Environmental Engineering Reference
In-Depth Information
discussions below, separation is referred to as the capacity, speed of analysis, and the
resolution. We will define these concepts in Section 10.1.3, but let us first examine
how qualitatively these factors affect separation. Both column-related parameters
(column length, internal diameter (i.d.), stationary film thickness, particle size of
column packing materials) and operational parameters are included. The latter
includes the flow velocity of mobile phase, and more importantly, the control of
temperature for GC and selection of mobile phase for HPLC. A summary of such
effects is listed in Table 10.2. The reader should be aware that exceptions to these
generations do exist as a result of a variety of chromatographic conditions.
Table 10.2 Major factors affecting separation in column chromatography
Effects on separation if
Capacity
Speed
Resolution
Optimal stationary phase
Optimal
Optimal
Optimal
a
Increase in column length
þ
þ
Increase in column internal diameter (i.d.)
þ
þ
d
Increase in stationary phase film thickness
þ
b
Increase in particle size
þ
? c
e
Increase in mobile phase flow velocity
þ
Increase in column temperature (GC)
?
þ
Increase in solvent polarity (RP-HPLC)
?
þ
a Increase; b Decrease; c Minor or negligible effect; d Increase or decrease depending on whether the
compound(s) are the low or high boiling points in GC; e Increase or decrease depending on whether the
flow velocity is below or above the optimal flow velocity.
From Table 10.2, the capacity, analytical speed, and resolution respond to
various column parameters differently, and oftentimes a compromise has to be made
with regard to the optimal conditions. Sample capacity can be increased by the use of
a longer or wider column with a thicker film of stationary phase, but doing so will
increase the analytical time. Resolution is proportional to the square root of column
length and the square of column internal diameter, so the resolution can be improved
with the use of a longer column or a column with a larger diameter. The use of longer
column in HPLC, however, may be limited by the pressure constrain. The effect of
film thickness on separation is dependent upon the type of chemicals. For example,
thin-film columns offer a higher resolution for high-boiling point chemicals but a
lower resolution for more volatile components in GC (Grob and Barry, 2004).
The effect of mobile phase flow velocity on separation is not straightforward. A
higher flow velocity will certainly reduce the analytical time, but its effect on
resolution depends on the range of flow velocity. A quantitative examination on how
flow affects chemical diffusion in various types of columns is beyond the scope of
this text. The interested reader should consult the references listed in this chapter for
a full account of the van Deemter equation, originally developed for packed columns
and the Golay equation for capillary columns. With these equations, an optimal flow
velocity can be justified. Understanding these quantitative mathematical expressions
is helpful for the chromatographer to effectively manipulate the chromatography
 
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