Page 228 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

P1: ZCK/MBQ P2: GQT Final Pages
Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

Liquid Chromatography 695

silica, Si O Si(CH 2 ) 3 OCH 2 CH (OH)CH 2 (OH), is
preferred for the separation of proteins. The selection of
an SEC packing is dependent on the range of components
with different molecular weights desired to be separated.
Calibration curves of polystyrene standards versus
retention volume for packings of different pore sizes are
˚
well documented. For 60– A silica, a linear MW range of
˚
4
2
10 to 10 is possible. For a 750- A silica, a linear MW
6
4
range of 10 to 10 is found. To expand the range of
molecular weights that can be separated, it is necessary
to connect in series columns of two different pore-sized
packings. For the previous example, a linear fractionation
6
2
range from 10 to 10 MW would be possible.
Unlike all other modes of LC separation, the mobile
phase is not chosen to control peak separation but to
ensure sample solubility and minimize solute-stationary
phase adsorption effects. Therefore, the sample solvent
determines whether the mobile phase is predominantly
organic or aqueous. In general, to minimize adsorption
effects, a mobile phase that is more strongly adsorbed
to the packing than the solute is advised. For example,
for the separation of polyurethanes on silica, dimethyl- FIGURE 24 Separation of protein standards by SEC. Col-
umn is Micropak TSK 3000 SW, 7.5 × 300 mm. Mobile phase
formamide is preferred over tetrahydrofuran. The ionic
is 0.1 M potassium phosphate and 0.1 M potassium chlo-
strength should generally be greater than 0.05 M when
ride, pH 6.8. Flow rate = 0.7 ml/min. (1) Glutamate dehydroge-
using aqueous mobile phases with silica. One important nase (290,000); (2) lactate dehydrogenase (140,000); (3) eno-
application for SEC is for the initial exploratory separa- lase (67,000); (4) adenylate kinase (32,000); (5) cytochrome c
tion of an unknown sample to indicate how complicated (12,400). [From Hearn, T. W., Regnier, F. E., and Wehr, C. T.
(1983). American Clinical Products Review, May/June. Reprinted
it might be. For example, SEC could easily separate a bi-
with permission by International Scientific Communications, Inc.]
ological sample into high-molecular-weight proteins and
low-molecular-weight peptides and amino acids. These
peaks could be collected for further HPLC study using is better suited to the separation of nonvolatile polar con-
ion exchange or reversed phase. A typical example of an stituents in less complex aqueous samples. Knowledge or
SEC protein separation is shown in Fig. 24. Another major experimental determination of the molecular weight, sol-
use of SEC is for the separation of polymeric oligomers ubility, and ionic nature of the sample is necessary before
such as polystyrene. In general, the peaks of an SEC chro- the appropriate LC method can be selected. Such, a guide
matogram are quite broad and resolution is only modest. to the selection of an HPLC mode is given in Fig. 25.
However, the important role that SEC can play in an over- Most separations of a modest number of components can
all HPLC separation scheme will be elaborated in the next be accomplished using a mobile phase of constant compo-
section. sition. Isocratic elution as compared to a gradient permits
better reproducibility and a higher sample throughput as
well as the use of simpler equipment. Two primary prac-
tical considerations when choosing solvents for a mobile
IV. DEVELOPMENT OF
SEPARATION STRATEGY phase are reactivity and purity. The mobile phase should
not react with itself or with the sample. Halide salts as
mobile-phase additives should be avoided since they can
A. Choosing a Separation Technique
corrode stainless steel tubing and fittings. Impure solvents
Knowledge of the physical and chemical properties of due to their absorbance at sensitive detector settings can
a sample is imperative for selecting the preferred sepa- prevent quantitation of trace solutes. Since large volumes
ration method or any analytical method for that matter. of solvent are pumped through the column, trace impu-
HPLC can be considered complementary, not competi- rities can also concentrate on the column, impairing its
tive, to GC. If the components of a very complex sample performance. Spectrograde or HPLC grade solvents are
can be volatilized (i.e., gasoline), use of capillary GC with highly recommended. In addition, the UV cutoff and vis-
MS detection would often be the method of choice. HPLC cosity of solvents should be considered (see Table III).