33
Figure 7. Changes with selectivity with pressure on a 5 cm C18 column of
Figure 6. Comparison of efficiency losses on columns of different length and i.d. on Acquity classic UHPLC system and Agilent 1100 for peaks of k = 1 to 10.
The instrumental bandspreading of the Acquity system (AQ) was taken as σ2 3.5 µL2
and for the Agilent 1100 system (AG) as σ2 = 25 µL2 Frictional Heating Effects.
Frictional heating is caused by percolation of the mobile phase through the packed bed. The power P generated in watts within the column is given by;
Power = ΔP x F ) and F is the volumetric fl ow rate (m3 (2)
where ΔP is the pressure drop (in SI units N/ m2
/s)
through the column. The heating effect produced can give rise to both axial and radial temperatures gradients in the column. Axial temperature gradients are formed with temperature generally increasing further down the column length. These can lead to changes in solute retention; in reversed- phase separations, a decrease in retention is usually observed. Radial temperature gradients are caused by loss of heat through the column walls causing the centre of the column to be at higher temperature than the wall region. Thus, a spread of velocities across the column radius occurs, that can lead to serious band broadening. Usually, the column is maintained in as adiabatic an environment as possible, to restrict heat losses and minimise the radial temperature gradient, although such an approach tends to maximise the axial gradient. The use of narrower bore columns (e.g. 2.1 mm i.d. rather than 4.6mm) is common as the power generated is less, and the surface area to volume ratio of the column is greater, promoting heat dissipation. Shell particles also promote heat dissipation due to the higher thermal conductivity of the solid core compared with porous materials containing typical HPLC solvents.
. =
5 µm particles. Mobile phase: 25% acetonitrile in phosphate buffer pH 2.7. Peak identities: 1 = uracil, 2 = aniline, 3 = naphthalene-2-sulphonic acid, 4 = propranolol, 5 = prednisone, 6 = acetophenone, 7 = diphenhydramine, 8 = nitrobenzene.
Effect of Pressure and Temperature on Selectivity
Pressure considered in isolation from other effects can cause important selectivity changes. For example, pressure increases from 100-1100 bar can cause increases in retention that range from 8-30 times for insulin (MW ~6 kDa) to myoglobin (30 kDa) in isocratic RP separations [6]. The effect of pressure in the absence of frictional heating can be measured by attaching restriction capillaries of small i.d. to the end of the column while maintaining the fl ow rate constant. The increase in retention with pressure may be caused by the compressibility of solutes which result in them occupying less volume in the stationary phase than in the mobile phase. Large molecules may be more compressible than small molecules, explaining the greater increases in retention that have been noted. Nevertheless, increases in k of 50% for 500 bar pressure increase can occur also for smaller molecules (MW < 500, [7]). Figure 7 shows some pronounced changes in selectivity for a mixture of small neutral molecules (e.g. nitrobenzene and acetophenone) together with some larger polar or ionised compounds (e.g. prednisone and diphenhydramine) on a short (5 cm) ODS column containing 5 µm particles at low pressure (no restriction capillary) and at pressures up to 811 bar achieved by adding restriction capillaries to the end of the column. The use of a short, relatively large particle column ensures that the pressure drop across the column itself
is small, regardless of the total pressure, and thus that frictional heating effects are negligible. While for example nitrobenzene shows relatively little increase in retention with pressure, diphenhydramine shows considerable increases. Thus it elutes just after nitrobenzene at low pressure, but co-elutes with nitrobenzene at the highest pressure used. Other studies have increased the pressure merely by increasing the fl ow rate, which results in a concurrent increase in the column temperature by frictional heating, especially when very small particle columns are used. As increased temperature generally results in reduced retention in reversed-phase chromatography, the effects act in opposite directions, giving some moderation in changes in retention. However, in hydrophilic interaction chromatography, increased pressure can reduce retention, giving pronounced loss of retention when fl ow rate is increased, as both effects operate in the same direction [8].
Monolithic Columns.
This section will concentrate on monolithic columns based on silica. Organic monoliths have found much success for the analysis of large biological molecules, but have not seen much application in the separation of small molecules. Monolithic silica columns were developed in the 1990s and commercialised in 2000. They had the potential to outperform the packed columns of the time. The fi rst commercialised monolithic silica column clad with PEEK,
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