Bioinstrumentation Essentials

Cheatsheet Content

1. Chromatography & Separation Techniques 1.1 Kd Volume (Size Exclusion Chromatography) The distribution coefficient ($K_d$) in Size Exclusion Chromatography (SEC) represents the fraction of the stationary phase volume that is accessible to a given solute molecule. For small molecules (can enter all pores): $K_d \approx 1$ For large molecules (excluded from all pores): $K_d \approx 0$ For molecules of intermediate size: $0 1.2 Retention Factor (Rf) The Retention Factor ($R_f$) in planar chromatography (e.g., TLC, paper chromatography) is the ratio of the distance traveled by the solute to the distance traveled by the solvent front, both measured from the origin. $R_f = \frac{\text{distance traveled by solute}}{\text{distance traveled by solvent front}}$ $R_f$ is always between 0 and 1. It is characteristic for a given compound under specific conditions (solvent, stationary phase, temperature). 1.3 Mobile and Stationary Phases (Chromatography) Chromatography separates components of a mixture based on their differential distribution between two phases: Mobile Phase: The solvent that moves through the stationary phase, carrying the sample components. It can be a liquid or a gas. Stationary Phase: The fixed medium (solid or liquid supported on a solid) through which the mobile phase passes. It can be a column packing, a thin layer on a plate, or paper. Example: In Thin Layer Chromatography (TLC), the stationary phase is typically silica gel on a plate, and the mobile phase is a solvent mixture that moves up the plate by capillary action. 1.4 Cation and Anion Exchange Chromatography Ion Exchange Chromatography (IEC) separates molecules based on their net charge. Feature Cation Exchange Chromatography Anion Exchange Chromatography Stationary Phase Negatively charged resin (binds cations) Positively charged resin (binds anions) Functional Groups Sulfonate ($-\text{SO}_3^-$), Carboxylate ($-\text{COO}^-$) Quaternary amines (e.g., $-\text{N}^+(CH_3)_3$) Binding Positively charged molecules bind Negatively charged molecules bind Elution Increasing salt concentration or decreasing pH (to protonate protein) Increasing salt concentration or increasing pH (to deprotonate protein) 1.5 Thin Layer Chromatography (TLC) TLC is a simple, rapid, and inexpensive planar chromatography technique used to separate non-volatile mixtures. Principle: Components separate based on differential adsorption to the stationary phase and solubility in the mobile phase. Stationary Phase: A thin layer of adsorbent material (e.g., silica gel, alumina) coated on a flat, inert substrate (glass, plastic, aluminum foil). Mobile Phase: A solvent or solvent mixture that moves up the plate by capillary action. Procedure: Spot sample on the origin line. Place plate in a developing chamber with mobile phase. Allow solvent to ascend. Visualize separated spots (UV light, chemical stains). Applications: Monitoring reaction progress, checking purity of compounds, identifying components in a mixture. 2. Radioactivity and Decay 2.1 Alpha, Beta, and Gamma Rays These are types of radiation emitted during radioactive decay. Property Alpha ($\alpha$) rays Beta ($\beta$) rays Gamma ($\gamma$) rays Nature Helium nucleus ($^4_2\text{He}^{2+}$) High-energy electron ($^0_{-1}e^-$) or positron ($^0_{+1}e^+$) High-energy electromagnetic radiation Charge +2 -1 (electron) or +1 (positron) 0 (neutral) Mass Relatively heavy (4 amu) Very light (negligible) Massless Penetration Low (stopped by paper/skin) Medium (stopped by aluminum) High (stopped by thick lead/concrete) Ionization High Medium Low 2.2 Alpha, Beta, Gamma Decay These are modes of radioactive decay where an unstable atomic nucleus transforms into a more stable one by emitting radiation. Alpha Decay: Emission of an alpha particle. Atomic number decreases by 2, mass number decreases by 4. Example: $^{238}_{92}\text{U} \rightarrow ^{234}_{90}\text{Th} + ^4_2\text{He}$ Beta-minus Decay: A neutron converts into a proton, emitting an electron (beta particle) and an antineutrino. Atomic number increases by 1, mass number remains unchanged. Example: $^{14}_{6}\text{C} \rightarrow ^{14}_{7}\text{N} + ^0_{-1}e^- + \bar{\nu}_e$ Beta-plus Decay (Positron Emission): A proton converts into a neutron, emitting a positron (beta particle) and a neutrino. Atomic number decreases by 1, mass number remains unchanged. Example: $^{22}_{11}\text{Na} \rightarrow ^{22}_{10}\text{Ne} + ^0_{+1}e^+ + \nu_e$ Gamma Decay: Emission of gamma rays from an excited nucleus. No change in atomic or mass number, only energy is released. Often follows alpha or beta decay. Example: $^{60}_{27}\text{Co}^* \rightarrow ^{60}_{27}\text{Co} + \gamma$ (where * denotes an excited state) 2.3 Electron Capture Theory and Positron Emission Electron Capture (EC): An inner orbital electron is captured by the nucleus, converting a proton into a neutron. An X-ray is emitted as an outer electron falls to fill the vacancy. Example: $^{40}_{19}\text{K} + ^0_{-1}e^- \rightarrow ^{40}_{18}\text{Ar} + \nu_e$ Effect: Atomic number decreases by 1, mass number unchanged. Positron Emission: (See Beta-plus Decay above) A proton converts into a neutron, emitting a positron. Effect: Atomic number decreases by 1, mass number unchanged. Both EC and positron emission occur in proton-rich nuclei to increase the neutron-to-proton ratio. 3. Electrophoresis 3.1 APS and $\beta$-Mercaptoethanol in SDS-PAGE SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) separates proteins primarily by molecular weight. APS (Ammonium Persulfate): An initiator for the polymerization of acrylamide and bis-acrylamide to form the polyacrylamide gel. It produces free radicals that catalyze the reaction. $\beta$-Mercaptoethanol ($\beta$-ME): A reducing agent used in the sample buffer. It breaks disulfide bonds (S-S) in proteins, causing them to unfold into their linear polypeptide chains. This ensures that the protein's migration rate is solely dependent on its size, not its complex 3D structure. 3.2 Stacking and Resolving Gels in SDS-PAGE Polyacrylamide gels in SDS-PAGE typically consist of two distinct layers: Stacking Gel (Top Gel): Lower acrylamide concentration (e.g., 4%), larger pores. Lower pH (e.g., pH 6.8). Contains a lower concentration of chloride ions. Function: To concentrate the protein sample into a narrow band before it enters the resolving gel. This is achieved by an ion-front effect (isotachophoresis) where proteins are "stacked" between leading chloride ions and trailing glycine ions. Resolving Gel (Separating Gel / Main Gel): Higher acrylamide concentration (e.g., 8-20%), smaller pores. Higher pH (e.g., pH 8.8). Function: To separate the proteins based on their molecular weight as they migrate through the dense gel matrix. The smaller pores provide resistance, allowing smaller proteins to migrate faster than larger ones. 3.3 Isoelectric Point (pI) The isoelectric point (pI) of a molecule (e.g., an amino acid, peptide, or protein) is the pH at which the molecule carries no net electrical charge. At this pH, the molecule's positive and negative charges are equal, making its net charge zero. At pH values below the pI, the molecule carries a net positive charge. At pH values above the pI, the molecule carries a net negative charge. The pI is crucial in techniques like isoelectric focusing (IEF), where molecules migrate in a pH gradient until they reach their pI and become stationary. 3.4 Native vs. SDS-PAGE Feature Native PAGE SDS-PAGE Denaturation No (proteins maintain native structure) Yes (SDS denatures proteins, $\beta$-ME reduces disulfide bonds) Charge Based on intrinsic charge of protein Uniform negative charge due to SDS binding Separation Basis Size, shape, and charge Primarily molecular weight (size) Purpose Studying protein activity, complex formation Determining molecular weight, assessing purity 4. Microscopy 4.1 Resolving and Resolution Power in Phase Contrast Microscopy Resolution: The ability of a microscope to distinguish between two closely spaced objects as separate entities. It is inversely proportional to the wavelength of light used and directly proportional to the numerical aperture of the objective lens. Resolving Power: A quantitative measure of resolution. The smaller the resolvable distance, the higher the resolving power. $d = \frac{0.61 \lambda}{\text{NA}}$ (Abbe's diffraction limit) where $d$ is the minimum resolvable distance, $\lambda$ is the wavelength of light, and NA is the numerical aperture. Phase Contrast Microscopy: Enhances contrast in unstained, transparent specimens by converting phase shifts (due to variations in refractive index and thickness within the specimen) into amplitude (brightness) differences. It improves the visibility of internal structures without staining but does not inherently increase the resolving power beyond the limits of bright-field microscopy. Its primary role is to improve contrast, making structures distinguishable that would otherwise be invisible. 4.2 Phase Contrast Microscope A type of light microscope that uses phase differences in light to create contrast in transparent specimens. Principle: Light passing through a specimen undergoes phase shifts (retardation) proportional to the refractive index and thickness of the sample. The phase contrast microscope converts these invisible phase differences into visible differences in brightness. Components: Annular Diaphragm: Located in the condenser, it produces a hollow cone of light. Phase Plate: Located in the objective lens, it has a phase ring that retards or advances the phase of the direct (unscattered) light, while diffracted (scattered) light passes through unaltered. Working: Unscattered light passes through the phase ring, undergoing a phase shift. Scattered light bypasses the phase ring. When these two sets of waves recombine, their interference patterns create variations in amplitude (brightness), making transparent structures visible as darker or brighter areas against a background. Applications: Observing living, unstained cells, cell division, and intracellular structures. 4.3 Fluorescent Microscopy A type of light microscopy that uses fluorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances. Principle: Specimens are stained with fluorophores (fluorescent dyes) that absorb light at a specific wavelength (excitation wavelength) and then emit light at a longer, lower-energy wavelength (emission wavelength). Working: An excitation light source (e.g., mercury lamp, LED, laser) emits light of the appropriate wavelength. An excitation filter selects only the excitation wavelength to illuminate the specimen. The fluorophores in the specimen absorb this light and emit fluorescent light. A dichroic mirror reflects the excitation light towards the specimen and allows the longer-wavelength emitted fluorescent light to pass through to the eyepiece/detector. An emission filter blocks any remaining excitation light and passes only the emitted fluorescent light, creating a bright image of the fluorescent structures against a dark background. Diagram (Conceptual): Light Source Excitation Filter Dichroic Mirror Specimen (Fluorophore) Emission Filter Detector/Eyepiece Applications: Immunofluorescence, live-cell imaging, tracking molecules, disease diagnosis. 5. Centrifugation 5.1 Centrifugation and its Types Centrifugation is a process that uses centrifugal force to separate components of a mixture based on their density, size, and shape. Principle: A centrifuge spins samples at high speeds, creating a strong centrifugal force that pushes denser particles away from the axis of rotation and towards the bottom of the tube (pellet), while less dense particles remain in the supernatant. Types: Differential Centrifugation: Separates particles of different sizes and densities by increasing centrifugal force in a stepwise manner. Used for subcellular fractionation. Density Gradient Centrifugation: Separates particles based on their buoyant density in a pre-formed or self-forming density gradient (e.g., sucrose, cesium chloride). Rate-Zonal Centrifugation: Separates based on size and shape, with particles sedimenting through a gradient until they reach a position where their rate of sedimentation slows. Isopycnic Centrifugation: Separates based on buoyant density. Particles migrate until they reach a point in the gradient where their density matches the surrounding medium (their isopycnic point). 5.2 Sedimentation Coefficient The sedimentation coefficient ($s$) is a measure of the rate at which a particle sediments in a centrifugal field. It is a characteristic property of a particle and depends on its mass, shape, and the density and viscosity of the medium. $s = \frac{v}{\omega^2 r}$ where $v$ is the sedimentation velocity, $\omega$ is the angular velocity of the rotor, and $r$ is the distance from the center of rotation. Units: Svedberg (S), where $1 S = 10^{-13}$ seconds. Larger, denser, and more compact particles generally have higher sedimentation coefficients. Used to characterize macromolecules (e.g., proteins, nucleic acids, ribosomes) and to determine their molecular weight and shape. 5.3 Applications of Centrifuge in Biotechnology/Food Industries Biotechnology: Cell Harvesting: Separating microbial cells (bacteria, yeast) from fermentation broths. Subcellular Fractionation: Isolating organelles (nuclei, mitochondria, ribosomes) from cell homogenates. Protein Purification: Removing insoluble aggregates, separating proteins from contaminants. DNA/RNA Isolation: Separating nucleic acids from cellular debris and other components. Virus Purification: Concentrating and purifying viruses for vaccine production or research. Blood Component Separation: Separating plasma, red blood cells, white blood cells for medical diagnostics and therapies. Food Industries: Clarification of Liquids: Removing suspended solids from fruit juices, wines, beers, and oils. Dairy Processing: Separating cream from milk, concentrating milk proteins. Oil Production: Separating oil from water and solids in vegetable oil and olive oil extraction. Starch Production: Separating starch granules from other components in agricultural products. Waste Treatment: Dewatering sludge in wastewater treatment plants. Quality Control: Detecting contaminants or impurities in food products.