Benzoic acid on Nova-Pak C18

[zabka]

We have troubles to analyze benzoate on a new Nova-Pak C18. Our mobile phase is: 10 mM KH2PO4, 1.5 mM TBAOH, pH adjusted with H3PO4 to 2.90. Linear gradient 10-78% of ACN in 17 minutes.
Benzoate peak tails extremely. Yesterday I called Waters' technician and he suggested to saturate the column with benzoate (10 injections of 500 ppm @ 100 ul). It really helped but in other gradient profile (same buffer) we had huge unexpected peaks at the same time where we have our target compound.
When we flushed the column with water and acetonitrile the extraneous peak disappeared. But today we started with benzoate and the performance is very low and with tailing again.
It is a kind of mystery. We have been using this type of buffer for more than 5 years, this is our fifth Nova-Pak C18. The old column has no problem with benzoate though it has less plates for nonpolar compounds. This is the first Nova-Pak with this problem.
Any suggestions how to solve it?

Thanks, Vojtech

[Uwe Neue]

Considering that you started with a brand-new column, have a low ion-pair concentration and run a gradient, my bet is that the brand-new column is not yet equilibrated with the ion-pair reagent. Therefore I recommend to run it for a while with the mobile phase A for at least half a liter, and then start running your gradient.

If I am right, you buy me a beer the next time I am in your neighborhood.

[Kostas Petritis]

In addition to Uwe comments, I would start with a new bottle of TBAOH, especially if you are using the same bottle for the last 5 years (your deal of unexpected peaks implies either somekind of degradation or your benzoate injections did not only contained benzoate...). Finally, it is a common practice for me to do a full regeneration of the column before starting using it. Normally I shouldn't have to do it but I was supriced with some colums to see what is coming out of them the first time of their use(using an ELSD detector).

I have one more question. Have you ever used in the past a brand new Nova-Pak C18 column for benzoate analysis?

[zabka]

The bottle of TBAOH was open in April 2004.
We run over 2 liters of buffer through the column.
This is fourth brand-new column we started with in past 6 years. Some of the columns have label "Division of Millipore".

Another of our analytes are cumol sulfonate and naphthalene sulfonate. In other gradient we analyze saccharin (pKa<2.90). All of these compouds have stable RTs, sharp peaks and high efficiency (on this column).

My guess is that RT of benzoate should not be influenced by ion-pair reagent since the pKa is 4.20 and pH of buffer is 2.90. I know that TBAOH stabilizes RT of saccharin. Without it the RT is in very broad window.

I tried to modify ion-pair reagent concentration. It shows linear plot (RT vs. conc.) from 0,75 to 3,00 mM. The column does not have to be saturated with the buffer. Couple of column volumes can do the job. I think 1,5 mM is huge concentration and all columns I used with it stabilized immediately.

Last month I tried Symmetry C18 with this buffer and benzoate had more tailing than "old" Nova-Pak C18. It made me to buy another one...

Sorry, no beers so far.
Thanks, Vojtech

[zabka]

I adjusted pH to 2.20 and nothing happened. I increased concentration of phosphate to 20 mM - no change. I prepared buffer without ion-pair reagent - the benzoate peak remained ugly. It reveals that TBAOH is not source of the problem.
Is there anything wrong with the column?

Thanks, Vojtech

[Uwe Neue]

Let me see if I understand you correctly!

You had run this assay before on 3 Nova-Pak columns without a problem, and this latest brandnew column is the first one that shows this problem.

You have also recently (a month ago) tried to run the same assay on a Symmetry column, but you had even more tailing than what you have now. Based on this, you bought this new Nova-Pak column that you are having a problem with right now.

Please confirm, if this is correct!

With respect to the rest of the information:

I would be careful to make a judgement, if ion-pairing is happening or not, based on the pKa and pH measured in water. You indicate that the retention increases with the TBA concentration. I would take this as evidence that ion-pairing is happening. A consequence of this is that the equilibration of the column with the ion-pair reagent is important. For one thing, 1.5 mM is a low concentration, and on top of this, you are running a gradient.

The fact that the peak remained ugly AFTER you removed the ion-pair reagent from the mobile phase indicates to me that the question of equlibration with the ion-pair reagent is on the right track. It is even more difficult to get an ion-pair reagent out of a column than to get it on the column.

I assume that you did not do the experiment with the increasing concentration of the ion-pair reagent with this column, but that this is an older experiment. Your lengthy equilibration (2 liters) with the mobile phase speaks against the suspicion that the column may not yet be equilibrated. On the other hand, if your ion-pair reagent contains junk (TBA usally does), an equilibration at a MUCH high concentration might help to get the column equilibrated with the junk. The thought is that once the column is full of the junk, the tailing will go away.

Keep me posted, how you are doing!

[zabka]

Yes, this is the fourth brand-new column we use with this buffer.
This is the first column with this problem.

Yes, I tried my sample on the Symmetry (a month ago) and I compared benzoate performance vs. Nova-Pak (column was in use for about 1.5 years).

The performance was:

Efficiency As. (based on 50% W)
Nova-Pak C18 (12/04) 40 000 1,50
Nova-Pak C18 (new - 4/2003) 45 500 1,50

Symmetry C18 (new) 39 300 3,10

The perfomance "NP -new" is taken from calibration file (i.e. one of first analysis on this column). The new columns were not equlibriated with buffer for more than a few col. volumes.


The format of the columns was not the same.
NP - cartidge 3.9x150mm with Sentry
Symmetry - 3.5 um, 4.6x75 mm, column


It is even more difficult to get an ion-pair reagent out of a column than to get it on the column.

It might be true, but in my experience when the column is flushed with water and acetonitrile then in the same gradient with water the retention of ionic molecules is very low. When low pH buffer without TBA is employed, the retention of benzoate is exactly the same.

I assume that you did not do the experiment with the increasing concentration of the ion-pair reagent with this column, but that this is an older experiment.
No, all experiments were performed on this particular column. TBA is not employed to help benzoate but to separate charged molecules (sulfonated aromates).

On the other hand, if your ion-pair reagent contains junk
We know it could contain "junk" and to prevent contamination of the buffer we "filter" TBAOH trough polymeric SPE sorbent. Some batches of TBAOH are significantly colored and without SPE cause baseline deterioration. We also observed that filtration of the reagent added life to guard columns.

We tested the same buffer on old column, new column, brand-new column which we keep as benchmark (i.e. it is not used for routine analyses but for problem solving). The NP column supplied recently was the only one with this problem.

I spent on this column four days and I cannot afford more. I called the manufacturer and they would supply another one.

Thanks, Vojtech

[Bill Tindall]

It is easy to get good retention and peak shape for benzoic acid. About any LC column will work, or at least it has done well on anyting I have tried. Use 0.3 % phosphoric acid and either methanol or acetonitrile. No ion pair reagent necessary. All that is important is to have the pH low.

[Uwe Neue]

Vojtech,

Well, I am glad that you will be getting a new column. Just in case that it does the same thong as the last one, keep in mind the things that were proposed here. You can also always contact me cia e-mail.

Good luck

Uwe

[SIELC_Tech]

Zabka,

We have a few methods and columns for benzoic acid. Mixed mode columns allow you to achieve various retentions for benzoic acid (or any other ionizable compound)by choosing appropriate mobile phase. In case of ACN/water/TFA (or other strong acid in the mobile phase) benzoic acid will retain on Primesep columns (with basic ion-pairing reagent embedded on the surface of silica) mainly by hydrophobic mechanism. If you use ACN/water/formic acid then benzoic acid will retain by ion-exchange and reverse phase mechanisms. By adjusting amount and type of the buffer in the mobile phase and amount of ACN you can obtain any retention time you want. Peak symmetry is close to 1.0.
Check the following link, as you can see on the bottom chromatogram you can even change the elution order of compounds.

http://allsep.com/makeCmp.php?cmp=Cmp_010