Chromatography Forum

i am currently working with sample matrix which contains plasidone s-630 which is polymer and its water soluble .

i need to remove this polymer from the sample matrix so as to get a clear chromatogram.

This elutes with a broad peaks right from 2 minuts to 30 minutes .

Here are some suggestions:

sample preparation, for example with a specific SPE method for your analyte

change of wavelength or detection scheme

Wavelength of analysis is 200 nm , my all impurities having a spectra of 210-195 nm . doseof the drug is about 0.25 mg so in formulation drug product while analysing the sample for related substances complete chromatograph is beling coverded by the plasidone-s630 . so can you please guide how to remove plasidone-s630 moleculer weight is about 10000-45000.

If your analytes are small molecules you may use an ultrafilter or SEC to get rid of the polymer.

I think this problem is related to my problem in http://www.sepsci.com/chromforum/viewto ... sc&start=0

To solve the problem, you may use:
- Acetonitrile instead of methanol as organic solvent in mobile phase. The disturber will be eluted completely near the front.
- Higher wavelength where the disturber cannot affect the baseline.
- Lower injected amount
- Additional hint: change the formula! (Formulator may be disagree with this suggestion though it is simplest method for analysts)

Whats this Ultrafilter can you suggest vendor and how it works. SEC i thought of doing it what range cart i should use and who is the vendor who provides cart for this kind of applications.

An ultrafilter has pores small enough to filter based on molecular size. The molecular weight cut-off (MWCO) starts around 3000 and goes up from there. Typically they are disposable devices that go into a high-speed centrifuge (10000 rpm), and process a few hunded microliters of sample. Millipore (www.millipore.com) is one popular brand.

One feature of this process is that the filtrate is (very nearly) at equilibrium with the polymer in the retentate. In other words, if your small molecule binds to the polymer, you will only recover the free molecule in solution. Whether this is good or bad depends on what you are trying to do.

Pall Filtron (or is it Pall Gelman?) has, among others, also 1000 mw cutoff filters. Sartorius and Whatman (also something else now?) have ultrafilters. Some are very convenient for using in centrifuges.
It is usually quite easy to shift equilibria away from proteins by adding MeOH, etc.

Thanks for your brillient suggestion.

I tried with the 5 KD and 10 kd Filters from various vendors , Due to presense of acetonitrile in a sample matrix pasidone also elutes from the filters and filterate found to be same as without ultrafilteration.

So i changed the sample preparation matrix to water and finds a wonder almost 80% of the inteferance due to this polymer is being removed by both the filters .

i am still worried about 20% to make a chromatogram looks normal as its a related substances chromatograph.

I had question that if i do not remove those 20% inteferance there can be unknown below it who know, even authorities may ask like this.

Do you have suggestion to improve filteration efficiency or to do something to remove complete inteferance.

Filter cutoffs are based on globular molecules, so if part of your polymer is nearly linear it could slip through. Another possibility is that there are lower weight fractions present. In either case a 1000 cutoff filter might be advantageous (or maybe even dialysis?). Or, for the case that the polymer is uniform (homogenous) at the given weight, maybe you can influence its shape with some addidtives?

Another option if you can't remove your polymer is simply to change your chromatographic technique. We have analyzed Tween in the presence of small molecules and protein by using a HILIC technique on a 25cm x 4.6mm ID TSK Gel Amide 80 column with a linear gradient from about 90-10% acetonitrile in 0.1%trifluoroacetic acid solution ( reverse gradient causes the small molecules and protein to retain at high ACN while tween elutes around 3-5 minutes, then other compounds elute as gradient moves into a more reverse phase type eluent mix). For more info on HILIC techniques, search on author Risley as one resource.