Instrumental Analysis II

Compiled by: S. Louw and K.M. Kalili

 Content covered in Instrumental Analysis I

• Flame emission and atomic absorption spectrometry

• Ultraviolet spectroscopy
• Infrared absorption spectroscopy
• Molecular fluorescence and phosphorescence
• Introduction to 1H NMR spectroscopy
• Mass spectrometry
 Course content
• Solvent extraction; Introduction to chromatographic methods of separation and
their applications
• Band broadening and column efficiency; optimization of column performance
• Applications of chromatography: qualitative and quantitative analysis by
chromatography
• Gas chromatography (GC); GC instrumentation, columns and stationary phases;
Applications of GC and advances in GC
• High performance liquid chromatography (HPLC) and column efficiency; LC
instrumentation
• Partition chromatography, adsorption chromatography; ion-exchange
chromatography; size exclusion chromatography
• 1H NMR revision: NMR theory, experimental methods and interpretation of spectra
• 13C NMR
• Advanced 1H NMR
• 2-dimensional NMR
 Instrumental Analysis II

Introduction to Analytical Separations

Compiled by: S. Louw and K.M. Kalili

The importance of Separation Science
in Analytical Chemistry

“In the vast majority of real analytical problems,

we must separate, identify and measure one or
more components from a complex mixture”.
– D. C. Harris, Quantitative Chemical Analysis, 8th Edition
Comprehensive analysis of a complex sample

Complex sample

Separation

Identification

Quantification
Comprehensive analysis of a complex sample

Complex sample

• Distillation • Masking
Separation • Extraction • Ion-exchange
• Precipitation • Chromatography
• Filtration • Electrophoresis, etc

• UV/vis
Identification • IR
• NMR
• MS

Quantification
 What are separation methods used for?

• Preparation/isolation of individual components in a

mixture

• Qualitative and quantitative analysis of components in a

mixture
 Solvent extraction
• Extraction is the transfer of a solute from one phase to
another, for example to:
 Isolate or concentrate the desired analyte
 Separate it from species that would interfere in the analysis
• Commonly, compounds may be extracted from an
aqueous solution using an organic solvent, e.g. diethyl
ether or hexane
• In the two-phase mixture: one phase is predominantly
water and the other phase is predominantly organic
• If a solute, S, is partitioned between phases 1 and 2

[S]2 Phase 2

⇌
[S]1 Phase 1

then the partition coefficient, K, is the equilibrium constant

for the reaction
𝑆 𝑖𝑛 𝑝ℎ𝑎𝑠𝑒 1 ⇌ 𝑆(𝑖𝑛 𝑝ℎ𝑎𝑠𝑒 2)

(𝑎𝑠 )2 [𝑆]2
𝐾= ≈
(𝑎𝑠 )1 [𝑆]1

where 𝒂𝒔 is the activity coefficient and 𝑺 is the concentration of the

solute. (For low concentrations of solute or when non-ionic species
are involved, the activity may be substituted by concentration).
• Suppose S is dissolved in a volume of solvent 1 (water), V1 (in mL), and
it is extracted using a volume, V2, of solvent 2 (toluene), and
n = number of moles of S
q = fraction of S remaining in phase 1 at equilibrium, then
[𝑆]1 = 𝑞𝑛 𝑉1
The solute transferred to phase 2 is (1 − 𝑞) and therefore:
[𝑆]2 = 1 − 𝑞 𝑛 𝑉2
Substituting the concentrations into the equation for the partition coefficient
and solving for q gives:
𝑉1
𝑞=
𝑉1 + 𝐾𝑉2

So q is the fraction of S remaining in phase 1 after 1 extraction.

• After x extractions, each with volume V2, the fraction remaining in water
is: 𝑥
𝑥
𝑉1
𝑞 =
𝑉1 + 𝐾𝑉2
 Example: Extraction efficiency
• Solute A has a K = 3
• Suppose 100 mL of a 0.010 M aqueous
solution of solute A is extracted with toluene
• What fraction of A remains in the aqueous
phase
a) if one extraction with 500 mL is performed?
b) if five extractions with 100 mL are performed?

See Harris, p. 538

 pH effects
• If the solute is an acid or base, its charge changes as the
pH of the solution is changed
– charged species are more soluble in the aqueous phase than
in the organic phase
 To extract a base into water, use a pH low enough to convert
B into BH+ Kb

 To extract the acid (HA) into water, use a pH high enough to

convert HA into A- Ka
• Distribution coefficient,
for instance for B, is defined as:
𝑡𝑜𝑡𝑎𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑝ℎ𝑎𝑠𝑒 2
• 𝐷=
𝑡𝑜𝑡𝑎𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑝ℎ𝑎𝑠𝑒 1
[𝐵]2
• 𝐷=
[𝐵]1 +[𝐵𝐻 + ]1

if it is assumed that BH+ only goes into the aqueous phase

 Introduction to Chromatography

• In 1903, a Russian botanist

Mikhail Tswett applied
adsorption chromatography
to the separation of plant
pigments.

• The separation of coloured

bands led to the name
chromatography, coined
from Greek words
chromatos, meaning colour,
and graph, meaning writing
 “colour writing”
 Tswett’s experiment

Stationary phase: Calcium carbonate (polar)

Mobile phase: petroleum ether (non-polar)
 Basic principles of chromatography
Extraction (static):
Phase 2
Phase 1
𝑋2 8
𝐾𝐷 = = =4
𝑋1 2

The chromatographic process (dynamic):

Phase 2 Stationary

Phase 1 Mobile

Phase 2 Stationary
𝑋𝑆
𝐾𝐷 =
Separatory funnels 𝑋𝑀
 Basic principles of chromatography
• In chromatography, a continuous equilibration of solute
occurs between two phases, mobile phase and stationary
phase
• Mobile phase:
 Is either a liquid, gas or
supercritical fluid

• Stationary phase
 Solid particles packed inside a column (usually porous or
superficially porous, different particle sizes are available)
 Liquid coating on particles or on the inside of a capillary
column
 Basic principles of chromatography
 In chromatography, analytes are separated based on their
differential partitioning between two phases i.e. mobile phase
and stationary phase.
 Solute with higher affinity for the stationary phase remains on
the column for longer
 Chromatographic techniques are classified based on the physical
nature of the mobile phase.
 The mobile phase can be a gas, a liquid or a supercritical fluid,
which gives rise to the three basic forms of chromatography,
namely, gas chromatography (GC), liquid chromatography (LC)
and supercritical fluid chromatography (SFC). 19
 Basic principles of chromatography
• The process of passing a liquid, gas or supercritical fluid
through a chromatographic column is called elution.
• The fluid entering the column is called the eluent, and the
fluid emerging at the end of the column is called the
eluate.

Elution modes
 Isocratic/isothermal conditions: mobile phase
composition/temperature stays constant
 Gradient/programmed conditions: mobile phase
composition/temperature is gradually changed during
the separation
 Basic principles of chromatography

• Retention time: the time required for a solute to pass

through the column
• Partition coefficient, K:
𝐶𝑠
𝐾=
𝐶𝑚
where Cs and Cm is the concentration of the solute in the
stationary and mobile phases, respectively.
 Chromatographic methods overview
• Preparative chromatography ( for large-scale applications)
 Column chromatography - Column packed with
stationary phase
 Planar chromatogaphy – paper stationary phase or a
sheet of material coated with stationary phase – i.e. thin
layer chromatography (TLC)
• Analytical chromatography (for small-scale applications)
 GC used for analysis of volatile and thermally-stable
compounds
 LC used for semi-volatile and non-volatile compounds
 SFC – semi-volatile and non-volatile compounds
Column chromatography (conventional)

Step 1: Load sample (black)

Step 2: Elute yellow pigment
Step 3: Elute red pigment
Step 4: Elute blue pigment
 Modern Column chromatography

detector
 Components of a chromatographic system
Injector

Column Chromatogram

Mobile phase Detector

delivery unit
 Planar chromatography
• Paper chromatography

• Thin layer chromatography

𝑏
𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑅𝑓 =
𝑎
Adsorption vs. absorption
 Types of chromatography

Adsorption chromatography. A solid

stationary phase and a liquid or
gaseous mobile phase are used.
Solutes are selectively adsorbed on
the surface of the solid particles.

Partition chromatography. A liquid

stationary phase is bonded to a solid
surface. Solutes selectively partition
between the liquid stationary and
the mobile phase
 Types of chromatography
Ion-exchange chromatography. Anions or
cations are covalently attached to the solid
stationary phase, usually a resin. Solute
ions of the opposite charge are attracted
to the stationary phase. The mobile phase
is a liquid.

Molecular exclusion chromatography.

Also known as size exclusion, gel
filtration or gel permeation
chromatography: Separates molecules by
size; larger solutes passing through most
quickly. No attractive interaction
between the stationary phase and the
solute.
 Types of chromatography
Affinity chromatography. Highly selective. Employs specific
interactions between one kind of solute molecule and a
second molecule that is covalently attached (immobilized) to
the stationary phase, e.g. antigen and antibody, enzyme and
substrate, or receptor and ligand
 Typical applications of chromatography
• Chromatography is used for the analysis of:
– pharmaceuticals
– forensic samples
– environmental samples
– petrochemicals
– food and drink
– flavours and fragrances
– explosives
– ecological samples, e.g. pheromones
– etc
 References
• D. C. Harris, Quantitative Chemical Analysis, Eighth Edition
• Analytical Chemistry 2.0 by David Harvey (electronic revision
of Modern Analytical Chemistry by David Harvey)

Further reading:
• D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of
Instrumental Analysis, Sixth Edition