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In the world of chromatography, separation is only half the battle. The other half: detecting what's coming off the column. After all, what good is separation if you cannot tell it occurred? To paraphrase the classic riddle, if a compound emerges from a column, and a detector isn't there to see it, did it really happen?
Fortunately, LC vendors now offer a wide range of detectors and detector modalities to ensure you can always monitor your separations. The fundamental question you have to ask is, what sorts of compounds do you expect to be looking for?
"That's the first thing: What is the chemical nature of the compounds you are looking at?" says Helmut Schulenberg-Schell, marketing manager for liquid chromatography at Agilent Technologies. "In probably 80% of cases, there is a UV-absorbing compound in there, so you go for the UV detector."
Indeed, at Agilent Technologies, Shimadzu, and Waters, UV/Visible absorbance detectors—which monitor what fraction of the intensity of a light source makes it to the detector at any given wavelength—are by far the most common detectors sold with LC systems, according to company representatives." Absorbance detectors are on 90-95% of the systems we sell," estimates Ed Sprake, product manager for high-volume quadrupole MS detectors at Waters. Fluorescence, refractive index (RI), and evaporative light scattering detectors (ELSD) occupy the next three spots on all three companies’ lists, though their order varies from company to company. Another popular choice: mass spectrometry.
"People are still using LC detectors, but I believe the market share on MS is slowly growing," says Simon Robinson, HPLC product manager at Shimadzu Scientific Instruments.
UV/Visible detectors actually come in two flavors: tunable detectors and photodiode array (PDA) detectors. The former measures absorbance at one or more discrete wavelengths; the latter enables a scan across a wide swatch of the electromagnetic spectrum (generally from the UV into the visible or near-infrared) using an array of individual photodiodes.
According to Schulenberg-Schell, PDA detectors are particularly helpful during methods development, when trying to identify the most sensitive way to detect one or more compounds. "You try to look at where the maximum absorbance is, so even if there are small amounts you can still detect traces of the compounds." Tunable devices are straightforward for routine production work, when you know exactly what you are looking for and a single wavelength at a time is enough. In some cases, however, when multiple compounds require that different wavelengths be tracked in parallel, or if you can confirm identity based on full UV spectral information, PDA detectors are a better option, even in production environments, he says.
The second question to answer in selecting LC detectors, says Schulenberg-Schell: "Would you like also to be prepared to detect non-UV-absorbing compounds?" If so, you probably should consider either an RI detector or an ELSD.
Both RI and ELSD are especially useful for compounds that lack chemical structures that enable them to absorb UV or visible radiation—compounds such as carbohydrates, petrochemicals, and polymers. RI detectors measure the change in the speed of light that occurs as it passes through a mixture of compound and mobile phase compared to its speed in mobile phase alone—the same effect that causes a straw to seem to bend in a glass of water. ELSD, on the other hand, measures light scattering. As sample emerges from the LC column, it is nebulized into tiny droplets and allowed to evaporate as it passes down a flight tube. At the end of that tube, the resulting particles—whose sizes are a function of concentration—are interrogated based on the degree to which they scatter a beam of light.
Of the two, ELSD is a newer technology. Robinson calls it a "universal detector." Adds Sprake, ELSD "is broadly complementary to absorbance techniques, because you don't need a chromophore for it to work." RI is also useful on a broad range of compounds, but is somewhat less sensitive, Robinson says. On the other hand, says Schulenberg-Schell, "In general, RI detectors are a little less expensive than ELSD."
Another key consideration: Is your LC system based on HPLC or UHPLC? Detectors have to be specially built to accommodate the higher pressures, smaller sample volumes, and lower flow rates inherent in UHPLC, says David DePasquale, product manager for LC detectors at Waters. For instance, of the six most popular LC detectors Waters offers, just three, in addition to mass spectrometry, are available for UHPLC: absorbance, ELSD, and fluorescence.
Fluorescence detectors are highly sensitive detectors best suited for cases where the compounds either are inherently fluorescent, or have been modified to be so.
James Curlett, advanced instrumental methods team leader for the Maine Health & Environmental Testing Laboratory, has four LC systems in his facility, one PerkinElmer and three Agilent 1200s, one of which is coupled to a fluorescence detector. The others are hooked to a PDA, an Applied Biosystems 4000 QTRAP hybrid tandem mass spectrometer, and an ICP-MS.
Because most environmental testing protocols call for gas chromatographic separations, Curlett estimates just 10% of the facility's work is run on LC, including detection of drugs, pesticides, and terrorism agents. Nevertheless, his lab's LC systems are seeing increased activity, he says.
"Primarily, we use [the fluorescence detector] for carbamate analysis," says Curlett. As these pesticides are not inherently fluorescent, his lab treats the LC eluent with hydroxide to form free acids, making the molecules reactive to a fluorophore, which they then couple to the pesticides prior to fluorescent analysis.
Curlett also plans to develop methods to use the fluorescent reader for chlorophyll pigment analysis, he says—an indicator of algal blooms in water. "The common method is to extract [chlorophyll] into a solvent and run on UV/Vis spectrophotometers, but there are like 23 different pigments in different algae, so we plan to extract the chlorophyll from other pigments and getting a handle on what's in there," he says.
The MS/MS, Curlett says, is used mostly for LSD, steroids, and ricin, and the PDA for drug testing. The ICP-MS, which is not yet online, will be used for elemental analyses, for instance in arsenic testing.
Other LC detectors represent a smaller share of the market, company representatives say. At Waters, for instance, the most commonly purchased detectors after absorbance, RI, fluorescence, and ELSD, are electrochemical detection and conductivity detection, says DePasquale. Electrochemical detection is sometimes used to quantify neurochemicals, while conductivity detectors are used for water analysis (e.g., of chloride ions). Another emerging detection modality, corona CAD (charged aerosol detection) (commercialized by ESA and recently purchased by Dionex), is similar to ELSD except that the nebulized particles are measured not based on light scatter, but on their ability to be electrically charged.
Even if you anticipate that all your experiments will involve UV absorbing or fluorescent molecules, it sometimes makes sense to have a second detector handy, says Robinson. "Say you have a sample mix where everything absorbs UV. There's the chance one of those molecules can be seen by refractive index detection while the others cannot," he says. "At that point, you can see the one molecule out of 20."
Nevertheless, as with the gas chromatography detection market, demand seems to be shifting towards using mass spectrometry as a universal detector—a reflection of the fact that MS offers both the most sensitive and comprehensive datasets (including structural information) of any detector, and that technology, software, and ease-of-use have all improved while costs have dropped, says Robinson.
Yet not everyone either wants or needs a mass spec, he adds. R&D labs may want the most information they can get. But in production facilities, where methods have already been established using UV/Visible detectors, MS is generally limited to identifying impurities and resolving QC issues.
"It's a balance," he says. "If you want the most information possible then you're willing to deal with a more complex detection [modality]. If you are spending 24/7 watching a reaction you've done for years, then there's no point having that complexity, because all [you] want is an affordable solution to what [you]'ve already been doing."
Please check out our Liquid Chromatography section for more information or to find manufacturers that sell these products.