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Photodiode Array Detectors: An Array of Possibilities for (U)HPLC Detection
by Josh P. Roberts
hotodiode array detectors—variously abbreviated as “PDA detec- tors” or simply “DADs”—are essentially spectrophotometers that transiently measure the absorbance of light by a liquid flowing past. They are the dominant detector type used in applications such as liquid chromatography (LC) to yield information about the identity, quantity, and purity of sample separations as they exit the column.
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As the name implies, the PDA itself is an array of photodiodes that convert the light impinging upon it into a proportional electrical signal that is then processed and recorded. DADs make use of “reverse optics” in which the full spectrum of light from a source interrogates the sample as it traverses what’s called a “flow cell.” The light that is not absorbed by the sample is then linearly dispersed—typically by a diffraction grating—into its dif- ferent wavelength components, with a specific bandwidth falling on a given pixel, explains John Gilmore, technology manager for Hamamatsu Corporation, which manufactures photonics components for original equipment manufacturers (OEMs). So, for example, pixel 1 might be exposed to light around 200 nm, while pixel 1024 (in a 1024-diode array) would see 600-nm light.
DADs contain no moving parts and (unlike fixed-, variable-, and scanning wavelength detectors) simultaneously interrogate the entire spectrum.
There is no doubt that the versatile DAD is the detector of choice for any research lab developing methods, says Helmut Schulenberg-Schell, direc- tor of business development for the liquid phase separations division at Agilent. “It gives you the ability to look at all the wavelengths at the same time. You can select spectra, you can overlay spectra, and so compare the absorption from different compounds you have separated … and you can compare to a reference spectrum from a reference compound.”
Enlightened detectors As manufacturers improved LC systems’ performance capabilities, there
was a “paradigm change” in the detectors as well. Ten years ago most de- tectors had a deuterium lamp for the UV range and a tungsten lamp for the visible range, say Holger Franz, senior product manager, HPLC Detectors, in the chromatography and mass spectrometry division of Thermo Fisher Scientific. “But with two light sources you had less freedom in optimizing your optics—it’s much more favorable to just go with one light source.” So ultrahigh-performance detectors from all the market-leading
companies use only a deuterium lamp, resulting in a wavelength range limited to about 600 nm, he explains. These companies “still offer a more legacy line with the two light sources and an extended wavelength range, but it’s not very popular anymore.”
For the past few years, higher-end PDAs have typically (but not exclu- sively) had 1024 diodes, Franz notes. “Now that the best detectors only cover the 190–600 nm range, that enables a better spectral resolution,” since any given diode queries a narrower bandwidth.
Another component of the system is the flow cell, through which the sample travels as it is being interrogated by the DAD. Conventional flow cells have a relatively short pathlength (typically ≤10 mm) to allow for enough light to illuminate the sample and maintain a high signal-to-noise ratio. Yet the shorter pathlength results in a lowered sensitivity (remem- ber that the Beer-Lambert law states that absorbance is proportional to pathlength × concentration).
A relatively new type of flow cell based on the principle of total internal reflection (like fiber optics) allows for a small volume of sample to be interrogated over a longer pathlength, yet without any loss of signal. It “definitely does things better,” with a wider linear range and less band broadening, according to Franz, who notes that all the leading DAD manu- facturers offer a “light pipe” flow cell (it goes by different names) either as an optional alternative or as a replacement to a conventional flow cell. (One such option offered by Shimadzu, for example, boasts an 85-mm optical pathlength.) The downsides of light pipe flow cells, says Franz, are the cost—the cells are about threefold higher in price—and that (unlike conventional flow cells) if you drop them they’ll break.
A three-dimensional spectrum DADs are typically used after an LC separation. For routine procedures,
where it’s just a matter of assuring that the eluate displays the expected retention times and absorbances for a known compound, it’s enough to examine a discrete set of wavelengths. Waters’ ACQUITY DADs, for ex- ample, can simultaneously monitor absorbance in up to eight individual 2-D channels (time vs absorbance at a given wavelength). DADs can be used to trigger fraction collectors and other devices based on time, ab- sorbance, or criteria such as ratio of spectral peaks.
AMERICAN LABORATORY • 40 • SEPTEMBER 2014
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