UNIVERSITY OF TECHNOLOGY, JAMAICA

FACULTY OF HEALTH AND APPLIED SCIENCES

Laboratory Instrumentation

Unit 7: Chromatography

7.1 What is chromatography?

A broad range of physical methods used to separate and/or analyse complex mixtures. The
components in a mixture are distributed between two phases: a stationary phase bed and a
mobile phase which percolates through the stationary bed. The method relies on differences in
partitioning behaviour between the flowing mobile phase and the stationary phase to separate
the components in a mixture.

A column (or other support for TLC) holds the stationary phase and the mobile phase carries the
sample through it. Sample components that partition strongly into the stationary phase spend a
greater amount of time in the column and are separated from components that stay
predominantly in the mobile phase and pass through the column faster.

Applications
Wide range of applications include:
Qualitative analysis
• Used as criteria of purity for organic compounds – contaminants if present are revealed
by the appearance of additional peaks
• Used to evaluate the effectiveness of purification procedures
• Retention times or volumes are employed for qualitative identification
Quantitative analysis
• peak areas or heights provide quantitative information
• Industrial- PAH, aromatics, pesticides
• Pharmaceutical/therapeutic drug testing
• Trace analysis in biological and environmental samples
• Quantification of aromatic, molecules present at trace concentrations in biological and
environmental samples
• Extended to a wide variety of organic and inorganic compounds via chemical labelling
and derivatization procedures

Factors that affect analyte separation and analysis of mixtures:

• Compound volatility
• Contamination of mobile phase
• Gas flow rate
• Oven temperature programme
• Stationary phase:

Stationary phase Trade name Maximum Common applications

temperature
Polydimethyl siloxane HP 1/ OV 1 350 Non polar phase;
hydrocarbons; polynuclear
aromatics; drugs; steroids;

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 1
Bartley-Hynes (University of Technology, Jamaica)
 PCBs
Stationary phase Trade name Maximum Common applications
temperature
Poly HP 3 / OV 3 350 Fatty acid methyl esters;
(phenylmethyldimethyl) alkaloids; drugs;
siloxane (10% phenyl) halogenated compounds
Poly (phenylmethyl) HP17/ OV17 250 Drugs; steroids; pesticides;
siloxane (50% phenyl) glycols
Poly OV 210 200 Chlorinated aromatics;
(trifluoropropyldimethyl) nitroaromatics; alkyl-
siloxane substituted benzenes
Polyethylene glycol Carbowax 20 M 250 Free acids; alcohols; ethers;
essential oils; glycols

7.3 Classification of Column Chromatographic Methods

General Classification Specific Method Stationary Phase

Gas Chromatography 1. Gas-liquid (GLC) - Liquid adsorbed or
(GC) bonded to a solid surface
- Solid
2. Gas-solid
Liquid Chromatography 1. Liquid-liquid or - Liquid adsorbed or
(LC) partition bonded to a solid surface
2. Liquid-solid or - Solid
adsorption
3. Ion exchange
- Ion exchange resin
4. Size Exclusion
- Liquid in interstices
5. Affinity of a polymeric solid
- Group specific liquid
bonded to a solid surface
Supercritical Fluid - Organic species
Chromatography (SFC) bonded to a solid surface

7.3 Types of Chromatography Methods

(source: http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm)

• Adsorption Chromatography
o one of the oldest types of
chromatography.
o utilizes a mobile or gaseous
phase that is adsorbed onto the surface
of a stationary solid phase.
o The equilibration between the
mobile and stationary phases accounts
for the separation of different solutes.

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 2
Bartley-Hynes (University of Technology, Jamaica)
 • Partition Chromatography
o Based on a thin film formed on the
surface of a solid support by a liquid
stationary phase.
o Solute equilibrates between the mobile
phase and the stationary liquid.

• Ion Exchange Chromatography

o A resin (solid stationary phase) is used
to covalently attach anions or cations
onto it.
o Solute ions of the opposite charge in
the mobile phase are attracted to the
resin by electrostatic forces.

• Molecular Exclusion Chromatography

o Also known as gel permeation or gel
filtration.
o This type lacks an attractive interaction
between the stationary phase and the
solute.
o The liquid or gaseous phase passes
through a porous gel which separates
molecules by size.
o The pores are small and exclude larger
solute molecules, causing the larger
molecules to pass through the column
at a faster rate than the smaller ones.
• Affinity Chromatography
o The most selective type of
chromatography.
o Utilizes the specific interaction between
one kind of solute molecule and a
second molecule that is immobilized on
a stationary phase.
o The specific solute molecule is bound
to the stationary phase and later
extracted by changing ion strength or
pH.

7.4 Basis of Chromatography

The Mobile and Stationary Phases

• The mobile phase is comprised of a solvent into which the sample is injected. The
solvent and sample flow through the column together; thus the mobile phase is often
referred to as the "carrier fluid."
• The stationary phase is the material in the column for which the components to be
separated have varying affinities.

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 3
Bartley-Hynes (University of Technology, Jamaica)
 • The materials which comprise the mobile and stationary phases depend on the general
type of chromatographic process being performed.

The Column
• Most modern applications of chromatography employ a column. This is where the
separation takes place.
• Usually a glass or metal tube of sufficient strength to withstand the pressures applied
across it.
• Contains the stationary phase. The mobile phase runs through the column and is
adsorbed onto the stationary phase.

Flow Meter
Detector

C
O Oven
L
U
M
N

Feed Injection
Pump

Solvent Tank

Basic Layout of a Chromatograph

Gas Chromatography
• The mobile phase is generally an inert gas.
• The stationary phase is generally an adsorbent or liquid distributed over the surface of a
porous, inert support.

Liquid Chromatography
• The mobile phase is a liquid of low viscosity which flows through the stationary phase
bed. This bed may be comprised of an immiscible liquid coated onto a porous support, a thin
film of liquid phase bonded to the surface of a sorbent, or a sorbent of controlled pore size.

7.5 Theory of GC and HPLC

Gas Chromatography (GC)

• Gas chromatography involves a sample being vapourised and injected onto the head of
a chromatographic column. The sample is transported through the column by the flow of
inert, gaseous mobile phase.
• In GLC, the column itself contains a liquid stationary phase which is adsorbed onto the
surface of an inert solid.

Schematic of a gas chromatograph

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 4
Bartley-Hynes (University of Technology, Jamaica)
Carrier Gas
• The carrier gas must be chemical inert. Commonly used gases include N, He, Ar and
CO2. The choice of carrier gas often depends on type of detector used.
• Carrier gas system also contains a molecular sieve to remove water and impurities.

Sample Injection Port

• For optimum column efficiency, the sample should not be too large and should be
introduced onto the column as a “plug” of vapour – slow injection of large samples leads to
band broadening and loss of resolution.

vs.
• Most common injection method is where a microsyringe is used to inject sample through
a rubber septum into a flash vapouriser port at head of column. Temperature of port usually
~50° > BP of least volatile sample.
• Sample size for packed columns 0.1 – 20 µ L. Capillary columns ~10-3 µ L.
• Injector contains a heated chamber – sample vapourises to form a mixture of carrier gas,
vapourised solvent and vapourised solutes:

Columns

Packed Bed Columns

• Comprised of a finely divided, inert, solid support material coated with liquid stationary
phase (GLC). This stationary phase completely fills the column.
• 1.5 – 10 m long; internal diameter of 2 – 4 mm.

Capillary or Open Tubular Columns

• The liquid stationary phase is a thin film or layer on the column wall. There is a
passageway through the centre of the column.
• Internal diameter < 1 mm.

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 5
Bartley-Hynes (University of Technology, Jamaica)
Column temperature

• For precise work, column temperature must be controlled to within 0.1°.

• The optimum column temperature depends on the boiling point of the sample. As a rule
of thumb, a temperature slightly above the average boiling point of the sample results in an
elution time of 2 – 30 minutes. Minimal temperatures give good resolution, but increase
elution times.
• If a sample has a wide boiling range, then temperature programming can be useful. The
column temperature is increased (either continuously or in steps) as separation proceeds.

Detectors

• Different detectors will give different types of selectivity:

o A non-selective detector responds to all compounds except the carrier gas.
o A selective detector responds to a range of compounds with a common physical
or chemical property.
o A specific detector responds to a single chemical compound.

• Detectors can also be grouped into concentration dependant detectors and mass flow
dependant detectors:
o The signal from a concentration dependant detector is related to the
concentration of solute in the detector, and does not usually destroy the sample.
o Mass flow dependant detectors usually destroy the sample, and the signal is
related to the rate at which solute molecules enter the detector.

Detectors must be able to respond quickly to low solute concentrations (a few ppt) as they are
eluted from the column.

Other properties include:

• Linear response
• Stability
• Uniform response to wide variety of species or predictable responses to one or more
classes of chemicals.

No one detector possesses all the desirable properties but the three most popular are:

• Flame ionisation detectors

• Thermal conductivity detectors
• Electron capture detectors

GC may also be coupled with other methods such as mass spectrometry (GC-MS) and infrared
spectrometry (GC-IR).

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 6
Bartley-Hynes (University of Technology, Jamaica)
High-Performance Liquid Chromatography (HPLC)

In early liquid chromatography, separation times were long – several hours. Column packings
(solid coated with thin liquid film) were 50 – 500 cm in length, with internal diameter of 10 – 50
mm and particle sizes >150 – 200 µ m.
For increased column efficiency, decrease particle size (decrease plate height or increase plate
count). Since the 1960,s HPLC was developed.

HPLC – most popular of the analytical separation techniques.

• High sensitivity
• Good adaptability
• Ease of automation
• Can be used for non-volatile compounds

Different methods are used depending on the nature of solutes to be separated:

• High molecular mass compounds (>10,000 g mol-1) utilise size-exclusion
chromatography.
• Low molecular mass ionics utilise ion-exchange or reverse-phase chromatography.
• Non-polar species utilise adsorption chromatography.

Instrumentation

• Pumping pressures of several hundred atmospheres are employed.

• Column particle sizes ≤ 10 µ m.
• The instruments are elaborate and expensive.

Mobile-Phase Reservoirs and Solvent Treatment Systems

• One or more mobile-phase reservoirs used.
• The treatment systems remove bubbles and particulates.
• Sparging is a process in which dissolved gases are swept out of a solvent by bubbles of
an inert, insoluble gas.
• Isocratic (simple) or gradient elution is used.
Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 7
Bartley-Hynes (University of Technology, Jamaica)
Pumping Systems
A pump should have the following characteristics:
• Pressures up to 6000 psi
• Pulse-free output
• Flow rates 0.1 – 10 mL/min
• Good flow reproducibilities
• Resistant to corrosion by a variety of solvents

Two types of pumps used: mechanical or pneumatic pumps.

NOTE: High pressures are not an explosion hazard because liquids are not very compressible.
If there is a rupture in a component, leading to leakage then there is a fire hazard.

Sample Injection Systems

• Syringe injection through a septum is used. However, it is are not very reproducible and
used for pressures <1500 psi. Includes, stop flow injection.
• Sampling loops are also employed.

o These are used in modern HPLC equipment.

o Sample size 5 – 500 µ L
o Sampling loops are very precise.

Columns
• Usually stainless steel tubing but heavy-walled glass tubing is also used.
The glass tubing can only be used for lower pressures (<600 psi).
• Column lengths vary from 10 to 30 cm with inside diameter of 4 – 10 mm.
• Packing sizes of 5 – 10 µ m. Packing usually made of silica particles
coated with a thin organic film chemically or physically bonded to the surface. Other
packing materials include alumina, porous polymers and ion-exchange resins.
• Plate count of 40,000 – 60,000 plates/m.
• New microcolumns are 3 – 7.5 cm in length with internal diameter of 1 –
4.6 mm. These columns have about 100,000 theoretical plates/m. (compare to GC
columns with 500 – 4,000 plates/m)

Detectors
• These are dependent on the nature of the sample.
• A number of detectors are used:
o Absorbance LC detector - based on the absorption of UV/VIS radiation (most
popular)

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 8
Bartley-Hynes (University of Technology, Jamaica)
 o Refractive index LC detector measures changes in the refractive index of the
solvent.
• Others include:
o Fluorescence; Conductivity; Mass spectrometry; Photoionization LC detectors.

LC methods include: partition, adsorption, ion-exchange and size exclusion.

High-performance partition chromatography

• This is the most widely used of LC methods.
• Divided into liquid-liquid or bonded-phase chromatography.

• Liquid-liquid packing: retention occurs by physical adsorption.

• Bonded-phase packing: covalent bonds are involved.

Normal vs. Reversed Phase Packings

Normal phase:
• Early LC was based on highly polar stationary phases, e.g, triethylene glycol or water.
• A non-polar solvent served as the mobile phase.
• The least polar component is eluted first. An increase in the polarity of the mobile phase
leads to a decrease in the elution time.
Reversed-phase:
• The stationary phase is typically a non-polar hydrocarbon while the mobile phase is a
polar solvent e.g., water, methanol.
o The most polar component is eluted first.
o An increase in the polarity of the mobile phase leads to an increase in elution
time.
o This type is usually used in modern instruments.

Comparison of GC and HPLC

• Both methods:
o Highly efficient, selective
o Widely applicable
o Utilise small sample sizes
o Usually non-destructive
o Easily adapted
• Advantages:
HPLC GC
• Used for non-volatile, thermally • Relatively simple and inexpensive
ustable samples
• Rapid
• Iorganic ions
• Superior resolution, particularly with
capillary columns
• Easily interfaced with mass
spectroscopy

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 9
Bartley-Hynes (University of Technology, Jamaica)
 • Column Efficiency in Chromatography

Example of a Chromatogram:

The effectiveness of a chromatographic column to separate solutes is dependent on the relative

rates at which the species are eluted. These rates are determined by the partition ratios of the
solutes between the two phases. Ideally, the partition ratio for a component should be constant
over a wide range of concentrations.
An equilibrium describes the partitioning of a solute A between the mobile and stationary
phases: Amobile  Astationary

The partition ratio (equilibrium constant) is defined as:

Cs ns / Vs
Kc = =
Cm nm / Vm

Cs, ns = concentration of solute in stationary phase

Cm, nm = concentration of solute in mobile phase
Vs, Vm = volume of stationary phase and mobile phase

Retention Time – can easily be measured and is a function of Kc.

NOTE: tR’ = tS

The first peak relates to a solute that is not retained by column, hence, tM is called dead or void
time.
Retention time = tR = tS + tM
L
Rate of migration of solute (cm/s) = v = Rate of migration of mobile phase =
tR
L
u=
tM
v = u ( fractionof timesolute spend sin mobilephas e )

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 10
Bartley-Hynes (University of Technology, Jamaica)
  molesofsol uteinmobil ephase 
v = u
 

 totalmoles ofsolute 

u
v=
1 +KcVs / Vm

Retention or capacity factor, k, is not dependent on column geometry or volumetric flow rate:
KAVs u
kA = ⇒ v=
VM 1 + kA
and
tR −tM tR '
kA =
tM
=
tM
tR’ = adjusted retention time

Selectivity factor, α – a measure of the relative migration rates of two solutes, i.e., how well the
peaks are separated.
KB kB (tR ) B −tM
α= = =
KA kA (tR ) A −tM

Description of Column Efficiency – Plate Theory

σ2
Plate Height = H =
L
Plate Count/number of theoretical plates,
L
N =
H
L = length of column packing

2 2
 tR   tR 
N =16   =5.54   W = width at base of peak; W1/2 = width at half height of peak
W  W 1 / 2 

NOTE: Efficiency of the column increases as N increases and H decreases.

• Band broadening reflects a loss in column efficiency.

It depends on the flow rate of the mobile phase:

,u
Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 11
Bartley-Hynes (University of Technology, Jamaica)
Column Resolution, Rs
• Tells how far apart two bands are relative to their widths.
2[(tR ) B − (tR ) A] ( RS )1 N1
RS = and =
WA +WB ( RS ) 2 N2

General Elution Problem:

1 2 3 4 5 6 (a)

1,2 3 4 5 6 (b)

1 2 3 4 56 (c)

For the above diagram:

a) Good retention factors for components 1& 2.
b) Changing conditions to optimize separation of components 5 & 6 bunches the peaks for
components 1-4.
c) Good retention factors for components 3 & 4.

To compensate for this problem, conditions can be changed as the separation proceeds:
Immediately after components 1 & 2 are eluted, the conditions can be changed to elute
components 3 & 4 and then changed again to elute components 5 & 6.
In liquid chromatography, this is effected using gradient elution (or solvent programming) –
composition of the mobile phase is varied during the elution. Elution with constant mobile phase
composition is called isocratic elution.
In gas chromatography, temperature programming is employed – temperature is varied.

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. 12
Bartley-Hynes (University of Technology, Jamaica)