Basic compunds (nortriptyline) at intermidiate pH

[Koen Hollebekkers]

Much is already written about the separation of basis compounds with reversed phase chromatography. Older types of columns use mobile phases with pH (with addition of TEA). The latter type of reversed phase columns can be use at low pH, but a drawback is the low retention.
There are nowadays also commercial columns available which show good at intermediate pH. At these conditions these columns give raise to retention due multiple retention mechanisms (hydrophobic/silanolic).
My question is the next.
This good peak shape at intermediate pH, is it due to the use of high purity Silica?
What is the best buffer at this pH? Which buffer will give the best peak shape?

[Uwe Neue]

I assume that you are talking about pH 7. AT pH 7, you do not have a lot of options with respect to buffers. Phosphate is the buffer commonly used.
The improvement in peak shape for basic compounds at pH 7 is for the most part due to the use of high-purity silicas, but this is not the only factor. Other improvements play a role as well, such as coating level, endcapping techniques etc.
The ultimately best results are obtained from hybrid materials. This may be due to additional factors not available with silica, such as a shift of the pK of the packing into the alkaline pH range. In addition, with a hybrid packing, you can also go to high pH, where basic compounds are not or only weakly ionized.

[SIELC_Tech]

One of the alternatives to consider, if you would like to analyze basic compounds at moderate pH (2-7), s mixed mode chromatography. With Primesep columns you don't need to go to very high or very low pH. You will find a lot of buffers which you can use in this range (ammonium acetate, ammonium formate, formic acid, TFA, etc.). You can choose your buffer based on the detection technique or the objective of your experiments. Mobile phases with pH=2-7 are not going to do any damage to the pump, detectors, valves, etc.). You can retain basic analytes with dual mechanism-reverse phase and ion-exchange and obtain retention from void to indefinite retention by playing with nature if the buffer and pH of the mobile phase (within the range of 2-7)
In addition to ultra pure silica you have a "masking" effect of the stationary phase which is usually have lower pKa value then silica and is more controllable due to the universal nature of the ligand.
Here is the link for "Improving of Peak Shape of Charged Analytes"

http://www.sielc.com/pdf/SIELC_ImprovingPeakShape.pdf

and a link with a few reason why you don't need to go above pH-7 in order to retain basic analytes in controllable fashion.

http://www.sielc.com/pdf/SIELC_June_2005.pdf

Leader column manufacturers are making columns with very pure silica and doing end capping in order to improve peak shape (Zorbax and Luna in my experience showed very good peak shape).
IN a lot of cases regular LC column with ion-pairing reagent will give you good peak shape (tailing factor of 1.5 or less).

Regards,

Vlad

[Victor]

Mr Neue gives a good answer. However, I might add that if you are specifically interested in analysing tricyclic antidepressants, you will have no problem analysing these at low pH since they are sufficiently hydrophobic to give k> 5 using perhaps 20% acetonitrile. Then you will have less problem finding a column that will work. It's not necessary to use intermediate pH. However, if you want to analyse more hydrophilic bases then you might well have a retention problem at low pH. Sometimes you can get round this by using very low modifier concentrations at low pH in conjunction with an embedded polar group column, which will not de-wet (no phase collapse) at such concentrations. These are just alternatives to the intermediate pH strategy.

[Koen Hollebekkers]

First of all, thanks for all the replies.
Concerning the topic. My question was not which pH is the most suited for the separation of basic compounds, but what it solution for the separation for basic compounds at pH 7.
I have read the articles of SCIELC. In these cases the have made the mobile phase acid with addition of TFA what will protonate the basic groups on the surface and therefore will give good peak shapes at low pH. The same principle like polar embedded columns.
The answer of Dr Neue is satisfying and, I know that this is the approach of Waters. But what is interesting in my opinion is the shift in pK for hybrid particle stationary phases. Are there papers published about this topic? And where can I find detailed literature about multiple reaction mechanisms in reversed phase LC?
Concerning buffers, if I understand it right, a phosphate buffer is well suitable for pH 7.
I hope to get some

[Uwe Neue]

Here are three publications that cover the subject of the shift in pK of the silanols:
1) U. D. Neue, C. H. Phoebe, K. Tran, Y.-F. Cheng, Z. Lu, “Dependence of Reversed-Phase Retention of Ionizable Analytes on pH, Concentration of Organic Solvent and Silanol Activityâ€