Various analytical techniques used in Pharmacology

1. Analytical Techniques in
Pharmacology
Capt Htet Wai Moe
04/03/17

2. Introduction
• From the stage of drug development to
marketing and post marketing, analytical
techniques play a great role

3. Common Analytical Techniques
• Titrimetry
• Chromatography
• Spectroscopy
• Capillary Electrophoresis
• Hyphenated techniques

4. Titrimetric techniques
Principle
• Addition of a reactant to a solution being
analyzed until some equivalence point is
reached
• Most familiar one is the acid-base titration
involving a color changing indicator

5. Fig. Titrimetry

6. Applications of Titrimetric techniques
• Provide standard pharmacopeial methods for
the assay of unformulated drugs and
excipients and some formulated drugs
• Used for standardization of raw materials and
intermediates used in drug synthesis
• Captopril, Albendazole, Gabapentin,
Sparfloxacin

7. Chromatography
Principle
• Separation technique used for separation of
mixture of low molecular weight compounds
based on their distribution in stationary phase
and mobile phase

8. Chromatographic techniques
• Thin Layer chromatography
• High performance thin layer chromatography
• High performance liquid chromatography
• Gas chromatography

9. Thin Layer chromatography (TLC)
Principle
• Chromatography technique used to separate
non-volatile mixtures
• Particles are separated based on partition or
adsorption

10. Thin Layer chromatography
• Performed on a sheet of glass, plastic , or
aluminum foil coated with a thin layer of
adsorbent material
• Adsorbent material – silica gel, aluminum
oxide or cellulose
• Adsorbent shows extreme selectivity toward
the substances being separated so as to the
dissimilarities in the rate of elution be large

12. Application of TLC
• Qualitative, quantitative, small scale and
preparative separation of amino acids,
steroids and nucleic acids
• Separation and isolation of large number of
inorganic compounds
• Various impurities of pharmaceuticals can be
identified and determined using TLC

13. High performance thin layer
chromatography (HPTLC)
Principle
• Enhanced form of thin layer chromatography
• Enhanced to automate the different steps, to
increase the resolution achieved, and to allow
more accurate quantitative measurements
• Automation is useful to overcome the
uncertainty in droplet size and position when
sample is applied to TLC plate by hand

14. Fig. HPTLC

15. Differences between TLC and HPTLC
Parameter TLC HPTLC
Chromatographic plate
used
Hand made/ pre-coated Pre-coated
Sorbent layer thickness 250 µm 100-200 µm
Particle size range 5-20 µm 4-8 µm
Application of sample
Manual/ Semi
automatic
Semi automatic/
Automatic
Sample volume 1-20 µl 0.2-5 µl
No. of samples/plate 15-20 40-50
Reproducibility of
results
Difficult Reproducible

16. High Performance Liquid
Chromatography (HPLC)
Principle
• Highly sensitive separation technique
• Sample and purified mobile phase are applied
to the column under high pressure
• Sample compounds are separated based on
adsorption, partition, ion exchange, molecular
exclusion or affinity principle

17. HPLC
• Relies on pumps to pass a pressurized liquid
solvent containing the sample mixture
through a column filled with a solid adsorbent
material
• Each component in the sample interacts
differently with the adsorbent material

18. HPLC
• Different flow rates for different components
lead to separation of components as they flow
out of the column
• Separate the complex mixture and recognize
the role of individual molecules

19. HPLC

20. Detectors in HPLC
• Choice of detectors is critical to guarantee that
all the components are detected
• Common types of detectors
– UV detector
– Photodiode array (PDA) detector
– Refractive index detector
– Fluorescence detector

21. UV detector
• Capable of monitoring several wavelengths
concurrently
• Can detect all the UV-absorbing components

22. UV detector

23. Photodiode array (PDA) detector
• Can sense a range of wavelengths
concurrently
• Wavelength range can be programmed and all
the compounds that absorb within this range
can be identified in a single analysis

24. Fig. Photodiode array detector

25. Refractive index detector
• Detector of choice to detect analytes with
restricted or no UV absorption such as
alcohols, sugars, carbohydrates, fatty acids,
and polymers

27. Fluorescence detector
• Most sensitive detectors
• 10-100 times higher than UV detector for
strong UV absorbing materials

28. Fluorescence detector

29. Applications of HPLC
• Manufacturing (e.g. during the production
process of pharmaceutical and biological
products)
• Legal (e.g. detecting performance
enhancement drugs in urine)
• Research (e.g. separating the components of a
complex biological sample)
• Medical (e.g. detecting Vitamin D levels in
blood serum)

30. Limitations of HPLC
• Price of columns and solvents
• Lack of long term reproducibility due to
proprietary nature of column packing

31. Gas Chromatography
Principle
• Used in analysis of the compounds that can be
vaporized without decomposition
• Testing the purity of a particular substance
• Separating the different components of
mixture

32. • Mobile phase is a carrier gas, usually an inert
gas such as helium or an unreactive gas such
as nitrogen
• Stationary phase is a microscopic layer of
liquid or polymer on an inert solid support
• Gaseous compounds being analyzed interact
with the walls of column
Gas Chromatography

33. Gas Chromatography

34. Applications of Gas Chromatography
• Assay of isotretinoin, cocaine and residual
solvents in betamethasone,
• Analysis of impurities of pharmaceuticals
• Identification of functional group of
compounds

35. Spectroscopy
• Analytical techniques used for quantification,
characterization and structural analysis of
macromolecules by measuring the absorption,
transmission, emission of radiation by the
molecules

36. Spectroscopic techniques
• UV-visible spectroscopy
• Mass spectrometry
• Near infrared spectroscopy
• Nuclear magnetic resonance spectroscopy
• Fluorimetry
• Phosphorimetry

37. UV-visible Spectroscopy
Category
Colorimetry Spectrophotometry

38. Beer’s Law
• Amount of light absorbed by a colored
solution is proportional to the concentration
of the solution
A α C
A = absorbance
C = concentration of the color in the solution

39. Lambert’s Law
• Amount of light absorbed by a colored
solution is proportional to the depth through
which the light passes in the solution
A α L
A = absorbance
L = depth through which light passes in the
solution

40. Colorimetry
• Quantification of colored compounds in
solution
Irradiating with visible light (400 – 700 nm)
Measuring the absorbance

41. Colorimetry

42. UV- Visible Spectrophotometry
• Quantification and characterization of
colorless compounds
Irradiating with UV (185-400 nm) or
visible light (400-700 nm)
Measuring the absorbance

43. UV-visible Spectrophotometry

44. Fig: Spectrophotometer

45. Applications of UV-visible
Spectroscopy
• To quantify protein, carbohydrates, Hb,
Chlorophyll
• To detect impurity in biological compounds
(Impurities are displayed as additional peaks
in spectrum)

46. Common Drugs analyzed by UV-
visible spectrophotometry
• Acetaminophen, Amlodipine, Ampicillin,
amoxycillin, Lisinopril, Flunarizine,
Carbenicillin, Diclofenac sodium, Diltiazem,
Nicorandil, Pantoprazole sodium, Zidovudine,
Verapamil

47. Mass Spectrometry
Principle
• Sample is ionized by bombarding it with high
energy electrons
• Some of the sample’s molecules to break into
charged fragments
• These ions are then separated according to
their mass-to-charge ratio

48. Mass Spectrometry (MS)
• Ions of the same mass-to-charge ratio will
undergo the same amount of deflection
• The ions are detected by electron multiplier
which can detect charged particles
• Results are displayed as spectra

49. Application of MS
• Elucidation of structure of organic and
biological molecules
• Determination of molecular mass of peptides,
proteins and oligonucleotides
• Identification of drugs abuse and metabolite
of drugs of abuse in blood, urine and saliva

50. Principle
• Uses light over the infrared range (700-15000
nm) of electromagnetic radiation spectrum
• The energy curve of an oscillating molecule is
affected by intramolecular interactions
• Vibrations around the equilibrium position
are non-symmetric and the spacings between
energy levels that the molecule can attain are
not identical
Near Infrared spectroscopy (NIRS)

51. • Easy preparation without any pretreatments
• Identification and qualification of raw
materials and intermediates
• Analysis of intact dosage forms
• Process monitoring and process control
Applications of (NIRS)

52. Near Infrared spectroscopy

53. Nuclear magnetic resonance
spectroscopy (NMR)
Principle
• The important property of nucleus is spinning
• Compound is subjected to external magnetic
field and radio wave
• Nucleus is promoted to higher magnetic
energy level and resonance occurs NMR
signal recorded as NMR peak gives
structural information about the compound

55. Applications of NMR
• To study the structure of antibiotics,
interleukin and nucleic acids
• To study drug metabolism
• For conformational analysis of
macromolecules

56. Fluorimetry
Principle
• Fluorescence is a phenomenon of emission of
radiation when the molecules are excited by
radiation at certain wavelength
• Fluorimetry measures the fluorescence
intensity at a particular wavelength with the
help of a filter fluorimeter

57. Fluorimetry

58. Fig. FLuorimeter

59. Applications of Fluorimetry
• High sensitive technique to identify the
presence and amount of specific molecules
• Quantitative analysis of various drugs in
dosage forms and biological fluids

60. Phosphorimetry
Principle
• Phosphorescence is also a phenomenon of
emission of radiation when the molecules are
excited by radiation at certain wavelength
• Difference from fluorimetry – emission of
radiation continues after light source is cut off
• Applications – Analysis of Quinolone,
Riboflavin, Anticancer drugs, Naproxen

61. Capillary Electrophoresis
Principle
• Separation of compounds that are capable of
acquiring electric charge in conduction
electrodes
• Based on difference in migration of charged
particle through a solution under the
influence of an external electrical field

62. Capillary Electrophoresis
• Solutes are perceived as peaks as they pass
through detector and the area of individual
peak is proportional to concentration
• Quicker time scale, requirement of only a
small amount up to Nano liter injection
volumes
• Applications – Separation of Atropine
sulphate, Codeine phosphate, Ketamine HCL,
Phenylephrine HCL

63. Fig. Capillary electrophoresis

64. Fig. Capillary electrophoresis

65. Hyphenated techniques
• Provides more information than could be
obtained by using the individual techniques in
isolation
• Time saving
• Additional information

66. Hyphenated techniques
• Hyphenated techniques combine
chromatographic and spectral methods to
exploit the advantages of both
• Chromatography produces pure or nearly pure
fractions of chemical components in a mixture
• Spectroscopy produces selective information
for identification using standards or library
spectra

67. • GC-MS
Gas chromatography–mass spectrometry
• LC-MS
Liquid chromatography–mass spectrometry
• LC-IR
Liquid chromatography–Infrared Spectroscopy
• LC-NMR
Liquid chromatography–Nuclear magnetic
resonance spectroscopy
• CE-MS
Capillary electrophoresis–mass spectrometry
Hyphenated techniques

68. GC-MS
• Compounds that are adequately volatile, small
and stable in high temperature in GC conditions
can be easily analyzed by GC-MS
• A carrier gas propels the sample down the
column
• GC separates the components of a mixture in
time
• MS detector provides information that aids in the
structural identification of each component

69. LC-MS
• Separated sample emerging from the column
can be identified on the basis of its mass
spectral data
• Combines chemical separating power of LC
with the ability of MS to selectively detect and
confirm molecular identity

70. LC-IR
• Coupling of LC and detection method infrared
spectrometry (IR)
• IR is useful spectroscopic technique for
identification of organic compounds

71. LC-NMR
• Analysis of complex mixtures of all types,
particularly the analysis of natural products
and drug-related metabolites in biofluids
• Reversed-phase columns are used in LC-NMR

72. CE-MS
• Almost all molecules can be separated using
this powerful method
• Separates species by applying voltage across
buffer-filled capillaries
• Generally used for separating ions that move
at different speeds when voltage is applied,
depending on their size and charge

73. References
• Julia C Drees, Alan H B Wu. Analytical
techniques. In: Michael L Bishop, editor.
Clinical chemistry. 5th Ed, Lippincott Williams
& Wilkins 2010; 130-165.
• Glen L Hortin, Bruce A Goldberger.
Chromatography and Extraction. In: Tietz
Textbook of Clinical Chemistry and Molecular
Diagnostics. 5th Ed, Elsevier 2012; 307-328.

74. THANK YOU!