Chemistry Reference
In-Depth Information
technology are available from a number of commercial vendors. A few easy steps
using equations that geometrically scale the original method to the new column
packed with sub-2-µm particles using exactly the same mobile phase composition
are necessary to achieve equivalent results. These equations take into account the
changes in the gradient time (unless using isocratic conditions), flow rate, and injec-
tion volume.
The gradient is scaled using
L
2
/L
1
× t
g1
= t
g2
where L
1
and L
2
are the lengths of the original and new columns, and t
g1
and t
g2
are
the times of each gradient step, respectively.
The flow rate is scaled taking into account the difference in the diameter of the
two columns:
(d
2
)
2
/(d
1
)
2
× F
1
= F
2
where d
2
and d
1
are the column diameters and F
1
and F
2
the flow rates.
To keep the column volumes proportional, the gradient steps should be readjusted
for the new flow rate:
(F
2
× t
g2
)/F
3
= t
g3
where F
2
and t
g2
are the flow rate and gradient time of the geometrically scaled values
and F
3
and t
g3
are the optimized values. (F
3
is usually increased above that calculated
for F
2
[0.5 mL/min in the example below], to better approximate the optimum linear
velocity for a sub-2-µm particle.)
The injection volume is scaled taking into account the volumes of the two columns:
V
1
× [(r
2
2
× L
2
)/(r
1
2
× L
1
)] = V
2
where r
2
2
and r
1
2
are the radii of the columns, L
1
and L
2
are the lengths of the col-
umns, and V
1
and V
2
are the injection volumes, respectively.
Laboratories might be interested in implementing UHPLC to save time and
expense compared to existing standard methods. Figure 5.6a shows an HPLC sepa-
ration of a series of related caffeic acid derivatives from
Echinacea purpurea
, a natu-
ral product. When column reequilibration is taken into account, the run time exceeds
40 min. When properly scaled for injection volume, flow rate, and gradient time, the
separation illustrated in Figure 5.6b is obtained. The run time is complete in less
than 6 min, including reequilibration, increasing throughput fold approximately 7
´
,
while using about a factor of 10XX less solvent. Proper scaling results in a new sepa-
ration that is accomplished without changing the look of the original separation; if it
was not for the time scale in Figure 5.6, it would be difficult to distinguish between
the two separations.
