column chromatography and ultra high performance liquid chromatography M.PHARM SEM-1 MORDEN PHARMACEUTICAL ANALYSIS TECHNIQUES

1. SEMINAR/ASSIGNMENT-I(MAT101T)
COLUMN CHROMATOGRAPHY
NAME:-VINIT NAI
M.PHARM(PQA) SEM 1
SUBJECT:-MORDERN PHARMACEUTICAL ANALYSIS TECHNICS
SUBJECT CODE :-MAT101T
GUIDED BY- DR.SHAILESH KORADIA
KRISHNA SCHOOL OF PHARMACY & RESEARCH(KSP)
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2. CONTENTS
Introduction
History
Principle
Experimental aspects of column chromatography
Packing techniques in C.C
Development technique (Elution)
Detection of components
Factors affecting column efficiency
Applications
Advantages and disadvantages of C.C
2

3. CHROMATOGRAPHY
 Chromatography is the term used to describe a separation technique
in which a mobile phase carrying a mixture is caused to move in
contact with a selectively absorbent stationary phase.
 There are a number of different kinds of chromatography, which
differ in the mobile and the stationary phase used.
3

4. HISTORY
 Column Chromatography was developed by the American chemist
in 1901.
 Mikhail Tswett, the Polish botanist, in 1906 used adsorption
columns in his investigations of plant pigments.
4

5. Column chromatography is one of the most useful methods
for the separation and purification of both solids and
liquids.
This is a solid - liquid technique in which the stationary
phase is a solid & mobile phase is a liquid.
5

6. PRINCIPLE :-
 Adsorption
 Mixture of components dissolved in the M.P is introduced in to the
column. Components moves depending upon their relative affinities.
 Adsorption column chromatography, the adsorbent, packed in a glass
column, and a solvent, the mobile phase, that moves slowly through the
packed column. A solvent used as a mobile phase is called an eluent.
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7.  A compound attracted more strongly by the mobile phase will move
rapidly through the column, and elute from, or come off, the column
dissolved in the eluent.
 In contrast, a compound more strongly attracted to the stationary
phase will move slowly through the column.
Partition:-
 The liquid is coted on the column
 the separation of components between two liquid phases viz original
solvent and the film of solvent used in the column.
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8. Experimental aspects of column chromatography:-
Adsorbents:-
 The usual adsorbents employed in column chromatography are silica,
alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc.
 Alumina is generally suitable for chromatography of less polar
compounds. Silica gel gives good results with compounds containing
polar functional groups.
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9. Adsorbent in C.C should meet following criteria:-
Particles should be spherical in shape & uniform in size
Mechanical stability must be high
They shouldn’t react chemically
It should be useful for separating for wide variety of
compounds
It should be freely available & inexpensive
The particle size of the commercially available grade is in the
range 50 – 200 µm.
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10. Selection of Stationary Phase
Success of chromatography depends upon proper selection of
S.P, it depends on the following:
1. Removal of impurities
2. No. of components to be separated .
3. Length of the column used
4. Affinity differences b/w components
5. Quantity of adsorbent used
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11. Mobile Phase
They act as solvent, developer & eluent. The function of a
mobile phase are:
As developing agent
To introduce the mixture into the column – as solvent
To remove pure components out of the column – as eluent
The choice of the solvent is depend on the solubility
characteristics of the mixture.
11

12. The solvents should also have sufficiently low boiling
points to permit ready recovery of eluted material.
However, polarity as seen the most important factor in
adsorption chromatography.
Different mobile phases used:(in increasing order of
polarity)
Petroleum ether, carbon tetrachloride, cyclohexane, ether,
acetone, benzene, toluene, esters, water, etc
It can be used in either pure form or as mixture of solvents
12

13. COLUMN CHARACTERISTICS
The main function of all the columns is to support the
stationary phase.
The material of the column is mostly good quality neutral
glass since it shouldn’t be affected by solvents. An ordinary
burette can also be used as column for separation.
Column dimensions - length & diameter ratio (10:1,30:1 or
100:1).
Various accessories are attached to the top and bottom of
the column for maintenance of the elution process.
13

14. The length of the column depends upon:-
Number of compounds to be separated
Type of adsorbent used
Quantity of the sample
Affinity of compounds towards the adsorbent used
Better separation will be obtained with a long narrow
column than short thick column because number of plates
will be more.
14

15. PREPARATION OF THE COLUMN
It consists of a glass tube with bottom portion of the
column – packed with glass wool/cotton wool or may
contain asbestos pad,
Above which adsorbent is packed
After packing a paper disc kept on the top, so that the
adsorbent layer is not disturbed during the introduction of
sample or mobile phase.
15

16. Packing techniques in C.C
There are two types of preparing the column, they are…
1.Dry packing / dry filling
2.Wet packing / wet filling
The column should be free from impurity, before using
column, it should be washed properly and dry it.
Before filling column with stationary phase, cotton/glass
wool is kept
It should be uniformly filled
16

17. DRY PACKING TECHNIQUE
Adsorbent is packed in the column in dry form
Fill the solvent, till equilibrium is reached
After filling tapping can be done to remove void spaces.
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18. 18

19. WET PACKING TECHNIQUE
ideal & common technique
The material is slurried with solvent and generally added
to the column in portions.
S.P settles uniformly & no crack in the column of
adsorbent.
solid settle down while the solvent remain upward.
this solvent is removed then again cotton plug is placed.
19

20. Introduction of the Sample
The sample which is usually a mixture of components is
dissolved in minimum quantity of the mobile phase.
The entire sample is introduced into the column at once
and get adsorbed on the top portion of the column.
From this zone, individual sample can be separated by a
process of elution.
20

21. 21

22. Development technique (Elution)
 By elution technique, the individual components are separated out
from the column. The two techniques are:-
(i) Isocratic elution technique:-
(ii) Gradient elution techniques:-
(i) Isocratic elution technique:-
 In this elution technique , same solvent composition or solvent of
same polarity is used throughout the process of separation.
 Example: chloroform only
22

23. (ii) Gradient elution techniques:-
 Solvents of gradually ↑ polarity or ↑ elution strength are used during
the process of separation.
 E.g. initially benzene, then chloroform, then ethyl acetate then
chloroform
23

24. DETECTION OF COMPONENTS
 If the compounds separated in a column chromatography procedure
are colored, the progress of the separation can simply be monitored
visually.
 If the compounds to be isolated from column chromatography are
colorless.
 In this case, small fractions of the eluent are collected sequentially
in labelled tubes and the composition of each fraction is analyzed by
TLC.
24

25. Eluting the sample:-
Components a, b, and c separate as column progresses.
Fractions can be collected in test tubes, vials, beakers, or
Erlenmeyer flasks.
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26. Analyzing the fractions: .
Analyze the fractions by thin-layer chromatography
26

27. FACTORS AFFECTING COLUMN
EFFICIENCY
1.Dimension of the column:- column efficiency has been improved
by increasing length/width ratio of the column.
2. Particle size of column packing:- separation to be improved by
decreasing the particle size of the adsorbent.
3.Temperature of the column:- The speed of the elution increases at
higher temperatures.
4.Packing of the column
5.Quality of solvents:- solvents having low viscosities is giving better
results.
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28. APPLICATIONS
 Separation of mixture of compounds
 Purification process
 Isolation of active constituents
 Estimation of drugs in formulation
 Isolation of active constituents
 separation of diastereomers
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29. • Advantages of C.C
» Any type of mixture can be separated
» Any quantity of mixture can be separated
» Wider choice of Mobile Phase
» Automation is possible
• Disadvantages of C.C
» Time consuming
» more amount of Mobile Phase are required
»Automation makes the techniques more complicated &
expensive
29

30. Reference:-
 https://www.slideshare.net/shaisejacob/column-chromato
 https://www.slideshare.net/krakeshguptha/column-chromatography-26966949
 S.RAVI SHANKAR, Text book of pharmaceutical analysis, Rx publications,
2001.Pg no : 13-4 to 13-13
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31. ULTRA HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
(UHPLC)
31

32. CONTENTS
 INTRODUCTION
 PRINCIPLE
 COMPARISON
 INSTRUMENTATION
 ADVANTAGES
 DISADVANTAGES
 APPLICATION
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33. INTRODUCTION:-
 UHPLC refers to ultra high performance liquid chromatography.
 It improves in three areas, chromatographic resolution, speed,
sensitivity.
 UHPLC is a rising chromatographic separation technique whose
packing materials have smaller particular size lesser than 2.5µm.
 The technology takes full advantage of chromatographic principles
to run separation using column packed with smaller particles and
higher flow rates.
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34. It can withstand high system back-pressure.
Special analytical columns UHPLC BEH C18 packed with
The factor responsible for development of UHPLC
technique was evolution of packing material used to effect
the separation.
The technology takes full advantage of chromatographic
principles to run separations using columns packed with
smaller particles.
It decreases analysis time and solvent consumption 1.7µm
particles are used in connection with system
34

35. PRINCIPLE:-
 The Principle of UHPLC is based on van deemeter equation which
describes the relationship between flow rate and HETP or column
efficiency.
 H=A + B/v + Cv
when,
 A= Eddy diffusion
 B= Longitudinal diffusion
 C= equilibrium mass transfer
 v= flow rate
 Van deemeter equation that describes the relationship between
linear velocity (flow rate) and plate height (HETP or column
efficiency)
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36. COMPARISON BETWEEN HPLC AND
UHPLC
36
Parameters HPLC UHPLC
Column XTerra,C18,50 ×
4.6mm
AQUITY UHPLC
BEH C18,50 ×2.1mm
Particle size 4µm particles 1.7µm particles
Flow rate 3.0 ml per min 0.6 ml per min
Injection volume 20 µl 3 µl partial loop fill
or 5 µl full loop fill
Total run time 10 min 1.5 min
Theoretical Plate
count
2000 7500
Column temperature 30 °C 65 °C

37. Parameters HPLC UHPLC
Maximum back
pressure
35-40 Mpa less 103.5 Mpa more
Resolution Less High
Analysis time More Less
sensitivity Less Higher
37

38. 38

39.  A completely new system design with advanced technology in the
pump, auto sampler, detector, data system, and service diagnostics
was required.
 Achieving small particle, high peak capacity separations requires a
greater pressure range than that achievable by HPLC system.
39

40. INSTRUMENTATION:-
 SOLVENT RESERVOIR:
 The most common type of solvent reservoir is glass bottle.
 Most of manufacturers supply these bottles with special caps,
tubing and filters to connect to the pump inlet and so the purge gas
(helium) used to remove dissolved air
40

41. PUMP:-
CONSTANT PRESSURE PUMP: Constant pressure is
used only for column packing.
CONSTANT FLOW PUMP: This type is mostly used in
all common UPLC application.
RECIPROCATING PISTON PUMP:
DUAL PISTON PUMP
41

42. SAMPLE INJECTION:-
In UHPLC , Sample introduction is critical. Conventional
injection valves, either automated or manual, and hardened to
work at extreme pressure.
To protect the column from extreme pressure fluctuations, the
injection process must be relatively pulse-free and the swept
volume of the device also needs to be minimal to reduce
potential band spreading.
Low volume injections with minimal carryover required to
increase sensitivity.
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43. UHPLC COLUMN:
 Resolution is increased in a 1.7µm particle packed column because is
better.
 Separation of the components of a sample requires a bonded phase
that provides both retention and selectivity.
 Four bonded phases are available for UHPLC separations:
 ACQUITY UHPLC BEH C18 and C8 (straight chain alkyl columns),
 ACQUITY UHPLC BEH shield RP 18 (embedded polar group
column)
 ACQUITY UHPLC BEH (phenyl group tethered to the silyl
functionality with a C6 alkyl)
 ACQUITY UHPLC BEH Amide columns
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44. 44

45. CHEMISTRY OF SMALLPARTICLES
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As the particle size decreases to less than 2.5µm, not only
there is significant gain in efficiency, but the efficiency
doesn't diminish at increased flow rates.
By using smaller particles, speed and peak capacity
(number of peaks resolved per unit time in gradient
separation) can be extended to new limits, termed ultra
performance liquid chromatography.

46. 46

47. DETECTORS:-
 UV Detectors
 Fluorescent detector
 Refractive index detector
 Light scattering detector
 Electrochemical detector
 Mass spectrometric detector
47

48. ADVANTAGES:-
 Decreases run time and increases sensitivity.
 Reducing analysis time so that more product can be produced with
existing resources.
 Provides the selectivity, sensitivity and dynamic range of LC analysis
 Maintains resolution performance
 Fast resolving power quickly quantifies related and unrelated
compounds.
 Operation cost is reduced.
 Less solvent consumption.
48

49. DISADVANTAGES:
 Due to increased pressure requires more maintenance and reduces
the life of the columns of this type.
 In addition, the phases of less than 2µm are generally non-
regenerable and thus have limited use.
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50. APPLICATION:
 Analysis of natural products and traditional herbal medicine.
 Identification of metabolite
 Study of metabonomics/metablomics
 Bio analysis/bioequivalence studies.
 Manufacturing/QA/QC
 Impurity profiling
 Forced degradation studies
 Dissolution testing
 Toxicity studies
50

51.  It provides high speed, accuracy and reproducible results for analysis
of drugs and their related substance. Thus method development time
decrease.
 UPLC used for accurate, reliable and reproducible analysis of amino
acids in area of protein characterization, cell culture monitoring and
nutritional analysis of food.
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52. Reference:-
 https://www.slideshare.net/pavi96/ultra-high-performance-liquid-chromatography
 https://www.slideshare.net/YendaManishankar/uhplc
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53. Thank you
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