Spectroscopy Flashcards
What is the difference between spectroscopy, spectrometry and spectrophotometry?
Spectroscopy → the study of the interaction of matter with light ie. electromagnetic radiation
Spectrometry → the method used to acquire a spectrum
Spectrophotometry → spectrometry in the UV-visible-near IR range
What is spectrophotometry used for?
Analysis of materials
Quantitation of components including in complex mixtures eg. determine protein structure
Can be time-resolved eg. to monitor rates of changes of reactants and products
How does spectroscopy work?
Photons in the UV/visible/near IR cause electrons to be excited from one orbital to another one of a higher energy; this is called an electronic transition.
This can only occur if the energy of the absorbed photon is equal to the energy required to move the electrons between the orbitals.
This results in bands of absorptions at specific wavelengths. The wavelengths, shapes and intensities of the bands of absorptions are characteristic of the absorbing substance.
What are the basic components of a spectrophotometer?
Light source
Entrance slit
Dispersion device such as a prism or diffraction grating to separate the different wavelengths of light
Exit slit - a narrow slit to select a single wavelength
A cuvette containing the sample
A detector to measure the light intensity
How does a prism and a diffraction grating separate light?
Prism → A triangular glass block in which different wavelengths of light will travel at different speeds. The light will disperse into the colours of the visible spectrum, with longer wavelengths (red, yellow) being refracted less than shorter wavelengths (blue, violet)
Prisms are highly efficient over the visible range and transmit 100% - however, prisms do not give a linear dispersion
Diffraction grating → A surface with closely spaced grooves on it. The periodic structure splits and diffracts light into several beams, but the groove period must be on the order of the wavelength of interest. The spectral range covered by a grating is dependent upon the groove spacing.
Wavelength dispersion is essentially constant, but it gives a lower transmittance.
How does the detector measure light intensity?
The detector is usually a photomultiplier or a photodiode.
These convert streams of photons into streams of electrons so converted light intensity into an electrical current which can then be measured.
What is the Beer-Lambert law?
A = εcl = log[I0/I]
Where
A = unitless
ε = molar extinction coefficient (M-1cm-1)
c = concentration of absorbing species (M)
l - pathlength (usually 1cm)
I0 = incident light intensity
I = exit light intensity
Why are standard curves often used to measure unknown concentrations?
Plots of absorbance against concentration can deviate from a straight line because of factors such as stray light.
Atrue = log[I0/I]
Ameasured = log[[I0+s][I+s]]
As absorbance increases the deviation caused by I+s becomes greater, so Ameasured becomes increasingly less than Atrue.
If a measurement is required over a wide range of concentrations, a standard curve is usually plotted alongside the experimental data. The absorbance of the unknown sample and its concentration can then be read from the standard curve.
What is an isosbestic point and what can it be used for?
Isosbestic point → a specific wavelength at which two chemical species have the same molar absorptivity.
The absorbance at the isosbestic point will remain constant regardless of the ratio of the two components.
The absorbance at this point can be used to quantitate the total amount of the two species combined, but it cannot be used to determine the individual concentrations.
How can you determine the concentrations of a two component mixture?
The extinction coefficients of each component must be known at two different wavelengths. They must have a different extinction coefficient at either one or both of these wavelengths.
A pair of simultaneous equations with two unknowns (the concentration of each species) can be derived from this.
For a 3 component mixture, 3 simultaneous equations would be needed, and so on.
How can rates of reactions be followed using spectrophotometry?
If substrates and products have different spectra then the reaction can be followed spectrophotometrically.
The rate of reaction can be monitored by plotting absorbance at a certain wavelength (the absorbance of which different between the reactants and products) against time.
What is fluorescence?
A molecule (fluorophore) absorbs light and this causes an electron to be excited to a higher state.
Some of this energy is released as heat, relaxing the electron down to a lower vibronic state.
The rest of the energy is emitted as light, and the electron relaxes back down to its original ground state.
The light emitted is always at a lower energy than the light absorbed, meaning that the emitted light has a longer wavelength.
Why are fluorescent methods extremely sensitive?
They are extremely sensitive because there is a very low background signal.
The technique also has the ability to detect small numbers of emitted photons.
What is the difference between intrinsic and extrinsic fluorescence?
Intrinsic → molecules that contain groups which have fluorescent properties
eg. proteins that contain tryptophan can be fluorescent
Extrinsic → molecules that are made fluorescent by adding fluorophores
What is GFP and what can it be used for?
GFP → Green Fluorescent Protein
It is a naturally occuring fluorescent protein originally isolated from jellyfish.
It can be attached to proteins of interest, so that when the gene is expressed GFP is covalently bonded to it.
GFP spontaneously folds into its active flouorescent form, allowing the protein of interest to be located.
GFP can be mutated to flouresce at different wavelengths and show different colours.
