At present, there are five common methods for detecting plant extracts: high performance liquid chromatography (HPLC), ultraviolet absorption spectroscopy (UV), thin layer chromatography (TLC), and gas chromatography (GC). And atomic absorption spectroscopy (AAS).
    The principle of action and application of each method are different. Among them, HPLC and UV are common detection methods for standard plant extracts, TLC is used for the detection of proportional plant extracts, GC is used to detect volatile liquids or oils, and AAS is used for the detection of heavy metal content of extracts.
    1. High performance liquid chromatography (HPLC)
    The whole process of HPLC is High Performance Liquid Chromatography, also known as “high pressure liquid chromatography”, “high-speed liquid chromatography”, “high resolution liquid chromatography”, “modern column chromatography” and the like. High performance liquid chromatography (HPLC) is an important branch of chromatography. With a liquid as the mobile phase, a high-pressure infusion system is used to pump a mobile phase with a single solvent of different polarity or a mixture of different solvents and buffers into a stationary phase. The column is separated from the components in the column and then entered into the detector for detection, thereby realizing the analysis of the sample. This method has become an important separation and analysis technology in the fields of 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 supplements, vitamins, and proteins in health foods. About 80% of the world’s organic compounds can be analyzed by HPLC.
    1.1 Process of high performance liquid chromatography
    The solvent in the reservoir is pumped into the chromatograph by the pump, then output, measured by flow and pressure, and introduced into the injector. The analyte is injected by the injector and passes through the 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 device, and the chromatogram is recorded. The waste liquid flows into the waste bottle. In the case of complex mixture separations (wider polarity ranges), gradient controllers can also be used for gradient elution. This is similar to the temperature programming of gas chromatography, except that the gas chromatograph changes the temperature.
    While HPLC changes the polarity of the mobile phase, the components of the sample are separated under optimal conditions.
    1.2 High performance liquid chromatography separation process
    As with other chromatographic processes, HPLC is also a continuous multiple exchange process of solute between the stationary phase and the mobile phase. It allows the separation of different solutes by the difference in the exclusion effect caused by the difference in partition coefficient, affinity, adsorption force or molecular size between the two phases.
    The starting sample is applied to the column head, assuming that the sample contains three components, A, B and C, entering the column with the mobile phase and starting to partition between the stationary phase and the mobile phase. Component A with a small partition coefficient is not easily blocked by the stationary phase and flows out of the column earlier. Component C, which has a large 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, and the second is eluted from the column. If a mixture containing multiple components enters the system, the components in the mixture flow out of the column in the order of their partition coefficients between the two phases for separation purposes.
    The separation of different components in the chromatographic process depends firstly on whether there is a difference in the partition coefficient, adsorption capacity, affinity, etc. of the two components. This is a thermodynamic equilibrium problem and the primary condition for separation. Secondly, when different components move in the column, the band broadens with the column length, and the separation is related to the diffusion coefficient between the two phases, the size of the stationary phase, the filling of the column, and the flow velocity of the mobile phase. Therefore, the final effect of separation is the comprehensive benefits of both thermodynamics and kinetics.
    2. Ultraviolet absorption spectroscopy (UV)
    UV detection is also one of the commonly used detection methods for plant extracts. UV is the abbreviation of English name ultraviolet, UV detection is also called ultraviolet detection method, ultraviolet spectrum detection method. The UV detection method is mainly used for the determination of complex composition and its stability constant. Quantitative analysis Structural analysis Qualitative analysis The application range defines that the ultraviolet spectrum is an electromagnetic wave in which some valence electrons in the molecule absorb a certain wavelength, from a low energy level to a high energy level. A spectrum produced when electrons in a molecule absorb energy and then transition from a ground state to an excited state, then emit energy (radiating out of the characteristic line) and return to the ground state; and the wavelength of the characteristic line is radiated in the ultraviolet region. Ultraviolet spectrum (UV)
    Ultraviolet light comes from a mercury UV lamp, which is obtained through a sapphire window and process flow. In addition to the specific UV wavelength, the narrow band blocks all transmitted light through the UV filter. These UV light can record only specific UV wavelengths through the filter as well as the test detector.
    The UV light passes directly through the UV filter of the same specification close to the lamp. The reference detector is placed behind the filter to test the current UV intensity. The reference detector signal is used to compensate for fluctuations in the intensity of the ultraviolet resource due to lamp aging, extreme temperature changes, and the like. The photocurrent results obtained by the measurement and reference detectors will be amplified, modified, and processed by the transmitter. The transmitter provides the calculated measurements in real time and is capable of transmitting multiple output values ​​to the process control system.
    2.1 UV qualitative analysis
    In the qualitative analysis of organic compounds, the UV-Vis spectrum is suitable for the identification of unsaturated organic compounds, especially conjugated systems, to infer the skeletal structure of the unknown. In addition, it can be qualitatively identified and structurally analyzed by infrared spectroscopy, nuclear magnetic resonance spectroscopy and mass spectrometry, so it is still a useful auxiliary method. There are generally two qualitative analysis methods, comparing the absorption spectrum curve and calculating the maximum absorption wavelength λmax using empirical rules, and then comparing with the measured values. Structural Analysis ?? Structural analysis can be used to determine the configuration and conformation of a compound. Such as the identification of cis and trans isomers and tautomers.
    2.2 UV quantitative analysis
    The basis for quantitative analysis of UV-visible spectrophotometry is Lambert-Beer’s law, that is, the absorbance of a substance measured at a certain wavelength is linear with its solubility. Accordingly, the concentration and content of the substance in the solution can be determined by measuring the absorbance of the solution to the incident light of a certain wavelength. Commonly used measurement methods are: one-component quantitative method, multi-component quantitative method, dual-wavelength method, differential light photometry and derivative spectroscopy. Determination of Complex Composition and Stability Constants 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 constants? Photometry is a common method for determining the dissociation constant of indicators or developers used in analytical chemistry. This method is especially suitable for weak acids or weak bases with low solubility.
    3. Thin layer chromatography (TLC)
    Thin layer chromatography (Thin Layer Chromatography) is commonly used for TLC, also known as thin layer chromatography, which belongs to solid-liquid adsorption chromatography. It is a small, fast and simple chromatography method developed in recent years, which combines the advantages of column chromatography and paper chromatography. On the one hand, it is suitable for the separation of small samples (several to several tens of micrograms, or even 0.01 μg); on the other hand, if the adsorption layer is thickened and the sample is spotted into a line, it can be separated as much as possible 500 mg of sample. It can therefore be used to refine the sample. Therefore, this method is particularly suitable for materials that are less volatile or subject to change at higher temperatures and cannot be analyzed by gas chromatography. Further, in the case of performing a chemical reaction, the gradual disappearance of the spot of the raw material is often observed by thin layer chromatography to judge whether or not the reaction is completed.
    Thin layer chromatography is to uniformly coat a layer of adsorbent or support agent on the cleaned glass plate (about 10×3cm). After drying and activating, the sample solution is added to the thin layer plate by capillary tube flattening. The starting line is about 1 cm at one end, dried or dried, and then placed in a developing tank containing a developing agent, and the immersion depth is 0.5 cm. When the leading edge of the developing agent is about 1 cm from the top, the chromatography plate is taken out, dried, sprayed with a color developing agent, or developed under an ultraviolet lamp. Thin-layer chromatography, also known as thin-plate chromatography, is one of the most important experimental techniques for the rapid separation and qualitative analysis of small substances. It is a solid-liquid adsorption chromatography with both column chromatography and paper chromatography. Advantages, on the one hand, for the separation of a small amount of sample (a few to a few micrograms, or even 0.01 micrograms); on the other hand, when making a thin layer, the adsorption layer is thickened and enlarged, so that it can be used to refine the sample. It is suitable for substances with low volatility or high temperature which are easy to change and cannot be analyzed by gas chromatography. In addition, TLC can be used to track organic reactions and a “pre-test” prior to column chromatography.
    4, gas chromatography analysis (GC)
    GC: Gas Chromatography Gas chromatography uses gas as the mobile phase of chromatography. Depending on the stationary phase used, it can be divided into two categories: the stationary phase is solid, called gas-solid chromatography; the stationary phase is liquid, which is called gas-liquid chromatography.
    The gas chromatograph system consists of an adsorbent contained in a column, or a stationary phase coated with a liquid on an inert solid and a mobile phase of a gas continuously passing through the column. After the sample to be separated and analyzed is added from one end of the column, the adsorption or dissolution ability of each component in the fixed sample is different, that is, the partition coefficient of each component between the stationary phase and the mobile phase is different, when the component is in When the two phases are repeatedly distributed and moved forward with the moving phase, the speed of movement of each component along the column is different, and the component with a small partition coefficient is short-lived by the stationary phase, and can be quickly removed from the end of the column. Flow out. The concentration c of each component flowing from the end of the column is plotted against the time t after the injection, and the resulting image is called a chromatogram. When the chromatographic process is flush mode, the retention time tR required for the component to flow out of the column after injection to the maximum concentration, the time tM of the component passing through the column space, and the component being retained in the column Adjusting the retention time t annoying relationship is:
    The ratio of t annoyance to tM in the formula indicates how many times the component stays in the mobile phase compared to the fixed phase, called the capacity factor k:
    It can also be seen from the chromatogram that the chromatographic peak flowing out from the column is not a rectangle, but a curve with an 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. Factors, thus causing regional expansion. There are two ways to store the stationary phase in the column. One is to hold the granular adsorbent in the column, or to hold the inert solid particles coated with the fixing solution [carrier or carrier (Table 2)]; The fixing solution is coated or chemically cross-linked to the inner wall of the capillary column. The column prepared by the former method is called a packed column, and the column prepared by the latter method is called a capillary column (or an open column).
    The efficiency of the column is usually expressed by the concept of the column of the distillation method, for example, using “the height “H or the number of pieces” n equivalent to a theoretical column to indicate the efficiency of the column. For packed columns:
    For open tubular columns:
    Where λ is a factor related to filling uniformity, called a filling irregularity factor; γ is a factor that causes the gas diffusion path to bend in the column packing, and is called a bending factor; dp is a filler average particle diameter (ie, particle size); u is the line speed of the carrier gas at column temperature and column pressure; Dg is the molecular diffusion coefficient of the component in the gas phase; Dl is the diffusion coefficient of the component in the liquid phase; df is the liquid film thickness of the fixed solution; dc is on The inner diameter of the column. Therefore, the number of columns in the column is n=L/H, where L is the length of the column; the value of n can be calculated by a given substance, and the chromatogram obtained by the experiment (Fig. 1) is calculated:
    In the formula, ω┩ is the full width at half maximum of the chromatographic peak. Since the distribution isotherms of the components of the gas chromatograph in the fixed solution are mostly linear, if the injection amount is small, the obtained peak elution curve is initially Gaussian normal distribution. To describe, its mathematical expression is:
    It is now experimentally and theoretically proved that the chromatographic peak shape of the substance is asymmetric and tail-tailing. If the Gaussian distribution corrected by the exponential decay is used as a distribution function describing the shape of the chromatographic peak, it is more precise:
    Where A represents the peak area; tG represents the center position of the Gaussian peak; σ represents the standard deviation of the Gaussian peak; τ represents the time constant of the exponential decay function; t’ is the integral variable.
    It has been pointed out above that the partition coefficients of the two components must be different and their peaks can be separated. With the difference, the column efficiency n required for separation is different, so to determine the separation of the two peaks (Figure 2), it is also necessary to use the column total separation efficiency index R:
    The relationship between n and R is:
    Where α’ is the component relative retention value; α is the component corrected relative retention value. It can be seen from the above formula that after selecting a suitable fixing solution and a column having a given number of columns, the α’ value should be adjusted by changing the temperature of the column to satisfy the degree of separation of the two components to a given R value.
    5. Atomic Absorption Spectroscopy (AAS)
    Atomic Absorption Spectroscopy (AAS), the full name is Atomic Absorption Spectrometry
    AAS basic principles:
    The atoms of each element can not only emit a series of characteristic lines, but also absorb and emit line wave atomic absorption spectroscopy schematics.
    The same characteristic line is long. When the light of a characteristic wavelength emitted by the light source passes through the atomic vapor, that is, the frequency of the incident radiation is equal to the energy frequency required for the electrons in the atom to transition from the ground state to the higher energy state (generally the first excited state) The outer electrons in the atom will selectively absorb the characteristic lines emitted by their isomers, weakening the incident light. The degree to which the characteristic line is weakened by absorption is called absorbance A, which is proportional to the content of the element to be measured: 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 line after absorption. According to the above formula, the absorbance of the unknown sample can be quantitatively analyzed against a standard series curve of known concentration. Since the atomic energy level is quantized, in all cases, the atom’s absorption of radiation is selective. Since the atomic structure of each element and the arrangement of the outer electrons are different, the energy absorbed by the element from the ground state to the first excited state is different, and thus the resonance absorption lines of the elements have different characteristics. The atomic absorption spectrum is located in the ultraviolet and visible regions of the spectrum.

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