chm 12901 general chemistry with a biological focus unit 10 study guides

Key Lab Safety Protocols

• Always wear appropriate personal protective equipment (PPE) including lab coats, safety glasses, and gloves when working with chemicals or biological samples
• Familiarize yourself with the location and proper use of safety showers, eyewash stations, and fire extinguishers in case of emergencies
• Handle chemicals and biological samples with caution, using fume hoods when necessary to minimize exposure to hazardous vapors or aerosols
• Properly label all containers with the chemical name, concentration, date, and any relevant safety warnings (flammable, corrosive, toxic)
• Dispose of chemicals and biological waste according to established protocols, using designated waste containers for different types of hazardous materials
  • Never pour chemicals down the drain or discard them in regular trash bins
  • Follow specific guidelines for disposing of sharps (needles, broken glass) and biohazardous waste
• Maintain a clean and organized workspace to prevent accidents and cross-contamination
  • Clean up spills immediately using appropriate absorbent materials and disposal methods
  • Disinfect work surfaces before and after handling biological samples

Essential Lab Equipment and Glassware

• Beakers are cylindrical containers used for mixing, heating, and storing liquids, available in various sizes (50 mL, 100 mL, 250 mL)
• Erlenmeyer flasks have a conical base and narrow neck, making them ideal for swirling and mixing solutions without spilling
• Graduated cylinders are used for accurately measuring and dispensing liquid volumes, with graduations marked in milliliters (10 mL, 25 mL, 100 mL)
• Volumetric flasks have a precise volume capacity and are used for preparing solutions of known concentration by diluting to the calibration mark
• Pipettes are used for transferring precise volumes of liquids, with different types available for specific volume ranges (micropipettes, serological pipettes, volumetric pipettes)
  • Micropipettes are adjustable and typically used for volumes between 0.1 μL and 1000 μL
  • Serological pipettes are graduated and used for larger volumes (1 mL to 25 mL)
• Burettes are long, graduated glass tubes with a stopcock at the bottom, used for precise delivery of liquids during titrations
• Thermometers measure temperature and are essential for monitoring chemical reactions and biological processes

Basic Measurement Techniques

• Accurately measure liquid volumes using appropriate glassware (graduated cylinders, pipettes, burettes) by reading the meniscus at eye level
  • The meniscus is the curved surface of a liquid in a container, and the volume should be read at the bottom of the meniscus for most liquids
• Use analytical balances for precise mass measurements of solids, ensuring the balance is properly calibrated and leveled
  • Tare the balance with a weighing paper or boat before adding the solid to be measured
  • Close the balance doors gently to prevent air currents from affecting the measurement
• Determine the density of a substance by measuring its mass and volume, then calculating density using the formula $density = \frac{mass}{volume}$
• Measure the pH of a solution using pH paper, pH meters, or color-changing indicators (phenolphthalein, bromothymol blue)
  • pH paper and indicators provide a color change corresponding to a specific pH range
  • pH meters offer a more precise numerical value for pH
• Record measurements with the appropriate number of significant figures based on the precision of the measuring device
  • For example, a graduated cylinder with graduations every 1 mL would yield measurements with 3 significant figures (e.g., 23.0 mL)

Solution Preparation and Dilutions

• Prepare solutions of known concentration (molarity) by dissolving a calculated mass of solute in a specific volume of solvent
  • Molarity ($M$) is defined as moles of solute per liter of solution: $M = \frac{moles,of,solute}{liters,of,solution}$
  • Use the molar mass of the solute to convert between grams and moles
• Perform dilutions to create less concentrated solutions from a stock solution of higher concentration
  • Use the formula $M_1V_1 = M_2V_2$, where $M_1$ and $V_1$ are the concentration and volume of the stock solution, and $M_2$ and $V_2$ are the concentration and volume of the diluted solution
  • Pipette the appropriate volume of stock solution into a volumetric flask, then fill to the calibration mark with solvent
• Prepare buffer solutions to maintain a stable pH by combining a weak acid and its conjugate base, or a weak base and its conjugate acid
  • The Henderson-Hasselbalch equation relates pH to the pKa of the acid and the ratio of the concentrations of the acid and its conjugate base: $pH = pKa + log\frac{[base]}{[acid]}$
• Make serial dilutions to create a series of solutions with progressively lower concentrations, often used in biological assays
  • Each dilution step typically involves a fixed ratio (e.g., 1:10) between the volume of the previous solution and the total volume of the new solution

Titration Methods

• Titration is a quantitative analysis technique used to determine the concentration of an unknown solution (analyte) by reacting it with a solution of known concentration (titrant)
• Acid-base titrations involve the reaction of an acid with a base, using color-changing indicators (phenolphthalein) or pH meters to detect the endpoint
  • The endpoint is the point at which the reaction is complete, indicated by a sharp color change or a rapid change in pH
• Redox titrations are based on oxidation-reduction reactions, often using potentiometric methods (redox electrodes) to detect the endpoint
• Complexometric titrations involve the formation of stable complexes between metal ions and ligands, with indicators (Eriochrome Black T) that change color when the metal ions are fully complexed
• Calculate the concentration of the analyte using the titration data and stoichiometric relationships
  • The general formula is $M_aV_a = M_tV_t$, where $M_a$ and $V_a$ are the molarity and volume of the analyte, and $M_t$ and $V_t$ are the molarity and volume of the titrant
• Perform titrations using a buret to accurately dispense the titrant into the analyte solution, with constant swirling or stirring to ensure complete mixing

Spectroscopy Basics

• Spectroscopy is the study of the interaction between matter and electromagnetic radiation, used to identify and quantify substances based on their absorption or emission of light
• UV-Visible spectroscopy measures the absorption of ultraviolet and visible light by a sample, with applications in quantifying the concentration of substances (proteins, nucleic acids) using the Beer-Lambert law: $A = \epsilon bc$
  • $A$ is the absorbance, $\epsilon$ is the molar attenuation coefficient, $b$ is the path length, and $c$ is the concentration
• Infrared (IR) spectroscopy detects the absorption of IR light by molecules, providing information about functional groups and molecular structure
  • IR absorption bands correspond to specific vibrational modes of bonds within the molecule
• Atomic absorption spectroscopy (AAS) is used to quantify the concentration of metal ions in a sample by measuring the absorption of light at wavelengths specific to each element
• Fluorescence spectroscopy measures the emission of light by a sample after it has absorbed light of a specific wavelength, with applications in studying biological molecules (fluorescent proteins, FRET)
• Prepare samples for spectroscopic analysis by dissolving in appropriate solvents, filtering if necessary, and using cuvettes or sample cells with a defined path length

Chromatography Techniques

• Chromatography is a separation technique that distributes components of a mixture between a stationary phase and a mobile phase based on their relative affinities
• Thin-layer chromatography (TLC) separates compounds on a thin layer of adsorbent material (silica gel, alumina) coated on a glass or plastic plate
  • The sample is spotted near the bottom of the plate, which is then placed in a chamber with a solvent (mobile phase) that travels up the plate by capillary action
  • The separation of compounds is based on their relative solubility in the mobile phase and their affinity for the stationary phase
• Column chromatography separates compounds using a glass or plastic column packed with a solid adsorbent (stationary phase), with the sample loaded onto the top of the column and eluted with a solvent (mobile phase)
  • Compounds are collected as separate fractions based on their retention time in the column
• High-performance liquid chromatography (HPLC) is an automated, high-resolution version of column chromatography, using high-pressure pumps to force the mobile phase through a densely packed column
  • HPLC is coupled with UV-Vis, fluorescence, or mass spectrometry detectors for sensitive and specific detection of separated compounds
• Gas chromatography (GC) separates volatile compounds using a gaseous mobile phase (helium, nitrogen) and a liquid or solid stationary phase coated inside a long, narrow column
  • GC is often coupled with mass spectrometry (GC-MS) for identification of separated compounds based on their mass spectra

Biological Sample Handling

• Use aseptic technique when handling biological samples (cell cultures, tissues) to prevent contamination by microorganisms
  • Work in a biosafety cabinet or laminar flow hood, using sterile pipettes, media, and glassware
  • Sterilize tools and glassware by autoclaving (high-pressure steam) or filtering (0.22 μm filters for liquids)
• Store biological samples at appropriate temperatures to maintain their integrity and prevent degradation
  • Short-term storage (days to weeks) at 4°C for whole cells, tissues, or purified proteins and nucleic acids
  • Long-term storage (months to years) at -20°C or -80°C, using cryoprotectants (glycerol, DMSO) to prevent ice crystal formation
• Extract proteins, nucleic acids, or other biomolecules from cells or tissues using specific lysis buffers and separation techniques
  • Protein extraction often involves mechanical disruption (sonication, homogenization) followed by centrifugation to remove insoluble debris
  • Nucleic acid extraction uses chaotropic agents (guanidinium salts) to denature proteins and silica-based spin columns to purify DNA or RNA
• Quantify the concentration of extracted biomolecules using spectroscopic methods
  • Proteins can be quantified using the Bradford or BCA assays, which produce a colorimetric change proportional to the protein concentration
  • Nucleic acids are quantified by measuring the absorbance at 260 nm ($A_{260}$), with an $A_{260}$ of 1.0 corresponding to 50 μg/mL for double-stranded DNA
• Analyze biological samples using techniques such as gel electrophoresis, Western blotting, or PCR
  • Gel electrophoresis separates proteins or nucleic acids based on their size and charge, using a porous gel matrix (agarose, polyacrylamide) and an electric field
  • Western blotting involves transferring separated proteins onto a membrane and detecting specific proteins using antibodies
  • Polymerase chain reaction (PCR) amplifies specific DNA sequences using primers, dNTPs, and a heat-stable DNA polymerase enzyme