Hello,
Thank you for response.
tom jupille wrote:
1. Do you have access to an advisor / mentor / prof with HPLC experience who can be your resource when problems arise (and they will)?
Yes, of course.
My situation:
The department where i will do my thesis is specialized in antimicrobial peptide synthesis. Due to the fact that i have been visiting this department for about 3 years as student's scientific circle member, i have become little familiar with synthesis methods, peptide analysis (mainly in terms of purity) and prep-hplc purification. Normally thesis subjects are presented by Professor, but because of the fact that i was a member of this student's circle i had an opportunity to choose my own topic. I'm quite interested in HPLC applications, so i chose topic which is connected both with peptides and HPLC. Professor is a peptide synthesis specialist, but he also is a owner of HPLC shop/service company so in case of any problem he can help me (i wanted to plan everything first and than show it to him)
tom jupille wrote:
2. You shoild definitely log the plate counts, but plates have a relatively small impact on resolution (square root).The greatest impact comes from retention [k'/(1+k')]; if k'is low, you will never get good resolution. Finally, the most useful parameter is selectivity [(alpha-1)/alpha]; that's where the solvent and column chemistry come in.
I figured that since resolution equation consists of both selectivity and retention it would be suitable for description of peak 'surroundings', and N would rather focus on peak. (Of Course N is also part of Rs equation but alone it also describes peak shape)
tom jupille wrote:
3.Flow rate does not have much impact for small molecules on today's columns.I generally recommed running at the highest flow rate consistent with your baseline noise and your paranoia about back pressure.
Sorry, my mistake i should have written that melittin, apamin and MCD are peptides, phospholipase A2 is protein. Ok, so check what flow will be most suitable for my hplc back pressure - and then i will do all analysis on that flow?
tom jupille wrote:
4. You can sidestep the issue of adjusting the solvent strength to get a reasonable k' by screening your columns with a gradient.Bear in mind that selectivity depends not only on the column, but also on the organic solvent, the buffer, the pH, and the temperature.
Yes, yes of course i know that organic solvent and buffer pH are also important, but as the analysed substances are peptides i assumed that I will use 'golden standards' of peptide analysis -> (A)H2O + 0,1% TFA (B) ACN + 0,1% TFA (or (A)H2O + 0,1% FA (B) ACN + 0,1% FA if i decide for MS detector) in high gradient (10-90% to make sure that every peptide is eluted). I completely forgot about temperature, so probably i will try to do analyses for every column in two options 25 st. C and 40 st. C
Regards,
Dorian