protein sample on AEX HPLC

[pg2000]

HI there,

this is the first time i run protein samples using anion exchange HPLC. the sample is a purified acidic protein (PI=3.8, so i use paperizine (PH5.6) as mobile phase, the gradient is 0.05-1M of NaCl within 40 min. the strange thing is that when the samples are injected in high quantity (200ug) and low quantity (20ug), the chromatogram differs from each other--- there are three split peaks in the chromatogram of 20ug injection, which is similar to blank profile, however there is only a single peak from 200 ug injection. the retention time of the product peaks are around 2.8 min. The column i use is DEAE 5pw (7.5 X75), UV monitor is set at 280 nm.

Could anybody have any thought on it? or you can give me any advice to improve the analytical protocol?

[tom jupille]

The most obvious explanation is overload.

More worrisome is the low retention time. It looks like your peak(s) may be almost unretained. For best results, your retention time should be at least twice your dead time.

What are the units on your column size? (I assume mm, but not sure) And what is your flow rate?

[pg2000]

Thanks Tom. I am fortunate to find this forum and could get unselfish help from the experts like you.

Your are right, the unit for the column size is 7.5 X 75 mm, and the flow rate i used is 0.9ml/min.

you point out the most posibility that may cause the problem of my IEX HPLC. I also doubted that the sample were not retained on the column at all, but if is not retained, why the 200ug and 20ug sampel injection give different peak profile?

i searched from this forum and find a topic (posted by SSGG in August 2005) similar to mine. in that topic, you advised that the weak base exchanger might give confusing effects on AEX, do you think that i should change to a strong column (such as Q-sepharose)? AS we just bought the column, that is no reason to replace it without further testing, i will try to decrease the ion strength and run at step salt gradient from zero, hope it will result in longer retention time.

[danko]

Hi pg2000,

From what I see, you can’t reduce the ion strength so much further, though you should try the zero chloride option. But there are 2 things you can do: First, check/evaluate the buffer (paperizine) concentration – it contributes to the elution strength, as you know. Second and maybe the best: Increase pH – it is almost certain that it will induce more negative sites which will lead to stronger binding.
So just change the buffer to phosphate at pH ca. 7 and see if it doesn’t help.

Good luck.

[tom jupille]

f is not retained, why the 200ug and 20ug sampel injection give different peak profile?

I can speculate on a few possibilities:

A baseline upset at or near t0 is not unusual; it's the result of the equilibrium disturbance caused by injecting a sample. At low concentration, you are seeing an unretained peak for your analyte superimposed on the baseline upset, which makes it look like multiple peaks. At high concentration, your sample peak is so much larger that it "buries" the t0 noise.

It is also possible that your sample is slightly retained, so that you are seeing a hint of separation at low concentration which is swamped by overload effects at higher concentration.

I would go ahead and try danko's suggestions before changing columns. Even at pH 7, you should be far enough below the pKa of DEAE groups that decreasing column capacity should not be an issue.

[pg2000]

Thanks again, Tom and Danko. You are really experts.

I am happy to say that we finally solved the problem, actually it is caused by a very silly mistake.

AS Tom indicated the possibility of overloading the sample, i tried a median loading (40 ug) and used a step gradient from zero salt. Starting with blank injection, unexpectly i saw a very sharp peak at 25% B (that is the correct retention time), this confirmed what Tom said about the overloading, in fact all the previous loading have occupied the binding sites of the new loadings, and before this step gradient trial, our focus are always in gradient between 2-15% buffer B (the salt concentration corresponding to the early eluting peaks from previous runs), we did not run the full gradient of B or sometimes cut the run just when we saw the early peaks. Not until this run, i know that my assisstant did not apply equilibration step between runs which i suppose he should know, so you can understand why my samples can not bind to the IEX column?

I posted this concluding masseage, First to appreciate the helps from warmhearted and knowledgeble persons in the forum, second I hope that usch silly mistakes will not happen again for any scientists doing HPLC in future!

[tom jupille]

Thanks for getting back to us with the solution. The only way we learn is from mistakes (either our own or someone else's)!