Determination Of Components From Herba Cistanehes By Analytical Supercritical Fluid Extraction With GC/MS
Mar 08, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Intro
Supercritical fluid extraction (SFE) technology, with its unique advantages, is attracting attention as a new sample preparation method in analytical chemistry and began to form an analytical or micro SFE technology. It has been proved to be an efficient separation method through the separation and determination of trace organic compounds in the environment, food, natural compounds, and other complex substrate samples, During the extraction process, the components in the sample are neither polluted nor lost.
In this experiment, supercritical carbon dioxide (or adding a small amount of methanol as a modifier) was used as the fluid, analytical SFE system was used, and combined with gas chromatography and mass spectrometry, the chemical components of volatile components in Cistanche deserticola were determined. The results were significantly different from what could get via the traditional methods.
Experimental part
1 supercritical fluid extraction system
The analytical SFE device was designed and installed by our laboratory, and the process is shown in Figure 1. The microextraction tank was composed of stainless steel liquid chromatography column tubes. The throttle tube was elastic quartz or stainless steel capillary with an inner diameter of 0.1mm. Its main function is to maintain and adjust the flow rate and pressure in the extraction process, and the extracted product was introduced into the collector or chromatographic system. The extract was collected by solvent collection for chromatographic/mass spectrometry analysis. The solvent was isopropyl alcohol.
The fluid used in the experiment was carbon dioxide (Beijing Experimental Plant) and the modifier was methanol (analytical purity, Beijing chemical plant)
Cistanche deserticola was some dark brown plant stems purchased from the market. It was broken and ground into granules to increase the surface area before the extraction.
Weighted 0.5g of sample into the extraction tank and adopt the dynamic extraction process at the selected pressure and temperature. The extraction pressure and flow rate are controlled by the resistance of the pump and throttle pipe, and the volume of fluid was measured.
Cistanche deserticola supplement
2 separation and identification of the extraction components
The extracted products were separated and identified by capillary gas chromatography (CGC) and gas chromatography/mass spectrometry (GC/MS)
The type of the gas chromatograph was Perkin Elmer F17. The capillary column was 50mx0.24mm i.d., SE-30 column (Shimadzu. Co.). FID, split injection, and temperature-programmed operation. The Varian 3400 GC was connected with the ITD 800 ion trap detector (Finnigan mat CO) through the open split. The chromatographic column was 30mx0.25mln i.d., DB-5 column (J & W Scientific). The linear velocity of carrier gas (He) was 30cm/s. When there was no split injection, the temperature of the injection port is 250 ℃. The initial temperature of the column was 50 ℃, maintained for 5min, and then increased to 300 ℃ at a heating rate of 5 ℃ / min.
The ion trap detector worked in full-scan electron impact (EI) mode, with a mass range of 40 ~ 450u, and took 1 second for each scan. The ion trap and transmission line temperatures are 215 ℃ and 250 ℃ respectively. In addition to El, chemical ionization (CI) mass spectrometry was obtained under the same chromatographic conditions. The CI reaction gas was methane. Considering the existence of homologs and isomers, the GC total ion flow diagram, and mass spectrum of the C10-C14, C17-C20 n-alkane mixture were used as qualitative auxiliary external standards.
Cistanche deserticola extract
Results and discussion
Cistanche deserticola is a Yang promoting medicine with complex components, and there are few reports on its volatile components. One of the methods to extract its components is to reflux the ethanol solution containing hydrochloric acid and then through a series of chemical treatment processes to obtain samples that can be directly analyzed by chromatography. Its main chemical components are hydrocarbon compounds such as heptadecane. The extraction process is very simple by the CO2-SFE method, Under the condition of 20MPa pressure and 50 ℃, only tens of milliliters of CO2 fluid is required, which can be completed within 1h. The separation results are shown in Fig. 2
It can be seen from Figure 2 that there are more than 30 main volatile components separated by gas chromatography. The molecular weight is determined by CI mass spectrum. The components can be roughly divided into three categories:
The first category is normal alkanes from C16 to C28. Low carbon hydrocarbons and their isomers also exist in the extract, but their content is low.
The second category is esters. The three main components are dibutyl phthalate, dibutyl sebacate, and dioctyl phthalate, accounting for more than 90% of the esters.
The third category is oxygen-containing and nitrogen-containing compounds with low molecular weight. Eugenol is the most important component. The matching degree of mass spectrometry retrieval of other compounds such as vanillin and isoeugenol is also more than 97%, and their chromatographic retention data are also consistent with the literature. The CI mass spectrometry shows that the MH+ ion masses of compounds with peak numbers 8 and 12 are 144 and 180 respectively, It shows that both of them are compounds containing one nitrogen atom. They are 5-oxyproline methyl ester and 1-Methyl-2 (lH) - pyridine ethyl acetate respectively. From the above results, it is not difficult to see that the extraction products of SFE are different from those in the literature.
Cistanche deserticola benefit
Carbon dioxide can dissolve and extract polar compounds under supercritical conditions, which have the characteristics of polar solvents. However, adding methanol, NO2 and other modifiers to CO2 can improve the efficiency of CO2 extraction of polar compounds. In this experiment, adding 3% methanol to pure CO2 significantly increases the yield of polar compounds (Fig. 3), and the extraction amount of alkanes are relatively inhibited. This is one of the methods to improve the extraction selectivity of supercritical fluid by adding modifiers. Because the type and amount of modifiers are controllable, the selectivity can also be controlled according to the sample.
The selectivity of SFE also depends on its state. The solvent strength of supercritical fluid is proportional to its density (, in this formula ρ Is the density of the fluid ρL is the density of the fluid material in the liquid state, and Pc is the critical pressure). When the temperature is constant, the fluid density increases nonlinearly with the increase of the system pressure, and the solubility increases. On the contrary, when the pressure is low, the density decreases, and the extraction efficiency decreases significantly. Fig. 4 shows that when the pressure is low to 9 MPa, only a small amount of solutes such as dibutyl phthalate are extracted from the Cistanche deserticola sample, The extraction efficiency of other components is very low. It also shows that the solubility of the solute in a supercritical fluid is also related to its structure and properties.
Cistanche deserticola supplement
The experimental results of SFE of Cistanche deserticola also show that in CO2 fluid, whether the modifier is added or not, increasing the temperature is not conducive to the improvement of extraction efficiency. Unless the density remains unchanged, the high temperature may be conducive to the extraction of some components. The basic conditions for CO2 fluid extraction of volatile components from Cistanche deserticola samples are that the pressure exceeds 20MPa and the temperature is at 50 ℃. Adding a small amount of methanol (3%)as a modifier has a better extraction effect on polar compounds.
