Chromatography

Chromatography

Chromatography

The history of modern chromatography

What is a chromatographic method
Classifying Analytical Separations
General Theory of Column Chromatography
Applications

Chromatography

The history of modern chromatography

1872 - 1919 The Russian botanist Mikhail Tswett
used a column packed with a stationary phase of
calcium carbonate and a mobile phase of petroleum
ether to separate colored pigments from plant
extracts.
1941 Martin and Singe established the
importance theory for chromatographic
separations (Nobel Prize in 1952)
Since then, chromatography in its many forms has
become the most important and widely used
separation technique
Chromatography

What is a chromatographic method

Chromatography is a physical method of
separation in which the components to be
separated are distributed between two phases,
one of which is stationary (stationary phase),
while the other (the mobile phase) moves in a
definite directions

Chromatography

Classifying Analytical Separations

Analytical separations may be classified in
three ways:
(1) by the physical state of the mobile phase
and stationary phase;
(2) by the method of contact between the
mobile phase and stationary phase;
(3) or by the chemical or physical mechanism
responsible for separating the samples
constituents.

Chromatography

Classifying Analytical Separations

(1) Analytical separations by the physical
state of the mobile phase and stationary
phase:

The mobile phase is usually a liquid or a gas, and

the stationary phase is a solid or a liquid film
coated on a solid surface.
Chromatographic techniques are often named
by listing the type of mobile phase, followed by
the type of stationary phase. (For example,
gasliquid chromatography: the mobile phase is
a gas and the stationary phase is a liquid.)
If only one phase is indicated, as in gas
chromatography, it is assumed to be the mobile
phase.

Chromatography

GC instrument

Chromatography

Schematic diagram for a typical gas chromatograph

Chromatography

GC

Chromatography

Chromatography

Chromatography

Chromatography

Stationary Phase
Packed columns
Capillary columns the most widely used
Wall-coated open-tubular (WCOT)

wall-coated open tubular consist of a capillary tube whose

walls are coated with liquid stationary phase.

Support coated open-tubular (SCOT)

support coated open tubular consist of a capillary lined

with a thin layer of support material such as diatomaceous
earth, onto which the stationary phase has been adsorbed.

Porous layer open-tubular (PLOT)

porous layer open tubular columns in which a thin layer of

adsorbent is affixed to the inner walls of the capillary.

Chromatography

Stationary Phase

Chromatography

Stationary Phase

Glass
surface

An example of a trimethylsilyl deactivating group.

Chromatography

Stationary
Phase

Chromatography

Classification of Chromatographic
Techniques

GC

Typical gas chromatogram of comples mixture using a capilary column

Chromatography

Gas Chromatography

Mobile Phase
Chromatographic Columns
Stationary Phases
Sample Introduction
Temperature Control
Detectors for Gas Chromatography
Quantitative Applications

Chromatography

What compounds Can Be

Determined by GC
- All gases
- Most nonionized organic molecules, solid or liquid,
containing up to about 25 carbons
- Many organometallic compounds (volatile
derivatives of metal ions may be prepared)

Chromatography

Chromatography

Schematic diagram of a high-performance

liquid chromatograph
Chromatography

Chromatography

Chromatography

High-Performance Liquid
Chromatography

Mobile Phases
HPLC Columns
Stationary Phases
Sample Introduction
Detectors for HPLC
Quantitative Applications

Chromatography

Classifying Analytical Separations

(2) Analytical separations by the method
of contact between the mobile phase and
stationary phase
Column chromatography:

The stationary phase is placed in a narrow column

through which the mobile phase moves under the
influence of gravity or pressure
The stationary phase is either a solid or a thin, liquid
film coating on a solid particulate packing material or
the columns walls.

Planar chromatography:

The stationary phase coats a flat glass, metal, or

plastic plate and is placed in a developing chamber.
A reservoir containing the mobile phase is placed in
contact with the stationary phase, and the mobile
phase moves by capillary action.

Chromatography

Chromatography

Classifying Analytical Separations

(3) Analytical separations by the chemical
or physical mechanism responsible for
separating the samples constituents
Adsorption chromatography:

solutes separate based on their ability to adsorb to a

solid stationary phase.

Partition chromatography:

A thin liquid film coating a solid support serves as the

stationary phase.
Separation is based on a difference in the equilibrium
partitioning of solutes between the liquid stationary
phase and the mobile phase.

Chromatography

Classifying Analytical Separations

(3) Analytical separations by the chemical

or physical mechanism responsible for

separating the samples constituents.

Adsorption chromatography: solutes separate based on

their ability to adsorb to a solid stationary phase.
Partition chromatography: a thin liquid film coating a
solid support serves as the stationary phase. Separation
is based on a difference in the equilibrium partitioning of
solutes between the liquid stationary phase and the
mobile phase.

Chromatography

Classifying Analytical Separations

(3) Analytical separations by the chemical

or physical mechanism responsible for

separating the samples constituents.

Ion exchange chromatography: Stationary phases

consisting of a solid support with covalently attached
anionic (e.g., SO3) or cationic (e.g., N(CH3)3+) functional
groups. Ionic solutes are attracted to the stationary
phase by electrostatic forces.
Size-exclusion chromatography: Porous gels are used as
stationary phases. Separation is due to differences in
the size of the solutes. Large solutes are unable to
penetrate into the porous stationary phase and so quickly
pass through the column. Smaller solutes enter into the
porous stationary phase, increasing the time spent on the
column.

Chromatography

Classifying Analytical Separations

Not all separation methods require a stationary
phase.
Electrophoretic separation: charged solutes migrate
under the influence of an applied potential field.
Differences in the mobility of the ions account for
their separation.

Chromatography

Classifying Analytical Separations (3)

a) Adsorption
chromatography

b) Partition
chromatography

c) Ion-exchange
chromatogIonraphy

d) Size-exclusion
chromatography

Chromatography

General Theory of
Column Chromatography

Progress of a
column
chromatographic
separation
showing the
separation of
two solute
bands.

Chromatography

General Theory of Column

Chromatography

chromatogram

A plot of the detectors signal as function of elution

time or volume.

retention time

The time a solute takes to move from the point of

injection to the detector (tr).

retention volume

The volume of mobile phase needed to move a solute

from its point of injection to the detector (Vr).
Dividing the retention volume by the mobile phases
flow rate, u, gives the retention time.

baseline width

The width of a solutes chromatographic band

measured at the baseline (w).

Chromatography

General Theory of Column

Chromatography
void time
The time required for unretained solutes to move
from the point of injection to the detector (tm).

void volume
The volume of mobile phase needed to move an
unretained solute from the point of injection to the
detector.

Chromatography

General Theory of Column

Chromatography
tr : retention time
tm : void time
W : baseline width

Chromatography

Chromatographic Resolution
resolution
The separation between two
chromatographic bands (R).

Chromatography

Three examples of chromatographic

resolution

Chromatographic Resolution- Example

In a chromatographic analysis of lemon oil a peak
for limonene has a retention time of 8.36 min
with a baseline width of 0.96 min. g-Terpinene
elutes at 9.54 min, with a baseline width of 0.64
min. What is the resolution between the two
peaks?

Chromatography

capacity factor
capacity factor
A measure of how strongly a solute is retained by the
stationary phase (k ).

adjusted retention time

The difference between a solutes retention time and
columns void time (tr).

Chromatography

capacity factor
In a chromatographic analysis of low-molecularweight acids, butyric acid elutes with a
retention time of 7.63 min. The columns void
time is 0.31 min. Calculate the capacity factor
for butyric acid.

Chromatography

Column Selectivity
selectivity factor
The ratio of capacity factors for two solutes showing
the columns selectivity for one of the solutes ().

Chromatography

Column Selectivity - Example

In a chromatographic analysis of low-molecularweight acids, butyric acid elutes with a retention
time of 7.63 min. The columns void time is 0.31 min.
The retention time for isobutyric acid is 5.98 min.
What is the selectivity factor for isobutyric acid
and butyric acid?
butyric
isobutyric

Chromatography

Column Efficiency

Column efficiency provides a quantitative

measure of the extent of band broadening
band broadening
the increase in a solutes baseline width as it moves
from the point of injection to the detector.

theoretical plate
Martin and Synge treated the chromatographic
column as though it consists of discrete sections
(theoretical plate) at which partitioning of the solute
between the stationary and mobile phases occurs.
With N: theoretical plates
H: the height of a theoretical plate
L: the column length
Chromatography

Column Efficiency
The number of theoretical plates
The number of theoretical plates depends on both the
properties of the column and the solute.
The number of theoretical plates for a column is not
fixed and may vary from solute to solute.
Columns with more theoretical plates are more likely
to separate a complex mixture.

tr: the retention time

W: the width of the
chromatographic peak

W1/2: the width of the

chromatographic peak
at half its height
Chromatography

Theoretical plates - Example

A chromatographic analysis for the chlorinated
pesticide Dieldrin gives a peak with a retention
time of 8.68 min and a baseline width of 0.29
min. How many theoretical plates are involved in
this separation? Given that the column used in
this analysis is 2.0 meters long, what is the
height of a theoretical plate?

Chromatography

Nonideal Behavior
fronting
A tail at the beginning of a chromatographic peak,
usually due to injecting too much sample (a).

Tailing
A tail at the end of a chromatographic peak, usually
due to the presence of highly active sites in the
stationary phase (b).

Chromatography

Optimizing Chromatographic
Separations

NB: the number of

theoretical plates
(the effect of
column efficiency)

(1)

Chromatography

: the influence of
column selectivity

(2)

kB, (the effect of

solute Bs capacity
factor)

(3)

Using the Capacity Factor to

Optimize Resolution (3)
Increasing kB (when kB small)

when the original value of kB is 1, increasing its

value to 10 gives an 82% improvement in
resolution; a further increase to 15 provides a
net improvement in resolution of only 87.5%.
However, improvement in resolution obtained by
increasing kB generally comes at the expense of
a longer analysis time.

Chromatography

Using the Capacity Factor to

Optimize Resolution (3)
Increasing kB

by decreasing the columns temperature in gas

chromatography
At a lower temperature a solutes vapor pressure
decreases (it spends more time in the stationary
phase). Therefore, its capacity factor increases.

Temperature programming in gas chromatography

The process of changing the columns temperature to
enhance the separation of both early and late eluting
solute accomplished.

Chromatography

A typical temperature program

(c)
(b)

(a)

(a) = initial temperature and time

(b) = ramp (C/min)
(c) = final hold time and temperature
Some GCs will allow for far more complex temperature programming
Chromatography

Using the Capacity Factor to

Optimize Resolution (3)
Increasing kB

By decreasing the solvent strength in liquid

chromatography
When the mobile phase has a low solvent strength,
solutes spend proportionally more time in the
stationary phase, thereby increasing their capacity
factors.

Gradient elution in liquid chromatography

The process of changing the mobile phases solvent
strength to enhance the separation of both early and late
eluting solutes.
Chromatography

Using the Capacity Factor to

Optimize Resolution (3)

Chromatography

Using Column Selectivity to Optimize

Resolution (2)
A second approach to improving resolution is to
adjust alpha, a.
when a is nearly 1, it usually is not possible to improve
resolution by adjusting kB or N.
changing a from 1.1 to 1.5 improves resolution by
267%.

In gas chromatography, adjustments in a are

usually accomplished by changing the stationary
phase,
In liquid chromatography, changing the
composition of the mobile phase is used.
Chromatography

Using Column Selectivity to Optimize

Resolution (2)

The variation in retention time with mobile phase pH

Chromatography

Using Column Selectivity to Optimize

Resolution (2)

the change in alpha with mobile phase pH

Chromatography

Using Column Efficiency to Optimize

Resolution (1)
Increase the length of the column (increase
retention time)
Decrease the height of a theoretical plate
To determine how the height of a theoretical plate
can be decreased, it is necessary to understand the
experimental factors contributing to the broadening
of a solutes chromatographic band.
The height of a theoretical plate is determined by
four contributions: multiple paths, longitudinal
diffusion, mass transfer in the stationary phase, and
mass transfer in the mobile phase.

Chromatography

Chromatography Effiency The van

Deemter Equation

The net height of a theoretical plate is a

summation of three terms:

Multiple Paths
Longitudinal dispersion term
Longitudinal Diffusion
Mass transfer
Mass Transfer
term

H A

Multiple path term

C u

A, B and C are
constants for
a given system

u: the average linear velocity of the carrier gas in cm/s (or

the liquid mobile-phase velocity for liquid chromatography)
Chromatography

Multiple Paths

Molecules passing through a stationary phase that differ in

path and length

Hp 2dp

A term

Where Hp= contribution to theoretical plate

= constant associated with consistency of packing
dp= average diameter of packing material
Chromatography

Open tubular column Hp=0

Longitudinal Diffusion

Hd

2Dm

B term

u
Where Dm = the solutes diffusion coefficient in the mobile phase
= constant relating to column packing
m = mobile phase velocity

B
Chromatography

decreases by increasing the flow rate

Mass Transfer
Diffusion of the
solute between
the mobile and
stationary phase
interface

Hs
C term

Hm

qk 'd 2f
2

Where

(1 k ') Ds

fn ( d 2p ,d c2 )

Chromatography

Dm

df =thickness of stationary phase

dc= the columns diameter
dp= average diameter of packing material
Ds= solutes diffusion coefficient in stationary phase
Dm= solutes diffusion coefficient in mobile phase
q = constant related to packing material
k = capacity factor

the exact form of Hm is unknown

Mass transfer
2
dp

1
C
6 DM

Cu decreases by
Where decreasing the flow rate

DM = the solutes diffusion

coefficient in the mobile phase
dp= average diameter of
packing material

The type and amount of liquid phase (of the

stationary phase), temperature
For example, C decreases : thin stationary liquid
phase film to minimize diffusion within this phase

Chromatography

Gas Chromatography Effiency The van

Deemter Equation
The net height of a theoretical plate is a
summation of three terms:
Multiple Paths (Eddy diffusion)
Longitudinal dispersion term
Longitudinal Diffusion
Mass Transfer
Mass transfer

H A

Multiple path term

H: The height of a theoretical plate

C u

term

A, B and C are constants

for a given system

u: the average linear velocity of the carrier gas in cm/s (or the liquid
mobile-phase velocity for liquid chromatography)
Chromatography

Multiple Paths

Hp: The contribution of multiple paths

to the height of a theoretical plate,

dp is the average diameter of the

particulate packing material

Chromatography

is a constant accounting for the

consistency of the packing

Longitudinal Diffusion
longitudinal diffusion
One contribution to band broadening in which solutes
diffuse from areas of high concentration to areas of
low concentration.
Because a solutes diffusion coefficient is larger in a
gaseous mobile phase than in a liquid mobile phase,
longitudinal diffusion is a more serious problem in gas
chromatography

Dm: the solutes diffusion

coefficient in the mobile phase

u: the mobile phase velocity

: a constant related to the
column packing
Chromatography

Mass transfer
The constant C term is the interphase mass transfer term and is due
to the finite time required for equilibrium of the solute to be
established between the two phases as it moves between the mobile
and stationary phases.
Diffusion of the solute between
2
the mobile and stationary phase
p
interface

1 d
C
6 DM

Where

The term Cu decreases by

decreasing the flow rate

DM = the solutes diffusion coefficient in the mobile phase

dp= average diameter of packing material

The type and amount of liquid phase (of the stationary phase), temperature
For example, C decreases : thin stationary liquid phase film to
minimize diffusion within this phase
for LC, smalle particles, thin stationary phase films,
low-viscosity mobile phases and high temperatures.
Chromatography

Van Deemter Curve

Chromatography

van Deemter Curves

Chromatography

van Deemter Curves

Chromatography

HPLC - Example
A 2.013-g sample of dried soil is extracted with
20.00 mL of methylene chloride. After filtering to
remove the soil, a 1-mL portion of the extract is
removed and diluted to 10 mL with acetonitrile.
Injecting 5 mL of the diluted extract into an HPLC
gives a signal of 0.217 for the PAH fluoranthene.
When 5 mL of a 20.0-ppm fluoranthene standard is
analyzed using the same conditions, a signal of
0.258 is measured. Report the parts per million of
fluoranthene in the soil.

Chromatography

Quantitative Measurements

Peak area ratio

(EtOH/PrOH)
0.24945
0.619154
1.1918
1.86534
2.39374
1.17868

The blood alcohol concentration is 0.142% (wt/vol)

Chromatography