Having seen the usual shifts (under one minute) of negative H2O peaks there was quite a surprise generated, recently, by shifts of several minutes:
Column: 4.6x250mm phenomenex prodigy (a C-18); mobile phase: 88%PBS (0.15M NaCl + 0.15M phosphate, pH = 7.2) + 12%ACN; flowr.: 0.45mL/min; H2O retention time: 8.2min.
The same column at the same flow rate gave RT = 6min for H2O when the m. phase was H2O + MeOH (estimated to or tm for this column: 5.9min).
The NaCl and phosphate are at ionic strength where these salts should have chaotropic characteristics. May it be possible that distribution (between the stationary and mobile phase) is obtained as explained for immobilized PEG (polyethyleneglycol) at http://bama.ua.edu/~rdrogers/webdocs/ABS/sld010.htm ? (Content in short: Chaotropic salts shift ionic substances toward an aqueous PEG-rich liquid or immobilized phase from an aqu. PEG-poor phase).
To top this off, pertechnetate (99mTcO4-) had a retention time of 11.8 under the first mentioned conditions.
Is there anybody who has seen something like this, or has refs, or an explanation?

What wavelength are you at. I think you are seeing a disruption of the organic adsorption equilibrium not the retention of water.

I agree: the thing at 3.7 mL is not your water peak. Too much retention.

The wavelength is 220nm. The peak is a bit large for a disruption?? But then again, there must have been quite a bit of dirt (which got disrupted?) somewhere to give such a large negative peak.
But: What about the retention of pertechnetate? MAG3 (a complex of 99mTc, apparently doubly negatively charged at pH = 7) appears at tm. (At a pH = 2.5 this complex is, expectedly, strongly retained).
Looks like I have to dig up some T2O.

HW-are you expecting the water peak to be unretained? If so, why not inject uracil or thoiurea which also should be unretained. Maybe this will help to identify your water peak. I agree there is a lot of debate as to the suitability of these compounds for unretained peak determination.

David

David, why would you expect the water peak to be retained on a C18 column with a ton of water in the mobile phase?

Recently we have seen the same behavior. We originally had some very elaborate explanations. We determined that the culprit was dissolved air. We were able to remove the peak by: vacuum degassing and helium sparging. If you want to test this just inject a very small volume (10ul) of an empty vial and look for the peak. I still don't understand the mechanism of retention but I am convinced that our observations are real.

David:
The reason for this post is that this was not at all expected, I donīt recall ever seeing this kind of thing, irrespective of whether itīs due to water or a disruption.
dan:
There is extensive experience, here, with air in all forms. "Dissolved" air, also air bubbles, have always given a positive peak. This is the first time I hear of a negative air peak. It appears they are either due to light dispersion of air or complexation of O2.

What about pertechnetate??

Iīll try to attach two chromatograms, one UV the other gamma of the same injection.

Mag3_019.cdf mag3_019.cdf (20 k)

Mag3_019g.cdf mag3_019g.cdf (11 k)

If someone succeeds in opening the above attachments (I didnīt, even with the original software) they need to know that the starting injection was a mixture of 99mTC-MAG3 (mag3_019g.cdf, gamma chrom., first big peak) and 99mTcO4- (2nd peak, therein). The mag3_019.cdf is the UV version (UV detector is first after coulumn, then gamma detector, timewise a few seconds later). The UV chrom shows two later injections of pure H2O. MAG3 and pertechnetate do not show in the UV.

To open the files, you just need to save them as mag3_019.cdf instead as mag3_019_cdf.unk suggested during download. Also they are of fairly different scale.

What is the magnitude of your peaks on UV in AU? All UV detectors are RI sensitive to some extent, thus you may see just the elution of your buffer salts (or better the lack of it in the sample). It can be confirmed by injecting NaCl or phosphate at higher concentration than in your eluent, the corresponding peaks should be positive then.

Ivan Vins

OK, just finished running tritium water (T2O) under the above conditions with the last bit of that mobile phase. Anon Monday and Uwe are right. The tritium peak is at 5.1 minutes, no other peaks are visible in the beta chrom. This means that tritium did not exchange to anything else. The negative peak(s) has the same shape as before but do come earlier (6.1 on the UV chrom, the UV detector is plumbed first), but that doesnīt seem to be unusual for disruptions (disturbances). So a chaotropic effect does at least not operate for H2O here.
David, on uracil, etc.: have not used it as there are ~always positive peaks at tm.....
Ivan: I did change the unk to cdf, but my software is a bit curious, there are not many that can handle radioactivity detectors, so ...
The hight in AU is ~0.03. If this was due to RI it would mean that NaCl or phosphate would be retained, now, to see pertechnetate even later than the negative peak is surprising to me, but NaCl at ~8 min? It could be that NaTcO4 is somewhat associated?
Hans

30 mAU is a quite big peak for RI effect, so this may be wrong idea. Single injection of 0,3 M NaCl can confirm this.

Has your column been ever used for ion pair methods? Some anion exchange groups present on your column can explain the retention of pertechnate.

Ivan,
the trouble is that practically anything but mobile phase will give this negative peak.
The column has not been used in an ion pair method.
In the link given in the initial post the pertechnetate goes into the PEG rich phase. It must have a tendency to associate. Nobody around who has more info on this?

Although I've no direct experience with this system, at this point I suppose I should contribute something to this conversation. One possibility not discussed so far is the well-known tendency of PEG to associate with metal ions. In a former life, I used to use a metal ion titration method for quantitating nonionic surfactants based on this principle. In principle, this is nothing more than a variant of the well-known tendency of cyclic polyethers to complex with cations. In this case the complexation is considerably weaker but it certainly isn't zero. Of course, once cations have bound to this phase, anions can be expected to associate with the immobilized cations. Perhaps this is the source of the phenomenon. If so, it can be tested by varying the cation in the eluent. Lithium salts should have considerably less affinity and thus reduce the effect.

Chris, do you have a ref on PEG and cations? It looks like I have to check into this, just canīt leave the TcO4- retention unexplored.

It's been more than 20 years since I worked in this area so I couldn't lay my hands on any of the original documentation but combining my vague recollection with a Web search I found the following which might be of some help: http://www.kcsnet.or.kr/publi/dh/dh92n5/dh92n5.html (this one describes a system involving calcium and iodine);
A Calorimetric Study on Thiocyanato Complexes of Cobalt(II) Ion in Micellar Solutions of a Nonionic Surfactant, S.Ishiguro, T.Sotobayashi, K.Satoh and K.Saito, Inorg. Chem., 27, 1152-1155 (1988).; Spectrophotometric Study of Thiocyanato Complexation of Cobalt(II) and Nickel(II) Ions in Micellar Solutions of a Nonionic Surfactant Triton X-100, Y.Umebayashi and S.Ishiguro, J. Solution Chem., 25, 731-746 (1996).; Unusual Behavior of Thiocyanato Complexation with Copper(II) and Zinc(II) Ions in Micellar Solutions of a Nonionic Surfactant Triton X-100.Y.Umebayashi, K.Nagahama and S.Ishiguro, J. Chem. Soc. Faraday Trans., 93, 1377-1381(1997).; Thiocyanato and Iodo Complexation of Cadmium(II) Ions in Micellar Solutions of a Nonionic Surfactant Triton X-100. Y.Umebayashi, K.Nagahama and S.Ishiguro, J. Colloid Interface Chem., 191, 391-397 (1997).

The method I used involved a titration with iron and thiocyanate but that's about all I can remember about it after all these years. Maybe the above will provide some clues, at least.

Thanks Chris, maybe some of this can be related to RP phases?

That brings up one more question to Evan:
Did you ever see retention of NaCl in RP HPLC?

By an interesting coincidence, I was visiting colleagues at Oak Ridge National Laboratory last week and they told me there is a commercial resin from EIChrom which involved immobilization of PEG on a stationary phase specifically for the purpose of retaining pertechnetate. Furthermore, they told me that it was known that the retention mechanism involved weak binding of a cation to the PEG and the association of the pertechnetate. I checked out their website but they don't list any resin of that sort any more. Nonetheless, armed with that information I found the following presentation which contains specific mention of retention of pertechnetate in a PEG biphasic system: http://bama.ua.edu/~rdrogers/webdocs/ABS/tsld001.htm

H.W.

to achieve retention of Cl- or other anions on RP-column is relatively simple by bonding some hydrophobic amine to your column - its called ion pair chromatography. If the amine is hydrophobic enough, in low organic content eluent it will stick to the column even if it is no more present in the mobile phase. The pertechnate I suppose can have significant affinity to anion exchangers (based on properties of similar anions). However, considering the ionic strenght of your eluent, the column will have to have quite a high anion exchange capacity to cause significant retention of chloride. If your column was new, its probably wrong trace, if it was used, I will recommend to check its anion exchange capacity somehow.

Negative peaks are usually caused by something in your eluent, what is missing in your sample. The absence of them, when injecting mobile phase, supports this theory. I will try to inject mobile phase components separatedly (i.e. NaCl, NaH2PO4, acetonitrile), to see which one is responsible for the contamination. (Quite commonly overlooked source of possible contamination is pH adjustement).

Chris,
check my first entry again, that link is there already. Itīs the one that got me thinking in the direction of the chaotropic effect to retain pertechnetate.
Ivan,
the conditions are, hopefully, as far from the ion exchange method as possible, though I donīt know what the original samples composition is, it is considerablay diluted by the time it is injected. By now this mobile phase is gone, whether this negative peak can be repeated is not clear, again, even if it is repeated, I donīt see how to add or leave out ions in the mobile phase without giving rise to a disturbance. Iīll try some things, if anything interesting comes up for another post....lets see. . .

Sorry for the embarrassing oversight. I should have checked your link in the first place. Anyway, this has motivated me to look a bit further and I found some rather solid references which detail the behavior of pertechnetate in a biphasic system. The first one is available on the Web: http://wwwsoc.nii.ac.jp/jsac/analsci/pdfs/a16_1039.pdf This one led me to a detailed reference which also cites a review article which I didn't check: R. D. Rogers, A. H. Bond, C. B. Bauer, J. Zhang, S. T. Griffin, J. Chromatogr. B, 1996, 680, 221. This second article gives a nice accounting of pertechnetate partition behavior along with the influence of both anions and cations on the behavior. It looks like sodium salts of highly hydrated anions such as sulfate or phosphate will result in maximal retention of pertechnetate. On the other hand, minimal retention of pertechnetate is achieved in the presence of salts of chaotropic anions and cations. The negative peak you observe at 8.2 min. must be a vacancy peak associated with another anion or cation impurity in the eluent system which absorbs in the ultraviolet and is retained by the same mechanism, rather than a "water" peak.

It looks like some more digging is in order. Thanks for your refs, they should help. There are some interesting relevant radioisotopes on the market, maybe some can be obtained. Presently, though, I am convinced that some organic dirt got into the mobile phase (I know it had a high absorption as mentioned) and caused the disturbance (disruption) peak, as discussed above. If strongly absorbing inorganic ions were present in the PBS and pertechnetate solutions some people would be in real trouble (is for human injection).