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Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

Liquid Chromatography 679

The wet-fill or slurry packing techniques are recom- be well shaped with no shoulders or split tops. This is
mended for particles less than 20 µm in diameter. In a sign of channeling and the column must be repacked.
this approach, the slurry is pumped quickly under high Excessive peak width is usually a sign of a void volume
pressure into the column blank. The solvent chosen to and again the column should be repacked.
prepare the slurry must wet the particles and keep them
well dispersed. In addition, it is desirable to choose a sol-
G. HPLC Detectors
vent with a density that approaches the particle density,
the so-called balanced density situation. Although sus- A wide variety of HPLC detectors have been developed to
pected cancer causing agents such as tetrabromoethane try to fill both the high-sensitivity and universal-detection
or chloroform can be used, this method is recommended requirements. Some of these are adaptations of well-
primarily for 10 µm particles. Particles 5 µm or less set- known GC detectors such as flame ionization and electron
tle very slowly, and other solvents should be considered. capture. This discussion will focus primarily on the design
Methanol or isopropyl alcohol are commonly considered and appropriate applications of the seven most common
for silica packings or polymers. For small reversed-phase and commercially available HPLC detectors. A listing of
silica particles, acetone-hexane mixtures, because of their these detectors is shown in Table II. The HPLC detectors
low viscosity, have been employed. Because of the possi- can generally be classified as either responsive to a change
bility of charging nonpolar reversed-phase packings due in the property of the mobile phase when a solute (sample
to the rapid liquid flow from the packing pump, 80% component) is present or to a property of the actual solute
methanol–20% sodium acetate (0.1%) in water has been itself.
recommended. An example of the latter is the most widely used HPLC
A high-pressure pneumatic pump capable of being pres- detector, the UV–VIS spectrophotometer (Fig. 7). Its pop-
surized to 8,000 to 10,000 psi by closing a shutoff valve ularity is due to a wide range of applicability, excellent
is generally necessary to pack efficient columns. Upon stability, and low cost. This detector is designed to be
opening the valve, the released pressure forces the pack- equipped with a low-volume (8 µl or less) flow cell usually
ing slurry from a stainless steel reservoir into the column 1 cm in pathlength. The low volume of the cell is impor-
blank in a few seconds. This “slamming” process gener- tant to minimize bandbroadening, as is the radius of the
ally will permit a uniform packed bed of particles in the connecting tubing to the column. Lenses are used to focus
column. The column bed is considered stable if the liquid as much light as possible through the 1- or 2-mm diameter
flow from the column is constant after several repressur- cell. Most filter HPLC spectrophotometers are equipped
ization cycles. After no liquid is observed to drip from the with a mercury lamp. This source emits 90% of its radia-
column, it is carefully removed from the packing reser- tion at 254 nm, which is an excellent wavelength for the
voir. The column ends are cleaned off with a razor blade detection of aromatic compounds. Different filters and/or
to permit the fittings to be attached. The column should special phosphor converters placed after the lamp allow
be checked with a test mixture and mobile phase chosen other wavelengths to be selected. The zinc lamp is used for
to give peak retention times from 5 to 15 min. A column shorter wavelengths such as 214 nm. Usually the light is
plate height two to three times the particle diameter should focusedthroughdualflowcells,oneconsideredthesample
be obtainable if the column is well packed. Peaks should side and the other the reference side. If the mobile phase

TABLE II Characteristics of Liquid Chromatography Detectors a
Temperature Linear Noise Maximum d
Detector basis Type b sensitive? range level c sensitivity
UV absorption S Low 5 × 10 3 2 × 10 −4 AU 2 × 10 −10
Refractive index G ±10 −4 ◦ C 5 × 10 5 2 × 10 −6 RIU 1 × 10 −7
Fluorometry S Low 6.4 × 10 3 0.005 v 10 −11
◦
Electrochemical S 1.5%/ C 5 × 10 3 2 × 10 −9 µamp 10 −12
◦
Conductometric S 2%/ C 1 × 10 6 0.05 µMho 10 −8
IR absorption S Low 10 3 0.01 AU 10 −6
Mass spectrometry G None 10 3 — 10 −10
a
Most of these data were taken from Snyder, L. R. and Kirkland, J. J. (1979). “Introduction to Modern Liquid Chromatography,
2nd ed.” Wiley-Interscience, New York, p. 162. Reprinted by permission of John Wiley & Sons, Inc.
b
G = General; S = selective.
c
AU = Absorbance units; RIU = refractive index units.
d
Sensitivity for a favorable sample in g/ml.