Ghost Peaks, Spikes, And Bubbles

Ghost Peaks, Spikes, And Bubbles

An infrequent but annoying problem wellknown to HPLC (and also to CE) is the appearance of "ghost peaks" and "spikes". Troubleshooting sections of leading publications for HPLC and CE are replete with suggestions (1-5) as to how these artifacts might be eliminated. The problems are normally seen with various optical detectors and are usually ascribed to the appearance of microbubbles within the eluate moving into the detector. Unfortunately, we see the same phenomenon with radioHPLC detectors which, after all, are sensitive detectors of light. In fact, with our scintillators which themselves are light generators when subjected to stimulus, we may have even more pronounced problems than with other detectors. Here, we propose to discuss the problem as it pertains to radioHPLC detection, its origins, how to recognize it, and what can be done to overcome it.

What It Isn't
In any system dependent upon light detection, whenever there are artifacts that are not easily explained, stray light must be considered (6). In a radioflowthrough detector, most often stray light is not the cause of the spikes or ghost peaks but, as it is easily checked, it is probably prudent to do so. Making a run after covering the instrument with a black cloth is a quick and simple means to establish whether or not light is causing a problem. Should the problem persist with the instrument covered, look to the scintillator reservoir or the waste line through which light may pipe for remarkable distances. Again, there is a simple test operation of the instrument with the room lights off. If during either of these tests the artifacts do disappear, the cause and the cure should be obvious and needn't be considered here. In further discussion, we assume that stray light is not the cause of the problem.

What It Is
At times, the spikes and ghost peaks are random, at other times quite repetitive. They have been observed with a radio HPLC detector even when no radioactivity is present. Some instrument users have reported them with a particular solvent or solvent pair, but not with others; it has been found that one manufacturer's solvents exhibit the phenomenon while another's do not. When one of these is the case, we must look to the chemicals rather than the instrument. In some instances, these artifacts always appear at a certain time in a gradient. When such non-random phenomena occur, it should be taken as a further strong indication that in some way the problem originates with the chromatography since the instruments are free running and do not have cumulative timers. So, where do the spikes and ghost peaks come from? Bubbles are the accepted cause. It has been known since as far back as 1934 that collapsing bubbles emit light. This phenomenon (7) results in light bursts with a duration of ~ 50 picoseconds during which time possibly as many as a million photons are emitted. They cover a broad spectrum from the infrared through the visible, and into the ultra violet. Most of that light is absorbed by the surrounding liquid but enough must get out, especially in the narrow crosssection of a flow-through detector cell, to activate a photomultiplier. Here, our coincidence counting is not helpful. With the entire burst lasting only 50 picoseconds, and with coincidence times measured in nanoseconds, both PMs are affected within the resolving time of the system and, if they pulse, there will always seem to be a coincidence. In the referenced article, one of several suggestions is that the temperature within the bubble climbs to "many tens of thousands of degrees"; elsewhere it is said that this might be as high as 500,000 degrees C. Such high temperature, if in fact the case, provides a plausible explanation for the origin of the light; we know from the sun and other stars that extreme heating of a gas results in a glow.

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Ghost Peaks, Spikes, And Bubbles

What is certainly clear is that such bubbles contain enormous energy concentrated over a very small about 500 nanometers cross-section. It is this energy which ultimately eats away the bronze propeller of an ocean liner or a supertanker in the process called "cavitation".

Coping With the Problem

Why are gradients a bigger problem than isocratic measurement and why is the appearance of spikes or ghost peaks occasionally so reproducible? It is suggested (1) that when air saturated solvents are mixed, at some point the mixture may have a lower capacity for air than either pure component and bubbles form; this appears to be just as true and as reproducible for a diodearray detector as for a radioflow detector. If this is so, one might expect, as has been shown, that for both detectors, ghost peaks are often eliminated by the addition of a backpressure regulator; presumably, the added backpressure keeps bubbles from forming. And to add further confirmation, with radioHPLC detectors and packed cells, the problems seem to be fewer for large cells with more packing and more backpressure than for smaller ones. The need for backpressure regulation has not escaped the manufacturers of fittings. An Upchurch Backpressure Regulator or their MicroMetering Valve at the system exit sometimes proves useful but care must be exercised to ensure that the increased backpressure does not rupture the HPLCflow cell (below). For most such cells, 75? psi is a safe upper limit. If more is needed, discuss the problem with the manufacturer of your equipment. And, you must keep in mind that not only may the cells not be meant for elevated pressure but your scintillator pump surely has severe limitations in that regard. Even better than preventing bubble formation is eliminating the source. Sometimes it is as simple as changing solvent vendors. Or, it may be necessary to degas solvent(s), and liquid scintillator too, by sparging with helium or applying vacuum, or both.

Some Notes of Caution

The addition of a radioflow detector to an existing HPLC system may make a spiking problem disappear from the HPLC detector and move to the radioflow detector. That's because the radio-flow detector serves to add backpressure to the HPLC detector with the point of pressure release moved further downstream. Under these circumstances, while it is tempting to suggest that there is a problem with the radioflow detector, the reality remains that the problem is with the chromatography solvents and it is there that an answer must be found. Bear in mind that increasing the backpressure, while possibly advantageous for reasons that have been suggested, must be done with a careful view to the entire system. Many mass detectors are not meant to operate under substantial backpressure and may be irreparably damaged when it is too high. And, a radio-flow detector with its transparent thinwall tubing may impose an even lower maximum pressure; if the backpressure must be elevated to overcome spiking, to eliminate concern about possible cell rupture, you may have to use a special high-pressure flow cell, which does exist.

References
(1) Dolan, J.W., LC-GC, 10, No. 4, 294 (1992) (2) Dolan, J.W., LC-GC, 11, No. 4, 270 (1993) (3) Dolan, J.W., LC-GC, 13, No. 12, 940 (1995) (4) Dolan, J.W., LC-GC, 21, No. 10, 968 (2003) (5) Weinberger, R., American Laboratory, 29, 24U (1997) (6) Goodman, D., Optics & Photonics News, January 1992, p. 52 (7) Knight, P., Nature, 381, 736 (1996) [ Home ] [ Contact us ] [ News ] [ Products ] [ Exhibits ]
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