Spectrophotometry, Fluoro/Nephelo/Turbimetry

transmittance equation

T = Is / I0 (Is < I0)

absorbance equation

A = 2 - log(%T)

Beer’s law is

concentration of a substance is directly proportional (must be linear) to the amount of light absorbed or inversely prop to the log of the transmitted light

Beer’s law equation

A = abc

a = constant

b = light path (cm)
c = conc of absorbing cmpd (g/L)

if b = 1cm, c is in mol/L → a becomes E

calibration constant (K)

cu = Au*K

plug in from standard curve, a conc and absorbance reading to get K

spec light sources

incandescent lamps

• tungsten: visible light

• hydrogen & deuterium lamps: UV

laser

monochromator function

isolates radiant energy of desired wavelength

types:

• filters

• prisms

• diffraction gratings

prism vs grating vs filter

sep white light into continuous spectrum by refraction

diffraction grating: thin layer of alloy on flat glass plate

holographic grating: v accurate

filter: wide/narrow bandpass, sharp cutoff

fiber optics

aka light pipes

transmit light throughout their lengths by internal reflections

photodetectors

converts light into an electric signal

prop to photons striking detector

photodiode array

2-D arrays of diodes

allows 2nm resolution per diode from 200-340nm

allows a 1nm resolution per diode from 340-800nm

single beam spec

beam of light passes through a monochromator

→ ligth passes through cuvette

→ any light not absorbed is transmitted to a detector

→ detector converts light energy to electrical energy

→ electrical energy registered on a meter

double beam in space spec

all components are x2 except for light source

double beam in-time

uses a light beam chopper inserted after exit slit → mirrors → onto common detector

**compensates for a light source variation & sensitivity changes of detector

fluorometry

measurement of visible spectrum

light source → light absorbed → electrons excited → energy re-emitted at longer wavelength (lower energy) → electron returns to ground state

Stoke’s shift

a constant that is difference in E or wl b/t max excitation & fluor emission

fluorescence polarization

smaller → faster rotation → light depolarized

bigger when bound → slower → polarized

nephelometry

meas of light scattered produced by soluble immune complexes suspended in solution at right angles to the incident beam

• depends on: particle size, wavelength, concentration of particles,

light scatter detected at 90 degree angle

target concentration & light scatter are directly prop

turbidimetry

light transmit detected at 0 or 180 degrees

light transmitted dec => analyte conc inc → inversely prop

nephelo/turbidimetry clinical applications

immunoglobulins

proteins

drugs

nephelo/turb limits

meph more sensitive

antigen xs (post-zone)

lipemia & hemolysis