This experiment is from the official website of Wuhan University School of Pharmacy

Operation method

Separation of barbiturates from phenobarbital by high performance liquid chromatography experiment

Principle

High performance liquid chromatography (HPLC) is an important chromatographic separation technique. According to the different stationary phases used and separation mechanisms, HPLC is generally categorized into partition chromatography, adsorption chromatography, ion exchange chromatography, and space exclusion chromatography. In partition chromatography, the degree of retention of components on the column depends on their partition coefficient K between the stationary phase and the mobile phase: the ratio of the concentration of the component in the stationary phase to the concentration of the component in the mobile phase. Obviously, the larger the value of K, the longer the retention time of the components on the stationary phase, and the larger the difference in polarity between the stationary phase and the mobile phase. Therefore, the normal phase chromatography method, in which the mobile phase is non-polar and the stationary phase is polar, and the reversed phase chromatography method, in which the mobile phase is polar and the stationary phase is non-polar, have emerged. The most widely used stationary phase is the so-called bonded stationary phase, in which the stationary phase solution is bonded to the silica gel surface by means of a chemical reaction. If non-polar substances such as n-alkanes (e.g., n-C18 alkanes) are bonded to a silica gel matrix, and a polar solvent (e.g., methanol and water) is used as the mobile phase, non-polar or weakly polar compounds can be separated. Accordingly, alkylbenzenes can be separated using reversed-phase liquid chromatography. In reversed-phase liquid chromatography, the stationary phase is less polar than the mobile phase, and the elution order depends on the hydrophobicity of the solute molecules, with the more hydrophobic ones having a longer retention time. HPLC can also be used for quantitative analysis, where the concentration of the substance to be measured is determined from the relationship between concentration and peak area. This experiment is based on the separation and quantitative analysis of barbiturates and phenobarbital based on their polarity differences.

Move

1. Prepare a standard solution of barbital and phenobarbital, both at a concentration of 0.15 mg-mL-1, using the mobile phase as the solvent.

2. Turn on the instrument and set the parameters.

3. After the instrument was stabilized, 20 μL of each mixed sample was injected with a syringe, and the retention time and retention distance were recorded at the same time as the sample was injected.

4. Into the unknown sample 20 microliters, write down the retention time of the chromatographic peaks of each component.

5. Take the retention time of the standard as the benchmark, and characterize each component of the unknown sample.

6. According to the peak area of the standard, estimate the content of the corresponding component in the unknown sample.

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