In the case of column chromatography of plant extract, which is the appropriate length of packed silica of a one-inch diameter and 18 inches long column, and what speed should be used for solvent flow?
Its a depends upon ur plant extract which secondary metabolite you are focussing, in which solvent that is soluble ....everything depends ..on ur target compound ...For example, one of the most widely used silica gel grades in the former technique is mesh 230 – 400 (40 – 63 µm), while the latter technique typically requires to mesh 70 – 230 (63 – 200 µm) silica gel ..
If you look at column dimensions and flow rates, you'll probably see a solvent velocity of ~7 cm/minute, this will be fairly close to the van Deempter flow rate since they want their users to have good resolution. This way of stating the flow rate allows one to more easily scale the column as the column width changes, and you'll note the reply above mine uses solvent velocity as well. For a 5 cm diameter column, that is around 137 mL/min assuming I did my algebra and math correctly. Make sure you run the column isocratically to determine the optimal flow rate.
If you can get a copy of How to Use Excel in Analytical Chemistry and General Scientific Data Analysis by Robert de Levie, Cambridge University Press, you'll find the calculations that you need (Chapter 6.9 in the 2001 edition).
SolventVelocity = (FlowRate * 4)/( PI * ColumnDiameter^2) ..you may use this link.. Roni Roy
https://www.sensorsone.com/volumetric-flow-rate-and-diameter-to-flow-velocity-calculator/
How much material are you purifying? How pure is it already? These will determine the size of your column. Do you know which compound you will be purifying in the extract, or is this a potentially new compound? These have a role in how you run your column.
I tend to consider initial chromatography of crude extracts to be a fast clean-up step because 99% of the material isn't active for a given biology, so I tend to use a very heavy loading. It is important to pack the column well, so the bands are horizontal; my experience is this is more important to resolution and peak width than hitting a particular flow rate.
Could you please mark the solvent front? Also, do you know which spot you want?
It appears a spot runs about 1/2 way up the plate. If this is true, the Rf is 0.5, and the compound will elute in about 2 columns volumes. Hexane-ethyl acetate 8:2 is a little bit strong solvent system (too much ethyl acetate) if that is the desired compound because the second compound will be eluting closely behind it. You will want you compound to elute about 0.1 to 0.3 Rf, causing elution in 3 to 10 column volumes. A column volume is roughly the void volume in HPLC. You can determine the elution in isocratic runs by CV = 1/Rf. The resolution otherwise looks good, so I would consider about 50 g silica (2% loading). I would dry-load the sample to maintain the resolution.
It's a TLC image of Solvent ratio Hexane: Ethyle acetate-8:2. Extract is about 300mg, Sir, will you please suggest me an appropriate length of packed silica for a one-inch diameter column, and flow rate in traditional column chromatography?
I'm targeting the first 2 spots. Jack Silver
The solvent is too strong, get a TLC such that the first spot has an Rf of 0.25, and that will give goos chromatography.more silica and solvent than such a purification should use.
The solvent is too strong, get a TLC such that the first spot has an Rf of 0.25, and that will give good chromatography.
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