Chromatography Forum

Hello,

my name is Dorian and I am V year Pharmacy student. Although the thesis is meant to be conducted at next semester, i need to submit my subject's title till the end of October.

I'm quite interested in HPLC analysis and because of that i was thinking about comparing various hplc columns (regular c18,c8, embedded c18, core shell, wide pore c18, pfp, monolithic etc.) in their bee venom analysis efficacy.

The efficacy would be expressed as a theoretical plate number and resolution of main component peaks (probably 4 - melittin, apamin, MCD peptide and phospholipase A2) and time of whole analysis.

And here come the questions:

1) How to determine what time (and flow) would be most suitable for my analysis? (just conduct analysis for every column at 3 different lengths [ for example 20, 40, 60 minutes] and 3 different flows [for example 0,5, 1 and 2 ml/min] and see for which options parameters are the best?)

2) In case i use single Quadrupole detector (in place of regular UV) - would mass chromatogram smoothing be a controversial interference in resolution parameter?

If there are some terrible mistakes in my reasoning, or you got some advice (or better idea for masters thesis ) - please help me.

Regards

Dorian

Several comments here:
1. Do you have access to an advisor / mentor / prof with HPLC experience who can be your resource when problems arise (and they will)?

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.

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.

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.

Hello,

Thank you for response.

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)

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)

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?

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