REPETIBILITY PROBLEMS WITH NORMAL PHASE

[nellyzugo]

I use the USP method to analize Isotretinoin capsules by normal phase, the selectivity was perfect buy I got problems with reproducibility of samples, however I take special care with the preparation ( Samples are in hexanes). What can I do to obtaine a good RSD?

Thanks,

Nelly Gómez

[Mark Tracy]

Is the problem quantification or retention time?

Retinoids are sensitive to light and oxygen. Keep the lights turned down low, use amber glassware, refrigerate the samples, degas the solvents, prepare only a few samples at a time, work quickly.

Normal phase is touchy about residual water in the mobile phase, and the equilibration times are longer than for reversed-phase. Watch out for stabilizers in the solvents. If your pump has piston seal wash, change it to something compatible. If your pump is a gradient pump, make sure the proportioning valve does not leak from an unused channel.

If the pump vendor offers special seals for normal-phase work, get some. Seals for reversed-phase don't last long in normal-phase solvents.

[nickster]

We had problems with reproducability but found that slowing the injector down when drawing up sample to eliminate the possibility of air being drawn up cured our problem .
Most of the time por reproducability is caused by a bad injector system unless your chromatography is poor.