Column Details
particle size :3um, pore size: 120 A, Dim. : 4.6 mmx100 mm
Dear Maghesree,,
Although it's possible doing a thing like that, all depends on how much peptides you want to purify in a run as in an analytical column you can load only so much and if you overload you'll end up with a bad separation, fronting and masked peaks.
Hi,
it all depends on your need. If in case you need to have a few mg or even less than that of pure peptide then you can definitely use analytical columns. The only major problem is the time you will need to purify a sample. This is because there will be some upper limit of the injectable volume of your analyte (100ul for the machine that I use). Hence, from a practical point of view, semiprep purification is always the preferred one. I personally had to use analytical column to purify a sample (tedious it is !) because there was a peak merging problem while using a semi-prep column. The protocol doesn't differ much as such as the difference in two modes is the amount of the analyte yo can handle.
Hope iy helps !
Ok. Thanks ! From what you say it looks like I just have to use a higher injection volume. I am also not sure how to determine time window do collect the desired fraction. I plan to conduct an analytic run with small injection volume (5 ul) and determine the fraction collection time window from the analysis result. Will that work ?
this again depends on your analyte volume. e.g. if you have say 200 microlitre of sample then to determine the time window (i guess you mean to optimize the gradient for the best separation and isolation of your desired peptide) you can inject 15-20 microlitre each time. If the crude profile is not too messy, then 2-3 test injection for this purpose is sufficient. Don't discard any collected fraction as finally after mass spec confirmation you can simply pool them and concentrate. On a general note, since you are handling only a few micrograms of peptide, it is not at all advisable to use any semi-prep column at all.
I am not sure. May try out Preparative Thin Layer Chromatography first. Micrograms of peptide may get lost in the column. Chances of sample loss in Prep TLC presumably is quite less.
not the best of ideas. There are reasons why people use expensive RP-HPLC instruments.
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