Clinical Lab Techniques

◑☞High-Performance Liquid Chromatography (HPLC): HPLC is an advanced analytical technique used for separating, identifying, and quantifying compounds in a mixture. It is widely used in pharmaceuticals, food science, environmental analysis, and biochemistry. ☞Principle of HPLC HPLC operates on the principle of separation based on the interaction between the analyte, the stationary phase, and the mobile phase. Components in a mixture are separated as they pass through the column due to differences in their affinity towards the stationary phase. ☞Key Components of HPLC ➤Mobile Phase ⇨A liquid solvent or a mixture of solvents that carries the sample through the column. ⇨Examples: Water, methanol, acetonitrile, and buffers. ➤Pump ⇨Delivers the mobile phase through the system at high pressure (up to 400 bar). ⇨Maintains consistent flow rates for reproducible results. ➤Injector ⇨Introduces the sample into the HPLC system, either manually or automatically. ⇨Sample volume typically ranges from 1 to 100 µL. ➤Column ⇨Packed with the stationary phase (e.g., silica particles) that separates the analytes. ⇨Types: C18 (Reverse phase), C8, Ion-exchange, Size exclusion. ➤Detector ⇨Identifies and quantifies the separated components. ⇨Common types: UV-Vis, PDA (Photodiode Array), Fluorescence, Refractive Index, and Mass Spectrometry (LC-MS). ➤Data System Software that records, processes, and analyzes the chromatographic data. ☞Basic HPLC Workflow 1.Prepare the sample and mobile phase. 2.Load the sample into the injector. 3.Pump the mobile phase through the system. 4.Separate the analytes in the column. 5.Detect and record the signal with the detector. 6.Analyze the chromatogram using software. ☞Types of HPLC Techniques ➤Normal-Phase HPLC 1.Stationary Phase: Polar (e.g., silica). 2.Mobile Phase: Non-polar (e.g., hexane). ➤Reverse-Phase HPLC 1.Stationary Phase: Non-polar (e.g., C18). 2.Mobile Phase: Polar (e.g., water, methanol). 3.Most commonly used technique. ➤Ion-Exchange HPLC 1.Separates ionic compounds based on their charge. 2.Used for amino acids, proteins, and nucleotides. ➤Size-Exclusion HPLC 1.Separates molecules based on their size. 2.Used for polymers and large biomolecules. ☞Applications of HPLC ➤Pharmaceuticals ~Drug formulation, purity testing, and dissolution studies. ➤Food and Beverage ~Detection of contaminants, preservatives, and nutritional components. ➤Environmental Analysis ~Monitoring pollutants in water, soil, and air. ➤Biochemistry ~Analysis of proteins, peptides, and metabolites. ☞Advantages of HPLC 1.High resolution and sensitivity. 2.Fast and efficient separation. 3.Can handle a wide variety of samples (polar, non-polar, ionic). 4.Quantitative and qualitative analysis in a single run. ☞Disadvantages of HPLC 1.Expensive instrumentation and maintenance. 2.Requires high purity solvents. 3.Complex method development for new samples.

🧬 Molecular amplification techniques: 1. Polymerase Chain Reaction (PCR) Principle: Uses DNA polymerase to amplify specific DNA sequences through repeated cycles of denaturation, annealing, and extension. Types: Conventional PCR: Standard method for DNA amplification. Real-Time PCR (qPCR): Monitors amplification in real-time using fluorescence. Reverse Transcription PCR (RT-PCR): Converts RNA to DNA before amplification. Multiplex PCR: Amplifies multiple targets in a single reaction. Nested PCR: Uses two sets of primers to improve specificity. 2. Loop-Mediated Isothermal Amplification (LAMP) Principle: Amplifies DNA at a constant temperature using multiple primers and DNA polymerase with high strand displacement activity. Advantages: Faster and highly specific; does not require thermal cycling. 3. Transcription-Mediated Amplification (TMA) Principle: Amplifies RNA using reverse transcriptase and RNA polymerase. Applications: Commonly used for detecting infectious agents like HIV and hepatitis viruses. 4. Nucleic Acid Sequence-Based Amplification (NASBA) Principle: Amplifies RNA at a constant temperature using reverse transcriptase, RNA polymerase, and RNase H. Applications: Used in viral diagnostics and gene expression studies. 5. Rolling Circle Amplification (RCA) Principle: Uses a circular DNA template and a DNA polymerase to produce long single-stranded DNA. Applications: Used in detecting pathogens and in nanotechnology applications. 6. Strand Displacement Amplification (SDA) Principle: Utilizes a DNA polymerase with strand displacement activity to amplify DNA sequences at a constant temperature. Applications: Used in clinical diagnostics. 7. Helicase-Dependent Amplification (HDA) Principle: Uses helicase enzymes to unwind DNA, eliminating the need for thermal cycling. Applications: Portable and suitable for point-of-care testing. 8. Recombinase Polymerase Amplification (RPA) Principle: Uses recombinase proteins to facilitate primer binding and amplification at a constant temperature. Advantages: Rapid and works at low temperatures. Applications of Molecular Amplification Techniques Medical diagnostics (e.g., COVID-19, tuberculosis, HIV detection) Forensic science (e.g., DNA fingerprinting) Genetic research (e.g., mutation analysis, gene expression studies) Agriculture and food safety (e.g., GMO detection, pathogen identification.

This coming week, I will jump back into technical processes, this time chromatography! I've used chromatography for separation and purification since the late 90s (that was harder than I thought to put in writing)! We will focus primarily on these techniques! Column Chromatography Column chromatography is a fundamental technique to purify individual chemical compounds from mixtures. It operates on the principle of differential adsorption of substances to a stationary phase (often silica gel or alumina) packed in a column versus their solubility in a moving liquid phase. This versatile method allows for the separation of volatile organic compounds to complex biomolecules, making it invaluable in research labs across pharmaceuticals, environmental science, and biochemistry for tasks such as drug purification, toxin removal, and the isolation of natural products. Scaling it up can be difficult, but lots of industries have done it. Ion Exchange Chromatography Ion exchange chromatography specializes in separating molecules based on their net charge. It uses a charged stationary phase that attracts oppositely charged molecules as a liquid mobile phase carries them. This technique is particularly effective for purifying proteins, nucleotides, and other charged biological molecules, playing a crucial role in biotechnology and pharmaceutical development. Ion exchange chromatography is essential for studying disease mechanisms, developing new medications, and producing biologics, demonstrating its critical role in advancing healthcare and therapeutic interventions. IEC was instrumental for extraction of acids from fermentations. Size Exclusion Chromatography Size exclusion chromatography, also known as gel filtration chromatography, separates molecules based on their size and shape, allowing larger molecules to elude faster than smaller ones through a matrix with pores of a specific size. This technique is crucial for the purification and characterization of polymers, proteins, and nucleic acids, facilitating the study of molecular weight distribution, complex formation, and protein folding. Its application extends to material science, biotherapeutics, and diagnostic research industries. Affinity Chromatography Affinity chromatography stands out for its high specificity, separating compounds based on a reversible interaction between the target molecule and a specific ligand bound to the stationary phase. Based on biological function or structure, this method is highly effective for purifying biomolecules, such as enzymes, antibodies, and receptors. Its precision and efficiency make it indispensable in the biopharmaceutical industry for drug purification, diagnostics, and therapeutic protein production, showcasing the power of leveraging biological specificity for advanced scientific applications. #science #intothedark #biomimicry #purification #flavor #fragrance #cannabis

Serial Dilution 1. Objective: The objective of the serial dilution was to progressively reduce the concentration of a substance (e.g., bacteria, virus, chemical, or antibody) in a stepwise manner to obtain countable colonies or accurate assay results. ________________________________________ 2. Principle: The test was based on the principle of repeated dilution of a sample using a constant dilution factor (usually 10-fold). Each dilution decreased the concentration of the original solution systematically, allowing enumeration or sensitivity testing. ________________________________________ 3. Materials: • Test sample (e.g., bacterial culture) • Sterile test tubes or dilution bottles • Sterile pipettes or micropipettes • Diluent (e.g., sterile saline or nutrient broth) • Vortex mixer • Petri dishes and agar medium (for plating) • Marker and rack • Gloves and lab coat ________________________________________ 4. Procedure: 1. Nine test tubes were labeled for dilutions from 10⁻¹ to 10⁻⁹. 2. Each tube was filled with 9 mL of sterile diluent. 3. 1 mL of the original sample was added to the first tube (10⁻¹) and mixed thoroughly. 4. Then, 1 mL from the 10⁻¹ tube was transferred to the 10⁻² tube, and so on until the final dilution. 5. Selected dilutions (e.g., 10⁻⁴ to 10⁻⁷) were plated on agar to count colonies. 6. Plates were incubated at appropriate temperature (usually 37°C for 24 hours). 7. Colonies were counted, and CFU/mL (colony-forming units per mL) was calculated using the dilution factor. ________________________________________ 5. Result: • Countable colonies (30–300 per plate) appeared at specific dilution levels. • The original concentration was calculated by multiplying colony count × dilution factor. ________________________________________ 6. Uses: • It was used in microbial enumeration in water, food, and clinical samples. • Applied in antibiotic sensitivity, toxin testing, and enzyme activity assays. • Essential in standard curve generation for molecular and biochemical tests. ________________________________________ 7. Consultation: Results were interpreted by microbiologists or lab technologists. For pathogenic organisms, results were communicated to clinicians for diagnosis and treatment decisions. The technique also ensured quality control in industrial microbiology labs.