevent the generation that waterfall boils.So the liquor alcoholic strength is adjusted into 45～55 °, preferably the liquor alcoholic strength is adjusted into about 52～53 °, extracts again.
3.1 the content of Volatile Phenols in the Chinese famous-brand and high-quality liquor of part
Adopt the analytical approach of setting up, the famous-brand and high-quality liquor of centering state is analyzed, and the content of part wine appearance Volatile Phenols compounds is seen table 2.9.
The content (μ g/mL) of Volatile Phenols in the famous-brand and high-quality liquor of table 2.9 part China
Figure BDA00001148423800171
Food fermentation research institute of Ministry of Light Industry was a research object with Maotai in 1980, adopted chemical derivatization to combine GC-FID/GC-MS that Volatile Phenols compounds in Maotai and the five-Grain Liquor is analyzed, and wherein the content of part Volatile Phenols is seen table 2.10.
Part volatile phenol concentrations presented in table 2.10 Maotai and the five-Grain Liquor (μ g/mL)
Visible by table, adopt the famous-brand and high-quality liquor of this analysis method centering state to analyze, the data of the data of the Volatile Phenols compounds that obtains and food fermentation research institute of Ministry of Light Industry report in 1980 are on the one magnitude and in same scope.
3.2 the Volatile Phenols source is explored in the liquor
Because the Volatile Phenols compounds is the important aroma constituent of alcoholic drink; This type component all has important effect for the aspects such as news perfume, taste and stability of wine; So find the source of Volatile Phenols, instruct production technology just to have certain meaning to a certain extent.
Get vinasse and bent medicine from certain brewery, use 60% edible alcohol water solution, soaked 2 hours by quality 1: 1 constant temperature in 35 ℃ of water-baths; After the filtration; Get 100ml solution and handle, analyze wherein whether contain volatility phenols material, see table 2.11 according to the method for setting up.
Volatility source in table 2.11 liquor
Figure BDA00001148423800181
Analysis result shows that in vinasse and bent medicine, all have volatility phenols material, wherein the kind of the volatility phenols material in the vinasse is more than the volatility phenols material in the bent medicine.Explain that the Volatile Phenols in the liquor is in brewageing the process of fermentation, just to produce; Consistent with bibliographical information: this type of Volatile Phenols is changed by hydroxycinnamic acid; Hydroxycinnamic acid normally combines with polysaccharide with the form of ester, and is present in the plant cell wall; In brewing process; It can discharge from polysaccharide; Through decarboxylation, the enzyme effect of pyrolysis or special yeast strain, these hydroxycinnamic acids; Particularly two kinds of hydroxycinnamic acids of p-Coumaric Acid and forulic acid can be transformed into denseer Volatile Phenols 4-vinylphenol of smell and 4-vinyl guaiacol.And the existence of yeast can make these vinyl phenol compound reduction, is transformed into corresponding ethyl compound 4-ethyl-phenol and 4-ethyl guaiacol.
3.3 the adjustment experiment of wine appearance amount
The sampling amount of wine appearance is adjusted to 50mL from 100mL, carried out the adjustment experiment of wine appearance amount, promptly get 50mL wine appearance, use CH 2Cl 2Or CHCl 3Carry out liquid-liquid extraction, 10～15mL/ time, extract 3 times; Merge organic phase and form extract, add 5～10mL1% glacial acetic acid solution in the extract of gained and do the restraining barrier, the about 7～9min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions; Flow out to no longer including distillate; Concentrate is settled to 25mL with 1% glacial acetic acid, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively; The result can realize satisfactory results to the recovery of 10 kinds of phenol shown in table 2.12 equally, is 80.13～101.21%.
Table 2.12 sampling amount is to the influence of the recovery
4 conclusions
Above-mentioned experimental result shows: the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor; Utilize fluorescent characteristic, the chiral resolution agent beta-schardinger dextrin-of aldehydes matter to split gordian techniquies such as overlap peak, eluent gradient wash-out and extract interpolation restraining barrier concentrate again; The high performance liquid chromatograph of having set up the band fluorescence detector is measured the method for 10 kinds of volatility phenols content of material in the China white wine; Chromatographic condition is following: chromatographic column, Synergi 4u Hydro-RP 80A (150 * 4.6mm); Fluorescence detector wavelength: Ex=270nm, Em=315nm; Mobile phase A: the WS of glacial acetic acid/beta-schardinger dextrin-(1/100); Mobile phase B: the WS of glacial acetic acid/acetonitrile/beta-schardinger dextrin-(1/40/60), the concentration of the WS of beta-schardinger dextrin-: 7.05 * 10 -3～8.81 * 10 -3Mol/L adopts the gradient elution program.The recovery that this method is measured 10 kinds of Volatile Phenols compounds is 83.8%～101.6%, and relative standard deviation is 0.9%～2.5%, detects to be limited to: 0.0025～0.0506 μ g/mL.This method accuracy, sensitivity and good reproducibility are described, and sample do not need chemically derivedly, use the high performance liquid chromatograph of relative low price, easy and simple to handle.Be expected to be used for the daily detection of China white wine Volatile Phenols compounds.
Description of drawings
Fig. 1 adds 14 kinds of Volatile Phenols standard specimen mixed solution chromatograms behind the beta-schardinger dextrin-in moving phase;
Fig. 2 adds beta-schardinger dextrin-and buffer salt and the back 14 kinds of Volatile Phenols standard specimen mixed solution chromatograms of alcohol in moving phase;
Fig. 3 is certain liquor direct injected chromatogram;
Fig. 4 adds 10 kinds of Volatile Phenols standard specimen mixed solution chromatograms behind the beta-schardinger dextrin-in moving phase;
Fig. 5 is the chromatogram of liquor;
Fig. 6-9 is 10 kinds of Volatile Phenols standard specimens of different beta-cyclodextrin concentration mixed solution chromatogram.
Embodiment
Embodiments of the invention 1.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Precision take by weighing phenol, guaiacol, p-Cresol ,-cresols, neighbour-cresols, 4-methyl guaiacol and 4,4-vinylphenol, 4-ethyl-phenol, 4-vinyl guaiacol, each 25mg of 4-ethyl guaiacol reference substance; Be settled to 25mL (4-vinylphenol, 4-vinyl guaiacol are used methanol constant volume) with 1% glacial acetic acid solution, be made into the standard reserving solution of 1mg/mL.Carry out doubling dilution with 1% glacial acetic acid solution, be made into mass concentration and be followed successively by 0.02,0.2, the phenolic compound of 1.0,2.0 and 4.0 μ g/mL mixes reference substance solution.With peak area (Y) mass concentration (X, μ g/mL) is carried out linear regression, set up the calibration curve (also claiming regression equation) of Volatile Phenols compounds in 10, be used for the external standard method quantitative Analysis.
Get 100mL wine appearance, use CH 2Cl 2Carry out liquid-liquid extraction, each 30mL extracts 3 times, and jolting was 100 times about the standard extraction was moved in separating funnel at every turn; Leave standstill, tell organic phase, merge organic phase and form extract; The gained extract is in adding 1% glacial acetic acid solution 20ml as the restraining barrier, and the about 15min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions is till no longer including the distillate outflow; Concentrate is settled to 50ml with 1% glacial acetic acid solution, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Moving phase is by mobile phase A phase and Mobile phase B phase composition, mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3-8.81 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
The alcohol concentration of wherein, getting wine appearance should be the 45-55 degree.
With 10 kinds of phenolic compounds to the standard items mixed solution; Setting up concentration range is the concentration of 0.02～4.0 μ g/mL10 kind phenolic compound and the calibration curve of high performance liquid chromatography chromatographic peak peak area; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.Reduction formula is following.
Figure BDA00001148423800211
Embodiments of the invention 2.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps: in the liquor
Get 100mL wine appearance, use CH 2Cl 2Carry out liquid-liquid extraction, 20mL/ time, extract 3 times; Merge organic phase and form extract, the gained extract add 20mL concentration be 1% glacial acetic acid solution as the restraining barrier, the about 15min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions again; Till no longer including distillate and flowing out; Concentrate is settled to 50mL with 1% glacial acetic acid solution, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
The alcohol concentration of the wine appearance of getting is that 52～53 degree are best.For the liquor of variable concentrations, when the liquor alcoholic strength is higher than 55 °, wine appearance is carried out a certain proportion of dilution with ultrapure water, making the liquor alcoholic strength is about 45～55 °, extracts again; When the liquor alcoholic strength is lower than 45 °, after extraction finishes, before the extract rotary evaporation concentrates, in extract, add a spot of ethanol, making the liquor alcoholic strength is about 45～55 °, revolves steaming again.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 3.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get the liquor wine appearance of 100mL degree, use CH 2Cl 2Carry out liquid-liquid extraction, 20mL/ time, extract 3 times; Jolting was 100 times about the standard extraction was moved in separating funnel at every turn, merged organic phase and formed extract, and the gained extract adds the 20mL ultrapure water as the restraining barrier; Rotation evaporation and concentration 15min under 35 ℃ of 0.075MPa conditions again, till no longer including distillate and flowing out, concentrate is settled to 50mL with 1% glacial acetic acid solution; After crossing 0.45 μ m filter membrane, supply the analysis of high-efficient liquid phase color spectrometer;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 4.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get 100mL 52 degree liquor wine appearance, use CH 2Cl 2Carry out liquid-liquid extraction, 20mL/ time, extract 3 times; Merge organic phase and form extract, the gained extract adds the 10mL ultrapure water as the restraining barrier, under 35 ℃ of 0.075MPa conditions, rotates evaporation and concentration again; Till no longer including distillate and flowing out; Concentrate is settled to 50mL with 1% glacial acetic acid solution, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3/ L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 5.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get 100mL 52 degree liquor wine appearance, use CH 2Cl 2Carry out liquid-liquid extraction, 30mL/ time, extract 3 times; Merge organic phase and form extract, the gained extract adds 10mL 3% aqueous acetic acid as the restraining barrier, under 35 ℃ of 0.075MPa conditions, rotates evaporation and concentration again; Till no longer including distillate and flowing out; Concentrate is settled to 50mL with 1% glacial acetic acid solution, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 6.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get 100mL 52 degree liquor wine appearance, use CHCl 3Carry out liquid-liquid extraction, 20mL/ time, extract 3 times; Merge organic phase and form extract, the gained extract adds 10mL 1% aqueous acetic acid as the restraining barrier, under 35 ℃ of 0.075MPa conditions, rotates evaporation and concentration again; Till no longer including distillate and flowing out; Concentrate is settled to 50mL with 1% glacial acetic acid solution, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 7.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get 50mL 52 degree liquor wine appearance, use CHCl 3Carry out liquid-liquid extraction, 10mL/ time, extract 3 times, jolting was 100 times about the standard extraction was moved in separating funnel at every turn; Leave standstill, tell organic phase, merge organic phase and form extract; The gained extract adds 5mL 1% aqueous acetic acid as the restraining barrier, and the about 7min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions again is till no longer including the distillate outflow; Concentrate is settled to 25mL with 1% glacial acetic acid solution, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 8.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get 50mL 52 degree liquor wine appearance, use CH 2Cl 2Carry out liquid-liquid extraction, 15mL/ time, extract 3 times, jolting was 100 times about the standard extraction was moved in separating funnel at every turn; Leave standstill, tell organic phase, merge organic phase and form extract; The gained extract adds 10mL 1% aqueous acetic acid as the restraining barrier, and the about 8min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions again is till no longer including the distillate outflow; Concentrate is settled to 25mL with 1% glacial acetic acid solution, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 9.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get 100mL 52 degree liquor wine appearance, use CH 2Cl 2Carry out liquid-liquid extraction, 30mL/ time, extract 3 times; Jolting was 100 times about the standard extraction was moved in separating funnel at every turn, left standstill, and told organic phase; Merge organic phase and form extract, the gained extract rotate the about 15min of evaporation and concentration under 35 ℃ of 0.075MPa conditions, till no longer including the distillate outflow; Concentrate is settled to 50mL with 1% glacial acetic acid solution, behind the 0.45 μ m filter membrane, supplies the analysis of high-efficient liquid phase color spectrometer excessively;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 10.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get 80mL 52 degree liquor wine appearance, use CH 2Cl 2Carry out liquid-liquid extraction, 24mL/ time, extract 3 times; Jolting was 100 times about the standard extraction was moved in separating funnel at every turn, left standstill, and told organic phase; Merge organic phase and form extract, the gained extract rotate the about 12min of evaporation and concentration under 35 ℃ of 0.075MPa conditions, till no longer including the distillate outflow; Concentrate is settled to 25 or 50mL with 1% glacial acetic acid solution, cross 0.45 μ m filter membrane after, supply the analysis of high-efficient liquid phase color spectrometer;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.
Embodiments of the invention 11.The high-performance liquid chromatogram determination method of Volatile Phenols compounds comprises the steps in the liquor,
Get 80mL 52 degree liquor wine appearance, use CH 2Cl 2Carry out liquid-liquid extraction, 24mL/ time, extract 3 times, jolting was 100 times about the standard extraction was moved in separating funnel at every turn; Leave standstill, tell organic phase, merge organic phase and form extract; The gained extract adds 15mL 1% aqueous acetic acid as the restraining barrier, and the about 12min of rotation evaporation and concentration under 35 ℃ of 0.075MPa conditions is till no longer including the distillate outflow; Concentrate is settled to 25 or 50mL with 1% glacial acetic acid solution, cross 0.45 μ m filter membrane after, supply the analysis of high-efficient liquid phase color spectrometer;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate, when 0min begins: contain mobile phase A 80%, Mobile phase B 20%; During 30min: mobile phase A 40%, Mobile phase B 60%; During 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm.
According to the concentration range of being set up concentration and the calibration curve of high performance liquid chromatography chromatographic peak peak area of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL; The peak area of HPLC being surveyed 10 kinds of phenol in the sample introduction calculates the concentration of 10 kinds of phenol in the sample introduction with calibration curve, is converted into the concentration of 10 kinds of phenol in the wine appearance again.

Claims (6)

1. the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor is characterized in that: comprises the steps,
Get 50～100mL wine appearance, use CH 2Cl 2Or CHCl 3Carry out liquid-liquid extraction, the consumption of each extractant is that the percent by volume of wine appearance is 20～30vol%, extracts 3 times; Merge organic phase and form extract; The extract of gained rotates evaporation and concentration under 35 ℃ of 0.075MPa conditions, flow out to no longer including distillate, and concentrate is settled to 25 or 50mL with 1% glacial acetic acid; After crossing 0.45 μ m filter membrane, supply the analysis of high-efficient liquid phase color spectrometer;
High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Moving phase is made up of mobile phase A and Mobile phase B, mobile phase A: the glacial acetic acid/beta-schardinger dextrin-WS=1/100; Mobile phase B: the glacial acetic acid/acetonitrile/beta-schardinger dextrin-WS=1/40/60, the beta-schardinger dextrin-concentration of the beta-schardinger dextrin-WS is: 7.05 * 10 -3-8.81 * 10 -3Mol/L; Gradient elution moving phase is provided with: by volume calculate 0min: contain mobile phase A 80%, Mobile phase B 20%; 30min: mobile phase A 40%, Mobile phase B 60%; 40min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;
Fluorescence detector condition: Ex=270nm, Em=315nm;
Concentration with 10 kinds of phenol of external standard method quantitative Analysis.
2. the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor according to claim 1 is characterized in that: the beta-schardinger dextrin-concentration of the said beta-schardinger dextrin-WS is: 7.05 * 10 -3Mol/L.
3. the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor according to claim 1; It is characterized in that: said extract adding 1-3% glacial acetic acid solution or ultrapure water are as the restraining barrier; Rotary evaporation concentrates again, and the addition on restraining barrier is that the percent by volume of wine appearance is 10-20vol%.
4. the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor according to claim 3 is characterized in that: said restraining barrier is 1% glacial acetic acid solution.
5. the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor according to claim 1, it is characterized in that: the alcohol concentration of said wine appearance is adjusted into the 45-55 degree.
6. the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor according to claim 5, it is characterized in that: the alcohol concentration of said wine appearance is adjusted into the 52-53 degree.
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CN107179368A (en) * 2017-06-15 2017-09-19 云南中烟工业有限责任公司 A kind of detection of fragrance component for lifting flue gas sensory scores and decision method
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