By shirish patel on Tuesday, September 16, 2003 - 08:15 am:
i am devloping method for a impurity analysis which is higly polar in nature whichi eluting in void in almost all rp mobile phase with higher number of carbon load so i decided to go my normal phase it does not having any acidic or amine group which can be retained by ion pair i am facing unexpected probleam in development by normal phse my conditions are as follows
mobile phase :
A :99% hexane B: 1%( 60IPA:40METHANOL)
FLOW RATE : 1.5ML/MIN
WAVEENGTH : 225
I AM DOING THIS DEVELOPMENT FOR THE formulation product so my sample preparation is as follows whichis sr matrix.
dose is 0.4mg avg wt is 330mg so
for sufficient impurity response i have to take minimum of 10mg equivalent
so i am taking 330 x 12.5 = 4.3gms in 50ml so it will be 5.2mg active in 50ml.
my diluent is : 500 IPA :10ML TRIETHYL AMINE TO ERODE THE HPMC AND EUDRAGIT MATRIX .
AFTER EXTRACTION I AM MAKING PH OF THE SAMPLE 4-5 WITH 2% H2SO4 IN IPA .
I AM INJECTING 25ul i am not getting proper peak shape its giving very much fronting , as on reducing injection volume i am getting little better peak still fronting is there .
so any body can throgh light what can be the cause and solution to this probleam actually i need to inject 100ul to have good impurity respose and better establishment of LOD AND LOQ .
you can talk to me also on
shi_ri@indiatimes.com
By A.Mouse on Tuesday, September 16, 2003 - 06:01 pm:
Your injection solvent is too strong. Try to dilute with hexane. If this does not work, evaporate to dryness and make up the sample in a solvent aht is more compatible with you mobile phase.
By Mokhtar on Thursday, September 18, 2003 - 05:46 am:
Your sample is diluted in a solvent which is more polar than your mobile phase and this may cause your peak to destort , try to make a dilution of the extract with the mobile phase.
By the way is the adjustment of pH nicessary? I think you may not need pH adjustment
By Anonymous on Thursday, September 18, 2003 - 07:54 am:
Actually i can not do evaporation to dryness as my mobile pahse almost 99% hexane and 1% mixture of ipa and methonol , .
and my sample solubility is not there in hexane its totally insoluble in hexane .
shirish patel
By Anonymous on Saturday, September 20, 2003 - 10:15 pm:
Primesep columns from Allsep Technologies (www.primesep.com) are designed to retain very polar compounds (amines, amidines, polyamines, strong organic acids etc.) by combination of reverse phase and ion-exchange mechanism. The ion pairing reagent is embedded on the column. Columns separate wide veriety of compounds (incluiding organic and inorganic compounds) in a single run with ACN-Water-TFA (or ammonium acetate or ammonium formate). You can use same column for normal. reverse-phase or ion exchange separation depending on the conditions. Check the website or call them, they will give you method development hints.
By A.Mouse on Monday, September 22, 2003 - 02:38 pm:
There is not need to change your method. Even if your sample is insoluble in hexane, if you try to make up the sample with something that is at least more compatible with your mobile phase, you should get a better peak shape. Think about it: If the sample would be completely insoluble in your mobile phase, it would not come out of your column at all.