mix consists of propylparaben as internal standard and glycyrrhizic acid, when i inject the mix only peak of internal std appear but when i inject each one alone each peak appear. using HPLC.
my column:4.6mm x 15cm packed with ODS particle size 5 µm
mobile phase: acetic acid(31%):acetonitrile 3:1
when inject each one alone:
RT of internal std = 12
RT of glycyrrhizic acid = 10
1- Glycyrrhizin contains an aglycone of five-fused rings attached with a disaccharide from one hand on the other hand propylparaben is a propyl ester of p-hydroxybenzoic acid.
2- Glycyrrhizin is used as emulsifier and in gel-formation of cosmotics.
3- there will be very strong intermolecular interaction (hydrogen bonding with sugar and van der waals with rings A and in addition to the hydrogen bond between carbonyl at C-11 in glycyrrhizin with the phenolic OH ) between them at least in mole : mole ratio.
4- You detect the paraben alone, I think because the molar ratio of paraben is higher than being consumed in complexation; thus could be detected, because it's found in excess relative to glycyrrhizin.
I change mobile phase ratio and nothing happen.then Chang column dimensions and also the same problem
Which kind of detector you used? UV detector? The way you can try to identify if those two compounds can combine as one is LC-MS. Check m/z value when you inject mixture.
Why is your mobile phase acetic acid concentration so high (31% in 3:1 with ACN)?
It might be the concentration problem reduce the both solution conc.if possible ,other wise if may be inter-convertible if spiking with each other.
Acid is added to the mobile phase to keep the acidic analyte in the protonated form. If you look at published methods, typical use level is ~0.1%. When the pH is too low, the bonds between the silica and the C18 of the stationary phase can be hydrolyzed, destroying your column and your peaks will broaden and tail very badly. It may not solve your problem with the missing peak, but you should change your mobile phase to more typical acid concentration.
I agree with Robert - reduce the acid concentration but do not exceed 0.5% (v/v). You may also try formic acid (0.1 - 0.2% (v/v)) which might give you better results.
I would suspect that your column is losing resolution, due to the amount of acetic acid in the mobile phase - pH below 2 is not usually recommended for analytical ODS columns - so your peaks are tending to co-elute. I would reduce the acetic acid concentration to a minimum and try using an ion-pair reagent to improve the separation instead. If you have access to a second HPLC pump, you could try running a gradient of increasing acetonitrile concentration, shallower in the zone where you expect the 2 peaks to elute without ion-pair reagent, unless they don't resolve well.
Hi Medhat Emad,
If you ask me to develop a method for such a high molecular weight drug, I will surely go on Gel permeation Chromatography.
I think such a high % of Acetic Acid is not needed as it will decrease the Ph of your mobile phase well below 2.0.
I will suggest you to go for pH 4.5. Your drug is having 3 pKa values. So it will be better to for the middle pKa so that you will at least get some good peak shape.
Secondly, what is the concentration of you internal standard? And what is the concentration of glycyrrhizic acid. If the response of your propyl paraben is less than that of your drug then it will definitely disappear when you combine both if them or vice versa.
I think with this method you might not be getting the good peak shape for glycyrrhizic acid.
I think you should change the column and go for a column which is having high % carbon loading and less silanol activity. Secondly Adjust the pH of Ammonium/Sodium acetate buffer to 4.5. instead of Acetonitrile use methanol as it will definitely improve your peak shape. Keep the proportion of mobile phase to 85:15. (buffer:methanol). Also have a look at the detection wavelength.
Please share your progress...
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