CHROMATOGRAPHY

The general name given to methods by which two or more compounds in a mixture are physically separated by distributing themselves between two phases: - a stationary phase (solid or liquid supported on a solid) and - a mobile phase (gas or liquid) The mobile phase continuously flows around the stationary phase. WHAT IS CHROMATOGRAPHY? Separation results from differences in their affinity for the stationary phase.

LIQUID CHROMATOGRAPHY Small volume of sample introduced to one side of the column. The mobile solvent phase moves the sample through the column packing. The individual components undergo adsorption and desorption on the packing  slowing their motion by different amounts depending on their affinity for the packing Each component is distributed between the stationary phase (s) and the mobile phase (m) as it passes down the column according to: X m  X s The distribution coefficient of component X: Large K x  X favours the stationary phase and will move slower through the column

Separation of Mixtures by Paper Chromatography Stationary phase Mobile phase

Separation of Mixtures by Column Chromatography Stationary phase Mobile phase Component with greater affinity for stationary phase Can collect the different fractions eluted

t 0 = time for solvent to traverse the column t r = retention time t w = peak base width h = peak height Can constantly monitor the eluted solution as a function of time or volume – produce a chromatogram.

Synthetic chemists use columns to separate their products They collect different fractions at different times

Mobile phase: generally an inert gas such as Helium Stationary phase: generally an adsorbent or liquid distributed over the surface of a porous, inert support. GAS CHROMATOGRAPHY Suitable for vapour phase samples To vaporise sample solutes and keep them in the vapour phase

COLUMNS Two general types of column: 1) Packed columns  contain a finely divided, inert, solid support material coated with liquid stationary phase.  length ~ m and internal diameter ~2 - 4mm. 2) Capillary columns (or Open tubular columns)  internal diameter ~ few tenths of a mm and length ~ up to 100 m  wall-coated columns: walls act as support for the stationary liquid phase  support-coated columns: wall is lined with a thin layer of support material onto which the stationary phase is adsorbed.

As before:

Column temperature: For precise work, column temperature must be controlled to within tenths of a degree. Optimum temperature  dependant on boiling point of the sample. Temperature programming - if a sample has a wide boiling range, column temperature is increased (either continuously or in steps) as separation proceeds. Capillary column in GC oven

For optimum column efficiency: - sample should not be too large packed columns:  L to 20  L capillary columns: ~1  L - sample should be introduced as a "plug" of vapour slow injection of large samples causes band broadening and loss of resolution. SAMPLE INJECTION Most common injection method - a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

Capillary GC split/splitless injector: Sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes. A proportion of the gas mixture passes into the column Inject sample To the column

DETECTORSELECTIVITY Thermal conductivity Universal Electron capture Halides, nitrates, nitriles, peroxides, anhydrides, organometallics Photo-ionization Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics Flame ionization Most organic compounds Flame photometric Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium Hall electrolytic Halide, nitrogen, nitrosamine, sulphur conductivity DETECTORS There are many detectors used in GC. Different detectors will give different types of selectivity.

Examples of some GC chromatograms

Effect of different columns on “separation ability”:

Equilibration of the sample between the stationary and mobile phase occurs in each plate. The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next. THEORETICAL PLATE MODEL Divide a column into a large number of separate layers, called theoretical plates. NB: this is a theory to help us understand and explain what is happening  the plates do not really exist

The number of theoretical plates (N) in a column can be found by examining a chromatographic peak after elution: t r = retention time w 1/2 = peak width at half-height Different retention times for different solutes  different numbers of plates for different solutes in a mixture w 1/2

To obtain optimal separations  sharp, symmetrical peaks must be obtained.  band broadening must be limited COLUMN EFFICIENCY The column is more efficient in separation if: - there are more plates - the plate height is smaller Calculate the Height Equivalent to a Theoretical Plate (HETP): L = length of column

u = average velocity of mobile phase A, B, C = factors which contribute to band broadening RATE THEORY OF CHROMATOGRAPHY  takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (plate model assumes infinitely fast equilibration).  resulting peak shape of a peak affected by the rate of elution Also the different paths available to solute molecules as they travel through the stationary phase affects peak shape. Van Deemter equation for plate height:

C - Resistance to mass transfer If velocity of mobile phase is high and analyte has a strong affinity for stationary phase  analyte in mobile phase will move ahead of that in stationary phase  band broadening A, B, C = factors which contribute to band broadening A - Eddy diffusion Solute molecules will take different paths through the stationary phase at random. Different paths have different lengths  band broadening B - Longitudinal diffusion Concentration of analyte is more at the center than the edges of the column,  analyte diffuses out from the center to the edges  band broadening High velocity of the mobile phase  analyte spends less time on the column  decreases the effects of longitudinal diffusion

Van Deemter plot: plot of plate height vs. average linear velocity of mobile phase  useful in determining the optimum mobile phase flow rate

Increase the selectivity by: - changing mobile phase composition - changing column temperature - changing composition of stationary phase - using special chemical effects (e.g. including complexing agents in stationary phase to complex with one of the solutes) But FIRST optimise capacity! Increase the number of theoretical plates. - Lengthening the column  an increase in retention time and increased band broadening - not desirable. - Instead reduce the height equivalent to a theoretical plate (HETP) by reducing the size of the stationary phase particles. Improve capacity  In GC by changing the temperature  In LC by changing the composition of the mobile phase 1 To obtain high resolution, the three terms must be maximised: 2 3