Separation

• Samples are usually complex mixtures. In order

to identify and quantify the components of a
Ch. 23 Fundamentals of Analytical mixture, we have to separate the components in
Separations the mixture.
• Separation methods
– Extraction
– Chromatography
– Electrophoresis

Solvent Extraction Solvent Extraction

• The transfer of an analyte from
K=
[S ]2 =
(1 − q )m / V2
one phase to a second based on
the relative solubility of the [S ]1 qm / V1
analyte in two immiscible liquids.
Fraction remaining in V
q= 1

[S ]
K= 2 =
(1 − q ) m / V2 phase 1 after 1 extraction V + KV
1 2

[S ]1 qm / V1 Fraction remaining in V
n

phase 1 after n extractions q = q ⋅ q ⋅ ...q =

n 1

K: the partition coefficient for distribution of S between n

V + KV 1 2

the two phases at equilibrium;

If q = 1/4, then 1/4 remains in phase 1 after one extraction
m: the moles of S in the system
K: the partition coefficient; q: the fraction of S remaining in
q: the fraction of S remaining in phase 1; phase 1; n: the # of extractions.

Extraction Efficiency
pH Effects
• A solute S has a partition coefficient of 3 between • The charge changes of an acid or base as the pH is
toluene and water. If you have 100 mL of a 0.010 M changed.
solution of S in water.(1)What fraction of the solute • Distribute coefficient (D): an alternate form of the
remains in H2O after a 500 mL extraction with toluene? partition coefficient.
(2) What fraction of the solute remains in H2O after a 5-
Total conc. in phase 2 C2
100 mL extractions with toluene? D= =
Total conc. in phase 1 C1
100
q= = 0.062 ≈ 6% • A basic amine whose neutral form, B, has partition
100 + (3)(500) coefficient, K, between phases 1 and 2. The conjugated
5 acid BH+ is soluble only in phase 1 and the acid
100
q= = 0.00098 ≈ 0.1% dissociation constant is Ka.
100 + (3)(100)
[ B] [ B] [ H ] +

It is more efficient to do several small extractions K= ; 2

Ka = 1

than one big extraction. [ B] 1

[ BH ] +
1

1
 pH Effects pH Effects
Total conc. in phase 2 C2 [ B] [ H ] +
[ B] • K for an amine B is 3.0 and the Ka for BH+ is 1.0×10-9. If
D= = Ka = 1
K= 2
50.00 mL of 0.010 M aqueous amine is extracted with 100
Total conc. in phase 1 C1 [ BH ] +
[ B]
1 1
mL of solvent, calculate the % remaining the in aqueous
[ B] KKa phase in M at (1) pH 10.00; (2) pH 8.00.
D= 2
= = Kα KKa 3.0 × 1.0 × 10 −9
[ B] + [ BH ] Ka + [ H ]
+ + B
1 1 pH = 10.00 : D = = = 2.73
Ka + [ H ] 1.0 × 10 + 1.0 × 10
+ −9 −10

V 50
[ HA]2 K[H + ] q= = 1
= 0.15 15%
D= = = Kα HA V + DV 50 + 2.73 × 100
[ HA]1 + [ A ]1 Ka + [ H + ]
− 1 2

[ A− ]1[ H + ] KKa 3.0 × 1.0 × 10 −9

( Ka = ;K =
[ HA]2
) pH = 8.00 : D = = = 0.273
[ HA]1 [ HA]1 Ka + [ H ] 1.0 × 10 + 1.0 × 10
+ −9 −8

V 50
: fraction of the species (P.191) q= =1
= 0.65 65%
V + DV 50 + 0.273 × 100
1 2

pH Effects Extraction with a Metal Chelator

• The weak acid HA with Ka = 1.0×10-5 is to be quantitatively
extracted from an aqueous sample (100 mL sample volume)
using 100 mL of a suitable organic solvent, by doing a single •Usually neutral complexes can be extracted into organic
extraction. The partition coefficient is 30.0. What is the solvents. Charged complexes (e.g. MEDTA2-) are not very
maximum pH allowed in order that no more than 5% of the acid soluble in organic solvents.
remains in the aqueous solution? •Commonly used: dithizone, 8-hydroquinoline, and
V 100 cupferron.
q= = 1
= 5%
V + DV 100 + D × 100
1 2

D = 19
K[H ] 30.0 × [ H ]
+ +

D= = = 19
Ka + [ H ] 1.0 × 10 + [ H ]
+ −5 +

[ H ] = 1.73 × 10 M
+ −5

pH = 4.76

Extraction with a Metal Chelator Extraction with a

Metal Chelator
•Commonly used: dithizone, 8-hydroquinoline, and
cupferron. •Each ligand can be presented
•Crown ethers can extract alkali metal ions and can bring as a weak acid, HL.
them into non-polar solvents. •Mn+ is in the aqueous phase
and MLn is in the organic
phase
•The distribution coefficient
(D) for metal ion extraction
depends on pH and [ligand].
•By select a pH, you can bring
the metal into either phase.

2
 Chromatography Type of Chromatography
• A separation process based on the various partitioning
coefficients of different solutes between the two phases. • Based on the mechanism of interaction of the solute
• Involving the interaction of solute(s) and two phases; operates on
with the stationary phase
the same principle as extraction, but one phase is held in place
while the other moves past it.
• Mobile phase: A gas or liquid that moves through the column. (1) Adsorption chromatography
- Solute is adsorbed on the surface of
• Stationary phase: A solid or liquid that remains in place. the stationary phase (solid).
- The stronger a solute adsorbs, the
longer it takes to travel through the
chromatography column

Type of Chromatography Type of Chromatography

• Based on the mechanism of interaction of the solute • Based on the mechanism of interaction of the solute
with the stationary phase with the stationary phase

(2) Partition chromatography (3) Ion-exchange

chromatography
- GC
-Retention is based on the
- the partitioning of solutes attraction between solute ions and
between a mobile phase (gas) charged sites bound to the
and bonded liquid stationary stationary phase (ionic
phase interactions to separate ions).
- A stationary phase of cations
will separate anions and vice
versa.

Type of Chromatography Type of Chromatography

• Based on the mechanism of interaction of the solute • Based on the mechanism of interaction of the solute
with the stationary phase with the stationary phase
(5) Affinity chromatography
-Specific interactions of one kind of solute molecule to a second
(4) Molecular Size exclusion molecular that is covalently attached to the stationary phase
chromatography -Most selective (e.g. use antibodies to select out one protein from a
-size exclusion, gel filtration, or gel mixture of hundreds)
permeation chromatography
-separate molecules by size
-large molecules pass through
faster (they do not get caught up in
pores)