Protein separation by reverse phase HPLC

[cgodse@gmail.com]

I am trying develop method for protein separation by reverse phase HPLC by using reported method using C4 300oA x 5 um x 2mm x 250 mm. The gradient was used as per the reported method. We do not get any peak of protein. Initially blank was injected showed good baseline and later sample injected 5 ul containing 10 ug protein in guanidine HCl and DTT. On subsequent injections a small peak 0.1 height was seen at 55 min having UV spectra with max 220. Later on, this peak came consistently in blank with same RT, height and UV absorption. Is this a issue of dwell volume or the slope of gradient? The reported method is on Agilent HPLC 1100 and I am using Waters Corp. However, the column is same.

[HPLC chemist]

You do not use a gradient with a 250 mm column! It is far to sluggish to changes in the current mobile phase composition. Try a 50 mm (5 cm) column.

Are you sure there is protein in the sample? Did you run a spectrum on the sample from 200-400 nm on a spectrophotomer? Their are some proteins that are totally insoluble in both the extract and the mobile phases (A or B). Thus, they can precipitate and clog your column.

[cgodse@gmail.com]

Thanks for your suggestions. I selected the 250 mm column as given in the reported method for the same protein. Yes! I did check spectra of my sample on Spec giving 220 nm absorbance maxima. Initially I loaded 20 ug/ml sample on column (5ul) then it was increased to 2mg/ml i.e 10ug going on the column. with this modification the peak was seen at 56 min.

Thanks

[danko]

250 mm column is not the problem - gradient or not gradient.
The problem is most probably the mobile phase!
Keep in mind that orotein is not just a protein. There are many upon many proteins and their solubility etc. differes a lot. Fortunately..... Other wise it wouldn't be possible to separate proteins frtom one another.
You don't tell the other parametets apart from the column specs. So it's not possible for the pannel to identify potential culprits.
Best regards

[mattmullaney]

Hi cgodse@gmail.com,

Danko has good points--knowing the mobile phases, gradient elution scheme and flow rate would be helpful. Note also, this would not be the first time that a published method doesn't succeed in someone else's lab.

The problem seems to be on of elution from the column, and it is possible that the protein of interest elutes in subsequent injections of Diluent. Perhaps a chaotropic agent or a bit of MeOH would help?

[Tom Waeghe]

What are your analysis conditions for this method? Mobile phase A? Mobile phase B? Temperature?

Frequently with a brand new column that has never seen protein at all or the protein of interest, it is beneficial to make several bolus injections onto the column with a fast up and down gradient.

Set the column temperature at at least 40 C or even 50 or 60 C, if possible.

For a column of your size, I'd recommend something like 100 uL of 1-2 mg/mL protein. Ensure that the protein is soluble in your sample solvent by dissolving it in 25:75 or 50:50 ACN/water with 0.1% (v/v) TFA (if you're using TFA as additive). A gradient from 10:90 to 80:20 ACN/water (with 0.1% TFA in each, or 0.08% in B and 0.1% in A) in 10 minutes with a hold for 5 minutes at 80:20, should be fine. After 2-3 of those injections, carry out a blank gradient, injecting 10 uL of 25:75 ACN/water or the sample solvent used for the bolus injections.

Assuming you're not seeing too much carryover, try injecting 10 uL of 1 mg/mL of your protein in an appropriate sample diluent. Monitor at 214 nm and also 230 nm and 280 nm if you have a diode-array detector.

Hope this helps,

Tom

Thomas J. Waeghe, Ph.D.
Senior Scientist
MAC-MOD Analytical, Inc.
103 Commons Court
Chadds Ford, PA 19317
800-441-7508
610-358-9696
610-358-5993 (fax)
twaeghe@mac-mod.com
http://www.mac-mod.com

[mbreslav]

I agree with Tom. Temperature is the key. Try maximum temperature that column can handle. Also use 300A pores for the stationary phase. Also take into account that a) proteins are denatured during RP separation, and b) separation mechanism of proteins on RP is very different from small molecules. Basically, there is very little mass exchange between mobile and stationary phase. Imagine protein sticking to the column, and then, when %organic is high enough it will just "take off". In other words there is no linear adsorption isotherm.

[Multidimensional]

As is so often the case with these posts, not enough information is provided to render any intelligent responses. It sounds like the user is also just starting out with HPLC so they may not even know what questions to ask or what information should be shared. Weird comments such as, "You do not use a gradient with a 250 mm column! It is far to sluggish to changes in the current mobile phase composition. Try a 50 mm (5 cm) column." ?-? These just confuse the issue as such statements are wildly inaccurate and of no value in this case.

To the original poster: Get some help and training with the HPLC basics first. Is there someone at your place of work with experience ? Ask them for help. A colleague can help you with such basic issues better than a web group can. You need training to better understand what is happening and to learn yourself how to run and check a method. Honestly, it sounds like you are not cleaning your column properly after each run (or poor sample prep), but only someone physically in your lab would be able to check everything to know for sure.