CHINESE
| Five common detection methods of plant extracts |
| Release time ：2020-07-20 Number of hits： |
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At present, there are five common detection methods of plant extracts, which are: high performance liquid chromatography (HPLC), ultraviolet absorption spectrometry (UV), thin layer chromatography (TLC), gas chromatography (GC) and atomic absorption spectrometry (AAS).
The principle and application of each method are different. Among them, HPLC and UV are the common detection methods of standard plant extracts, TLC is used to detect the proportion of plant extracts, GC is used to detect volatile liquids or oils, and AAS is used to detect the content of heavy metals in the extracts.
1. High performance liquid chromatography (HPLC)
The whole process of HPLC is high performance liquid chromatography (HPLC), also known as "high pressure liquid chromatography", "high speed liquid chromatography", "high resolution liquid chromatography", "modern column chromatography", etc. High performance liquid chromatography (HPLC) is an important branch of chromatography. With liquid as the mobile phase, the mobile phases such as single solvent with different polarity or different proportions of mixed solvent, buffer solution and other mobile phases are pumped into the chromatographic column with fixed phase. After the components in the column are separated, they enter the detector for detection, so as to realize the analysis of samples. This method has become an important separation and analysis technology in chemistry, medicine, industry, agronomy, commodity inspection and legal inspection. High performance liquid chromatography (HPLC) is an analytical method developed in the late 1960s. In recent years, it has been widely used in the separation and determination of functional ingredients, nutritional fortifiers, vitamins and proteins in health food. About 80% of the organic compounds in the world can be analyzed and determined by HPLC.
1.1 process flow of high performance liquid chromatography
The solvent in the liquid storage bottle is sucked into the chromatographic system by the pump, and then output. After flow and pressure measurement, it is led into the injector. The detected substance is injected by the injector and passes through the chromatographic column with the mobile phase. After separation on the column, it enters the detector. The detection signal is collected and processed by the data processing equipment, and the chromatogram is recorded. The waste liquid flows into the waste liquid bottle. In the case of complex mixture separation (wide polarity range), gradient controller can also be used for gradient elution. This is similar to the temperature programming of gas chromatography, except that gas chromatography changes the temperature,
However, HPLC changed the polarity of mobile phase and separated the components under the best conditions.
1.2 separation process of high performance liquid chromatography
Like other chromatographic processes, HPLC is also a process of solute exchange between stationary phase and mobile phase. It can separate different solutes by the difference of partition coefficient, affinity, adsorption force or molecular size between two phases.
At first, the sample is added to the column head. Assuming that the sample contains three components, a, B and C, they enter the column together with the mobile phase and begin to distribute between the stationary phase and the mobile phase. Component A with small partition coefficient is not easy to be retained by the stationary phase and flows out of the column earlier. Component C with high partition coefficient has a long residence time on the stationary phase and flows out of the column later. The partition coefficient of component B is between a and C. the second elution column. If a mixture containing more than one component enters the system, the components in the mixture flow out of the chromatographic column according to the different partition coefficients between the two phases to achieve the purpose of separation.
The separation of different components in the chromatographic process, first of all, depends on whether there are differences in the partition coefficient, adsorption capacity and affinity of each component in the two phases. This is a thermodynamic equilibrium problem and the primary condition for separation. Secondly, when different components move in the column, the spectral band broadens with the column length. The separation is related to the diffusion coefficient between the two phases, the size of the stationary phase, the filling condition of the column and the flow rate of the mobile phase. Therefore, the final effect of separation is the comprehensive benefit of thermodynamics and kinetics.
2. Ultraviolet absorption spectrometry (UV)
UV detection method is also one of the commonly used detection methods of plant extracts. UV is the abbreviation of the English name ultraviolet, UV detection is also known as UV detection, UV spectrum detection. UV detection method is mainly used to determine the composition and stability constant of complexes, quantitative analysis of structure analysis and qualitative analysis of application scope. The definition of UV spectrum is that some valence electrons in molecules absorb electromagnetic waves of a certain wavelength, A spectrum produced by jumping from a low energy level to a high energy level. When electrons in a molecule absorb energy, they will jump from the ground state to the excited state, and then emit energy (radiate characteristic lines) to return to the ground state. The wavelength of the characteristic line radiated is called ultraviolet spectrum (UV) in the ultraviolet region
Ultraviolet light comes from a mercury ultraviolet lamp, which is obtained through sapphire window and process flow. Except for certain UV wavelengths, narrow bands block all transmitted light through UV filters. These ultraviolet rays can pass through filters and test detectors to record only specific UV wavelengths.
UV light passes directly through the same size UV filter close to the lamp. The reference detector is placed behind the filter to measure the current UV intensity. The reference detector signal is used to compensate for the fluctuation of UV resource intensity caused by lamp aging and extreme temperature changes. The photocurrent results from the measurement and reference detector will be amplified, modified and processed by the transmitter. The transmitter can provide the calculated measurement results in real time, and can send multiple output values to the processing control system.
2.1 UV qualitative analysis
In the qualitative analysis of organic compounds, UV Vis spectroscopy is suitable for the identification of unsaturated organic compounds, especially conjugated systems, so as to infer the skeleton structure of unknown compounds. In addition, it can be combined with infrared spectroscopy, nuclear magnetic resonance spectroscopy and mass spectrometry for qualitative identification and structural analysis, so it is still a useful auxiliary method. Generally, there are two qualitative analysis methods: comparing the absorption spectrum curve and calculating the maximum absorption wavelength λ Max by empirical rules, and then comparing with the measured value. Structural analysis? Structural analysis can be used to determine the configuration and conformation of a compound. Such as the identification of CIS trans isomers and tautomers.
2.2 UV quantitative analysis
The quantitative analysis of UV-vis spectrophotometry is based on Lambert Beer law, that is, the absorbance of a substance at a certain wavelength is linearly related to its solubility. Therefore, the concentration and content of the substance in the solution can be determined by measuring the absorbance of the solution to the incident light at a certain wavelength. There are three kinds of common determination methods: single component quantitative method, multi-component quantitative method, dual wavelength method, differential spectrophotometry and derivative spectrometry. Determination of the composition and stability constant of complexes? There are two common methods for measuring the composition of complexes: molar ratio method (also known as saturation method) and equimolar continuous change method (also known as job method). Determination of acid-base dissociation constant? Photometric method is a common method for determining dissociation constant of indicator or chromogenic agent applied in analytical chemistry. This method is especially suitable for weak acid or weak base with low solubility.
3. Thin layer chromatography (TLC)
Thin layer chromatography (TLC) is often expressed by TLC, also known as thin layer chromatography, which belongs to solid-liquid adsorption chromatography. It has the advantages of both column chromatography and paper chromatography. On the one hand, it is suitable for the separation of a small amount of samples (several to dozens of micrograms, even 0.01 μ g); on the other hand, if the adsorption layer is thickened and the sample is dotted into a line, up to 500 mg of sample can be separated. Therefore, it can be used to refine samples. Therefore, this method is especially suitable for the materials with low volatility or easy to change at high temperature and cannot be analyzed by gas chromatography. In addition, thin layer chromatography is often used to observe the gradual disappearance of raw material spots to judge whether the reaction is completed.
Thin layer chromatography is to apply a layer of adsorbent or supporting agent on the washed glass plate (about 10 × 3cm). After drying and activating, the sample solution is added to the starting line about 1cm away from one end of the thin layer plate with a flat capillary tube. After cooling or blowing dry, the thin layer plate is placed in the development tank containing the developer, and the immersion depth is 0.5cm. When the front edge of the developing agent is about 1cm from the top, take out the chromatographic plate, spray the developer after drying, or develop the color under the ultraviolet lamp. Thin layer chromatography, also known as thin plate chromatography, is a kind of chromatography. It is a very important experimental technology for rapid separation and qualitative analysis of a small amount of substances. It belongs to solid-liquid adsorption chromatography. It has the advantages of both column chromatography and paper chromatography. On the one hand, it is suitable for the separation of a small amount of samples (several to a few micrograms, even 0.01 micrograms); on the other hand, when making thin-layer plates, the adsorption layer should be thickened Therefore, it can be used to refine samples. This method is especially suitable for the quality that can not be analyzed by gas chromatography because of its low volatility or high temperature. In addition, TLC can also be used to track organic reactions and a "pre test" before column chromatography.
4. Gas chromatography (GC)
GC: gas chromatography a chromatographic method using gas as the mobile phase. According to the different stationary phases used, they can be divided into two categories: the stationary phase is solid, which is called gas-solid chromatography; and the stationary phase is liquid, it is called gas-liquid chromatography.
The gas chromatography system consists of adsorbent in the string, a stationary phase coated with liquid on an inert solid, and a mobile phase of gas continuously passing through the column. After the sample to be separated and analyzed is added from one end of the column, due to the different adsorption or dissolution capacity of each component in the fixed relative sample, that is, the distribution coefficient of each component in the stationary phase and the mobile phase is different, when the component is repeatedly distributed in the two phases and moves forward with the moving phase, the velocity of each component along the column is different, and the component with small partition coefficient is fixed The retention time of the phase is short, and it can flow out quickly from the end of the column. The time t after injection is plotted with the concentration C of each component flowing out from the end of the column, and the resulting graph is called chromatogram. When the chromatographic process is washing method, the relationship between the retention time (TR) and the time (TM) of the component passing through the column space and the adjusted retention time (T) of the component retained in the column are as follows:
Where the ratio of t to TM represents the number of times the retention time of the component in the stationary phase is longer than that in the mobile phase, which is called the capacity factor K:
It can also be seen from the chromatogram that the chromatographic peak flowing out from the back of the column is not a rectangle, but a curve of approximate Gaussian distribution. This is due to the existence of eddy current diffusion, longitudinal diffusion and mass transfer resistance when the components move in the column, resulting in regional expansion. There are two ways to store stationary phase in chromatographic column: one is to hold granular adsorbent or inert solid particles coated with stationary solution (carrier or support (Table 2)); the other is to coat or chemically cross-link the stationary phase on the inner wall of capillary column. The chromatographic column prepared by the former method is called packed column, and the chromatographic column prepared by the latter method is called capillary column (or open tube column).
The column efficiency is usually expressed by the concept of column plate in distillation method, for example, the column efficiency is expressed by "the height of a theoretical column" h or "number of plates" n. For filled columns:
For open string:
In the formula, λ is the factor related to filling uniformity, which is called filling irregularity factor; γ is the factor that makes the gas diffusion path bend by filling in the column; DP is the average particle diameter (i.e. particle size); u is the linear velocity of carrier gas at column temperature and pressure; DG is the molecular diffusion coefficient of component in gas phase; DL is the diffusion coefficient of component in liquid phase; DF is fixed DC is the inner diameter of the open string. Therefore, the number of column plates is n = L / h, where l is the length of chromatographic column; the value of N can be tested with a given substance and calculated from the chromatogram obtained from the experiment (Fig. 1)
Where ω┩ is the half height width of chromatographic peak. Because the distribution isotherms of components in gas chromatography in stationary solution are mostly linear, if the injection volume is small, the peak outflow curve obtained is initially described by Gaussian normal distribution, and its mathematical expression is as follows:
It has been proved experimentally and theoretically that the shape of chromatographic peaks is asymmetric and trailing. It is more accurate to use the Gaussian distribution modified by exponential decay as the distribution function to describe the shape of chromatographic peaks
Where a is the peak area; TG is the central position of the Gaussian peak; σ is the standard deviation of the Gaussian peak; τ is the time constant of the exponential decay function; t ′ is the integral variable.
It has been pointed out that the partition coefficients of the two components must be different in order for their chromatographic peaks to be separated. If there is a difference, the required column efficiency n will be different. Therefore, to distinguish the separation of two chromatographic peaks (Fig. 2), the total separation efficiency index R: should be used
The relationship between N and R is as follows
Where α′ is the relative retention value of components and α is the relative retention value of group correction. It can be seen from the above formula that after selecting the appropriate stationary solution and the chromatographic column with a given number of plates, the α′ value should be adjusted by changing the column temperature to meet the separation degree of separating the two components to the given r value.
5. Atomic absorption spectrometry (AAS)
Atomic absorption spectrometry (AAS), the full name is atomic absorption spectrometry
Basic principle of AAS:
The atoms of each element can not only emit a series of characteristic spectral lines, but also absorb and emit line wave atomic absorption spectrum schematic diagram
Characteristic spectral lines of the same length. When the light of a characteristic wavelength emitted by the light source passes through the atomic vapor, that is, the incident radiation frequency is equal to the energy frequency required for the electron in the atom to transition from the ground state to the higher energy state (generally, it is the first excited state), the outer electrons in the atom will selectively absorb the characteristic spectral lines emitted by the same elements, so as to weaken the incident light. The degree to which the characteristic spectral line is weakened due to absorption is called absorbance a, which is directly proportional to the content of the tested element: where k is a constant; C is the concentration of the sample; i0v is the intensity of the original light source; and IV is the intensity of the characteristic spectral line after absorption. According to the above formula, the absorbance of the unknown sample can be quantitatively analyzed against the standard series curve of known concentration. Because the atomic energy level is quantized, in all cases, the atomic absorption of radiation is selective. Due to the different atomic structure and the arrangement of outer electrons, the absorption energy of each element is different when it transits from the ground state to the first excited state. The atomic absorption spectrum is located in the ultraviolet and visible regions of the spectrum.
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