BITC 1402

1
BITC 1402

• General, Organic and Biochemistry or GOC,
  Chapters 3, 4, 5
• Review of General Chem 1
• Laboratory Methods or LM, 24, 14, 15

2
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?

3
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

4
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

5
Chapter 7pg 201Le Chateliers PrincipleIf an
external stress is applied to a system in
equilibrium, the system reacts in such a way as
to partially relieve that stress
6
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

7
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)

8
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

9
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

10
Other Methods

• UV oxidation removes organic contaminants and
  may kill bacteria
• Ozone kills bacteria

11
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?

12
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

13
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)

14
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

15
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 schoolwhy is this a problematic process
  in todays lab?)

16
Sterilization and Storage of Solutions

• Review Tables 24.9 and 24.10
• Activity problem 1 and 3

17
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

18
Certain Requirements Must be Met

• The instruments response must have a consistent
  and predictable relationship with the property
  being measured
• The instruments 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

19
Calibration of Common Bioinstrumentation

• pH meter
• Balance
• Centrifuge
• (How is calibration different from validation?)

20
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
  Hz1cycle/sec (why is AC delivered by power
  companies and NOT DC?)
• Rectification- change AC to DC

21

• 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.

22

• 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?

23

• Ohms Law
• VIR
• Application of Ohms 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.

24
Power, Work, and Circuits

• Power voltage x current or
• W (V)(I)
• A good equation to know if youre trying to
  figure out how many instruments you can run in a
  lab at one time

25
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

26

• 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

27
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 100/1100

28

• 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

29
BITC 1402The Measurement of Weight

• Chapter 15

30
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?

31
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?

32
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)

33
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!)

34
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

35
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!

36
Mechanical Balance

• Must be calibrated by a trained technician (ASTM
  Standard E 319-85)

37
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)

38
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

39
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.

40
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

41
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)

42
Writing SOPs for the Use of Balances

• Examples are found on page 292

43
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