UV/ Vis spectroscopy Flashcards
What are the components of a UV/VIS spectrophotometer?
UV—> deuterium lamp Vis—> tungsten lamp Monochromator Optics- direct light to detector Director
How does a spectrophotometer work?
Polychromatic light enters monochromator- prism splits light into various wavelengths
Light leaves exit slit with selected wavelength and passes through sample
Sample absorbs certain intensity of light
What is a dual beam instrument?
2 cuvettes- light entering spilts into two- 1 passes through sample and 1 thought reference
Why use reference sample?
Compensated for other factors affecting abs such as solvents
How does Single beam instrument avoid detecting other factors?
Tared to 0- calibrated
What solvent is the sample dissolved in?
A solvent with minimum abs in the wavelength range used
What cuvettes are used for Vis?
Plastic
Glass
Cuvettes used for UV?
Quartz
Silica
cuvettes should be handled with which side?
From frosted side- to avoid fingers proteins being detected
Which drugs have abs in UV?
Drugs with aromatic rings and double bonds
What should be the maximum abs of a sample?
Below 1.5- need dilution otherwise
What is difference spectrometry
Taking spec of one sample- making a change and calc abs again to see how this change affects abs
What is derivative spectrophotometer?
If there is a big change- amplify it
If small change- flatten it
What is the process of luminescence?
1- excitation- electron excited to higher energy state by light abs
2- excited state lifetime- the time E stays up before returning
3- luminescence- emission of photons- E return to ground state
Why is fluorescence emitted at a diff wavelength?
As E- return to ground state- some energy lost- as energy changes so does wavelength
What does a jablonski diagram show?
Energy diagram that describes the process of photon emission
What is stokes shift?
The difference in wavelength between abs and emission
Are drugs with longer stokes shirtfront easier to understand?
YES
What is a fluorophore?
A molecule that is fluorescent
Is fluorescence more than UV for detection of analytes?
YES
What is the instrument used to measure fluorescence?
Spectrofluorometer
Components of a spectrofluorometer?
Xe lamp/ laser
Cell holders- 4 sided and transparent
Monochromator
Detector
Fluorescence spectrum x and y axis?
X axis- wavelength
Y axis- fluorescence intensity
Does sample need to be diluted and why?
YES- conc 10-100x less than abs spec
Very sensitive —> better for potent drugs
