Key words: supercritical fluid chromatography , lycopene
1 Introduction Lycopene is a kind of carotenoid, widely distributed in various plants such as tomato, watermelon, grape, etc. As a polyene aromatic hydrocarbon, lycopene is a strong antioxidant, which can eliminate the freedom in blood vessels. Base, quenching singlet oxygen has a certain effect on inhibiting cancer. In recent years, research on the analytical methods of lycopene has also increased. Commonly used methods are HPLC , TLC, and ultraviolet spectrophotometry . These methods have their own characteristics, HPLC accuracy is high, but organic solvents are expensive; TLC equipment requirements are not high, but the analysis time is long, the precision is poor; UV spectrophotometry is relatively simple, but due to p. Interference with carotene and the like is likely to cause large errors.
Analysis of carotene by supercritical fluid chromatography has been reported. Lesellier E column and Aubert analyzed α-carotene and β-carotene by supercritical fluid chromatography. However, the special analysis of lycopene by supercritical fluid chromatography has not been reported. Supercritical fluid has the advantages of high diffusibility and strong solubility, low dosage of organic solvent, low operating temperature, etc. This paper examines the temperature of the column, the pressure of the supercritical fluid, the composition of the supercritical fluid, and the carrier concentration. The influence of factors on the separation of lycopene, a powerful analytical separation method for the study of lycopene.
2 Experimental part
2.1 Instruments and reagents The laboratory's self-assembled supercritical fluid chromatograph includes: two ISCO 260DM syringe pumps for carbon dioxide delivery, one ISCO100DM syringe pump for carrier, and three pumps controlled by a single controller. It can accurately control the flow rate of the column front pressure and the carrier; the freezer (Chongqing Sida Experimental Instrument Factory) freezes the carbon dioxide to a 6 °C; the incubator (Haian Petroleum Instrument Factory); the TSP-100 high pressure UV-VIS detector (US TSP) Company); Rhendyne 7125-shaped six-way injection valve with 20μL quantitative tube and other parts.
Carbon dioxide (Beijing Yupu North Gas Industry Co., Ltd., purity 99.99%); anhydrous ethanol (Beijing Chemical Plant, analytical grade); n-hexane (Beijing Chemical Plant, analytical grade).
2.2 Samples and treatment samples include: lycopene standard, β-carotene, oxidized lycopene standard after half a month at room temperature, oxidized lycopene standard added with β-carotene; The critical propane extracts the tomato product, the extracted tomato material, and the raffinate. The above samples were weighed and dissolved in n-hexane, respectively.
2.3 Chromatographic conditions
Spherisorb Ctg column (Dalian Institute of Chemical Physics, Chinese Academy of Sciences, size: 250mm × 4.5mm, 10μm packing); mobile phase is carbon dioxide - ethanol, carbon dioxide - n-hexane; detection wavelength: 472nm; injection volume: 20μL; temperature, pressure , mobile phase flow rate and composition are described below.
3 Results and discussion
3.1 Qualitative analysis of lycopene The sample of lycopene used in this experiment is a supercritical propane extraction tomato product, in which the main impurity is β-carotene, and because lycopene is easily oxidized, lycopene, The lycopene oxide and carotene were qualitatively analyzed. Under the same chromatographic conditions, lycopene standard solution, oxidized lycopene standard solution, and lycopene standard solution containing β-carotene were separately injected. As can be seen from the results, the retention time of lycopene and its oxide, β-carotene, increased with decreasing polarity.
3.2 Determination of zui good conditions In order to ensure the quantitative accuracy of lycopene, by examining the effects of pressure, temperature, mobile phase composition and concentration on the separation of lycopene and its oxides, the good conditions for the separation of lycopene were determined.
3.2.1 Influence of pre-column pressure Change the pre-column pressure. When the pre-column pressure is increased from 17.0 MPa to 20.0 MPa, the capacity factor of lycopene and its oxide is gradually reduced, and the retention time of both is shortened, but Lycopene and its oxides can be separated.
3.2.2 Influence of post-column pressure When the pre-column pressure, temperature and carrier flow rate are unchanged, the post-column pressure is increased from 15 MPa to 19 MPa, and the capacity factor of lycopene and its oxide are reduced, but lycopene The relative retention value of its oxide decreases with the increase of post-column pressure, and the degree of separation also tends to decrease.
3.2.3 Influence of temperature The change trend of capacity factor with temperature is shown in the figure. As the temperature increases, the capacity factor of lycopene and its oxide decreases. The relative retention of lycopene and its oxide is large at room temperature. It can also be seen from the figure that when the analysis temperature is low, the retention time of lycopene and its oxide is longer, but the degree of separation is larger, so the temperature of separation can be selected from room temperature.
3.2.4 Effect of carrier When the concentration of ethanol increases from 5% to 8%, the lycopene capacity factor decreases rapidly. When the concentration increases to 16%, the relative retention of lycopene and its oxide The concentration of ethanol is suitably 8% to 10%.
If n-hexane is used as a carrier, the change trend is the same as that of ethanol. The relative retention value of lycopene and its oxide is not much different from that of ethanol as a carrier, about 1.2. However, at the same concentration, the capacity factor for separating lycopene by n-hexane as a carrier is smaller than that of ethanol.
3.3 Quantitative analysis of lycopene
3.3.1 Drawing a standard working curve of lycopene Prepare a series of concentrations of lycopene standard solution, respectively, and take 20 μL of the above standard solution into the chromatogram, and according to the concentration. The peak area is used as a standard curve. The standard curve equation is Y = a 0.049 + 7.42 × 0.0000001X (the unit of Y is g / L), the fitness is 0.9990, and the linear relationship is good. Linear range: 3 ~ 240mg / L.
3.3.2 Supercritical extraction of lycopene sample chromatograms Select appropriate chromatographic separation conditions, take 20 μL of lycopene product in n-hexane solution into the chromatogram, the peak area of ​​lycopene in the product into the standard curve, you can The concentration of lycopene in the solution was determined, and the lycopene content in the product was determined.
3.3.3 Determination of precision and parallelity Weigh an appropriate amount of the same batch of tomato products, raw materials and raffinate, respectively, and dissolve in 10 mL of n-hexane. Each of the above solutions was measured in parallel for 4 times. The results can be seen in the table. The relative standard deviations of the measurement results are all within 6%, with good precision, and the parallelism of the results is also good.
According to the combined content and total amount of materials, it can be seen that the total amount of lycopene in the raw materials is consistent with the total amount of lycopene in the product and the raffinate, and the obtained results are reliable and accurate.
4 Conclusions (1) Using supercritical fluid chromatography to qualitatively analyze lycopene on a C18 column, the separation conditions can be improved by changing the temperature, pressure, and carrier concentration. The optimized conditions determined in this study are 20.0 MPa for column pressure, 3.0 to 4.0 MPa for column pressure, room temperature for separation, and 8% to 10% for carrier. The retention time of lycopene was approximately 3 min and the analysis time was shorter than HPLC. (2) Quantitative lycopene was quantified by supercritical fluid chromatography with a relative standard deviation of less than 6%, and the repeatability and parallelism were good.
References
1 Cheng Jian (Zheng Jian), Zeng Qingxiao (Zeng Qingxiao). Food and Fermentation lndustr/ez (Food and Fermentation Industries), 1999, 26(2): 75-78
2 Wang Qiang (Wang Qiang), Han Yashan (Han Yashan), Dai Yunqing (Dai Yunqing). Chinese J. Chromatogr. (Chromatography), 1997, 15(6): 534-535
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Production Specification Sheet
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Product Name |
Country of Origin |
Ningxia in China |
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ANALYSIS |
DESCRIPTION |
TEST METHODS |
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Product Name |
Organic Goji Juice |
Conventional Goji Juice |
Conventional Contracted Goji Juice |
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BRIX |
NLT 13 |
NLT 36 |
Organoleptic Inspection |
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Color |
Bright auburn or Purple red |
Organoleptic Inspection |
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Smell and Taste |
Characteristic |
Organoleptic Inspection |
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Texture |
The fruit pulp contains, a period of few days the juice will appear pulp precipitation |
Organoleptic Inspection |
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Total plate count(cfu/ml) |
NMT 1000 |
GB4789.2 |
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Salmonella |
Absence |
GB/T 4789.4 |
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Staphylococcus |
Absence |
GB 4789.10 |
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Pb, mg/kg |
NMT 0.5 |
GB 5009.12 |
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As, mg/kg |
NMT 0.5 |
GB/T 5009.11 |
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Cu, mg/kg |
NMT 10.0 |
GB/T 5009.13 |
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Pesticide Residue |
Absence |
NMT 0.2ppm |
GB/T 19648-2006, GB/T 200769-2008 |
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Shelf Life |
12 months if stored in a cool ventilated dry place |
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Package |
210kg/drum.,Internal: double aseptic bag. External: Drum |
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Storage |
It should be stored under the dry and ventilated environment |
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