Instant Notes: Analytical Chemistry

Solute detection Detectors are based on a selective responsefor the solute, such as UV-
absorbanceor fluorescence, or on a bulk propertyof the mobile phase which is
modified by the solute, such as refractive index. Ideally, detectors should have
the following characteristics:

● a rapid and reproducible response to solutes;

● high sensitivity, i.e. able to detect very low levels of solutes;

● stability in operation;

● a small volume cell to minimize band broadening, i.e. 8ml or less for a

conventional column, 1ml or less for a microbore column;

● a signal directly proportional to solute concentration or mass over a wide

range (linear dynamic range);

● insensitivity to changes in temperature and flow rate;

● a cell design that does not entrap air bubbles that outgas from the mobile

phase at the end of the column.

Many types of detector have been investigated, and the most widely used are

summarized below and in Table 4.

● UV-visible absorbance detector. This type, which is the most widely used, is

based on the absorbance of UVor visibleradiation in the range 190–800 nm

by solute species containing chromophoric groupsor structures (Topics E8

and E9). Detector cells are generally 1 mm diameter tubes with a 10 mm

optical path length and designed so as to eliminate refractive index effects

which can alter the measured absorbance. There are three types of UV-

visible absorbance detector:

(i) Fixed-wavelength filter-photometers, which are the simplest, employing

mercury-vapor lamp sources and optical filters to select a limited number

of wavelengths, e.g. 254, 280, 334 and 436 nm, and a phototube detector.

They have a limited use, lacking versatility, but they are cheap.

(ii) Variable-wavelength spectrophotometers (Fig. 5) are much more

162 Section D – Separation techniques

Table 4. Characteristics of HPLC detectors

Detector Sensitivity g cm-^3 Linear range Characteristics

UV-visible absorbance Good sensitivity, most widely used, selective
Filter-photometer 5 ¥ 10 -^10104 for unsaturated groups and structures. Not
Spectrophotometer 5 ¥ 10 -^10105 significantly flow or temperature sensitive.
Diode-array Can be used with gradient elution.
Spectrometer >2 ¥ 10 -^10105

Fluorescence 10 -^12104 Excellent sensitivity, selective, including
fluorescent derivatives. Not flow or temperature
sensitive.

Refractive index 5 ¥ 10 -^7104 Almost universal, but only moderate sensitivity.
Very temperature sensitive (control to ±0.001°C).
Cannot be used with gradient elution.

Electrochemical
Conductimetric 10 -^8104 Flow and moderately temperature sensitive.
Amperometric 10 -^12105 Cannot be used with gradient elution. Detects
only ionic solutes. Excellent sensitivity, selective
but problems with electrode contamination.