Page 9 - BR20828_E Accucore 0713S

However, in practice the peak capacity is
generally calculated using gradient time and
baseline peak width, and assuming constant
peak width during the gradient run (Equation
8, [7]). The calculated peak capacities for a
mixture of seven phenones (Table 1) are
similar for the fully porous sub-2µm and
solid-core materials and approximately 50%
higher than that on the fully porous 5µm
material, for the same column dimensions,
run under the same conditions. As discussed
above, solid-core materials suffer less
diffusional effects (C- terms of the van
Deemeter equation) and are more
homogeneously packed (A- term of the van
Deemeter equation) than fully porous
materials of similar particle size. Therefore,
peak widths are greatly reduced and peak
capacities increased for this type of column
packing material. The observed high peak
capacity of the fully porous sub-2µm can be
attributed to the small particle size (A- term
of the van Deemter equation). Narrower
chromatographic peak widths have other
advantages, such as improved resolution and
improved sensitivity. In practice, resolution is
calculated by dividing the distance (in
minutes) between peaks by the average
width of those peaks (Equation 9). From
Table 1 it is evident that the fully porous sub-
2µm and the Accucore 2.6µm columns
provide the highest resolution for the closely
eluting compounds in the phenone mixture,
4.39 and 3.87 respectively. When analytes are
eluted from the column in narrow
chromatographic bands, or in other words in
low volume peaks, the sensitivity of the
analysis in increased as the solute mass is
concentrated into a smaller volume. Table 1
also compares the signal-to-noise ratios
(S/N) obtained under the same
chromatographic conditions for 4 columns of
the same dimensions, packed with 5, 3, sub-
2µm fully porous and 2.6 µm solid-core
particles. The highest S/N is observed for the
latter material. This is particularly important
in trace analysis, where a narrow peak is
more likely to “appear” above the baseline
noise.
Equation 9
R s – Resolution between a pair of peaks
t x – retention time of peak x
w x- peak x width at baseline
Loading
Despite the low surface area characteristic of
solid-core materials, the performance of
Accucore is comparable to that of fully
porous materials for the same sample
loading. On Figure 6, the loading on
Accucore is compared with that on a sub-
2µm material. The plot of peak area as a
function of the amount of solute loaded on
the column (Figure 6a) shows a linear
relationship for both the solid-core 2.6µm
and the sub-2µm columns, with a high
correlation coefficient (0.999) for both, which
is indicative of no overload. Monitoring of
peak asymmetry, efficiency and retention
time at the peak apex as the loading on the
solid-core column was increased revealed no
significant change of the normalised values
of asymmetry efficiency and retention time as
a function of load on the column, Figure 6b.
If the columns were mass overloaded there
would be a loss of peak asymmetry and
efficiency and a decrease in the retention
time at the peak apex.
Working with solid-core particle
packed columns
Method transfer from 5 µm fully porous
columns
There are several reasons for scaling down a
method from a conventional 4.6mm ID
column packed with fully porous 5 or 3µm
particles to short, narrow-bore columns
packed with fully porous sub-2µm or solid-
core particles. As discussed above, fully
porous sub-2µm and solid-core particles
facilitate improvements in resolving power,
sensitivity and peak capacity. Furthermore,
reducing the column internal diameter also
facilitates sensitivity improvements and
shorter columns can often deliver the
required resolution. Figure 3 demonstrated
that columns packed with fully porous sub-
2µm or solid-core particles are run at high
linear velocities to achieve their optimal
performance compared to equivalent
columns packed with larger particles,
therefore providing faster run times and
increased sample throughput. The faster
separations reduce the quantity of mobile
phase per run compared with separations of
the same efficiency with longer columns of
larger particles. This has cost implications in
terms of solvent consumption and also waste
disposal and therefore significant savings can
be achieved by scaling down methods.
When transferring methods to fast LC,
several approaches can be taken, depending
on the analytical needs. If column
dimensions are maintained and only particle
size is reduced then an improvement in
efficiency and, therefore, resolution,
sensitivity and peak capacity is obtained. A
second, more common approach is to
reduce not only particle size but also column
dimensions, which has the benefit of
reducing analysis time.
In Figure 7, a gradient method run on a fully
porous 5µm, 150 x 4.6mm ID column is
Table 1. Comparison of the peak capacity, resolution of a critical pair, signal-to-noise ratio and column
backpressure for a mixture of phenones on fully porous 5, 3, <2µ m and solid-core 2.6µ m particle packed
100x2.1mm columns.
Figure 7. Example of method transfer from fully porous 5µm 150x4.6mm column to Accucore 2.6µm, 100x2.1
ID mm column (similar column chemistry)
5µm 3µm <2µm Accucore 2.6µm
Resolution critical pair 2.57 3.26 4.39 3.87
Peak capacity 32 43 51 51
Signal-to-noise ratio 122 152 211 228
Pressure (bar) 31 67 268 133
Rs =
2 (t2 -t1)
(w1-w2)