Drug Research and Instruments used in Research Methodology

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S.J.G AYURVEDA MEDICAL COLLEGE & HOSPITAL KOPPAL
PHYTOCHEMISTRY – AN OVERVIEW
Phytochemistry is the name given to the study
of the chemistry of plants.
Motivation for life Academy by Dr Khalid B.M 1
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STEPS IN PHYTOCHEMICALANALYSIS
Biological activity
1. Selection of promising plant material Folk medicine
2. Proper collection of selected plants.
3. Authentication of plant
4. Drying of plant
5. Grinding of dried plants
6. Packing, storage & preservation
7. Extraction
8. Purification
9. Identification of bioactive compounds.
Wild plant
Cultivated
Establishing identity by
taxonomy experts
Field botanist
Maceration
Percolation
Digestion
Hot continuous extraction
Super critical fluid
Extraction
Microwave
Solid phase extraction
Chromatography
Simple qualitative analysis
Advanced qualitative /
quantitative analysis
Spectroscopy UV-VIS,
NMR, MS
Chromatography
X ray crystallography Motivation for life Academy by Dr Khalid B.M 2
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Simple Qualitative Analysis
Objective: Detection of bio-active compounds
Example: Testing alkaloids
Mayer’s Test: Filtrates were treated with Mayer’s reagent
(Potassium Mercuric Iodide).
Formation of a yellow coloured precipitate indicates the presence
of alkaloids.
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Objective: Detection of bio-active compounds
Example: Testing alkaloids
Dragendroff’s Test: Filtrates were treated
with Dragendroff’s reagent (solution of
Potassium Bismuth Iodide).
Formation of red precipitate indicates the
presence of alkaloids.
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Molisch’s Test: Filtrates were treated with 2 drops
of alcoholic alpha-naphthol solution in a test tube.
Formation of the violet ring at the junction indicates
the presence of Carbohydrates
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Benedict’s Test: Filtrates were treated
with Benedict’s reagent and heated
gently.
Orange red precipitate indicates the
presence of reducing sugars..
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Modified Borntrager’s Test:
Extracts were treated with Ferric Chloride
solution and immersed in boiling water for
about 5 minutes. The mixture was cooled and
extracted with equal volumes of benzene. The
benzene layer was separated and treated with
ammonia solution.
Formation of rose-pink colour in the ammonical
layer indicates the presence of anthranol
glycosides.
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PERCOLATION
Continues flow of solvent through the crude
drug to get the extract
Done using percolator
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C H R O M A T O G R A P H Y – AN OVERVIEW
Learning Objectives:
 To understand the fundamental Concepts, Principal, Method, Applications,
Advantages & Disadvantages
 CCIM Syllabus - Awareness of Chromatography technique
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CHROMATOGRAPHY
 Most popular TOOL in BIOCHEMISTRY.
 CHROMATOGRAPHY– Chromo – colour
Graphy – measure or to write.
 20th century this technique used for separation of plant pigments such as
chlorophyll
 Introduced by Russian botanist MIKHAIL ISWETT
 “Chromatography is an analytical technique used for separation of coloured
substance from the plants is now the most extensive technique of separation of
coloured colourless organic compounds”.
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CHROMATOGRAPHY
PRNICIPLE:
In chromatography substance are separated due to their relative affinities for the
stationary & mobile phase, distribution co-efficient ‘K’ governs the distribution.
K = Concentration of a component in stationary phase
Concentration of a component in moving phase
K=CS/CM
If K=1, the substance will distribute equally in two phase.
If K<1, the substance will have lesser affinity for stationary phase & more affinity for mobile phase.
If K>1, the substance will have more affinity for stationary phase & compared to that with mobile phase
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CHROMATOGRAPHY
K = Concentration of a component in stationary phase
Concentration of a component in moving phase
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CHROMATOGRAPHY
STATIONARY PHASE:
May be a solid or liquid supported as a
thin film on the surface of inert solid
through which the sample contained in
the mobile phase Percolates.
MOBILE PHASE:
Mobile phase flowing over the surface of
The stationary phase may be a gas or liquid.
It may be called as ELUENT.
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CHROMATOGRAPHY
TYPES OF CHROMOTORAPHY:
1. Partition Chromatography: Stationary phase = liquid
Paper chromatography
Column chromatography
Gas liquid chromatography
2. Adsorption Chromatography: Stationary phase = solid
Thin layer chromatography/ High performance thin layer chromatography
High performance liquid chromatography
Flash chromatography
Gas solid chromatography
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CHROMATOGRAPHY
PAPER CHROMATOGRAPHY:
 It is type of Partition chromatography
 Filter paper – stationary phase
 Solvent = Mobile phase
METHOD:
 Papers: whatmann 31 whatmann 20
 Solvents: chloroform, benzene, isopropnal,
ammonia, cyclohexane etc
Stationary phase is immersed in mobile phase.
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CHROMATOGRAPHY
PAPER CHROMATOGRAPHY
APPLICATION
1. Mainly for separation of amino acids
2. For separation of mixtures of plant, animal origin
3. For identification & separation of impurities
4. For identification of drugs
5. For analysis of metabolites of drugs in body fluids
6. For analysis of mixtures of sugar, vitamins,
antibiotics etc
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CHROMATOGRAPHY
COLUMN CHROMATOGRAPHY
Column chromatography is a separation technique in which a column of stationary phase
is used. The stationary phase used in column chromatography may be solid or liquid.
Types:
1. Adsorption column chromatography
2. Partition column chromatography
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CHROMATOGRAPHY
APPLICATION:
1. Adsorption column chromatography
Column chromatography has limited application compared to other analytical techniques.
 Separation of mixture compounds
 Removal of impurities from a compound
 Isolation of active constituents from crude extracts of plants, animals etc.
 Isolation of metabolites from biological fluids like blood, serum or plasma etc.
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CHROMATOGRAPHY
APPLICATION:
1. Partition column chromatography
 separation of wide range of biological compounds
 Separation of closely related substance which as amino acids formed in the hydrolysis of
protein
 Separation closely related aliphatic alcohols, sugar derivatives and carboxylic acids.
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CHROMATOGRAPHY
FLASH CHROMATOGRAPHY
FC also known as medium pressure chromatography, this type of chromatographic
technique uses compressed gas[nitrogen or air] or a pump to push solvent through
column. This technique advantages because it allows for faster flow rates of th solvent,
as opposed to simple gravity flow.
APPLICATION:
1. Purifiction of synthetic products
2. Purification of plant pigments
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CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY
Thin layer chromatography was first introduce by
Izmailov & shraiber
Principle:
TLC is a solid- liquid from of chromatography
where the stationary phase is normally a polar
absorbent & the mobile phase is single solvent or
combination of solvents.
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CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY
APPLICATION:
1. Separation of mixtures of drugs of both chemical
& biological origin
2. Identification of substances
3. Identifying the related compounds in drugs
4. The detection of the decomposition products in drugs
5. Examination of a chemical reaction & purification processes.
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CHROMATOGRAPHY
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
HPTLC is a from of TLC that provides superior separation power using optimized
coating material, novel procedures for mobile phase feeding, layer conditioning, &
improved sample application.
KEY FEATURES OF HPTLC:
 Simultaneous processing of sample & standard
 Several analysts work simultaneously
 Lower analysis time & less cost per analysis
 Low maintenance coast
 No prior treatment for solvents like filtration & degassing
 Visual detection possible – open system
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CHROMATOGRAPHY
Advancement made on TLC leading to the development of HPTLC:
 HPTLC is modern technique, combined with automated sample application &
densitometric scanning, is sensitive & completely reliable , suitable for use in
qualitative & quantitative analysis.
 HPTLC is a valuable tool for reliable identification because it can provide
chromatographic fingerprints that can be visualized & stored as electronic images.
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CHROMATOGRAPHY
APPLICATION
1. HPTLC method is applied for qualitative & quantitative analysis.
2. Qualitative analysis was used for identification of drugs/chemicals or certain toxic
residuals compounds in food or other preparation, & fingerprinting of herbal
drugs/biological matrixes
3.Quantitave analysis was used for the determination of drugs/ metabolites in
pharmaceutical preparations or biological matrixes, impurities, residual toxic
compounds, drug stability testing, & quality control purpose.
4.HPTLC now a days applied to obtain finger print patterns of herbal formulations.
Quantification of active ingredients & also detection of adulteration
5. In clinical laboratories to estimate the level drugs in plasma.
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CHROMATOGRAPHY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC was developed in early 1970’s & originally
referred as HPLC because, high pressure was used to
increase the efficiency of separation.
PRINCIPLE:
HPLC is a solid liquid form of chromatography &
the separation principle employed is adsorption
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CHROMATOGRAPHY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
APPLICATION:
1. For both for qualitative & quantitative analysis of components in a sample
2. For checking purity of components & presence of impurities.
3. For isolation & identification of mixture of components of both natural & synthetic
origin.
4. In biopharmaceutical, pharmacokinetic & stability studies.
5. For analysis of drug & metabolites in body fluids.
6. To separate closely related compounds efficiently
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CHROMATOGRAPHY
GAS CHROMATOGRAPHY
Gas chromatography is an analytical technique used for separation of thermally stable
& volatile substances. In GC, the mobile phase used is gas & the stationary phase is
either liquid or solid.
PRINCIPLE:
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CHROMATOGRAPHY
GAS CHROMATOGRAPHY
APPLICATION:
1 For qualitative & quantitative analysis of liquids, gases & vapours & particularly
organic compounds.
2. To isolate & identify metabolites in body fluids
3. In multi component analysis of mixture of drugs
4. To determine purity of the compounds.
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SPECTROPHOTOMETRY – AN OVERVIEW
Spectrophotometry deals with the measurement of light absorption or transmission. This
one of the most valuable analytical technique in biochemistry. Unknown compounds
may be identified by their characteristics absorption spectra in the ultraviolet, visible or
infrared regions of the electromagnetic spectrum.
PRINCIPAL:
All spectrophotometric measurements are based on upon BEER-LAMBERT’S LAW.
BEER-LAMBERT’S LAW
It is liner relation between absorbance and concentration of absorbing species
It is combination of two laws beers and lamberts.
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COLORIMETER
This is the instrument used for measurement of coloured substances. This instrument is
operative in the visible range [400-800nm] of electromagnetic spectrum of light.
Light filter cuvette detector display
Source [photodiode cell] [galvanometer]
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SPECTROPHOTMETER
Spectrophotometer is an instrument that measure the amount of light absorbed by a
substance.
Spectrophotometer is a modified & advanced instrument of colorimeter for measurement
of light intensity. When absorption characteristics of a compound are unknown,
spectrophotometer can be employee
Basic principle :Interaction light with matter
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AUTOMATION IN BIOCHEMISTRY – AN OVERVIEW
IUPAC describes automation an replacement of human manipulative efforts &
facilities in the performance of given process by mechanical & instrumental devices
that are regulated by feedback of information so the apparatus is self monitoring or
self adjusting.
Auto analyser is the instrument which performs analysis of a specimen by means
of automation.
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ADVANTAGES OF AUTOANALYSERS
1. Large number of samples can be tested in a short time
2. Variety of tests can be performed by using various methods such as endpoint &
rate of reaction
3. By using ELISA EMIT, TURBIDIMETRY, NEPHELOMETRY etc. it is
possible to determine concentration of hormones, drugs, tumour markers &
various types of antibodies & proteins
4. Most of the methods performed on automation are accurate, precise, sensitive
& specific
5. In case of fully automated analyser, the laboratory staff member do nto come in
contact with specimen & reagents[bio-hazards material] & hence working on
these analyser are safe.
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SEMI-AUTOMATED BIOCHEMICALANALYZER
In this analyser the initial stages of specimen analysis are
performed by the laboratory technician, including pipetting
of reagents, specimen & mixing, incubating reaction mixtures.
Features:
 Almost all routine biochemical tests can be performed
 Economical methodology, consumes less amount of
reagents test
 Plots absorbance verses wavelength for selected range
 Performs statistical analysis & plots levey-jenning charts
 Performs immunoassay, coagulation & drug assay
 Printing facilities is available Motivation for life Academy by Dr Khalid B.M 35
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FULLY-AUTOMATED DISCRETE BIOCHEMICALANALYZER
These auto analysers perform all the functions of
semi-auto analysers & in addition to that they also
perform following additional functions.
 automatic dispensing of reagents
 Automatic dispensing of samples
 Automatic mixing of reaction mixture
 Incubating of reaction mixture
 Two types 1. batch analyser
2. random access analyser
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Steps
1. Specimen identification
2. Specimen preparation, handling and delivery
3. Reagent handling, storage, identification & delivery
4. Mixing, incubation & reading
5. Measurement device:
photometry & spectrophotometry: for biochemical analyser Eg: blood glucose,
urea, creatinine, lipid, enzymes estimation etc.
Turbidimetry- Nephelometry: for determination of various proteins & antibodies
in the saple Eg. CRP, ASLO, ASLO titre, RA test, Immunoglobulins etc.
Ion selective electrode[ISE]: for determination of electrolytes Eg: sodium,
potassium, chloride, ionic calcium, lithium etc.
6. Signal processing, handling of data & control of microprocessors.
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EXAMPLES OF RANDOM ACCES ANALYZER
ERBA XL – 600
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EXAMPLES OF RANDOM ACCES ANALYZER
HITACHI 704 & 705
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DRY CHEMISTRY ANALYZERS
Eastman Kodak company introduced dry chemistry
analyser, which do not use wet chemicals but the
chemicals are incorporated into the series of thin
films on single use slide. Patient samples permeated
through the various layers on the slide and the end
results are determined calorimetrically.
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ELECTROLYTE ANALYZER
Electrolyte are measured by a process know as potentiometry.
This method measures the voltage that develops between the
inner & outer surface of an ion selective electrode[ISE].
ISE is a sensor that converts the activity of a specific ion
dissolved in a solution or serum into an electric potential,
which is measured by a voltmeter & pH meter.
APPLICATION:
To measure electrolytes like Na*,K*,Cl, Ca etc
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BLOOD CELL COUNTER
Automation in haematology is based on coulter principal ,
which states that particles pulled through an orifice,
concurrent with an electrical current, produce a change
in impedance that is proportional to the size of the particles
traversing the orifice.
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BLOOD CELL COUNTER
Coulter principal can be applied to count & size the various cells that make up
whole blood.
Complete blood count performed by an automated analyser that counts number &
types of different cells within the blood
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NEPHELOMETRY & TURBIDOMETRIC ANALYSIS
Nephelometry & turbidometric are closely related to colorimetric techniques. Both
techniques are based on measuring the scattered radiations by non transparent
particles which are suspended or dispersed in a solution, but they differ in the way
of measuring the scattered radiation.
Nephelometry deals with the analysis of colloidal systems[suspension or dispersion]
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NEPHELOMETRY & TURBIDOMETRIC ANALYSIS
APPLICATION:
1.Clinical application: determination of immunoglobulins
[ total IgG, IgE, IgM, IgA] in serum & other biological
fluids.
2. Analysis of organic compounds
3. Analysis of inorganic compounds
4. Analysis of biochemical compounds
5. Analysis of water
6. Drug development
7. Air pollutants
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ELISA Technique
Enzyme linked immunosorbent assay
ELISA is used as diagnostic tools in medicine & as quality control measures in
various industries, it is also used as analytical tools in biochemical research for the
detection & qualification of specific antigens & antibodies in a given sample.
Principal:
ELISA uses the basic immunology concept of an antigen binding t its specific
antibody, which allows detection of vary small quantities of antigen such as proteins,
peptides, hormones, or antibody in a fluid sample
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ELISA Technique
TYPES
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
APPLICATION:
ELISA is widely used for the determination of small quantities of hormones,
antigens, antibody & other biological substances. Most commonly used in pregnancy
testing, diagnosis of AIDS, detection of tumour markers etc.
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pH METER
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HOT AIR OVEN
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CENTRIFUGE
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INCUBUTOR
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Be a fellow traveller………………………………….
13-07-2023

Drug Research and Instruments used in Research Methodology

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