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Theory Notes

Introduction

A colorimeter is a device used in colorimetry. The word generally refers to the device that measures the absorbance of particular wavelengths of light by a specific solution. This device is most commonly used to determine the concentration of a known solute in a given solution by the application of the Beer-Lambert law.

Principle of colorimetry

Colored solutions have the property of absorbing light of definite wave lengths. The amount of light absorbed or transmitted by a colored solution is in accordance with the Beer-Lambert law.

Beer’s law- The intensity of the color is directly proportional to the concentration of the colored particles in the solution.

Lambert’s law- The amount of light absorbed by a colored solution depends on the length of the column or the depth of the depth of the liquid through which the light passes.

Equations

When a monochromatic light with an original intensity ‘Io’, passes through a solution that can absorb radiant energy, Is will be less than the Io.

Some of the radiant energy is reflected back by the cell containing the solution, or absorbed by the cell wall or the solvent.

The amount of radiation absorbed may be measured in a number of ways:

  1. By measuring transmittance
  2. by measuring absorbance

1. By measuring transmittance-  The transmittance (T) is defined as-

T= Is/ Io

The ratio is expressed as percentage, thus

% T= 100 x Is/ Io

As the concentration of the compound increases, less light is transmitted.

%T varies inversely and logarithmically with the concentration.

 2) By measuring absorbance-  Absorbance measurement is convenient than transmittance.

Absorbance (A) or optical density is directly proportional to the concentration.

The relationship between absorbance and transmittance can be expressed as –

A = – log Is/ Io

   = – log T

   = log 1/T

To convert T to % T,

A = log 1/T x 100/100

   = log (100)/%T

   = log 100-log %T

    = 2-log %T

Thus,

A = 2-log %T

In other words, absorbance (Optical density) and Transmittance (T) are reciprocally related.

So, if all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite (Figure-1)

Absorbance versus Transmittance

Figure 1: Transmittance and absorbance are reciprocally related.

Lambert -Beer’s law

The mathematical expression at a given wavelength can be represented as follows-

OD = A = Ʃcl

Since,

OD = – log Is/ Io

(Absorption has no units, since it is a ratio)

Thus:

– log Is/ Io = cl

or Is/ Io = e Ʃcl

Where =Ʃ is a Constant- It is the molar extinction coefficient( Molar absorptivity) with units of L mol-1 cm-1

C = Concentration of the colored substance, expressed in mol L-1

l = is the path length of the sample – that is, the path length of the cuvette in which the sample is contained.

e- base of the natural logarithm.

Since Is/ Io is known as transmittance (T)

Thus,

T= e Ʃcl

Taking logarithm:

-log 10T =Ʃcl

As per equation:

-log T= A

Hence

A = OD = Ʃcl

Since the thickness of the layer of solution is constant in the instrument, optical density is proportional to the concentration.

When optical density is plotted against concentration “c”, a straight line passing through the origin should be obtained, because the absorbance is directly proportional to the concentration. (Figure-2)

Absorbance versus concentration

Figure 2: Relationship of absorbance and concentration of a solute in a solution

 

The concentration of an unknown solution can be readily determined by measurement of its absorbance and interpolation of its concentration from the graph of the standards.

When % T is plotted versus concentration, a curvilinear relationship is obtained.

The linear relationship between concentration and absorbance is both simple and straightforward, which is why it is preferred to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T.

Calculation of unknown concentration in the test sample

Since there is a linear relationship between absorbance and concentration, it is possible to calculate the unknown concentration of a substance in the test sample by simple proportional equation-

Absorbance of unknown         Concentration of unknown

———————————- = ————————————–

Absorbance of standard          Concentration of Standard

                                                            Absorption of unknown

Concentration of unknown = ————————————— x Concentration of Standard

                                                            Absorption of standard

Thus,

Concentration of Unknown (Test sample T)

                                                            OD of Test

                                                = ————————– x Concentration of Standard

                                                            OD of standard

Some of the incident energy may be reflected by the cell containing the solution or absorbed by the cell wall or the solvent. To eliminate these factors and to consider the absorption by the compound, a blank solution or a reference solution having everything but the compound to be measured is used.

Thus The concentration of unknown can be expressed as-

Concentration of Unknown (Test sample T)

                                                            OD of Test – OD of Blank

                                                = —————————————— x Concentration of Standard

                                                            OD of standard – OD of Blank

Deviations from Beer’s law are observed when a very large concentration of an unknown substance is measured or when the incident light is not mono chromatic light.

 

Components of a photo colorimeter

1) Light source

The light source is usually a tungsten lamp for wavelength in the visible range (320-700 nm) and a deuterium or hydrogen lamp for ultraviolet light (below 350 nm). Hydrogen lamp is usually preferred to UV range.

2) Monochromators

This is for the selection of sufficiently narrow wave band. The monochromator consists of an entrance slit to exclude unwanted, followed by absorption or interference filters, prisms or diffraction grating for wave length selection. (Figure-3)

Colorimeter

Figure 3: Components of a colorimeter.

 

The interference filters consist of thin layer of magnesium fluoride crystals with a semitransparent coating of silver on each side. The interference filters have a bandpass of 5-8 nm. The band pass is defined as the width of the spectrum that will be isolated by a monochromator. The choice of filter depends upon the final color of the  solution formed.

Wave length (nm) Filter used/Color absorbed Color of solution
350-430 Violet Yellow Blue
430-475 Blue Yellow
475- 495 Green blue Orange
495-505 Blue green Red
505-555 Green Purple
555-575 Yellow green Violet
575-600 Yellow Blue
600=650 Orange Green blue
650-700 Red Blue green

3) Lens

Instruments using filters as wavelength selectors require lenses to focus correctly the light from the source through the filter and cuvette to the detector. In the ultraviolet range, quartz or fused silica is essential because the glass does not transmit light efficiently at wave length shorter than 340 nm.

An exit slit at the end of monochromator allows only a narrow fraction of the spectrum of reach the sample cuvette.

4) Sample cuvette

For accurate and precise reading, cuvette must be transparent, clean, devoid of any scratches. The optical path of the cuvette is always 1 cm. Glass cuvettes are used for reading in the visible light range while quartz or fused silica cuvettes are used for UV range.

5) Photosensitive detectors

These detectors contain a light-sensitive surface that releases electrons in number proportional to the intensity of light on it, converting light energy into electrical energy. Different detectors used are-

a) Barrier layer cells

b) Photosensitive tubes

c) Photomultiplier tubes

d) Photoconductive cells

6) Read out devices– The detector response can be measured by any of the following read out devices-

a) Galvanometer

b) Ammeter

c) Recorder

d) Digital read out.

The signal may be transmitted to computer or print out device. Most modern instruments are of direct reading type where the amplified detector signal operates a galvanometer.

 

 

 

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The word Chromatography is derived from two Greek words –Chroma means – color and graphein to write. Chromatography is the collective term for a family of laboratory techniques for the separation of mixtures.

Chromatography, literally “color writing”, was used—and named— in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll.

Chromatography is a misnomer since it is no longer limited to the separation of the colored substances.

Principle- Chromatography is based on the principle of the partition of the solute between two phases/solvents. It usually consists of a mobile phase and a stationary phase. The mobile phase usually refers to the mixture of the substances to be separated dissolved in a liquid or a gas. The stationary phase is a porous solid matrix through which the sample contained in the mobile phase percolates. The interaction between the mobile and the stationary phases result in the separation of the compounds from the mixture. These interactions include the physico chemical principles such as the adsorption, ion- exchange, molecular sieving and affinity

 Classification-There are two ways to classify the methodology of chromatography-

A) Based on nature of interactions between the sample components and the stationary phase,whereby the components are retarded to different degrees in their migration with the mobile phase and are consequently separated from each other- Following are the different types of chromatographic procedures-

1) Partition chromatography

2) Adsorption chromatography

3) Ion – Exchange chromatography

4) Gel filtration chromatography

5) Affinity chromatography

6) High performance liquid chromatography

B) Base on nature of stationary phase or mobile phase

It is of two types

1)      Planar- It may be Paper or Thin layer

2)      Column- it may be Gas or Liquid

 Planar chromatography

  • Planar chromatography is a separation technique in which the stationary phase is present as or on a plane.
  • The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography).
  • Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase.
  •  The specific Retardation factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.

 Column Chromatography

  • Column chromatography is a separation technique in which the stationary bed is within a tube.
  • The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column).
  • Differences in rates of movement through the medium are calculated to different retention times of the sample.

 1. Partition Chromatography

 This is more commonly used for the separation of mixture of amino acids and peptides. The molecules of a mixture get partitioned between the stationary and the mobile phase depending on the relative affinity of each one of the phases.

 a) Paper chromatography- Paper chromatography is an analytical technique for separating and identifying mixtures that are or can be colored, especially pigments. This method has been largely replaced by thin layer chromatography, however it is still a powerful technique. It is a liquid- liquid partition chromatography. The stationary phase is water held on a solid support of filter paper (cellulose). The mobile phase is a mixture of immiscible solvents which are mixtures of water, a non polar solvent and an acid or base e.g. Butanol, acetic acid water or phenol-water-ammonia.

Technique(Figure-1)

  • A small concentrated spot of solution that contains the sample of the solute is applied to a strip of chromatography paper about two centimeters away from the base of the plate.
  • This sample is absorbed onto the paper and may form interactions with it.
  • Any substance that reacts or bonds with the paper cannot be measured using this technique.
  • The paper is then dipped in to a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container.
  • The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper. (Figure-1)
  • As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample.
  • Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibers in the paper.
  • Paper chromatography takes anywhere from several minutes to several hours.
  •  In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same values in a particular solvent.
  •  In these cases, two-way chromatography is used to separate the multiple-pigment spots.

Paper chromatography

Figure-1 showing paper chromatography(Ascending)

Ascending Chromatography

In this method, the solvent is in pool at the bottom of the vessel in which the paper is supported. It rises up the paper by capillary action against the force of gravity (Figure-1).

Descending Chromatography

In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper. The liquid moves down by capillary action as well as by the gravitational force. In this case, the flow is more rapid as compared to the ascending method. Because of this rapid speed, the chromatography is completed in a comparatively shorter time.

Descending paper chromatography

Figure-2- showing descending paper chromatography. The developing solvent is placed in a trough at the top which is usually made up of an inert material. The paper is then suspended in the solvent. Substances that cannot be separated by ascending method, can be separated by the above descending method.              

 AnalysisAfter development, the spots corresponding to different compounds may be located by their color, ultraviolet light, ninhydrin or by treatment with iodine vapors. The paper remaining after the experiment is known as the Chromatogram.

Composition of Filter Paper

The original work in paper chromatography was carried on whatman no.1 filter paper. These days paper for chromatography is made from cotton cellulose.

Rƒ value

The components which have been separated differ in their retention factor i.e Ratio of distance traveled from the spot or origin by the solute component to that of the distance traveled from the spot or origin by the solvent. Retention Factor can never be greater than one.

 Rf, – Distance traveled by sample/ distance traveled by solvent.

The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes using the Rf value in an experiment. The retention values found can be compared to known values, and from that conclusions can be drawn.

Rƒ values are usually expressed as a fraction of two decimal places.

 If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front.

Two dimensionalchromatography–  Sometimes, it is difficult to separate a complex mixture of substances by a single run with one solvent system. In such a case, a second run is carried out by a different solvent system, in a direction perpendicular to the first run. This is referred to as- two dimensional chromatography which enhances the separation of a mixture in to individual components. (Figure-3)

 Two dimensional paper chromatography

Figure-3- showing two dimensional paper chromatography

Importance-

1) Paper chromatography is a very easy, simple, rapid and highly efficient method of separation.

2) It can be applied even in microgram quantities of the sample.

3)It can also be used for the separation of  a wide variety of materials like amino acids, oligopeptides, sugars, oligosaccharides, glycosides, purines and pyrimidines, steroids, vitamins and some alkaloids like penicillin, tetracyclin and streptomycin.

4) It is not preferred for separating proteins because they are not soluble in many of the solvent systems and are also denatured by them. Paper chromatography is inferior to thin layer chromatography in resolving power.

b) Thin layer chromatography (TLC) is a chromatographic technique used to separate mixtures. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose immobilized onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated is then dissolved in an appropriate solvent and is drawn up the plate via capillary action, separating the experimental solution based on the polarity of the components of the compound in question. .                                   

Importance of TLC– Its wide range of uses include

  • determination of the pigments a plant contains
  • detection of pesticides or insecticides in food
  • identifying compounds present in a given substance
  • monitoring organic reaction

Technique

The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.

A small spot of solution containing the sample is applied to a plate, about one centimeter from the base. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and placed in a sealed container. The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent.(Figure-4).

Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the most polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value).

 Thin layer chromatography

Figure-4- showing the mechanism of thin layer chromatography

Analysis

As the chemicals being separated may be colorless, several methods exist to visualize the spots:

  • Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254)..
  • Iodine vapors are a general unspecific color reagent
  • Ninhydrin is used for amino acids and proteins
  • Sulphuric acid is used for phospholipids

Once visible, the Rf value , or Retention factor, of each spot can be determined .These values depend on the solvent used, and the type of TLC plate, and are not physical constants. The Rf value for compounds (amino acids, peptides ,sugars, fatty acids, phospholipids etc.) for commonly used solvent systems have been calculated and available for comparison.TLC can also be used for two dimensional chromatography  using the same plate with two solvent systems, as in the case of paper chromatography.

Advantages of TLC over paper chromatography-

1) In case of paper chromatography , it takes 14-16 hrs for the separation of the components, but in TLC, It takes only 3-4 hrs.

2) TLC has the advantage that the corrosive reagents like sulphuric acid can also be used which pose a limitation for the paper chromatography.

3) It is easier to separate and visualize the components by this method.

4) It has the capacity to analyze multiple samples in a single run.

5) It is relatively a low cost.

2)Adsorption chromatography

  • In this technique the separation is based on differences in adsorption at the surface of the solid stationary medium.
  • The adsorbents such as silica gel, charcoal powder and calcium hydroxyapatite are packed in to a column in a glass tube.
  • This serves as the stationary phase. The sample mixture in a solvent is loaded on this column.
  • The individual components get differentially adsorbed on to the adsorbent (Figure-5).
  • The elution is carried out by a buffer system (mobile phase).
  •  The most weakly held fraction moves fastest, followed by others, according to the order of tightness in adsorption.
  • The individual compounds come out of the column at different rates which may be separately collected and identified

 

 Adsorption chromatography

Figure-5- showing adsorption chromatography

 3)Ion – Exchange chromatography

  • Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules (Figure-6).
  • It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
  • The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.
  • Ion exchange chromatography retains analyte molecules based on coulombic (ionic) interactions.
  • The stationary phase surface displays ionic functional groups  that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consisting of the cationic species  and the anionic species can be retained by the stationary phase.

 

 Ion exchange chromatography

 

Figure-6 -showing ion exchange chromatography

Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group.

Cation Exchange resins-  Polysterene sulfonate resins, CM- Sephadex gel, CM – cellulose, These bear acidic groups and immobilize cations from adjacent solutions.

Anion exchange chromatography retains anions using positively charged functional group.

Anion exchangers-  DEAE cellulose, Trimethyl amino polysterene, DEAE- sephadex. All these bear basic groups ionizing into fixed positions and immobilize anions from neighboring solutions.

The ionic groups in cation exchange resins are sulphonic and carboxyl groups, while the anion exchange resins have a quaternary nitrogen.

 4) Gel filtration chromatography or Molecular Sieve chromatography-

  • This is extremely useful  in separating ribosomes, viruses, nucleic acids and proteins depending on their particle sizes and shapes.
  •  In Gel filtration chromatography, the separation of the particles is based on their size, shape and molecular weight.
  •  This technique is also referred to as molecular exclusion chromatography.
  • The apparatus consists of a column packed with sponge like gel beads( usually cross- linked polysaccharides) containing pores.
  • The gels serve as molecular sieves for the separation of smaller and bigger molecules
  • The solution mixture containing molecules of different sizes( say protein) is applied to the column and eluted with a buffer. (Figure-7).
  •  The larger molecules cannot pass through the pores of a gel and therefore move faster.
  • On the other hand, the smaller molecules enter the gel beads and are left behind which come out slowly.
  • By selecting the gel beads of different porosity, the molecules can be separated.
  • The commercially available gels include (G-10, G-25,G-100), Bio gel(P-10,P-30,P-100) and Sepharose(6B,4B,2B).
  • The gel filtration  chromatography can be used for an approximate determination of molecular weights.
  • This is done by using a calibrated column with substances of known molecular weights.

 

 Molecular sieve chromatography

Figure-7- showing mechanism of molecular sieve chromatography.

 5) Affinity chromatography

  • The principle of affinity chromatography is based on the property of specific and non-covalent binding of proteins to other molecules, referred to as substrates or cofactors.
  • The technique involves the use of ligands covalently attached to an inert and porous matrix in a column.
  • The immobilized ligands act as moleculal hooks to selectively pick up the desired protein while the remaining proteins pass through the column.
  • The desired protein captured by the ligand, can be eluted by using free ligand molecules.
  • Alternatively, some reagents that can break protein ligand interactions can also be employed for the separation (Figure-8)
  • Affinity chromatography is useful for the purification of enzymes, vitamins, nucleic acids, drugs , hormone receptors, antibodies etc.
  • For example NAD is used to purify dehydrogenases. By using antibodies, antigens can be easily separated.
  • Conversely, antibodies can be purified by passing through a column containing the antigen.
  • It is also widely used for the estimation of glycated Hb. Normal Hb does not bind and comes out first, while glycated Hb binds with the Boronic acid used as a ligand. Sorbitol is then added to elute the glycated Hb which can be quantitated then.

Affinity chromatography

Figure-8- Showing the mechanism of Affinity chromatography

6) High performance liquid chromatography

  • In general chromatographic techniques are slow and time consuming.
  •  The separation can be greatly improved by applying high pressure in the range of 5000-10,000 pounds per square inch, hence this technique is also referred to as high pressure liquid chromatography.
  •  HPLC requires the use of non compressible resin materials and strong metal columns.
  • The eluents of the columns are detected by methods such as UVabsorption and fluorescence.
  •  It can be applied in the form of partition, adsorption, ion exchange or molecular sieve chromatography  .
  • The stationary phase consists of an immobilized thin layer of a liquid on the micro glass or plastic beads, tightly packed in to a narrow column. (Figure-9)
  • The mobile phase consists of a buffered solvent system which is passed under high pressure through the column for eluting the solutes of the sample.         

 High performance liquid chromatography

Figure-9- Showing the apparatus for high performance liquid chromatography

Due to rapidity in action it is used for assaying amino acids, peptides, proteins, carbohydrates, lipids, nucleic acids and related compounds, vitamins, hormones, metabolites and drugs such as antiarrytmics, antibiotics, antiepileptics,analgesics, bronchial smooth muscle relaxants and anti-depressants.

 6) Gas liquid chromatography-                                      

  • This is the method of choice for the separation of volatile substances or volatile derivatives of certain in volatile substances.
  • In GLC. the stationary phase is an inert solid material(diatomaceous earth or powdered firebrick), impregnated with a non volatile liquid(silicon or polyethylene glycol).
  • This is packed in a narrow column and maintained at high temperature (around 200 degree C).
  • A mixture of volatile material is injected in to the column along with the mobile phase, which is an inert gas (argon, helium or nitrogen).
  • The separation of the volatile material is based on the partition of the components between the mobile phase (gas) and stationary phase( liquid), hence the name gas liquid chromatography. (Figure-10)
  • The separated compounds can be identified and quantitated by a detector. Gas liquid chromatography is sensitive, rapid and reliable.
  •  It is frequently used for the quantitative estimations of biological materials such as lipids, drugs and vitamins.

 

 Gas liquid chromatography

Figure-10- showing apparatus for gas liquid chromatography.

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ELISAEnzyme Linked Immuno Sorbent Assay

The ELISA techniques are widely used for-

1) Quantitative estimation  of-

  • Hormones
  • Amino acids
  • Growth factors
  • Tumor  markers and
  • Other analytes which are present in very small concentration in biological fluids

 2) Qualitative detection of-

  • Bacterial or viral antigens
  • Antibodies against microbes
  • Any antigen, or antibody present in small concentration  in the biological fluid/tissues

 Advantages of ELISA

  • The test can be undertaken to detect antigens or antibodies present in very small quantities in tissues or blood.
  •  It is highly economical and a sensitive method.
  •  An enzyme is used as a label, so it is non-isotopic immune assay.
  •  It is more sensitive than Radio-immuno Assay and there is no risk of radiations hazards.

Principle

 ELISA is based on the Immuno chemical principles of Ag-Ab reaction. The specific interactions between specific antigens and antibodies are used here to detect/ quantitate the specific components of a biological fluid, using the enzyme labeled antibodies. The substrate of the enzyme is added, coupled with a colour reagent; which is colourless in the beginning but during the course of the reaction becomes coloured. The intensity of the colour formed predicts the concentration of the Ag/Ab present in the given sample.

Steps of ELISA

A) Antigen detection

  Sandwich ELISA (Figure-2)

A specific antibody is fixed to the well of a micro titer plate(Figure-1) The patient’s serum is added in the well and incubated for 30 minutes at 37 degrees Celsius. During this time, if the serum contains the antigen, it is fixed on the antibody. Excess antigens and other unwanted proteins are washed out. Then antibodies tagged with horse-radish peroxidase(HRP) enzyme are added. If the antigen is already fixed, the Ab-HRP conjugate will be fixed in the well. Then a colour reagent, containing hydrogen peroxide (H2O2) and diamino benzidine (DAB) are added. The reaction is as follows-

 

i) H2O2   ————————–>      H2O  +  Nascent oxygen [o]

The reaction is catalyzed by Horse radish peroxidase enzyme

ii) Diamino benzidine—————————>   Oxidized DAB

   (colorless)                                                            (brown color)

 The reaction takes place in the presence of Nascent oxygen [o]

Thus in the above reaction, antigen is sandwiched between Ab ( solid phase) and the enzyme  labeled antibody. Development of brown colour indicates that the antigen is originally present in the patient’s serum. Colour developed is proportional to the antigen in the serum. Therefore intensity of the colour is measured from which the concentration of the antigen can be calculated. Other chromogens that can be used in ELISA are NBT (Nitro blue tetrazolium-blue colour) and NPP (Nitro phenyl phosphate-yellow color).

 ELISA plate

Figure-1- Showing microtitre plate

B) Antibody detection

Indirect ELISA (Figure-2)

This is useful to detect small quantities of antibodies in the blood e.g. to detect HIV antibodies in the patient’s serum, this test can be undertaken by the following method-

Antigen from HIV is coated in the well of a multiwell microtitre plate. Patient’s serum is added and incubated. If it contains the antibodies, it is fixed. The wells are washed. This is done to remove excess unbound antibodies. Then a second antibody (Antibody against human immunoglobulin) conjugated with HRP is added. Then colour reagent containing hydrogen peroxide and diamino benzidine is poured over. Development of   brown colour indicates the presence of antibodies in the patient’s serum. The colour developed is proportional to the antibody concentration. Thus from the intensity of colour, the concentration of Ab can be determined(Figure-1).

ELISA is less specific for HIV so the confirmation by other methods is necessary for the final diagnosis of AIDS. 

 Steps of ELISA

Figure-2-Showing the steps of Sandwich and Indirect ELISA

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Electrophoresis is the movement of charged particles through an electrolyte when subjected to an electric Field-

  • Cations move towards cathode
  • Anions move towards anode 
  • By this technique solutes are separated by their different rates of travel through an electric field.
  • Commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids 

Factors affecting Electrophoresis

The rate of migration of a solute in an electric field depends on  the following factors-

  1. Net charge on the particle
  2. Mass and shape of the particles 
  3. p H of the medium
  4. Strength of electric field
  5. Properties of supporting medium
  6. Temperature

Electrophoretic Mobility 

 Electrophoretic mobility is defined as the rate of migration (cm/sec)per unit field strength(Volts/cm)

 µ=Q/6πrη

Where µ- Electrophoretic mobility

Q-Net charge on the ion

r-Ionic radius of the solute

η- Viscosity of the medium

  • The Electrophoretic  mobility is directly proportional to net charge and inversely proportional to molecular size and viscosity of the electrophoresis medium
  • The p H of solution affects the mobility of the ion by determining the amount and nature of charge
  • Proteins, nucleic acids, nucleotides and amino acids  bear charged polar groups making them suitable groups for electrophoresis
  • Carbohydrates carrying no charged groups are first bound to charged groups like Borate or Sulfite ions and then electrophoresis is carried out. 
  • Lipids are not electrophoresed because electrophoretic current requires polar solvents in which most lipids are insoluble

Types of Electrophoresis

1) Horizontal

2) Vertical

  Vertical electrophoresis is mainly used for Polyacrylamide gel electrophoresis.

Electrophoresis Apparatus

  Electrophoresis apparatus consists of-

  1. Buffer tank -to hold the buffer
  2. Buffer
  3. Electrodes- made of platinum or carbon
  4. Power supply
  5. Support media

 

Figure-1- Showing apparatus for Horizontal electrophoresis.

Note-Choice of buffer depends on the nature of substance to be separated and the electricity is supplied at a constant current and voltage. 

The electrophoresis support on which separation takes place may contact the buffer directly or by means of wicks.

  The entire apparatus is covered to minimize separation

Support media for electrophoresis

  1. Filter Paper
  2. Cellulose acetate membrane
  3. Agar or Agarose gel
  4. Starch Gel
  5. Polyacrylamide gel

A)  Paper Electrophoresis

 The support medium is a filter paper frequently used in isolating proteins, amino acids and oligopeptides.

Procedure

  1. A long strip of filter paper is moistened with a suitable buffer      solution of the desired p Hand the sample is applied transversely across the central part of the strip
  2. Ends are fixed to dip in buffer solutions in two troughs fitted with electrodes
  3. Electric field of about 20 volts/cm is established
  4. The charged particles of sample migrate along the strip towards respective electrodes of opposite polarity, according to net charges, sizes and interactions with the solid matrix
  5. Homogeneous group of particles migrate as a separate band
  6. The electrophoresis is carried out for16-18 hours
  7. Separated Proteins are fixed to a solid support using a fixative such as Acetone or Methanol
  8. Proteins are stained to make them visible
  9. The separated proteins appear as distinct bands
  10. Drawback-long time interval and blurring of margins

B) Cellulose Acetate Membrane Electrophoresis

  • Preferred solid support medium
  • Less time-consuming 
  • Excellent separation 
  • Membranes can be stored for a longer time 
  • Widely used for separation of lipoproteins, isoenzymes and hemoglobin

C) Gel Electrophoresis

  • The term “gel” in this instance refers to the matrix used for containing, and then separating the target molecules
  •  In most cases the gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. 
  • A  gel block made of Polyacrylamide, Agarose or substituted starch gel is used in this method as the solid support 
  • Agar gel is used for separation of different types of protein mixtures as well as nucleic acids 
  • Polyacrylamide is most suitable for separation of nucleic acids. It is also frequently used in separating proteins, peptides and amino acids from microgram quantities of mixed samples

a) Agarose gel electrophoresis

  • Commonly used support medium 
  • Less expensive than cellulose acetate 
  • Equally good separation 
  • Agar is a complex acidic polysaccharide containing monomers of sulfatedgalactose 
  • Agarose is  a sulfate freefraction of Agar 
  • Gel is prepared in  buffer andspread over a microscopic slide 
  •  A small sample of serum or biological fluid is applied by cutting in tothe gel with a sharp edge 
  • The electrophoretic run takes about 90 minutes

b) Poly Acrylamide Gel Electrophoresis-PAGE

  • Most popular type 
  • Polyacrylamide is a polymer formed when acrylamide is heated with avariety of catalysts with or without cross linking agents 
  • It is thermostable, transparent, strong and relatively chemically inert
  •  Gels are uncharged and are prepared in a variety of pore sizes 
  • Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving

Types of PAGE

PAGE can be classified according the separation conditions into:

  Native-PAGE:

  •  Separation is based upon charge, size, and shape of macromolecules
  •  Useful for separation and/or purification of mixture of proteins 
  • This was the original mode of electrophoresis.

Denatured-PAGE or SDS-PAGE

  •  Separation is based upon the molecular weight of proteins
  • The most common method for determining MW of proteins 
  • Very useful for checking purity of protein samples

PAGE-Procedure

  1. The gel of different pore sizes is cast in to a column inside a vertical tube, often with large pore gel at the top and small pore gel at the bottom(figure-2)
  2. Micrograms quantity of the sample is placed over the top of the gel column and covered by a buffer solution having such a p H so as to change sample components in to anions
  3. The foot of the gel column is made to dip in the same buffer in the bottom reservoir
  4. Cathode and anode are kept above and below the column  to impose an electric field through the column
  5. Macromolecular anions move towards the anode  down the gel column
  6. There is no external solvent space, all the migratory particles have to pass through the gel pores 
  7. Rate of migration depends on the charge to mass ratio
  8. Different sample components get separated in to discrete migratory bands along the gel column on the basis of electrophoretic mobility and gel filtration effect
  9. PAGE may yield 20 or more fractions and may be used to study individual proteins and nucleic acids in serum especially genetic variants and isoenzymes

 Figure-2- Polyacrylamide gel electrophoresis

Slab PAGE

  • The Polyacrylamide gel is cast as thin rectangular slab inside a plastic frame and this slab is placed vertically on a buffer solution taken in a reservoir 
  • Several samples dissolved in dense sucrose solution or glycerol are placed in separate wells cut in to the upper edge of the slab and are covered by the same buffer solution. Cathode and anode are above and below to produce electric field effect. Different components migrate simultaneously down parallel lanes in the slab and get separated in to bands

SDS PAGE

  • SDS-PAGEsodium dodecyl sulfate Polyacrylamide gel electrophoresis, is technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility 
  • The SDS gel electrophoresis of samples having identical charge to mass ratios results in fractionation by size and is probably the world’s most widely used biochemical method  
  • When a detergent  SDS(Sodium-Dodecyl-Sulfate)is added to PAGE the combined procedure is termed as SDS PAGE SDS coats protein molecules giving all proteins a constant charge-mass ratio 
  • Due to masking of charges of proteins by the large negative charge on SDS binding with them, the proteins migrate along the gel in order of increasing sizes or molecular weights 
  •  Molecular weight of a given protein can be determined by comparing the relative electrophoretic mobility of sample with that of standard protein of known molecular weight, when both the sample and the standard proteins are electrophoresed side by side in the same gel slab (figure-3)
  •  In oligomeric proteins, SDS PAGE usually gives the molecular weight of separated monomer chains of the proteins because SDS cleaves the non covalent bonds interlinking the monomer chains in the intact molecule.     

 Figure-3- SDS PAGE

VISUALIZATION

  • After the electrophoresis is complete, the molecules in the gel can be stained to make them visible 
  • Ethidium bromide, silver, or coomassie blue dye may be used for this process 
  • Other methods may also be used to visualize the separation of the mixture’s components on the geL 
  • If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions .If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.

OTHER TYPES OF ELECTROPHORESIS

 Capillary Electrophoresis

 Immuno electrophoresis

 Isoelectric focusing (Isoelectrophoresis)

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