MAAC: Analytical Spectrophometry 2

Question 1

What can UV/Vis absorption be used for in relation to drug analysis?

Answer 1

Quantification of drugs in formulations (without excipient interferences):

–Determine pKa of some drugs

–Determine partition coefficients and solubilities of drugs

–Determine release of drugs from formulations with time (dissolution testing)

–Monitor reaction kinetics of drug degradation

–UV spectrum used as one of the pharmacopoeial identity checks

Question 2

What happens when polychromatic light passes through a sample?

Why is this?

Answer 2

The intensity decreases due to 3 processes:

1. Reflection at phase boundaries, i.e. liquid/air, glass/liquid etc. This is caused by differences in the refractive index of the different materials through which the light is passing.
2. Scattering of light caused by non-homogeneity of the sample.
3. Absorbance by atoms or molecules in solution.

Question 3

How do you prevent these?

Answer 3

1. Use matched pair cuvettes (Cuvettes normally come in pairs) for the reference and sample. This way the cuvette is the exact same material for both so refractive indexes for these are both taken into account. The use of the same solvents in both refence and sample (bar drug) takes into account the effect of the liquid on the reflection of light too.
2. Sample may not be completely dissolved therefore particles remain, air bubbles in cuvette, finger prints on cuvette. These will scatter light away from the detector and it will be assumed the light has been absorbed and hence give incorrect reading. To remove airbubbles the cuvette can be tapped on the table and the bubble with float to the top. To remove particles the solution must be filtered using fine filter paper. Cuvettes must be made sure they are clean inside and outside and when handling them should be held at the top corner or edges.

Question 4

What wavelength is selected for the analysis?

Answer 4

•Usually use the wavelength corresponding to the absorption peak (λmax)

–Scan sample over region of interest and note the wavelength at highest absorption

–Can also use British Pharmacopoeia to find λmax of a particular drug or clarks if not medicinal sample

Wavelength max gives greatest sensitivity and peaks tend to be fairly flat

Question 5

The following gives a reminder of molecular orbitals.

Things to remember, the larger the change in energy, the shorter the wavelength and larger the frequency

Question 6

What is Beer’s Law and what is Lambert’s Law?

What is the equation that combines them?

Answer 6

1. Lambert - Exponential decrease in light intensity as Path length increases
2. Beer’s - Exponential decrease in light intensity as concentration increases

A= log I₀/I = kcl

A= Absorbance

k= constant

c= concentation

l = Path length

Question 7

What is the relationship between A, L and C

Answer 7

A is directionally proportional to L

A is directionally proportional to C

Question 8

the Absorbance constant can be expressed in 2 different ways depending on the units of concentration.

When are is molar absorpitivity constant and specific absorbance used?

Answer 8

1. If the units of concentration are molarity (i.e. number of moles per litre), constant is ε (the Greek letter epsilon) and is known as the molar absorptivity.

–ε has units of L mol -1 cm -1 although the units are seldom expressed.

–ε is equal to the absorbance of a 1 M solution in a cell of path length 1 cm

1. if the concentration of the sample is expressed in percentage weight by volume (% w/v), then the constant used is A 1%, 1 cm usually written as A11

–% w/v = 1 g in 100 mL

–Called the specific absorbance, with units of dL g-1 cm-1

dL = decilitre (1/10 L) = 100 mL

Question 9

When can the A11 valvue in beers law be useful?

Answer 9

•The A11 value is useful in pharmaceutical analyses where the molecular weight of the sample may be unknown

–e.g. when analysing a protein or DNA, or

–where a mixture of several components is being analysed in the same sample (e.g. an ointment or suspension).

Question 10

What equation relates A11 and E (episilon)?

Answer 10

E = A11 x Mr / 100

Question 11

What are examples where there may be deviations from Beer’s Law of A is linear with C?

Answer 11

1. Instumental:
   
   • The law is obeyed when monochromatic light (e.g. lazer) is used. A lot of the time polychromatic light is used and a monchromatic used to pick a particular wavelength. This therefore means there is a slight deviation in the results.
   • Stray light in the instrument at high absorbances can lead to underreporting value of A (and curve in graph). Stray light is ight not suppose to reach the detector that may bounce of the multiple opti in the instrument. Above a value of 3 this becomes a very big factor there for low Absorbances sould be worked at
2. Chemical Deviations:
   
   • Also arise since ε depends on refractive index, n, of the medium.
     • ε is only constant for the absorbing molecule or ion in a particular solvent at a particular wavelength
   • pH causes the greatest deviation
     • Some chromophores will shift wavelength with a change in pH
3. Concentration affects
   
   • Beer’s law describes the absorption behaviour of dilute solutions only
   • Below 10 mM, concentration effects not usually observed
   • Above 10 mM the close distance between absorbing molecules can alter ability to absorb a given λ

Question 12

1. How do you stop pH effects?
2. How do you stop concentations effects?

Answer 12

1. pH effects can be overcome in 2 ways:
   
   • All solutions are buffered
   • Take measurements at the isosbestic point (the point where the 2 overlapping curves intersect) as opposed to lamda max of both as they share a common point.
2. If graph starts to curve due to conc, dilute sample further

Question 13

What are the 2 methods used when using spectroscopic measurements in drug analysis?

They are different in the US and EU

Answer 13

Comparative (US):

• The sample concentration is worked out if you have the concentration of the standard
• Absorbance Test/ A Std = [Test]/ [std]
• Adv - Can be used even if drug undergoes chemical reaction during the assay
• Dis - Need authentic sample of drug in question for comparision (expensive)

Absolute (EU)

• Absorbance measured and BL equation used to calculate [Drug]
• Range of standard solutions created and absorbance of each measured. Calibration curve created and unknown worked out using ththis

Question 14

Why should at least one other analysis method be used with Lamda max to determine the drug?

Answer 14

• Lamda max is characterisitic of a particular chromophore - not a particular drug. Therefore if 2 drugs have a similar structure and contain the same chromophores these can be the same.
• Lamda max can move position as the drug is ionised

Question 15

What is a chromophore?

What is an auxochrome?

Answer 15

1. The part of the molecule responsible for absorbing light - conjugated part.
2. This is a functional group attached to the chromophore which does not absorb light energy itself, but which influences the wavelengths of light absorbed by the chromophore.

Question 16

What is it called when an auxochrome causes a shift to longer wavelength?

Answer 16

Bathochromic effect or red shift

Question 17

What are examples of auxochromes and what do they require? What does their presence do?

Answer 17

• Examples include the NH2, OH and SH groups.
• Possess lone pairs of non-bonded electrons which can interact with the p electrons of the chromophore and allow light of longer wavelength to be absorbed.

Question 18

Read and undertands: example of auxochrome

Answer 18

Question 19

What is it called when an auxochrome causes a shift to shorter wavelength?

When does this occur?

Answer 19

Hypsochromic effect or blue shift

• Occurs when compounds with a basic auxochrome ionise and the lone pair is no longer able to interact with the electrons of the chromophore.
  
  • Also seen when use different solvents or at elevated temperatures

Question 20

Hyposochrmoic and bathochromic effects rarely happen in isolation. The intensity of light absorbed also changes.

What are these effects called?

Answer 20

Bathochromic effect usually associates with increases in light absorption (hyperchromic effect)

Hypsochromic effects usually associated with decreased light absorption (hypochromic effect)

Question 21

How do these 4 effects affect the lamda max?

Answer 21

Bathochromic - shift to longer wavelenth

Hyperchromic - bigger peak

Hypsochromic - Shorter wavelenth

Hypochromic - smaller peak

Question 22

What can bathochromic/ hypsochromic shifts be used to identify?

Answer 22

Amines - put in acid and base

Question 23

Read