I am intend to buy a SEC-HPLC column that is suitable to used with the peptide Glucagon(29 amino acids) at a pH=2.5. Can anyone recommend a column that might be suitable? The company, APEX, that I regularly order from recommended the column TSKgel SuperSW2000 that, as per the documentation, can be used with peptides at pH=2.5. Does anyone have any experience with this particular column?
Patrick: Your compound is small enough ( ~ 3,500 Mw) that you can even use a conventional RP C8 or C18 silica column (~100A to 300A) with acidified buffer and ACN to retain and resolve from a range of peptides from 500 to 10,000 Mw too. A strictly size exclusion column (SEC) like the TSKgel SuperSW2000 is better suited for molecules that start at ~ 5,000 Mw and go up to ~ 150K in size, so no, IF you are only going to look at (resolve) molecules that are less than 5,000 Mw in size, then that may not be a very good choice. If your goal is to retain and analyze a wider Mw range of compounds, say a 5K and 75K, then something like that might make more sense.
Tosoh's TSK column line is widely used and suitable for many applications, but wait, we really do not know what you intend to use it for... "pH" is not an application or goal. Suggesting a column type w/o knowing what question is being asked is not very helpful in guiding you to useful knowledge. The first question to ask is what do you wish to resolve your sample from? What is your goal? Do you hope to see some structure (peaks) of this ~ 3,500 Mw molecule? Retaining a sample on any HPLC column is a good start, but what do you wish to learn from this analysis? Do you need to separate it from one or more compounds or will it only be analyzed in a very pure form, alone, on the one column? Does the compound need to be intact or in a specific conformation in solution (pH, choice of buffers or salts and/or the use of any organic solvents will effect this. It "size" will change. We refer to this as it hydrodynamic volume and it is important to understand when choosing column types)? Besides selecting an appropriate column, be sure and also select a few other peptide and/or small proteins, above and below the target's Mw to calibrate the column against (another topic for another day). These are examples of extremely important questions which should be addressed early. Once the answers have been found, then you can review the HPLC product catalogs or literature to find a suitable column to start with. After that, run some experiments to see what works best for your sample and be sure and get lots of help from an experienced local chromatographer too.
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