UV-VIS SPECTROPHOTOMETRY Flashcards
Is uv-vis a spectrometry, spectrophotometry, or spectroscopy
spectrophotometry
is this a qualitative or quantitative method
quantitative
what is this used for
to determine the concentration of an ion in a solution
name three industries that use this process
- clinical chemistry to study enzyme kinetic
- pharmaceuticals for dissolution texting of tablets
- biomedical and genetic to quantify DNA and protein/enzyme activity and
what is a chromophore and how does it relate to uv-vis spec
chromophores are sections in a molecule that consists of alternating single and double bonds and absorbs uv ligh and visible light.
- since they can absorb light this absorbance can be sued to identify compounds and concentrations of compounds in the molecule
list the 5 components of a uv-vis spectrophotometry
- source
- monochrometre (prism)
- beam splitter
- sample cell
- detectors
what is the function of the source
provides a stable continuous spectrum of sufficient light intensity
what is the function of the monochrometer
- aka prism
used to separate light into a range of individual wavelengths (make a rainbow) - also selects a specific wavelength to be passed through the sample
what is the function of the beam splitter
splits the wavelength into equal intensities,
- a sample beam passes through the sample
- a reference beam that passes through a reference which is often a blank solvent
what is the purpose of the reference beam
to account for any fluctuation in the light source or the instrument itself
what is the function of a sample cell
samples placed in cells or cuvettes for the light to pass through
what is the function of the detectors
- measures the intensity of light that has passed through the sample of reference
- calculate the absorption
- convert a light signal into an electric signal (current)
define ‘error’ and give example in relation to uv-vis spec
- difference between measured value and “actual” value
- types of error: systematic, random
- contamination of sample in cuvette in form of residue or fingerprints could cause absorbance that interferes with the system
define ‘uncertainty’ and give example in relation to uv-vis spec
- +/- maximum difference from the mean
- a range of values within which the true value is expected to lie
- caused due to:
– limitation of the measuring instrument (systematic error)
– the skill of the experimenter (random error)
give two examples of uncertainty in this process
1) the detector is very sensitive and any background noise could cause interference
–> cause variations in measurements introducing uncertainty into the absorbance/transmittance measurement
2) wavelength chosen by the instrument is slightly off from the true value, the absorbance measurement may not accurately
define ‘limitation’ and give example in relation to uv-vis spec
limitations are restrictions
- uv-vis spec can only be used for low conc. after a certain point the absorbance will be constant
what is the format of a calibration graph
plot absorbance over concentration (mol/L)
what is the shape of the calibration curve
linear –> Beer Lambert Law which states absorbance is proportional to concentration
what is the formula for absorbance
A = 3cl
A = absorbance
3 = molar absorption cell (different for each)
c = molar conc.
l = diameter of the cuvette in cm
what is the purpose of using a distilled water reference blank?
it’s used to calibrate the spectrophotometer
–> it tells spectrophotometer to ignore any absorbance that is caused by the solvent (water) or the cuvette material
–> Distilled water is chosen because it is typically free of impurities that could absorb light at the wavelengths you’re using in your measurement.
Other than repeating the experiment, list 4 ways reliability of the investigation can be improved?
- ensure proper calibration
- control temperature and light source
- consistent process
- use blank to eliminate impurities interference
how can temperature effect absorbance
higher temp –> molecules have more kinetic energy –> cause shift in absorption wavelength as molecules may absorb light at slightly different wavelengths due to changes in their energy states.
