Chromatography Forum

Hi all,
i have a chromatographic problem. I would like some suggestions if it is possible.
Well, i have a pharmaceutical formulation and i have to separate two APIs (choline and succinylcholine). The first one has reduced retention using C8 columns. I tried ion pairing with the C8 column, using HFBA. I have managed to have efficient retention times for both the compounds, but the main problem is the separation of choline from NaCl (excipient). It co-elutes very early (even with 5% ACN) (I use a C8 250x4.6 mm, 5um) using 30% ACN.
Is there any way to separate choline from NaCl using the C8 column (using the ion-pair) or any other RP column even CN?( I don't have any HILIC or a more polar column available).
Is it possible to increase the retention of choline without increasing the elution time of NaCl simultaneously?

My suggestion is using an aqueous AQ column or HILIC. Send my a email, I can suggest you couple HPLC columns.
Charles

C18 columns that work in 100% water will work for this application. They are often called AQ columns. Atlantis T3 from Waters is an example of such a column.

SiliaChrom AQ C18 is an other example with a broad pH range (1.5-9.0).
Charles

there are many AQ to choose from
I would try as suggested AQ column first (Cyano also shows increased retention of polar compounds, so if you have then try it)

You can use Acclaim Trinity P1 column to overcome retention and selectivity challenges easily.

The Figure 1 in the file (http://www.dionex.com/en-us/webdocs/707 ... 239-01.pdf) shows a separation of 11 commonly cations and anions in pharmaceutical drug formulation, in which choline is well separated from Na+ and Cl- ions.

Acclaim Trinity P1 column is a reversed-phase, cation-exchange and anion-exchange trimodal silica column specially designed for pharmaceutical active ingredients (APIs) and respective counterion analysis, including:
1. simultaneous separation of API and its counterions
2. simultaneous separation of a mixture of APIs and respective counterions
3. simultaneous separation of anionic and cationic pharmaceutical counerions

One of the main benefits is the selectivity of this column can be adjusted by mobile phase ionic strength, pH and organic solvent content. As the result, the Trinity P1 column provides ideal selectivity and greater flexibility in method development.

This column will work for your application. Please let me know if you have any questions.

I use the Atlantis T3 quite often with highly aqueous mobile phases. Works like a charm.

I've used a Phenomenex 250x4.6 mm PFP-2 column for highly polar compounds, with decent retention using 100% water mobile phase (nitroguanidine was the analyte).

You could try switching from ACN to MeOH and varying the organic %.... If that doesn't work with the ion pairing, you might have to beg your boss to buy either a T3 or a HILIC (or both!!)

Unfortunately, MeOH is not an option. The detector wavelength is set at 205 nm, and is not possible to have a smooth baseline. I think that i will start begging for a HILIC!!

What about the sample preparation?
Did you try to get rid of the NaCl before you inject?

Maybe you could try to "preciptate" the NaCl by a high content of organic (e.g. ACN) and then dilute the supernatant or dry an aliquote and re-disolve in water?

Did you try solid phase extraction?

Beside this: if your analyte coelutes with NaCl than it's questionable that you really have enough retention at all. What is the capacity factor of your subtance?
So even if you can improve the sample preparation, it still would be better to switch to an AQ (e.g. Atlantis T3) or a HILIC column.