Chromatography Forum

There is a split peak observed by Quality and I am helping with their investigation. It was believed the split peak is a new impurity and that a new column can separate the compounds. However when I tried using my system and a new column, I was unable to observe the split peak. In addition, I did another trial using the questionable column and the mobile phases (which produce the split peak in Quality's system) in my system, it also revealed no split peak. No one else seems to be able to reproduce the split peak except for Quality.

I did realize that there is more than 1 split peak, around 2 to 3 other peaks have split too.

It is a gradient program - Mobile phase A: 10mM phosphate buffer: ACN (95:5); Mobile phase B: ACN:H2O (80:20). Diluent is methanol.

What could cause this split? Is it really a new impurity? What else can I do to eliminate other causes?

Appreciate any help given.

Hi sfu, a few of questions:
Where are your split peaks eluting in the chromatogram, are they all eluting early?
What is your injection volume?
Is everyone using the same make & model of HPLC?
Is it only showing up on one of the HPLC systems in the Quality department?

Looking at what you have posted so far my first thought is that the splitting is due to having a strong sample solvent.

Mike

Hi Mike, the split peaks are spread throughout the run. The peak of interest elutes early and there are another 2 peaks that elute out in the middle of the run and nearer to the end of the run.
Inj volume is 10 ul.
Quality and I are using the same model and make of HPLC.
Quality has tried on two other systems and they are able to reproduce the split peaks.

Initially I had the same thought on strong sample solvent. But this method has been validated and used for a few years without any issues. If I want to verify this is a cause, what can I do to prove it?

Since this split peak split from an important impurity (say Impurity X), Quality had ran this Impurity X standard at concentration similar to the sample. It was obvious that the split peak lies out of Impurity X peak. I wonder if this would confirm that it is a different impurity.

Sarah

Hi Sarah, if you have markers of the impurities and can run them at the concentration seen in the samples, this should show if the peaks are splitting or if you have poorly resolved peaks, from two or more related substances. I would run the markers on the quality instrument and your own.

Based on what you said split peaks should go lower down the list of possibilities, and sounds more like a very small change in a condition (mobile phase prep, system set-up etc.) is causing this effect.

Mike

as others, my first thought would be too strong on sample diluent.

Will the peak split gets better if the injection volume is reduced?
->try a injection linearity e.g. 5, 3, 2, 1 µL and see if the response (Area/Vinj) is stais the same or if there is a deviation from linearity.
If you have a PDA detector than you probably compare the spectra of the two peaks (hopefully the impurity has some different sepctra)

If you're using the same model of HPLC in the two labs, then are the diameter of the capillary (especiall inj - column) also the same or is QC using smaller id capillaries (on all of their systems). Where they changed since validation?
-> smaller capillary would make the system more sensitive to strong samples solvents effect.

Another possibility would be void volumes somewhere in the systems of QC.
Where they recently maintained? ->check, re-fit every connection.

Are they using adjustable fittings and do the installation of the column properly?
Or do they have fixed fittings that were set for some other column type (eg. Waters vs. Agilent columns have different depths of the connection)?

are you using a high temp in the oven
something like 40 or above?

Yes. The column T is 40°C.

Hollow, Quality is using metal capillary while I'm usign PEEK capillary. According to my vendor, metal capillary is slightly smaller than PEEK. Quality is using adjustable fittings.

Mike, I ran markers of the impurity at concentration seen in the sample. However in both Quality and my system, only 1 peak was observed for this impurity marker. This seems to suggest that I have poorly resolved peaks.

when the temp is very high
then you need to make sure that you have enough tubing present in the oven that is heating the mobile phase to the correct temp prior to being in the column
otherwise you get a decrease in efficiency that can go as bad as a wide system peaks splitting all over.