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BITC 1402 General, Organic and Biochemistry or GOC, Chapters 3, 4, 5 Review of General Chem 1 Laboratory Methods or LM, 24, 14, 15 Chapter 3 What is the octet rule? Define electronegativity Compare and contrast ionic and covalent bonding, which is stronger etc. Compare and contrast nonpolar and polar covalent bonding What is a Lewis structure and what does it show for a molecule? Compare and contrast Lewis structure and VSEPR models What is a dipole? How are binary molecular compounds named? Chapter 4 Be able to calculate formula, molecular weights Be able to write chemical equations including a net ionic equation Be able to determine limiting reagent, percent yield Be able to determine spectator ions Be able to determine what is oxidized and what is reduced in a redox rxn Define heat of reaction, exothermic and endothermic, and heat of combustion Chapter 5 Define intermolecular forces of attraction and be able to list them in order of increasing strength Define surface tension, vapor pressure, boiling point, melting point, and crystallization Define phase, phase change, sublimation, heat of fusion, heat of vaporization, triple point, and a phase diagram Chapter 7 pg 201 Le Chatelier’s Principle: If an external stress is applied to a system in equilibrium, the system reacts in such a way as to partially relieve that stress Activities Chapters 3, 4, and 5 Ch3: 53, 58, 69, 72, 77, 102, 105 Ch4: 29, 41, 46, 50, 53, 73, 90, 92 Ch5: 51, 56, 57, 88 LM: Chapter 24 Contaminants of water (table 24.1) Dissolved inorganics Dissolved organics Suspended particles Dissolved gases Microorganisms Pyrogens/endotoxins Measure total organic carbon (TOC) Measure total solids (TS) Types of Water (Table 24.3) I (highest purity) used for analytical procedures, tissue culture, some HPLC, electrophoresis buffers, immunology assays II suitable for microbiology procedures III used for initial glassware rinses Biologically pure- tissue culture Organically pure- HPLC, GC/MS WFI- water for injection Methods of Water Purification Review table 24.4 What method(s) will remove pyrogens, dissolved ionized gases? Distillation Ion Exchange Deionization (remove all ionic contaminants) Carbon Adsorption –remove dissolved organic compounds Filtration Depth Microfiltration Ultrafiltration Reverse osmosis Other Methods UV oxidation – removes organic contaminants and may kill bacteria Ozone – kills bacteria Systems in the Lab RO Deionization distillation What type(s) of water are possible and how do we determine what type(s) of water are being produced? Handling of Reagent Water Highly purified water is an extremely aggressive solvent Readily leach contaminants from any vessel and will also dissolve carbon dioxide from the air (Is this why the pH value continually changed?) Type 1 cannot be stored for any length of time –Type II can be stored for short periods of time Monitoring Resistivity – The theoretical max ionic purity for water is 18.3 megohm-cm and 17 is acceptable Bacterial counts Pyrogens Organic carbon (potassium permanganate) Maintenance Distilled water: frequent cleaning prepared daily Ion exchange/ deionization: regenerated and sanitized Filters: tested, flushed and sanitized Activated carbon: washed/ recharged UV: replaced annually DOCUMENTATION Characteristics & Cleaning of Glass and Plastic Labware Review tables 24.5 and 24.6 Cleaning Prerinse Contaminant removal (review table 24.7) Rinse Final rinse Drying (I used chromic acid to clean pipettes in graduate school—why is this a problematic process in today’s lab?) Sterilization and Storage of Solutions Review Tables 24.9 and 24.10 Activity: problem 1 and 3 Chapter 14: Introduction to Instrumental Methods and Electricity Mechanical measurement instrument has these components: Interface Transducer (Sensor) Signal Processor Display (Readout) Identify these components in a balance Certain Requirements Must be Met: The instrument’s response must have a consistent and predictable relationship with the property being measured The instrument’s response must be related to internationally accepted units of measurement Calibration is the process by which the response of an instrument is related to internationally accepted measurement units Calibration of Common Bioinstrumentation pH meter Balance Centrifuge (How is calibration different from validation?) Basic Terminology and Concepts of Electricity Define the following: Current Amperes or amps (6.25 x 1018 electrons/sec = 1amp) DC (delivered by batteries) AC (USA- frequency is 60 times/sec) 1 Hz=1cycle/sec (why is AC delivered by power companies and NOT DC?) Rectification- change AC to DC Voltage What is the definition of energy? Potential energy? Electrical potential is also called electromotive force (EMF) or voltage (V). The voltage supplied by the power company is either in the range of 110 to 120 or 220. Resistance Impedance to electron flow The units are ohms (1 ohm is the value of resistance through which 1 V maintains a current of 1 A) What is a conductor? What is a semiconductor? An insulator? Ohm’s Law V=IR Application of Ohm’s Law Gel electrophoresis: The sample mixture is placed in a gel matrix. The gel is placed in a box, buffer is poured over it, and a current (I) is applied. The positive and negative ions in the gel and the buffer conduct the current. A power supply provides the voltage (V), source of the I. The gel provides R, which increases with time as the ions are run out of the gel. Therefore, to maintain I, V must be increased BUT this also increases the heat in the gel-thus, it is better to maintain constant V instead of constant I TO prevent excessive heating of the gel. Power, Work, and Circuits Power = voltage x current or W = (V)(I) A good equation to know if you’re trying to figure out how many instruments you can run in a lab at one time Equipment in the Laboratory Power supplies convert AC to DC Transducers or detectors generate an electrical signal in response to a physical or chemical property of a sample (review table 14.1) Detection limit, sensitivity or range of the equipment is the minimum level of the material or property of interest that causes a detectable signal Electrical noise is an important factor in determining range Noise Short-term is defined as random, rapid “spikes” Long-term or drift is a relatively long-term increase or decrease in readings due to changes in the instrument Expressed as signal-to-noise ratio: the higher the ratio, the better the performance (root mean square noise- RMS) The detection limit of a detector is therefore often defined in practice as the minimum level of sample that generates a signal at least twice the average noise level The dynamic range of a detector is the range of sample concentrations that can be accurately measured by the detector Signal Processing Units Amplifier boosts the voltage or current from a detector in proportion to the size of the original signal Gain is the degree to which a signal can be increased or decreased For example, if input is 1 mV and the output is 100 mV then Gain = 100/1=100 Attenuator reduces a signal in order to best display it by a readout device Readout devices Meters, strip chart recorders, computer screens Quality and Safety Issues Preventive maintenance Performance verification Instrument validation Environmental Factors that Affect the Performance of Instruments (table 14.4) Electrical Safety (table 14.5) Problems: 1, 2, 8 BITC 1402 The Measurement of Weight Chapter 15 Chapter 15: Weight The force of gravity on an object Balances are instruments used to measure this force Mass The amount of matter in an object expressed in units of grams Why are these instruments named balances? How is a mechanical balance different from an electronic balance? Which one would you use to balance tubes for centrifugation? Characteristics and Types of Balances Range (highest to lowest) Capacity (the heaviest sample that can be weighed) Sensitivity (or readability)- the smallest value of weight that will cause a change in the response of the balance (determines the no. of places right of the decimal point) Which is more sensitive, an analytical balance or a pan balance? (review figure 15.4) How about the balances in the lab? How Does an Electronic Balance Work? The weighing pan is depressed by a small amount when an object is placed on it. The balance has a detector that senses the depression of the pan An electromagnetic force is generated to restore the pan to its original (“null”) position. This force is measured as an electrical signal that is in turn converted to a digital display of weight value (i.e. the balance compares the electrical signal of the unknown sample to the signal of standard(s) of known weight) General Procedure for Weighing a Sample with an Electronic Analytical Balance Make sure the balance is level Adjust the balance to zero (the pan should be clean and doors shut to avoid air currents) Tare the weighing container or weigh the empty container Place the sample on the weighing pan & read the value for the measurement Remove the sample; clean the balance and area around it (I will fail you if you do not!) Factors That Affect the Quality of Weight Measurements Temperature (samples are heavier when they are cold) “Warming up” period for the balance? Static charge (ionizing blower, antistatic brush) Ground yourself (hold the sample on the spatula and touch the balance) Review table 15.1 Calibration and Maintenance of a Balance Must be checked periodically Must be checked when a balance is moved For microbalances (5 places pass the decimal to the right), they must be calibrated when the weather changes! Mechanical Balance Must be calibrated by a trained technician (ASTM Standard E 319-85) Electronic Balance: Two-point Calibration The balance is set to zero using the appropriate knob, when the weighing pan is clean and empty The first weight is added and the balance is set to the value of the standard The second weight (usually heavier than the first) is added and the balance is set to the value of the standard A quick check is to add exactly 1mL of water to a tared weigh boat-it should weigh exactly 1g (of course your micropipettor needs to be measuring exactly 1 mL) Standards Purchased with a certificate showing their traceability to NIST The appropriate weights for a balance have been established by ASTM Standard E 617 There are different classes: 1, 2, 3 and 4 with 1 being the most rigorous (4 is recommended for student use) Handle with tongs because they are damaged by skin oils and by cleaners that remove such oils (Keep two sets: one for use and one for storage; every 6 months measure the one for use against the one for storage Review the Appendix Quality Programs and Balances Laboratories that meet the requirements of a quality system, such as ISO 9000 or cGMP, have procedures that detail how to operate each balance and how to maintain, calibrate, and check their performance. Verifying Balance Accuracy, Precision, and Linearity Accuracy is tested by weighing one or more mass standards Precision is measured by weighing a sample multiple times and calculating the standard deviation Linearity is tested by weighing subsets of weights and comparing the sum of the subsets to the weight of the objects all together Checking the Linearity of a Balance Select 4 items whose weight is about the capacity of the balance and label them A, B, C, D Weigh all 4 items separately and added up the values Weigh all 4 items together and compare that value to the summed value Do this for a low weight (25%), the midpoint weight (50%) and for a weight close to capacity of the balance (75%) Writing SOPs for the Use of Balances Examples are found on page 292 Mass versus Weight The major force measured in weighing is the force of gravity. However there is also a slight buoyant force from air. Therefore objects weighed in a vacuum are heavier than weighed in air. This is the priniciple of buoyancy. (Note 1 g of metal weighs differently than 1 g of water) The discrepancy between mass and weight is called the buoyancy error-usually this error is ignored! Exercises: 4, 5, 6