I need to purify the peptides from protein hydrolysates. We have RP-HPLC from water. C18 column is used. How to optimize the separation/purification?
A good starting point would be to use 0.1% TFA in water with an MeCN (with 0.1%TFA) linear gradient from 0% to 60% over 40 mins, with a 5 min re-equilibration at the end. Monitor by UV at 214nm and at 40DegC. Depending on what you get, you can adjust the gradient parameters accordingly.
A good starting point would be to use 0.1% TFA in water with an MeCN (with 0.1%TFA) linear gradient from 0% to 60% over 40 mins, with a 5 min re-equilibration at the end. Monitor by UV at 214nm and at 40DegC. Depending on what you get, you can adjust the gradient parameters accordingly.
you can purify peptides from protein hydrolysates with a mixed system:
A: 0.1% TFA/water
B: 0.1% TFA/ACN or 0.1% TFA/Me-OH
Good luck.
please take a look to these 2 article. It will be helpful.
1) Purification and Characterization of Antioxidative
Peptides from Protein Hydrolysate of Lecithin-Free Egg Yolk
2) Purification and Characterization of Antioxidant Peptide
from Sunflower Protein Hydrolysate
Hi! You may try a bit modified method which basically has the same approach as George suggested.
1. 0.1% Formic Acid in Water: Methanol
2. Gradient:: 2% MeOH to 60% MeOH in 60 min and a hold up of 10 min (as you are developing the protocol for the first time)
3. Flow rate: 1.5ml/min (Assumung that you have analytical column)
4. Detection system: UV/Vis detector set at 280 nm.
Hope that works..Best of Luck!!
In continuation with my earlier post, if your machine happens to be equipped with a diode array detector(DAD), then you may consider monitoring the end products in multiple wavelengths. I would consider 220 nm (Abs maxima for peptide bonds and most of the amino acids) as well s 280 nm (Abs maxima for tyrosine and tryptophan).
I also appreciate the links of Shulamit Levin, they may prove quite helpful.
Cheers!
Reverse phase chromatography utilizing C-18 or C-8 columns can be one of the ways to separate and purify peptides that result from hydrolysis of proteins. Reverse phase using C-18 or C-8 means solvent system must be combination of organic and aqueous phase, i.e., methanol or acetonitrile in combination with water. Each of these solvents may have 0.1% acetic acid or formic acid or trifluoro acetic acid. Regarding detecting the separated/eluted compounds, UV detection can be used. Wavelength of 214 nm or 220 nm or 226 nm corresponds to detection of peptides. 254 nm, 275 nm and 280 nm may also be used, which would help in determining the presence of phenylalanine (254 nm), tyrosine (275 nm) and tryptophan (280 nm). Furthermore, reverse phase chromatography can be coupled to electrospray ionization mass spectrometry. Therefore, by combining the UV data along with that of mass spectrometry, it is possible to get some details on the identity of the peptides.
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