Chromatography and Polymerase Chain Reaction

1. Chromatography &
PCR
Dr. Yash N. Panchal
Resident doctor
Pharmacology Department
AMC MET Medical college
08/10/2021 1

2. Presentation Layout
 Chromatography TLC : Principle
 Principle of Chromatography : Instrumentation
 Types of Chromatography : Procedure
 HPLC : Type : Application
: Components : Advantages & Disadvantages
: Procedure
: Coupling with MS PCR : Principle
: Application : Procedure
: Advantages & Disadvantages : Types , Application , Advantages

3. What is Chromatography ?

4. Chromatography
 It is defined as analytical method in which separation of
active constituent from complex mixture occurs, while
mixture is distributed in two phases.
 This technique is used in :
SEPERATION IDENTIFICATION QUANTIFICATION

5. Principle of Chromatography
ADSORPTION AND SEPERATION

6. Liquid Chromatography
Mobile Phase is Liquid

7. HPLC (High Performance
Liquid Chromatography)
High Pressure
Chromatography
Liquid
Chromatography
Column
Chromatography
Qualitative and
Quantitative
analysis
Applicable for
Volatile and Non-
volatile compound

8. HPLC MACHINE
8

9. INSTRUMENTATION OF
HPLC
1- Solvent storage bottle (Mobile phase)
2- High pressure pump
3- Sample mixture
4- Column
5- Detector
6- Recorder

12. Mobile Phase

13. Mobile Phase
As it moves, so called as Mobile Phase
Liquid mobile phase is used in HPLC
Is mixture of solvent- polar/non-polar
Contained is glass of stainless steel, capable of holding 1L of solvent
Solvent selection, depends on Stationary phase

14. Degassing of Mobile Phase
 De-gassing is done to prevent
formation of gas-bubbles in
the pump or detector prior to
use
 De-gassing is done by vacuum
or sparing with helium gas
 Then, mobile phase are filtered to remove any
particulate matter that may clog the system

15. Tubing System in HPLC
Ability to
withstand
pressure
Should be
Inert
Able to
carry
sufficient
volume

16. PUMP
Pump
material is
chemically
inert to
solvent
Can generate
pressure up
to 40 MP
Solvent
reservoir
coupled to
pump

17. 1- Timing issue
2- Small particle size
of stationary phase
increases resistance

18. Sample Injector System
 It is automated sample injection system
 Injector device can be :
SEPTUM INJECTOR STOP FLOW INJECTOR RHEODYNE INJECTOR

19. Stationary Phase- Column

20.  Made of stainless steel
 Can withstand pressure up to 50 MP
 Length : 5-25 cm
 Internal diameter : 4.6 mm
 Flow of solvent : 1-3 ml/minute
 Contains adsorbent (Stationary phase ) of smaller particle size

21. Reason for smaller particle size
?
Small Particle Size
More surface/ volume ratio
More surface area for
Interaction with Solvent
More efficient Separation

22. Heart of the
HPLC
Actual
separation
occurs here
Success or
failure of HPLC
depends on it

23. Stationary Phase material
 Silica or Alumina ( of 5-10 micron ) commonly used
 The separation is the result of different components
adhering to or diffusion into the packing particles when
the mobile phase is forced through the column
 Selection of mobile phase depends on stationary phase
material

25. Normal Phase HPLC

26. Reverse Phase HPLC

27.  Here Silica is made non-polar by adding long
Hydrocarbon chain of 8 to 18 Carbon atoms (C8 or C18)
 C18 being more hydrophobic than C8, commonly used
 Mobile phase used is polar (Methanol)
 First elute will be Polar
 Now a days, Reverse phase HPLC is commonly used

28. Size Exclusion Chromatography

29. Ion Exchange Chromatography

30. Detector System
 Is coupled to column and detects substances
 It sends signals to processing unit (Computer)
 Ideal detector must be :
Sensitive
Non-
destructive
Insensitive to
temperature
Reliable

31.  Most commonly used detector system is UV detector
system
 Other detector system :
1- Electro chemical
2- Florescence
3- Chemiluminence
4- Optical rotation
5- Refractive index

32. Processing Unit
Coupled
to
Detectors
Computers
Forms
Chromato
gram on
display
First,
Baseline is
formed

33. Chromatogram

34. Interpretation of
Chromatogram
 It is the time required for individual compound to pass
from the column
 Measured from time of sample administration to peak of
that particular compound
 Different compounds have different unique R.T.

35. Continued…
 Retention time depends on
1- Pressure applied by pump
2- Nature and size of stationary phase molecules
3- Composition of solvent
4- Temperature of Column

37. Quantification of
Compounds
 Concentration of compound
is measured using AUC
of that particular peak
 More AUC More is the
concentration
 Measured automatically by computer
 Computers are connected to printers

38. Coupling of HPLC with Mass
spectrometer
 When detector is showing the peak, some of what
substance is passing through the detector at that time
can be diverted to a mass spectrometer
 There it will form Fragmentation pattern
 Pattern can be compared against a computer database
of known pattern
 Identity of substance can be found without having to
know their retention time

40. Application of HPLC
Sugar analysis in fruit
juices
To detect
pharmaceuticals
in water
To detect
pesticides in
drinking water
To ensure quality
of soft drinks
Analysis of
alcohol
percentage in
beers
To analyze
polycyclic
compounds in
vegetables
To detect
Phenolic
compound in
drinking water

41. Continued…
Pharmaceutical
quality control
studies
Shelf-life
determinations
of
pharmaceutical
products
To detect
anabolic
steroids in
serum, urine
Forensic
analysis of
textile dyes
Estimation of
bilirubin and
biliverdin in
hepatic
disorders
Quantification
of ions in
human serum,
urine
Detection and
quantification
of drug in
biological
samples

42. DISADVANTAGES ADVANTAGES

43. TLC (Thin layer
chromatography)
Planar
chromatography
Qualitative method
only
HPTLC more
precise version

45. Principle of Action
Separation
depends on
relative affinity
of compounds
towards 2 phase
Compound with higher
affinity for Stationary
phase will travel slowly
Compound
under influence
of mobile phase
travel over
stationary phase

46. TLC MACHINE

47. Instrumentation of TLC
1- TLC Plates
2- TLC Chamber
3- Mobile phase
4- Filter paper

48. TLC Plates of different size

49. TLC Plates
1
• Readymade available in market, where aluminum base is used
2
• Can also prepare in lab by using glass slide as a base, where
Stationary phase is applied over it
3
• Readymade plate is preferred
4
• Any side is taken as base, and above 1 cm from base line by pencil is
made
5
• TLC plate, after preparing kept in TLC Chambers

50. TLC Chamber

51. TLC Chamber
 Contains mobile phase as a solvent
 Mobile phase used, should be particulate free and of
highest purity for proper development of Spots
 Solvent used, should be chemically inert to sample and
stationary phase
 A moistened filter paper is placed onto wall of chamber
to maintain humidity and Edge effect

52. Procedure
TLC plates are
prepared
Filter paper is
applied over the
wall of chamber
Mobile phase is
added and chamber
is saturated
Lid is closed, to
allow movement of
mobile phase over
stationary phase
Spots are
developed. After
washing, the
process is repeated

53. Mistake to avoid !!!
 Side of plate with base line is placed facing mobile phase
 Prevent immersion of TLC plate’s sample line into mobile
phase , to prevent its dissolution into mobile phase

54. Movement of
Mobile Phase
over
stationary
phase is by
Capillary
action

55. Outcome
 TLC plate are removed and allowed them to dry
 Spots are visualized by various techniques

56. Polyphenol are
visualized by putting
it in UV light
chamber
For Basic substance,
dip in acid, allow to
dry and then heat it
Brown spots
Iodine vapor
treatment for C=C
compounds
Valinine stain for
alcohol and
aldehyde
Ninhydrin test for
amino acids

57. How to identify compound
?
 Measured using Retention Factor (RF)
 Range from 0 to 1
 RF = Distance covered by individual compound / Distance
covered by solvent
 Calculated RF is matched with Standards and substance is
identified
 In Normal TLC, larger RF value will be of less polar substance

58. How to identify compound
?

59. Types of TLC
Stationary phase is Polar Stationary phase Non-polar
1st elute – Non-polar 1st elute - Polar

60. Application of TLC
To check
purity of
sample
To identify
sample like
acid,
alcohol,
amines To identify
pesticides
in drinking
water
For rapid
determinati
on of
petroleum
products in
sample
To identify
drug in
biological
samples

61. DISADVANTAGES
• Time consuming
• Spots larger than 2mm
overlap
• Long streak sometimes
formed rather than spots
ADVANTAGES
• Simple process
• Cheaper
• Purity standards of given
sample can be assessed

62. HPLC VS TLC
FEATURES : HPLC TLC
INSTRUMENTATION Much Minimal
SAMPLE APPLICATION Automated, injection Manual, spotting
MOBILE PHASE MOVEMENT By High pressure Capillary action
FORM OF RESULTS Peak Spot
COST Costly Cheaper
ANALYSIS Qualitative and quantitative Qualitative only
COMMON METHOD Reverse phase HPLC Normal Phase TLC
SAMPLE PREPERATION NO YES
TIME 3-6 Minutes 3-4 Hours

63. DNA STRUCTURE

64. PCR (Polymerase Chain
Reaction)
Used to amplify single copy of DNA to thousands
of copies
Amplify specific DNA fragments from minute
quantity, even that DNA is of poor quality
On of the those few scientific revolutionary
development

65. HISTORY OF PCR

66. Concept already given in 1971
by Dr. Gobind Khurana

67. Principle of PCR

68. Components required

69. Properties of DNA Template
Sample
DNA
It contains DNA region we need to amplify
Concentra
tion
Required
30- 50 ng with 50-55 % of GC content,
Low concentration – False negative result,
High concentration - False positive result
Purification
Required
Before procedure, template is purified and
impurities are removed by either anion exchange
technique or phenol-chloroform technique

71. Thermus Aquaticus

73. Is short sequence of
Nucleotide
20 Nucleotide
containing single DNA
strand
< 10 and > 20
Nucleotide are not
recommended
2 Primers are used in
each PCR reaction
They are designed
(given sequence) that
they bind to opposite
strand of template

74. DNTP is building block of DNA molecule
• Consist of Phosphate group, deoxyribose sugar, and
nitrogenous base – adenine, guanine (Purine), thymine
and cytosine (Pyrimidine)
Function of DNTP is to expand DNA strand in
presence of Taq polymerase
• It binds with complementary DNA strand by Hydrogen
bond

75. STEP OF PCR
PCR
Ingredients
assembled in
PCR Tube
Denaturation
Annealing
Extension

76. Denaturation
Heat is applied to
reaction (70-75 °C)
To separate DNA strands
Single stranded template
formation occur

77. Annealing
Now Taq
Polymerase
will extend
the primer ,
synthesizing
new strand
of DNA
Taq
Polymerase
Primer
binds to its
complimen
tary
sequence
on the
single
stranded
DNA
Binding
After
heating,
reaction is
cooled (55
to 60 °C to
allow
primer to
bind
Cooling

79. Extension
ELONGATION
Synthesis of new DNA Strand occurs with extension of primer
TAQ POLYMERASE
Being enzyme of DNA replication, assembles nucleotide and so extends primer
RAISING OF TEMPERATURE
After Annealing, temperature is raised to 70 °C for action of Taq Polymerase

82. Cycle and Timing
 Cycle repeats 25-35 times in one PCR reaction
 One PCR reaction takes 3-4 hrs. to complete
 Time duration, depends upon length of DNA
region to be copied
 If reaction is efficient, target region can go from
one or a few copies to billions

84. APPLICATION
Genetic engineering
Diagnosing genetic
disorder and so
preventing disease
by screening before
birth, e.g - CF
Genetic
fingerprinting –
from sample of
blood, semen, hair
In Archaeology to
identify human or
animals from
samples
Detection of
Infection in the
Environment
Detection of
infectious diseases,
e.g – Covid 19
Personalized
medicine

85. Types of PCR
 Here Complimentary DNA (CDNA) is created by
transcription from RNA
 Then formation of dsDNA done, then by PCR, Gene
expression is qualitatively studied using RTPCR
 Test for qualitative detection of nucleic acid from SARS-
CoV-2

86.  Aka Real time PCR
 Allow quantification of target species, that is total
bacterial count in chemical sample
 By adding dye like Sybr green, the florescence signal is
proportional to amount of DNA synthesized

87.  Used for the identification of species that inhabit humans
, e.g- Malaria
 Multiple primer pairs are used for concomitant
detection of different species

88.  Is modification of PCR, that reduces non-specific binding
of primary product, limiting non specific products

89. References
• Murray, Robert K. Harper's Illustrated Biochemistry. New York: McGraw-Hill, 2003. Print.
• Harvey, Richard A., Ph. D. Lippincott's Illustrated Reviews: Biochemistry. Philadelphia :Wolters Kluwer
Health, 2011.
• Kumar, Vinay, et al. Robbins Basic Pathology. 10th ed., Elsevier - Health Sciences Division, 2017.
• Postgraduate Pharmacology 1st Edition 2020 by Sougata Sarkar.
• Hearn, Milton TW. "High–Performance Liquid Chromatography and Its Application to Protein
Chemistry." Advances in chromatography (2021): 1-82.
• Jalil, Abduladheem Turki, et al. "Polymerase chain reaction technique for molecular detection of HPV16
infections among women with cervical cancer in Dhi-Qar Province." Materials Today:
Proceedings (2021).

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