BE 450 PPT 8 Chemical and Biological Properties - Exam 2

spectroscopy, chromatography

characterization of biomaterial properties relating to the chemical composition can be accomplished through what 2 techniques?

spectroscopy

measure the interaction between light and matter

chromatography

molecules are physically separated on the basis of chemical characteristics (charge or MW)

collimator

red

wavelength selector

green

monochromator

blue

sample solution

orange

detector

purple

energies

spectroscopy: tune input light to evaluate specific attributes of sample based on spectrum of _____________

detector

spectroscopy: tune output ___________ to determine specified characteristics

frequency, wavelength

higher energies result in higher _____________ and shorter _____________

orbitals, matches

UV-VIS light can push electrons to higher energy _________ only when energy ____________ the energy difference between orbitals

absorbed

UV-VIS: when electrons are pushed to higher energy orbitals, that energy is ____________ by the compound

wavelengths

different compounds will absorb different _______________

light

UV-VIS measurements: expose sample to increasing wavelengths of __________

absorbance

UV-VIS measurement: capture the ________________ of light (done by detecting the amount of transmitted light)

peaks

UV-VIS measurement: ________ correspond to specific chemical compounds

UV-VIS measurement

can also be useful to quantify the concentration of a specific compound

beer-lambert's law

equation name?

A

beer-lambert's law: absorbance

E

beer-lambert's law: molar attenuation coefficient

c

beer-lambert's law: concentration

l

beer-lambert's law: optical pathlength

protein

BCA assay quantifies __________ content

infrared

type of spectroscopy? radiation of wavelengths of 0.78-1000nm

ball and spring model

represents a bond between 2 atoms, single, double, and triple bonds have characteristic vibrational behavior

detection

infrared (IR) spectroscopy: different bonds will absorb specific wavelengths of light, allowing for ____________

nuclear magnetic resonance (NMR)

uses radiation in the radio-frequency region (0.5-75nm) to excite molecules

nucleus

NMR: induces changes in the _________ rather than electrons

transitions

NMR: the presence of a strong magnetic field is needed to observe the nuclear ____________

positively

nuclei are _____________ charged

magnetic

nuclei spin creates a small __________ field

2

nuclei have ___ orientations to the spin

unequal

nuclei must have _________ numbers of protons and neutrons to produce a signal in NMR analysis

spin

NMR: when protons are placed in a larger, external magnetic field, H0, they ______ one of two ways

favorable

NMR: proton in magnetic field spinning the same direction as the field is energetically ________________

unfavorable

NMR: proton in magnetic field spinning the opposite direction as the field is energetically ________________

lower

NMR: proton in magnetic field spinning the same direction as the field has a _________ energy state (alpha)

higher

NMR: proton in magnetic field spinning the opposite direction as the field has a _________ energy state (beta)

hv

the difference in energy between these 2 states is

increases

the difference in these 2 energy states ___________ with increasing strength of the magnetic field

resonance frequency

proton spin will flip from alpha to beta, or vice versa, if exposed to radio energy of frequency (n) ____________ _____________

energy

when exposed to the same magnetic field (H0), various molecules require different amounts of ___________ to resonate

local environment

the resonance frequency depends strongly on the ______ _____________ of each nucleus

compound

the local environment changes depending on the chemical ____________

electrons

energy dispersive X-ray spectroscopy: X-ray ejects ___________

characteristic energy

higher energy electrons fill ejected electron position, leading to emission of ___________________ __________

elemental

energy dispersive X-ray spectroscopy: ____________ information gathered about material

high performance liquid chromatography

provides information based on separation of substances by chemical properties (size or charge)

size-exclusion chromatography

high-performance liquid chromatography: SEC meaning?

gel permeation chromatography

size-exclusion chromatography (SEC): GPC meaning

gel filtration chromatography

size-exclusion chromatography (SEC): GFC meaning

refractive

size-exclusion chromatography (SEC): eluted compounds change the ___________ index of solvent

mass spectroscopy

does NOT involve measurement of absorption of electromagnetic radiation

mass spectroscopy

determines atomic and molecular masses of various species

high energy

mass spectroscopy: first the sample is ionized through bombardment with _____ _________ particles (usually electrons)

magnetic field

mass spectroscopy: charged species are forced through a ____________ ________

linear

mass spectroscopy: interaction deflects species from a ________ path

more

mass spectroscopy: lighter species deflected _______ than heavier ones

mass

mass spectroscopy: species are separated by differences in ______

isotopes

mass spectroscopy: extremely sensitive to difference between __________ of the same element

inorganic, organic

information given from mass spec: qualitative and quantitative assessment of ____________ and ___________ molecules

polymers

information given from mass spec: primarily used for natural and synthetic ___________

isotopes

information given from mass spec: used to identify ____________ ratios within a substance

bonds

information given from mass spec: can provide information on strength of various _______ in a molecule

exposure, delivered

two types of biocompatibility testing considerations

exposure (in vivo tests)

amount the while animal/person is exposed to (inhaled, skin exposure, swallowed, injected, or ingested)

delivered dose (in vitro)

amount arriving at the cell: absorption > circulation and distribution > delivery to organs > delivery to cells

toxicity

cell death through inhibition of key metabolic pathways

in vitro

(cell exposure tests) target cell doses using delivered dosage

cytotoxicity assays

screening tool for raw materials or component products before they are put into the design of a medical device

direct contact, agar diffusion, elution

three types of cytotoxicity assays

direct contact

confluent monolayer + sample > 24 hr > assay

agar diffusion

agar added above culture + sample > 1-3 days > assay

elution

extract of the material using PBS or media added to culture > evaluate after 24 hr > stain for viability

leachable

elution: determines whether a product or compound will have any toxic effect due to _______________ on living cells

cell

evaluate effect through: ______ cytotoxicity and behavior

cell behavior

adhesion, spreading, migration, invasion, changes in mRNA and protein

Calcein-AM

live/dead stain: enters cytoplasm and is cleaved by esterases (only present in living cells) > cleavage leads to green fluorescence

ethidium homodimer

live/dead stain: enters nucleus of compromised (dead) cells, intercalating agent, fluoresces red

vital stain

taken up and retained by healthy cells (dead cells appear colorless)

MTT assay

cytotoxicity assessment assay - indication of mitochondrial activity

adhesion and spreading

cell behavior?

cell mobility

cell behavior?

migration assay (Boyden chamber)

cell behavior?

scratch wound assay

cell behavior?

phenotype

toxins can alter the cell ________________, which can be determined through DNA/RNA, Protein Assays

DNA/RNA

type of assay? PCR/qPCR

DNA/RNA

type of assay? micro-array testing

protein

type of assay? western blotting

protein

type of assay? immuno-staining

protein

type of assay? ELISA

polymerase chain reaction (PCR)

quick, easy method for generating unlimited copies of any fragment of DNA

denaturation, annealing, extension

3 major steps in PCR repeated in 30 or 40 cycles?

denaturation

open strands, stops enzymatic actions > ready to copy (94-95C)

annealing

using polymerase (enzyme which assembles DNA base pairs) using primers (base pairs which fit exactly) (54-60C)

extension

ideal working temperature for polymerase, bases are added complementary to the template from 5' to 3' (68-74C)

gene expression microarrays

to determine which genes are suppressed and which are turned on after a treatment

gene expression microarrays

based on tracing mRNA, a chip made from an array of DNA sequences corresponding to known gene

electrophoresis

western blotting - ____________________