The “general” elution problem: a single set of conditions is often unsatisfactory for good separation of all components in a complex mixture in an acceptable time (wide range of k values)

a) all components separated but time is long and peak widths are broad at long times b) a faster separation and good peak shapes for 5,6 but 1,2 and 3,4 are not resolved c) intermediate case where 1,2 still not resolved common solution: change separation conditions dynamically – e.g. temperature gradient, solvent gradient

Partition Chromatography: Liquid stationary phase bonded either by adsorption (non-covalent) or covalent bonding. Ex. of covalent bonded phase

R = C-8, C-18 hydrocarbon (often) Bonded phases have longer lifetimes and are more amenable to gradient elution. Generally, lower sample loading capacities, however.

The analogy to temperature programming in GC is the variation in solvent composition to dynamically alter partitioning during elution, Fixed solvent composition: isocratic elution Variation of solvent composition: gradient elution

Normal-phase HPLC polar stationary phase, non-polar mobile phase least polar solute elutes first, most polar solute last Reverse-phase HPLC non-polar stationary phase (e.g., hydrocarbon) polar mobile phase (e.g., H2O, CH3OH, CH3CN) most polar solute elutes first, least polar last Gradient elution: change of solvent composition to change k values during a given separation normal phase: polar solutes more strongly retained in stationary phase and therefore they come out later. To reduce the time for elution of slow eluting species, we want to decrease the k values. To do this, the solvent gradient must increase polar composition of mobile phase with time. reverse phase: To decrease k values for slow eluting solutes (non-polar), the solvent gradient must decrease polar composition of mobile phase with time. Solvent Polarity Index, P Solvent P fluoroalkane <-2 tetrahydrofuran 4.0 cyclohexane 0.04 chloroform 4.1 n-hexane 0.1 ethanol 4.3 carbon tetrachloride 1.6 dioxane 4.8 diisopropyl ether 2.4 methanol 5.1 toluene 2.4 acetonitrile 5.8 diethyl ether 2.8 nitromethane 6.0 dichloroethylene 3.1 water 10.2

A variety of means for detection of species that elute from the column are available, some of which we have studied in this course:

Liquid Chromatography – variations: liquid-liquid (partition) adsorption (liquid-solid) ion-exchange size-exclusion affinity chiral

Gas chromatography (GC) and liquid chromatography (LC):

The parameters describing chromatograms for both GC and LC are calculated in the same way. The concepts developed thus far for extractions and column chromatography apply to both. The species for which GC and LC are useful, as well as technological aspects, differ. (some overlap)

GC – elements of a gas chromatograph system

Carrier gases (mobile phase) – usually He, sometimes Ar, N2, H2 flow rates – packed columns 1-25 mL/min open tubular 25-150 mL/min Columns

length – 2-50 m elution times – typically 2-30 min.

Temperature – since partitioning is between the gas and a liquid, efficiencies and elution times are highly dependent upon temperature. It is usually controlled to within a few tenths of a degree. Effect of temperature:

a) isothermal at 45 oC b) isothermal at 145 oC c) programmed 30-180 oC

short column + temperature programming can lead to fast GC (with compromise in resolution)