Chapter 34

Miscellaneous Separation Methods

34 A Supercritical fluid separations

Supercritical fluids are an important class of solvents that have unique solvating properties which are useful in chromatography and solvent extraction.

In supercritical fluid chromatography (SFC), the supercritical fluid acts as the mobile phase.

Properties of Supercritical Fluids

A supercritical fluid is formed whenever a substance is heated above its critical temperature.

The critical temperature is the temperature above which a substance cannot be liquified.

The density of a supercritical fluid is 200 to 400 times that of its gaseous state, and it is nearly as dense as its liquid state.

Supercritical fluids are able to dissolve large nonvolatile molecules.

Instrumentation and Operating variables

Instruments for supercritical fluid chromatography are similar in design to high-performance liquid chromatographs.

In addition, the pumping system must include:1. a chilled pump head to keep the fluid in the liquid state 2. a provision for controlling and measuring the column pressure.

Effects of Pressure

The density of a supercritical fluid increases rapidly and nonlinearly with pressure increases.

Density increases also change retention factors (k) and thus elution times.

Gradient elution can be achieved in SFC by systematically changing the column pressure or the density of the supercritical fluid.

Columns

Both packed columns and open tubular columns are used in supercritical fluid chromatography.

Very long columns can be used in SFC because the viscosity of supercritical fluids is so low.

Mobile Phases

The most widely used mobile phase is carbon dioxide.

It is an excellent solvent for a variety of nonpolar organic molecules, transmits in the ultraviolet, is odorless, nontoxic, widely available, and inexpensive.

Its critical temperature of 31°C and its pressure of 73 atm at the critical temperature permit a wide selection of temperatures and pressures without exceeding the operating limits of modern HPLC equipment.

Detectors

Sensitive and universal detectors of gas chromatography are applicable to this technique as well.

UV-visible absorption and light-scattering detectors are also used.

Supercritical Fluid Chromatography versus Other Column Methods

34 B Planar chromatography

Planar chromatographic methods include thin-layer chromatography (TLC), paper chromatography (PC), and electrochromatography.

This method makes use of a flat, relatively thin layer of material that is either self-supporting or is coated on a glass, plastic, or metal surface.

The mobile phase moves through the stationary phase by capillary action.

Thin-layer chromatography (TLC) can be considered a form of liquid-solid chromatography.

TLC has found widespread use in clinical laboratories and many biochemical and biological studies as well as industrial laboratories.

Principles of Thin-Layer Chromatography

A thin-layer plate is prepared by spreading an aqueous slurry of the finely ground solid onto the clean surface of a glass or plastic plate or microscope slide.

The plate is then allowed to stand until the layer has set.

High-performance plates usually have film thicknesses of 100 m and particle diameters of 5 m or less.

The sample is applied as a spot 1 to 2 cm from the edge of the plate by touching a capillary tube containing the sample to the plate or by use of a syringe.

Plate development is the process by which a sample is carried through the stationary phase by a mobile phase.

The plate is placed in a closed container saturated with vapors of the developing solvent.

Locating Analytes on the Plate

The process of locating analytes on a thin-layer plate is often termed visualization.

Several methods are used to locate sample components after separation.

Two common methods that can be applied to most organic mixtures involve spraying with a solution of iodine or sulfuric acid.

Both of these reagents react with organic compounds to yield dark products.

Paper Chromatography

Paper chromatography is performed in the same way as those on thin-layer plates.

The papers are manufactured from highly purified cellulose with specific porosity and thickness and sufficient adsorbed water to make the stationary phase aqueous.

Paper treated with silicone or paraffin oil permits reversed-phase paper chromatog-raphy in which the mobile phase is a polar solvent.

34 C Capillary electrophoresis

Electrophoresis is a separation method based on the differential rates of migration of charged species in an applied dc electric field.

It was first developed by the Swedish chemist Arne Tiselius in the 1930s for the study of serum proteins.

It has the unique ability to separate charged macromolecules such as proteins (enzymes, hormones, and antibodies) and nucleic acids (DNA and RNA).

Electrophoretic separations are performed in a flat stabilized medium such as paper or a porous semisolid gel.

A unique feature of capillary electrophoresis is electro-osmotic flow.

When a high voltage is applied across a fused-silica capillary tube containing a buffer solution, electro-osmotic flow usually occurs in which the solvent migrates toward the cathode.

Electro osmosis leads to bulk solution flow that has a flat profile across the tube because flow originates at the walls of the tubing.

This profile is essentially flat, hence, electro osmotic flow does not contribute significantly to band broadening the way pressure-driven flow does in liquid chromatography.

Although analytes migrate according to their charges within the capillary, the electroosmotic flow rate is usually sufficient to move all positive, neutral, and even negative species toward the same end of the capillary.

The Basis for Electrophoretic Separations

The migration rate v of an ion in an electric field is given by

where E is the electric field strength in volts per centimeter, V is the applied voltage, L is the length of the tube between electrodes, and e is the electrophoretic mobility.

Electrophoretic mobility is proportional to the charge on the ion and inversely proportional to the frictional retarding force on the ion.

In electrophoresis, plate count N can be calculated as:

where D is the diffusion coefficient of the solute (cm2/s).

L

VEv ee

D

VN e

2

Applications of Capillary Electrophoresis

Capillary electrophoretic separations are performed in several ways called modes.

These modes include isoelectric focusing, isotachophoresis, and capillary zone electrophoresis (CZE).

Applications include:

1.Separation of Small Ions2.Separation of Molecular Species

34 D Capillary electrochromatography

Capillary electrochromatography (CEC) is a hybrid of HPLC and capillary electrophoresis (CE).

Like HPLC, it is applicable to the separation of neutral species.

Like CE, it provides highly efficient separations on microvolumes of sample solution.

In CEC, a mobile phase is transported across a stationary phase by electroosmotic flow.

Packed Column Electrochromatography

In this method, a polar solvent is usually driven by electroosmotic flow through a capillary that is packed with a reversed-phase HPLC packing.

Separations depend on the distribution of the analyte species between the mobile phase and the liquid stationary phase held on the packing.

Micellar Electrokinetic Capillary Chromatography

In this technique, a surfactant is introduced at a concentration level at which micelles form.

Micelles form in aqueous solutions when the concentration of an ionic species having a long-chain hydrocarbon tail is increased above a certain level called the critical micelle concentration (CMC).

At this point, the surfactant begins to form spherical aggregates.

Micelles constitute a stable second phase that can incorporate nonpolar compounds in the hydrocarbon interior of the particles, thus solubilizing the nonpolar species.

Capillary electrophoresis carried out in the presence of micelles is termed micellar electrokinetic capillary chromatography (MEKC).

In this technique, surfactants (usually sodium dodecyl sulfate) are added to the operating buffer in amounts that exceed the critical micelle concentration.

34 E Field-flow fractionation

Separations in FFF occur in a thin ribbon-like flow channel.

In the presence of the field, sample components migrate toward the accumulation wall at a velocity determined by the strength of the interaction of the component with the field.

The separation results are revealed by a plot of detector response versus time, called a fractogram, which is similar to a chromatogram.

FFF Methods

Different FFF subtechniques result from the application of different types of fields or gradients.

The methods that have been used are sedimentation FFF, electrical FFF, thermal FFF, and flow FFF.

Advantages of FFF over Chromatographic Methods

1.No packing material or stationary phase is needed for separation to occur.

2. The geometry and flow profiles of FFF are well characterized.

The effects of most external fields can be readily modeled, thus giving fairly exact theoretical predictions of retention and plate height.

3. The external field governs FFF retention. With electrical, centrifugal and flow FFF, the perpendicular forces can be varied rapidly and in a time-programmed fashion making it versatile in adapting to different types of samples.

4. FFF methods are best suited at present for macromolecules and particles that are for the most part beyond the molecular mass range of chromatographic methods.

On the other hand, chromatographic methods are superior for low-molecular-mass substances.