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4.

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.

4.4.4 High-Performance Liquid Chromatography (HPLC)


High-performance liquid chromatography (HPLC), sometimes called high-pressure
liquid chromatography, attacks problems associated with traditional column chro­
matography with great success.9
In traditional LC chromatography the stationary phase consists of a particle size
which is large relative to that used in HPLC. In order to speed the diffusion of sample
into the stationary phase, very fine particles of stationary phase are used in HPLG
These particles are on the order of a few micrometers in size. The small size of such
fine particles produces a new problem, such as potentially slow flow rates; pressures
well above atmospheriC pressure are necessary to push the mobile phase through tightly
packed columns of fine particles. P\lmps delivering thousands of pounds per square
inch push mobile phases through columns of very fine particles. Detectors that differ­
entiate samples by refractive index, by UV absorption, and by fluorescence are
commonly used.
High-performance liquid chromatography offers the advantage of high speed,
reusable columns, automatic and continuous solvent addition, reprodUcible pro­
grammed gradients of solvents, and automatic and continuous monitoring of the eluted
samples. Less than 1 mg of sample is commonly analyzed. Preparative-scale instruments
separate between several milligrams and a few grams of sample. The main disadvantage
of preparative-scale separations is that large amounts of solvents are used.

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.

-
Helium - - - - ,

Solvent reservoirs

Figure 4.44 Schematic of


a high-performance liqUid
Injector chromatography column.
110 t> Chapter 4. Separation of Mixtures

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.

Analysis of Chromatograms The retention times and percent composition of each


component are determined from the chromatogram. The retention times are calculated
by dividing the distance by the chart speed (Figure 4.35). The distance is measured
from the zero time mark, when the sample is injected, to the top of the peak. The zero
time mark can be made by moving the pen on the recorder up and down before the
sample is injected into the column. The chart speed is determined by looking at the
recorder. The units of the chart speed may be in cm/min or mm/min. Care must be taken
so that the units cancel out properly, leaving behind only the unit of time.

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