2. DEFINITION
It is a physical separation method in
which the components of a mixture are
separated by differences in their
distribution between two phases, one
of which is stationary (stationary phase)
while the other (mobile phase) moves
through it in a definite direction .
3.
4. Stationary phase
(Steady Phase)
(Solid or liquid)
Mobile phase
(Moving Phase)
(Liquid or Gas)
Detectors
If Solid stationary
phase- Adsorption
Liquid- Partition
5. HISTORY
The subject of Chromatography was introduced into scientific
world in a very modest way by M. Tswett in 1906.
He employed a technique to separate various pigments such as
chlorophylls and xanthophylls by passing the solution of these
compounds into the glass column which was packed with
finely divided calcium carbonate.
After the later, Thompson and Way had realized the Ion
Exchange properties of soils.
In 1935, Adams and Holmes give Ion exchange
chromatography.
The concept of Gas-Liquid Chromatography was first
introduced by Martin and Synge in 1941.
In 1944, from Martin laboratory, the separation of amino acid
by paper chromatography was reported.
In 1952, the importance of the chromatography was observed
when both Synge and Martin were awarded with Nobel Prize.
6. In 1959, a technique known as Gel Filtration chromatography
was observed which is used to separate low molecular weight
substances from high molecular substances.
In 1960, further improvement in liquid chromatography led to
the development of High Performance Liquid Chromatography.
The following decade of 1970’s saw an improvement in the
field of adsorption chromatography in the form of Affinity
chromatography which was mainly based on biological
interactions.
A new field was originated which was supercritical fluid
chromatography.
Supercritical fluid chromatography is a hybrid of gas and liquid
chromatography and combine advantageous feature of the both
gas and liquid chromatography.
It will not be wrong to say that the entire twentieth century can
be named as the century of chromatography.
7. BASICPRINCIPLE
The samples are subjected to flow by
mobile phase onto or through the stable
stationary phase. The sample components
are separated into fractions based on their
relative affinity towards the two phases
during their travel.
8.
9.
10. CLASSIFICATION
Different methods were attempted for
classification of chromatography:
1) Based on nature of SP & MP
2) Based on Principle of separation
3) Based on modes of chromatography
4) Based on Physical mean
11. 1. BASEDONNATUREOFMP&SP
MP & SP
Liquid chromatography
Gas chromatography
Supercritical fluid
chromatography
(SFC)
SP= always solid or liquid coating on solid surface
MP= liquid or gas
12. Sr. No. SP MP CHROMATOGRAPHY
1 Solid Gas GSC
2 Liquid Gas GLC
3 Solid Liquid Paper
TLC
HPLC
4 Solid Resin Liquid Ion exchange
5 Solid Matrix Liquid Gel Permeation
6 Liquid Liquid TLC,
Partition Chrom
14. a. Partitionchromatography
PRINCIPLE:
• MP= Liquid or Gas
• SP= Liquid coated on solid support
• Partition - based on the relative solubility of
analyte in mobile and stationary phases
• Analytes are distributed according to their
partition coeffient.
• K= Cs/Cm
• If K is higher, comp retained more strongly by
SP.
15.
16.
17.
18. b. Adsorptionchromatography
PRINCIPLE:
SP= Solid on which sample components are adsorbed by
week ionic forces.
The small energy is required to break this forces which is
achieve by flow of MP
MP= Liquid in SLC and Gas in GSC
Separation is based mainly on differences between the
adsorption affinities of the sample components for the
surface of an active solid.
More affinity towards SP-travel slower-retain more
Less affinity towards MP-travel faster-elute 1st
19.
20. c. Size-exclusionchromatography
PRINCIPLE:
Separation is based mainly on exclusion effects, such as
differences in molecular size and/or shape or in charge.
•The term Size-Exclusion Chromatography may also be used
when separation is based on molecular size.
• The terms Gel Filtration and Gel-Permeation
Chromatography (GPC) were used earlier to describe this
process when the stationary phase is a swollen gel.
•SP= polymers which have been cross linked to yield an open
network with number of pores of same size
21.
22.
23. d. Affinitychromatography
PRINCIPLE:
SP= specific ligand
Many biological molecules having unique structure,
so they form strong covalent bond with SP.
The process is an entrapment in which the target
molecule becomes trapped on stationary phase.
The Stationary phase can then be removed from the
mixture, washed and then target molecule is
released from the entrapment.
Example- antigen with antibody
31. PARAMETER NP RP
SP Polar Non Polar
MP Non Polar Polar
Comp eluted 1st &
retain less
Non Polar Polar
Comp eluted last &
retain more
Polar Non Polar
Ex of SP Silica gel C8, C18
32. 4. BASED ON PHYSICAL MEANS
I. Column
chromatography
II. Planner
chromatography
SP=placed in narrow tube
MP=allow to flow under
pressure
Ex-Column chrom.
SP=supported on stainless
steel plate, glass, paper
MP=moves through SP
Ex-TLC, Paper chrom.
34. TERMINOLOGIES
1) Partition coefficient:K
K=
𝐶𝑠
𝐶𝑚
=
𝐶𝑜𝑛𝑐.𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑖𝑛 𝑆𝑃
𝐶𝑜𝑛𝑐.𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑖𝑛 𝑀𝑃
K is changed by changing temp, nature of SP & MP.
If K value is larger then comp. retain more in SP
If K value is smaller then comp.retain more in MP.
35. TERMINOLOGIES
2) Retention time: tr
Definition= Its time required to elute sample
component from SP by MP. OR
The distance of peak maximum from the origin in
chromatogram is expressed as tr.
It is Qualitative measurement.
36. TERMINOLOGIES
3) Retention volume: vr
Definition= It’s a volume of MP required to elute
sample component from SP.
Vr= F * tr
Its also defined as a product of flow rate & retention
time.
It’s quantitative measurement.
37. TERMINOLOGIES
4) Dead time t0 &
It’ is defined as a
time required for MP
to flow from one end
to another end of SP.
V0= F * t0
Dead volume v0
Definition= the volume
of MP corresponding to
dead time.
38. TERMINOLOGIES
5) Retention factor or capacity factor (k’)
It is used to describe migration rate of sample onto the
column.
K’=
𝑣−𝑣0
𝑣0
=
𝑡−𝑡0
𝑡0
It is independent of flow rate.
Ideal value=2-10
If value is less than 1 then elution becomes faster and
accurate determination of time becomes difficult.
A high k’ value indicates that the sample is highly
retained and has spend a significant time interacting
with SP.
39. TERMINOLOGIES
6) Asymmetric factor (Af)
It is defined as a ration of peak width of right side of tr
to the peak width of left side of tr.
Af=
𝐵𝐶
𝐶𝐴
=
𝑝𝑒𝑎𝑘 𝑤𝑖𝑑𝑡ℎ 𝑜𝑓 𝑟𝑖𝑔ℎ𝑡 𝑠𝑖𝑑𝑒
𝑝𝑒𝑎𝑘 𝑤𝑖𝑑𝑡ℎ 𝑜𝑓 𝑙𝑒𝑓𝑡 𝑠𝑖𝑑𝑒
Ideal value=1 or nearer to 1
If value is more than 1 then tailing observed
If value is less than 1 then fronting observed
40. TERMINOLOGIES
7) Resolution Rs
It is defined as quantitative measure of column which
provides it’s ability to separate two analytes.
Resolution is directly propotional to separation.
41. TERMINOLOGIES
8) Relative Retention α:
It is also known as selectivity factor.
defined as ability of chromatographic system to
chemically differentiate between two samples.
α =
𝑡−𝑡0
𝑡𝑟𝑒𝑓
−𝑡0
OR α =
𝑣−𝑣0
𝑣𝑟𝑒𝑓
−𝑣0
α =
𝐾2
𝐾1
where K=partition coefficient
α =
𝑘′
2
𝑘′
1
where k’=capacity factor
42. THEORIES OF CHROMATOGRAPY
There are two theories to explain
chromatography
1) Plate theory - older
developed by Martin & Synge in 1941
2) Rate theory - currently in use
Proposed by van Deemter in 1956
Accounts for the dynamics of the separation
43. In this theory, chromatographic system
(column) considered as divided into a
number (N) of adjacent imaginary
segments called theoretical plates
Within each theoretical plate analyte(s)
completely equilibrate between stationary
phase and mobile phase
Column Theoretical plate
PLATE THEORY- Martin & Synge in
1954, nobel price
44. The movement of solute is considered as a
series of stepwise transfer from one plate to
another plate.
The efficiency of separation is depends on
1. No. of plates α efficiency of separation
2. Width of plate α
1
𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦
Greater separation occurs with:
–greater number of theoretical plates (N)
–as plate height (H or HETP) becomes smaller
L = N H or H = L / N
where L is length of column, N is number of plates,
and H is height of plates or height equivalent to
theoretical plate (HETP)
45. Asumption:
1. Elution peak will be symmetrical
2. Band broadening directly related to no. of
plates.
Limitation:
1. It does not describe column performance
with respect to velocity of MP.
46. To overcome the limitation of plate theory,
rate theory was proposed by Van Deemter.
According to this theory, MP is
continuously passes over SP at fix rate. So
that solute molecules are partitioned &
transported in chromatographic system.
The rate theory has resulted in a number
of different equations like
Van Deemter equation
RATE THEORY- Van Deemter 1956
47. H = A + B/u + µ [CM +CS]
H: Height of theoretical plates/HETP
A: Eddy’s diffusion, Which depends on column packing
material size & shape. It is due to different path followed
by solute
B: Molecular or longitudinal diffusion
H will increased due to diffusion.
Diffusion will decreased by increasing MP flow rate
C: Mass transfer of solute from SP to MP
CM= Mobile phase mass transfer
CS= Stationary phase mass transfer
µ : average linear velocity
To obtain maximum efficiency, H value must be less. For
that constant A, B & C must be minimum.
Van Deemter equation
48. Term A
-molecules may travel
unequal distances
-independent of u
-depends on size of SP
particles
-It can be minimized by
using small & uniform
particles.
Term A:Eddy’s diffusion
time
Eddy diffusion
MP moves through the column
which is packed with stationary
phase. Solute molecules will take
different paths through the
stationary phase at random. This
will cause broadening of the
band, because different paths
are of different lengths.
49.
50.
51. Term B Longitudinal diffusion
Band broadening increases when time for diffusion
within column increases.
So we must reduces the time available for solute for
diffusion
For that purpose, Increases MP flow rate.
So B is minimized by increasing MP flow rate.
One of the main causes of band broadening is DIFFUSION of solute from
concentrated region to diluted region
The diffusion coefficient measures the ratio at which a substance
moves randomly from a region of higher concentration
to a region of lower concentration
52. MOBILE PHASE
SAMPLE
CONC. OF GAS- MORE IN MIDDLE- SO SAMPLE WHICH IS PRESENT IN MIDDLE WILL
ELUTE FIRST-
CONC. OF GAS- LESS AT EDGES- SO SAMPLE WHICH IS PRESENT IN EDGES-WILL
ELUTE LAST-
so PEAK WIL BE BROAD
In column chromatgrpahy- value of B is 0
B. Longitudinal diffusion:
53.
54. Term C (Mass Transfer)
Bandwidth
Stationary
phase
Mobile
phase
Elution
It is understand by time required to established
equilibrium of solute between SP & MP.
The faster the MP moves, the less time is
available for the equilibrium for mass transfer.
If flow rate increases, less time exist for
equilibrium.
Slow
equilibration
Broadened bandwidth
