Page 75 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN002E-79 May 17, 2001 20:28

Capillary Zone Electrophoresis 361

employ UV or UV–Vis absorbance as the primary mode of throughthecapillaryisdispersedbyagratingontoanarray
detection. The simplest approach is the use of line-source of photodiodes, each of which samples a narrow spectral
lamps or continuum-source lamps with wavelength selec- range. In fast-scanning detectors, monochromatic light is
tion by filters. Better flexibility is obtained using a con- collected from the source using a movable grating and slit
tinuum (e.g., deuterium lamp) source with wavelength se- assembly and directed to the capillary; light transmitted by
lection by a grating monochromator. the capillary is detected by a single photodiode. Scanning
All commercial CE absorbance detectors employ on- is accomplished by rapidly rotating the grating through an
tube detection, in which a section of the capillary itself angle to “slew” across the desired spectral range. Photodi-
is used as the detection cell. This permits detection of ode array detectors are more sensitive in full-scan mode,
separated zones with no loss in resolution. Most capil- but the high light intensity can damage coated or gel-filled
laries used for CE are coated with with a polymer (usu- capillaries.
ally polyimide) which protects the fused silica capillary
and provides it with mechanical stability. Since the poly-
2. Fluorescence
mer is not optically transparent, it must be removed from
the detection segment to form a “window,” and this win- Fluorescence detection offers the possibility of high sen-
dow must be accurately positioned in the optical path to sitivity and, in the case of complex samples, improved
achieve good sensitivity. This segment of bare capillary selectivity. However, this mode of detection requires that
is very fragile and is subject to breakage during manipu- the analyte exhibit native fluorescence or contain a group
lation and installation of the capillary. Capillaries with a to which a fluorophore can be attached by chemical deriva-
UV-transparent coating are commercially available which tization. The number of compounds that fall into the for-
eliminate this problem; however, the coating is not resis- mer category are small, and while many analytes contain
tant to some coolants (e.g., fluorinated hydrocarbons) used derivatizable groups (e.g., amino, carboxyl, hydroxyl),
in liquid-cooled CE systems. most derivatization chemistries are limited by one or more
In on-tube detection, the internal diameter of the cap- disadvantages (slow reaction kinetics, complicated reac-
illary forms the detection light path. In accordance with tion or cleanup conditions, poor yields, interference by
Beer’s law, the sensitivity of a concentration-sensitive de- matrix components, derivative instability, interference by
tector is a direct function of the length of the light path. reaction side products or unreacted derivatizing agent).
Therefore, in comparison to an HPLC detector with a When compared to fluorescence detectors for HPLC,
1-cm path length, detector signal strength should be re- the design of a fluorescence detector for CE presents some
duced 200-fold in a CE system equipped with a 50-µm- technical problems. In order to obtain acceptable sensitiv-
i.d. capillary. Concentration sensitivity can be improved ity, it is necessary to focus sufficient excitation light on the
by employing focusing lenses to collect light at the capil- capillary lumen. This is difficult to achieve with a conven-
lary lumen, by detecting at low wavelengths (where most tional light source, but is easily accomplished using a laser.
analytes have greater absorbance), and by using sample- The most popular sources for laser-induced fluorescence
focusing techniques during the injection process. How- (LIF) detection are the argon ion and helium–neon lasers,
ever, even under ideal conditions, the concentration limit which are stable and relatively inexpensive. These lasers
of detection (CLOD) is about 10 −6 M. Sensitivity in op- produce emission lines close to the excitation wavelengths
tical detectors can be improved by the use of extended- for several common fluorophores. The CLOD for a laser-
pathlength capillaries. These include capillaries with in- based fluorescence detector can be as low as 10 −12 M.
creased diameters in the window segment (“bubble cell” The most successful applications of CE–LIF detection
capillaries) or in-line cells with a section of capillary po- have been in the analysis of carbohydrates and nucleic
sitioned axially along the light path (“Z-cells”). acids. Carbohydrates can be derivatized with agents such
Several commercial CE systems incorporate scanning as aminopyrene trisulfonic acid, which introduces a flu-
absorbance detectors. Scanning detection enables on-the- orophore and also confers electrophoretic mobility to the
fly acquisition of spectra as analytes migrate through the analyte. Double-stranded nucleic acids can be detected
detection point; this information can assist in the identi- using intercalating dyes and by covalent attachment of
fication of peaks based on spectral patterns, in detection nucleotides carrying fluorophores. The fluorescently la-
of peak impurities by variation in spectral profiles across beled primers or chain terminating nucleotides which are
a peak, or in determation of the absorbance maximum of widely used in automated slab gel-based DNA sequencers
an unknown compound. Two different designs are used are also used in capillary-based sequencers. In one multi-
to accomplish scanning detection in CE instruments. In capillary sequencer, a novel sheath flow system is used for
photodiode array (PDA) detectors, the capillary is illu- LIF excitation of separated bands with imaging of emitted
minated with full-spectrum source light; the light passing fluorescence on a charge-coupled device (CCD) detector.