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4 Chromatography 109
Using the Microscale Chromatography Columns The microscale columns are used
the same day they are prepared and are used in the same manner as the larger-scale
columns. Once moistened, they should not be allowed to run dry.
Solvent System In the schematic (Figure 4.44), the apparatus is eqUipped with one
or more glass or steel solvent reservoirs. The solvent may be heated or stirred, or the
9Consult Chapter 12 for analytical chemistry books that explain this method in detail.
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Helium - - - - ,
Solvent reservoirs
solvent reservoirs may contain attachments inlets for inert gases for solvent degassing.
The presence of dissolved gases results in poorer resolution of the peaks.
Pumps The pump for the HPLC instrument must be pulseless and able to generate
reproducible flow rates. The pump must be able to drive the mobile phase through
long, narrow columns which are packed with very fine particles and to generate pres
sures up to 6000 psi and a solvent flow of 0.1-10 mLimin.
Sample Introduction The sample is introduced into the system via a syringe. A sam
pling loop is the most common method of sample introduction. The sample is injected
into a small loop. As a lever is moved, the loop is closed off from the outside and the
solvent is allowed to pass through the loop, thus introducing the sample into the system.
Columna Columns for HPLC are usually constructed from stainless steel tubing.
Fittings and plugs must be inert and. should not detract from the homogeneity of the
flow. Analytical columns range in length from 10 to 150 cm long. The inside diameters
vary from 1 to 20 mm. Columns of less than 8 mm are difficult for a novice to pack.
The dimensions of a preparative LC column are typically 30 cm by 5 cm.
Silica is the most common type of packing material. The silica may be coated with
a thin organic film, which is chemically bonded to the surface of the silica. By using a
reverse-phase type of adsorbent, compounds may be separated on the basis of
nonpolar-nonpolar interactions rather than polar-polar interactions.
Detectors The most commonly used detector for HPLC is an ultraviolet spec
trophotometer or a refractometer. Other detectors such as infrared and electrochemi
cal have also been used. The spectrophotometers are considered to be much more
versatile and can detect a wider range of compounds. By using a refractometer, changes
in the solvent refractive index can be detected.