You can use preparative HPLC with prep columns available at the vendor and fraction collect them. On how much do you need to isolate depends on the scope of the work. You also need to know the stability of the collected fraction and what happens when you strip the solvents after the collection. Pay attention at the pH changes while concentrating and the effect of salts.

In some cases analytical HPLC could work. Depends on how complex is your sample. If it is a simple separation 2-3 compounds and you have good resolution you can overload the column (high concentration injection) and collect the fraction of interest after your detector. Secondly the term "analytical" relates to HPLC system or column? For most HPLC analytical instruments you can set a flowrate of 5mL/min or even 8-10 mL/min - thus adequate to use with preparative (or semi-preparative) HPLC column.

best regsards,

GB

Yes. I used HPLC in isolating and detecting my RS's. As per ICH Q3 you are required to have an LOQ of 0.05% wt/wt of the API's concentration. For a 10 mg tablet this is 0.005 mg or 5 ug, not including dilution and extraction volumes. Typically, my LOQ was 50 ng/mL when using a PDA/DAD as a detector. However, you will have to increase the injection volume to the maximum and also use the wavelength maximum.

@andrei blasko thank you so much sir for helpful answer . Stability after isolation is important part that I will keep in mind.

@grzegorz boczkaj thank you for answer. Here analytical means lab HPLC those have flow rate of typically 1 ml or 1.2 ml /min maximum.

@bruce neagle sir thank you for answer, as injection volume is fixed to 20 micro litre i have to work in that range and lLoQ will not be a problem.

Usually for a 5 cm column the flow rate is ~1 mL/min. and the flow rate for a 25 cm is ~0.5 mL/min. Otherwise the back pressure will be over 3000 psi and the column will die.

Pragati, the fixed 20uL is based 9n loop volume - change the loop in the injwction valve to bigger volume - simple :)

The pressure drop depends on particle size and solvent viscosity ( beside the flowrate). For preparative applications you don't need 1,7 or 3um particles. 5um particles will work ok.

Regards

GB

Thank you all for your time and valuable comments. some of the points are useful and will keep in mind.