CN102621239B - High performance liquid chromatography determination method for volatile phenolic compounds in white spirits - Google Patents

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 been found to be present in beer, and grape wine also has in the Spirit.The Volatile Phenols compounds is the important aroma constituent of alcoholic drink, because this class component all has important effect for the aspects such as news perfume (or spice), taste and stability of wine, 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 in conjunction with in the analysis and research that Volatile Phenols unit compound carries out in the liquor of GC-FID/GC-MS, 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 phenol (University Of Qingdao's journals in the liquor with solvent extraction, 1990,3 (1), 102～105), use to simulate wine sample 500mL, through adding the antioxidant sodium thiosulfite, add 2M NaOH solution and regulate PH=12.5, make phenol form phenates and enter water, ethanol is removed in distillation, uses CH ₂Cl ₂Alkalescence and neutral compound are removed in extraction, use 4M HCl and 6%NaHCO again ₃Aqueous solution is regulated PH=8.0, uses CH at last ₂Cl ₂Extraction phenol carries out GC-FID and analyzes.As seen its pre-treatment step is loaded down with trivial details, and only test simulating the wine sample, 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%, can not reach the requirement that the detection method recovery is 80～120%, RSD value＜5%, therefore not set up the analyzing detecting method of Volatile Phenols in the liquor.

The Zhu Yan of bioengineering institute of Southern Yangtze University in 2010. the model document received. Xu Yan. use and immerse solid-phase microextraction (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, namely 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 sample, uses CH ₂Cl ₂Or CHCl ₃Carry out liquid-liquid extraction, the consumption of each extractant is that the percent by volume of wine sample is 20～30vol%, extract 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, analyze for the high-efficient liquid phase color spectrometer;

High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Flow and formed mobile phase A by mobile phase A and Mobile phase B: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3-8.81 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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;

Quantitatively calculate the concentration of 10 kinds of phenol with external standard method.

The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, the beta-schardinger dextrin-concentration of described beta-schardinger dextrin-aqueous solution 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, described 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 sample is 10-20vol%.

The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, described restraining barrier are 1% glacial acetic acid solution.

The high-performance liquid chromatogram determination method of Volatile Phenols compounds in the above-mentioned liquor, described wine sample 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, described wine sample 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 described 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, described liquor is: the distinctive a kind of Spirit of China.Be saccharifying ferment with bent class, distiller's yeast, utilize 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 high performance liquid chromatography-fluoroscopic examination (HPLC-FLD) method to the mensuration of 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).

Acetic acid (is analyzed pure, Kingsoft, Chengdu chemical reagent company limited), acetonitrile (chromatographically pure, MERCK company), methylene chloride (analyze pure, Tianjin Fu Yu Fine Chemical Co., Ltd), 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), the neighbour, 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 sample, use CH ₂Cl ₂Or CHCl ₃Carry out liquid-liquid extraction, the consumption of each extractant is that the percent by volume of wine sample is 20～30vol%, extract 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 sample 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, analyze for HPLC.

1.2.2HPLC chromatographic condition

Mobile phase A: glacial acetic acid/beta-schardinger dextrin-aqueous solution (1/100); Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution (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 the concentration of 10 kinds of phenolic compound standard items of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area, peak area with 10 kinds of phenol surveying in the HPLC sample introduction, quantitatively calculate the concentration of 10 kinds of corresponding phenol with external standard method, be converted into former concentration of getting 10 kinds of phenol in the wine sample 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 may interact with surrounding environment, make:

A. the part particle on the excited state, its energy is partly dissipated, 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 the frequency that excites and energy.

The relation of fluorescence intensity and solution concentration: fluorescence is afterwards to be launched and go out by light absorbing substance, 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 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)

$e^x=1+x+\frac{x^2}{2!}+\mathrm{\Λ}+\frac{x^n}{n!}+\mathrm{\Λ}$

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 namely 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, are conducive to improve 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, under this wavelength condition, and the fluorescence intensity maximum of compound, detection sensitivity is the highest.Great majority have the compound of fluorescence at the 230nm place absorption to be arranged, so excitation wavelength is made as 230nm and is 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 sees Table 2.1.

The optimum detection wavelength of table 2.114 kind of Volatile Phenols

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) with diode array (DAD) detecting device in the experiment, be above-mentioned 10 kinds of phenolic compound mixed standard solutions of 1 μ g/mL with concentration, under same chromatographic separation condition, use diode array detector (280nm) and fluorescence detector (Ex=270nm respectively, Em=315nm) measure, find that it is 17.3～43.0 that diode array detector 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, 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 diode array detector sensitivity.Because the volatile phenol concentrations presented in the liquor is lower, high sensitivity is conducive to accurate quantitative test, and considers complicated component in the liquor, and fluorescence detector has higher selectivity, be conducive to get 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 carry out in conjunction with Volatile Phenols compounds in the Maotai of GC-FID/GC-MS, 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 detect in alcoholic drink 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 by interpolation beta-schardinger dextrin-paracresol isomeride in flowing mutually, 5 easy overlap peaks are groped the chromatographic condition that 14 kinds of Volatile Phenols separate simultaneously.After the factors such as the flow velocity by the concentration that changes the beta-schardinger dextrin-aqueous solution, the phase that flows 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

As seen from the figure, the fractionation of paracresol isomeride can be finished, but the peak overlap of 4-ethyl guaiacol and 2-ethyl-phenol.Continue to grope the method that both may 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 during liquid chromatography (especially reverse-phase chromatography) flows mutually, also is the object solvent of polarity.Alcohol exists down, envelope form when the chirality guest molecule forms inclusion complex with beta-schardinger dextrin-is different when pure with nothing, pure exists the appreciable impact cyclodextrin to the chiral selectivity of 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 alcohol 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, the characteristic of its influence degree and alcohol, 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, for 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 is conducive to form 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 added beta-schardinger dextrin-flow mutually in add buffer salt with pure.Groped after different buffer salts, different alcohol and both different proportions in flowing mutually, obtain that buffer salt biphosphate ammonium concentration is 40mmol/L in flowing mutually, 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, see 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 fully with the 2-ethyl-phenol, 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 sample 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 sample of having analyzed.So Volatile Phenols to be analyzed in the liquor is defined 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.

As seen from Figure 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 sample is concentrated sample introduction analysis more afterwards.Select which kind of organic solvent that the wine sample 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 (%)

Annotate: not stratified after the jolting when " ... " expression joins in 45～55 liquor of spending as extractant, therefore can not extract the Volatile Phenols in the liquor.

By table 2.2 as seen, methylene chloride and methenyl choloride obviously are better than 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 samples are housed, add ether and tetrahydrofuran, carry out extracting operation, leave standstill 30mim and do not see layering, it is not stratified to add 30mL water, 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 samples are added ultrapure water, and to be down 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 the phase that flows is very big to the retention time influence.P-Cresol and-the cresols structure is very close with character, can't separate in flowing mutually common, need beta-schardinger dextrin-can to split it as mobile phase adjuvant.The phenolic hydroxyl group easily ionizable has dual retention mechanism at fixing phase surface.Non-polar solvent is difficult to the separating effect that reaches desirable.Employing contains 1% acetic acid to be done to flow and mutually the ionization of phenol is suppressed, and becomes the hydrophobic association thing of neutral molecule under anti-phase condition, and separating effect and peak type are greatly improved.Under mobile equality elution requirement, 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 saving 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; Flowing changes mutually, solution be prevent from flowing evaporate mutually, 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 by operations such as exhausts; It is mutually improper to flow, and solution is suitably mixed in the pulpit for changing to flow mutually or make to flow.

2.6 chromatographic peak is qualitative in the liquor sample

Qualitative for chromatographic peak in the liquor sample, it is qualitative to adopt the retention time at contrast standard specimen and liquor sample chromatogram peak and fluorescence spectrum 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 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 the quality volumetric 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 sees Table 2.3.Be 3 to determine detectabilities with signal to noise ratio (S/N ratio).

Regression equation and the detectability of table 2.3 Volatile Phenols compounds

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 that a class is made of by 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 by the coupling wedging function with the hydrophobic effect of guest molecule and shape size, optionally inorganic, organic, the 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 the reversed-phase high-performance liquid chromatography separable cresols isomeride of beta-schardinger dextrin-as the phase adjuvant that flows, 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, methyl substituted phenyl ring in the cresols structure will embed in the cavity of beta-schardinger dextrin-owing to lipophilicity, and phenolic hydroxyl group will with the beta-schardinger dextrin-chamber outside hydroxyl generation hydrogen bond action, 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 the cresols isomeride can be split.According to the eluting order of cresols isomeride on reversed-phase column, can infer that the cresols isomeride enters the beta-schardinger dextrin-cavity, the host-guest complex of formation stability order 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 that different beta-cyclodextrin concentration is to the influence of 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

By Fig. 6～9 as can be known, when beta-schardinger dextrin-concentration be 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

The result shows that the degree of separation of cresols isomeride increases along with the increase of beta-schardinger dextrin-concentration.This may be because the increase of beta-schardinger dextrin-concentration is conducive to strengthen 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, owing to be subjected to the restriction of solubleness, in flowing mutually, can not dissolve fully, make the phase transformation muddiness that flows, and easily stop up chromatographic column, therefore through relatively selecting 7.05～8.81 * 10 ^-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 sample (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, rotate evaporation and concentration under 35 ℃ of conditions, 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, analyze for HPLC.Investigate its recovery.

Table 2.5 extraction times and volume are to the recovery influence (%) of Volatile Phenols compounds

Annotate: 3 ' expression is to 100mL wine sample CH ₂Cl ₂Extraction 30mL/ time, extracts 3 times; 3 " expression is to 100mL wine sample CHCl 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 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, extracts 3 ' items during the recovery sees Table 3 times.As seen 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, in seeing 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 sample (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, sample introduction analysis, external standard method is quantitative.The recovery sees 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

By last table 2.7 contrast as can be seen, 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 do the restraining barrier, the recovery of 10 kinds of Volatile Phenols reaches be increased to 8 more than 90%, all the other 2 also is 87.7% and 88.8%, illustrate and revolve before the steaming, add the restraining barrier in extract, the recovery of 10 kinds of Volatile Phenols obviously is better than not adding steaming is directly revolved on the restraining barrier to extract result.Reason may 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 evaporate by the restraining barrier; And contain hydroxyl in the phenolic compound molecule, by 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, therefore the accuracy of measuring is improved, and analysis result is stable.

Investigated the influence of different blocking layer consumption to the recovery, by table 2.7 as can be known, add 4mL, the recovery that 10mL and 20mL restraining barrier can obtain, when the restraining barrier of adding just when extract forms thin overlayer, 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, determine that therefore the addition on restraining barrier is 10～20ml, i.e. the 10-20vol% of wine sample percent by volume.

The recovery when investigating variable concentrations glacial acetic acid aqueous solution, ultrapure water as the restraining barrier the results are shown in Table 2.7.

Table 2.7 revolves and adds the recovery that the different blocking layer is measured in the steaming process

As seen from table, ultrapure water is relative consistent as the measured recovery in restraining barrier with the variable concentrations glacial acetic acid aqueous solution, for keeping sample with mobile mutually identical, selects 1% glacial acetic acid as the restraining barrier, examines or check its recovery, sees Table 2.8.

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, easily cause the waterfall phenomenon of boiling to cause the loss of extract.Analyze reason, because ethanol can dissolve each other with aqueous solution, also can dissolve each other with methylene chloride, 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 restraining barrier aqueous solution 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 the wine sample 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 energy 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 sample Volatile Phenols compounds sees 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

Food fermentation research institute of Ministry of Light Industry was research object with Maotai in 1980, adopted chemical derivatization to analyze in conjunction with Volatile Phenols compounds in the Maotai of GC-FID/GC-MS and the five-Grain Liquor, and wherein the content of part Volatile Phenols sees Table 2.10.

Part volatile phenol concentrations presented in table 2.10 Maotai and the five-Grain Liquor (μ g/mL)

By table as seen, 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 an order of 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 class component all has important effect for the aspects such as news perfume (or spice), 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 according to the method for setting up, analyze wherein whether contain volatility phenols material, see Table 2.11.

Volatility source in table 2.11 liquor

Analysis result shows that all have volatility phenols material in vinasse and bent medicine, wherein the kind of the volatility phenols material in the vinasse is more than the volatility phenols material in the bent medicine.Illustrate that the Volatile Phenols in the liquor is just to produce in brewageing the process of fermentation, consistent with bibliographical information: this type of Volatile Phenols is changed by hydroxycinnamic acid, hydroxycinnamic acid normally is combined with polysaccharide with the form of ester, and is present in the plant cell wall; In brewing process, it can discharge from polysaccharide, pass 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 the 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 of wine sample amount experiment

The sampling amount of wine sample is adjusted to 50mL from 100mL, carried out the adjustment experiment of wine sample amount, namely get 50mL wine sample, 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, analyzes for the high-efficient liquid phase color spectrometer excessively; The result can realize satisfactory results equally to the recovery of 10 kinds of phenol shown in table 2.12, 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 as follows: chromatographic column, Synergi 4u Hydro-RP 80A (150 * 4.6mm); Fluorescence detector wavelength: Ex=270nm, Em=315nm; Mobile phase A: the aqueous solution of glacial acetic acid/beta-schardinger dextrin-(1/100); Mobile phase B: the aqueous solution of glacial acetic acid/acetonitrile/beta-schardinger dextrin-(1/40/60), the concentration of the aqueous solution 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 the daily detection for China white wine Volatile Phenols compounds.

Description of drawings

Fig. 1 is 14 kinds of Volatile Phenols standard specimen mixed solution chromatograms add beta-schardinger dextrin-in flowing mutually after;

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 flowing mutually;

Fig. 3 is certain liquor direct injected chromatogram;

Fig. 4 is 10 kinds of Volatile Phenols standard specimen mixed solution chromatograms add beta-schardinger dextrin-in flowing mutually after;

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 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 external standard method and quantitatively calculate.

Get 100mL wine sample, use CH ₂Cl ₂Carry out liquid-liquid extraction, each 30mL extracts 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 is in adding 1% glacial acetic acid solution 20ml as the restraining barrier, rotation evaporation and concentration about 15min under 35 ℃ of 0.075MPa conditions, till no longer including the distillate outflow, concentrate is settled to 50ml with 1% glacial acetic acid solution, after crossing 0.45 μ m filter membrane, analyze for the high-efficient liquid phase color spectrometer;

High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6mm; Flow by mobile phase A phase and Mobile phase B phase composition, mobile phase A: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3-8.81 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Wherein, the alcohol concentration of getting the wine sample 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 sample again.Reduction formula is as follows.

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 sample, 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, analyzes for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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 sample of getting is that 52～53 degree are best.For the liquor of variable concentrations, when the liquor alcoholic strength is higher than 55 °, with ultrapure water the wine sample is carried out a certain proportion of dilution, 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 sample 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, 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, analyze for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 samples, 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, rotates evaporation and concentration again under 35 ℃ of 0.075MPa conditions, 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, analyzes for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3/ L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 samples, 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, rotates evaporation and concentration again under 35 ℃ of 0.075MPa conditions, 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, analyzes for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 samples, 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, rotates evaporation and concentration again under 35 ℃ of 0.075MPa conditions, 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, analyzes for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 samples, 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, rotation evaporation and concentration about 7min under 35 ℃ of 0.075MPa conditions again, till no longer including the distillate outflow, concentrate is settled to 25mL with 1% glacial acetic acid solution, after crossing 0.45 μ m filter membrane, analyze for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 samples, 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, rotation evaporation and concentration about 8min under 35 ℃ of 0.075MPa conditions again, till no longer including the distillate outflow, concentrate is settled to 25mL with 1% glacial acetic acid solution, after crossing 0.45 μ m filter membrane, analyze for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 samples, 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, leave standstill, tell organic phase, merge organic phase and form extract, the gained extract rotates the about 15min of evaporation and concentration under 35 ℃ of 0.075MPa conditions, 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, analyzes for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 samples, 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 rotates the about 12min of evaporation and concentration under 35 ℃ of 0.075MPa conditions, 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 after, analyze for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample 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 samples, 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, rotation evaporation and concentration about 12min 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, after crossing 0.45 μ m filter membrane, analyze for the 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: glacial acetic acid/beta-schardinger dextrin-aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/beta-schardinger dextrin-aqueous solution=1/40/60, the beta-schardinger dextrin-concentration of beta-schardinger dextrin-aqueous solution is: 7.05 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: 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.

Be the concentration of 10 kinds of phenolic compound reference substances of 0.02～4.0 μ g/mL and the calibration curve of high performance liquid chromatography chromatographic peak peak area according to the concentration range of setting up, 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 sample again.

Claims (4)

1. the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor is characterized in that: comprises the steps,

The wine sample is adjusted its alcoholic strength to the 45-55 degree with ethanol or ultrapure water, gets the wine sample after 50 ~ 100 mL adjust, and uses CH ₂Cl ₂Or CHCl ₃Carry out liquid-liquid extraction, the consumption of each extractant is that the percent by volume of wine sample is 20 ~ 30 vol%, extract 3 times, merge organic phase and form extract, the extract adding 1-3% glacial acetic acid solution of gained or ultrapure water are as the restraining barrier, the addition on restraining barrier is that the percent by volume of wine sample is 10-20vol%, under 35 ℃ of 0.075MPa conditions, rotate evaporation and concentration, flow out to no longer including distillate, concentrate is settled to 25 or 50 mL with 1% glacial acetic acid, after crossing 0.45 μ m filter membrane, analyze 10 kinds of volatility phenols materials for the high-efficient liquid phase color spectrometer: phenol, guaiacol, p-Cresol, between-cresols, neighbour-cresols, the 4-methyl guaiacol and 4, the 4-vinylphenol, the 4-ethyl-phenol, 4-vinyl guaiacol and 4-ethyl guaiacol;

High performance liquid chromatography detects chromatographic condition: chromatographic column: Synergi 4u Hydro-RP 80A, 150 * 4.6 mm; Flow and formed mobile phase A by mobile phase A and Mobile phase B: glacial acetic acid/β -Cyclodextrin aqueous solution=1/100; Mobile phase B: glacial acetic acid/acetonitrile/β -Cyclodextrin aqueous solution=1/40/60, β -The β of cyclodextrin aqueous solution -Cyclodextrin concentration is: 7.05 * 10 ^-3-8.81 * 10 ^-3Mol/L; Gradient elution flows and arranges mutually: by volume calculate 0min: contain mobile phase A 80%, Mobile phase B 20%; 30min: mobile phase A 40%, Mobile phase B 60%; 40 min: mobile phase A 30%, Mobile phase B 70%; Column flow rate: 1.5 mL/min; Column temperature: 25 ℃; Sample size: 20 μ L;

Fluorescence detector condition: Ex=270nm, Em=315nm;

Quantitatively calculate the concentration of 10 kinds of phenol with external standard method.

2. the high-performance liquid chromatogram determination method of Volatile Phenols compounds in the liquor according to claim 1 is characterized in that: described β -The β of cyclodextrin aqueous solution -Cyclodextrin concentration 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: described restraining barrier is 1% glacial acetic acid solution.

4. 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 described wine sample is adjusted into the 52-53 degree.

Images