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Photodiode array detectors—variously abbreviated as “PDA detectors” 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.
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 different 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, director 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 detectors had a deuterium lamp for the UV range and a tungsten lamp for the visible range, says 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 exclusively) 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 (remember 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 manufacturers 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 example, 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, absorbance, or criteria such as ratio of spectral peaks.
Yet since DADs are able to capture the full spectrum they can also be used in 3-D mode (time vs wavelength vs absorbance). This allows the researcher to better determine an unknown compound, for example, or to assess the purity of the sample. But, warns Franz, be careful to order 3-D-enabled software.
The frequency at which a DAD queries the sample can be adjusted to accommodate, for example, low- versus high-pressure separations, or larger versus smaller expected bands. It’s certainly important to use a data rate that’s fast enough to adequately define the shape of peaks; Waters recommends at least 15 data points across the narrowest peak,1 but faster means more data being collected and processed, which can slow the system. Most systems made today have a maximum data rate of at least 80 Hz, which Schulenberg-Schell says is “good enough” for even UHPLC, but 10-year-old DADs may not be able to meet those demands.
What to look for in a diode array detector
Although DADs can generally be purchased separately, “I would definitely see the detector not just as an isolated box, but in the context of the entire system and the software,” says Franz. There’s not really an advantage in cherry-picking.
That being said, some systems come with options, and available detectors may be part of the purchasing decision.
Cost and performance differences of (at least higher-end) DADs have been narrowing. Wavelength accuracy isn’t as important as it is in spectrometry, because in LC analysis the same slight deviation from exact wavelength would be seen in both the sample and reference standard to which it is compared, points out Schulenberg-Schell. Yet, as discussed above, parameters such as the technology type and pathlength of the flow cell, data rate, whether there is a second lamp (and thus capabilities to detect in the visible range, as well as spectral resolution), and number of diodes in the PAD, can have an effect.
Table 1 – Purchasing considerations for diode array detectors
The sensitivity—which allows the detector to identify smaller peaks faster—is principally driven by the noise. While vendors will typically list this specification, “the user has to be careful of the conditions under which the noise is specified,” such as the time filter and the solvent used, says Franz. “When a vendor refers to ASTM conditions, the noise should be comparable, but not every supplier sticks to it.”
Another spec is drift, which refers to the stability of the detector, Schulenberg-Schell notes. Many vendors such as Agilent build in electronic thermal controls to help mitigate drift. A list of purchasing considerations for diode array detectors is given in Table 1.
Dynamic range solutions
One of the main challenges faced by DADs is that a choice typically needs to be made between sensitivity to low light levels and the ability of the detector to quantitate higher levels without becoming saturated. A variety of strategies have recently been introduced to deal with dynamic range.
Table 2 – Providers of diode array detectors
For example, with variable integration time, which combines software and hardware, “you can identify pixels that have a lot of signal in them, and read them more quickly and integrate those for shorter periods of time than pixels that have less signal on them,” explains Gilmore.
Agilent’s 1260 Infinity High Dynamic Range DAD strategy is to “basically cluster two DADs” together—one with a 3.7-mm-pathlength flow cell and the other with a 60-mm-pathlength flow cell—into a single unit, and computationally combine the signals, says Schulenberg-Schell.
Shimadzu uses its Intelligent Dynamic Range Extension Calculator (i-DReC) to detect when a peak becomes saturated and automatically shift the profile to a wavelength with lower absorption, generating a correction factor to calculate the peaks at the original target wavelength.
Although there are many specialized detectors, querying various parameters that can be used to interrogate samples coming off a liquid separation—from fluorescence to electrochemical to light scattering to radioactivity—the photodiode array detector is arguably the most versatile, and most necessary, tool for the research laboratory.
Table 2 lists some providers of diode array detectors.
Reference
- ACQUITY UPLC Photodiode Array and eλPhotodiode Array Detector Operator’s Overview and Mainenance Guide. http://www.waters.com/ webassets/cms/support/docs/715002209ra.pdf.
Josh P. Roberts has been a full-time biomedical science writer for more than a decade. After earning an M.A. in the history and philosophy of science, he went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology; e-mail: [email protected]
Please see our HPLC PDA Detector section to find manufacturers that sell these products