CN101173915B - Method for measuring free phenol content in grape organization using high efficiency liquid chromatography - Google Patents
Method for measuring free phenol content in grape organization using high efficiency liquid chromatography Download PDFInfo
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- CN101173915B CN101173915B CN2007100187427A CN200710018742A CN101173915B CN 101173915 B CN101173915 B CN 101173915B CN 2007100187427 A CN2007100187427 A CN 2007100187427A CN 200710018742 A CN200710018742 A CN 200710018742A CN 101173915 B CN101173915 B CN 101173915B
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Abstract
The invention relates to a method for determining the mono-phenol content in grape tissue with high performance liquid chromatography, which comprises the following steps: firstly, adopting ZORBAX SB-C18 4.6Mum x 250Mum, 5Mum chromatographic column; secondly, adopting 1.3% acetic acid solution (A) and acetonitrile (B) as the mobile phase to perform gradient elution; thirdly, 0.8 ml/min flow speed, 20Mu l injection volume, and 30 DEG C column temperature; fourthly, determining the maximum absorption wavelength of the target substances, and then changing the testing wavelength to enable all thetest substances to be tested under their maximum absorption wavelength; fifthly, changing the testing wavelength in testing process, so as to enable all the substances to be tested under their maximum absorption wavelength. The invention enables the tested substances to be tested under their maximum absorption wavelength through changing the testing wavelength, presents the mono-phenol content objectively and truly on the determining level, and has the advantages of accurate determination, simple and sensitive method, and good repeatability.
Description
One, technical field:
The present invention relates to the method that a kind of chromatogram detects, be specifically related to a kind of method with free phenol content in the high effective liquid chromatography for measuring grape tissue, promptly a kind of high performance liquid chromatography change detection wavelength is measured the method for 6 kinds of free phenol content in the grape tissue simultaneously.
Two, background technology:
High performance liquid chromatography (HPLC) is widely used in a plurality of actual measurements fields such as residues of pesticides, hormone, polyphenols and organic acid because of the validity and the accuracy of its detection.Generally, need be when utilizing high performance liquid chromatography to carry out the mensuration of sample through the extraction and the purifying procedure of a series of complexity, and mostly be under constant wavelength, to detect.For single-component system, it is effective measuring under constant wavelength.But when multicomponent system is measured, because the maximum absorption wavelength of each component is not quite similar, loaded down with trivial details in addition pre-treatment process can make test substance that certain loss is arranged, and therefore utilizes the constant wave regular way to be difficult to the real content that complete detection goes out each component in the multicomponent system.For sensitivity that improves the multicomponent system detection and the real content that reflects tested component in the polycomponent objectively, should simplify the pre-treatment step of sample as much as possible and under the maximum absorption wavelength of each material, detect, especially to trace, trace materials.
Plant polyphenol (plant polyphenol) be a class be present in plant skin, root, stem, leaf and fruit in the native compound of biologically active, be widely used in fields such as chemical industry, medicine, forestry, food.As everyone knows, grape is rich in multiple polyphenols, it is one of fruit of cultivated area maximum in the world, and vine polyphenol has become a very important source of the required polyphenol of production practices, can measure truely and accurately that polyphenol content has important practical significance in the different grape tissues.
Three, summary of the invention:
The object of the present invention is to provide a kind of method with free phenol content in the high effective liquid chromatography for measuring grape tissue, wavelength switching makes institute's material of surveying all obtain detection under its maximum absorption wavelength, thereby its content of objective reality ground reflection on the mensuration aspect, measure accurately, method is simple, sensitive, favorable reproducibility.
For achieving the above object, the technical solution used in the present invention is:
A kind of method with free phenol content in the high effective liquid chromatography for measuring grape tissue is characterized in that, said method comprising the steps of:
A, employing ZORBAX SB-C18 4.6mm * 250mm, 5 μ m chromatographic columns;
B, the acetic acid aqueous solution with 1.3% (A) and acetonitrile (B) are moving phase, the gradient elution program is 0.00～0.01min, 0～14% B, 0.01～6.50min 14%～11% B, 6.50～10.00min 11%B, 10.00～10.01min 11%～16%B, 10.01～30.00min 16%～30%B, 30.00～35.00min, 30%～0B;
C, flow velocity 0.8mL/min, sample size 20 μ L, 30 ℃ of column temperatures;
D, utilize diode array detector and ultraviolet spectrophotometer to determine the maximum absorption wavelength of each target substance, and then the change detection wavelength make that all test substances all obtain detecting under its maximum absorption wavelength
Change detection wavelength in e, the mensuration process, make institute's material of surveying all under its maximum absorption wavelength, obtain detection, the wavelength program is 5.72～6.02min 272nm, 7.17～7.60min 260nm, 8.58～9.18min 280nm, 12.82～13.74min 280nm, 20.39～21.22min 275nm, 29.59～30.4min 306nm, the detection wavelength of all the other time periods is 360nm;
Compared with prior art, the advantage and the effect that have of the present invention is as follows:
1, the present invention measures accurately, and method is simple, sensitive, favorable reproducibility.
2, linear test and detectability test findings show, in the finite concentration scope, and the good (r of the linear relationship of the calibration curve of 6 kinds of free phenols
2〉=0.9990); Compare with the detection method of constant wavelength, the detectability of 6 kinds of free phenols under this method (LOD, S/N 〉=3) all has reduction in various degree, and sensitivity is improved.
3,6 kinds of free phenol recovery of standard addition 〉=89.5%, retention time and peak area are the relative standard deviation (RSD) in (n=10) and (n=10) experiment in the daytime respectively≤0.3%, 2.9% and 0.3%, 3.1% in a few days.
Four, description of drawings:
Fig. 1 is contour map (the peak sequence number: 1-gallic acid, 2-protocatechuic acid, 3-catechin, 4-epicatechin, 5-L-Epicatechin gallate, 6-resveratrol of mixing standard specimen.)
Fig. 2 is the spectrogram of catechin.
Fig. 3 is the qualitative chromatogram of mark-on of resveratrol (RES), and wherein a is for adding standard specimen, and b is not for adding standard specimen.
Fig. 4 is the comparison diagram of 6 kinds of free phenols under different chromatographic conditions in the bast, and wherein a is the change detection wavelength, and b is constant detection wavelength.
Five, embodiment:
The present invention is the method that a kind of high performance liquid chromatography change detection wavelength is measured 6 kinds of free phenol content in the grape tissue simultaneously, and concrete peak sequence is gallic acid (Gallic acid), protocatechuic acid (Protocatechuic acid), catechin ((+)-Catechin), epicatechin ((-)-Epicatechin), L-Epicatechin gallate ((-)-Epicatechin-3-O-gallate) and resveratrol (Trans-Resveratrol).Wavelength switching makes institute's material of surveying all obtain detection under its maximum absorption wavelength, thereby objective reality ground reflects its content on the mensuration aspect.
Carry out a large amount of comparison tests repeatedly by investigating different test parameterss, finally selected best chromatographic condition for use.Compare with the method for constant detection wavelength, this method has reduced the detection limit of measured matter significantly, and in measuring actual sample, obtained baseline steadily, separating effect and all good chromatogram of peak shape.
Further explain the present invention with specific embodiment below, but protection scope of the present invention is not limited to these embodiment.
Embodiment one, the foundation of chromatographic condition and sample detection
Instrument: LC-2010A
HTType is equipped with the high performance liquid chromatograph of automatic sampling apparatus, and LC-6AD type high performance liquid chromatograph type is equipped with SPD-10MA
VPDiode array detector (DAD), all parts are controlled by Tianjin, island CLASS-VP 6.12 workstations, UV-1700 type ultraviolet spectrophotometer (day island proper Tianjin company).
Reagent: standard specimens such as gallic acid, protocatechuic acid, catechin, epicatechin, L-Epicatechin gallate and resveratrol are all available from Sigma and Fluka company; Methyl alcohol and acetonitrile are chromatographically pure (U.S. TEDIA company); Acetate is that homemade analysis is pure.
Adopt ZORBAX SB-C18 4.6mm * 250mm, 5 μ m chromatographic columns;
Flow velocity 0.8mL/min, sample size 20 μ L, 30 ℃ of column temperatures;
The selection of moving phase is to the methyl alcohol and the acetic acid aqueous solution of different acid concentrations, and acetonitrile and acetic acid aqueous solution are investigated as moving phase, through after the test of many times, finds that acetic acid aqueous solution (A) and the acetonitrile (B) with 1.3% is that the moving phase effect is preferable.The gradient elution program is 0.00～0.01min, 0～14%B, 0.01～6.50min 14%～11% B, 6.50～10.00min, 11% B, 10.00～10.01min, 11%～16%B, 10.01～30.00min 16%～30% B, 30.00～35.00min, 30%～0B;
The selection that detects wavelength utilizes the ultraviolet diode array detector that the mixed mark of 6 kinds of free phenols solution is scanned in 190～800nm scope and obtains each material maximum absorption wavelength band (Fig. 1), determine the maximum absorption wavelength (Fig. 2) of each material then with ultraviolet spectrophotometer.The maximum absorption wavelength of gallic acid, protocatechuic acid, catechin, epicatechin, L-Epicatechin gallate and resveratrol is respectively: 272nm, 260nm, 280nm, 280nm, 275nm and 306nm.Select suitable wavelength switching time point according to relative retention time, all the other, section was constant wavelength 360nm (can avoid the appearance of solvent peak and many impurity peaks under 360nm) detection time.In the machine running process, with interior any change detection wavelength, the response of wavelength and stabilization time are no more than 0.1 second (these data are the performance parameter of instrument, and island proper Tianjin company provides by day) at 190～370nm, the present invention is chosen in wavelength switching in the 0.01min, and obtains good effect.The wavelength program is 5.72～6.02min 272nm, 7.17～7.60min 260nm, 8.58～9.18min 280nm, 12.82～13.74min 280nm, 20.39～21.22min 275nm, 29.59～30.4min 306nm, the detection wavelength of all the other time periods is 360nm;
Qualitative experiment
Utilize relative retention time method and interpolation standard substance method qualitative.Comparison mixes mark solution and chromatogram and the relative retention time of sample test liquid under identical chromatographic condition; In sample, add an amount of various standard substances respectively, relatively the chromatogram of mark-on sample and mark-on sample not under the identical chromatographic conditions.As shown in Figure 3, add before and after the standard specimen in sample, the position that resveratrol (trans-Resveratrol is abbreviated as RES) goes out the peak is consistent.
Typical curve and related coefficient
Accurately take by weighing 6 kinds of each 5mg of standard specimen, in the brown volumetric flask of 10mL, dilute 20,40,100,200 and 400 times successively, mix the sealing of mark solution and be stored in-20 ℃ of refrigerators standby with methanol constant volume.To mix standard specimen solution and by concentration auto injection is set from low to high, each concentration repeats sample introduction 3 times, with the average peak area (A) of each free phenol concentration (C) is drawn calibration curve, and calculates related coefficient (seeing Table 1).
The regretional analysis of 6 kinds of free phenol typical curves of table 1
Recovery of standard addition, precision, accuracy experiment
Accurately take by weighing grape fruit and hibernaculum and carry out the recovery of standard addition experiment, add the mixing standard specimen of high, medium and low 3 levels respectively in sample, every group is repeated sample introduction 6 times, measures its recovery, and calculates the relative standard deviation (RSD) of content; Certain density mixed mark solution is carried out in a few days and experiment in the daytime, and in a few days continuous sample introduction is 10 times, every 1d sample introduction 1 time and lasting 10d, calculates the relative retention time of each free phenol and the RSD of peak area (seeing Table 2) in the daytime.
The recovery of standard addition of 6 kinds of free phenols of table 2, the precision of method and accuracy
Determination on content in the sample
Take by weighing 1～2g sample,, add 15mL 95% methyl alcohol and stir evenly (solid-liquid ratio is 1: 10) to Powdered with liquid nitrogen grinding, be transferred to rapidly in the centrifuge tube that is with black paper, 15min, the centrifugal 10min of 10000r/min (4 ℃) are extracted in sealing in ultrasonic drilling machine under 4 ℃.Repeat to extract three times, merge extract, be evaporated to 15mL and be liquid to be measured, enter HPLC excessively behind the 0.45 μ m filter and detect.The results are shown in Table 3
The average content (mg/kg) of 6 kinds of free phenols in the table 3 grape tissue
The comparative experiments of embodiment two change detection wavelength and constant detection wavelength
Instrument is with embodiment one
Reagent is with embodiment one
The chromatographic condition of two kinds of methods of chromatographic condition is except that wavelength setting program difference, and the detection wavelength of constant detection wavelength method is 280nm, and other parameters are all with embodiment one
The comparison of chromatogram and minimum detectability (LOD): analyzing identical sample under two kinds of methods, is minimum detectability with signal to noise ratio (S/N ratio) (S/N) greater than 3 o'clock detected various amount of substances, and chromatogram is seen Fig. 4, and detection limit sees Table 4
The detection limit of two kinds of following 6 kinds of free phenols of condition of table 4
Claims (2)
1. the method with free phenol content in the high effective liquid chromatography for measuring grape tissue is characterized in that, said method comprising the steps of:
A, employing ZORBAX SB-C18 4.6mm * 250mm, 5 μ m chromatographic columns;
B, the acetic acid aqueous solution with 1.3% (A) and acetonitrile (B) are moving phase, the gradient elution program is 0.00～0.01min, 0～14% B, 0.01～6.50min 14%～11% B, 6.50～10.00min 11%B, 10.00～10.01min 11%～16% B, 10.01～30.00min 16%～30%B, 30.00～35.00min, 30%～0B;
C, flow velocity 0.8mL/min, sample size 20 μ L, 30 ℃ of column temperatures;
Change detection wavelength in d, the mensuration process, make institute's material of surveying all under its maximum absorption wavelength, obtain detection, the wavelength program is 5.72～6.02min 272nm, 7.17～7.60min 260nm, 8.58～9.18min 280nm, 12.82～13.74min 280nm, 20.39～21.22min 275nm, 29.59～30.4min 306nm, the detection wavelength of all the other time periods is 360nm.
2. a kind of method with free phenol content in the high effective liquid chromatography for measuring grape tissue according to claim 1 is characterized in that: adopt diode array detector and ultraviolet spectrophotometer to determine the maximum absorption wavelength of each test substance.
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Non-Patent Citations (7)
| Title |
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| 刘志勇 |
| 刘志勇;汪军;岳霞丽;董元彦.高效液相色谱切换波长法测定多组分体系.《分析化学》.2006,第34卷(第6期),894. * |
| 刘志勇;汪军;岳霞丽;董元彦.高效液相色谱切换波长法测定油菜内源激素.《光谱实验室》.2006,第23卷(第2期),285-288. * |
| 岳霞丽 |
| 汪军 |
| 董元彦.高效液相色谱切换波长法测定多组分体系.《分析化学》.2006,第34卷(第6期),894. |
| 董元彦.高效液相色谱切换波长法测定油菜内源激素.《光谱实验室》.2006,第23卷(第2期),285-288. |
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