Introduction to Chromatography

Chromatography is used to separate, identify and quantitate the

components in a mixture.

* Operates on the same

principle as extraction,
but one phase is held in
place while the other
moves past it.

The stationary phase is the substance to which solvents and the

analyte travels through or binds to.

The mobile phase is the analyte and solvent mixture which travels
through the stationary phase.
 A chromatogram is the visual output of the chromatograph. Different
peaks or patterns on the chromatogram correspond to different
components of the separated mixture

The retention time is the characteristic time it takes for a particular

molecule to pass through the system.
The components that are strongly retained by the stationary
phase move only slowly (i.e. longer retention time) with the
flow of mobile phase.
Two ways of categorizing chromatographic methods:

1. Based upon the physical means by which the stationary and

mobile phase are brought into contact.

Column chromatography - stationary phase is held in a narrow tube

Planar chromatography - stationary phase is supported on a flat plane
2. Based upon the types of mobile and stationary phases and the
kinds of equilibria involved in the transfer of solutes between
phases.

General Classification Specific method Stationary phase Type of Equilibrium

Liquid-liquid partition Liquid adsorbed on a solid Partition between immiscible liquid

Organic species bonded Partition between liquid and

Liquid- bonded phase
to a solid surface. bonded surface
liquid chrom. Liquid-solid, or
(mobile phase: adsorption
Solid Adsorption

liquid)
Ion-exchange Ion- exchange resin Ion-exchange

Liquid in interstices of
Size exclusion Partition/sieving
polymeric solid
Liquid absorbed on a Partition between gas and
Gas- liquid
solid liquid
gas chrom. Organic species bonded Partition between liquid and
Gas- bonded phase
(mobile phase: gas) to a olid surface bonded surface

Gas- solid Solid Adsorption

Supercritical fluid chrom(SFC) Organic species bonded Partition between supercritical

(mobile phase: Supercritical fluid) to a solid surface. fluid and bonded surface
Elution involves transporting a species through column by continuous
addition of fresh mobile phase.

Separation of Mixture of A and B

A+ B

packed column B

Detector
t0 t1 t2 t3 t4

A B
Detector signal

t0 t1 t2 t3 t4
time
Normal Phase Chromatography
- Polar stationary phase
- Nonpolar mobile phase
*** More nonpolar solute elutes first

Reversed Phase Chromatography

- Nonpolar stationary phase
- Polar mobile phase
*** More polar solute elutes first
Introduction of additional mobile phase (eluent) forces the mobile
phase containing a part of the sample down the column, where
further partition between the mobile phase and fresh portion of
the stationary phase occurs.

Concentration Profiles of Analytes bands A and B at two

different times in their Migration

t1 t2
Concentration

The effectiveness of a chromatographic column in separating 2 analytes

depends in part upon the relative rates at which the 2 species are eluted.
 These rate of elution is determined by the magnitude of the equilibrium
constant for the reaction by which the species distribute itself
between the mobile and stationary phase.

Partition Coefficient or Partition Ratio, K

For the analyte species A

Amobile ⇔ Astationary K= CS/CM

CS is the molar concentration of the analyte in stationary phase
CM is the molar concentration of the analyte in mobile phase

Ideally, the partion ratio is constant over a wide range of

concentrations: that is CS is directly proportional to CM.

Chromatography in which the partition coefficient is constant is

termed as linear chromatography.
Retention time, tR
Retention time (tR) is the time it takes after sample injection for the analyte
peak to reach the detector.

The time for the unretained species to reach the detector is called dead
time (or injection time) (tM).
The rate of migration of the unretained species is the same as the
average rate of the mobile phase.

tR of B

tR of A
tM

Typical Chromatogram for a Two-component Mixture

Average linear rate of molecules of solute migration:

  L / tR L = column length
Average linear velocity (flow rate) of molecules of mobile phase:

u  L / tM
* Distance traveled by
solvent per unit time

Relationship between Migration Rate and Partition Ratio

 1 
   u 
1  KVS / VM 
where
VS is volume of analyte in stationary phase
VM is volume of analyte in mobile phase

Capacity Factor, k’: The rate of solute migration
- widely used to describe the migration rates of solutes on columns
For solute A, the capacity factor, k’A is defined as
K AVS
k'A 
VM
 The capacity factor, k’A can also be expressed in terms of tR:

 The longer a component is retained by the

tR  tM
k'A 
column, the greater is the capacity factor
 One of the parameters used to check integrity
tM of column is to measure the capacity factor of
a standard

The Selectivity Factor, α: Relative migration rates (Relative retention)

Theasselectivity factor α of a column for the two species A and B is defined
KB

KA
where KB is the partition ratio for the more strongly retained solute B and KA
is for the less retained solute A.
By definition, α is always greater than unity.
can also be expressed in terms of k’:
Selectivity factor,α,
k'B

k'A
 Selectivity factor,α, can also be expressed in terms of tR:

t R B  t M  The greater the relative retention, the greater

 the separation between 2 components.
t R A  t M  Independent of flow rate, thus can be used to
help identify peaks when flow rate changes.

 A Quantitative Description of Column Efficiency

Two related terms are widely used as quantitative measures of
chromatographic column efficiency:
(1) plate height H and
(2) plate count or number of theoretical plates N

The two are related by the equation

N = L/H
where L is the length (usually in cm) of the column packing
Column Efficiency (Plate Height), H is defined as

 2
where σ2 is variance of
H the analyte peak
Number of Molecules L

(L - 1σ) (L + 1σ) LW

 4t R
W = width of base

L
Distance migrated

•The smaller the plate height, the narrower the bandwidth
• The ability of a column to separate components of a mixture is improved by
decreasing the plate height

Substituting the expression of σ LW 2

H
16t R 
into the H equation gives: 2
But N = L/H. Thus 2
t R 
N  16 
W 
Alternatively, the width at half-peak height, W 1/2 can also be used.
2
 t R 
 N  5.55 
W1/2 

The efficiency of chromatographic column increases as the number of

plates become greater and as the plate height becomes smaller.

Minimum H, maximum N = maximum efficiency
 Variables that affect column efficiency
 Linear velocity of mobile phase (u)
 Diffusion coefficient in mobile phase (DM)*
 Diffusion coefficient in stationary phase. (DS)*
 Capacity factor (k’)
 Diameter of packing particle (dP)
 Thickness of liquid coating on stationary phase (df)

Effect of mobile phase flow rate:

Kinetic effects on column efficiency depends upon the length of time the
mobile phase is in contact with the stationary phase, which is dependent to
flow rate of mobile phase.

Liquid Chrom Gas Chrom

H (mm) H (mm)

0.1 3.0
0 0.5 1.0 0 2.0 8.0
Flow rate (cm/s) Flow rate (cm/s)
HPLC column
 Gas
Chromatographic
column
 - both show minimum in H at low flow rates
- LC are obtained at lower flow rates than GC
- H in LC are an order of magnitude smaller than those in GC

Disadvantage of LC:

- it is impractical to employ LC column longer than 50 cm (because

of high P drop)
- GC column may be 50 cm or more in length.
- greater number of plates N, more superior
 Theory of Band Broadening
The efficiency of most columns can be approximated by the
van Deemter equation.
* It tells us how the column and
H = A + B/u + Cux flow rate affect the plate height

“A” Term : Multiple path term

“B” Term : Longitudinal diffusion term

“C” Term : Mass transfer term / Equilibrium time

Packed columns: A, B, C  0
Open tubular columns : A = 0
Capillary electrophoresis : A = C = 0
“A” Term: Multiple path term
—solute molecules can take different paths
through the stationary phase, so some take longer to go
through column than others.
--- This is term is independent of flow rate.

residence time in the column for

molecules of same species is variable

This effect is called eddy diffusion.

“B” Term: Longitudinal diffusion term
—the longer time a solute stays on the
column the more time it has to diffuse, therefore
spreading out its concentration.
---This term is inversely proportional to flow rate
(i.e. longer time on column with slower flow rate and vice
versa)
“C” Term: Mass transfer
Cux = (Cs + Cm)ux
term
—this arises from slow
equilibrium for solutes
partitioning between the mobile
and stationary phases. If the
equilibrium is slow, while a
solute molecule is in the
stationary phase other
molecules will have traveled
down the column a certain
distance.
---The overall result is
band spreading that is
proportional to the flow rate.
The Stationary Phase Mass Transfer Coefficient term (CSu)

- directly proportional to the thickness of the film (stationary phase)

and inversely proportional to the diffusion coefficient, DS

- with thick films molecules travel farther to reach the surface and
with small DS, they travel slower.

Consequence: slower rate of mass transfer and an increase in plate

height, H.

Decreasing stationary phase thickness reduces plate height and

increases efficiency because the solute can diffuse faster

The Mobile Phase Mass Transfer Coefficient term (CMu)

- CM is inversely proportional to DM

- zone broadening in the mobile phase is due in part to

the multitude of pathways by which a molecule can find
its way through a packed column.
Effect of Mobile Phase Velocity on terms in van Deemter equation

H
the curve is the summation
Contribution CS u of various effects
to H, cm
CMu
an optimum flow rate exists at
which H is a minimum and
B/u the separation effieciency
is a maximum,
Mobile Phase velocity, u (cm/s)

Summary of Methods for Reducing Band Broadening

at same velocity, the narrower the column the more efficient

with gaseous mobile phase, lowering T and thus DM increases

efficiency

with liquid mobile phase, thickness of layer should be minimized

Asymmetric Bandshapes
• Overloading produces a gradual rise and an abrupt fall of the
chromatographic peak
• A long tail occurs when some sites retain solute more strongly than
other sites.
Efficiency of Separation
Two factors contribute to how well compounds are separated by
chromatography:

1) Difference in elution times between peaks

- The farther apart, the better their separation

2) How broad the peaks are

- The wider the peaks, the poorer their separation

Column Resolution, Rs
The resolution R, of a column provides a quantitative measure of its
ability to separate two analytes.

The resolution of each column is defined as

Z 2t R B  t R A 
RS  
WA  WB WA  WB
 The resolution of a column can also be expressed in terms of Capacity
and Selectivity Factors:
N  1 k'B 
RS    
4  a 1 k'B 
where k’B is the capacity factor of slower-moving solute

This equation can be rearranged to give the number of plates, N.


2
   1 k'B 
2

N  16R 
2
  
 1  k'B 
s

Effect of Resolution on Retention Time

The time (tR)B required to elute to species with a resolution of Rs is
 1 k'B 
2 3

16R H 
2
 u = linear velocity of
t R B  s
  mobile phase
u  1 k'B 2
Example
Substance A and B have retention times of 16.40 and 17.63 min,
respectively, on a 30.0-cm column. An unretained species passes
through the column in 1.30 min. The peak widths (at base) for A and B
are 1.11 and 1.21 min, respectively. Calculate (a) the column resolution,
(b) the average number of plates in the column, (c) the plate height, (d)
the length of column required to achieve a resolutions of 1.5 and (e) the
time required to elute substance B on the column that gives RS = 1.5

Solution
2t R B  t R A  217.63 16.40
a) RS    1.06
WA  WB 1.11 1.21

b) t R 2 16.40 2

N  16   16   3493
W  1.11 
 t R 2 17.63 2
and
N  16   16   3397
W  1.21 
 Nave = (3493 + 3397)/2 = 3445 = 3.4 x 103


c) H = L/N = 30.0/3445 = 8.7 x 10-3 cm

(d) k’ and α do not change greatly with increasing N and L.

Thus, substituting N1 and N2 into the RS equation and dividing one
of the resulting equations by the other yield
RS 1 N1

RS 2 N2
where the subscripts 1 and 2 refer to the original and longer columns,
respectively.

Substituting the appropriate values for N1, (RS)1, and (RS)2 gives
1.06 3445

1.5 N2

1.5 2
N 2  3445   6.9x10
3

1.06 

But L = NH = (6.9 x103) (8.6 x10-3) = 60 cm

(e) Substituting (RS)1 and (RS)2 into N equation and dividing yield

t R 1 RS 1 17.63 1.06

2 2

  
t R 2 RS 22 t R 2 1.52
(tR)2 = 35 min

Thus, to
obtain the improved resolution, the column length and
consequently the separation time must be doubled.