Late eluting peaks Spiltting

[rc_12321]

Dear members i need your help here.


Mobile phase -A: 0.1% OPA; Mobile Phase-B: Acetonitrile (100%)

Flow: 0.25 ml/min; Temp=50 C Wavelength: 235 nm

Gradient: Time---------%A----------%B
0 ---------75 -----------25
2 ---------75-------------25
7 ----------40-------------60
12-----------20-------------80
12.1----------75-------------25
14------------75------------25

Sample Pka= 3.8, Diluent: M.P-A: M.P-B= 1:1

The problem is with late eluting peaks .We have four known impurities.The main elutes at about 2.8 min .The first imp elutes at 1.8 min , second at 7.1 min,third at 8.2 min and fourth at 9.1 min.During method development every thing is fine ,but problem starts when it was put into use for regular analysis.The imp at 1.8 min and Principal peak are still good.But the rest of three impurities are eluting with improper peak shapes .We increased buffer concentration ,but it was not worked out.Any suggestions from you?

[zokitano]

Did you use the same column/column type in regular analysis as during method development?
Did you try to run placebo solution or blank solution (only the solvent)?
Have you noticed any other peaks that can coelute with your peaks of interest?

Regards

[rc_12321]

The method is by UPLC.The column 50x2.1 mm-C18-1.8 u.

We are using same column as used in method development.We have found no interference of blank i.e diluent , solvent and placebo .We have not found any coluting peaks also.During development we performed degradation studies and that the method is stability indicating one.

[zokitano]

But the rest of three impurities are eluting with improper peak shapes

Explain, please what do you mean for improper peak shape. Does it mean that you observe peak tailing, fronting, splitting or something else?
If you observed peak splitting as you said in your topic name, than it could be due to: unfiltered (dirty) samples, blocked frit, coelution from the previous injections, contamination of the system...

Are you using that column on an UPLC system or on a regular HPLC? Extracolumn effects can cause distorting (broadening) of the peaks if you have transferred the method on regular HPLC with greater dwell volume.

If you can post a chromatogram that would say enough for a start.

[AdrianF]

What does OPA stand for? What is the buffer you refer to?

[rc_12321]

But the rest of three impurities are eluting with improper peak shapes

Explain, please what do you mean for improper peak shape. Does it mean that you observe peak tailing, fronting, splitting or something else?
If you observed peak splitting as you said in your topic name, than it could be due to: unfiltered (dirty) samples, blocked frit, coelution from the previous injections, contamination of the system...

Are you using that column on an UPLC system or on a regular HPLC? Extracolumn effects can cause distorting (broadening) of the peaks if you have transferred the method on regular HPLC with greater dwell volume.

If you can post a chromatogram that would say enough for a start.

Of the three improper peak shapes , the first peak has gone bad just before reaching apex, the second peak has small spilt at the apex and the third one has parabolic shape .

We are using same column.

[rc_12321]

What does OPA stand for? What is the buffer you refer to?

OPA is an abridged form of ORTHOPHOSPHORIC ACID.

[unmgvar]

have you checked the assymetry-tailing of the first 2 peaks as well?
check the width of those peaks as well.
are they the same as during method development or are they worst.
are you using the same instrument? or is it a second instrument?

[rc_12321]

have you checked the assymetry-tailing of the first 2 peaks as well?
check the width of those peaks as well.I verified the width of those two peaks as well are they the same as during method development or are they worst.There is no much difference .
are you using the same instrument? or is it a second instrument?we have only one UPLC right now.

[unmgvar]

still if there is difference and it is bigger, then i would go for those possibilities:
your column if you have not checked that yet. maybe it's time to change it
could be dead volume somewhere. especially the fittings of the column.
the temperature difference since you are working at 50 degrees. have you shorten the tubing in there?
maybe the oven is not heating homegenous enough anymore, that is an important issue with small column IDs.

[jthornburg]

The easiest and most-likely solution is probably to change to a new column (after checking for dead volume).
Look back at some earlier runs with the column to look for a trend in worsening asymmetry and dropping plate counts (N). My experience has been that columns usually go bad gradually, with shoulders developing and then peaks starting to split. Mobile phase adjustments won't help much at this point. Doing a thorough cleaning could buy you some time if you don't have a replacement column on hand. Good luck!