Spectrophotometry Flashcards
what does spectroscopy mean?
use light to probe matter - how a sample absorbs light
what is the set up of absorbance spectroscopy?
power in with certain wavelength with photons of light enter sample and sample has molecules that will absorb that wavelength and unabsobed light is transmitted (power out)
how does concentration sample relate to power out/transmitted light?
high conc = lower transmittence bc more light absorbed by sample
how do you calculate % transmittence?
[power (out)/ power (in)] x 100
how do you calculate absorbance?
A = 2 - log(%T)
what is absorbance spectrum?
graph that shows absorbance vs wavelength of light
different for each molecule
shows you what wavelength is best absorbed by the molecule
relationship between transmittance and absorbance?
inverse
parts if a spectrophotometer?
light source-> slit (select a band) -> monochrmator (separate white light) -> slit (to select 1 color) -> sample -> detector
spec 20 vs advanced spectrophotometer?
spec 20 - large volume required, manually set 0% transmittance, blank in sample holder (test tube vs cuvette)
both 1 sample at a time
characteristics of a blank?
contain everything besides substance you are trying to measure (DI water)
reblank everytime the wavelength is changed
how to make absorbance spectrum with spec 20?
measure absorbance at every wavelength (blank each time) with the same sample
difference between spec 20 and microplate reader?
reads multiple samples at once, small samples
load as triplicate (this is a technical control!)
samples for standard curve? making standard curve?
standard with unknown conc serial dilution with standard of known conc
put each sample to microplate or test tube, measure absorbance at each wavelength
graph wavelengths, line of best fit (R^2 tells you how accurate line of best fit is)
colorometric assay>
used if lamba max can’t be measured directly by spec, add color which can be measured
