CN105510475A - Raw Pu'er tea grade and quality evaluation method - Google Patents
Raw Pu'er tea grade and quality evaluation method Download PDFInfo
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Abstract
A Pu'er tea grade and quality evaluation method comprises the steps that a raw Pu'er tea test sample liquid to be tested is prepared; efficient liquid-phase chromatographic conditions are adopted; a chromatogram of nutgall catechin gallate is made; the raw Pu'er tea test sample liquid to be tested is used as a detection object, and the positions of reference ingredients in a raw Pu'er tea test sample are identified; chromatographic peaks are acquired, the number of the chromatographic peaks marked in the test sample is at least eight, and the calculated sum of the content percentages of the chromatographic peak areas in the whole chromatographic peak area is greater than or equal to 65%. The Pu'er tea grade and quality evaluation method can relatively and objectively evaluate the Pu'er tea grade and quality, enables the raw Pu'er tea grade evaluation and quality analysis to be objective and accurate, is convenient and easy to operate and can be widely applied to grade and quality analysis of raw compressed Pu'er tea and products containing above 40% of raw Pu'er tea usage amount so as to meet the demand for modernized Pu'er tea production.
Description
Technical field
The present invention relates to a kind of grade and quality evaluating method of tealeaves, particularly adopt traditional Chinese medicine fingerprint analytical approach to the grade of Pu'er raw tea and quality evaluating method.Grade of the present invention and Quality analysis and evaluation method may be used for grade and the quality analysis of the raw tea of Pu'er tea and converted products thereof.
Background technology
Pu'er tea is with the big-leaf species in yunnan solar dried green tea in Yunnan Province's certain area for raw material, the loose tea processed through after fermentation and compressed tea, and Appearance color is brown red, and endoplasm soup look red is dense bright, the unique Chen Xiang of fragrance, and mellow time of flavour is sweet, brown red at the bottom of leaf.Have raw tea and ripe tea point, raw tea spontaneous fermentation, ripe tea artificial repening.
Tealeaves in state-owned a lot of years drink history, along with people are to food security pay attention to day by day, the quality standard that varieties of food items comprises tealeaves is also more and more higher, standard performed by current Pu'er tea is the national standard of national geography famous special product, from the place of production, fresh leaf, technique, multiple project such as physical and chemical index specifies Pu'er tea, but graded index wherein carries out by means of only sense organ contrast, lack objective evaluation index, define the detection of Tea Polyphenols in standard simultaneously, in tealeaves, other index compositions are as theanine, the detection method of the existing national standard of caffeine composition, but do not have product standard to specify its limitation etc. at present, all be based upon more subjective flavour to the evaluation of tea grades and quality, in the indexs such as fragrance.The traditional Chinese medicine fingerprint research related in the present invention is the hot fields of traditional Chinese medicine quality research, and it formulates characteristic fingerprint pattern mainly through after sample collection widely, then carries out the control of traditional Chinese medicine quality by the relative retention time specifying each peak.Although the research of traditional Chinese medicine fingerprint is comparatively general, be transplanted in the research of tealeaves and quality control more rarely seen.Existing literature research is mainly for the research of the caffeine in green tea, the single composition of Tea Polyphenols, or be more specifically for composition Study wherein, the content balance etc. of such as composition, there is not yet the grade about Pu'er tea overall composition formation situation and Quality analysis and evaluation research.The work such as the grade of Pu'er tea product and quality assessment deciding grade and level complete primarily of the experienced person of evaluating, especially endoplasm organoleptic indicator project wherein, need the person of evaluating by virtue of experience to judge completely, the evaluation method that this subjectivity is very strong is easy to the subjective factor such as diet, the mood impact being subject to evaluating personnel.There is this problem equally in the grade evaluation of other teas at present, generally verifies as master with subjectivity, and the specificity of Objective Test On Numberical project is not strong again, so relatively comprehensively teas product objective evaluation system is urgently set up.
Summary of the invention
The object of the invention is for evaluating at present the strong technological deficiency of Pu'er tea standard subjectivity, a kind of a kind of grade of Pu'er raw tea based on high performance liquid chromatography and the technical scheme of quality evaluating method are provided.The method is fully in conjunction with existing Natural products research technical method, and in conjunction with the standard-required of existing Pu'er tea product, considering can the method for relatively objective evaluation Pu'er tea grade and quality.
Technical scheme of the present invention; A kind of grade of Pu'er tea and quality evaluating method, the uv absorption chromatogram of Pu'er tea sample is obtained by high performance liquid chromatography, utilize the percentage of characteristic peak in normalization method computer chromatography, carry out grade evaluation according to the ratio of wherein one or more characteristic peaks, its step is as follows:
A. the pre-treating method of sample: get Epigallo-catechin gallate (EGCG) and make solvent with methyl alcohol, is mixed with the solution of every 1ml containing 0.5mg, obtains with reference to composition/reference substance solution; Get the raw tea of Pu'er tea crosses 40 mesh sieves powder 3-5g for sample, add the solvent of 12-20 times amount in 70-80 DEG C of water-bath lixiviate 10-15 minute, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains the raw tea test sample of Pu'er tea liquid to be measured to 10ml;
B. the chromatographic condition of efficient liquid phase: Stationary liquid is octadecyl silane, mobile phase by organic phase, aqueous phase, correctives phase composition, determined wavelength 245nm-360nm; Mobile phase elution requirement is aqueous phase and organic phase gradient elution, and aqueous phase is down to 80%-84% at 0-15min by volume ratio 85%-90%, is down to 65%-70% at 15-35min by volume ratio 80%-84%;
C. adopt the chromatographic condition of the efficient liquid phase described in step b, with reference composition/reference substance for detected object, analyze according to chromatographic condition, obtain the chromatogram of Epigallo-catechin gallate (EGCG);
D. the chromatographic condition of the efficient liquid phase described in step b is adopted, with the raw tea test sample of Pu'er tea liquid to be measured for detected object, analyze according to chromatographic condition, obtain the chromatogram of the raw tea sample of Pu'er tea, chromatogram finds the peak consistent with Epigallo-catechin gallate (EGCG) retention time, points out out with reference to the position of composition in the raw tea sample of Pu'er tea;
E. the chromatographic peak within 5-30 minute is gathered, using Epigallo-catechin gallate (EGCG) as reference composition, have 8 by the characteristic peak labeling method mark in the raw tea characteristic fingerprint pattern of Pu'er tea at least for the characteristic peak in sample respectively, calculating the wherein percentage composition sum of each characteristic peak area in all peak areas should be more than or equal to 65%.
Organic phase in described step b in mobile phase is at least a kind in methyl alcohol, acetonitrile, acetone, and correctives is at least a kind in formic acid, acetic acid, trifluoroacetic acid, phosphoric acid mutually.
In described step b, the organic phase of mobile phase is methyl alcohol, acetonitrile a kind of or two kinds, correctives be one in acetic acid, trifluoroacetic acid or two kinds.
In described step b, mobile phase elution requirement is aqueous phase and organic phase gradient elution, and aqueous phase is down to 81%-82% at 0-15min by volume ratio 89%-90%, is down to 66%-68% at 15-35min by volume ratio 81%-82%.
In described step a, test sample lixiviate solvent is be at least a kind in methyl alcohol, ethanol, acetone, normal butyl alcohol, water, phosphoric acid.
Characteristic peak labeling method in described step e in Pu'er tea characteristic fingerprint pattern is, be with reference to peak with Epigallo-catechin gallate (EGCG), marker peak S, specify that its retention time is 1.000, calculate other characteristic peaks relative to reference to the relative retention time at peak, its relative retention time should setting ± 5% within, the relative retention time setting of each characteristic peak is characteristic peak 1(P1) setting 0.429, characteristic peak 2(P2) setting 0.556, characteristic peak 3(P3) (caffeine) setting 0.576, characteristic peak 4(P4) setting 0.642, characteristic peak 5(P5) setting 0.723, characteristic peak 6(P6) setting 0.769, characteristic peak 7(P7) setting 0.849, characteristic peak 8(S) setting 1.000, characteristic peak 9(P9) setting 1.134, characteristic peak 10(P10) setting 1.183, characteristic peak 11(P11) setting 1.389.
The present invention relates to one with the raw tea of Pu'er tea for research object, adopt efficient liquid phase chromatographic analysis to carry out objective analysis in conjunction with the method for traditional Chinese medicine fingerprint analysis to the sense organ endoplasm composition that tea sample given birth to by Pu'er tea, the inventive method may be used for the raw tea product of Pu'er tea or gives birth to grade and the quality analysis of the product that tea is processed into primarily of Pu'er tea.Under HPLC chromatographiccondition of the present invention, carry out detection to the Pu'er tea sample of multiple batches of multiple grade to analyze, obtain corresponding chromatogram, traditional Chinese medicine fingerprint similarity evaluation software is utilized to carry out the comparative analysis of chromatogram, obtain the chromatogram that the raw tea of Pu'er tea has common feature, i.e. the characteristic fingerprint pattern of the raw tea of Pu'er tea; Recycle same HPLC chromatographiccondition, carry out detecting the chromatogram analyzed and obtain the raw tea sample of Pu'er tea to sample to be evaluated, the chromatogram of sample and characteristic fingerprint pattern are compared, can carry out judging its Quality Grade situation according to the raw tea classification of the Pu'er tea specified in the present invention and quality evaluating method.
The present invention passes through many batches, after the raw tea sample of the Pu'er tea in multiple place of production carries out constituent analysis, obtain the HPLC analytical method of a raw tea component of blanket Pu'er tea, in conjunction with traditional Chinese medicine fingerprint analytical approach, the chromatogram obtained is analysed and compared again, determine the characteristic fingerprint pattern of the raw tea of Pu'er tea, present 11 characteristic peaks altogether, through identifying that wherein the 8th peak is Epigallo-catechin gallate (EGCG), this composition can be specified out the relative retention time of other 10 characteristic peaks as reference, this composition is with reference to may be used at each characteristic peak of test sample constituent analysis tense marker simultaneously.
The present invention, first by the research of HPLC analytical method, determines the parameter of chromatographiccondition.Adopt the chromatographic condition of this efficient liquid phase, with reference composition/reference substance for detected object, analyze according to chromatographic condition, obtain the chromatogram of Epigallo-catechin gallate (EGCG); Again with the chromatographic condition of this efficient liquid phase, test sample liquid to be measured is produced for detected object with Pu'er tea, analyze according to chromatographic condition, obtain the chromatogram of testing sample, chromatogram finds the peak consistent with Epigallo-catechin gallate (EGCG) retention time, points out out with reference to the position of composition in the raw tea sample of Pu'er tea.The raw tea characteristic fingerprint pattern of Pu'er tea is set up again based on the detect delay of characteristic sample.Analysis sample is respectively, S1, Yun County, Lincang; S2, ShiShimonoseki, Dali; S3, version Na Puwen; S4, Fengqing, Lincang; S5, Pu'er Man Wan; S6, Yunlong, Dali; S7, Changning, the Baoshan; S8, Pu'er billows deep blue; S9, version receive Yi Wu.Each sample obtains corresponding chromatogram after high-efficient liquid phase chromatogram technique analysis, chromatographic fingerprints of Chinese materia medica similarity evaluation system is used to analyze, similarity more than 0.9, generate contrast collection of illustrative plates by sample, after extracting contrast spectrum data, obtain the data such as retention time, peak area that each becomes swarming.Fig. 1 is shown in chromatogram comparative analysis.Using Epigallo-catechin gallate (EGCG) as with reference to peak, calculate each characteristic peak relative to the relative retention time with reference to peak according to the information of characteristic fingerprint pattern.Define the relative retention time setting of each characteristic peak.Then, get the test sample preparation test sample liquid to be measured that will detect, analyze according to above-mentioned high performance liquid chromatography, obtain the chromatogram of test sample, with Epigallo-catechin gallate (EGCG) mark with reference to peak, according to the regulation of other characteristic peaks in Pu'er tea contrast characteristic spectrum, find other characteristic peaks, gather the chromatographic data of 5-30 minute, calculate the ratio of peak area in all peak areas of 8 characteristic peaks, this numerical value should be more than or equal to 65%, otherwise does not belong to the qualified raw tea of Pu'er tea.
As follows to the situation of different sample analysis when setting up the raw tea characteristic fingerprint pattern of Pu'er tea:
Table 1: the quality assessment of three raw tea samples of rank Pu'er tea
The present invention is used for the raw grade of tea of Pu'er tea and the concrete operation step of quality classification and explanation:
Sample 1 respectively, superfine Puer tea sun fixation tea (Y1); Sample 2, level Four Puer tea sun fixation tea (Y2); Sample 3, eight grades of Puer tea sun fixation tea (Y3), carry out efficient liquid phase chromatographic analysis according to the inventive method and see Fig. 3, national standard method and the inventive method evaluate illustrated in table 2.
Table 2: the quality assessment of three raw tea samples of rank Pu'er tea
Method of the present invention can be used for the Quality analysis and evaluation of the raw tea of Pu'er tea and raw material or its processed goods.The inventive method can be used for Pu'er tea raw tea raw material, Pu'er tea ripe tea raw material, the grade of Puer tea sun fixation tea and Quality analysis and evaluation, for the assorted guidance of Pu'er tea production run Raw.The inventive method can be used for grade and the quality analysis of the raw tea compressed tea of Pu'er tea and the raw tea consumption of Pu'er tea product more than 40%.
In the inventive method high-efficient liquid phase chromatogram condition, the selection of mobile phase and elution requirement can be: mobile phase is by organic phase, aqueous phase, correctives phase composition, correctives can be dissolved in organic phase or aqueous phase mutually, and the ratio that correctives accounts for organic phase or aqueous phase is mutually 0.05%-5%.Mobile phase elution requirement is the aqueous phase and the organic phase gradient elution that contain correctives phase.
A is organic phase, B is aqueous phase, correctives phased soln is in aqueous phase.A is acetonitrile, B is that 2% acetic acid aqueous solution carries out gradient elution by table 3 official hour and volume ratio.
Table 3
Illustrate: wherein 0-15min, mobile phase A (%) 15 → 20, represent that, when 0-15min wash-out, the volume that mobile phase A part accounts for mobile phase is increased to 20% by 15%.Lower same.
A is organic phase, B is aqueous phase, correctives phased soln is in aqueous phase.A is methyl alcohol, B is that 2% trifluoroacetic acid aqueous solution carries out gradient elution by table 4 official hour and volume ratio.
Table 4
A is organic phase, B is that aqueous phase, correctives are dissolved in organic phase and aqueous phase mutually respectively.A be containing the acetonitrile solution of 10% water and 2% acetic acid, B for containing 10% acetonitrile and 2% second aqueous acid, carry out gradient elution by table 5 stipulated time and volume ratio.
Table 5
The inventive method only uses Epigallo-catechin gallate (EGCG) single component to contrast/reference, without the need to using the standard items of all the components to contrast, can realize becoming swarming composition situation to carry out assay to other, saving testing cost; Need when carrying out the evaluation of sense organ endoplasm according to national standard method to rely on experienced tea judgement personnel to carry out, evaluation result is often by the impact of the conditions such as the person's of evaluating subjective emotion, diet, evaluation environment, the inventive method uses HPLC checkout equipment, its good reproducibility of analysis results, the subjective feeling impact of not examined personnel, evaluation result is more objective credible.The present invention fully in conjunction with existing Natural products research technical method, in conjunction with the standard of existing Pu'er tea product, can relatively objective evaluation Pu'er tea grade and quality.Make the ranking of the raw tea of Pu'er tea and quality analysis objective and accurate, and be conveniently easy to operation, the inventive method can be widely used in grade and the quality analysis of the raw tea compressed tea of Pu'er tea and the raw tea consumption of Pu'er tea product more than 40%, to meet the needs of Pu'er tea modern production.
Accompanying drawing explanation
Fig. 1 is that Pu'er tea sample finger-print of the present invention compares
Fig. 2 is Pu'er tea contrast characteristic spectrum
Fig. 3 is the HPLC chromatogram quality assessment of the raw tea sample of three rank Pu'er teas.
Embodiment
Below in conjunction with embodiment, the present invention is described further, but be not limited to scope of embodiments.
The mass analysis method of a kind of Pu'er tea that the present invention relates to, Pu'er tea reference fingerprint is obtained with traditional Chinese medicine fingerprint method for evaluating similarity, be with reference to composition with Epigallo-catechin gallate (EGCG), regulation is 1.000 with reference to the retention time at peak, other characteristic peaks relative to reference to peak relative retention time should setting ± 5% within, the relative retention time setting of each characteristic peak is characteristic peak 1(P1) setting 0.429, characteristic peak 2(P2) setting 0.556, characteristic peak 3(P3) (caffeine) setting 0.576, characteristic peak 4(P4) setting 0.642, characteristic peak 5(P5) setting 0.723, characteristic peak 6(P6) setting 0.769, characteristic peak 7(P7) setting 0.849, characteristic peak 8(S) setting 1.000, characteristic peak 9(P9) setting 1.134, characteristic peak 10(P10) setting 1.183, characteristic peak 11(P11) setting 1.389.
Sample classification and quality corresponding relation can refer to table 6 evaluation:
Table 6: the raw tea classification of Pu'er tea and quality assessment
Quality evaluating method of the present invention according to the following steps and method carry out determination and evaluation:
A. the pre-treating method of sample: get Epigallo-catechin gallate (EGCG) and make solvent with methyl alcohol, is mixed with the solution of every 1ml containing 0.5mg.Get 40 mesh sieves crossed by Pu'er tea powder 3-5g for sample, add the solvent of 12-20 times amount in 70-80 DEG C of water-bath lixiviate 10-15 minute, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains test sample liquid to be measured to 10ml.
B. the chromatographic condition of efficient liquid phase: Stationary liquid is octadecyl silane, mobile phase by organic phase, aqueous phase, correctives phase composition, determined wavelength 245nm-360nm; Mobile phase elution requirement is aqueous phase and organic phase gradient elution, and aqueous phase is down to 80%-84% at 0-15min by volume ratio 85%-90%, is down to 65%-70% at 15-35min by volume ratio 80%-84%.Detect column temperature 35 DEG C; Flow velocity 1ml/min.Number of theoretical plate calculates should be not less than 3000 by Epigallo-catechin gallate (EGCG).Organic phase in mobile phase be in methyl alcohol, acetonitrile, acetone one or more, correctives is formic acid, acetic acid, trifluoroacetic acid, one or more in phosphoric acid mutually.
C. the chromatographic peak within 5-30 minute is gathered, using Epigallo-catechin gallate (EGCG) as reference composition, press the characteristic peak in the characteristic peak labeling method mark test sample in Pu'er tea characteristic fingerprint pattern respectively, should have 8 characteristic peaks at least, the percentage composition sum calculating wherein each characteristic peak area should be more than or equal to 65%.
The inventive method can be used for the quality analysis of the raw tea raw material of Pu'er tea, the raw tea compressed tea of Pu'er tea, the product of the raw tea consumption of Pu'er tea more than 40%.
Below in conjunction with embodiment, the present invention is described further, but be not limited to embodiment.
Embodiment 1:
Get the raw tea raw material of Pu'er tea to pulverize, got 3 grams, the powder of 40 mesh sieves, and added the methyl alcohol of 12 times amount in 70 DEG C of water-bath lixiviates 10 minutes, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains test sample liquid to be measured to 10ml.Get Epigallo-catechin gallate (EGCG) reference substance appropriate, accurately weighed, add methyl alcohol and be mixed with the solution of every 1ml containing 0.5mg the most with reference to ingredient solution.Take octadecyl silane as filling agent (C18 reverse-phase chromatographic column); Mobile phase is divided into A, B two parts, carries out gradient elution by the official hour in table 7 and volume ratio; Determined wavelength is 245nm; Column temperature 35 DEG C; Flow velocity 1ml/min.Number of theoretical plate calculates should be not less than 3000 by Epigallo-catechin gallate (EGCG).Gather the chromatographic peak within 5-30 minute.
Table 7
Using Epigallo-catechin gallate (EGCG) as with reference to peak (peak S), according to the regulation to characteristic peak in the raw tea characteristic fingerprint pattern of Pu'er tea, point out out the characteristic peak in sample chromatogram figure and calculate the peak area of each characteristic peak.This sample tool 11 characteristic peaks, the number percent that peak area accounts for total peak area in 5-30 minute chromatogram is 86.3%, and wherein the area of peak 1-6 and accounting are 11.2%, and it is 8.3% that peak 11 accounts for.This sample should belong to the raw tea raw material of superfine Pu'er tea.
Embodiment 2:
Get the little Tuo tea of the raw tea of Pu'er tea to pulverize, got 5 grams, the powder of 40 mesh sieves, and added the acetone of 20 times amount in 80 DEG C of water-bath lixiviates 15 minutes, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains test sample liquid to be measured to 10ml.Get Epigallo-catechin gallate (EGCG) reference substance appropriate, accurately weighed, add methyl alcohol and be mixed with the solution of every 1ml containing 0.5mg the most with reference to ingredient solution.Take octadecyl silane as filling agent (C18 reverse-phase chromatographic column); Mobile phase is divided into A, B two parts, carries out gradient elution by the official hour in table 8 and volume ratio; Determined wavelength is 360nm; Column temperature 35 DEG C; Flow velocity 1ml/min.Number of theoretical plate calculates should be not less than 3000 by Epigallo-catechin gallate (EGCG).Gather the chromatographic peak within 5-30 minute.
Table 8
Using Epigallo-catechin gallate (EGCG) as with reference to peak (peak S), according to the regulation to characteristic peak in the raw tea characteristic fingerprint pattern of Pu'er tea, point out out the characteristic peak in sample chromatogram figure and calculate the peak area of each characteristic peak.This sample tool 11 characteristic peaks, the number percent that peak area accounts for total peak area in 5-30 minute chromatogram is 80.8%, and wherein the area of peak 1-6 and accounting are 8.5%, and peak 11 accounting is 5.9%.This sample should belong to the little Tuo tea of the raw tea of secondary Pu'er tea.
Embodiment 3:
Get Pu'er tea raw tea cake tea to pulverize, got 4 grams, the powder of 40 mesh sieves, and added 95% ethanol water of 15 times amount in 75 DEG C of water-bath lixiviates 12 minutes, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains test sample liquid to be measured to 10ml.Get Epigallo-catechin gallate (EGCG) reference substance appropriate, accurately weighed, add methyl alcohol and be mixed with the solution of every 1ml containing 0.5mg the most with reference to ingredient solution.Take octadecyl silane as filling agent (C18 reverse-phase chromatographic column); Mobile phase is divided into A, B two parts, A be containing the acetonitrile solution of 10% water and 2% acetic acid, B for containing 10% acetonitrile and 2% second aqueous acid, carry out gradient elution by the official hour in table 9 and volume ratio; Determined wavelength is 250nm; Column temperature 35 DEG C; Flow velocity 1ml/min.Number of theoretical plate calculates should be not less than 3000 by Epigallo-catechin gallate (EGCG).Gather the chromatographic peak within 5-30 minute.
Table 9
Using Epigallo-catechin gallate (EGCG) as with reference to peak (peak S), according to the regulation to characteristic peak in the raw tea characteristic fingerprint pattern of Pu'er tea, point out out the characteristic peak in sample chromatogram figure and calculate the peak area of each characteristic peak.This sample tool 9 characteristic peaks, the number percent that peak area accounts for total peak area in 5-30 minute chromatogram is 76.3%, and wherein the area of peak 1-6 and accounting are 6.9%, and peak 11 accounting is 4.5%.This sample should belong to level Four Pu'er tea raw tea cake tea.
Embodiment 4:
Get the raw tea ointment of Pu'er tea to pulverize, got 3 grams, the powder of 40 mesh sieves, add the butanol solution containing 2% phosphoric acid of 18 times amount in 70 DEG C of water-bath lixiviates 10 minutes, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains test sample liquid to be measured to 10ml.Get Epigallo-catechin gallate (EGCG) reference substance appropriate, accurately weighed, add methyl alcohol and be mixed with the solution of every 1ml containing 0.5mg the most with reference to ingredient solution.Take octadecyl silane as filling agent (C18 reverse-phase chromatographic column); Mobile phase is divided into A, B two parts, carries out gradient elution by the regulation in table 10; Determined wavelength is 280nm; Column temperature 35 DEG C; Flow velocity 1ml/min.Number of theoretical plate calculates should be not less than 3000 by Epigallo-catechin gallate (EGCG).
Table 10
A in mobile phase is be aqueous solution containing 10% methyl alcohol and 2% trifluoroacetic acid containing the methanol solution of 10% water and 2% trifluoroacetic acid, B.Gather the chromatographic peak within 5-30 minute.Using Epigallo-catechin gallate (EGCG) as with reference to peak (peak S), according to the regulation to characteristic peak in the raw tea characteristic fingerprint pattern of Pu'er tea, point out out the characteristic peak in sample chromatogram figure and calculate the peak area of each characteristic peak.This sample tool 9 characteristic peaks, the number percent that peak area accounts for total peak area in 5-30 minute chromatogram is 70.2%, and wherein the area of peak 1-6 and accounting are 6.4%, and peak 11 accounting is 7.2%.。
Embodiment 5:
Ripe tea raw material and the Puer tea sun fixation tea of getting Pu'er tea are pulverized, and get 3.5 grams, the powder of 40 mesh sieves, and add the methyl alcohol of 12 times amount in 75 DEG C of water-bath lixiviates 13 minutes, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains test sample liquid to be measured to 10ml.Get Epigallo-catechin gallate (EGCG) reference substance appropriate, accurately weighed, add methyl alcohol and be mixed with the solution of every 1ml containing 0.5mg the most with reference to ingredient solution.Take octadecyl silane as filling agent (C18 reverse-phase chromatographic column); Mobile phase is divided into A, B two parts, carries out gradient elution by the official hour in table 11 and volume ratio; Determined wavelength is 287nm; Column temperature 35 DEG C; Flow velocity 1ml/min.Number of theoretical plate calculates should be not less than 3000 by Epigallo-catechin gallate (EGCG).Gather the chromatographic peak within 5-30 minute.
Table 11
Using Epigallo-catechin gallate (EGCG) as with reference to peak (peak S), according to the regulation to characteristic peak in the raw tea characteristic fingerprint pattern of Pu'er tea, point out out the characteristic peak in sample chromatogram figure and calculate the peak area of each characteristic peak.This sample tool 8 characteristic peaks, the number percent that peak area accounts for total peak area in 5-30 minute chromatogram is 69.8%, and wherein the area of peak 1-6 and accounting are 4.2%, and peak 11 accounting is 4.6%.。
Embodiment 6:
Get the tea in bag containing Puer tea sun fixation tea and chrysanthemum, wherein Puer tea sun fixation tea accounts for 60%, sampling is pulverized, got 5 grams, the powder of 40 mesh sieves, add 80% ethanol water of 18 times amount in 80 DEG C of water-bath lixiviates 15 minutes, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains test sample liquid to be measured to 10ml.Get Epigallo-catechin gallate (EGCG) reference substance appropriate, accurately weighed, add methyl alcohol and be mixed with the solution of every 1ml containing 0.5mg the most with reference to ingredient solution.Take octadecyl silane as filling agent (C18 reverse-phase chromatographic column); Mobile phase is divided into A, B two parts, carries out gradient elution by the official hour in table 12 and volume ratio; Determined wavelength is 275nm; Column temperature 35 DEG C; Flow velocity 1ml/min.Number of theoretical plate calculates should be not less than 3000 by Epigallo-catechin gallate (EGCG).Gather the chromatographic peak within 5-30 minute.
Table 12
Using Epigallo-catechin gallate (EGCG) as with reference to peak (peak S), according to the regulation to characteristic peak in the raw tea characteristic fingerprint pattern of Pu'er tea, point out out the characteristic peak in sample chromatogram figure and calculate the peak area of each characteristic peak.This sample tool 8 characteristic peaks, the number percent that peak area accounts for total peak area in 5-30 minute chromatogram is 67.2%, and wherein the area of peak 1-6 and accounting are 4.7%, and peak 11 accounting is 2.6%.
Claims (6)
1. the grade of a Pu'er tea and quality evaluating method, it is characterized in that, the uv absorption chromatogram of Pu'er tea sample is obtained by high performance liquid chromatography, utilize the percentage of characteristic peak in normalization method computer chromatography, carry out grade evaluation according to the ratio of wherein one or more characteristic peaks, its step is as follows:
A. the pre-treating method of sample: get Epigallo-catechin gallate (EGCG) and make solvent with methyl alcohol, is mixed with the solution of every 1ml containing 0.5mg, obtains with reference to composition/reference substance solution; Get the raw tea of Pu'er tea crosses 40 mesh sieves powder 3-5g for sample, add the solvent of 12-20 times amount in 70-80 DEG C of water-bath lixiviate 10-15 minute, lixiviate 2 times, centrifugal, merge supernatant evaporate to dryness in 80 DEG C of water-baths, namely residue methanol constant volume obtains the raw tea test sample of Pu'er tea liquid to be measured to 10ml;
B. the chromatographic condition of efficient liquid phase: Stationary liquid is octadecyl silane, mobile phase by organic phase, aqueous phase, correctives phase composition, determined wavelength 245nm-360nm; Mobile phase elution requirement is aqueous phase and organic phase gradient elution, and aqueous phase is down to 80%-84% at 0-15min by volume ratio 85%-90%, is down to 65%-70% at 15-35min by volume ratio 80%-84%;
C. adopt the chromatographic condition of the efficient liquid phase described in step b, with reference composition/reference substance for detected object, analyze according to chromatographic condition, obtain the chromatogram of Epigallo-catechin gallate (EGCG);
D. the chromatographic condition of the efficient liquid phase described in step b is adopted, with the raw tea test sample of Pu'er tea liquid to be measured for detected object, analyze according to chromatographic condition, obtain the chromatogram of the raw tea sample of Pu'er tea, chromatogram finds the peak consistent with Epigallo-catechin gallate (EGCG) retention time, points out out with reference to the position of composition in the raw tea sample of Pu'er tea;
E. the chromatographic peak within 5-30 minute is gathered, using Epigallo-catechin gallate (EGCG) as reference composition, have 8 by the characteristic peak labeling method mark in the raw tea characteristic fingerprint pattern of Pu'er tea at least for the characteristic peak in sample respectively, calculating the wherein percentage composition sum of each characteristic peak area in all peak areas should be more than or equal to 65%.
2. the grade of a kind of Pu'er tea as claimed in claim 1 and quality evaluating method, it is characterized in that, organic phase in described step b in mobile phase is at least a kind in methyl alcohol, acetonitrile, acetone, and correctives is at least a kind in formic acid, acetic acid, trifluoroacetic acid, phosphoric acid mutually.
3. the grade of a kind of Pu'er tea as claimed in claim 1 and quality evaluating method, is characterized in that, in described step b, the organic phase of mobile phase is methyl alcohol, acetonitrile a kind of or two kinds, correctives be one in acetic acid, trifluoroacetic acid or two kinds.
4. the grade of a kind of Pu'er tea as claimed in claim 1 and quality evaluating method, it is characterized in that, in described step b, mobile phase elution requirement is aqueous phase and organic phase gradient elution, and aqueous phase is down to 81%-82% at 0-15min by volume ratio 89%-90%, is down to 66%-68% at 15-35min by volume ratio 81%-82%.
5. the grade of a kind of Pu'er tea as claimed in claim 1 and quality evaluating method, it is characterized in that, in described step a, test sample lixiviate solvent is be at least a kind in methyl alcohol, ethanol, acetone, normal butyl alcohol, water, phosphoric acid.
6. the grade of a kind of Pu'er tea as claimed in claim 1 and quality evaluating method, it is characterized in that, characteristic peak labeling method in described step e in Pu'er tea characteristic fingerprint pattern is, be with reference to peak with Epigallo-catechin gallate (EGCG), marker peak S, specify that its retention time is 1.000, calculate other characteristic peaks relative to reference to the relative retention time at peak, its relative retention time should setting ± 5% within, the relative retention time setting of each characteristic peak is characteristic peak 1(P1) setting 0.429, characteristic peak 2(P2) setting 0.556, characteristic peak 3(P3) (caffeine) setting 0.576, characteristic peak 4(P4) setting 0.642, characteristic peak 5(P5) setting 0.723, characteristic peak 6(P6) setting 0.769, characteristic peak 7(P7) setting 0.849, characteristic peak 8(S) setting 1.000, characteristic peak 9(P9) setting 1.134, characteristic peak 10(P10) setting 1.183, characteristic peak 11(P11) setting 1.389.
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