Environmental Engineering Reference
In-Depth Information
LSC has limited choices of stationary phases, using either silica or alumina, and
is best suited to samples that are soluble in nonpolar solvents (i.e., low solubility in
aqueous solvents). One particular application of LSC is its ability to differentiate
isomeric mixtures.
Most of the liquid chromatography are performed by LLC, which is based on
the partition between liquid mobile phase and a liquid stationary film attached on a
support surface (packing material). The liquid stationary film can be retained on
surface through physical adsorption or by chemical bonding (the bonded phase).
There are two types of bonded phase packing, the normal phase and the reverse
phase. Normal phase HPLC, as the name implies, was considered to be normal
during the early stage of HPLC development, whereas the reverse phase HPLC has
become the most popular. Approximately three quarters of HPLC instruments are
operated in the reverse phase mode.
In normal phase HPLC (NP-HPLC), the stationary phase is highly polar,
whereas the mobile phase is a relatively nonpolar solvent, such as hexane, methylene
chloride, or chloroform. Polar stationary phases for normal-phase chromatography
in increasing order of polarity include cyano [(CH
2
)
3
CN], diol [(CH
2
)
2
]CH
2
(OH)-
CH
2
OH], amino [(CH
2
)
3
NH
2
], and dimethylamino [(CH
2
)
3
N(CH
3
)
2
]. In reverse
phase HPLC (RP-HPLC), the stationary phase is a nonpolar liquid, whereas the
mobile phase is relatively polar solvent such as water, methanol, acetonitrile, or a
mixture of water with one of the organic solvents. The most common nonpolar
bonded phases are hydrocarbons such as n-decyl (C
8
¼C
8
H
17
)orn-octyldecyl
(C
18
¼C
18
H
37
). C
8
is intermediate in hydrophobicity and C
18
is very nonpolar.
One should bear in mind different elution sequences of chemicals between the
two types of liquid chromatography. In normal phase, the polar stationary phase and
nonpolar mobile phase favor the retention of polar compounds so that the least polar
component elutes first. The least polar component is also the most soluble
component in the hydrophobic mobile phase solvent. Increase in the polarity of the
mobile phase will decrease the elution time. By contrast, in the reverse phase mode,
the most polar component elutes first, and increasing mobile phase polarity increases
the elution time.
10.1.3 Other Parameters Important to Compound
Separation
Up to this point, we have learned that an appropriate column with the right chemistry
is essential for the separation of a given set of compounds. In the worst case scenario,
separation of compounds may be impossible when the compounds have the same
affinity with the selected column stationary phase. This is where chemistry can play
a critical role, and understanding the chemistry fundamentals is crucial to the
success of chromatographic analysis.
There are, however, other factors that are also important to separation. Even
though they may not be as critical as the stationary phase, it is likely that separation
can be greatly improved if these parameters are selected at the optimal values. In the
Search WWH ::
Custom Search