Primary peak too close to Solvent Front

[olaniyi]

Hi all,

We are using a highly concentrated Sodium Dodecyl sulphate solution ( ion pair reagent of 0.04M ) as our Mobile Phase A and 100% Acetonitrile as Mobile Phase B to analyse 3 compounds. One is a neutral sugar, the second is a steroid and third is polar compound. We obtain good chromatography for the three compounds, good resolution between the 3 compounds. The only problem is that the neutral sugar which is the first compound to elute is too close to the solvent front.

What are the various ways one can try to move a primary peak away from the solvent front bearing in mind we are at the limit of organic content in the mobile Phase which can't be changed further.

[Steve]

Do you have the ability to change the temperature of the column (lower)?

[Mark Tracy]

Sugars are inherently hydrophilic, and will always elute near the void in RP-HPLC. One option would be to use a column that tolerates 100% aqueous mobile phase. Actually, SDS is a powerful wetting agent, and dewetting of the C18 phase does not occur even at 0% organic.

You could derivatize the sugar, but that is a real nuisance.

[Cliff Mitchell]

What is your ratio of A to B? If the % organic is low enough, SDS will form micelles in your mobile phase. If this is the case, you could decrease the SDS concentration and neutrals will retain longer. If if if ...

[SIELC_Tech]

Is your compound just underivatized sugar or it is something like glucose pentaacetate?
Considering the fact that you have steroid and sugar (underivatized) it is hard to develop isocratic method. You need amino column to retain sugars but your polar compound will come in a void on amino column.
Do you need to quantitate all three; are you planning to do LC/MS or preparative work?
If you provide me with your email we will develop method fro you (free of charge)

Regards,

Vlad

[JA]

I don't understand why the retention of neutral compounds is decreased if [SDS] > cmc.

If [SDS] < cmc, and the "free" surfactant aids solvation of the sugar wouldn't it have less retention?
If [SDS] < cmc, and a monomeric coating is formed on the stationary phase, wouldn't this effectively yield and ionic surface layer which prevents retention of any hydrophobic function on the sugar.

Would somebody kindly explain?

[Uwe Neue]

Interesting problem...
Do you need the SDS to get retention for your polar compound, or is it only used to make the stationary phase wettable for retention of the sugar? If the latter is the case, there are some other potential solutions available.

If this is the case, you need a water wettable reversed-phase column that does not encounter dewetting in 100% water. There are two solutions available that I can think of. One is the use of the (modern) Atlantis dC18 column, which has been designed to not suffer from dewetting, while providing optimal retention in 100% water. Another solution is to use a non-endcapped C18 column. I used to run myself the good old Resolve C18 column for carbohydrates in 100% water. So this column might to the job for you.

[Rob Burgess]

Just to enlighten you all, Ola is a colleague of mine.

What he meant to add was it is not just a pure sugar, but rather a sugar ester (acetate in fact).... if this adds anything to the posting answers we'd both be very welcome to any revised responses.

PS. We are using a 40-50 % AcN mobile phase to elute our compounds from a short 50mm Zorbax C18 column. Currently we're aiming for a fast run time (below 3 minutes).

Please also note that we are strictly limited to a 40-50% dissolving solvent for our samples also as we need to recover the drugs from cascade impaction plates. Dervitisation and other fancy techniques are probably out as this would reduce sample throughput too much!

[Cliff Mitchell]

I don't understand why the retention of neutral compounds is decreased if [SDS] > cmc.

If [SDS] < cmc, and the "free" surfactant aids solvation of the sugar wouldn't it have less retention?
If [SDS] < cmc, and a monomeric coating is formed on the stationary phase, wouldn't this effectively yield and ionic surface layer which prevents retention of any hydrophobic function on the sugar.

Would somebody kindly explain?

My thoughts were that, if they were above the CMC of SDS, and reduced the concentration of SDS while still staying above the CMC, the concentration of micelles in the mobile phase will decrease and neutrals/nonpolars will spend more time in the stationary phase.

In this case, above 40% organic, I don't think SDS will form micelles regardless of SDS concentration. I believe that as you add organic to the solution, the CMC shifts to higher concentration and eventually becomes unreachable. Furthermore, even if above the CMC, the two conditions you described, monolayer on stationary phase and solvation enhancement in mobile phase, both by surfactant molecules, will still occur. When above the CMC, not all surfactant molecules are in micelles. In fact, I believe the same amount of surfactant molecules are "free" at surfactant concentrations from just below the CMC to many many times that concentration.

Even if those two situations do occur, when there are micelles in the mobile phase, neutrals and nonpolars bind to the micelle, spend less time in the stationary phase (and more in the mobile phase) and are retained less.

As to explaining whether or not those two situations do occur...

[Uwe Neue]

Rob and Ola,

You have a sugar acetate, a steroid and a polar compound. If the polar compound is non-ionic, why do you need the SDS in the mobile phase?

If the polar compound is a base, do you need the SDS to bring it into the correct retention window?

I am also still not sure now if you are running a gradient or not. Are you running from 40 to 50% MeCN? Or is this isocratic at a composition around 40-50% MeCN.

If the problem is that you need 40% organic to dissolve your sample (the steroid?), there are some tricks that you can play with the injection to avoid peak distortion.

Please give more information so that we understand this better!

[Rob Burgess]

Uwe,

The polar compound is a 2° amine, and has virtually no retention when used in the conventional reversed phase mode.

We have intended to keep the method isocratic and have varied the % B composistion between 40 and 50% AcN to 1) obtain adequate retention and 2) good peak shapes for the amine.

I've also looked at fast gradients (no SDS, just acid modified MP) but the peak shape is still terrible for the amine.

[Uwe Neue]

You need the SDS to get the polar base retained. You need the 40% acetonitrile to match the sample composition. You need more retention for your sugar ester.

Here is another suggestion: you are concerned that the steroid will not dissolve any more if you make up the sample in less than 40% acetonitrile. How about dissolving the sample in 40% acetonitrile, but diluting it with an SDS solution 1:1 (may less might be enough). The idea is that the SDS will keep the hydrophobic analyte dissolved, or at least suspended, but now you could reduce the amount of organic at the beginning of the assay down much further to get the sugar ester to the retention that you want.

[SIELC_Tech]

I am just reviving the topic to update interested parties on approaches to separate mixture of compounds by mixed mode chromatography.
The mixture contains amine, sugar ester and steroid. The claim on the thread (which is locked) was that neutral sugar (glucose pentaacetate) and steroid co-elute on a Primesep column. The modeling separation was performed on a common steroid (prednisolone) and glucose pentaacetate.
Amines are retained well on all cation exchange mixed mode columns.

(http://allsep.com/Applications_By_Publish_Date.php)

With the actual structure of the steroid and amine unknown, we used common compounds to show the applicability of Primesep mixed mode approach to the retention control of neutral compounds in the mixture. The goal was to develop an isocratic method, which will provide adequate retention and separation for all three compounds.
Another objective was to develop an analytical method without an ion-pairing reagent. This approach will allow using a different detection technique (ELSD, LC/MS and UV) and potentially have conditions scaleable to the preparative chromatography.

The desired separation was achieved with different columns and mobile phases:

http://allsep.com/makeChr.php?chr=Chr_089

http://allsep.com/makeChr.php?chr=Chr_090