Introduction to Analytical Techniques in Phaese III, Spectrophotometry, Reflectance photometry, Nephelometry & Turbidimetry, Osmometry, Potentiometry, Flowcytometry, Densitometry, Electrophoresis, LC-MS, ICP-MS Presented by B. Kranthi Kumar Department of Pharmacology

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
B. kranthi kumar
(Reg. No. 20L81S0108)
Under the guidance/Mentorship of
Dr. P. Ramalingam, M.Pharm, Ph.D,
Research Director& Professor of Pharmaceutical and Medicinal Chemistry
ANALYTICAL TECHNIQUES
IN PHASE III

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
 Spectrophotometry
 Reflectance photometry
 Nephelometry & Turbidimetry
 Osmometry
 Potentiometry
 Flowcytometry
 Densitometry
 Electrophoresis
 LC-MS
 ICP-MS
References
Contents

3. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Spectrophotometry is the quantitative measurement of how much a
chemical substance absorbs light by passing a beam of light through
the sample using a spectrophotometer.
• In clinical laboratories, spectrophotometers are used for many
applications such as quantitative analysis, kinetics, spectrum
scanning and a multiple components - DNA/Protien testing .
Spectrophotometry

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
1. VIS Spectrophotometer
2. UV-VIS spectrophotometer
3. Infrared spectrophotometer
4. Fluorescence spectrophotometer
5. Atomic absorption spectrophotometer
Types of spectrophotometers

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
• Beam of light is directed at a flat reaction surface & the reflected
light is quantified.
• Reaction mixture in carrier is illuminated with diffuse light & the
intensity of the reflected light from the chromogen is compared with
the intensity of the light reflected from reference surface.
• Used as quantitative measurement of surface reaction such as dipstick
or dry film chemistry system.
Reflectance photometry

6. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
• These are the analytical techniques used to measure scattered light.
Principle of Nephelometry
• Intensity of light scattered by a suspension is measured at 90 degrees
angle.
• Intensity of scattered light ά concentration of suspension.
Nephelometry & Turbidimetry

8. RIPER
AUTONOMOUS
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NBA (UG)
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
• Quantitative nephelometry is a lab test to quickly and accurately
measure levels of certain proteins called immunoglobulins in the
blood.
• Immunoglobulins are antibodies that help in fighting infection.
• This test specifically measures the Immunoglobulins IgM, IgG and
IgA.

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
• Principle of Turbidimetry :
• Measurement of decrease in light transmitted through a turbid
solution is measured.
• Immunoturbidimetry is an important tool in the broad diagnostic field
of clinical chemistry.
• It is used to determine serum proteins not detectable with classical
clinical chemistry methods.
• Immunoturbidimetry uses the classical antigen-antibody reaction.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
• The antigen-antibody complexes aggregate to form particles that can
be optically detected by a photometer.

11. RIPER
AUTONOMOUS
NAAC &
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
• Osmometry is an advanced analytical method for determining the
osmotic concentration of solutions.
• The osmotic-or solute-concentration of a colloidal system is
expressed in osmoles(Osm) per unit of volume (Osm/L) or weight
(Osm/kg).
• In clinical and formulation contexts, osmolarity/osmolality refers to
solutes as small as ions which require exceptional sensitivity down to
the milli osmole(mOsm) range.
Osmometry

12. RIPER
AUTONOMOUS
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NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
• An Osmometer is a device for measuring the osmotic strength of a
solution, colloid, or compound.
• Several research laboratories utilize osmometry routinely in screening
for potential fluid or electrolyte inbalances.

13. RIPER
AUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
• Potentiometry is an electrochemical measurement technique .
• Mainly, the measuring setup consists of two electrodes(galvanic
cell).These half cells are called indicator (or measuring) and
reference electrode.
• Potentiometric titration is a laboratory method to determine the
concentration of a given analyte. It is used in the characterization of
acids.
Potentiometry

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• In Potentiometric measurements the potential between two electrodes
is measured using a high impedence voltmeter.
• Potentiometer is essentially a voltage divider used for measuring
potential.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
• The basic principle of flow cytometry is the passage of cells in single
file in front of laser so they can be detected, counted and sorted.
• Other instruments using flow cytometry include cell sorters which
physically separate and thereby purify cells of interest based on their
optical properties.
• The analysis of samples using flowcytometry can provide cell data to
support early determination of drug efficacy.
Flowcytometry

16. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
• In the clinical hematology laboratory, flowcytometry is used for
immunophenotyping of a variety of specimens, including whole
blood and bone marrow.

17. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
• Densitometry is the quantitative measurement of optical density in
light-sensitive materials, such as photographic paper or photographic
film, due to exposure to light.
• Spot densitometry: the value of light absorption is measured at a
single spot.
• Bidimentional densitometry: The values
of light absorption are expressed as a 2D
synthetic image.
• Special devices have been developed
for clinical diagnosis, called bone
densitometers.
Densitometry

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
• It is defined as the migration of the charged particle through a
solution under the influence of external electrical field.
• It is used to seperate DNA,RNA or protein molecules based on their
size and electric charge.
Electrophoresis

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
• Liquid chromatography-mass spectrometry(LC-MS) is an analytical
chemistry technique that combines the physical separation
capabilities of liquid chromatography(or HPLC) with the mass
analysis capabilities of mass spectrometry (MS).
• The LC-MS/MS is a powerfull tool for the detection of residual
chemical compounds, confirmatory identification of small organic
molecules, and confirmation and quantitation of contaminants and
adulterants in pharmaceutical and food samples.
LC-MS

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21. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
• Inductively coupled plasma(ICP)-Mass spectrometry(MS)
• ICP-MS is a type of mass spectrometry that uses an inductively
coupled plasma to ionize the sample.
• ICP-MS used to measure elements at trace levels in biological fluids.
• ICP-MS used to measure the individual isotopes of each element.
ICP-MS

22. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
• Albanese, C V, Diessel, E, Genant H K.Clinical applications and
measurments using DXA. J Clin Densitom.2003;6;75-85.
• Gbylik, M, Posyniak, A, Mitrowska, K, Bladek, T, Zmudzki, J.
Principles of liquid chromatography-tandem mass spectroscopy.
2013;5;940-948.
• Smith, S.B., Aldridge, P.K., & Callis, J.B. Observation of individual
DNA molecules undergoing gel electrophoresis. Science. 1989;243;
203-206.
Reference

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23