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what is spectroscopy?
the study of the interaction of energy with matter. Energy applied to matter can be absorbed, emitted, cause a chemical change.
what are some examples of spectroscopy?
infrared IR spectroscopy
UV visible Spectroscopy
Nuclear magnetic resonance NMR
Mass spectrometry MS
What is electromagnetic spectrum?
different molecules absorb at different wavelengths of light. it is the complete spectrum (or continuum) of all forms of “light”
what is the composition of white light in UV-Vis spectroscopy?
sunlight = white light; wavelength range = 380-750nm
composition of a range of colors
when white light falls on an object the color detected by the eye depends on ABSORPTION/REFLECTION properties of material in the object
if material completely REFLECTS all light = looks white
if material completely ABSORBS all light = looks black
what does a spectrophotometer do?
if a molecule absorbs in the UV-Visible region, you can find the wavelength that is absorbed using a spectrophotometer. Shines light within range of wavelengths 200-800 nm
what is the difference between a single-beam and double-beam spectrophotometer?
single-beam: it only has one beam of light to shine through the spectrophotometer, so it can only read one sample at a time.
double-beam: it has a beam splitter and mirrors that split the beam of light in two, so it can read the reference cell and sample cell at the same time.
what does a UV spectroscopy measure?
It measures the absorption, emission, or scattering of electromagnetic radiation by atoms or molecules.
when UV/Vis light strikes an atom/molecule, it can either be transmitted or absorbed
energy absorbed corresponds to the amount of necessary to promote an electron form low energy orbital to higher energy orbital
an optical spectrometer records the wavelengths at which absorption
what is absorbance?
A = log Io/I
A = absorbance
Io = the intensity of the light before passing through the sample
I = the intensity of the light after passing through the sample
absorbance values typically range from 0 (no abs) to 2 (99% abs)
λmax = wavelength of maximum absorbance
what are the different cuvettes used in spectroscopy?
plastic cuvettes
glass cuvettes
quarts cuvettes
what are plastic cuvettes?
they dissolve in most organic solvents and absorb light in the ultraviolet region. They can only be used for aqueous solutions
what are glass cuvettes?
they can be used with most solvents, however they too absorb in the UV range
what are quartz cuvettes?
they don’t absorb in the UV-Vis spectrum and can be used with virtually all samples (very expensive)
what are the uses of UV spectroscopy?
give information about presence and absence of unsaturated functional groups
for structural proof, usually not critical data, but essential for further studies
used to determine analyte concentration either at one time or often over a desired time period
measures the absorption of light across the UV and Vis light wavelengths through a liquid sample
what sample did we use for the UV/Vis lab?
salicylic acid
what did we use to measure out our stock solutions in the UV/Vis lab?
volumetric flask: used to make up solutions with fixed volumes (100mL)
accuracy within 0.04%
why is it necessary to determine water content in pharmaceuticals?
pharmaceutical: compound manufactured for use as a medicinal drug
many API and adjuvants contain water in an absorbed form (surface water) or bound as a hydrate (water of crystallization)
quality and usability of raw materials, intermediates and substances necessary for manufacturing of high-grade pharmaceutical products
water content of pharmaceuticals strongly influences their quality, shelf life, and stability as well as the release of active substances
the determination of water assumes great importance in pharmaceutical analysis
what methods are used for the determination of water content?
loss on drying: determines the amount of volatile matter of any kind that is driven off under the conditions specified (gravitational method)
moisture analyzer: uses infrared heating technology to perform a loss on drying method (moisture balance/meter)
Karl Fischer titration: uses coulometric or volumetric titration to determine trace amounts of water in a sample (automatized Karl Fischer titrator)
what two methods did we use for moisture analysis lab?
loss on drying using an oven: volatility of compound
moisture analyzer: >1% water content
how does loss on drying using an oven work?
samples are heated at certain temperatures (95-105°C) in an oven and then water is evaporated. The loss of weight is moisture content in the sample
how does the moisture analyzer work?
consists of 2 important components: Balance and Heater
the sample is placed in the moisture analyzer and the balance captures the initial weight
an infrared energy heater is used to heat the sample
during the test the balance records the weight
when sample no longer loses weight (water) the instrument shuts off the heat and uses the final weight to calculate moisture
why do we why do we use moisture analysis in pharmaceutics?
technique used in various stages like moisture in industry
means of qc: presence of water can allow for undesired microbial growth and purity check for APIs dry vs. wet product
effectiveness: crystal structure, dissolution rates, permeability, properties of both active ingredients and excipients
stability: manufacturing and shelf life
what is partition coefficient (P)?
defined as the ratio at equilibrium of the two concentrations of a dissolved substance in two-phase system consisting of two immiscible solvents
in the case of n-octanol and water
measure of the difference in solubility of the compound in these two phases
dimensionless and is usually given in the form of its logarithm to base ten (log P)
what does the partition coefficient do?
describes the equilibrium partitioning of a solute between two immiscible phases
are frequently used as a measure of lipophilicity in drug discovery
when one of the solvents is water and the other is a non-polar solvent, the log P value is a measure of lipophilicty or hydrophobicity
how is the partition coefficient obtained?
when a solution is placed in a separator funnel and shaken with an immiscible solvent
solution often dissolve in part into both layers
the solutes are said to “partition” between the two layers or “distribute themselves” between the two layers
when equilibrium is established, the ratio of concentration of solute in each layer is constant for each system
can be represented by a value K or P
K(P) = molarity in organic phase / molarity in aqueous phase
what is the equation of log P?
log P < 0: more drug in aqueous layer, hydrophilic
log P = 0: equally partitioned between lipid and aqueous
lop P > 0: more drug in organic layer
what is the Lipinski Rule of Five?
molecular weight <500
Log P < 5
< 5 H-bond donors (sum of NH and OH)
< 10 H-bond acceptors (sum of N and O)
otherwise absorption and bioavailability are likely to be poor. N.B. this is for oral drugs only
what is the effect of pH on Partition Coefficient?
non-polar phase is usually dominated by the un-ionized form of the solute
if a compound can ionize then the observed partitioning between water and octane will be pH dependent
knowledge how this value changes with the acidity (pH value) in the aqueous phase is important for understanding many biological activities
what solutions do we used for the partition coefficient lab?
salicylic acid solutions
what organic solvent do we mix our salicylic acid solutions with and in what?
mixing it with isopropyl myristate in a separatory funnel
where are we obtaining our final concentration of salicylic acid and how will we measure it?
after separation of aqueous and organic phases, the final concentration of salicylic acid in the aqueous layer will be determined using the colorimetric assay and visible spectroscopy
how is the equilibrium concentration of salicylic acid in the organic layer calculated?
its calculated by subtracting the final concentration of salicylic acid in the aqueous solution from it's starting concentration
how is the partition coefficient of salicylic acid calculated?
can be calculated through dividing the final concentration of SA in the organic layer by its final concentration in the aqueous layer
what color reagent do we use for the partition coefficient lab?
ferric nitrate color reagent: violet color
what is viscosity?
viscosity η is the property that describes a fluid’s resistance to flow
describes fluidity of substances
ratio of shearing tree to velocity gradient in a fluid
what is the formula for viscosity?
dvx/dz: velocity gradient, shear rate
F: force per unit area, shear stress
1 pascal.second = 10 poise
1 poise (P): g/(cm.s)
(SI) 1 centi-Oise (cP) = 1millipascal.second
what is a capillary viscometer?
single viscosity measure in which the flow rate of fluids in a circular narrow tube is used for viscosity determination
what is the equation for the viscometer?
∆P: pressure difference between the ends of the tube
l: length of the tube
r:radius of the tube
φ: volume flow rate
what is the viscosity of common fluids?
temperature dependence of viscosity
viscosity decreases with increasing temperature
what is rheology?
the study of the flow of materials that behave in an interesting or unusual manner
what is a fluid?
a fluid is a substance that deforms continuously under the action of a shearing force
what are the fluid classifications by flow properties?
newtonian systems (ideal and simple liquid)
non-newtonian systems (non-ideal fluid): Bingham plastic flow, pseudo plastic flow, dilatant flow
how do you distinguish the difference between newtonian and non-newtonian fluids?
if flow properties behave linearly in accordance with Newtons’s law of flow, then the system is a newtonian flow
otherwise, it’s a non-newtonian fluid or complex fluid
what are some examples of complex fluids in food?
emulsions (mayonnaise, ice cream)
foams (whipped cream)
suspensions (mustard, chocolate)
bread dough
what is an example of complex fluids in biofluids?
suspensions (blood)
what are some examples of complex fluids in personal care products?
suspensions (nail polish, face scrubs)
solutions/gels (shampoos, conditioners)
foams (shaving cream)
what are some examples of complex fluids in pharmaceuticals?
gels
emulsions
aerosols (nasal sprays)
what are some common non-newtonian behaviors?
shear-thinning: house paint, honey, ketchup
Bingham plastic: toothpaste, molten chocolate
shear-thickening: cornstarch paste
simple liquids (water, mineral oils, emulsions) follow newtonian flow properties
viscosity is a constant at constant temperature and is independent of shear stress or rate of shear
the slope in a newtonian liquid is the viscosity
what is the relationship between viscosity and stress in different liquids?
newtonian liquids: as stress increases, viscosity stays the same
shear thickening: as stress increases, viscosity increases
shear thinning: as stress increases, viscosity decreases
what can we use to measure viscosity?
capillary rheometer
cone and plate rheometer
how does a cone and plate rheometer work?
the fluid is placed on horizontal plate and a shallow cone placed into it
the plate is rotated and the force on the cone measured
the measured force and the degree of twist give the shear stress, while the rotational speed and cone dimensions give the shear rate
what is the power law model?
newton’s equation can be written as a power law that encompasses the simpler forms of non-newtonian behavior
η is replaced with K, the flow consistency index [Pa s^n], and the velocity gradient is raised to some power n called the flow behavior index [dimensionless]
from the power law model, how can the variable n determine the type of flow?
n=1, its a newtonian flow
n<1, shear-thinning
n>1, shear-thickening
what type of materials/fluid did we use in the rheology lab?
Vick’s Cough Syrup (cherry)
what are the four steps to a safe laboratory?
Recognize: (RTK, SDS)
Assess: (elimination, substitution, minimization)
Minimize the risks: (admin & eng controls, PPE, housekeeping)
Prepare for emergencies: (911, fire alarm, safety equipment)
what does this pictogram mean?
chemical is explosive
what does this pictogram mean?
chemical is oxidizing
what does this pictogram mean?
chemical becomes a gas under pressure
what does this pictogram mean?
chemical is flammable
what does this pictogram mean?
chemical is toxic
what does this pictogram mean?
chemical is corrosive
what does this pictogram mean?
chemical is harmful
what does this pictogram mean?
chemical is hazardous to health (carcinogen)
what are some applicable regulations?
OSHA 1910.1450
Georgia Rules & Regulations
RCRA
NFPA codes
ANSI Standards
what are some other guidelines?
BOR Laboratory Design Criteria
Prudent Laboratory Practices, National Research Council
Wiley Guide to Chemical Incompatibilities
UGA Environmental Health & Safety Policies 6.01 & 6.02
what is required on a door sign for a laboratory?
the door sign needs…
NFPA Ratings
quantities
Contact information
needs to be updated annually
what else is required in a laboratory?
emergency numbers: posted near or on back of door
hand-washing facilities: soap and paper towels must be present at one sink
what are some best practices in a laboratory?
food/drink prep, storage, and consumption are prohibited
never work alone
always notify instructor of accidents, injuries, spills
AAA-always add acid to water
what are some PPE practices?
proper PPE must be worn
no open-toed shoes or shorts
wearing respirators
working with pyrophoric?
what are some sink conditions you have to check in a laboratory?
evidence of sink disposal
check under sinks
leaky faucets
what are some chemical storage & documentation practices you need to do?
acid/bases: liquid corrosives in secondary containment
label everything
appropriate fridge for flammables
what are some chemical inventory practices you need to do?
make sure your chemical inventories are up to date
undefined storage locations are red flags —> caution signs based on this
bar codes should be on all chemicals
how do you store chemicals in a laboratory?
alphabetical storage is discouraged
store by general hazard: acids, bases, flammables, oxidizers, toxins
consult SDS
what are the chemical storage color codes?
stripes: same color incompatibles
blue: health hazards/toxins
red: flammable liquids
yellow: reactive materials
white: corrosives
what’s the difference between NFPA and GHS labels
GHS: lower the categorization number, the greater the severity of the hazard
NFPA: higher the categorization number, the greater the severity of the hazard
what is required on a GHS label?
chemical name
pictograms
supplier identification
product identifier
first aid statement
signal word
hazard statement
precautionary statement
what are legacy chemicals?
chemical stocks inherited from previous occupants or teams
what are the storage requirements of gas cylinders & lecture cylinders?
stored upright
tagged (full, in user empty)
anchored individually
safety caps
special requirements for health hazards (NFPA 3 or 4)
what is the difference between bench top, biosafety cabinet, and a chemical hood?
bench top: low risk, airflow build into the laboratory
biosafety cabinet: moderate to high risk, air flow is from top to bottom and recycles 70% of air back into the cabinet
chemical fume hood: moderate to high risk, airflow is up and out, used for volatile substances and air is not recycled it is discarded
what is needed for peroxide forming chemicals?
date upon receipt and upon opening
requirements vary depending on class
appendix I of the CLSM has more information
what are SDSs and what training is required?
Safety Data Sheets : must be readily available
Trainings to be completed…
RTK training (annually)
Hazardous Waste training (annually)
Lab Safety Basics (one time)
Biosafety & Rad Safety Training if necessary
what are the requirements for flammable volumes?
volume shouldn't exceed 20 gal/100ft²
no more than 120 gallons total
No more than 35 gallons should be outside a cabinet
what are the requirements of Hazardous Waste Storage?
label appropriately: “hazardous waste” + contents
closed unless adding wastes: port caps
liquids in secondary containment
hazard segregation
what are the requirements for First Aid and Spill Kits?
fill out your safety information sign
properly stocked
signage indicating location
what are the requirements of safety equipment?
EW/SS: unobstructed and annually tested
Fire extinguisher: mounted, properly charged, and certified annually
working with active metals?
what are the requirements for fume hoods?
uncluttered
no unattended spills
certified annually
avoid storage
working with perchloric acid radioisotopes?
what are the requirements for electrical safety?
no frayed chords
properly grounded (3-pronged)
temporary use of extension cords
outlet wiring: GFCI protection
power strip ratings: UL 1449, UL-TVSS, UL-SPD3
what are the requirements for motors and pumps?
covering moving belts
no leaving oil
secondary containment if near sink or drain
what are the sharps policy?
sharps must go into approved sharps container
considered full at ¾ level
autoclave prior to having picked up by vendor
plastic “sharps” & glass
what is the emergency procedure in case of a fire?
if you can use a fire extinguisher then it should be used
if not, pull the fire alarm and evacuate to nearest reasonable exit
keeps the exits clear
keep fire doors closed to contain the fire only in the laboratory
what are the top safety topics for laboratories?
substitution of elimination of hazardous agents, waste minimization
administrative controls
personal protective equipment (PPE)
engineering controls
safety equipment
housekeeping
what to do in an emergency
what are some methods of substitution of elimination of hazardous agents, waste minimization?
no radioactive materials, toxins, highly pathogenic biological materials
ethidium bromide users have seen a push to use safer products such as Sybrsafe and Gel Red
despite efforts to reduce risk, labs still must handle chemicals that are caustic, flammable, mutagenic or irritant
what are some methods for administrative controls?
Training on safety practices and chemical waste disposal and commitment to follow the rules. For example, do not work alone
Don appropriate lab coat, goggles/safety glasses, nitrile gloves, closed-toe shoes and pants/scrubs
Set the example, others observe your behavior
No eating, no drinking or application of cosmetics/contact lenses. Some volatile chemicals may dry out contact lenses; recommendation that you wear your glasses in the lab setting or on the ready
No admittance of students to lab session who fail to comply with rules.
what are some methods for PPE?
Primary factor for users to continue wearing PPE is the FIT!
Check to see if gloves are appropriate for intended use: chemicals or dry ice.
what are some methods for engineering controls?
Laminar air flow units, Chemical fume hoods or local exhaust (snorkels)
Room design, negative pressure with respect to the hallway
Process enclosure, dilution ventilation
what are some examples for safety equipment?
Location of eye washes and deluge shower
Fire extinguishers
Telephone/fire alarm
what are some methods for housekeeping?
Number one reason for accidents in laboratories: Trips, Slips and Falls
Keep aisles clear of clutter; do not place chemical waste containers on the floor in traffic areas.
Label chemical waste containers and no pouring of waste chemicals down the sink.
Caution with open flame use, Bunsen burners, smell of burnt hair, keep control of long hair
what are some methods to be prepared for an emergency?
Keep important contact numbers nearby, UGA emergencies dial 911 to get Campus Police.
Alarm to evacuate: secure materials, turn off flames, equipment, grab personal belonging and leave in an orderly manner via the nearest building exit. Go to planned rendezvous point.
Before you commence your duties and you have questions about Safety call the Office of Research Safety, 8AM-5PM, M-F, at 706-542-5288.
what are 10 things wrong with this image?
funnel is open
unknown stain on the carpet
bins are not labeled
there is spilled unknowns on the outside of the bin
idk the rest
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