HPLC Analysis of Biomolecules Technical Guide - Interscience

24
HPLC Analysis of Biomolecules
The anion exchange process may be represented by the
equilibrium [3]:
[3] ~NH + C - + X - = ~NH + X - + C -
where C - is the counter ion to the fixed ion exchange site, ~NH + ,
present in the eluent. Analyte X - can then displace C - to give the ion
pair NH + X - . For effective chromatography, this displacement reaction
must be at equilibrium. Retention is therefore controlled by the
equilibrium [3] for which the equilibrium constant K IE is given by
equation [4]:
[4]
K IE = [C- ] [~NH + X - ]
[X - ] [~NH + C - ]
The capacity factor, k, of an analyte (X - ) is a more useful
chromatographic parameter and is proportional to the distribution
coefficient, D IE . Its relationship to the equilibrium constant, K IE , is
given by equation [5]:
[5]
K ∝ D IE = [~NH+ X - ] = KIE • [~NH+ C - ]
[X - ] [C - ]
Since the concentration of the ion exchange sites, [~NH + X - ], is
constant and fixed by the rigid structure of the matrix, k is inversely
proportional to the concentration of the counter ion (ionic strength)
in the eluent: Desorption is then brought about by increasing the
salt concentration as shown by equation [6].
[6] K ∝ [C - ] -1
Particles for Ion Exchange
Both polymer- and silica-based particles are available for ion
exchange separations of biomolecules. Polymer-based particles
have the advantage of extended pH range (chemical stability), while
silica-based particles have higher efficiency and better mechanical
strength. However, there are hybrid particles that offer the benefits
of both. An example is the BioBasic ion exchangers that comprise
high purity silica with a polymeric coating. The polymeric coating
extends the usable pH range, and also shields proteins from adsorbing
to the silica surface.
Particle Size, Pore Size, and Column Length
The effects of these variables in ion exchange are largely the same
as for RP-HPLC, discussed previously. Efficiency (resolution) increases
with decreasing particle size and increasing column length, but at
the expense of higher back pressure for smaller particles and longer
columns. Longer columns also increase analysis time. Pore size
should be larger, 300Å or greater, for proteins and large peptides
to ensure access to the interior of the pores where the majority of
the surface area resides.
Flow Rate
High column efficiency (number of theoretical plates) is an important
factor to consider when maximizing the resolution of a separation.
In ion exchange chromatography the efficiency (sharpness) of a peak
is governed not only by how well the column is packed but also by
the mass transfer characteristics of the ion exchange mechanism
itself. The term mass transfer used here refers to the efficient
migration of the analyte from the mobile phase on to the stationary
phase ion exchange site and then back into the mobile phase from
the ion exchange site. If this process is not very rapid, broad peaks
can result with a possible loss in analyte resolution. Figure 41 illustrates
how the column efficiency increases as the flow rate is
reduced. Depending on the complexity of the analysis, a tradeoff is
required between time and efficiency.
Figure 41: Effect of flow rate on efficiency in ion exchange
Temperature
The mass transfer component of the Van Deemter equation can
often be improved through temperature control. The mass transfer
can be increased by increasing the temperature at which the separation
takes place. The way in which column temperature is
achieved is also of significant importance.
Figure 42 shows the effect that increasing column temperature
has on efficiency. It is important to note the role of preheating the
mobile phase on the separation. For example, heating only the
column itself and not heating the solvent supplied to it can actually
increase band spreading and reduce column efficiency as shown
in Figure 42. Heating the solvent prior to its arrival at the column
prevents this band spreading and improves the mass transfer associated
with the ion exchange mechanism. The result is an increase
in the column efficiency for any given flow rate.
Figure 42: Effect of T on efficiency in ion exchange