Need help, splited peak and SDS

[zeq]

Hi everyone, i hope somebody can help me, let me introduce you the problem:
I am developing a method to quantify a very lipophilic drug, the samples are dissolved in 0.5 % SDS, and the concentration is so low that i have to inject it without any dilution.
The mobile phase is ACN and 10 mM ammonium acetate.
The column is a Xterra c18 150 mm.
The problem appears after a number of inyections, the peak starts to be distorted(shoulder, split peak, etc).
I tried saturating the column adding SDS to the mobile phase but it didn´t work.
If you have any suggestions please let me know, it is driving me crazy.

[Noser222]

Are you observing any pressure increase?

[rhaefe]

I have experienced similar behavior with Tween 20 in samples on a polymeric RP column. The Tween 20 was building up on the stationary phase over a period of 10 to 25 injections resulting in shoulders on peaks, peak splitting and decreasing retention times.
A thorough wash with 80% ACN/20%Water at 85°C restored column performance. Use of a guard column helped but eventually there was of course break through of the surfactant onto the analytical column.

[Uwe Neue]

Does your analyte have ionizable functional groups? Then you may need to control the pH (around 4.75 for an acetate buffer).

The equilibration of the column with SDS may take a long time and a large volume, if your method is isocratic, and may be impossible if you are running a gradient.

[Bryan Evans]

Are these dissolution samples?

You can also try a less retentive phase (such as C8 or maybe phenyl).
SDS might come off easier.

[zeq]

Thanks for the quick responses!:
I am not experiencieg pressure increase.
The molecule has two inonizable groups, but they are not inoized( pka 4.2-5.6 and ph is 6., The method is gradient so i think i will change to isocratic.
These are not dissolution samples, maybe a will try a phenyl.
If you have any comments, please let me know.!!Thanks.

[Bryan Evans]

I just looked at the mobile phase conditions. I agree - you
should add acid - it may also help to break up the micelle caused
by the SDS.

[Uwe Neue]

If you do not use a proper buffer (pH 4.75 for an acetate buffer), you can easily get weird peaks. I would use a proper buffer first before doing anything else.

[mohan_2008]

Accumulation of SDS onto the column is a very slow process and a pressure increase will not be seen right away.

The SDS in addition can act as an ion pair (just like hexane sulfonate) and can alter the RP character of your stationary phase. As a result, you have all kinds of mixed mode mechanisms and peak distortions.

I work with drug entities with such formulations on a daily basis. Based on my experience - Never try to shoot the SDS or Tween or CMC straight onto the column without diluting it with a suitable diluent.

If your working concentration is low, it still doesn't matter - make it even lower as much as possible as long as the peak is on scale.

Otherwise make a a very low concentration and inject twice the amount.
For eg: If your nominal concentration is 5 ug/ml with an inj volume of 10 ul - try to make 1 ug/ml and then inject 50 ul.

The point is trying to lower the amount of SDS in your injection solvent.
Do not forget to add a gradient clean up step with atleast 80%ACN in the mobile phase.

[chemgeek]

I've used a Javelin pre-column in the past to clean up/remove the SDS from a sample injection with moderate success. the nice thing is to clean the pre-column you just run a normal wash with high aqueous and a slightly elevated temp (I never went above 50C).

Unfortunately I left the columns and contact info for the providers at my prior job, so I can't be of much help in getting the aquisition info...

Try a goolge search on Javelin (and various other mispellings) and SDS/SLS and you should find the supplier.

[zeq]

If you do not use a proper buffer (pH 4.75 for an acetate buffer), you can easily get weird peaks. I would use a proper buffer first before doing anything else.

What do you mean with a proper buffer?, do you think a ph=6.8 buffer is no t adequate??why??please tell me your opinion, thanks.

[Kostas Petritis]

What he means is that your buffer does not have any buffer capacity at pH of 6.8...

[Uwe Neue]

I think that you need to have control over the pH of the mobile phase. pH control means that you need to work at a pH close to the pKa of your buffer ion. For acetate, this means pH 4.75. If you do not have control over your pH, it is easily possible to get strange peaks for ionizable analytes. If you have a proper buffer, such problems go away.

So let us do this first: make a proper buffer, either at pH 4.75 with acetate, or with a phosphate buffer at pH 7. Afterwards we worry about your SDS, which may also be a part of your problem.

[chemgeek]

So let us do this first: make a proper buffer, either at pH 4.75 with acetate, or with a phosphate buffer at pH 7. Afterwards we worry about your SDS, which may also be a part of your problem.

I agree with you're assesment of the buffer, but the high SDS/SLS content is also a long term issue that needs fixed.

I don't think he should be using Acetate, mostly because that is in the middle of his 2 PKA's, I think something either above or below that will serve him better, either protonate or de-protenate fully otherwise. at pH=4.75 he will likely stabilize things with a split peat. go 1-2 units above or below the pka's for best effect, and either reduce the SDS/SLS being injected on the column through sample dilution, or use a pre/gaurd-column.

[Kostas Petritis]

I read your problem more carefully and it looks to me that your problem should be the addition of SDS as part of the injection solvent. My question is, do you really need the 0.5% SDS to solubilize your compound? Can't you solubilize your compound by increasing the concentration of ACN (or solublize it in 100% ACN and then add water)?

In the current conditions (among other), you will have a pH mismatch between your mobile phase and injection solvent.

What is the % of ACN when in isocratic mode?