Nucleic Acid Analysis with UV-vis and NMR - Spectroscopy

32 Spectroscopy 24(11) November 2009 www.spectroscopyonline.com
HETP (µm)
60
40
20
0
0 2 4 6 8
Flow Rate (mL/min)
liquid chromatography (HPLC) flow
rate for achieving the best separation
can vary from 0.7 mL/min to 2 mL/
min, as shown in the Van Deemter
plot in Figure 3. In this figure, the
HETP label on the y-axis refers to the
height equivalent to a theoretical plate
— the lower the HETP, the better
the separation. As shown in the Van
Deemter plot, the best separations are
achieved with 3-µm packings and a
flow rate of 1.5–2 mL/min. ICP-MS
can handle 1.5-mL/min sample introduction
rates without a problem, so
the best separations are achieved with
a 3-µm packing and an LC flow rate
of 1.5 mL/min. These conditions are
well suited for standard-bore
columns.
If a 3-µm packing is used in a narrow-bore
column, a flow rate of 1.5
mL/min will result in a very high back
pressure, typically too high to be run
safely. This is the result of the smaller
internal diameter of the column. As a
result, narrow-bore columns usually
are run with LC flow rates of 0.2–0.5
mL/min. As seen in the Van Deemter
plot, these low flow rates do not provide
the best separation, which means
that using a narrow-bore column may
involve sacrificing some separation
capability. This compromise may
or may not be a problem, depending
upon the spacing between peaks.
However, this usually can be
10m
5m
3m
Figure 3: Van Deemter plots for various LC stationary phase particle diameters.
compensated for by changing the
mobile phase conditions.
Narrow-bore columns offer two advantages
compared with standard-bore
columns: sharper peaks and less reagent
consumption. The sharper peaks
are a direct result of the narrower column
width — there is less radial dispersion,
which leads to sharper, more
intense peaks than those obtained
using standard-bore columns. The increased
peak sharpness may compensate
for the loss of resolution resulting
from the nonoptimal flow rates. The
increased peak height may also mean
that lower levels of the species can be
seen.
A main drawback of narrow-bore
columns versus standard-bore columns
is that their capacity is reduced.
Because fewer stationary phase particles
can physically fit into the narrower
column, there is less surface area available
for species to bind. As a result,
these columns can become overloaded
easily, which could be a problem when
analyzing unknowns.
Typically, this reduced capacity
is compensated for by diluting the
samples more or using smaller injection
volumes. Because less sample is
injected onto the column, this may
offset the advantage of increased peak
height. Therefore, it is not always possible
to measure lower levels with a
narrow-bore column.
Another potential disadvantage of
narrow-bore versus standard-bore columns
is sample throughput. Because
lower LC flow rates are used for narrow-bore
columns, separations tend to
take longer than with standard-bore
columns, which will affect sample
throughput. The advantage of a lower
LC flow rate is that there is less consumption
of reagents and less waste to
dispose.
When deciding whether to use a
standard-bore or narrow-bore column,
users must consider which combination
of factors is best for a particular
laboratory and application. There is no
single correct answer — each situation
is different.
HPLC Versus UHPLC for
Speciation
Within the past several years, a new
form of LC has emerged: ultrahighpressure
liquid chromatography
(UHPLC). The Van Deemter plot
shows that the smaller the particle size
in the column, the better the separation.
UHPLC uses columns with
particle sizes less than 3 µm, typically
1.5–2.5 µm. These smaller particles
provide more surface area for mobile
phase and species to interact, which
leads to better separation. Or, another
way to think of it is that shorter
columns with smaller packings can
provide the same separation as longer
columns with larger packings. The advantage
of the shorter columns is that
the time for separations are reduced
greatly.
The other signifcance of the smaller
packings is a much higher back pressure,
typically more than 10,000 psi.
This increased pressure requires a special
HPLC pump capable of handling
it. Normal HPLC pumps cannot be
used with columns containing packing
material with a diameter of