Analytical techniques - UV/Vis spectroscopy

Question 1

Define the terms in the beer Lambert law

Answer 1

A = absorbance - no units
A(1%,1cm) = specific absorbance of a 1 wt% solution of a sample in a cuvette at 1cm pathlength (dL cm-1 g-1) 

c = concentration in g/100ml (same as gdL-1) 
l = path length, always 1cm

Question 2

How do we calculate a concentration of a compound using UV?

Answer 2

Using the beer Lambert law = A= A(1%,1cm) cl

c = A / A(1%,1cm) l

Question 3

Define a chromophore?

Answer 3

A functional group that absorbs UV/vis light

Question 4

what happens to chromophores in UV?

Answer 4

irrigation or organic compounds with UV light can lead to promotion of an electron from either a molecular bonding orbital or a lone pair, to another orbital (excited state). This causes incident photons at that frequency to be absorbed

Unsaturated residues have HOMO/LUMO energy separations which correspond to the energy of photons in the UV/Vis region of the spectrum

Question 5

Define an auxochrome

Answer 5

modify the ability of the chromophore to absorb light, whilst not contributing significantly to absorption themselves (i.e. doesn’t absorb light)

Question 6

when do we determine the pKa of the ionisable group?

Answer 6

where a pH dependent UV shift is produced we use it to determine the pKa is the ionisable group responsible for the shift

Question 7

what Is the PKa equation?

Answer 7

PKa = pH + log Ai - A / A - Au

Question 8

Define the terms in the PKa equation

Answer 8

A = measured absorbance of the drug in a buffered solution of known pH at a specific wavelength

Ai = absorbance of fully ionised drug
Au = absorbance of un-ionised drug

Question 9

what happens in the rare case that the ionisation process leads to a decrease in absorbance or a shift to lower the wavelengths?

Answer 9

Log is subtracted from pH when calculating pKa -

pKa = pH - log Ai - A / A - Au

Question 10

how is UV/vis calibrated? (general)

Answer 10

1. absorbance scale
2. wavelength scale
3. resolution of instrument
4. assessment of optics

Question 11

describe absorbance scale calibration

Answer 11

uses potassium dichromate solution at select wavelengths

Question 12

Describe wavelength scale calibration

Answer 12

precision of a spectrometers output is assessed by analysing the specific wavelength maxima of a 5% w/v holmium perchlorate solution

Question 13

Describe resolution of instrument calibration

Answer 13

assessed using a 0.02% toluene in hexane - BP requires that the ratio of the ABS at 269nm to that recorded at 266nm is roughly 1.5

Question 14

Describe assessment of optics

Answer 14

this relates to the state of instrument and amount of stray light - stray light affords false ABS readings.
BP states the use of 1.3% KCl solution assessed at 200nm - sample absorbance must be <2

Question 15

advantages of UV/vis?

Answer 15

inexpensive and robust technique for analysing solutions
amenable to automation
don’t need extensive training to carry out
can be coupled readily to other separative techniques

Question 16

Disadvantages

Answer 16

limited selectivity as a result of multiple chromophores.

analysis of complex mixtures is not always viable