Peak Splitting on UPLC

[mmehta]

Hello all.
I am working on developing a new method for Pse/Ibu/CPM Related Substances.This Method Runs ok for Couple of Runs and all of sudden all my peaks starts de-shaping/Splitting.
If you have any suggestions for this Method. Below are the details for this method.

MP-A : 1.15g Ammo. dihydrogen phosphate + 10 ml TEA in 2L Water. pH 6.0 with Phosphoric Acid.
MP-B : 25% MP-A 75% ACN
Column = Kinetex C18 1.7µm, 2.1 X 100 mm (part #00D-4475-AN)
Temp : 40 Degrees
Initial Gradient: A:B:: 99:1 Goes up to 40::60 for eluting 4IBA.
Any help will be appreciated.

Thanks in advance,

[DR]

Only 2 things come to mind immediately

1) is something slipping, creating a void somewhere between the injector and detector (I'd check both ends of the column first)?

2) Wow, that's a lot of TEA - which is known to alter column chemistry substantially. It may be that the TEA and column have not reached equilibrium until you're several injections into a sample set and that your degraded peak shape is what you should expect in the long run. I'd look for another mobile phase modifier.

[mmehta]

Only 2 things come to mind immediately

1) is something slipping, creating a void somewhere between the injector and detector (I'd check both ends of the column first)?

2) Wow, that's a lot of TEA - which is known to alter column chemistry substantially. It may be that the TEA and column have not reached equilibrium until you're several injections into a sample set and that your degraded peak shape is what you should expect in the long run. I'd look for another mobile phase modifier.

I tried Using HSS C18 column(same dimentions as Kinetex) and reducing TEA to 2 mL/2L. But still same situation.

[carls]

Are you using the original Acquity UPLC? If so, what is the % organic of the weak needle wash solvent and what volume is specified in the method? Also, which injection mode are you using and what is the loop volume?

If you stop the flow for 10 minutes, leaving the column in place, then restart the analysis does the peak shape return to normal for the first few injections?

What is the flowrate?

[mmehta]

Are you using the original Acquity UPLC? If so, what is the % organic of the weak needle wash solvent and what volume is specified in the method? Also, which injection mode are you using and what is the loop volume?

If you stop the flow for 10 minutes, leaving the column in place, then restart the analysis does the peak shape return to normal for the first few injections?

What is the flowrate?

Sorry about the insufficient information.
Week Wash = 10% ACN Volume in Method = 600 µL
Strong Wash = 75% ACN Volume in Method = 200 µL
Inj. Volume = 2µL (Full Loop)
Flow Rate = 0.45 mL/min

Yes and I'm Using Original Acquity UPLC with TUV Detector.

If I restart the System on next day It gives me split peak. But once you start the system and if you have good peak shape, Peaks are not spliting through out the analyses.It's Only after three or four Sample set runs it is giving me split peaks.

[carls]

If you replace the column with a new one does the splitting go away? If so, you are likely fouling the inlet frit. To resolve frit fouling you can either improve your sample clean-up or use a guard column. The guard column will likely foul at the same frequency so if up-time is your primary concern then you'll still need to improve your sample clean-up.

[mmehta]

If you replace the column with a new one does the splitting go away? If so, you are likely fouling the inlet frit. To resolve frit fouling you can either improve your sample clean-up or use a guard column. The guard column will likely foul at the same frequency so if up-time is your primary concern then you'll still need to improve your sample clean-up.

I am Filtering my samples using 0.22µ PVDF FIlter. All the MP and Wash Solvents are filtered through 0.2 µm GHP filter. Problem is same between the kinetex and brand new HSS c18 column.

[Mattias]

I had these kind of problems when I used the mobile phase preheater.

[carls]

What is your sample matrix? If it is a biological matrix (blood, plasma, urine, etc.) then peptides and proteins are likely what is fouling the frit.

Also, what is your sample solvent?

[mmehta]

What is your sample matrix? If it is a biological matrix (blood, plasma, urine, etc.) then peptides and proteins are likely what is fouling the frit.

Also, what is your sample solvent?

Samples are solid dosage forms.(Tablets). Diluting Solvents is 30% ACN in Water.

[pharmaguy]

Your starting mobile phase is highly aqueous, are you dewetting the C18 phase? if start with higher organic does your peak shape improve (yes they will come out earlier)?

[carls]

Perhaps your injector or pump are shedding material that is plugging the columns. I would service both components if you don't know when they were last serviced or if has been a while.

A simpler explanation would be bacterial growth in either your mobile phase A or weak wash solvent reservoir. Try rinsing both channels (including the tubing, degasser, etc) with IPA and replacing the filters.

[DR]

30% ACN seems pretty strong for your diluent. I realize that your inj. vol. is pretty low, but using a more aqueous solvent may help.

[Hollow]

with 60% of 75% ACN = maximum 45% ACN on your column

is this enough to flush any strongly retained compounds off the column?
Maybe something is accumulating on your column/frit.

Does it depend on number of samples injected or volume of mobile phases (no. of blank gradients)?

Maybe you could increase to 95%B (=71% ACN) with the same slope of the gradient or as a step after the last compound of interest has eluted.

What about the re-equilibration to initial after each gradient? Is it sufficient e.g. >5 column volumes; ca. 3 min?

[mmehta]

Your starting mobile phase is highly aqueous, are you dewetting the C18 phase? if start with higher organic does your peak shape improve (yes they will come out earlier)?

If I increase the organic I am not getting resolution between Ephedrine HCl and Pseudoephedrine HCl