Chromatography Forum

Hello,
I would like to ask one question. I made a reproducibility test on my new column. I use ion-pairing agent in mobile phase to achieve retention. It is dibutylammonium acetate. There was slight retention decrease observed from analysis to analysis. It is just because the equilibration of column is not long enough or is the column being contamined with IP agent? Will retention stabilize after the column is fully saturated with IP ? Thanks. PHOBIUS

Phobius,

working with ion-pair reagents and running ion-pair chromatography method requires consideration of some factors that can affect chromatography (band spacing, retention time reproducibility, selectivity).

First of all, let the column to be equilibrated long enough to ensure better reproducibility between runs. Observe the baseline, especially its drift. When you're sure that the column is equilibrated (stable baseline, low drift) you can start your analyses. There is time required to saturate the column stationary phase with ion-pair reagent. Stationary phase uptake for different ion-pair reagents can vary, so it is required longer time for equilibration when using some of them or shorter when using other reagents. The time required for equilibration depends on the stationary phase also (different for RP and NP chromatography).

Because adsorption processes on the stationary phase are usually temperature dependent, I will suggest you to thermostat your column to ensure thermally stable environment for the equilibrium processes on the stationary phase. Slight change of the temperature (when the column is not thermostated) can cause retention time shift of the analytes that are ionized and are involved in to the ion-exchange process on the stationary phase.

Also, you should consider about proper column regeneration after running on it ion-pair separations. Because it is very important for the column life and also (for which you're also interested) for obtaining the same retention of your analytes when running analyses in different days.

Hope this helps,
Regards

thanks for your advices, I will try equilibrate the column longer between analyses. One more thing: after 10 runs, the peaks of two analytes co-eluate, but in the first ten analyses, they were separated nicely. What could be the problem? The mobile phase is 10 mM dibutylammonium acetate pH 7. Could it be, that the DBAA is evaporating from the mobile phase? btw I am allready using a column heater to keep the temperature constant.

Do you use premixed mobile phase? And do you sparge your mobile phase with helium when degassing it throughout all analyses?

I have seen such a situation, it is not surprising that you loose resolution during subsequent runs in ion-pair chromatography. Especially when there are close eluting peaks in the chromatogram. At the moment when you'll observe lost of resolution between critical peaks (bands), try to equilibrate the column longer before you proceed with your further analyses.

Best regards

I do not use premixed mobile phases, I mix it myself. I do not sparge it with helium, the HPLC machine I am using has its own degaser. I use helium only to make my voice funnier Thank you for advices again.

As mentioned above, it may take a long time until the column is equilibrated with the ion-pair reagent. It could easily be the 10 runs that result in a coelution of your peaks. With other words, the completely functional ion-pair method could result in a coelution of these two peaks. You may need to tweak the method.

I was afraid, that somebody writes something like you just have written.

I was wondering, if it would be possible to avoid the coelution by running a blank sample after every 10 runs or change the used buffer to a fresh one after 10 runs but if you are right and the equilibration takes so long (10 runs) the whole method needs to be improved.

The problem is, when I slow down the gradient or even make it an isocratic elution, the analytes will separate (I hope) but I will lost my beautiful peak shapes. They will be broad and not nice at all

Phobius,

could you post the mobile phase, column, instrument, gradient, column equilibration time between runs (and other conditions) that you're using in your ion-pair chromatography method? Maybe we'd know how to help you.

I had a similar problem when I was trying to validate an ion-pair chromatography method for determination of phosphates and phosphites in an active pharmaceutical substance. During subsequent runs I observed shifting of the RT of the both anions and decrease of the resolution between their peaks in the chromatogram. I tried to equilibrate the column longer, but in the first place that procedure gave a little improvement to the resolution, but later (after several days) I couldn't achieve that. Then I tried to regenerate the column when lost of resolution was firstly observed, then equilibrate it again with the mobile phase (with ion-pair reagent) and after that I observed the initial resolution between the phosphates and phosphites that I needed.

I was using tetrabutilammonium hydroxide as pairing reagent and inverse UV detection (I used potassium phtalate in my mobile phase). I regenerated my column (Purospher STAR RP18 endcapped 150x4.6, 5um) with a mixture of methanol:0.02M KH2PO4 = 50:50 for an hour (at 1mL/min). After that I reequilibtrated the column again with my initial mobile phase for 2 hours (until the baseline became less noisy).

Regards

OK, so here are my chromatographic conditions:

Mobile phase A: 10 mM DBAA pH=7,00
B: MeOH

Sationary phase: Synergi Hydro C18 250 x 2,0 mm, 4um particle size (Phenomenex)

Flow rate: 0,2 ml/min

Gradient: 0´-5´ 60% A : 40% B
45´ 70% B
46´ 60% A : 40% B
46´- 60´ 60%A : 40% B

The first peak appears at 17,25 last one at 23,93. They are quite narrow. (at the base they are cca 0,81 min wide)
In first 10 runs they are baseline-separated, but after 10. analysis the 4. and 5. coelute.

Column heating: 30°C,

Equipment: Alliance 2690 Separations Module linked simultaneously to a PDA 996 (Waters) and a ZMD 2000 single quadrupole mass
spectrometer equipped with an electrospray interface (Micromass).
Analytes: N6-substituted adenine nucleotides (mono- di- and triphospates)

Is it necessary to prolong your run up to 45th minute (with 70% B), when the last peak elutes at 24th minute? If there are no other peaks that come out after 24th minute you could shorten your method, keep the steepness of the gradient = 0.75 %B/min but shorten the range of the gradient (to keep the Rt of your analytes as in your previous runs) and give more time for column reequilibration with the initial mobile phase composition. Your last peak come out with a composition of methanol in your mobile phase of 58%.

Regards

You are right, it is not necessary to go up to 70% of MeOH, however, I´ll need to get rid of less polar compounds in real world samples.

By using a gradient with ionpairing you have an inherently unstable situation. When the methanol is increased the amount of DBAA bound to the column will change. Perhaps it would help to have DBAA in the methanol.

However I doubt you can get a totally satisfactory solution while using a gradient. Ion paired eluent take hours to equilibrate and as soon as you change the methanol conc you have disturbed the equilibrium

Do you find any solution for your problem?
Do those two peaks still co-elute?

well, sometimes they co-elute, but nevermind. The interbatch and intrabatch reproducibility is quite OK. With MS detection, it is not such a big problem. The method is good enough to measure some metabolites in cancer cells (I need only separate mono- di- and triphosphate of the same nucleoside in this case). More difficult separations (of different mono- di- and triphosphates) in plant material we will probably perform by capillary electrophoresis with MS detection.
But thanks for your advices anyway. PHOBIUS

Hi

Well recently we had a lengthy discussion with my colleague regarding matter falling to subject mentioned above…
He was observed a shift in R.T when he analysis large number of sample at a stretch (a single run is about 40 min..and many times he has about 20-25 samples to analyze )..he used TFA as an ion pairing agent ( ACN and Water as mobile phase)

Could this change in R.T due to the evaporation of ion pairing agent…although I am not convinced with the concept of evaporation of TFA…but still could this be a possibility??