The preparative chromatography is a kind of modular purification device with powerful functions. It improves the purification process in Chinese herbal medicine, chemical synthesis, and drug separation of biological protein, allowing the gradient elution of four different solvents. It is necessary to know how to use preparative chromatography correctly and properly for better use.
1. Point sample on the plate. Judge the polarity of the sample and determine the purification system through TLC plate. Preliminarily determine the polarity of target products.
2. Pretreatment of sample: The samples coming into the chromatography equipment must be filtered by sample filter device (disposable syringe and filter head). Before filtration, the sample must be weighed and treated by ultrasonic with a constant volume. The polarity of the solvent selected for constant volume should be as small as possible and the solubility should be as good as possible. The treated sample must completely dissolve to form a homogeneous solution and will not emulsify or precipitate out solids when it meets with the solvent used for the purification system. Put the treated sample into the test tube with stopper for later use.
3. Clean pipelines: There is normal phase and reverse phase chromatography in terms of cleaning methods. For normal phase chromatography, firstly use the large polar solvent to flush pipelines and then use the small polar solvent to flush them. The reverse phase chromatography generally uses acetonitrile and water as the eluent. When adopting the reverse phase chromatography for purification, firstly use water to flush pipelines and then use acetonitrile to rinse them.
4. Rinse columns: Firstly use the large polar solvent to flush columns and then use the small polar solvent to flush them to make columns rinsed. It should be ensured that columns are fully and reasonably rinsed, otherwise it will seriously influences the separation effect.
5. Set up the purification method: Determine the concentration gradient, time, flow rate and collection method based on analysis results or TLC results.
6. Load sample: When using the sampler, load sample at constant speed and pay attention to the system pressure to prevent overpressure, column blockage and the overpressure protection of the solvent pump that will stop the equipment. It also should avoid the column overpressure that will lead to the backflow and overflow of sample and the loss of sample. Loading sample at constant speed can avoid sample’s fault on the column and improve the purification effect.
7. Start purification: Observe the monitor whether there is any peak. If a peak begins, keep the concentration constant until the end of the peak. Then continue to increase the concentration according to the gradient. As long as a peak begins, keep the concentration constant until the end of the peak. Find the target product through TLC.
8. Continuously load sample for purification: Repeat the step four to rinse columns when the purification need continue after purifying last batch of sample.
9. Remove solvent: Remove the solvent by rotary evaporator to obtain pure product.
10. If there are higher requirements for product, the solvent should be completely removed, which requires a freeze dryer to remove residual solvent.