Chromatography Forum

I just recently started a new job in a QC lab for sanitizes and soaps. Currently we seem to be having issues with our HPLC's auto-integration of the C-16 peak of BAC/BKC in our soaps. Often times it doesn't even identify the C-16 peak and we have to manually integrate the peak. Due to the addition of an audit trail, we would like to have our program auto integrate the peak. One of our lab managers had the idea of increasing the concentration to extend away from the baseline to get more defined peaks. Usually we would create a 1/4 dilution. He tried using a 1/2 dilution and eventually decided on a straight injection of the soap. At a glance I would say this has been more successful than the 1/4 dilution; however, the auto-integration still has issues with the C-16 peak.

I am wondering if there are any tips to getting more defined peaks for our software to have an easier time handling the integration. Our specifications are as follows:

Column: MicroSorb-MV100-5 CN @ 5um partical size column,
250 x 4.6(LxID(mm))w/Guard
Eluent A: Acetonitrile
Eluent B: 0.075M Acetate Buffer pH 5.0 HOAC
Binary Isocratic Elution: 62% Eluent A/38% Eluent B
Flow: 1ml/min
Pump Pressure: 200 psi-8500psi
Detection: UV Flowcell @ 262nm
Software: Galaxie

Any help would be greatly appreciated!

Hi.
We have methods for BAK (you called BKC) in which we use Cyano column (same brand), but shorter column.
Please try:
1/ Use a lower wavelength, e.g. 210nm.
2/ Filter and discard the filtrate more. I have seen many kinds of filters that retained more of the BAK homologs.
Recommended: discard the first 2mL. Better: discard the first 3mL to saturate the syringe filter.
BTW, what is the intended concentration in the final product?

Good luck.
Alfred.

I'd say the first thing to do would be to investigate WHY the CDS has problems to auto-integrate the peak.
- Is the peak to small?
- Bad peak shape?
- Not enough resolution from neighbouring peaks?
- ...

"More defined peaks" can mean anything. The question is, is this a matter of improving the chromatography or do you "only" need to optimize the integration settings? Or both?

BTW, what is the intended concentration in the final product?
.

Final concentration is supposed to be at 0.12%

The head of R&D said that we should keep the wavelength at 262nm because we want to avoid coellution with other organic compounds in our product.


Our issue is that the C-16 peak is small and that is the reason for the issue with auto-integration.

Hello

Each software has feature (or at least it should have) that allows you to optimize integration parameters. You can play around with parameters as threshold and peak width to obtain correct auto integration even for small peaks and then (if you don't want to have these parameters for all peaks) save them for specific time range.
It should give you correct auto integration for small peaks.

Regards

Tomasz Kubowicz

Hi.
We have methods for BAK (you called BKC) in which we use Cyano column (same brand), but shorter column.
Please try:
1/ Use a lower wavelength, e.g. 210nm.
2/ Filter and discard the filtrate more. I have seen many kinds of filters that retained more of the BAK homologs.
Recommended: discard the first 2mL. Better: discard the first 3mL to saturate the syringe filter.
BTW, what is the intended concentration in the final product?

Good luck.
Alfred.

We also use cyano column here, gradient from buffer to ACN because the longer the alkyl the broader the peak is in isocratic mode. Gradient really helps a lot. Wavelength 265 nm is suitable for final concentrations as low as 10 mg/L.