HPLC Analysis of Biomolecules Technical Guide - Interscience

8
HPLC Analysis of Biomolecules
Column Length
Retention is generally proportional to column length in reversed phase
HPLC. However, protein separations tend to be an exception to this
rule. Figure 4 compares 150 mm and 50 mm C8 columns for the
separation of proteins. When the same 30 minute gradient program
is used for both columns, the retention of the last peak, ovalbumin,
is only slightly shorter for the 50 mm column. An explanation for
this result is that reversed phase gradient protein separations occur
primarily by an adsorption/desorption type mechanism, rather than
the typical partitioning mechanism. The adsorption and desorption
of proteins occurs at the head of the column with the proteins
interacting very little with the majority of the column length that
they encounter after desorption. If shorter columns are used with the
goal of reducing analysis time, it is necessary to reduce the gradient
length in proportion to column length, as shown in Figure 5.
Figure 4: Effect of column length on RP-HPLC separation of proteins
Figure 5: Column length comparison with gradient proportional to
column length
Flow Rate
Increasing flow rate is the easiest way to decrease the analysis time.
However, flow rate also affects column efficiency, which in turn affects
resolution and pressure. Figure 6 demonstrates the relationship
between column efficiency and flow rate. Note that the flow rate for
optimum efficiency is dependent on the particle size of the column
packing material. The optimum flow rate for a 3 µm column is
approximately 1.5 times greater than that of a 5 µm column of the
same I.D. This means a 3 µm column will not only provide more
efficiency, but can do so in less time than the same length of a 5 µm
column. The graph in Figure 6 could be generalized to columns of
any I.D. by converting the values on the x-axis from flow rates to
linear velocities. Linear velocity (cm/min) is the rate at which mobile
phase moves through the column and can be estimated by dividing
column length by the retention time of an unretained peak.
The main disadvantage of increasing flow rate is higher pressure.
Elevated pressures will shorten column lifetime as well as increase
wear on the HPLC system. Increasing flow rate above the optimum
will also reduce column efficiency and resolution. Decreasing the
flow rate reduces system wear. However, it also increases analysis
time. If the flow rate is reduced below optimum for the particle
size, it can reduce efficiency and resolution due to increased
band dispersion.
Figure 6: Effect of flow rate on RP-HPLC column efficiency
For isocratic separations, the relationship between flow rate
and retention time is generally a straightforward inverse correlation;
doubling the flow rate will reduce retention by one half. However,
this relationship does not hold for gradient separations of proteins
and peptides. As mentioned earlier, reversed phase gradient protein
separations occur primarily by an adsorption/desorption type mechanism,
rather than the classical partitioning mechanism. Because of
the adsorption/desorption mechanism, flow rate has less effect on
retention than might be expected.
Figure 7 shows a fast protein separation performed at flow
rates of 0.5, 1.0 and 1.5 mL/min with other conditions held constant.
Note that increasing flow rate threefold decreases retention by only
about a third because the rate of gradient formation determines rate
of protein migration. The benefits of reducing flow rate are decreased
pressure and lowered consumption of mobile phase. Figure 7 also
shows that flow rate can affect selectivity (elution order) possibly
due to the different partitioning rates.