Of course, there are verity of material, but i need essential material and buffers for this technique. maybe there are some condition for reagent selection that i dont know them.
Thank you!
Hi, it depends on the kind of analysis you want to do and the type of analyte(s) you are looking at. These will inform the type of mobile phase solvent you will use and so on. So if you give an idea of the matrix/analyte you are interested in, you may get more specific answers
Hi dear Gbolahan Iyiola Asejeje
what i need to purify by HPLC is protein.
Hi Ibrahim Farhani I think these docs can give you some guidance on how to narrow down on specifics for your application:
https://www.hplc.eu/Downloads/ACE_Guide_Peptides.pdf
That is a question that can't be answered in a forum such as this. HPLC covers preparative HPLC to purify compounds, as well as analytical HPLC. Although there is a lot of overlap, there is a lot of differences between the two.
Start by learning the basics of chromatography- the types of stationary phases, the concept of strong and weak solvents, and how those affect elution.
I find that from thin layer chromatography through flash and HPLC, the concepts are all the same.
Hi, the question is very general. You can detect most of the materials with Hplc technique. But the hplc method, buffers, preparation of the solutions etc are all depend on the materials you search for. So you should ask the question for an exact material, component, technique or something else.
best wishes
Back at you, Dear Fatma Demircan Yildirim
I want to purify protein about 75 KDa.
Ibrahim Farhani How much, what is the source? Peptides can be more difficult than smaller molecules. Solubility in water, perseveration of any secondary or tertiary structure become important.
Please look at the following below links which may help you in your analysis:
https://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=8658572
https://www.thermofisher.com/sa/en/home/industrial/chromatography/liquid-chromatography-lc/hplc-standards-reagents-solvents.html
Thanks
The source is supernatant solution of cell. I want to purify secreted protein.
Thanks.
Ibrahim Farhani Such samples are often run by filtering the supernatant, then loading on the column. The peptide are focused at the head of the column, and then eluted. You should look at solid phase extraction (SPE) methods, or use flash chromatography (reverse phase) as an initial concentration and purification method. You should spend some time researching the effects of modifiers, and method development since peptides do elute differently from one another, otherwise we couldn't purify them. Understand how the isoelectric point of a peptide affects how it is purified..
Ibrahim... The question is too big to be answered in few words. The sample preparation reagents and buffers together with the choice of the solvent system are highly dependent on the analyte to be determined. More details may help us to advise you for the required treatment procedures. ... Regards
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