Post on 16-Jul-2015
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Dispersion system is defined as a
heterogenous two phase system in which
internal (dispersed,discontinous ) phase is
distributed or dispersed within the
continuous (external) phase or vehicle.
The internal and external phase may be
solids, liquids and also gases.
Eg.suspensions,emulsions,aerosols
Continuous Phase
Disperse Phase
Solid Liquid Gas
Solid Glasses containing
finely dispersed
metals, e.g., ruby
glass containing
gold, pastes such
as toothpaste
Gels, suspensions
(Kaolin)
Smoke, dust
Liquid Solid emulsion
(mineral oil in wax),
Cold cream
Emulsions such as
milk, mayonnaise,
oil in water
Fog, mist, liquid
aerosol, throat and
nasal relief sprays
Gas Solid foam
(foamed plastic)
Foams
(carbonated soft
drinks)
none
A. Mixers
B. High speed dispersers
C. Rotor stator mixers
D. Combination mixers
E. In- line mixers
F. Non- mechanical disperse processing
G. Fine suspension and size reduction
equipment
The most often used mixing implement is marine
propeller mixer.
These machines use rounded,pitched,three blade
design that produces mostly axial flow
They provide good flow and blending capabilities
in small batches of low to medium viscosities
Propellers mixers can be installed on vertical
centerline or through the side wall of process vessel
They can be operated at around 300-400rpm
These are used mostly for liquid-liquid blending
applications in some easily producible suspensions.
The most versatile of all mixers in the entire span of
mixing equipments are the axial and radial flow
turbines
Turbines mixers can be made to handle huge
batches,evenupto 5,00,00gal & suitable for
emulsification process.
Radial Flow turbine Axial flow turbine
It is low speed and low capability
The anchor agitator is a slow (50rpm) device whose sole function is to rotate the contents of a batch in a radial direction without providing any significant shear
They are typically designed to be able to withstand a maximum viscosity beyond which they might actually blend or break.
A flexible or movable
blades are attached to the anchor for the
purpose of actually
scrapping the side walls,they are known as
scraped surface
agitators
These are definitely required in
emulsification
equipment where heat transfers are necessary
It consists of anchor agitator
and counterrotating set of
cross bars provides excellent
blending
These works well on materials with viscosities
from 5000-25,000cps
By installing a stationary baffle,some mixing
capabilities are improved
It is also called as saw blade disperser This machine consists of a variable speed
shaft connected to an impeller with a serrated edge
The tip speed is set around 4000 ft/min The diameter of impeller should be 1/3 of
diameter of vessel The impeller should be located one impeller
diameter off the bottom of vessel It can deagglomerating particles when the
viscosities between 10,000 to 20,000cps. Application: It is used for pigment dispersion,dye stuffs, carbon dispersion and paints
Limitations:
Air incorporation is
another problem so
it is best used for
suspensions and not
for emulsions
This mixing machine uses an impeller that is installed at a close tolerance to a stationary housing,which baffles and restrict the flow caused by the rotation of the impeller in the liquid.
Particles caught between rotor & stator are crushed and separated by mechanical action of the impeller.
They are three types:
1. Radial flow with stator
2. Rotating stator
3. Axial flow rotor/stator mixers
Better control of temperature and pressures
Eliminates line loses caused by pumping from tank to tank
Saves floor space Simplifies the process, save labor costs
Types : Anchor plus rotor/stator
Anchor plus disperser
Counter rotating with coaxial rotor/stator
It is combination of simple
anchor agitator with a
rotor/stator mixer
It provides a high shear rates
for dispersion and
emulsification
It is beneficial if there is
requirement to pull down
powders from the top
Limitation:
It is difficult to design the
anchor such that it allows the
placement of the high shear
mixer close to the bottom of
the vessel.
It is combination
of high speed disperser with an
anchor agitator
It works well on medium to high
viscosity
suspensions
This design is known as triple action mixer,combines
the high viscosity mixing capabilities of
counterrotating axial flow cross bars with a
standard anchor type scraped surface agitator
and also with a high speed rotor/stator mixer
It is capable of generating fine dispersions and fine
droplets of the internal phase for stable emulsions
It can operated at viscosity of 50,000cps
They are always jacketed for heating and cooling
because it is necessary in case of preparation of
creams and ointments
Advantages:
Handles wide range of viscosities from water thin to as high as 1million cps
Disperse and emulsifies very efficiently
Provides shortest mixing time
Disadvantages:
Difficult to clean due to complicated design
1.Rotor/stator mixer disperser emulsifier
2.Colloidal mill
3.Piston homogenizer
4.Ultrasonic vibrating homogenizer
5.Micro fluidizer
6.Low pressure cyclone emulsifier
7.Homogenizer/Extruders
8.Static mixer
These mixers acting as submerged pumps
design can be made that places the
rotor/stator in a pump housing and allows
for product to be pumped through itself.
The product inside the rotor/stator mixing
pump,the droplets and particles subjected
to a wide variety of high shear rates.
It is designed with fine tolerance rotor/stator
gaps that promotes high shear rates and
high amount of shear per pass through.
It is used to disperse the solids into liquids and to emulsify liquid-liquid systems.
These generally used as polishing machines for emulsions or suspensions because they produce fine particle or droplet size product to enhance a products stability.
The rotor/stator gap is generally set between 0.030 & 0.001 inch.
They are operated at speed of 3600 rpm
The rotor diameter is 10-30cm provides flow rates in the area of 4000-6000L/hr depending upon the viscosity.
Advantages :
Shear rates high than in rotor/stator machines
Colloidal mill produces emulsions and suspensions with particle size distribution smaller than the particle sizes obtainable using fixed gap rotor/stator mixers.
Limitations :
Flow rates lower than in rotor/stator machines
Not used for abrasive products
It is the most powerfull device for producing
emulsions and suspensions
It uses high power positive displacement
piston type pump to produce pressure of
3000-10,000 psig and then force the
premixed product through a specially
designed restricting wall where a extremely
high shear forces are exerted
Here turbulence and high shear are the
major parameters in size reduction
It having continuous
Capabilities of 2500L/hr at
15hp to 50,000L/hr at 150hp
Limitations:
They cannot handle the product feed above
200cps
High maintanence cost and down time
Lack product homogenity
and batch to batch variability
Has similarity with high pressure homogenizer effective device for dispersing and emulsifying
It uses a positive displacement pump to force the premixed liquid through an elliptical opening at a speed of 100m/sec
This high speed flow impinges on to the edge of blade shaped obstacle called a vibrating knife.
Capacities and
pressures of systems
range from laboratory
units, producing 4-
10L/min at 1200-1700 psig requiring 2-5hp,to
full scale productions
units with capacities of upto 450L/min at
350psig requiring 60hp.
This device uses a high pressure positive displacement pump operating at a pressure of 500-20,000psig which accelerate the process flow upto 500m/min through the interaction chamber
The interaction chamber consists of small channels known as micro channels having diameter narrow as 50μm and cause the flow of product to occur as very thin sheets.
The configuration of micro channels within the interaction chamber ressembles Y-shaped flow streams in which the process stream divides into these micro channels,creating two separate micro streams
The micro streams are then brought
together in an impingement area through
which all of the product must flow
At the impingement area the collision of
the two high speed flow streams in a very
tight spot creates various droplet size
reduction and different mixing mechanisms
such a cavitation,implosion,shears and
turbulence
The micro fluidizer technology satisfy the
requirements for producing finest emulsions
known as micro emulsions
It is used for formation of emulsions and suspensions
the shear arises from the difference in the velocity of the fluid,as the fluid travels in a spiral towards the center
They operates in the 200psig and capacities of 7.5-225L/min.
The recommended viscosity limit is 1-2000cps.
They are capable of producing emulsions in the
2-10μm range.
Working :
Product enters through
tangential entry port
Product is forced to
circulate in concentric
layers towards the
center and ends of
the chamber
Shear arises from the
difference in the
velocity of fluid,as the
fluid travels in a spiral
towards the center
Cross section of flow path
through the interaction
chamber
It is a high pressure homogenizer with an
adjustable valve having production
capacities from 8mL/hr to 12000mL/hr
A positive displacement pump produces
pressure up to 30,000psig.
The apparatus capable of producing
fine emulsions and liposomal dispersions
A true low shear and low energy requirement device for emulsifying immiscible liquid mixture is the static mixture.
It is also called as pipe line mixture,this device is actually a series of specially designed baffles in a cylindrical pipe.
These simple devices are extensively used for the preparation of unstable emulsions for liquid-liquid extraction purposes
Size distributions obtainable range from 100-1000μm
Critical fluids liposome process:
Near critical or super critical fluid solvents
with or without polar co-solvents for the
formation of uniform and stable liposomes
having high encapsulation efficiences.
Super critical fluids can be uniquely used to
encapsulate very hydrophobic molecules
Super critical fluids are gases such as
carbon dioxide & propane
When these fluids compressed at
conditions above their critical
temperature and pressure,these
substances become fluids and ability to
dissolve other materials.
The gaseous characteristics increases
mass transfer rates,thereby significantly
reducing processing time.
Small added amounts of visible polar co-
solvents such as alcohol can be used to
adjust polarity and to maximize the
selectivity and capacity of the solvents
Triple roll mill:
• Disperse small tightly bound
agglomerates and hard discrete particles
Particles are subjected to
High shear
Mechanical crushing
Smearing
It is used for size reduction fine solid
discrete particles or for
deagglomeration of very tightly bound
agglomerates.
The machine consists of cylindrical drum
into which a charge of heavy spherical
balls usually metal or ceramic is loaded
along with the components of the
dispersion.
A Modernized improvement of the ball mill is the
agitated bead mill
The bead mill uses a charge of inert small balls to
2-8mm diameter
Media mill- if beads are ceramic
Shot mill- if beads are steel
Sand mill- if grains of sand are used
The Design consist of a cylinder which can be
either vertical or horizontal,that has a high speed
agitator,which is capable of fluidizing the charge
of beads,causing them to collide at very high
speed.
The premix is pumped through the housing.There is a high probability that each particle must be repeatedly subjected to the high stresses that result when the beads collide with each other or the high speed impeller
Agitated bead mill is used in pigment dispersion industry due to its fine solids grinding and dispersing capabilities.It is not often used in pharmaceutical industry except when particle size requirements falls below 10μm.
Pharmaceutical dosage forms,disperse
systems volume 3 by
Lackman,Lieberman,Marcel
Dekker,NY.pg no:291-362
Pharmaceutical engineering(principles &
practices) by C.V.S.
Subrahmanyam.pg.no:155,161,229.
ENZYME LINKED IMMUNO SORBENT
ASSAY(ELISA)&
RADIO IMMUNOASSAY (RIA)
P.SAHITHI REDDY
M.PHARMACY
H.T.NO:256213886024
Under the guidance of
Mr Utham prasad
Contents
ELISA
OVERVIEW
PRINCIPLE
PROTOCOL
DIFFERENT TYPES
MATERIAL REQUIRED
MERITS&DEMERITS
APPLICATIONS
OVERVIEW:
Enzyme Linked Immuno sorbent Assay (ELISA) Term Was Coined By Engvall and Pearlmann in 1971 is a quantitative technique used in immunology to detect the presence of an antibody or an antigen in sample
• Antigen of interest is absorbed on to
surface sorbent
• Antigen is recognised only by the specific
antibody immuno
• This antibody is recognised by second
antibody (immuno) which has enzyme
attached (enzyme linked)
• Substrate reacts with enzyme to produce
product usually depicted by coloured
PRINCIPLE:
ELISA is based on specific interaction between Ag and
their corresponding Ab. One of the immuno reactant is
coated to a solid phase support such as polycarbonate,
polyvinyl polyacrylamide tubes or microwells.
To the respective antibody or antigen is added
To antigen-antibody complex so formed an enzyme
linked antibody is added. A colourless substance is
added to well. This substance is a substrate for the
enzyme which catalyses the conversion of colourless
substance to coloured product.
MATERIALS REQUIRED
• Test sample
• Antibody /Antigen
• Polystyrene microtiter plates
• Blocking buffer
• Washing buffer
• Substrate
• Enzyme
Enzymes Used in ELISA
Horseradish peroxidase(very
commonly used )
Alkaline phosphatase
Beta –galactosidase
Lacto peroxidase
Tetra Methyl Benzidine (substrate)
The enzyme substrate should be colourless
After degradation by the enzyme it should be strongly coloured
fluorescent
Sandwich ELISA direct method
2 Antibodies Required
Must Recognize Different Epitopes
1st Antibody Is Referred To As Capture Ab
2nd Antibody Detection Ab
2nd Antibody Is Biotinylated
Enzymes Commonly Used: HRP (Horse Radish
Peroxidase) And AKP (Alkaline Phosphatase)
Substrate is TMB (Chromogen)
Prepare a surface to which a known quantity of capture
antibody is bound
Block any non specific biding sites on the surface
Apply the antigen – containing sample to the plate
Wash the plate ,so that unbound antigen is removed
Apply enzyme linked primary antibodies as detection
antibodies which also bind specifically to the antigen
Protocol for sandwich
ELISA
Wash the plate, so that the unbound antibody-
enzyme conjugates are removed
Apply a chemical which is converted by the enzyme
into a coloured product
Measure the absorbency of the plate wells to
determine the presence and quantity of antigen
Indirect ELISA:
• The protein antigen to be tested for is added to each
well of ELISA plate, where it is given time to adhere
to plastic through charge interactions
• A solution of non-reacting protein is added to block
any plastic surface in the well that remains uncoated
by the protein antigen
• Then the serum is added, which contains a mixture of
the serum antibodies, of unknown concentration,
some of which may bind specifically to the test
antigen that is coating the well.
• Afterwards, a secondary antibody is added, which will bind to
the antibody bound to the test antigen in the well. This
secondary antibody often has an enzyme attached to it
• A substrate for this enzyme is then added. Often, this
substrate changes colour upon reaction with the enzyme. The
colour change shows that secondary antibody has bound to
primary antibody, which strongly implies that the donor has
had an immune reaction to the test antigen.
• The higher the concentration of the primary antibody that was
present in the serum, the stronger the colour change. Often a
spectrometer is used to give quantitative values for colour
strength •
Conti….
COMPETIVE ELISA
• The labelled antigen competes for primary antibody
binding sites with the sample antigen (unlabelled).
The more antigen in the sample, the less labelled
antigen is retained in the well and the weaker the
signal).
RESULTS
After reading the results the standard curve is drawn were the
concentration is plotted on the X-axis and the absorbance on
the Y-axis.
Advantages
• Reagents are relatively cheap& have a long shelf life
• Easy to perform and quick procedures
• No radiation hazards during handling
• Has wide usage to variety of infections
Limitations
To carry out the test antibody must be
available
Concentration may be unclear
Possibility of False positive
Possibility of False negative
Applications
The various sections devoted to the uses are
Detection of mycobacterium antibodies in tuberculosis
Detection of rotavirus in faeces
Detection of hepatitis B markers in serum
Detection of HIV antibodies in blood sample
Hormones
Proteins
Drug markers
Serum proteins
Vaccine quality control
Tumour markers
CONTENTS
RADIO IMMUNOASSAY (RIA)
OVERVIEW
PRINCIPLE
MATERIAL REQUIRED
PROTOCOL
INSTRUMENTAION
MERITS&DEMERITS
APPLICATIONS
Overview:
Radio immuno assay was developed by
Berson&yalow(1956) for the quantitative measurement of insulin in human plasma
RIA principles have found wide application in the
field of drug analysis kinetic studies, immuno
diagnosis
RIA is specific, sensitive &rapid
An immunoassay is a test that uses antibody and antigen
complex as means as a mean of generating a measureable
result
An antibody :antigen complex is known as a immune
complex.
Immunoassays are different from other types of laboratory
tests, such as colorimetric tests, because they use antibody:
antigen complexes to generate a signal that can be measured .
In contrast, most routine clinical chemistry tests utilize
chemical reaction between the reagent and sample to generate
a test result
Principle
RIA is a competitive binding assay.
The antibody & labelled antigen are always present as
limiting factors and the concentrations of unlabelled
antigens(sample) under examination is increased
continually
Uses an immune reaction
(Antigen –antibody reaction ) to estimate ligand
Unbound Ag* and Ag washed
Radioactivity of bound residue measured
Ligand conc is inversely related to radioactivity
[Ag: ligand to be measured ;Ag* radiolabelled ligand ]
Materials required
a) Preparation &characterisation of Antigen [Ligand to be
analysed ]
b) Radiolabelling of the Antigen
c) Preparation of the specific antibody
d) Development of Assay system
PREPARATION AND RADIOLABELLING
OF THE ANTIGEN
Antigens prepared by
synthesis of the molecule
isolation from natural sources
Radiolabelling [Tagging procedure ]
3H,14C,125I are used as radioactive tags
Antigens are tagged to 3H14c125I
tagging should NOT affect antigenic specificity and activity
PREAPRATION OF SPECIFIC
ANTIBODY
Antigen injected intradermal into rabbit
antibody production
Antibodies recovered from the serum
Some ligand are not antigenic
hormones ,steroids drugs HAPTENS
E.g.: castrin morphine
ASSAY Procedure
Add known amounts of the test sample+ labelled
antigen into the microtitre wells
incubate – allow the reaction to reach completion
Decant and wash the contents of the well-
removes all unbound antigens
Radioactivity remaining in the mocrotitre wells
measured by a counter [GM counter, scintillation
counter]
Conti…
Intensity of radioactivity is inversely correlated
with the conc of the antigens in the test sample
INSTRUMENTATION:
CENTRIFUGE:
swing bucket rotor : 1200-2500 rpm
Fixed angle head rotor :3500-4000 rpm
RADIOACTIVE counter
gamma counter : which is used for a gamma energy
emitting isotopes. E.g. 125I.
Scintillation counter : it is used for beta energy emitting
isotopes.eg. Tritium 3H and 14C isotopes
Merits and demerits
Merits :
highly specific : immune reactions are specific
high sensitivity : immune reaction are sensitive
Demerits:
radiation hazards :uses radiolabelled reagents
Requires specially trained personnel
Labs require special license to handle radioactive
material
Requires special arrangements for :requisition,
storage of radioactive material
radioactive waste disposal.
APPLICTIONS :
Used in the estimation of pharmaceutical
drugs like
Morphine
Clonazepam
Barbiturates
Neobentine
Flurazepam
And others
Insulin
Gastrin
Glucogon
Growth hormones
Limitations :
The limitations of the RIA are
Its expensive
Being hazardous Handling the radioactive material
The radio isotopes used 125I or 131I emit gamma
radiation these requires a special counting machine