EMR and UV-vis spectrophotometery

Question 1

What is the electromagnetic spectrum classified in order of?

Answer 1

decreasing wavelength

Question 2

Name the EM spectrum

Answer 2

Gamma rays, X-rays, UV, Visible, Infrared, Microwaves and radiowaves

Question 3

What are light waves

Answer 3

electromagnetic made of moving electric (E) and magnetic (B) fields vibrating 90 degrees to each other at the speed of light.
The energy from these fields (electromagnetic) makes the wave move

Question 4

What does the speed of light equal?

Answer 4

c=fλ
(f = frequency, cycles per second)
(λ = wavelength, length of one cycle)
c=3x10^8 m/s

Question 5

What does energy equal?

Answer 5

E=hf
(h = planks constant)
high frequency = high energy

Question 6

What does light behave as?

Answer 6

Can be energy carrying waves but also can behave as a particle travelling in discrete units called PHOTONS

Question 7

What does the energy of a photon equal?

Answer 7

E = hc/λ

When photons hit something, EMR waves interact like other waves.

Question 8

What is absorption of radiation?

Answer 8

• Selective removal of certain frequencies by transfer of energy to atoms or molecules
• Electrons promoted from lower-energy (ground) states to higher energy (excited) states.
• Energy of exciting photon must exactly match the energy difference between the ground state and one of the excited states of the absorbing species.

Question 9

What is emission of radiation?

Answer 9

• Electromagnetic radiation is produced when excited particles return to lower-energy levels or the ground state.

Question 10

What will cause light to scatter more?

Answer 10

If something hits a bigger particle

Question 11

What is total internal energy in a molecule a sum of energy of ?

Answer 11

1) Electrons
2) vibrations between the molecule’s own atoms
3) rotations of the molecule

Question 12

What are common spectroscopy techniques which rely on use of EMR in pharmaceutical analysis?

Answer 12

 UV-Vis absorption – electrons excited
 Infra-Red (IR) absorption- vibrations excited
 Fluorescence emission – electrons returning back

Question 13

What happens when photons hit a molecule/

Answer 13

• A molecule/atom changes its energy state by absorbing or emitting energy that is equal to the energy difference between ground E0 and excited E1state.

Question 14

What does each energy level have further levels of?

Answer 14

• Each energy level is discrete but has further vibrational and rotational levels which are also discrete.

Question 15

Therefore, what can excitation of a molecule be?

Answer 15

Can be electronic, vibrational and rotational

Question 16

Higher excited states need…

Answer 16

MORE energy e.g. UV (short wavelength)

Question 17

Lower excited states need…

Answer 17

LESS energy e.g. visible (long wavelength)

Question 18

Within vibrational levels need…

Answer 18

EVEN LOWER energy e.g. IR (longer wavelength)

Question 19

What is UV-VIS spectrum of a drug a plot of?

Answer 19

absorbance vs wavelength

Question 20

What does the wavelength light that a particular molecule absorbs depend on?

Answer 20

Differs depending on molecular structure because energy levels are discrete and different in different molecules

Question 21

Do most drugs absorb UV light?

Answer 21

YES - except few coloured ones

Question 22

What sort of spectrum does UV-VIS have?

Answer 22

BROAD, rather than sharp lines

Question 23

Why does it have a broad spec?

Answer 23

• Photons with slight difference in energy can still cause electronic transitions by exciting electrons from the many vibrational state that corresponds its energy (wavelength)
• Photons from light provides energy to excite electrons (non-bonding (n) and bonding π electrons) from ground state to excited state

Question 24

What is sigma bonding?

Answer 24

Sigma σ (bonding) molecular orbital – e.g. formed by electrons in 2S orbitals e.g. CH3 - CH3 (Ethane) – forming SINGLE bonds. Electrons most close to nucleus

Question 25

What is pi bonding?

Answer 25

π (bonding in double or triple bonds) molecular orbital : formed by electrons in p orbitals. forming double or triple bonds

Question 26

What is n bonding?

Answer 26

n (non-bonding) atomic orbital found on e.g. nitrogen , oxygen (electronegative) as lone pairs

Question 27

What happens when a bonding orbital (HOMO) is created?

Answer 27

When a bonding orbital (HOMO) is created there is also created a corresponding anti-bonding orbital (LUMO) that is normally unoccupied but lies at higher (less stable) state.
• So σ has σ* and π has π*

Question 28

What energy levels can be used in pharmaceutical analysis?

Answer 28

Only π to π* and n to π* are in the right region (low enough energy) for any use
in pharmaceutical analysis. The others are in far UV where air interferes.

Question 29

Can UV spec be measured easily?

Answer 29

NO - <200nm absorption – not much use in analysis.
σ to σ* n to σ* would require photons of λ below 200nm to get right level of excitation energy. In this far UV region molecules in medium (air/solvent) will have similar transitions so practically the spectrum can not be measured easily

Question 30

Uses of UV absorption?

Answer 30

1) Identification of drugs – by comparing and interpreting spectra. Not absolute ID.
2) Measuring reactions, ionisation (Pka determination, solubility, drug release – dependent on change in absorption spectrum (λmax and/or intensity)
3) Quantification of drugs – Most useful and widely used (Beer Lambert Law)

Question 31

What bonds have good absorption in UV >200nm/

Answer 31

DOUBLE BONDS

Question 32

Position and intensity of absorption changes depending on …?

Answer 32

substituent groups, degree of conjugation, nature of solvent, pH.

Question 33

What is a conjugated structure?

Answer 33

having double bonds alternating with single bonds

Question 34

What do conjugated structures tend to do to absorption ?

Answer 34

tend to shift absorption to longer wavelength (batochromic shift)

Question 35

What happens the longer the conjugated system?

Answer 35

The more extensive (longer) conjugated system the less is the separation in energy levels and thus longer the wavelength of absorption.

Question 36

Why do conjugated systems give bathochromic shift?

Answer 36

In conjugated systems electrons are easily delocalised making the energy gaps between the various orbitals smaller thus lowering the energy to excite them. This gives bathochromic shift.

Question 37

Delocalization (resonance stabilization) extends if;

Answer 37

1. benzene rings are present
2. alternating double and single bonds exist between C=C, C=O, C=N, N=N, N=O.
3. Lone pairs on substituent groups with nitrogen, oxygen or halogen become involved e.g. NO2 groups. (RAISES INTENSITY)

Question 38

What is an Auxochrome?

Answer 38

a functional group containing lone pair(s) (e.g. OH, NH2of electrons that does not absorb appreciable amount of UV/Visible light on its own but shifts peaks of molecules that it is attached with to longer λ (bathochromic shift) and makes the peak higher intensity (hyperchromic shift).

Question 39

What is hyperchromic shift/

Answer 39

peak higher intensity

Question 40

What is a chromophore?

Answer 40

a part of a molecule that absorbs UV or visible light (double bond , groups with lone pair atoms).

Question 41

What is the amount of light of intensity that leaves a sample given by?

Answer 41

T =   I / I0   or          
%T = 100 x T

(I = lower intensity)
(I0 = light of intensity)

Question 42

How much energy would a more concentrated sample absorb?

Answer 42

A more concentrated sample would absorb more and transmit less. However transmittance is not linear with increase in concentration instead it falls exponentially. To make this relationship linear it is best reported as absorbance which is the logarithm (base 10) of the reciprocal of the transmittance:
A = log (1/T) = log I0 / I

Question 43

What is the amount of light absorbed (A) dependent on?

Answer 43

the concentration of sample

Question 44

What is the Beer lambert law?

Answer 44

A= εbc
• where ε is molar extinction coefficient (M-1cm-1) or molar absorpitivity (i.e. the absorbance value of a 1M solution). It is constant for a particular compound at given λ.
• b is the path length of the cell in cm
• c is concentration in moles/litre

Question 45

ε units

Answer 45

• where ε is molar extinction coefficient (M-1cm-1) or molar absorpitivity (i.e. the absorbance value of a 1M solution). It is constant for a particular compound at given λ.
• b is the path length of the cell in cm
• c is concentration in moles/litre

Question 46

Measuring Conc:

Answer 46

c=A/A1%

Question 47

For absorbance to be directly proportional to c following assumptions must apply:

Answer 47

1. ) Incident radiation must be monochromatic (single wavelength)- this may not always be the case due to instrument calibration errors, stray light (without passing through sample) going directly to detector.
2. ) Absorbance checks can be made against standards such as potassium dichromate- the A must lie within specified range in BP. Stray light – KCL reference solution.
3. ) Each molecule in solution acts as an independent absorbing species in solution- not wholly true esp, at high conc
4. ) Absorption takes place in a homogenous solution – may not be true for turbid samples causing scatter of light.
5. ) Other factors: association, dissociation, photodegradation, solvation, complexation or adsorption, or if sample emits fluorescence, then positive or negative deviations from the Beer–Lambert Law may be observed.

Question 48

BP limits for testing equipment and apparatus:

Answer 48

To minimise errors BP recommends standard materials for testing equipment
and apparatus. The absorbance of the solvent used against air and at the prescribed wavelength shall not exceed 0.4 and is preferably less than 0.2.