Chromatography Forum

anyone know is there a general cause for negative peaks in RP HPLC, ammonium acetate buffer pH 7 as MP, DAD detector 235 nm, for creatinine determination, flow 1ml/min .

The peak of creatinine is negative at 2,5 min (Retention time).

What are your column dimensions? Does the negative peak interfere with your analysis? Depending on what you're injecting and your column dimensions, this may be an injection artifact, and is either a disturbance in the flow rate (valve switching) or an unretained component of the injection mixture that has less absorbance than the mobile phase.

Dear sir,

THe column is C18 (15 cm × 10 mm ) , i injecte a solution of creatinine 100 mg/l ( fivefold dilution in mobile phase)

where the peak of creatinine has to appear, the peak is negative, the cutoff of the MP is 205 nm , and the wavelength on the measurement of creatinine is 235 nm. The flow rate 1 ml/min. Pression 980 PSI.

I can not understand where is the problem.

Negative peaks with a DAD are often the result of an inappropriate reference wavelength setting. Check to see if reference wavelength is enabled. If it is, turn it off.

Dear sir,

In detector parameters 'Totalchrom' , i fixe the reference wavelength at 235 nm (creatinine), so If I have welle understand , i have to turn it off. Or i have to fixe the reference wavelength at 205 nm (MP cutoff).

Thank you

You want your reference wavelength to be at a wavelength where your compound and mobile phase both do not absorb. Try something higher - I think the default is something like 360-380nm. Or, turn it off, if possible.

Thank you very much.