Analytical Methods: Spectrophotometry

400-700 nm

visible spectrum

<400 nm

UV region

>700 nm

infrared region

• wavelength inversely proportional to frequency

• frequency directly proportional to energy

rel. between wavelength, frequency, and energy

• ROYGBV

• MIUXGC

longest wavelength → shortest wavelength in electromagnetic spectrum

spectrophotometry

measure of light transmitted by a solution

1. light / radiant source — source of polychromatic light

2. entrance slit — minimizes stray light

3. monochromator — isolates specific wavelength of light

4. exit slit — controls bandwidth / bandpass

5. cuvet / absorption cell / analytical cell / sample cell — holds the soln.

6. photodector — detects and converts transmitted light → electricity

7. meter / read out device → displays output

parts of spectrophotometry

Tungsten light bulb/incandescent tungsten/tungsten iodide lamp

most commonly used light / radiant source

hollow cathode lamp

light / radiant source in AAS

deuterium discharge lamp and the mercury arc lamp

most common UV light / radiant source

stray light

most common cause of loss of linearity

second order spectra

• Deteriorated optics

• Light dispersed by a darkened enveloped

• Extraneous room light

most common cause of stray light

absorbance error

major effect of stray light

sharp cutoff filter

filter best for measuring stray light

Neutral Density filter and Dichromate sol'n

verify absorbance accuracy on linearity

grating

most commonly used monochromator

prism

produces purest light in UV range

• Didymium or holmium oxide filter

• mercury vapor lamp

used for quality control of monochromator

square cuvette

cuvette has more advantage

discard

scratched optical surfaces in cuvette → ???

alumina silica

most commonly used cuvet

glass cuvets

cuvets for visible range BUT absorbs UV

quartz

cuvets for UV radiation

Beer’s law

states that unknown subs.

• directly proportional to absorbed light

• inversely proportional to transmitted light

Cu = Au/As x CS

• Cu = conc. of unknown

• Au = absorbance of uknown

• As = absorbance of standard

• Cs = conc. of unknown

concentration of unknown: Beer’s law

Lambert law

absorbance increases exponentially w/ an increase in light path (direct relationship)

photomultiplier tube

most common type of photodetector → excellent sensitivity (never expose to light → burn out)

barrier layer cell / photocell / photovoltaic cell

simplest detector (requires no external voltage source)

photodiode

photodetector that respond to a specific wavelength → UV visible

phototube

Same as photocell, differs in that an outside voltage is required for operatio

reagent blank

blanking technique → corrects for absorbance caused by COLOR OF THE REAGENT

0 Abs (100 transmission)

in reagent blank technique, adjust spectro to ???

sample blank

blanking technique → corrects for interfering chromogens / lipemia

ultracentrifuge

used to clear serum or plasma chylomicrons

sample blank

blanking technique → subtract intrinsic absorbance of the sample caused by hemolysis, icterus, turbidity, drug interference

saline (sample + saline)

sample blank: substitute ___ for reagent

single beam spectrophotometer

simplest type of spectrophotometer: 1 measurement at a time

double beam spectrophotometer

spectrophotometer permit automatic correction of sample & reference absorbance

double beam in space

double beam spectrophotometer: use 2 photodetectors

double beam in time

double beam spectrophotometer: one photodector & alternately passed the monochromatic light using chopper