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- How to choose solvent system used as flash column mobile phase?
- How to choose column size and solvent flow rate?
- Sample loading and wetting time
- Biotage system conversion kits
- More to come...
- How to choose solvent system used as flash column mobile phase?
- How to choose column size and solvent flow rate?
- Sample loading and wetting time
- Biotage system conversion kits
- More to come ...
Solvent selection as one of the key issues for flash column separation and purification depends on the polarity of the compounds and the type of sorbent packed inside the flash column. The polarity of the solvent that runs through the column affects the relative flow rates at which sample compounds move through the column. Typically, chemist uses TLC (thin-layer chromatography) to determine which solvent system to use. An appropriate solvent system is expected to give an Rf (retention distance) value between 0.2 and 0.3 from TLC analysis. The one-component solvent systems include pentane, petroleum ether, hexane, ether, dichloromethane, and ethyl acetate.
Solvent system generally consists of a binary mixture of higher and lower polarity solvents at different ratios. For example, hexane/ethyl acetate at ratio of 1/1 is widely used for the normal phase silica gel columns to separate ordinary compounds and some difficult-to-separate compounds. However, for a complicated mixture, the gradient elution is strongly recommended to effectively separate the compounds by linearly or step-by-step increasing the polarity of the solvent running through the column during the entire purification course. The ideal solvent system is the system that separates the components. Be noted that the solvent used for analysis must be of a high purity and should not be reused without purification.
| Two-component solvent systems | Application |
|---|---|
| Hexane/Ethyl Acetate | Ordinary compounds and difficult separations |
| Methanol/Dichloromethane (methanol ≤10%) | Polar compounds |
Flash columns size is dependent on the sample load and other parameters, such as solvent system and flow rate. Recommended sample load as listed depends on how hard your products separation on TLC and how clean your reaction. The recommended flow rates are based on hexane solvent and may vary with other solvent. The ratio of sample to silica gel typically varies from 0.1% to 10%, we recommend a ratio of sample load to silica gel at 1:40 (i.e. 1g sample : 40g silica gel) for better separation.
| Sample size, mg | Column size, g of SiO2 | Recommended solvent flow rate, ml/min | Luknova normal phase product catalog number |
|---|---|---|---|
| 4mg-0.4g | 4 | 15-20 | FC003004 (40/pkg.) FC003004-0 (480/pkg.) |
| 12mg-1.2g | 12 | 20-40 | FC003012 (30/pkg.) FC003012-0 (360/pkg.) |
| 25mg-2.5g | 25 | 25-50 | FC003025 (25/pkg.) FC003025-0 (300/pkg.) |
| 40mg-4g | 40 | 30-60 |
FC003040 (20/pkg.) FC003040-0 (240/pkg.) |
| 80mg-8g | 80 | 40-80 | FC003080 (10/pkg.) FC003080-0 (80/pkg.) |
| 0.12g-12g | 120 | 60-120 | FC003120 (8/pkg.) FC003120-0 (64/pkg.) |
| 0.24g-24g | 240 | 70-180 | FC003240 (4/pkg.) FC003240-0 (32/pkg.) |
| 0.33g-33g | 330 | 80-180 | FC003330 (4/pkg.) FC003330-0 (32/pkg.) |
For samples difficult to separate (as evidenced by TLC spots overlapping or very close), large or stacking columns will give better separation and purity. Relatively the separation resolution and purity increase with column length (i.e. stacking columns), but the pressure drop and separation time also increase with improved resolution and purity. The real flow rates also depend on your separation too. The reasonably low flow rate will result in better separation but take longer separation time. The more experiences you have on the column chromatography the more comfortable you feel to decide the size of column and to set up proper solvent gradient to give you a good separation. .
Sample loading is a process to load reaction mixture (i.e. sample) onto the column. Manually loading liquid sample with a pipette remains a conventional but effective method. However, loading liquid sample with syringe injection is more common and popular technique due to its simplicity and convenience. Typically, a reasonable sample volume (more than 2 ml) coupled with a minimum volume (i.e. less than 10 ml) of solvent to dissolve the sample will give better separation results. For sample with low solubility and/or high viscosity, solid loading method with Luknova solid load column is recommended to use. First, the hard-to-dissolve sample is dissolved in a certain amount of polar solvent and loaded onto the sorbent, such as silica gel. An approximate ratio of sample to silica gel is 1: 5 (i.e. 1 g sample : 5 g silica gel). After removing the solvent through evaporation (such as rotary evaporator), the sample is left on the silica gel sorbent via adsorption. Then load the sample adsorbed silica gel into an empty Luknova solid load column, fill sand to the near-column top position (leave about 3/8 inch empty space from the top edge), add an inlet frit onto the sand top, and insert the sealing insert. Firmly push a perforated cap through the sealing insert onto the column body and tightly screw the cap on the column body. Finally, connect the assembled column loaded with solid sample to the purification system.
The column should be completely wetted with solvent prior to sample loading. The wetting time varies with solvent system, flow rate, sorbent inside the column, and the column length. An acceptablely wetted column should be uniform and without bubbles inside the column. For instance, a complete wetting takes about 4 minutes for a 40-gram flash column at a hexane flow rate of 40 ml/min.
For Biogate Horizon, SP1, and SP4 systems, less expensive line connection as shown below is recommended by using luer fittings and lines to employ Luknova Flash Columns and Solid Load Columns. The top connection line which comes from the pump outlet connects to the columne inlet via red-black adapter. The column outlet is connected to the UV detector inlet.
