What is the best wave length for measuring the absorbance of peptides during HPLC run?
Dear Ebrahim Elkhtab,
214nm(nanometer) UV- detection is very best wavelength for measuring the absorbance of all peptides., As Am peptide researcher i have analyzed verity of peptides, 214nm is best, you just try this wavelength with gradient means you will get perfect result.
if you want more details download my research papers from my research gate ID, freely available it will gave up analytical information clearly
Best Luck & Regards
R.Selva......................
We predominantly use LC-PDA-ESIMS for our initial characterizations. In my experience, the peptides often do not have substantial absorbance outside from 215 nm. With that said, we have encountered a couple of compounds that appear invisible to the HPLC-PDA requiring the natural products chemist to perform the fraction collection almost blindly (he used peaks on either side of the peptide retention time as a hallmark). In this case fractions are collected in the time that corresponds to your peptide's retention time and then subsequently analyzed by direct inject or Laser-ablation ESIMS.
In the end, I prefer to collect all wavelengths and show three during the run 190nm (if possible), 210nm, and 280nm and follow up with MS.
Dear Ebrahim Elkhtab,
214nm(nanometer) UV- detection is very best wavelength for measuring the absorbance of all peptides., As Am peptide researcher i have analyzed verity of peptides, 214nm is best, you just try this wavelength with gradient means you will get perfect result.
if you want more details download my research papers from my research gate ID, freely available it will gave up analytical information clearly
Best Luck & Regards
R.Selva......................
i tried to figure out today the best wave length for measuring peptide response, i tried 220, 215 and 205. all of these wave lengths are supported by many reviews. but i found that 215 is giving the better response. i may try to measure at 214.
thank you all very much
Yah exactly 214nm UV will give best for almost all type of peptides
Hello Ebrahim,
I use 215 nm +/- 2 depending on the UVD and adjustment.
But if you have a conjugated double bond to one ore more of your peptide groups, e.g. a fatty acid or others, you will get a much higher UV-respons at that wawelenght.
If you are using a "pure" peptide back bone molecule as an Internal or external standard for assay analysis you will get problems wrt quantitation or % re.l chrom. purity of the particular specie. So - "what you see is NOT what you get". ( very bad yield if you in e.g. separation chemistry are isolating unknown peptides; the pool content is drastically decreased after vac-evaporation and freeze drying ).
WKR Torben Koch
Hello Torben Koch,
i tried 214 but i found that 215 is better for my peptides, in all cases i dont know yet what i have. thank you very much for your kind reply.
Oh Nice., We are regular peptide maker, Simple to Complicated peptide daily making and have verity of improvement in synthetic methods, but up to now i am using 214nm is best. also verity of scientists in this field suggested me best is 214. here i saw many of them said 215. if got good results good.
All the best for all Researchers., Have A Happy Day..,
Regards R.Selva.....
Dear R. Selvam,
I'am very interested in the peptide dosage, do you have a paper as a reference of the method?
Dear all
I am very interested in the peptides
please, could you help with few papers as reference of the methods ??
205-220 nm for neat peptides.
280 nm for proteins with potential peptide and amino acids interference.
Sir, R. Selvam, and other researchers
Could you please send the research papers as a reference to determine peptide concentration at 214nm.
kindly send the research paper title or link address or send to my mail id [email protected]
It will greatly help me.
Thank you
you are very welcome
https://www.journalofdairyscience.org/article/S0022-0302(17)30884-6/fulltext
This paper uses different substituted amides. I don't see why people go to 214 based on thee results from this paper.
Article The absorption spectra of simple amides and peptides
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