isoaltion of low level impurities

[aniket]

helow, i am currently working on a project of isolation of low level impurities from an API, i am very confused as what method i should use for the isoaltion of those impurites? i represnt an university and we dont have Prep HPLC (we can not afford it) then how do i isoalte those impurties, i need to do structural elucidaton of those impurities so how much i need to isoalte? please help me on this?

thank you
aniket

[Srinivas SK]

Dear Aniket:

A prep HPLC is not the only option for purification/isolation of API impurities, so don't worry if you don't have one. But one will need to know what elucidation studies you plan. NMR? FTIR? MS? Then one can decide how much of the test compound you will need to isolate and by what methodology.

Assuming you will do the usual elucidation studies, I would say, off-hand, that you will need at least 500 mg of each impurity to get started with elucidation.

For that, one will need to know how much of these "low-level impurities" exist in your API and what you think their functional groups could be - if you are in a position to share this information.

Also, if you could let me know what separation instruments are there in your lab, I'd be able to give you some pointers on how to go about this problem.

With warm rgds,

S.K. Srinivas
Bangalore

[DR]

Ask the person with the NMR. If it has a very small cell, a few mg may suffice. Without a semi-prep LC, you will need to figure out how much API you can load before compromising your resolution,
inject, collect fractions,
inject, collect fractions,
inject, collect fractions,
inject, collect fractions,
inject, collect fractions,
...
deliver fractions to NMR, MS etc.

[james little]

what about using a mixture of TLC to work out a method and doing flash chromatography, not too high tech, but cheap.

Often can find enriched impurities in filtrate from recrystallization

[aniket]

thank you srinivas for replying, i am planning to elucidate by NMR, and MS, in our lab we have a analytical HPLC with a normal column of 250 x 2.5 mm , also we ahve GC and HPTLC, i jsut wanted to know how do i know how much impurities are there in my sample using an HPLC method, also in what way i can use TLC for isoaltion experiments?
thank you
aniket

[JM]

To isolate impurity from API, First you have to see out of all these impurities , how many are degradation products ( stress testing will tell you this ) and than increase the % of these impurities by controlled degradation of API.
Identify whether other impurities are process related ( intermediate etc)LC-MS can give you direct information about these degradation products and other process impurities.

For NMR you atleast need 5 mg impurity , which should be isolated from enriched degraded samples. for process related impurities talk to your synthetic guy if he can spare some junk ML from API final ppt step. or els as said by DR inject and collect.

Good luck
JM

[Srinivas SK]

Hello Aniket:

I'll need some info on the nature of the sample before I could tell you how to use HPLC to do impurity profiling.

But, generally speaking, this is what I would do:

Assuming you're using a C18 column and assuming your API is a non-ionic species, I would run a gradient using ACN:Water, starting from about 20% ACN, going upto 100% ACN in about 20 mins. If you have a Waters system, you can try using one of the pre-programmed gradient profiles in the software - a concave gradient is preferable.

If you have any other system, say a Shimadzu LC 10ATVP, you can program the gradient yourself, but don't make the gradient too steep.

Run the system for an hour at least.

This will give you a decent idea of the impurities you may have in your sample. Of course, the synthesis protocol used for your API will give you an idea of what impurities you can expect.

The HPLC method has to be fine-tuned and optimised, especially if there are ionisable species present in your sample. Can't offer much details at this point, since it would take up too much space here, but I'd be glad to help out personally, if you feel the need.

W.r.t your isolation problem :

You can use your Camag system for isolation work. You can use the Linomat system to apply the sample as a streak on a prep tlc plate and develop it. I've provided a useful clickable link for you below on this technique.

In fact, you can make prep tlc plates by hand and isolate your compounds of interest.

We did a lot of hand-made prep tlc in my old days at UDCT and we got real good results. We couldn't afford a Camag, so we managed on our own - made our own plates and believe it or not, we even made our own silica gel for TLC !

I'd be happy to give you some pointers on old-fashioned tlc plate making and prep tlc, if you wish. Pls feel free to ask, if so.

Here's the useful link I mentioned:

http://www.chromatography.co.uk/TECHNIQS/TLC/cnote1.htm

Try it out and let me know.

Warm rgds,

S.K. Srinivas

sksrinivas.kprime@gmail.com
09845089865