2. Topics to be dealt Chromatography TLC HPTLC HPLC
3. Introduction Separation of mixture passed through medium
components move at different rates Physico-chemical method colour
to write Mobile phase mixture to be seperated Stationary phase the
material on which the separation takes place can be solid, gel, or
liquid Chroma Graphein
4. Principle Various constituents of mixture travel at
different speeds seperates basis differential partitioning M.P and
S.P Seperation formation of bands on S.P M.P liquid or gas S.P
solid or liquid
5. History Of Chromatography important technique 2 Nobel prize
Archer John Porter Martin and Richard Laurence Millington Synge
1952 Partition Chromatography 1906 Michael Tsvett Russian Botanist
concept of liquid chromatography to purify chlorophylls from plant
extracts. FATHER OF CHROMATOGRAPHY Plant pigments like xanthophyll,
chlorophyll, carotenes. CHROMATOGRAPHY or COLOUR WRITING
6. 1931 Richard Kuhn use of this method in biologically
important materials. 1941 British Chemists Archer.J.P.Martin and
Richard.L.M.Synge PARTITION CHROMATOGRAPHY one liquid firmly
attached to the granulated solid packed in a glass tube and second
immiscible with the first. Martin and Anthony .J.James gas liquid
partition chromatography moving phase could be gas.
7. 1940s lack of uniformity , lack of reproducibility in silica
gel sheet of filter paper is used. PAPER CHROMATOGRAPHY wide
application in the analysis of biologically important compunds such
as amino acids, steroids, carbohydrates and bile pigments. 1956
Nikolay.A.Imaylor and Maria.S.Shrayber thin film on the glass plate
THIN LAYER CHROMATOGRAPHY 1944 Erika Cremer compressed gas been
used GAS CHROMATOGRAPHY
8. Types of Chromatography 1) Based on physical state of both
phases homogenous same S.P and M.P heterogenous different S.P and
M.P 2) Based on the principle of separation used adsorption sample
gets separated due to greater affinity to adsorb partition get
separated due to relative differences of dissolution and partition
into different layers.
9. 3) Based on the chemical nature of stationary phase and
mobile phase normal phase chromatography S.P polar and M.P is non-
polar in nature. Used in column chromatographic technique. reverse
chromatography S.P is non-polar and M.P is polar in nature. Used in
routine analysis as most of these substances like drugs etc.
10. 4) Based on shape of stationary phase column chromatography
S.P is column in shape widely used in HPLC, GC planar
chromatography S.P is flat. TLC 5) Based on the purpose of
chromatography experiment preparative chromatography injected
sample is very small pure sample is collected. analytical
chromatography injected sample is very small aimed to identify the
components in the sample and individual concentrations in the
sample
11. 6) Based on the physical or chemical characters of the S.P
size exclusion C S.P has pores . molecules are allowed to pass
through large to small path under mobile phase influence and pass
through the column ion exchange C S.P has definite charged ions.
passed sample retains molecules with opposite charge leaves
molecules of same charge. to elute it need to pass through another
phase with similar charge to S.P is passed to recover the bond
molecules
12. Applications of Chromatography 1. Widely chemical industry
2. Environmental testing trace quantities pesticides as DDT in
ground water. 3. Growing use of pharmaceutical industry 4. Quality
control food industry analying additives, vitamins, preservative,
proteins, aminoacids. 5. Can separate and detect contaminants 6.
Finding drugs in urine or other body fluids traces of flammable
chemicals in burned material
13. Overall available techniques for regular analysis include
1. Column chromatography 2. HPLC 3. GC 4. Ion exchange
chromatography 5. Size exclusion chromatography 6. TLC 7. HPTLC 8.
Paper chromatography 9. Affinity chromatography 8. Paper
chromatography 9. Affinity chromatography 10. LC-MS (Liquid
Chromatography with Mass Spectroscopy) 11. GC-MS ( Gas
Chromatography with Mass Spectroscopy) 12. Ultra High Performance
Chromatography
14. Thin Layer Chromatography Introduction type of planar
chromatography Used by researchers field of phytochemicals,
biochemistry identify components compound mixture alkaloids,
phospholipids, aminoacids. Determine no. of components in mixture
Identity purity of a compound. Semi quantitative method
Sophisticated version - HPTLC
15. Principle Based on the principle of separation Separation
relative affinity of cpds towards S.P and M.P Cpds under the
influence of M.P travel over the surface of S.P During this cpds
with higher affinity to S.P travel slowly while others travel
faster. Separation of mixture achieved Separation completed cmpnts
visualized as spots at respective level on the plates.
16. TLC chromatographic system components 1)TLC plates o
Preferably ready made with S.P o Stable and chemically inert
plates. o S.P on the plate uniform thickness consists of fine
particle size 2) TLC chamber o Used development of TLC plate. o
Chamber maintains uniform environment proper development of spots.
o Prevents evaporation of solvent keeps process dust free.
17. 3) Mobile phase o Comprises of solvent or solvent mixture o
M.P should be particulate free - & highest priority for proper
development of TLC spots. o Solvents chemically inert with the
sample. 4) Filter paper o moistened in the M.P by placing inside
the chamber. o Helps uniform rise in M.P over the length of
S.P.
18. TLC procedure S.P applied onto the plate uniformly allowed
to dry and stabilize. Readymade plates preferred now-a-days. thin
mark made at the bottom of the plate pencil sample spots. samples
solutions are applied on the spots marked on the line at equal
distances. M.P poured TLC chamber few centimeters above the chamber
bottom.
19. filter paper moistened in M.P placed on the inner wall of
the chamber maintain equal humidity in the entire chamber avoid
edge effect. plate prepared with sample spotting placed in TLC
chamber side of the plate with sample line towards the mobile
phase. chamber closed with a lid. Plate is immersed such a way
sample spots above the level of M.P Sufficient time given spot
development Plates removed allowed to dry Sample spots visualized
UV chamber or any recommended methods.
20. Advantages of TLC 1. simple process with short development
time. 2. Separate compound spots visualized easily 3. identify the
individual compounds. 4. Helps isolating most of the compounds. 5.
Separation process faster, selectivity of compounds higher 6.
Purity sample assessed easily 7. cheaper
21. Applications of Thin layer chromatography check purity of
given samples. Identification of compounds like acids, alcohols,
proteins, alkaloids, amines, antibiotics etc. purify samples i.e
for purification process. keep a check on the performance of other
separation processes.
22. High Performance Thin layer Chromatography Introduction
HPTLC High Performance Thin layer Chromatography / High Pressure
Thin Layer Chromatography. sophisticated advancement TLC Advantages
better resolution, faster development of spots ,easy detection and
quantification of separated compounds.
23. Advancement due to Use of ready made HPTLC plates optimized
absorbent layers smaller particles size with uniform particle size
distribution as the stationary phase. use of pressure enables for
faster development of chromatograms even complex mixtures The HPTLC
systemautomated to a greater extent Auto sampling Online mixing of
solvents Auto detection of compounds Recording and storage of
data
24. Advantages of HPTLC over TLC: Samples in minute quantities
like in nano-gram range can be detected using HPTLC. Handling and
human errors are minimum due to automation. Better accuracy and
sensitivity than TLC. Disadvantages many folds expensive than TLC.
Bulky instrumentation large space requirement. Requires stringent
condition of operation ( dust free environment and temperature
controlled conditions.) Technically skilled person is required
25. Procedure i. preparation of test solution ii. Preparation
of standard solutions iii. Sample application and plate layout iv.
Preconditioning of the plate v. Preparation of the developing
chamber and development of plate vi. Derivatization procedure vii.
Visualization viii. system suitability ix. Evaluation and
acceptance criteria x. Documentation
26. Preparation Of Test Solution 100mg of powdered bacterial
ingredient / 10mg of dry extract Sonicated for 15 min with 1ml of
ethanol centrifuged Filtrate or supernatant is used SAMPLE
SOLUTION
27. Preparation Of Standard Solution Reference standard shaken
Sonicated in methanol conc. of 10mg/ml Essential oils R.M
(reference material) dissolved in toluene conc. of 50L/mL
28. Sample Application And Plate Layout Samples applied at
narrow bands 8.00.5mm L & D 8.00.5mm from the lower edge of the
plate Distance between tracks Marked with pencil close to one of
the edges of the plate before development
29. Preconditioning Of The Plate Following sample application
Plate is conditioned at Relative humidity of 33% Min of 10min
30. Preparation Of Developing Chamber And Development Of Plate
Twin trough is used rear trough is fitted with filter paper Chamber
is charged Sufficient volume of developing solvent 5mm in both the
troughs lid is closed Chamber left for saturation (20mins)
Contd
31. Contd Plate introduced in vertical position Into Front
trough of the chamber So that Coating layer faces the filter paper
When Coating layer reaches the distance of 6cm(development path)
Plate is removed from the chamber & Dried in vertical position
in the stream of cold air
32. Derivatization Procedure Where Derivatization reagents are
used Defined volumes of reagents in soln., (1-2mL) are Homogenously
sprayed onto the plate Or The plate is immersed into the reagent
solution Defined speed for defined time speed=50mm/s, time 1s
33. Visualization Observation and evaluation May be Performed
under UV 254nm, UV 366nm or white Light prior to and after
derivatization
34. System Suitability 2/more reference substances are selected
Similar but just seperable RF values under the chromatographic
conditions to be used eg:- chlorogenic (blue) & hyperoside
(yellow) used for flavonoids S.S for reference stds may be provided
or Substances designated to check system suitability for
resolution, position and colors of the bands included in the ref.
std Contd
35. Contd Description of the resolution, position and colors
for the key bands Of the reference material fingerprint should
Match the description in the MONOGRAPH within a specified tolerance
range S.S requirements in an individual monograph satisfied When
the results obtained comply with those specified in the
monograph
36. Evaluation And Acceptance Criteria Chromatograms (sample
solution & standard solution) compared Against the description
on the Acceptance Criteria Of the monograph With respect to one
position, zone separation, color and relative intensity
37. Documentation Documentation is necessary to record the
results In Auditable manner to comply with current good
manufacturing practices Proper documentation tools should be
employed
38. Application of HPTLC For detection and analysis of
components of phytochemsitry, medicinal chemistry & organic
chemistry. Complex and scarce compounds can be analysed.
39. HPLC High pressure liquid chromatography HPLC high pressure
used in the principle of its operation. due to its efficiency in
analysis of compounds regarded High performance liquid
chromatography. Also called High patience liquid chromatography due
to long human time requirement and patience widely used in the
fields of clinical research, biochemical research, industrial
quality control etc.
40. Principle separation of compounds in a mixture more
efficiently and also quickly than Column Chromatography The
separation of compounds due to their relative differences in travel
through the column on application of pressure exerted through
mobile phase or carrying liquid. The compounds of the mixture
travel with different rates due to their relative affinities with
the solvent and stationary phase.
41. Compound with higher affinity towards stationary phase of
the column travels slowly and vice-versa. The separation is more
effective greater surface area very small particle size stationary
phase than used in column chromatography. decrease in particle size
increases the disadvantage i.e., it proportionately enhances the
flow time and run time due to increased surface area. minimize this
obstacle the high pressure is applied to the flow of hplc mobile
phase through the column by use of pumps.
42. The HPLC method or process steps include mixture injected
into a stream of mobile phase flowing at a defined pressure. The
injected mixture now flow over the stationary phase inside the
columnunder the influence of pressurealong with the mobile phase.
During this flow based on the affinity of individual compounds
mixture towards stationary and mobile phase, some compounds get
eluted first out of the column others later.
43. Outside the column sent into a detector & individual
compounds are detected recorded in a computer chromatography
software installed. The recordings compared standard compound's
HPLC values and the individual compounds are identified. over all
theory of HPLC relative separation and detection of compounds.
44. Types of HPLC HPLC analysis is of differentiated based on
a) The stationary phase in the column used b) Based on purpose of
use
45. The stationary phase in the column used: Based on the
nature of stationary phase used it can be either normal phase or
reverse phase hplc.
46. Normal phase chromatography: column stationary phase is
made of polar compounds like silica gel, alumina etc.. The polar
compounds or molecules sample under analysis higher affinity to the
stationary phase they are retained longer in the column than
non-polar ones. non-polar compounds are eluted first under the
affinity to non-polar mobile phase polar ones are eluted
later.
47. Reverse phase hplc: exactly the opposite of normal phase
happens. The stationary phase is made of non-polar compounds like
C18, C8 type of organic compounds. The mobile phase used is polar.
compounds of high polarity or eluted first those of low polarity or
no-polarity are eluted last.
48. Most of the applications in HPLC require evaluation of
drugs biochemical molecules and other substances used by humans
they are polar (water soluble) in nature. So, reverse phase hplc is
widely used.
49. Based on purpose of use: Here HPLC is used for either
Analysis mode: done to estimate different types of molecules their
individual quantities in the mixture help of a detector.
Preparative mode: intention of process separate large amounts of
specific molecule from a mixture. The molecule or substance eluted
highest purity. The column size, sample size is comparatively large
than that of analytical mode.
50. Advantages 1. Includes aspects of analysis (quantitative or
qualitative). 2. Evaluates all the molecules same family. 3.
Substances low concentration nano and picograms detected
sensitivity of HPLC detector. 4. Due high seperation efficiency
quality high purity
51. Disadvantages expensive technique requires heavy processing
(mixing, homogenization, filtration, degassing, derivatization etc)
time consuming must have good amount of patience. systems operation
requires prior hplc training and effective hplc troubleshooting
skills.
52. Applications 1. Detection 2. Analysis determination 3.
Quantification 4. Derivation of molecules plant and medical
importance
53. TERMS Absorption; Process of retention in which the solute
partitions into a liquid like coating Active site; A reactive or
strongly attracting site on the surface of a chromatographic
packing that may bind analytes or cause peak tailing. Adsorbent;
Packing used in adsorption chromatography. Silica gel and alumina
are the most frequently used absorbents in chromatography and
sample preparation Contd
54. Contd Adsorption; Process of retention in which the
interactions between the solute and the surface of an adsorbent
dominate Band; Refers to the chromatographic peak as it moves along
and is eluted from the column Chromatogram; A plot of detector
signal output versus time or elution time during the
chromatographic process Contd
55. Contd Chromatograph; Device used to implement
chromatographic separation Column; Tube and stationary phase
through which mobile phase flows resulting in the chromatographic
separation Effluent; mobile phase that exits the column Eluate;
Solute mobile phase mixture
56. Eluent; Another word for mobile phase. Elution; process of
passing of mobile phase throught he chromatographic bed to
transport solution.
