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Calibration trouble Silybin
Posted: Thu Dec 30, 2004 11:20 am
by 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
Posted: Thu Dec 30, 2004 3:30 pm
by 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).
Posted: Thu Jan 06, 2005 8:31 am
by 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
Posted: Thu Jan 06, 2005 10:32 am
by 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
Posted: Sat Jan 08, 2005 11:48 am
by 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
Posted: Sat Jan 08, 2005 11:51 am
by 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