Page 213 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

P1: ZCK/MBQ P2: GQT Final Pages
Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

680 Liquid Chromatography

FIGURE 7 Diagram of a photodiode array instrument. For HPLC, the cuvette is replaced by a flow cell similar to that
used for the standard UV–VIS detector. [From Siouffi, A-M., Chapter 1, in “Food Analysis by HPLC.” (L. M. L. Nollet,
ed.), Marcel Dekker, New York.

does not absorb, which is often the case, the reference side which is a function of the solute concentration. Although
is usually air. However, occasions arise where it is advan- the RI detector is versatile, it is not particularly sensitive
tageous to pump the mobile phase through both cells for (microgram level) and is very prone to temperature fluc-
subtraction purposes. The detector is quite stable to tem- tuations. Careful temperature control of the detector cell
perature fluctuations; however, air bubbles that lodge or with a water jacket is crucial for maintaining a stable base-
pass through the flow cell will cause baseline spikes. A line. Modifications in the electronic design and the use of
UV–VIS HPLC detector equipped with a monochromator a laser source have also improved the detectability of the
for specific wavelength selectivity is more versatile but RI detector.
also more expensive. Deuterium and tungsten lamps are Luminescence detectors can be either based on fluo-
used to provide ultraviolet and visible light, respectively. rescence or chemiluminescence. The fluorescence HPLC
Often multiple wavelengths can be monitored simultane- detector is basically a fluorometer equipped with a flow
ously. The photodiode array (PDA) UV–VIS detector has cell slightly larger in volume than the UV–VIS detec-
now become quite common as the standard HPLC de- tor to permit more fluorescent light from the solute to
tector ordered with a new instrument. The PDA detector reach the photomultiplier. Again, the primary modifica-
employs a reverse optics design which allows the entire tion is the use of lenses to focus the excitation source light
spectrum of light to be dispersed onto a diode array (see onto the flow cell. Filter instruments are often more sensi-
Fig. 7). Each diode of the array is responsible for detect- tive than monochromator instruments because of greater
ing light of a narrow wavelength range depending on the light throughput. Again, lasers have allowed the use of
desired spectral range to be covered and the speed of data small volume flow cells with lower detection limits. The
acquisition. This instrument can take a spectrum over a fluorescence detector, although useful for only a select
preselected wavelength range in a fraction of a second as class of aromatic hydrocarbons or derivatives, is about
the solute elutes from the column. Therefore, qualitative 100 times more sensitive than a UV–VIS detector. For
(peak purity) as well as quantitative information about the example, polyaromatic hydrocarbons can be determined
unknown sample component can be obtained. at the ppb level. Signal stability with respect to temper-
The differential refractometer is perhaps the second ature is good. The coupling of fluorescence to HPLC
most widely used HPLC detector because of its universal has generally minimized background signals from im-
nature. Potentially, any substance with a refractive index purities and oxygen quenching effects. If the lamp of
(RI) different from the mobile phase is detectable. The the fluorescence detector is turned off, it can operate as
deflection type RI detector consists of a light source pass- an effective chemiluminescence detector. Postcolumn ad-
ing two beams of monochromatic light through a double dition of reagents such as luminol and a metal catalyst
prism that constitutes the sample cell and reference cell. for the detection of an oxidizing agent such as H 2 O 2 is
If the mobile phase composition changes, the altered re- necessary.
fractive index causes the beam to be deflected from its The electrochemical (EC) HPLC detector is basically
initial position on the photomultiplier detector. The elec- a small electrode composed usually of glassy carbon
trical signal produced is proportional to the light position, mounted in a flow cell (Fig. 8a). The auxiliary electrode is