I am trying to purify the bacterial protein (3kd size) by C18 column (NUCLEODUR® C18 ec, 250 mm, 4.6 mm, 5 µm, 110 A◦) on HPLC. Firstly, I am running semi preparative run and collect the fractions. The program is 20 to 80% Acetonitrile gradient for 30 min. But problem comes during repeat run of the collected fraction, sample does not bind with the same column, elute immediately after loading. I am loading the sample always in flow of water (1 ml/min). I tried it several times only single time it retained in column but most of the times sample eluted. Complete acetonitrile portion was removed from the collected fraction before loading. Now where is the problem, how to fix it, I need help.
Amended on 5 November 2017
Please, use our RP-HPLC method (please see file; Lysozyme by RP-HPLC). This general RP-HPLC method can elute peptides (Enkephalin; Mr 560, [Tyr1]-Somatostatin; Mr 1,730), and proteins (upto Serotransferrin; Mr. 90,000) reproducibly, since high-recovery analysis has been achieved. Lysozyme peak in this report has already indicated that the presence of glyco-lysozyme at leading portion. Usually pure protein shows symmetric peak-shape by this excellent method (our unpublished observation).
Other examples of proteins such as BSA (Mr 66,000) and Ovalbumin (Mr 45,000) has also reported in
"Hayakawa K, Okada E, Higashikuze H, Kawamoto T. Improved recovery of ovalbumin by reversed-phase high-performance liquid chromatography. J Chromatogr 256: 172-175, 1983".
I have preciously compared protein recovery between 300 A and 120 A silica gels, and the result has indicated that 120 A gel is superior than 300 A (my unpublished observation).
I wish you your success by using our method.
Hi dear
You must use amino column for more efficient separation of protein and amino acids
Are you re-equlibrating the column between runs? After the first run is finished, the column and system are filled with 80% acetonitrile, and this needs to be flushed out prior to the next injection.
I'm a little confused by what I read- I saw that the sample is injected into water at 1 mL/min, but the gradient is 20 to 80% acetonitrile. I read that the gradient really starts at 0% acetonitrile, in this case, because the sample is injected in water. Depending on how long the column sits in 0% acetonitrile, 100% water, you may be experiencing "phase collapse". Since HPLC systems usually run the column with the initial solvent system, the column could be in 100% water for a long time. Although this link refers to a flash column, phase collapse applies to any C18: http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/RediSep%20Rf%20Gold%20C18Aq%20for%20Highly%20Aqueous%20Mobile%20Phases%20App%20Note.pdf .
You should start the run with 20% acetonitrile, and inject into the column while it is running that solvent mixture.
At this point, the problem seems like improper equilibration- either too short, or in conditions that favor phase collapse.
One last question- which solvent are you using to dissolve the sample, and how much solution are you injecting?
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Thanks everyone for suggestions.
I just read about phase collapse. This may be the reason and I will try to overcome this by restoring the column at 50% or more acetonitrile at high pressure . Actually the reason behind the loading at 20% acetonitrile was immadiate elution after loading.
The sample was dissolved at water and the injection volume was 3 ml for 1st step and 500 ul of the fraction for 2nd step.
Now my another query is that possible that the target protein is not binding to the column due to its larger size in comparison to pore size of the column particles (110 angstrom)?
Try 5% acetonitrile instead of 0%. That should prevent phase collapse, and may help your retention compared to injecting at 20%.
My experience is that large peptides will bind to a column with 110 A pore size, but the loading is reduced. A simplified picture is that the peptides "bridge" the pores, so the stationary phase within those poses essentially doesn't exist. The peptide is adsorbed on the C18 that is accessible to it. The larger pore sizes allow entry, and create what appears to be larger surface area for molecules of that size.
80 Angstrom is recommended pore size for MW of less than 4000 so I think 110 A is big enough for a 3 kDa peptide.
I recommend you to go through this short guide on RP-HPLC for more facts and details.
https://www.google.co.in/url?sa=t&source=web&rct=j&url=https://www.agilent.com/cs/library/eseminars/Public/Method%2520Development%2520for%2520Peptides%2520and%2520Proteins.pdf&ved=0ahUKEwiIub2HoarXAhUCTI8KHX99DZ8QFggsMAA&usg=AOvVaw0JUOJ2SgN9uVXb2r0Dj0W2
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