Column chromatography

Presenting a presentation on the topic of Column chromatography with including basics of chromatography, principles, equations, graphs and data related to it.
Topics which covered in this ppt is
Principle of chromatography
classification of chromatography
partition coefficient
chromatogram
Resolution
plate theory
determination of N
band zone broadening
rate theory

https://www.linkedin.com/in/preeti-choudhary-266414182/
https://www.instagram.com/chaudharypreeti1997/
https://www.facebook.com/profile.php?id=100013419194533
https://twitter.com/preetic27018281

Please like, share, comment and follow.
stay connected

If any query then contact:
[email protected]

Thanking-You
Preeti Choudhary

1. Column chromatography
Submitted by: Preeti choudhary
MSC(applied physics)

2. PRINCIPLES OF
CHROMATOGRAPHY

3. 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 .
Principles of Chromatography

4. Chromatography classification

5. 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).
Principles of Chromatography

6. 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.
Principles of Chromatography

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

8. INDIVIDUAL SPECIES ARE
RETARDED BY THE
STATIONARY PHASE BASED
ONVARIOUS INTERACTIONS
SUCHAS:
•SURFACE ADSORPTION
•RELATIVE SOLUBILITY
•CHARGE
Principles of Chromatography

9. Partition/Distribution Coefficient
Asthemobilephasebearingthesoluteentersthe
column,thesolutedistributesitselfbetween
stationaryandmobilephase.
Thisdistributionbetweenthe2phasesisdescribed
bytheDistributionCoefficient‘K’,definedas
K=C
s/C
M
whereC
s&C
Mrefertotheconcentrationsofthe
soluteinthestationaryandmobilephases.

10. IFTHEVALUE OFK=1THEN THESOLUTE
IS EQUALLY DISTRIBUTED BETWEEN
STATIONARY ANDMOBILE PHASES .
FORK<1,THESOLUTE TRAVELS FASTER
THROUGH THE COLUMN BECAUSE IT
SPENDS MORE TIMEINMOBILE PHASE.
FOR K>1,THE SOLUTE WILL BE
RETAINED INTHESTATIONARY 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.
Partition Coefficient

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
t
M= retention time of mobile phase (dead time)
t
R= retention time of analyte (solute)
t
S = time spent in stationary phase (adjusted retention time)
L = length of the column

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

14. Retention time and volume
Retentiontime,t
R-timerequiredtoreachthepeak
maximumfromthepointofinjection.
Deadtime,t
M-timerequiredfortheunretained
speciestoreachthepeakmaximumfromthepoint
ofinjection.
Retentionvolume,V
R–volumeofmobilephase
requiredtoeluteasolutetoamaximumfroma
column.
.

15. Velocity RelationshipsMS
M
S
MMSS
SSMM
MM
VVK
v
c
c
K
VcVc
v
VcVc
Vc
v
/1
1
Constanton Distributi
/1
1











16. Capacity and Selectivity Factors
Capacity / Retention Factor (k
A)–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 FactorM
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


Adjusted retention time

18. Capacity/Retention Factor
wherek
AisthecapacityfactorforsoluteA.
Itsvalueshouldliebetween1and5.
Ifkislessthanunity,accuratedeterminationofits
retentiontimeisdifficult.
Ifitstoolarge,elutiontimebecomesinordinately
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









)(
)(
)()(



B retained more than A >1

20. Selectivity factor
Theselectivityfactorfortwoanalytesinacolumn
providesameasureofhowwellthecolumnwill
separatethetwo.
αisalwaysgreaterthanunity.
Greatertheselectivityfactor,greaterwillbethe
separationbetweenthetwocomponents.

21. Principles of Chromatography
Raising
V
S General increase in retention time
V
M General decrease in retention time
µ Increases speed of separation.
V
SandV
Mcanbealteredbychangingcolumn
diameterandlengthforspecificcolumn
packing.
µcanbealteredbychangingtheflowrate.
Alltermscanbefoundbyknowinghowthe
columnwasprepared.

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 andHeight
equivalent to theoretical plates (HETP)
Explained by Plate andRate 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 andRate Theories
sstandard deviation s
2
/Lvariance per unit length.
L = length of column packingL
H
H
L
N
N
H
2
plates ofnumber
height plate
s





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 t
R
W
This approach is taken because peaks evolve as
Gaussian-like shapes and can be treated
statistically.
In essence, we are taking 2 sor 4 s.
2

28. Determination of N
•Wecanmeasurethe
widthathalfheight.
•Thisinsuresthatwe
arewellabove
background noise
andawayfromany
detectorsensitivity
limitproblems.

29. Determination of N
Since the peak is Gaussian in nature, we end up with the
following modified formula.
N = 5.54 t
R
W
1/2
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
pates ofnumber

















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
BandBroadeningisamajorproblembecauseiteffectsthe
resolutionofsolutesthathavesimilarretentiontime.The
peakwidthincreaseswiththesquarerootofcolumn
length.Therefore,wejustcannotmakeacolumnlonger
toobtaina‘better’separation.

34. Rate theory
Platetheoryneglectstheconceptsofsolutediffusion
andflowpathswhichleadtobandbroadening.
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 DeemterEquation
Theoreticalstudiesofzonebroadeninginthe1950s
byDutchchemicalengineersledtothevan
Deemterequation,whichcanbewritteninthe
form
H=B+C
Su+C
mu
u
where
B–longitudinal diffusion
C
S–masstransfercoefficientinmobilephase
C
M-masstransfercoefficientinstationaryphase
u–velocity of mobile phase

36. LONGITUDINAL DIFFUSION
Longitudinaldiffusionterm(B/u)depends
upondiffusioncoefficientD
M.Solute
continuouslydiffusesawayfromthe
concentratedcenterofitszone.
Thelongerthesoluteisinthecolumn,
broadeningeffectincreases,
Zone of solute after short time on column
Zone of solute after longer time on column
Direction of travel

37. MASS TRANSFER TERM -C
Su
C
suis
 thickness of the stationary phase film on the support particles
 the flow rate
1/ diffusion coefficient D
Sof the solute in
the film
Slower rate of mass transfer increases plate height which is
undesirable

38. MASS TRANSFER TERM C
MU
 square of particle diameter of the packing
 square of column diameter
 flow rate
1/ diffusion coefficient of analytein the
mobile phase D
M
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)
Broadeningeffectsmaybeminimizedbycareful
controloftheflowrate.
Generally,theamountofbroadeningincreases
astheflowratedecreases.
Broadening1/µ
Sufficienttimemustbeallowedforthesoluteto
equilibratebetweenthetwophases.Foragiven
separationtherewillbesomeoptimumflowrate.
Thisoptimum flowrateisfound
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
NotmucheffectfromC
SuorC
Muasthereisno
packingandthephaseisverythin

47. Liquid chromatography
•Atfirst,LCrelied
on irregular
packing.Nowthe
packingarepretty
goodsotheC
Su
termisverylow.
•TheB/uandC
Mu
termsarelow
because liquids
diffusemuchmore
slowlythangases

48. Column Resolution
Resolution R, of a column provides a quantitative
measure of its ability to separate two analytes.
Resolution of 1.5gives 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[(t
R)
B(t
R)
A]
W
A+ W
B W
A+ W
B

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
Oftentheactualbandsobservedarenotsymmetrical
Gaussiancurvesbutrathershowoneoffollowing
behaviours.
Carefuladjustmentoftheoperationalparameters,
especiallythesizeofsamplemaycorrectthese
problems.
Theymayalsobeattributedtopoorcolumnpacking
orsampleinjection.

54. Fronting and Tailing

55. Chromatogram of Orange Juice Compounds

56. Thank-you