Post-column derivatization: static vs dynamic mixers

[adam]

Hi all

We are presently trying to transfer a post-column derivatization method and are having some problems with peak tailing and noise. Both of these things would seem to be related to the mixer. We have tried a glass bead mixer and a vortex mixer (which in my opinion, is really just a tee). Neither of these gives us the chromatography we need.

Does anyone have any experience with this situation. It is something of a paradox: the more dead volume you have in the mixer, the better the mixing; but the worse the peak shapes will be. Has anyone tried static vs dynamic mixers; and maybee can share their experience.

Since we know the problem is the mixer, I won't bother to post all the parameters. But here's a few key bits of info:

Column ID: 4.6 mm
particle diameter: 7 um
column flow rate: 1 ml/min
reaction line flow rate: 0.8 mL/min

[Uwe Neue]

No, mixing is not the problem in post-column derivatization. For most situations, a simple T is plenty. More sophisticated devices can be designed, but a T is good enough for most practitioners.

The second question is the length of the reaction time that you need, since you need to store the column effluent and the reagents for this reaction time. If the reaction time is very short, you do not need to worry about sophisticated reaction coils. On the other hand, if you do need some reaction time, than a more sophisticated coil is best to suppress bandspreading.

Excessive baseline noise may be related to pump pulsation or background reactions that show up in the detector. If you specify the reaction, maybe I can help.

[adam]

Uwe

The reaction is arsenazo (III) binding with gadolinium (Gd). The lab that developed this method has told us that the reaction is very fast.

Are you sure mixing is not a problem that could explain the noise?? In my experience mixing is a classic problem with respect to HPLC mobile phases: especially when either or both of the mobile phases are 100% (for example mobile phase A is 100% aqueous, and mobile phase B is 80/20 ACN/Water). In several cases we have revised a gradient so that neither mobile phase is 100%. And this has worked pretty well for us in giving smoother baselines. But with post-column derivatization, the mixing is occuring downstream of the column, so we don't have the column to act as a mixer. It would seem to me that mixing would be a huge problem in a situation like this.

Thanks
Adam

[Mark Tracy]

Uwe is quite right, the mixer is most likely not the problem, and a simple tee suffices in almost any circumstance.

The most probable cause of mixing noise is one of the pumps. What are you using for the reagent pump? Most HPLC pumps don't do a good job as reagent pumps because their pressure feedback control systems assume the existence of a column to provide backpressure, consequently, they are often unstable. Some reagent pumps from Eldex, SSI, etc. have pulse dampers, but the damper also requires the backpressure of a column in order to function. With some reagent pumps you will need to add the pulse damper. The simple fix is to put a restrictor between the reagent pump and mixer.

The next most likely problem is the reagent. Is it transparent at the detector wavelength? In a post-column system, noise is proportional to the background intensity.

Some detectors are more sensitive to refractive index noise than others. May not be much you can do here. If you are using a diode array detector, the choice of reference wavelength can improve the noise.

It could even be your analytical pump. Post-column techniques are less forgiving of ripple than standard UV. Your analytical pump should be in the best possible condition, reasonably modern, and well primed. Mobile phase degas is a good idea.

A short reaction coil might help. Try about 1-2 meter of 0.25 mm i.d. teflon tubing. This should cause very little band-broadening, but give a few seconds for the reagent to diffuse into the mobile phase.

If all the above have been optimized, then you might be able to see the difference between various mixer designs.

Just in case you are wondering, I used to work tech support for Pickering Laboratories (www.pickeringlabs.com) who are specialists in post-column techniques.

Best of luck,

[adam]

Thanks for the detailed information.

I'm still having trouble understanding why mixing would not be a problem if it's done post column; whereas it is often a substantial problem when done pre column (i.e. the typical low pressure gradiant system). Don't get me wrong. I believe you guys know what you're talking about. But for some reason I can't get my head around it.

Your point about pulsations due to the reagent pump makes a lot of sense. I had not thought of that. Do you suppose that instead of a restrictor, we might put a glass bead trap on that line (just before the tee).

Thanks again.

[Mark Tracy]

The whole idea is to make some back pressure, say 50-150 bar. As long as it is chemically and mechanically stable most anything will do. You could use a long piece of 0.12 mm i.d. tubing, or a packed bed.

In a LP mixing gradient pump, the mobile phases enter the pump head as a train of slugs. In general, there is not enough turbulence inside the pump to thoroughly mix these, expecially if you are trying to mix acetone or THF. Furthermore, during the gradient, each stroke of the pump is a slightly different composition. The external mixer smooths out these two kinds of inhomogeneity. Some pumps do not have explicit "mixers" but have other pieces that do that as a side effect.

In post-column mixing, the streams are already fairly uniform in composition, so the problem is reduced to keeping the flow rates uniform. The mixing requirements are just a lot smaller in scale.

[Uwe Neue]

I agree with most of Mark's propositions.

The mixing problems that you have with gradients working close to 100% A or B is that you need to mix in close to 0% B or A. This is not comparable to the problem that you have here, where both pumps work at a flow of around 1 mL/min.

Mark's suggestion to add a bit of backpressure to the reagent pump might be best, since most HPLC pumps do not like to work at their best when the backpressure is 0.

[HW Mueller]

This could be quite interesting if you told us what arsenazo is, exactly, which is analyte and which is reagent, details of the chromatography, how the complex(?) is detected, how you know that the tailing occurs post-column, whether the baseline is smooth when the reagent is not pumped, what the mobile phase is, in what the reagent is dissolved, pH of the solutions....
I can imagine that the Gd causes some troubles, but then one should expect bad repeatability (precision). If the tailing is indeed post-column it appears that the volume there is to large.

[Chris Pohl]

Hans,

Arsenazo III is a colorimetric metal chelating agent, used for "visualizing" metal ions.

Adam,

In general, when mixing two homogeneous liquids where each liquid is delivered at constant flow rate, mixing requirements are minimal as suggested by Mark and Uwe. The reason for significant focus on mixing in HPLC is connected to the use of the solvent valve and the details on how solvent is metered in during the pump stroke with a low pressure gradient pump. In the latter case, the mixer needs to be large enough to homogenized different eluent compositions supplied to the pump during different points in the pump stroke. However, your situation would appear to be more similar to a high pressure gradient HPLC pump. In this case, mixing requirements are typically minimal.

There are, however, a few possible scenarios where you would appear to need significant mixing:

1). Your reagent pump is not supplying pulseless flow. As Mark suggested above, you might not have adequate backpressure on your reagent flow and this can result in oscillating flow that could produce the symptom you describe. Corrective action would be to incorporate 4-500 psi backpressure between the pump outlet and the mixing tee.

2). You are using a standard HPLC pump with stainless steel components to deliver your Arsenazo III. Since your reagent can form colored complexes with metals in stainless steel and the reagent is undoubtedly at least mildly corrosive to stainless steel, oscillations in the background would be expected since the flow in your pumping system is actually oscillating with only one pump head delivering flow at any given time. During the time that flow is stagnant in a given pump head, the amount of metal contamination would be expected to rise, dropping again as flow resumes. A solution to this problem would be to use a reagent pump with nonmetallic fluidic components

3). Your eluent system is being generated using a low pressure gradient pump and one of your eluents contains significantly more trace metal contaminants than the other. In this case, failure to adequately mix the eluent as it exits the analytical pump can also produce symptoms you describe. If you make a premixed eluent in a single bottle, you should be able to determine whether or not contamination of one of your eluents is responsible for your problem.

4). One or more of your eluent components is mildly corrosive to stainless steel and you are using a stainless steel analytical pump. This scenario is similar to the second scenario described above. A solution to this problem would be to use a pumping system with nonmetallic fluidic components.

[adam]

Interesting. We've come to the conclusion that the tailing issue is coming from the column. But are still working on the noise issue. I will give some consideration to the above suggestions.

Thanks Mark, Uwe, and Chris for your helpful suggestions.

[HW Mueller]

So the complex absorbs quite differently than Arsenazo itself? If this is the case it seems obvious that if you have a noisy baseline, without a Gd injection, that some other metals must be complexing, intermittantly, with the reagent (or is its absorbance strongly influenced by pH?, temp.?). If only your Gd peak is choppy it seems clear that you are pumping badly or you are at a pH where the Gd complex is unstable.