Calibration trouble Silybin

[juaant]

I am Juan Manuel, at this moment I try to do a calibration line of silybin. My problem is: If I dissolve with methanol the silybin (20 mg/ 100 ml) I have four peaks, but If I dissolve with methanol/water (50%/50%) (20mg/100ml) I have two peaks. The sylibin have two diastereoisomers.

Other problem is when I dissolve with methanol (20mg/100ml- > 200 ppm) before I dilute with water 100 ppm (50ml of 200 ppm with 50 ml of water) and others dilute. I have not good calibration.

Why is it happening?

Thanks everybody

[MG]

As to your first problem, perhaps you are experiencing injection solvent mismatch with the methanol? I have seen injections in excessively strong solvent produce split peaks. It would help to know your conditions (column, mobile phase(s), flow rate, injection volume).

[Veronika R. Meyer]

Dear Juan Manuel, a similar problem has been published by Hoffman et al. in J. Chromatogr. 465 (1989) p. 189. They analyzed phenylalanine on a RP HPLC phase with buffer/acetonitrile 92:8. When the sample was dissolved in e.g. water/acetonitrile 1:1 the peak looked very crazy because the sample solvent was much too strong related to the eluent.

Veronika Meyer

[Veronika R. Meyer]

Heavy peak broadening, although no double peaks, were observed in vitamin analysis if the sample solvent was too strong, see Naish et al., Chromatographia 20 (1985) p. 335.

Veronika Meyer

[juaant]

As to your first problem, perhaps you are experiencing injection solvent mismatch with the methanol? I have seen injections in excessively strong solvent produce split peaks. It would help to know your conditions (column, mobile phase(s), flow rate, injection volume).

My colum is Kromasil C18 250*4,6, flow 1ml/min, injection volume 20 microlitres and mobile phase is 50% methanol 50% buffer to ph 2,8 phosphate

[juaant]

Dear Juan Manuel, a similar problem has been published by Hoffman et al. in J. Chromatogr. 465 (1989) p. 189. They analyzed phenylalanine on a RP HPLC phase with buffer/acetonitrile 92:8. When the sample was dissolved in e.g. water/acetonitrile 1:1 the peak looked very crazy because the sample solvent was much too strong related to the eluent.

Veronika Meyer

hello Veronika, thanks by your answer. At this moment I don´t acess to this articles if you can send me to "juaant@cartif.es" I was very wondeful