The document discusses the principles of chromatography. It describes how chromatography separates components in a mixture based on differences in their interactions with mobile and stationary phases. It discusses how Michael Tswett first demonstrated chromatography in 1903 and the key aspects of how it works. These include how retention time, partition coefficients, selectivity factors and efficiency parameters like plate number and height equivalent to a theoretical plate are used to characterize chromatographic separations.

1. Submitted by: Preeti choudhary
M.Sc.(Applied Physics)

2. PRINCIPLES OF CHROMATOGRAPHY

3. Principles of Chromatography
Chromatography is the process of
separating components in a mixture from one
another based on difference in their
properties.
A common feature to all chromatographic
methods is the distribution of the components
between two phases, the stationary phase and
the mobile phase.

4. Chromatography classification

5. Principles of Chromatography
The first detailed description of chromatography is
credited to Michael Tswett, a Russian biochemist, who
separated chlorophyll from a mixture of plant pigments
in 1903.
He placed a small amount of mixture on a column
packed with powdered calcium carbonate (the
stationary phase) and washed the sample through with
petroleum ether ( the mobile phase).

6. Principles of Chromatography
As the sample progressed down the column the
various components moved at different rates. Sample
components are carried by the mobile phase through
a bed of stationary phase.
Each component produced a band that had distinctive
color . Thus the Greek word chromatography for
colour and to write. Although the colored bands were
part of this first experiment, color is not important for
the method to work.

7. A + B
B
A B
B
B
A
A
Sample Mobile Phase

8. Principles of Chromatography
Individual species are retarded by the
stationary phase based on various
interactions such as :
• Surface adsorption
• Relative solubility
• Charge

9. Partition/Distribution Coefficient
As the mobile phase bearing the solute enters the
column, the solute distributes itself between stationary
and mobile phase.
This distribution between the 2 phases is described by the
Distribution Coefficient ‘K’, defined as
K = Cs / CM
where Cs & CM refer to the concentrations of the solute
in the stationary and mobile phases.

10. Partition Coefficient
 If the value of K = 1 then the solute is equally
distributed between stationary and mobile phases.
 For K < 1, the solute travels faster through the
column because it spends more time in mobile
phase.
 For K > 1, the solute will be retained in the
stationary phase or will exit the column after
longer time.
Different solutes will have different values of
distribution coefficients, so their movement
through the column will be of different rates.

11. Chromatogram
The detector produces a signal which is plotted
graphically on the chart of an electronic
recorder and is called a Chromatogram.
A chromatogram gives
• Qualitative information using retention time of
various peaks
• Quantitative data from peak area or peak height
of the components.

12. Chromatogram - Retention Times
tM = retention time of mobile phase (dead time)
tR = retention time of analyte (solute)
tS = time spent in stationary phase (adjusted retention time)
L = length of the column

13. Velocities : Linear rate of solute migration
M
R
t
L
t
L
v
=
=
µ
Velocity = distance/time  length of column/ retention
times
Velocity of solute:
Velocity of mobile phase:

14. Retention time and volume
Retention time, tR - time required to reach the peak
maximum from the point of injection.
Dead time, tM - time required for the unretained species
to reach the peak maximum from the point of
injection.
Retention volume, VR – volume of mobile phase
required to elute a solute to a maximum from a
column.
.

15. Velocity Relationships
MS
M
S
MMSS
SSMM
MM
VVK
v
c
c
K
VcVc
v
VcVc
Vc
v
/1
1
ConstantonDistributi
/1
1
+
×=
=
+
×=
+
×=
µ
µ
µ

16. Capacity and Selectivity Factors
• Capacity / Retention Factor (kA)– it describes
rate of migration of solute in a column or
relative indication of time spent by solute in a
column.
• Selectivity Factor (α) – It provides a measure
of how well a column separates the two
analytes

17. Capacity/Retention Factor
M
MR
A
AMR
A
MSAA
MS
t
tt
k
kt
L
t
L
k
v
VVKk
VVK
v
−
=
+
×=
+
×=
=
+
×=
1
1
1
1
Factor)(Retention/
/1
1
µ
µ
Adjusted retention time

18. Capacity/Retention Factor
where kAis thecapacity factor for solute A.
• Its value should lie between 1 and 5.
• If k is less than unity, accurate determination
of its retention time is difficult.
• If its too large, elution time becomes
inordinately long

19. Selectivity Factor: can you separate from your neighbour?
MAR
MBR
M
MBR
B
M
MAR
A
A
B
A
B
tt
tt
t
tt
kand
t
tt
k
k
k
K
K
−
−
=
−
=
−
=
=
=
)(
)(
)()(
α
α
α
B retained more than A  α >1

20. Selectivity factor
• The selectivity factor for two analytes in a
column provides a measure of how well the
column will separate the two.
• α is always greater than unity.
• Greater the selectivity factor, greater will be
the separation between the two components.

21. Principles of Chromatography
Raising
VS General increase in retention time
VM General decrease in retention time
µ Increases speed of separation.
• VS and VM can be altered by changing column
diameter and length for specific column packing.
• µ can be altered by changing the flowrate.
• All terms can be found by knowing how the
column was prepared.

22. All research in this field is aimed towards
maximum separation of components in minimum
time possible or in other words increasing the
efficiency of the column
Measure of column efficiency is given by
number of Theoretical Plates and Height
equivalent to theoretical plates (HETP)
Explained by Plate and Rate Theories

23. Plate Theory
Plate theory assumes that a column is
mathematically equivalent to a plate
column.
An equilibrium is established for the solute
between the mobile and stationary phase on
each plate.
It is a useful theory and can predict many
aspects of chromatographic performance.

24. Plates of fractionating column
• In a fractioning column
equilibrium is established
between the liquid and
gaseous phase at every
bubble cap plate.
• Likewise it is imagined that
in a chromatographic
column , solute equilibrium
is established between
stationary and mobile
phase at every imaginary
plate

25. Plate and Rate Theories
σ  standard deviation σ2
/L variance per unit length.
L = length of column packing
L
H
H
L
N
N
H
2
platesofnumber
heightplate
σ
=
=
=
=

26. Plate Theory
The number of plates ( N ) can be determined
from the retention time and peak width.
It doesn’t matter what units (minutes or
seconds) are used as long as they are same.

27. Determination of N
The number of plates is calculated as:
N = 16 tR
W
This approach is taken because peaks evolve as
Gaussian-like shapes and can be treated statistically.
In essence, we are taking + 2 σ or 4 σ.
2

28. Determination of N
• We can measure the
width at half height.
• This insures that we
are well above
background noise and
away from any
detector sensitivity
limit problems.

29. Determination of N
Since the peak is Gaussian in nature, we end up with the
following modified formula.
N = 5.54 tR
W1/2
For a fixed length column, we can calculate an additional term
– h (or HETP)
h = height equivalent of a theoretical plate
= column length / N
2

30. 2
2/1
2
54.5
16
patesofnumber






=






=
=
W
t
N
W
t
N
N
R
R

31. Summary of Plate Theory
• Successfully accounts for the peak shapes and
rate of movement
• Does not account for the ‘mechanism’ causing
peak broadening
• No indication of other parameters’ effects
• No indication for adjusting experimental
parameters

32. Band/ Zone broadening
• In this example, we have materials with the
same elution time but different numbers of
plates
• Zone broadening is related to Mass Transfer
processes

33. Band Broadening
Band Broadening is a major problem because it effects the
resolution of solutes that have similar retention time. The
peak width increases with the square root of column length.
Therefore, we just cannot make a column longer to obtain a
‘better’ separation.

34. Rate theory
Plate theory neglects the concepts of solute diffusion
and flow paths which lead to band broadening.
Rate theory accounts for these and presumes band
broadening is caused due to:
• Slow equilibrium of solute between mobile and
stationary phases
• Time is required for solute molecules to diffuse from
the interior of these phase to there interface where
transfer occur

35. Theory of Band Broadening
van Deemter Equation
Theoretical studies of zone broadening in the 1950s by
Dutch chemical engineers led to the van Deemter
equation, which can be written in the form
H = B + CSu + Cmu
u
where
B – longitudinal diffusion
CS–mass transfer coefficient in mobile phase
CM-mass transfer coefficient in stationary phase
u– velocity of mobile phase

36. LONGITUDINAL DIFFUSION
Longitudinal diffusion term (B/u) depends upon
diffusion coefficient DM. Solute continuously
diffuses away from the concentrated center of its
zone.
The longer the solute is in the column, broadening
effect increases,
Zone of solute after short time on column
Zone of solute after longer time on column
Direction of travel

37. MASS TRANSFER TERM- CSu
Csu is
α thickness of the stationary phase film on the support particles
α the flow rate
1/ α diffusion coefficient DS of the solute in
the film
Slower rate of mass transfer increases plate height which is undesirable

38. MASS TRANSFER TERM CMU
α square of particle diameter of the packing
α square of column diameter
α flow rate
1/ α diffusion coefficient of analyte in the
mobile phase DM
Zone broadening or band broadening occurs due to
a) eddy diffusion- different path lengths passed by
solutes
b) diffusion of solute from one stream of mobile phase
to another
c) stagnant or static pools of solvent formed within
stationary phase

40. Effect of flow rate (µ)
• Broadening effects may be minimized by careful
control of the flow rate.
• Generally, the amount of broadening increases as
the flow rate decreases.
• Broadening α 1 / µ
• Sufficient time must be allowed for the solute to
equilibrate between the two phases. For a given
separation there will be some optimum flow rate.
• This optimum flow rate is found experimentally.

43. Methods for Reducing Band Broadening
• Small packing diameter (of stationary
phase)
• Small column diameter
• For liquid stationary phase- thickness of
the layer should be minimized
• Optimum flow-rate of mobile phase
• Optimum temperature
• Variation in solvent composition

44. Packed GLC Column

45. Open tubular column

46. Capillary columns
Not much effect from CSu or CMu as there is
no packing and the phase is very thin

47. Liquid chromatography
• At first, LC relied on
irregular packing.
Now the packing are
pretty good so the
CSu term is very low.
• The B/u and CMu
terms are low
because liquids
diffuse much more
slowly than gases

48. Column Resolution
Resolution R, of a column provides a
quantitative measure of its ability to
separate two analytes.
Resolution of 1.5 gives almost complete peak
separation
The smaller the HETP or larger the N, the
higher the resolving power of the column.

49. Resolution

50. Resolution
R = 2∆Z = 2[(tR) B − (tR) A]
WA + WB WA + WB

51. Chromatographic
Separations with a twist

52. FACTORS FOR INCREASING RESOLUTION
1. Increase column length
2. Decrease column diameter
3. Decrease flow-rate
4. Pack column uniformly
5. Use uniform stationary phase (packing material)
6. Decrease sample size
7. Select proper stationary phase
8. Select proper mobile phase
9. Use proper pressure
10. Use gradient elution

53. Unsymmetrical bands
Often the actual bands observed are not
symmetrical Gaussian curves but rather show
one of following behaviours.
Careful adjustment of the operational
parameters, especially the size of sample may
correct these problems.
They may also be attributed to poor column
packing or sample injection.

54. Fronting and Tailing

55. Chromatogram of Orange Juice Compounds

56. Thank-you
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