Chromatography Forum

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.

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

Hey there -

Chromatography of peptides, even fairly small ones, is different from other small molecules. Under reversed phase conditions, isocratic HPLC of peptides is frequently not possible. Below a certain organic concentration, you'll have essentially infinite retention and above it you'll have none. I must admit that I do not entirely understand your motivation for having ammonium acetate, formic acid, and potassium phosphate in you mobile phase, either.

I would suggest a gradient between an aqueous phase containing a perfluorinated carboxylic acid ion pairing agent at low pH and either acetonitrile and methanol.

I don't have much time now to explain further - meeting - but will get back later on.

Cheers!
CJ

Here I am..
Well, my peptide is highly recommended to be in phosphate buffer conditions (pH 7.4). I have already thought of moving to sodium phosphate instead (especially after reading quite a few tips - LCResources). I am using ammonium acetate and formic acid as mobile phase modifiers since I have observed that they improve peptide chromatography (in general) and help out sample ionisation when there is the potential of coupling UPLC/HPLC methods to MS (as in my case).

Regards
T

Looks like you have gone from a system where the buffer level remains the same regardless of mix ratio to one where the level of buffering varies with %B present.

the Acetonitrile may be having an appreciable effect on the pH of your system which may acount for the retention problem.

any thoughts?? shall i go back to MeOH?(I went to AcN in order to gain better peak widths) or shall I go for a %B of lets say (50%AcN, 50% phosphate)? would you also think that this is not due to column "age" ? my mind is in such a mess!