High Performance Liquid Chromatography (HPLC)
- HPLC stands for “High-performance liquid chromatography”(sometimes referred to as High-pressure liquid chromatography).
- powerful tool in analysis, it yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase.
- HPLC is a chromatographic technique that can separate a mixture of compounds
- used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of a mixture.
- physical method in which separation of components takes place between two phases-a stationary phase and a mobile phase
- Stationary phase :
- The substance on which adsorption of the analyte (the substance to be separated during chromatography) takes place .
- It can be a solid, a gel, or a solid liquid combination
- Mobile phase :
- solvent which carries the analyte (a liquid or a gas)
- HPLC is a type of liquid chromatography where the sample is forced through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane by a liquid (mobile phase) at high pressure.
- Liquid chromatography is a separation technique that involves:
- the placement (injection) of a small volume of liquid sample into a tube packed with porous particles (stationary phase) where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity.
- The main principle of separation is adsorption .
- When a mixture of components are introduced into the column various chemical and/or physical interactions take place between the sample molecules and the particles of the column packing
- They travel according to their relative affinities towards the stationary phase.
- The component which has more affinity towards the adsorbent, travels slower.
- The component which has less affinity towards the stationary phase travels faster.
- Since no two components have the same affinity towards the stationary phase, the components are separated
- These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount.
- The output from the detector is called a liquid chromatogram In principle, LC and HPLC work the same way except the speed, efficiency, sensitivity and ease of operation of HPLC is vastly superior
TYPES OF HPLC
- I.BASED ON MODE OF SEPERATION
- 1.Normal phase chromatography – stationary phase is polar (hydrophilic) and mobile face is non-polar (hydrophobic).
- 2.Reverse phase chromatography– stationary face is non-polar (hydrophobic) and mobile face is Polar (hydrophilic).
- Reverse phase chromatography is more commonly used as drugs are usually hydrophilic
- BASED ON SCALE OF OPERATION
- 1.Analytical HPLC
- No recovery of individual components of substance
- 2.Preparative HPLC
- Individual components of substance can be recovered
- BASED ON TYPE OF ANALYSIS
- 1.Qualitative analysis
- Analysis of a substance in order to ascertain the nature of its chemical constituents
- 2.Quantitaive analysis
- Determining the amounts and proportions of its chemical constituents .
- Retention time (RT)
- In a chromatogram, different peaks correspond to different components of the separated mixture
- Time taken for the analyte to travel from the column inlet to the point of detection(maximum peak)
- Negative peaks à occur if mobile phase absorbance is larger than sample absorbance.
- Base line spikes à occur due to the air bubbles in the mobile phase and/or detector, column deterioration.
- HPLC is one of the most widely applied analytical separation techniques.
- • Tablet dissolution of pharmaceutical dosages.
- • Shelf life determinations of pharmaceutical products.
- • Identification of counterfeit drug products.
- • Pharmaceutical quality control.
ADVANTAGES OF HPLC:
- Separations fast and efficient (high resolution power)
- Continuous monitoring of the column effluent
- It can be applied to the separation and analysis of very complex mixtures
- Accurate quantitative measurements.
- Repetitive and reproducible analysis using the same column.
- Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment
- research purposes, detecting concentrations of potential clinical candidates
the amount of light absorbed is directly proportional to the concentration of the solute in the solution
the amount of light absorbed is directly proportional to the length and thickness of the solution under analysis.
Enzyme Linked ImmunoSorbent Assay
- widely used technique for detection of antigen (Ag) or antibody(Ab).
- based on the formation of Ag-Ab complex , which is detected by chromogenic detection using enzyme conjugated secondary antibody.
- The conjugated enzyme acts on a specific substrate called chromogenic substrate, and generates a colored reaction product.
- This product is qualitatively or quantitatively read using an ELISA plate reader.
TYPES OF ELISA
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
- It is used in the detection of antigen in the given biological sample.
- Microtiter wells are initially coated with antigen to be detected which is followed by an antibody linked to an enzyme conjugate.
- This follows the addition of substrate which produces color detected using ELISA detector
- It is used for detection of an antibody in the given sample.
- Microtiter wells are initially coated with antigen specific for antibody to be detected, followed by the
addition of sample.
- Enzyme conjugated Secondary Antibody is added followed by the substrate which forms a colored reaction product.
- It is used for detecting an antigen in the given sample.
- Microtiter wells are initially coated with monoclonal antibodies(called capture antibody) raised against antigen to be detected, followed by addition of sample.
- Any trace of antigen is detected by adding primary antibody (a MAb),followed by enzyme conjugated secondary Ab and a chromogenic substrate; or by directly adding an enzyme conjugated primary Ab
- This variation of ELISA is used to quantitatively estimate the amount of antigen in the given
- Ag and Ab are initially incubated so that they form Ag-Ab complex.
- This mixture is then added to microtiter wells coated with synthetic analogue of antigen to be detected, any free antibody binds to these antigens
- This complex is estimated by enzyme conjugated secondary antibody by chromogenic detection
- More the amount of antigen in the sample, lesser is the antibody available to bind to microtiter wells.
- Since ELISA can detect both antigen and antibody, it is a useful tool for determining serum antibody
- food industry à detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs.
- detection of Mycobacterium antibodies in tuberculosis
- detection of hepatitis B markers in serum
- detection of enterotoxin of E. coli in feces
- detection of HIV antibodies in blood samples
- ELISA in immunochromatography to detect àHGC in urine sample
- Sensitive assay à Equipments are widely available.
- No radiation hazards.
- Reagents are cheap with long shelf life.
- Qualitative and quantitative.
- ELISA can be used on most types of biological samples, such as plasma, serum, urine, and cell extracts
- Only monoclonal antibodies can be used as matched pairs
- Monoclonal antibodies can cost more than polyclonal antibodies
- Negative controls may indicate positive results if blocking solution is ineffective [secondary antibody or antigen (unknown sample) can bind to open sites in well]
- Enzyme/substrate reaction is short term hence color must be read as soon as possible.
- An immunoassay is a test that uses antibody and antigen complexes
- RIA is an elegant tech. in analytical chemistry.
- If substance to be analysed is in very low quantities, in the orders of micrograms, nanograms, conventional methods like gravimetric and colorimetric method fail.
- RIA finds extensive application in the assay of many substances which are present in trace amount in blood.
- Principle Of RIA
- The amount of Ab per tube is kept constant, the amount of antigen added (known or unknown) is the variable parameter.
- The added antigen will be distributed between a bound (B) and a free (F) fraction.
- This distribution is governed by the association constant (KA) of the Ab:
- Ab + Ag à AgAb
- K = [AbAg] /[Ab][Ag]
- Competitive binding of radiolabelled antigen and unlabelled antigen to a high affinity antibody.
- The labelled antigen is mixed with the antibody at a concentration that saturates the antigen –binding sites of the antibody.
- As the concentration of the unlabelled antigen increases more labelled antigen will be replaced from the binding site.
- The decrease in the amount of radiolabelled antigen bound specific antibody in the presence of the test samples is measured to determine the amount of antigen Present in the test sample.
Reagents used in RIA:
- A tracer i.e. a labelled ligand à radioisotopes used are à Beta emitters- 3H and 14C, Gamma emitters- 125 I
- A binder (Antibody) which is the specific antiserum à prepared by injecting (repeatedly) the antigen together with Freund’s adjuvant into suitable animal such as guinea pig, rabbit or goat.
- A separation system to separate to separate the ‘bound’ and ‘free’ phases à required because the bound fraction does not precipitate spontaneously at the low concentration. Variety of procedure are available à Physical method Filtration, chromatography, electrophoresis, chemical methods
- A standard (in highly pure form) à Drugs ,protein,hormone etc must be in pure form so they can be diluted
- A free human antiserum
General Procedure for Performing a RIA Analysis
- A known quantity of an antigen is made radioactive
- This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another.
- a sample of serum from a patient containing an unknown quantity of that same antigen is added
- This causes the unlabeled (or “cold”) antigen from the serum to compete with the radiolabeled antigen for antibody binding sites
- As the concentration of “cold” antigen is increase, more of it binds to the antibody
- by displacing the radio labelled variant and reduces the ratio of antibody-bound radio labelled antigen to free radio labelled antigen.
- The bound antigens are then separated from the unbound ones
- the radioactivity of the free antigen remaining in the supernatant is measured.
Advantages Of RIA
- very sensitive technique used to measure concentrations of antigen without the need to use a bioassay.
- can measure one trillionth (10-12) of a gram of material per milliliter of blood.
- structurally specific as antigen: antibody reaction are highly specific.
- indirect method of analysis.
- It is a saturation analysis as active reagent added in smaller quantity than that of analyte.
Disadvantages Of RIA
- Prolonged reaction time (in days) as a consequence highly diluted reagent is used.
- Radioactive Iodine used in is not a cheap reagent.
- Possible health hazards due to handling of radioisotopes.
- All the reagents must be added precisely.
- Difficulty of automation.
- Lengthy counting time
Uses for RIA
- Narcotics (drug) detection
- Blood bank screening for the hepatitis (a highly contagious condition) virus,
- Early cancer detection,
- Measurement of growth hormone levels,
- Tracking of the leukemia virus,
- Diagnosis and treatment of peptic ulcers, and
- Research with brain chemicals called neurotransmitters
- spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength
- in the spectrophotometer a prism (or) grating is used to split the incident beam into different wavelengths.
- By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on the test solution.
- The range of the wavelengths of the incident light can be as low as 1 to 2nm.
- The spectrophotometer is useful for measuring the absorption spectrum of a compound, the absorption of light by a solution at each wavelength.
Radiant Energy Sources:
- Ultraviolet radiation: Most commonly used sources of UV radiation are the hydrogen lamp and the deuterium lamp. Xenon lamp may also be used for UV radiation, but the radiation produced is not as stable as the hydrogen lamp.
- Visible radiation: “Tungsten filament” lamp is the most commonly used source for visible radiation. It is inexpensive and emails continuous radiation in the range between 350 and 2500nm.
- IR radiation: “Nernst Glower” and “Global” are the most satisfactory sources of IR radiation. Global is more stable than the nearest flower.
- A monochromator resolves polychromatic radiation into its individual wavelengths and isolates these wavelengths into very narrow bands
- Wavelength resolving device like a PRISM (or) a GRATING
- A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its ability to reflect different wavelengths to a different extent
- solutions and are put in cells known as “CUVETTES”. Cuvettes meant for the visible region are made up of either ordinary glass (or) sometimes Quartz.
WORKING OF THE SPECTROPHOTOMETER
- requires being calibrated first
- done by using the standard solutions of the known concentration of the
- standard solutions are filled in the Cuvettes and placed in the Cuvette holder in the spectrophotometer that is similar to the colorimeter
- ray of light with a certain wavelength that is specific for the assay is directed towards the solution.
- Before reaching the solution the ray of light passes through a series of the diffraction grating, prism, and mirrors.
- These mirrors are used for navigation of the light in the spectrophotometer and the prism splits the beam of light into different wavelength and the diffraction grating allows the required wavelength to pass through it and reaches the Cuvette containing the standard or Test solutions.
- It analyzes the reflected light and compares with a predetermined standard solution.
- The photodetector system measures the intensity of transmitted light and converts it into the electrical signals that are sent to the galvanometer.
- The galvanometer measures the electrical signals and displays it in the digital form.
- If the absorption of the solution is higher than there will be more light absorbed by the solution and if the absorption of the solution is low then more lights will be transmitted through the solution which affects the galvanometer reading and corresponds to the concentration of the solute in the solution.
- In double beam spectrophotometers, the beam splitters are present which splits the monochromatic light into two beams one for the standard solution and the other for test solution.
- In this, the absorbance of Standard and the Test solution can be measured at the same time and any no. of test solutions can be analyzed against one standard.
- It gives more accurate and precise results, eliminates the errors which occur due to the fluctuations in the light output and the sensitivity of the detector.
APPLICATIONS OF THE SPECTROPHOTOMETER
- for the determination of the concentration of colored as well as colorless compounds by measuring the optical density or its absorbance.
- can also be used for the determination of the course of the reaction by measuring the rate of formation and disappearance of the light absorbing compound in the range of the visible & UV region of electromagnetic spectrum.
- By spectrophotometer, a compound can be identified by determining the absorption spectrum in the visible region of the light spectrum as well as the UV region of the electromagnetic spectrum.