Chromatography Forum

I have inherited HPLC performance qualification duties from a coworker who recently left the company. I got very brief training before he left. I should note here that I am a biologist by education and training, not an analytical chemist. The procedure I was shown calls for 5 standards of butyl benzoate, ranging from 1.0 mg/L to 10.0 mg/L, prepared in HPLC-grade water, from an acetonitrile (ACN) stock. We also include a butyl benzoate QC sample at 5.0 mg/L, prepared the same way. I was having trouble getting consistent results until I changed from preparing the samples in water to preparing in 20:80 ACN:water. Still, I seem to be getting some unexpected variability, particularly at the higher concentrations (7.5 mg/L and 10.0 mg/L). My questions are: Are there stability/solubility issues with butyl benzoate that I should be aware of? Should I be preparing these samples immediately before analysis? Should I use a higher solvent concentration? Should I be using a different concentration range? Any general hints on this procedure?

Compare peak shapes and retention times - are the more concentrated standards eluting earlier and asymmetrically compared to the lower concentration injections?
If so, you may be overloading the column or detector.

DR - Thanks for the reply. Retention times are in excellent agreement, as are peak widths (two criteria for passing qualification). Peak shapes are good also. I should have explained better in my original post, but when I said I get unexpected variability, I mean that linearity falls apart at the higher concentrations. However, if I make them fresh, or get new samples from refrigerated stocks, it all seems to come into line. Hence my question about stability or solubility. But if butyl benzoate is so sensitive to stability or solubility issues, why use it for a standard? It seems to be referenced in many discussions of performance or operational qualification testing.

DR - Thanks for the reply. Retention times are in excellent agreement, as are peak widths (two criteria for passing qualification). Peak shapes are good also. I should have explained better in my original post, but when I said I get unexpected variability, I mean that linearity falls apart at the higher concentrations. However, if I make them fresh, or get new samples from refrigerated stocks, it all seems to come into line. Hence my question about stability or solubility. But if butyl benzoate is so sensitive to stability or solubility issues, why use it for a standard? It seems to be referenced in many discussions of performance or operational qualification testing.fnaf games

This is useful for me.

I have inherited HPLC performance qualification duties

Exactly what is the purpose of such performance qualification? Internal, or to serve as "proof" in event of an FDA audit????

After we had validated a test procedure as per cGMP, our management required a yearly Service/Performance Verification by an outside vendor (guy with a tie) and proper paperwork; we never did any in-between performance verifications unless after a repair (which increasingly had to be done by a guy with a tie, no matter how simple.

The procedure I was shown calls for 5 standards of butyl benzoate, ranging from 1.0 mg/L to 10.0 mg/L, prepared in HPLC-grade water, from an acetonitrile (ACN) stock.

I cannot fathom why a butyl benzoate standard would be prepared using water instead of an organic solvent. That's certainly not what I would've considered...

I wonder what is the better standard to do the HPLC validation testing ? and what kind of validation tests are needed ?

I wonder what is the better standard to do the HPLC validation testing ? and what kind of validation tests are needed ?

When I was working, I seem to remember Agilent using caffeine standards for HPLC qualifications.

PA/PH/OMCL (11) 04 R6
QUALIFICATION OF EQUIPMENT
ANNEX 1: QUALIFICATION OF LIQUID CHROMATOGRAPHY EQUIPMENT
https://www.edqm.eu/documents/52006/128 ... 8491788491