Guided by:

Dr.S.B.Bhanja

M.Pharm,Ph.D

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

Propeller mixers

Turbine mixers

Anchor mixers

Scraped surface agitators

Counter rotation

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.

Marine Propeller mixer Clamp mounted portable mixer

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

RADIAL FLOW WITH

STATOR

ROTATING STATOR AXIAL FLOW ROTOR/STATOR

MIXER

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.

Stage 1

Stage 2

Stage 3

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

Types:

1. Triple roll mill

2. Ball mill

3. Agitated bead mill

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.

Ball mill

Limitations

Typically time consuming process,milling time

are often measured in days.

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

TYPES:

a)Indirect ELISA

b)Sandwich ELISA

c) Competitive ELISA

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)

The ELISA plate is coated with Antibody to detect

specific antigen

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

The results obtained by plotting a graph

antibody bound to labelled antigen Unlabelled antigen

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

References :

A textbook of pharmaceutical analysis

Biotechnology and its applications in pharmacy