Spectroscopy etc Flashcards
(21 cards)
In ultraviolet visible spectroscopy, the ratio of which of the following pairs of wavelengths provides an estimate of DNA purity?
260-280 nm
Which amino acids have useful fluorescent characteristics?
Tryptophan and Tyrosine
A 2-D (ief/SDS-PAGE) gel analysis separates proteins on the basis of:
size followed by charge
Reverse phase chromatography separates molecules based on which of the following physical properties?
boh
In HPLC (high performance liquid chromatography) the stationary phase is typically which of the following?
boh
What term is given to the difference between the excitation wavelength and emission wavelength of a fluorophore?
boh
In ultraviolet/visible spectroscopy the Beer-Lambert Law states that, for a given substance, the absorption of radiation is proportional to which of the following?
Concentration of the absorbing solution
Path length of the absorbing solution
The intensity of the lamp
Lamp of UV/ Vis and cuvette
Deuterium and tungsten
Plastic or quartz
beer lambert’s law
A= log Io/I
A=ecl
Stokes shift
l(em)- l(ex)
l(em)> l(ex)
higher wavelength means lower energy!
At what angle is fluorescence measured?
90°
lamp for fluorescence
Xenon
How many monochromator for fluorescence spec.?
2 one for emitted and one for excitation
Molecules separate because they have different…
Distribution coefficients Kd which is determined by size, charge, hydrophobicity and shape
What is gel filtration based on?
Size of molecules (larger are faster and smaller slower)
Uses of gel filtration
Desalting/buffer exchange
What is ion exchange chrome. based on ?
Charge of molecules.
Bound molecules are then collected by eluting them with increasing gradients of salts
Uses of ion exchange
Protein separation
What is normal phase based on?
Hydrophobicity of molecules which bind to polar stationary phase
What is reverse phase based on?
Hydrophobicity of molecules which bind to non-polar stationary phase
What is affinity chrom. based on?
Shape of molecules. A ligand specific to component we want to isolate is immobilised in the stationary phase. The component binds and I then washed away by a mobile phase that breaks the interaction
