Page 321 - Instrumentation Reference Book 3E

Chromatography 305
non-polar liquid phases. Specially treated papers The apparatus used for measuring the separ-
are also available, such as those containing ion ated substances in both paper and thin layer
exchange resins. Papers for paper chromatog- chromatography is quite straightforward labora-
raphy can also be made of glass fibers or nylon tory-type equipment, for example, visibleiuitra-
as well as cellulose. violet spectrometers to determine the color
In thin layer chromatography; instead of using density or the UV absorbance of the spots.
paper, a thin layer of an adsorbing substance such Thin layer chromatography is generally found
as silica gel is coated onto a glass or plastic plate. to be more sensitive than paper chromatography.
A very small volume of sample (-30~1) is trans- development of the chromatogram is faster and it
ferred onto one end of the plate, which is then is possible to use a wider range of mobile phases
placed in a closed tank dipping into a solvent, and reagents to detect the position of the spots.
the mobile phase. As the mobile phase moves Uses include the determination of phenols. carci-
along the plate the components of the sample are nogenic polynuclear aromatic hydrocarbons.
separated into a series of spots at different dis- non-ionic detergents, oils, pesticides, amino acids.
tances from the sample starting position. Figure and chlorophylls.
15.1 shows alternative arrangements. The location
of the spots can be identified by their color, or if
colorless by spraying the plate with a reagent that 15.2.2. I High-perfonmiice liquid chronzatographj~
produces a visible color (or UV-detectable absorb- Although liquid chromatography in columns was
ance) with[ the compounds of interest. The position used by Tswett at the beginning of the twentieth
of the spots identifies the compound, the intensity century, an improved, quantitative version of the
of the color, the concentration. technique, high-perfoimance liquid chromatog-
To establish a method for a particular mixture raphy (HPLC), has been fully developed more
of compounds one has to select suitable adsorb- recently. By using precision instruments, deterniin-
ents, solvents. or mixtures of solvents, and a ation of trace organic and inorganic materials at
sensitive and selective reagent for detecting the concentrations of to 10-"g are possible.
separated compounds. There are many textbooks There are also several advantages of HPLC over
which discuss this in detail and give applications other chromatographic techniques. HPLC is more
of the teclhnique. rapid and gives better separations than classical
liquid chromatography. It also gives better repro-
ducibility: resolution, and accuracy than thin layer
chromatography. although the latter is generally
the more sensitive technique. A large variety of
separation methods is available with HPLC:
luant reservoir liquidiliquid; liquidisolid; ion exchange, and exclu-
Samples applied here sion chromatography; but, again, the sensitivity
Paper obtainable is less than with gas chromatography.
Classical column liquid chromatography. in
which the mobile liquid passed by gravity
through the column of stationary phase. was used
up to about 1946-50. In these methods a glass
column was packed with a stationary phase such
as silica gel and the sample added at the top of the
column. Solvent, the mobile phase, was then
added at the top of the column. and this flowed
through under the force of gravity until the sam-
Paper or TLC plate ple components were either separated in the col-
umn or were sequentially eluted from it. In the
latter case components were identified by refract-
ive index or absorption spectroscopy. This type of
elution procedure is slow (taking several hours).
Samples applied here and the identification of the components of the
sample is difficuit and time-consuming.
Eluant reservoir Modern high-performance liquid chroma-
tography equipment has considerably better per-
(b) formance and is available from many chemical
Figure 15.1 Apparatus forpaperor thin-layer measuring instrument manufacturers. The main
chromatography: (a) descending eluant used with paper parts of a general-purpose HPLC apparatus are
chromatography, (b) ascending eluant used with paper
chromatography or TLC. as shown in Figure 15.2.