Chapter 23

An Introduction toAnalytical Separations

Partition Between Two Phases A. Separatory Funnel two immiscible liquids -- solutes soluble

in both

Partition Coefficient, K K = CU / CL after equilibration

K depends upon: solute -- polarity, ionization, etc. 2 liquid phases -- polarity etc.

If K >> 1 or K << 1 for a particular solute ==> can purify the solute pretty well with a single extraction (or relatively few extractions).

But what if K is approximately equal to 1?

Let's look at two cases: (a) K = 1 (b) K = 3 if K > 1 ==> more material in right most

vessels --> peak moves to right quicklyif K < 1 == > more material in leftmost vessels --> peak moves to right more slowly(In the next diagram, lower phase stays in place while the upper phase is transferred to the next vessel to the right with fresh lower phase.)

a. K = 1

1/2

1/2

1/4 1/4

1/4 1/4

1/8 1/4 1/8

1/8 1/4 1/8

1/16 3/16 3/16 1/16

1/16 3/16 3/16 1/16

1/1 1/8 3/16 1/8 1/1

1/1 1/8 3/16 1/8 1/1

b. K = 3

3/4

1/4

3/16 9/16

1/16 3/16

3/1 18/64 27/64

1/1 6/1 9/1

3/256 27/256 81/256 81/256

1/256 9/256 27/256 27/256

3/1024 36/1024 162/1024 324/1024 243/1024

1/1024 12/1024 54/1024 108/1024 81/1024

General Principle - Differential Hold-up

Method - Column - Thin layer - Paper Columns can be of various sizes on the

benchtop, or smaller with smaller sized packing and used in an instrument.

Generally there is a MOVING PHASE and a STATIONARY PHASE

Principle (reason for hold-up)

Adsorption Partition Ion Exchange Size Exclusion Affinity

Liquid chromatography Liquid - liquid(adsorbed or bonded on

solid) Liquid - solid SFC - liquid (adsorbed or bonded on

solid) Gas chromatography Gas - liquid(adsorbed or bonded on

solid Gas - solid

Chromatography is the separation - we must also have a detection method to follow the separation

Basic detectors - give a peak at a characteristic time for each component.

The most desirable, but more expensive detectors, in particular mass spectrometers, give a unique spectrum of each compound as it comes off, from which it can be identified.

Ion Exchange Chromatography

In ion exchange chromatography, charged substances are separated via column materials that carry an opposite charge.

The ionic groups of exchanger columns are covalently bound to the gel matrix and are compensated for by small concentrations of counter ions, which are present in the buffer.

When a sample is added to the column, an exchange with the weakly bound counter ions takes place.

Resin

Two basic types of ion exchangers: those for binding positively charged ions

or cations, which display on their surface negatively charged groups

and those for binding negatively charged ions or anions, which display on their surface positively charged groups.

Polystyrene-divinylbenzene resin (PS-DVB) Most common polymer base for ion-exchange

chromatography. Ionic groups are incorporated by chemical reactions. Porosity and mechanical stability are altered by varying the cross-linking through the variation of the DVB content.

Low crosslinking – resin swells a lot, changes size with different ions.

High crosslinking – solubility decreases - selectivity increases

Anion exchangers

R-NR3+ OH- + A- R-NR3

+ A- + OH-

strong base pH 0-12

R-NH3+ OH- + A- R-NH3

+ A- + OH- weak

base pH 0-9

Apparatus

column support stationary phase mobile phase detector

Column copper tubing stainless steel tubing glass tubing Polymer (plastic)

Support finely divided solids

ground firebrickalumina, specially treated

walls of column for capillary columns

Stationary Phase stationary phase evenly dispersed on

surface of support

Mobile Phase sample mixture carried through

stationary phase by mobile phase non-reactive gas in glc (gas-liquid

chromatography, gc) non-reactive liquid in llc (liquid-liquid

chromatography, lc)