labs Flashcards
define spectrophotometry
used to measure conc of specific substances w/i body fluids
how does spectrophotometry work
EM radiation of particular freq passed through sample
under ordinary conditions, atoms exist in ground energy state and via incident radiation, e-s in outer shell jump to higher energy level. Each jump is unique and requires absorption of specific amount of energy
Transmitted radiation analyzed in terms of intensity at different frequencies by showing the relationship between wavelength of radiation stored and stimulated transmission on absorption spectrum
why is acid erythrogram used
to determine historical changes in number of cells that are hemolyzed under influence of weak acid like HCl
how to study acid hemolysis
the H+ from HCl will penetrate PM of erythrocytes via protonation of Hb and protein membranes = osmotic destruction
We use spectophotometry to find extinction at time intervals to find change in extinction and % hemolysisi
acid erythrogram of a healthy individual
left = older cells that 1st undergo hemolysis since they have low diameter/area/vol right= young cells that undergo lysis last and have high sphericity index
define hemosomes
liposomes containing entrapped Hb
describe liposomes
stable, closed systems, used to administer different drugs:
unilamellar liposomes fuse their membranes w/ target cell membrane and introduce contents directly into cytosol
multilamellar liposomes fuse w/ plasmalemma of target cells so liposomes inject content w/o outermost bilayer
substances can be taken up by adsorption as liposomes attach to cell surfaces via electrostatic attraction
preparation of hemosomes
- lipids dissolved and mixed in solvent of chloroform:methanol to a homogenous mixture of lipids
- solvent is removed by evaporation via dry nitrogen steam for small vol of solvent, for larger vol, at reduced pressure via rotary evaporation = this forms thin lipid film on sides of flask
- lipid film dried to remove residual solvent in vacuum pump overnight
- dry lipid film hydrated by adding buffer w/ water
- spin flask in warm water bath to hydrate lipid, temp must be above phase transition temp
- 1 hr hydration time = vigorous shaking, mixing, stirring = lipids self organized into closed vesicles as small amounts of solvent is entrapped
define molecular chromatography
separates and purifies components according to size and volume in mixture of molecules containing 2 phases, immobile and mobile phase
separation is based on ability of sample molecules to penetrate highly porous bead-like structure of stationary phase
define thin layer chromatography
used to separate non-volatile/dissolved substances by their migration.
stationary phase as silca/alumina gel applies to glass in thin later, gypsum (inert binder) added to maintain phase
mobile phase = suitable mix of solvents like acetic acid or alcohol
Kd
distribution coefficient sample conc in phase 1/sample cone in phase 2 = V1/V2
Rf
Retardation factor, distance run by solute/distance run by solvent front
electrophoresis
movement of charged particles relative to liquid it’s suspended in under influence of electric field
microelectrophoresis
a type of electrophoresis using zeta potential since it is more accessible than surface and stern potential
zeta potential
potential at surface of shear plane between particle w/ ion cloud and surrounding medium
hemodialysis
life-sustaining procedure for patients w/ end-stage renal disease
extra-corpeal process, filters waste products by removing extra fluid from blood, restores proper electrolyte balance and eliminates edema from body
hemodialysis circuit
hemodialyses, dialysate fluid, permanent vascular access
artificial membrane tube immersed in large vol of dialysate, blood pumped through tubing and back into vein. The membrane only allows blood solutes to pass out, no proteins or cells
hemodialysis fluid flows via
countercurrent flow, opposite to blood flow, to maintain conc gradient of solutes for max efficiency
ultrafiltration
hemodialysis fluid removal from blood by altering hydrostatic pressure of dialysate fluid
t0.5
time from which dialysis onset at which initial sol conc of urea decreases 2 fold, prescribes the adequate does of dialysis
describe Cu
trace element, co-factor for enzymes EG cytochrome oxidase, Cu-Zn-superoxide dismutase
is Cu good or bad for the body
can be bad, since an XS of Cu leads to Wilson’s disease and pathogenesis of Alzheimer’s
Cuprous ion participates in formation of ROS = damages lipids, NA, proteins and carbohydrates
Cu+ also leads to oxidative injury by and produces cupric ions and .OH to induce oxidaton of LDL/HDL by Cu+ = promotes atherosclerosis
how can we study Cu generation in membranes
during oxidative injury cuprous ions + hydrogen peroxide produce cupric ions and .OH.
We can add adrenaline as it can measure superoxide anion radical formation = oxidizes to adrenochrome = colour = so we use spectrophotometry
how to determine catalytic activity of Fe and Pb in lipid peroxidation of guinea pig liver
Fe involved in FR mediated oxidations = Haber-Weiss reactions that will produce SOR involved in LP
MDA is end product of LP, adding TBA/Thiobarbituric acid = will produce a colour= measure the extinction using spectophotometry
define optical activity
ability of substances to rotate plane of polarized light passing through
how to study acid-catalysed sucrose hydrolysis
sucrose will be hydrolysed to sucrase, since all sugars are optically active, we measure this reaction using polarimetry to accurately study sucrose inversion by observing angle of rotation of polarised light at regular time intervals
