Supercritical Fluid Chromatography Introduction:

Supercritical Fluid Chromatography (SFC) is a hybrid of gas and liquid chromatography that combines some of the best features of both

Supercritical Fluid exist at temperatures and pressures above its critical temperature and pressure and have densities, viscosities and other properties that are intermediates between those of the substance in its gaseous and liquid state.

Gas (STP) Supercritical Fluid Liquid

Density (g/cm3) (0.6-2) x 10-3 0.2-0.5 0.6-2

Diffusion coefficient (cm2/s) (1-4)x10-1 10-3-10-4 (0.2-2)x10-5

Viscosity (g cm-1 s-1) (1-3)x10-4 (1-3)x10-4 (0.2-3)x10-2

Critical temperature temperature above which a distinct liquid phase does not exist regardless of pressure

Important Properties of Supercritical fluids – remarkable ability to dissolve large, non-volatile molecules

‚ e.g., supercritical CO2 can dissolve n-alkanes containing over 30 carbon atoms

‚ related to their high densities– dissolved analytes are easily recovered

‚ equilibrate with atmosphere at relatively low temperatures ‚ e.g., analyte in supercritical CO2 can be recovered by reducing the pressure

and allowing the CO2 to evaporate

– no need for organic solvents ‚ environmentally friendly

– inexpensive, innocuous and non-toxic – higher diffusion coefficients and lower viscosities

relative to liquids ‚ faster and higher resolution separations

Advantages of SFC compared to LC and GC:- SFC can separate compounds that are not conveniently handled by GC or LC.

‚non-volatile or thermally labile and

‚ contain no functional group that makes possible detection in LC using spectroscopic or electrochemical techniques

up to 25% of all separation problems fall into this categoryexamples include: polymers, fossil fuels, pesticides, foods, drugs, etc

- Separations are faster then LC - Run at lower temperature than GC - Beneficial in industrial scale purification

Faster elution

Reduction in peak width (3)

Reduction in elution time (4x)

Instrumentation:

Instrumentation for SFC is very similar to ordinary HPLC equipment since the temperature & pressure requirements for supercritical fluids fall within the standard operation

Two Major Differences – thermostated column oven

‚ requires precise temperature control of mobile phase (typically supercritical CO2)

– restrictor or back-pressure device‚ required to maintain desired pressure in column ‚pressure change to convert from supercritical fluid to a gas for transfer to

detector

Effects of Pressure:

Pressure increases results in reduced elution time - increase in density of mobile phase- effects retention or capacity factor (k’)- pressure changes analogous to gradient elution in LC and GC

Example 15: What properties of a supercritical fluid are important in chromatography?

Introduction:

Electrophoresis is a technique in which solutes are separated by their different rates of travel through an electric field.

- commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids

Similar to chromatography, both involve separation by different rates of travel through

the system, but:electrophoresis different rates of travel are produced by an electric fieldchromatography different rates of travel due to chemical interactions

between the solutes and a stationary or mobile phase

Electrophoresis

Theory:

The migration rate of solutes in an electric field will depend on the size and charge of the solute, as well as other factors

Retention

The degree of retention, or rate of travel, of a solute in an electrophoretic system is given by:

v = E

where: E = electric field strengthv = Velocity of the solute in the electric field = Electrophoretic mobility of the solute

The value of is a constant for a given solute under a particular set of experimental

conditions and depends on the following: ‚ionic charge on the analyte ‚ inversely proportional to frictional retarding factors:

- size of analyte- shape of analyte- viscosity of medium

Note: charge-to-size ratio of analyte is an important factor in its mobility small/high charged molecules migrate faster; large/low charged molecules migrate slower

Efficiency:

Electrophoresis may be performed in an open-tubular system (capillary) or in a packed-bed (slab)

Packed-bed (slab) system Capillary system

Joule Heating: – uneven heating in the system by the electric field

‚causes different points in the system to have different temperatures ‚ non-uniform mixing of solute and solvent

– gives rise to peak broadening in electrophoresis– decrease band-broadening caused by Joule heating:

‚ use of packed-bed systems (or stabilizers) prevents mixing of solvent from different regions of the system ‚use of more efficient cooling prevents the formation of thermal gradients

Zone Electrophoresis:Involves applying narrow bands of sample to the electrophoretic system

- common type of electrophoresis- buffer composition is constant throughout system- solutes can be totally separated in a reasonable amount of time into separate

“zones” based on mobility- zones are separated by regions of buffer

Packed-bed (slab): – much work in zone electrophoresis uses packed-bed instead of open tubular supports

‚avoids band-broadening from Joule heating and unstable density gradients – packed beds “stabilize” solvent in the system

‚ prevents mixing between different regions – common supports:

‚ cellulose acetate, paper, agarose and polyacrylamide gels – band-broadening caused by diffusion around particles of the packed bed – porous matrix may also result in molecules being separated according to size in addition to electrophoretic mobility SEC

Capillary electrophoresis (CE): – another type of zone electrophoresis– use of narrow open tubular supports or capillaries

‚ typically 25-75 m I.D. – narrow bore capillaries allows efficient removal of Joule heat from the system

‚ decrease band-broadening‚ allows stronger electric fields to be used

faster migration of solutes decreasing analysis time – easy to automate – very small sample sizes (nl) – no particles no multiple paths – many types of detectors can be used in capillary electrophoresis

‚ Fluorescence or UV/Vis absorbance are commonly used

CE separation of DNA mixtures

Because of the extreme efficiency and narrow peaks produced by CE, it is an area of intense research

- especially in areas of biotechnology and biochemical separations- very small sample requirements:

- useful in separating and studying the ionic solutes present in single cells- screening of large chemical libraries for drug discovery- genome sequencing

Electroosmotic flow – all solutes, regardless of charge, will migrate towards the negative electrode of the system

- feature of capillary electrophoresis- produced by the presence of negative charge on the surface of the capillary

‚ negative charge is produced by ionization of silanol groups on the capillary’s surface ‚ occurs at pH > 4-5 ‚net positive charge must exist in solution to counter negative charge of capillary’s surface ‚ net positive charge of solution causes net migration toward the negative electrode

- ALL solutes eventually pass detector, regardless of charge

Electroosmotic flow – all solutes, regardless of charge, will migrate towards the negative electrode of the system

- flow profile is flat no peak broadening ‚ pressure-driven flow in LC causes parabolic profile caused by boundary effects and inertia

Direction of

Electroosmotic flow

Direction & velocity due to potential Overall direction & velocity(electrophoretic) (electrophoretic) + (electroosmotic)

Anode (+) Cathode (-)

Isoelectric focusing – pH affects charge which affects migration

HA H+ + A- Ka = [H+][A-]/[HA]

For a protein composed of amino acids

H3+N

O

OH

H2N

O

O-

H3+N

O

O-

Low pH (acidic) High pH (basic)pI

isoelectric point (pI) - pH at which net charge on molecule is zero

If plate has pH gradient, protein migrates until local pH = pI

Example 16: Three large proteins are ionized at the pH at which a capillary electrophoresis

separation is carried out. If the ions are designated A2+, B+ and C3+, predict the order of the elution?