1. Chromatography
is a technique in which the components of a mixture are separated based on
The differences in rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase.

2. 31E Chromatographic Separations
Chromatography is a widely used method for the separation, identification, and determination of the chemical components in complex mixtures.
No other separation method is as powerful and generally applicable as is chromatography.

3. 31E-1 General Description of Chromatography
The term chromatography is has a broad definition because the name has been applied to several systems and techniques.
All of these methods, however, have in common the use of a stationary phase and a mobile phase.
Components of a mixture are carried through the stationary phase by the flow of a mobile phase, and separations are based on differences in migration rates among the mobile-phase components.

4. The stationary phase in chromatography is a phase that is fixed in place either in a column or on a planar surface.
The mobile phase in chromatography is a phase that moves over or through the stationary phase carrying with it the analyte mixture.
The mobile phase may be a gas, a liquid, or a supercritical fluid.
Planar & column chromatography are based on same types of equilibria.

5. 31E-2 Classification of Chromatographic Methods
Chromatographic methods are of two basic types.
In column chromatography, the stationary phase is held in a narrow tube, and the mobile phase is forced through the tube under pressure or by gravity.
In planar chromatography, the stationary phase is supported on a flat plate or in the pores of a paper,
and the mobile phase moves through the stationary phase by capillary action or under the influence of gravity.

6. Chromatographic methods fall into three categories based on the nature of the mobile phase: liquid, gas, and supercritical fluid (table 31.4).
The second column of the table reveals that there are
five types of liquid chromatography and
two types of gas chromatography
that differ in the nature of the stationary phase and the types of equilibria between phases.

8. Gas chromatography and supercritical fluid chromatography require the use of a column.
Only liquid mobile phases can be used on planar surfaces.

9. 31E-3 Elution in Column Chromatography
Figure 31-6a shows how two components A and B of a sample are resolved on a packed column by elution.
The column consists of narrow-bore tubing that is packed with a finely divided inert solid that holds the stationary phase on its surface.
The mobile phase occupies the open spaces between the particles of the packing.

10. Figure 31-6 (a) Diagram showing the separation of a mixture of components A and B by column elution chromatography. (b) The detector signal at the various stages of elution shown in (a).

11. Ideally, the resulting differences in rates cause the components in a mixture to separate into peaks, bands, or zones, along the length of the column (Figure 31-7).
Isolation of the separated species is then accomplished by passing a sufficient quantity of mobile phase through the column to cause the individual bands to pass out the end (to be eluted from the column.

12. Elution is a process in which solutes are washed through a stationary phase by the movement of a mobile phase.
The mobile phase that exits the column is termed the eluate. An eluent is a solvent used to carry the components of a mixture through a stationary phase.

13. Chromatograms
If a detector that responds to solute concentration is placed after the column and its signal is plotted as a function of time (or of volume of added mobile phase.
Such a plot, called a chromatogram, is useful for both qualitative and quantitative analysis.
The positions of the peak maxima is used to identify the components of the sample. The peak areas provide a quantitative measure of the amount of each species.

14. A chromatogram is a plot of some function of solute concentration versus elution time or elution volume.

15. The Russian botanist Mikhail Tswett (1872–1919) invented chromatography shortly after the turn of the twentieth century. He used the technique to separate various plant pigments, such as chlorophylls and xanthophylls, by passing solutions of these species through glass columns packed with finely divided calcium carbonate. The separated species appeared as colored bands on the column, which accounts for the name he chose for the method (Greek chroma meaning “color” and graphein meaning “to write”).

16. Methods for Improving Column Performance
Fig shows concentration profiles for the bands containing solutes A and B. Because B is more strongly retained by the stationary phase than is A, B lags during the migration.
The distance between the two increases as they move down the column.
At the same time, however, broadening of both bands takes place, lowering the separation efficiency of the column.
While band broadening is inevitable, conditions can often be found where it occurs more slowly than band separation.

17. Figure 31-7 Concentration profiles of solute bands A and B at two different times in their migration down the column in Figure The times t1 and t2 are indicated in Figure 31-6.

18. Thus, as shown in Figure 31-7, a clean separation of species is possible provided the column is sufficiently long.
Several chemical and physical variables influence the rates of band separation and band broadening.
As a result, improved separations can often be realized by the control of variables that either
increase the rate of band separation or
decrease the rate of band spreading.
These alternatives are illustrated in Figure 31-8

19. Figure 31-8 Two-component chromatogram illustrating two methods for improving separation. (a) Original chromatogram with overlapping peaks. (b) Improvement brought about by an increase in band separation. (c) Improvement brought about by a decrease in band widths.

20. 31E-4 Migration Rates of Solutes
The effectiveness of a chromatographic column in separating two solutes depends in part on the relative rates at which the two species are eluted.
These rates in turn are determined by the ratios of the solute concentrations in each of the two phases.

21. Distribution Constants
All chromatographic separations are based on differences in the extent to which solutes are distributed between the mobile and the stationary phase.
For the solute species A, the equilibrium is described by the equation or

22. Retention Times
Fig is a simple chromatogram of a two-component mixture. The small peak on the left is for a species that is not retained by the stationary phase.
The time tM after sample injection for this peak to appear is sometimes called the dead or void time.
The dead time provides a measure of the average rate of migration of the mobile phase and is an important parameter in identifying analyte peaks.

23. Fig. 31-9 A typical chromatogram for a two-component mixture
Fig A typical chromatogram for a two-component mixture. The small peak on the left represents a solute that is not retained on the column and so reaches the detector almost immediately after elution is begun. Thus, its retention time, tM, is approximately equal to the time required for a molecule of the mobile phase to pass through the column.

24. To aid in measuring tM, an unretained species can be added if one is not already present in the sample or the mobile phase.
The larger peak on the right in Figure 31-9 is that of an analyte species.
The time required for this zone to reach the detector after sample injection is called the retention time and is given the symbol tR.
The analyte has been retained because it spends a time tS in the stationary phase.

26. The retention time, tR, is the time between injection of a sample and the appearance of a solute peak at the detector of a chromatographic column.

27. Volumetric Flow Rate and Linear Flow Velocity
Experimentally in chromatography the mobile phase flow is usually characterized by the volumetric flow rate, F (cm3/min), at the column outlet.
For an open tubular column, F is related to the linear velocity at the column outlet uo by