Hi, Does anyone know of a good general HPLC column (reverse phase) for the separation of glucose penta-acetate and derivatives thereof, such as the methoxyphenol-, the bromosugar. I am a masters student and do not have much experience of HPLC method development. Thanks.
By Bruce Freeman on Monday, December 10, 2001 - 11:51 am:
A good general HPLC column is any C18 column. Choose a 4-micron spherical partical, 15 cm x 2 mm to 5 mm and you can't go too far wrong.
The problem is the mobile phase. Try something like 40% acetonitrile in water and change the acetonitrile level to get the retention times you need.
Then play with substituting methanol for part of the acetonitrile (at maybe 7 parts methanol replacing 4 or 5 parts acetonitrile, to retain your retention times generally) in an effort to change the elution profile of the various compounds.
If acidic compounds elute too early, add a little acetic acid to the mobile phase. (Not a lot, but the amount can matter.)
Temperature can also matter. Start with 40C or so, but try as much as 70C to 90C to see what effect tht has.
You can probably get away with 2 mL/min on a 4.6 mm column. Slower is better.
Remember that if you are working with a known mixture (no surprizes) then the lowest resolution >1.5 is the best chromatography. Anything more just wastes time and mobile phase.
If you approach method development systemmatically, it's not too difficult. A good reference is Snyder and Kirkland's "Introduction ot Modern Liquid Chromatography". (Avoid THF as a MP ingredient, however, unless you can't get acetonitrile and methanol to do the job. THF leads to too many complications.)
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