Spectrophotometry (Experiment 3.1-3.4) Flashcards
Spectrophotometry
is the process that determined the amount of light absorbed by colored compounds.
What is the absorption of light due to in the spectrophotometer?
the absorption of light is due to the interaction of light with the electronic and vibrational modes of molecules (their chemical bonds).
What are the limitations of Spectronic 20?
- does not permit reliable readings much below A = 0.05 or above A = 0.9.
- Bandwidth of monochromator is quite broad.
Beer Lambert Equation (different forms)
A = -log10 (T) = Ecl = OD
There is an exponential __________ of temperature with respect to concentration.
decrease
There is a linear relationship between absorbance and concentration at ________ concentrations, at __________ there is a deviation.
There is a linear relationship between absorbance and concentration at low concentrations, at high concentration, there is a deviation.
There is a linear relationship between absorbance and concentration at ________ concentrations, at __________ there is a deviation.
There is a linear relationship between absorbance and concentration at low concentrations, at high concentration, there is a deviation.
What is the isobestic point?
No changes in absorbance occur, system in equilibrium.
Does acidic PNP absorb?
No
Does basic PNP absorb?
Yes
How do we determine the pKa of a compound based on absorbance?
pH at Amax/2
Acid dissociation constant
Ka = [H+][A-]/[HA]
Henderson-Hasselbach Equation
pH = pKa + log [A-]/[HA]
Henderson-Hasselbach Equation
pH = pKa + log [A-]/[HA]
How do we determine concentration of unknown PNP?
Optical Assay of PNP
y = mx
y = absorbance at 400 nm
m = slope
x = concentration of PNP
