peak in void volume

[trilobite]

Dear all, I am eagerly seeking for advice..

Initially, I have set up an UPLC method for peptide analysis using an Acquity BEH C18 column.
FYI:
Mobile phase A: 90% 25mM KH2PO4 (pH 7.4), 10% MeOH, 2 mM ammonium acetate, 0.1% f.a
Mobile phase B: 10% 25mM KH2PO4 (pH 7.4), 90% MeOH, , 2 mM ammonium acetate, 0.1% f.a
gradient conditions: 90%A (0-6 min), 50%A (6-9min), 90%A (9-12 min)
flow rate:0.5 mL/min
Tcolumn:25C
Tsample:10±5C
Peptide eluted at 4.594 min

Then, I got split peaks. Hence, I performed a thorough cleaning of the UPLC system and column following Acquity restoration guidelines. Split peaks were removed.

With the aim of getting a shorter run time than the one of 12 min, I changed MeOH to AcN
To avoid buffer precipitation, mobile phases were changed to:
Mobile phase A: 90% 25mM KH2PO4 (pH 7.4), 10% AcN, 2 mM ammonium acetate, 0.1% f.a
Mobile phase B: 100% AcN, 2 mM ammonium acetate, 0.1% f.a
Tcolumn:40C
I tried a number of isocratic conditions (60%A, 50%A, 30%A) and flow rates from 0.2-0.5mL/min but peptide eluted very early (max retention time of 0.6min at 0.2mL/min)
I tried a number of gradient conditions and flow rates of 0.6mL/min (since increasing flow rate would decrease run time) but peptide eluted similarly very early. Even when I performed the "MeOH gradient" described above (note different mobile phase conditions,Tcolumn and flow rate) peptide eluted at 0.3 min.
In all cases, drifts lead to peak elution within the void volume

I am in a mess really.Any feedback would be appreciated.
Regards,
T

[ym3142]

Dear T

It is interesting. First of all,

1) why you need three additives in you mobile phase? After you have potassium phos pH7.2 why you need ammonium acetate? and again why F.a.? Please shed some light on us.
2) what is you column demension?
3) since you pep elutes at 4.6 min why you 90%A continues to 6 min? can you stop at 5 min?
4) why you set 50% A for 3 min? how about 1 min? how about 10% A for 1min?
5) can you get flow 1ml/min? or 2?
6) why you change temp from 25 to 40?
7) why your peak split in the original conditions? because of the column? the conditions? or samples? Could you find the solution after you figure these out?
whatever the results you got, were they reproducible? Are you sure the peak is for your peptide or only front/void peak?
9) you original conditions says 90% A (0-6min) and the Rt was 4.6min. Therefore you peptide was eluted under 90%A. If you set up strong mobile phase you will get shorter RT 1min at 80% A, 0.5min at 70%A, 0.1min at 60% A, 0.01min at 50% A. (the last several are just kiddings. )

Thanks for sharing

[trilobite]

Well,

1)I use phosphate instead of H2O to establish constant pH conditions. I use ammonium acetate and formic acid since I have seen that both help towards compound ionisation (the UPLC method I will set up - if I manage to - is going to be coupled to an ESI-MS/MS)
2) 2.1 x 50 mm
3) probably
4) if I am correct, would you suggest 90%A (0-5min), 50%A (1min) and 90%A(3min) or alternatively:90%A (0-5min), 10%A (1min) and back to 90%A (equilibration)?it is true that I hesitate to go for rather steep gradients.am I wrong?
5) Acquity UPLC max flow rate is at 2 mL/min
6) I changed temperature from 25 to 40C in an attempt to get sharper peak shapes
7) I am pretty sure it was because of the rather poor column condition.after washing etc, chromatography is nice
peaks are totally reproducible (intra- and inter-day runs)
9) you are right. the funny thing though is that no matter the AcN conditions, my peptide was eluted at 0.345 min (isocratic or gradient runs)

This is the first time I am encountering an issue like this. Any feedback would be highly appreciated.
T

[ym3142]

Dear T

Interesting.

Are you sure early Rt 4.6 was true and not fake?

Could you reproduce the Rt 4.6? If yes 1) do gradual modification make sure the peak move as you expect after every change; 2) you should see peak around 4.6 min under isocratic 90% A.

by the way, How large is you peptide?

If not you sample or sample prep has problem.

Good Luck.

[trilobite]

Indeed, rt 4.6min was true and highly reproducible when using the MeOH gradient.
my peptide is 15 aminoacids long
I will have a go with 90% A isocratic and let you know.
Sample preparation??(I used a freshly made stock and made working solutions in initial mobile phase conditions allowing sufficient column equilibration every time)

Fingers crossed
Thank u

[Mattias]

I am also quite confused by your method description. Your peptide elutes in the isocratic part, so why use a gradient? Are you looking for impurities also?

If you want to couple this method to MS, you must avoid phosphate buffer, especially at the high concentration you are running (25 mM).

I assume that you want to run the MS in negative mode, hence the relatively high pH of the mobile phase? If so, I would choose to run with unbuffered ammonium acetate (5-10 mM = pH 6.9) in the A channel and acetonitrile in the B channel. The lowest organic you have tried is 10%, which may be to high for this peptide. With the BEH C18 column, you can just as well start with 0% organic.

If you want to run in positive mode (the most common for peptides), a simple solution of 0.05% TFA in water usually works really well for peptides (for the A-channel).

[trilobite]

Dear Mattias,

thank you so much for your comments and feedback.

May I clarify that I firstly run the gradient I mentioned (in MeOH) and then tried a variety of isocratic conditions with no luck (in AcN) with the aim of shorter run times.
In terms of impurities: If you mean from the peptide, I used a freshly made stock. If you refer to the UPLC system, I suspect that all this washing I performed (to remove split peaks that came up from nowhere after 15-20 runs) should remove any contaminants.

You are right about phosphates and MS - this one is also what it troubles me. I have been told that this peptide of mine is only soluble in "phosphate buffer conditions pH 7.4". This is the reason why I ended up with such a high pH of the mobile phase.
Also I must say that in MS terms I prefer formic acid instead of TFA (ionisation).

Regards,
T

[Mattias]

I sounds strange that the peptide is only soluble in phosphate buffer pH 7.4. I think you need to investigate the solubility and stability of the peptide at different pH values. Anyhow, the mobile phase does not have to perfectly match the sample solvent.

You have no choice but to remove the phosphate buffer in you want to run MS. pH 7.4 is not a good pH range for MS-compatible buffers. I have had quite good luck with unbuffered AmAc, even though it is not a buffer.

Acetonitrile is a stronger solvent than MeOH, that could maybe explain why you don't get any retention using acetonitrile (at 10%). Peptides are usually very sensitive to "elution power" of the mobile phase.

I think you need more background data of the peptide, and to try lower amount of organic in the mobile phase.

Good luck!