Introduction to the preparation of preparative columns
Introduction to the preparation of preparative columns
Selection of chromatographic separation mode For the selection of chromatographic separation mode, separation conditions should be tested using a variety of analytical columns. When multiple modes are available, the following factors should be considered, including:
Resolution: the selectivity of the chromatographic packing to the component being measured
Loading amount: the carrying capacity of the sample to be tested by the chromatographic packing
Analysis speed: the separation time of the sample to be tested
Column Specs Selection The following table provides general rules for column specification selection. The following factors should be considered when selecting, including:
1 Column inner diameter: The sample should be loaded with the cross-sectional area of ​​the column. The inner diameter of the column should be selected to suit the sample loading.
2 Particle size of the filler: The smaller the particle size, the higher the efficiency of the column, but the higher the price and the higher the column pressure, the higher the pressure tolerance of the hardware of the equipment. When the peak of the component being measured is very close to other peaks, a higher efficiency is required, and a smaller particle size chromatographic packing is very useful. Conversely, the larger the particle size, the lower the efficiency, but the price is relatively low, and the equipment requirements are not high.
3 Column length: The longer the column, the higher the efficiency and higher sample loading, but the column pressure will be higher and the separation time may be longer.
Steps for preparing column separation performance test
1 Considering the factors that directly increase the packing from the analytical stage to the preparative stage, if a large amount of compound is to be purified, the size of the analytical column and the particle size of the filler (10 μm or larger) which can be used as a preparative column need to be considered; For larger projects, consideration must be given to selecting a larger column size and larger internal diameter column to purify the product under the same packing as the analytical column.
2 The selectivity of the analytical column (4.6mm inner diameter or smaller) should be selected under a variety of fillers and a variety of chromatographic conditions. Some products can be compared with a variety of chromatographic modes using liquid chromatography conditions selection software. This can shorten the time for separation conditions.
3 After obtaining the best separation conditions, it is necessary to test the carrying capacity of the sample on the analytical column, compare the purity of the product under various loadings, and select the maximum loading of the most ideal product purity.
4 Select the specification of the preparative column, and perform the amplification separation according to the sample loading data obtained on the analytical column. Usually, the filler with the same particle size as the analytical column and the length of the column are selected to achieve the prediction of the preparative separation. The loading of the scale-up preparative column should be selected based on the ratio of the cross-sectional area of ​​the preparative column to the analytical column.
5 Check the results of the preparative chromatographic separation and the area and purity of the chromatographic distillate.
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