Chromatography Basics

Chromatography

• Chromatogram is the visual output of a chromatograph, which separates mixtures into individual components.
• Chromatograph is equipment that enables sophisticated separation of mixtures.
• Retention time is the characteristic time it takes for a particular analyte to pass through the system under set conditions.
• Eluent is the solvent that carries the analyte, while eluate is the fluid exiting the column.
• Elution is the process of passing the mobile phase through the column.
• Flow rate is the measure of how much mobile phase passes through the column per minute (ml/min).
• Linear velocity is the distance passed by the mobile phase per minute in the column (cm/min).

Uses of Chromatography

• Analyze: Examine a mixture, its components, and their relations to one another.
• Identify: Determine the identity of a mixture or components based on known components.
• Purify: Separate components to isolate one of interest for further study.
• Quantify: Determine the amount of the mixture and/or components present in the sample.
• Real-life examples: Pharmaceutical, hospital, law enforcement, and environmental agency applications.

Types of Chromatography

• Liquid Chromatography: Separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase).
• Gas Chromatography: Separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or solid beads (stationary phase).
• Paper Chromatography: Separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase).
• Thin-Layer Chromatography: Separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase).

Radioimmunoassay (RIA)

• RIA is a laboratory technique used to measure the concentration of a specific antigen or hormone in a sample.
• It is a highly sensitive and specific method that uses radioactivity to detect and quantify the binding of antibodies to antigens.
• Principle: Preparation, incubation, separation, measurement, and calibration.
• Widely used in various fields, including endocrinology, immunology, infectious disease diagnosis, and cancer research.
• Limitations: Radioactive material handling requirements, limited shelf life of radiolabeled tracers, and potential for non-specific binding.

Polymerase Chain Reaction (PCR)

• PCR is a laboratory technique used to make many copies of a specific DNA sequence.
• Steps: Denaturation, annealing, extension, and repeat.
• PCR is a powerful tool in molecular biology, used for DNA cloning, genetic testing, and forensic analysis.
• PCR has five core 'ingredients': DNA template, primers, DNA nucleotide bases, Taq polymerase enzyme, and a buffer.
• Types of PCR: Conventional PCR, Real-time PCR (qPCR), Reverse Transcription PCR (RT-PCR), and Multiplex PCR.
• Applications: Genetic testing, gene expression analysis, forensic analysis, cancer research, and viral load detection.
• Advantages: High sensitivity, high specificity, and fast results.
• Limitations: Contamination risk and inhibitors.

Enzyme Immunoassay (EIA)

• EIA is a type of immunoassay that uses an enzyme bonded to an antigen or antibody to detect specific molecules in a sample.
• Types of EIA: Enzyme-linked immunosorbent assay (ELISA), Enzyme multiplied immunoassay technique (EMIT), Fluorescent enzyme immunoassays (FEIAs), Chemiluminescent immunoassays (CLIAs), and Radioimmunoassays (RIAs).
• EIAs are widely used in medical and research applications for measuring protein, pathogens, and other molecules in blood samples.

Learn about the fundamental concepts of chromatography, including chromatograms, chromatographs, retention time, eluent, eluate, and elution.