Lecture Notes Wednesday April 11th 2007

Answers to quiz

High performance liquid chromatography is achieved by keeping the mass transfer in the liquid and mobile phases as rapid as possible. This is achieved by making the liquid spacing between the particles of stationary phase as small as possible commensurate with still being able to pump liquid through at a reasonable rate (1 mL/min) and by making the stationary phase a thin liquid coating on the surface of a spherical bead. The most popular form of this is to coat silica beads with a hydrocarbon "fur" consisting of molecules 18 carbons in length. The beads are also porous so that the cracks and fissures in the interior are also coated. This stationary phase is non polar and so the mobile phase is more polar. Typically it consists of a mixture of water, methanol, acetonitrile and buffer components. This combination gives reversed-phase chromatography.

The addition of an ion-pairing agent creates the possibility of the separation of charged species when the stationary phase is non-polar by embedding the non-polar part of the ion-pair agent in the C-18 chains leaving a charged head group exposed to the mobile phase.

Separation can also be based on ion exchange. The stationary phase contains ionizable groups so that either cautions are retained or anions are retained. This form of HPLC is called ion chromatography or IC (it should really be called high performance liquid chromatography of ions).

An HPLC set up will consist of a mobile phase reservoir, a high pressure pump, an injection valve, a column packed with stationary phase, and a detector. The whole apparatus may be under computer control which also collects and processes the data.

For a typical column 25 mm internal diameter and 30 cm long, the empty column has a volume of about 5 mL. If the beads, on which the stationary phase is coated, are 5 micrometers in diameter, then about 40 billion can be packed in under simple cubic packing conditions.

Simple cubic packing is not the most effective way of filling a given volume with spheres, but if the spheres pack as closely as they can (cubic close packing or face centered cubic packing), about half as much again of the available volume can be filled. Thus the number of spheres than can be accommodated in the column is about 60 billion. The volume of one sphere is 6.54 x 10^-8^ cubic mm, so the volume occupied by spheres is 3924 cubic mm or µL. Thus the volume not occupied by spheres is 1 mL to one significant figure.

As we want the separation to take place while the zone of analytes is traveling through the column and that the result will be several separated zones, the volume injected must be substantially less than the 1 mL of mobile phase in the column. Thus 10 µL is a good volume to inject.

If a peak basewidth is 1 min for a flow rate of 1 mL/min, then the volume of mobile phase in which the analyte is eluted is 1 mL. As the injected volume was 10 µL, the dilution is 100 on average across the eluted zone. But if the peak shape is assumed to be an isosceles triangle (a non uniform concentration profile across the zone), then the peak height will correspond to a dilution of 50 times. The peak area must be the same no matter what the concentration profile, and so the triangle will have a height twice that of the rectangle corresponding to a uniform concentration profile across the eluted zone.

Chromatographic separations are thus dilution techniques and this is a consideration when deciding if the detector can measure the analyte after the separation has taken place. To some extent, the choice of injection volume is governed by these issues. The dilution is less with a larger volume, but the separation is worse.

Detectors that are widely used are spectroscopic, often based on UV-vis absorption. Low-cost devices are fixed wavelength, often with a mercury lamp giving a bright emission at 254 nm. More expensive devices are variable wavelength and some (even more expensive) can measure all wavelengths simultaneously (with a diode array) so that the spectrum of an eluting compound can be obtained. This helps with qualitative analysis and in deciding whether there are co-eluting components present. Molecular fluorescence can also be used, as can atomic absorption (not very common) or atomic emission (also not very common as not many emission spectrometers can handle the continuously changing signal). The simplest form of optical detector is the refractive index detector, which although not very sensitive is useful for molecules (such as sugars) that do not absorb radiation. If the analyte is electroactive (i.e. can be oxidized or reduced), then votammetry or amperometry can be used (current is measured). For the separation of ions (ion chromatography), a conductivity detector is used. This is usually preceded by a device that decreases the conductivity of the mobile phase (by exchanging cations for H+ and anions for OH-, so that water is formed), allowing analytes to be detected above the decreased background signal.

Mass spectrometry can also be used. The interface is complicated. For atomic species the ICP can function as an interface. There is also a niche market for hydride generation with atomic fluorescence as a detection mode. HG-AFS would be reserved for As, Se and Hg.