High performance liquid chromatography experimental technical questions and answers
A-01000 Mouse anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (internal reference antibody) Anti-GAPDH (mouseAnti-GAPDHMonoclonal) Solution: The theoretical and technical achievements of gas chromatography have created conditions for the development of liquid chromatographs. From its high efficiency, high speed and high sensitivity, it is inspired by 5-10 particles to improve column efficiency and high pressure pump. Accelerate the flow rate of the liquid mobile phase; design high-sensitivity, low-density UV, fluorescence and other detectors to improve detection sensitivity and overcome the shortcomings of classical liquid chromatography, thus achieving high efficiency, rapidity and sensitivity. 2. What are the advantages and disadvantages of high performance liquid chromatography compared with gas chromatography? Solution: The object of gas chromatography analysis is that it has a certain volatility at school temperature and is stable to heat. It is therefore limited to the analysis of gases and compounds with low boiling points or volatile derivatives. High-performance liquid chromatography, because of the liquid as the mobile phase, can be analyzed as long as the substance to be analyzed has a certain degree of resolution in the selected mobile phase, so it has wide applicability and is not limited by sample volatility and thermal stability. It is especially suitable for compounds with high boiling point, strong polarity and poor thermal stability, such as biochemical substances and drugs, ionic compounds, Natural products with poor thermal stability, and the like. In the currently known organicizing stations, Only 20% of the samples can be satisfactorily separated by gas chromatography without chemical treatment, and 80% of the organic compounds are analyzed by high performance liquid chromatography. The mobile phase in the gas chromatograph is inert, it has no force on the components, only acts as a carrier, and the mobile phase of high performance liquid chromatography not only acts as a carrier, but also has a certain affinity for the flow relative components, which can be changed by changing the flow. The phase type and composition increase the selectivity of the separation, and there are many compounds which can be used as a mobile phase, and the selection is wide. Another advantage of high performance liquid chromatography compared to gas chromatography is that sample recovery is relatively easy, as long as the open container is placed at the end of the column, the separated components can be easily collected. Recovery is quantitative and can be used to purify and prepare a single substance of sufficient purity. Insufficient for high performance liquid chromatography, the sensitivity of the detectors is not as good as that of gas chromatography. Special attention must be paid to the effect of "extra-column effect" on column efficiency and chromatographic separation. 3. Compare the Hu curve of gas chromatography and liquid chromatography, and analyze the different causes. Solution: It can be seen that the Hu curve obtained by gas chromatography and liquid chromatography is very different in shape, and the flow velocity of the mobile phase has different effects on the efficiency of the column. On the Hu curve of the gas chromatograph, the height H of the tray varies with u. Hyperbolic curve. The curve has a low point of zui. At this time, the column effect is high, the plate height is small, and the flow rate is good. On the liquid chromatogram Hu curve, there is no phenomenon that the flow rate decreases the plate height, and since the flow velocity of zui is close to zero, the flow rate corresponding to the low plate height of the Zui is generally not observed. Under normal conditions, the flow rate is reduced and the plate height H is always reduced, which is significantly different from gas chromatography. In the gas chromatography, the flow velocity of the mobile phase increases, and the column efficiency decreases linearly. In the liquid chromatography, the flow velocity of the mobile phase increases, and the efficiency of the column decreases gently. The main reason is that the mobile phase of liquid chromatography is liquid, and the diffusion coefficient Dm of liquid phase is very small, usually only one of 104~105 of the gas diffusion coefficient, so the molecular diffusion term does not play much role in low u. Therefore, the liquid chromatogram Hu curve failed to show a decrease in flow rate and an increase in plate height. At high speed, although the line speed in the column is increased, the mass transfer of the stationary phase and the mobile phase can be performed very quickly, so the Hu curve rises slowly. 4. Briefly describe the main factors that cause chromatographic peak expansion in liquid chromatography. How to reduce the expansion of the ankle band and improve the efficiency of the column? Solution: The main factors causing the expansion of the chromene peak in liquid chromatography are eddy current diffusion, mobile phase mass transfer, retention mobile phase mass transfer and extra-column effect. In liquid chromatography, the band expansion should be reduced, the column efficiency should be improved, the diameter of the reclaimed particles should be reduced, the depth of the material hole should be reduced, and the uniformity of the filling should be improved. The low viscosity solvent should be used as the mobile phase, and the flow rate should be as low as possible. Try to use smaller dead volume injectors, detectors, fittings, and transfer lines. 5. Why do you want to propose a folding parameter? What are the characteristics? Solution: We know that chromatographic operating conditions have an effect on the plate height. Giddings found that the stationary phase is the same, the filling is good, and the total particle size dp is different, so the Hu curve is different. In liquid chromatography, the plate height is a function of the particle size. In order to compare the plate height H. under the chromatographic conditions, the folding parameters are included, including the height of the folded plate, the flow rate of the fold, and the length of the folded column. It is characterized by the same H-ur curve for different particle size fillers, so the column efficiency of different fillers can be compared under the same conditions. 6. The length of the liquid chromatography column A is 15cm, the carrier particle size is 5m. The other B column is 30cm long, and the carrier particle size is 10m. Is the column efficiency of the two columns equal? Solution: The length of the folded column of the A-pillar is 30,000, and the length of the folded column of the B-column is also 30,000, indicating that the components pass through 30,000 carrier particles from the column population to the outlet in the two columns. The efficiency of the two schools is equal. 7. What is the gradient elution, which samples are suitable for analysis? What is the difference between temperature and temperature? Solution: Gradient leaching is in the process of separation. The composition, polarity, pH value of the mobile phase are continuously changed according to the 'determination procedure'. The components in the sample can be peaked at a good k. For components with short retention times, overcrowding, and even overlap, components with a long retention time and a flat peak shape are well separated, and are particularly suitable for the analysis of complex samples with a wide range of k values ​​in the sample. Gradient elution is very similar to the temperature programming of gas chromatography, and the purpose of both is the same. The difference is that the temperature is programmed to change the column temperature. Liquid chromatography, by changing the composition, polarity, and pH of the mobile phase, achieves the goal of changing k. 8. Why should the mobile phase be degassed beforehand? What kinds of common degassing methods are there? Solution: The dissolved gas in the mobile phase has the following disadvantages: the bubble enters the detector, causing the light absorption to change into an electrical signal, the baseline jumps suddenly, and the interference is detected; when the gas dissolved in the solvent enters the column, it may flow with the flow. The phase or stationary phase undergoes a chemical reaction; dissolved gases can also cause oxidative degradation of certain samples. Errors in separation and analysis results. Therefore, degassing must be performed before use. Commonly used degassing methods are as follows: (1) heating degassing method; (2) suction degassing method; (3) blowing degassing method; (4) ultrasonic oscillation degassing method. 9. According to the size of the fixed phase pore size, what are the types of liquid chromatography stationary phases? What are the characteristics and scope of application? Solution: From the perspective of the pore depth of the stationary phase, the stationary phase of liquid chromatography is divided into two types: surface porous type (thin shell type) and fully porous type (full pore type). The surface porous type stationary phase porous layer has small thickness, shallow pores, relatively small dead volume, rapid peak output, high column efficiency, large particles, good permeability, and shallow phase, gradient elution, when the mobile phase composition changes. The mobile phase components inside and outside the pores can quickly reach equilibrium, high mechanical strength, and easy packing. The lack of porous layer is small, so the column capacity is small. The large sample size is limited, which is suitable for simple sample analysis and rapid analysis.
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A-01006 serotonin antibody Anti-5-HT (5-hydroxytryptamine)
A-01007 serotonin receptor 1A antibody Anti-5-HTR1A (5-HT1A (5-hydroxytryptamine/serotonin receptor 1A; 5HT1a; ADRBRL1; ADRB2RL1; HTR1A)
A-01008 5-HT/serotoninR1B antibody Anti-5-HTR1B (5-hydroxytryptamine/serotoninreceptor 1B)
A-01009 5-HT/serotoninR2B antibody Anti-5-HTR2B (5-hydroxytryptamine(serotonin)receptor2B)
A-01010 Serotonin Receptor 2A Antibody Anti-5-HTR2A
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1. How is high performance liquid chromatography efficient, fast and sensitive?