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Keywords: Microcolumn High Performance Liquid Chromatography; Solid Phase Extraction; Plant Pigment; Tobacco Leaf Introduction Plant pigment is one of the important natural chemical substances in tobacco. Whether the pigment degradation is complete and proper during the tobacco leaf preparation process will directly affect the flavor of tobacco And color. Therefore, it is of great significance to study the change of pigment during the tobacco preparation process to control the quality of tobacco. Tobacco pigments are usually determined by spectrophotometry and liquid chromatography. The accuracy of the photometric method is poor. The traditional liquid chromatography sample preparation is cumbersome and the separation time is long. Compared with conventional high performance liquid chromatography, microcolumn high performance liquid chromatography has the characteristics of less mobile phase consumption, short analysis time, can be splitless and can be directly used with mass spectrometry, etc., and has been rapidly developed in recent years. In this paper, we studied the plant pigments in tobacco using Liquids XterraTM RP18 (1.0 × 50mm, Φ2.5μm) microcolumn as the stationary phase. The main pigments in tobacco can reach baseline separation within 4.0min. This method is used for the analysis of plant pigments in tobacco samples, with satisfactory results.
1 Experimental part
1.1 Main instruments and reagents High-performance liquid chromatography-mass spectrometry (Waters, USA), including 2690 separation system (quaternary pump and automatic sampler), 996 (PAD) ultraviolet diode matrix detector, Millennium chromatography management Software; Milli-Q50 ultrapure water treatment instrument (Millipore Corporation, USA).
(1 + 1) Methanol-isopropanol solution, methanol and isopropanol are chromatographically pure reagents (produced by US Fisher); chlorophyll-a, chlorophyll-b, lutein, β-carotene, neoxanthophyll , Violaxanthin and pheophytin standard (produced by Fluka Company, content> 95%); the water is quartz sub-boiling distilled water and treated with Mili-Q50 ultra-pure water meter, and the conductivity of the water reaches the level 1 water requirement.
1.2 Chromatographic conditions The solid phase extraction column is: Waters Sep-Park-C18 solid phase extraction cartridge; the chromatography column: Waters XterraTM RP18 (1.0 × 50mm, Φ2.5μm) liquid chromatography column; the mobile phase is: A (1 +1) Methanol-isopropanol solution, B water, and the volume ratio is eluted by a gradient condition of linear increase in 0 min (90% A + 10% B) and 3.5 min (100% A + O% B). Flow rate: 0.2 mL / min; injection volume: 2.0 μL; detection wavelengths are: lutein, 444.9 nm;
β-carotene, 452.1 nm; chlorophyll-a, 649.5 nm; chlorophyll-b, 664.2 nm; violaxanthin, 441.2 nm; neoxanthophyll (neoxanthin), 442.4 nm; pheophytin, 445. 4nm; each component is detected at the maximum wavelength. Under the above chromatographic conditions, the chromatograms of the standard and tobacco samples at 450nm wavelength.
1.3 Sample treatment Fresh tobacco leaves are crushed into a slurry, and the dried tobacco leaves are crushed in a crusher and passed through a 0.1mm sieve. Weigh 0.2500g smoke sample, add 35ml90% acetone to shake and extract for 30 min, filter, filter residue and wash with lOml90% acetone 2-3 times until the residue is white. Combine the filtrates and make up to 50 ml. Take 10 ml, dilute to 50 ml with water, pass the pre-treated Sep-Park-C18 solid phase extraction cartridge at a flow rate of 10 ml / min. After the enrichment is completed, after centrifugal dehydration of the cartridge, use 2.0 ml at a flow rate of 10 ml / min Acetone elutes, and the eluent is filtered with a 0.45 μm syringe filter, and the sample is analyzed according to the above chromatographic conditions.
2 Results and discussion
2.1 Sample preparation
2.1.1 Extraction of pigment in the sample 90% acetone is used as the extraction solvent to extract the plant pigment in tobacco. At the same time, three methods of ultrasonic leaching, oscillating leaching and heating reflux extraction were used for leaching control experiment. The leaching rates of the three methods were not obvious. Oscillation leaching was selected for this experiment. Experiments have shown that for a smoke sample of about 0.25μm, the pigment can be completely leached by shaking extraction with more than 30ml of 90% acetone for 30min.
2.1.2 Solid-phase extraction conditions experiment The Waters Sep-Park-C18 solid-phase extraction cartridge was used to adsorb the pigment, and then the Waters SPE vacuum extraction device was used to extract 20 samples at a time. The small column was first activated with 5 ml of methanol, and then the residual methanol on the small column was washed with 30 ml of water. The flow rates for both small column activation and sample enrichment were 10 ml / min. The leaching solution is diluted with water to reduce the solvent's ability to elute the pigment by reducing the solvent strength. If 90% acetone leaching solution is passed directly through the small column, the pigment will not be in C due to the excessive solvent strength. The solid phase extraction cartridge is fully retained. The experiment shows that as the solvent strength decreases, the retention of the pigment on the small column is enhanced. When the dilution rate of the sample leaching solution is about 5 times and then passes through the column, the pigment can be completely enriched on the small column. The enriched pigment can be eluted with an eluent. Methanol, ethanol, acetone, and tetrahydrofuran were used as the eluent to elute the pigment on the small column. It was found that the pigment can be completely washed with tetrahydrofuran or acetone, but the strength of the tetrahydrofuran solvent is too strong. The qualitative and fatty substances are also eluted and can not achieve a good pre-separation effect. When acetone is used as the eluent, the amount of waxy and fatty substances eluted is relatively small, which can ensure that the pigment is completely eluted and better The interference components were separated, and then the pigment enriched in the small column can be completely eluted with about 2.0 mL of acetone.
2.2 Selection of separation conditions The separation of plant pigments requires a mobile phase with strong solvent strength. Tetrahydrofuran-water is the most commonly used system, but tetrahydrofuran is expensive and the solvent will corrode the filter membrane when filtering; when using methanol as the mobile phase The peak shape of β-carotene is poor, and other regulators such as ethyl acetate, isopropyl alcohol, chloroform, etc. need to be added. In this experiment, methanol and isopropanol (1 + 1) solution was used as the mobile phase. Experiments show that the gradient elution of each pigment can achieve baseline separation, and the analysis time is shorter. We tested different gradient conditions. The reasonable gradient conditions are: A (1 + 1) methanol-isopropanol solution, B water, according to 0min (90% A + 10% B), 3.5 min (100% A + 0% B).
2.3 Qualification of main pigments and selection of detection wavelength A total of 12 peaks were detected in the tobacco samples. The spectrum of each main pigment peak can be obtained by the ultraviolet diode matrix detector. Lutein, β-carotene, chlorophyll-a, chlorophyll-b, violaxanthin, and neoxanthophyll (neoxanthin) can be confirmed by comparison with the standard sample chromatogram and spectrogram.
In order to reduce the mutual interference and quantification error of each component, each component extracts the chromatogram at its maximum wavelength, and extracts the peak area of ​​the chromatogram at this wavelength for quantification. The detection wavelengths of the main pigments are: lutein: 444.9 nm, β-carotene: 452.1 nm, chlorophyll-a: 649.5 nm, chlorophyll-b: 664.2 nm, violaxanthin: 441.2 nm, neoxanthophyll: 442.4nm, pheophytin: 445.4 nm; only a few samples of pheophytin can be detected, the rest of the components are not purchased because of the corresponding standard, and because the content is too little, the spectrum is too noisy and distorted, so it can not be compared with the library It is qualitative; the chromatographic peak with a retention time of 0.304 is an unresolved mixed component. Its maximum absorption wavelength is between 340 and 420 nm. Its retention time on the column is very short. It is a more polar compound and is estimated to be polyphenol. Oxide or browning reaction product.
2.4 Working curves and detection limits were prepared with standard solutions of various plant pigments with concentrations of 100 μg / ml, 20 μg / ml, 8 μg / ml, 0.8 μg / ml, and 0.12 μg / ml, respectively. Integrate the peak area, calculate the regression equation, and calculate the lowest detection concentration of each component according to the signal-to-noise ratio S / N = 3.
2.5 Recovery rate and precision Accurately weigh two copies of the same kind of smoke samples A and B (a total of seven groups), add a known amount of different kinds of pigment standards, and measure 5 times under the same conditions. The measured amount is used to calculate the recovery rate, and the relative standard deviation is calculated based on the results of 5 parallel determinations.
2.6 Sample analysis results Tobacco samples are processed according to the 1.3 sample pretreatment process and then injected for analysis.
references:
[1] Jin Wenbo. Diya. Tobacco Chemistry [M]. Beijing: Tsinghua University Press. 1993.
[2] Zuo Tianjue, Zhu Zunquan. Tobacco production, physiology and biochemistry [M]. Shanghai: Shanghai Far East Publishing House, 1993
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Study on determination of plant pigments in tobacco samples by microcolumn high performance liquid chromatography
Abstract: The plant pigments in tobacco samples were determined by microcolumn high performance liquid chromatography; the pigments in tobacco samples were extracted by shaking with 90% acetone, and the extract was pre-separated and enriched by Waters Sep-Park-C18 solid phase extraction cartridge , Using Waters Xterra TM RP18 (1.0 × 50mm, Φ2.5μm) as the stationary phase, (1 + 1) methanol isopropanol solution-water gradient as the mobile phase, chlorophyll-a, chlorophyll-b, β in tobacco leaves -Carotene, lutein, neo-xanthophyll, violaxanthin and pheophytin are separated and extracted under the monitoring of their maximum absorption wavelength, and the peak area of ​​the chromatogram is quantified. The standard recovery rate of this method is 95% to 105%, and the RSD is 1.6% to 2.5%. It was used to determine the content of natural pigments in tobacco with satisfactory results.