Spectroscopy Flashcards
What is spectroscopy?
The study of the interaction of matter with electromagnetic radiation
What is absorption spectroscopy?
Light absorbed by atoms/molecules increases their energy and is perceived as colours on the EM spectrum is affected by the solvent/environment
What is meant by bathochromic?
A shift to a longer wavelength
What is meant by hypsochromic?
A shift to a shorter wavelength
What is the Beer-Lambert-Bouguer law?
The absorptivity of a substance is equal to the molar attenuation coefficient x the optical path length x the concentration of the attenuating species
What are the key assumptions of the B-L-B law?
Monochromatic/parallel illumination Homogenous solution No scatter from medium Linear absorbance change with concentration Linear response of the detector
What are the applications of spectroscopy?
Quantification of proteins/nucleic acids Protein/nucleic acid unfolding Characterisation of substrates/cofactors Binding to proteins Biochemical assays
Describe the quantification of proteins
A direct measurement A280
Tryptophan/tyrosine/dislfides absorb UV light at around 280nm wavelength
Describe the biuret method?
React protein with copper sulfate/ NaOH/ potassium tartrate and the colour changes to violet at 540nm wavelength
Describe the BCA assay method?
Cu is bound to the N in the protein
The complex is bound to bicinchronic acid
A purple colour change
Is very sensitive to N contaminants
What wavelength does pure RNA/DNA absorb?
260nm
