Flashcards in Spectrophotometry and Electrophoresis Deck (19):
What is electrophoresis?
a method of analysing molecules on the basis of charge by measuring their migration in an electric field.
What is spectrophotometry?
a way of analysing molecules on the basis of their spectral properties
How is electrophoresis set up?
- Electrophoresis buffer at pH 8.6 so that Hb has an overall negative charge and will migrate towards the anode
- The cellulose acetate strip is suspended above the buffer but electrically connected by “wicks” of wet filter paper (these are plastic rods keep strip in contact with wicks)
- E.m.f = 200v, A= 20mA
How does a spectrophotometer work?
Measures the proportion of light absorbed compared with a blank, expressed as a logarithmic number known as absorbance. The measurement can be made at any wavelength, but needs to be adjusted for the wavelength required and then calibrated using a blank solution.
How is absorbance calculated?
log10(light transmitted through the blank solution/light transmitted through the test solution)
When does the maximum absorbance of the coloured solution occur?
in the region of the opposite colour
e.g. orange (max is blue)
green (max is red)
purple (max is yellow)
What does the colour of the solution tell us?
That of the remaining light which is transmitted
What colour are most proteins and why?
Most proteins are transparent to visible light, though they absorb in the ultraviolet range
Why is haemoglobin different to other proteins?
It has a haem group - binding of oxygen alters the electric resonance properties of haem changing its absorbance spectrum
What does the Beer Lambert law tell us?
Shows that the absorbance of a solution is proportional to the concentration of the
absorbing solute, and to the distance (or path length) travelled by the light through the sample.
What is the Beer Lambert law equation?
A = E * c * ℓ
A = absorbance
C= concentration of the absorbing substance (typically in mol/litre)
ℓ = path length (in cm)
E = constant (the Extinction Coefficient) for the substance being measured at that particular
How can we use the Beer Lambert law practically?
- If a graph of absorbance against concentration is drawn, unknown concentrations can be determined
- If the substrate or product of an enzyme reaction absorbs light then the method can be used to follow the course of the reaction.
Why is there a difference in the motility of HbA and HbS?
difference in charge caused by a point mutation in one amino acid of the beta chain. Glutamate (-ve) is replaced by valine (uncharged)
What will be the difference in the migration of HbA and HbS?
HbA will migrate further towards the anode as it has more negative charge
What is gel electrophoresis?
Proteins migrate through the pores of a gel.
-If the charge differences of proteins can be masked (by including a substance called SDS in the
electrophoresis buffer) the speed of migration depends only on protein size, with the smaller proteins migrating faster since they pass more easily through the pores.
Why do some substances fail to obey the Beer-Lambert Law?
At high concentrations a protein might form dimers
with a different coefficient and linearity typically needs
to be checked by constructing a standard curve.
What is methaemoglobin?
Normal O2 binding does not change the oxidation state of the iron atom. However it can become oxidised to
Fe3+, in which case the molecule is known as methaemoglobin. This is the state which gives dried blood its brown colour.
At which wavelength was maximum absorption of Hb- what colour is that?
For the haemoglobin, the wavelength at which you got maximum absorption was 540 nm