UV spec

Question 1

What makes up a UV spectrophometer?

Answer 1

1) Lamps – deuterium/tungsten
2) Monochromator
3) Optics
4) detector

Question 2

How does the spectrophotometer work?

Answer 2

Polychromatic light enters a monochromator.
Monochromatic light leaves through exit slit with a selected wavelength and is made to pass through the sample.
The sample will absorb certain intensity of this light and the detector behind the sample records the light leaving the sample as a signal that is converted to a desirable format (Absorbance).

Question 3

What happens in a dual beam instrument?

Answer 3

In dual beam instrument I o is split into two beams by mirrors and lenses. One of these pass through sample while the other passes through reference.
Reference compensates for absorption effects other than sample – e.g. solvents/buffers.
In the case of single beam the machine is zeroed to make this correction at the start.

Question 4

How is the sample handled?

Answer 4

Sample is dissolved in a suitable solvent which should have minimum if any absorption in the λ range used.
e.g. for UV (>200nm): acetonitrile , ethanol, water.

Question 5

What are cells (cuvettes) made out of?

Answer 5

plastic or glass for visible region but only quartz or silica is transparent in the UV region.
• Cuvettes handled from frosted side – filled to level above the beam. DO NOT NEED TO FILL COMPLETELY.

Question 6

What is the ideal absorbance of the sample?

Answer 6

Ideally the absorbance of sample should be < 1.5 otherwise dilution is recommended

Question 7

What can be done to compensate for the presence of extraneous materials in a drug sample?

Answer 7

Measure a difference spectrum (or absorbance) in drug sample before and after adjusting certain condition e.g. pH. Only works if the change selectively affects drug only.

Question 8

What order is the plot of absorbance against wavelength?

Answer 8

ZERO ORDER

Question 9

In derivative spectophotometry, what is the absorbance differentiated in respect to?

Answer 9

wavelength

dA/dλ = f ′(λ), first derivative
d2A/dλ2 = f ″(λ), second derivative

Question 10

What does differentiating do to the spectra?

Answer 10

Results in sharp peaks (rapid change in A vs λ) becoming more amplified. Gives a more characteristic profile.
Broad bands become less prominent compared to sharp bands. This effect increases with increasing order of the derivative.
NB: Derivatives simply help identify peaks it does not increase the data.

Question 11

What is the process of luminescence?

Answer 11

(1) Excitation : electrons excited to higher energy state by light absorption
(2) Excited state life-time : ~ electrons stay in the excited state for a short time.
(3) Luminescence: Electrons return back to ground state and light is then emitted with less energy (some energy lost)

Question 12

What is the Jablonski diagram?

Answer 12

Energy diagram that describes the process of photon emission is called Jablonski diagram

Question 13

What is fluorescence?

Answer 13

return from excited singlet state to ground state; does not require change in spin orientation (more common of relaxation). The time spent from excitation back to So is called lifetime (τ) – typically few nanoseconds

Question 14

What is internal conversion?

Answer 14

radiationless transition but vibrational levels need to match

Question 15

What is intersystem crossing?

Answer 15

Molecules relax via a non-radiative transition to the T1

Requires spin orientation to change.

Question 16

What is phosphorescence?

Answer 16

return from a triplet excited state to a ground state; electron requires change in spin orientation. Lifetimes are slow (msecs, second. Usually forbidden but it does happen. Phosphorescence has a longer lifetime than fluorescence (milliseconds rather than nanosecs). Phosphorescence generally occurs at longer wavelengths than fluorescence because the energy difference between S0 and T1 is lower

Question 17

What is the process of fluorescence?

Answer 17

(1) Excitation – upon light absorption , a chromophore that was in lowest vibrational state (V0) of the ground state S0 is excited to some higher vibrational energy level (e.g. V1 or V2 etc) of S1 or S2
(2) Vibrational relaxations (non-radiative relaxation) takes place till the lowest (relaxed) vibrational level of S1 is reached. Molecule may undergo conformational change or interact with environment to achieve this state.
(3) The molecule then relaxes from this lowest vibrational energy level of the excited state S1 to one of the vibrational energy level of the ground S0 state. In doing so fluorescence is emitted.
takes about 10-15 s (2) 10-12 s, and (3) 10-9 s (or nanoseconds)

Question 18

Describe the fluorescence spectrum

Answer 18

A plot of fluorescence emission against wavelength.
The spacings of the energy levels for the first excited electronic states are similar for absorption and fluorescence processes. This implies that the fluorescence spectrum of molecules can be near to mirror image of absorption spectrum but only for simple molecules.

Question 19

What is Stokes shift?

Answer 19

The difference in wavelength between absorption and emission is called stokes shift.

as some of absorbed energy is lost due to processes that happen in the excited state lifetime. Examples of such processes :
Vibrational relaxation (heat), Collisional quenching, Energy transfer to other molecules (FRET), Intersystem crossing & Photobleaching
Hence the wavelength of light emitted by fluorescence is higher than the light that was absorbed.

Question 20

Fluorescence excitation spectrum

Answer 20

for dilute solutions this is identical to absorption spectrum. It is a plot of iintensity of emission as a function of exciting wavelength. This spectrum helps to identify suitable wavelengths that can be used to excite the fluorophore.

Question 21

Fluorescence emission spectrum

Answer 21

Fluorescence intensity against wavelength. The shape of this spectrum does not depend on which excitation wavelength was used whereas the intensity of emission does depend on the excitation wavelength.

Question 22

What is more sensitive, Fluorescence or UV-Vis for detection of analytes?

Answer 22

FLUORESCENCE

Question 23

Why is fluorescence more sensitive?

Answer 23

(1) The process is repeatable. Same molecule can be excited repeatedly thus producing many photons.
(2) Emission is at higher wavelength than absorption (stokes shift) so background signal can be quite low at detection wavelength. Thus better S/N ration

Question 24

What is the instrumentation that measures fluorescence called and what does it contain?

Answer 24

Instruments that measures fluorescence is called spectrofluorometer

1. ) Excitation source – Xe arc lamp, laser
2. ) Cell holder- cells have to be four sided
3. ) Monochromators
4. ) Detectors

Laser –> Excitation monochromator –> emission monochromator –> detector

Question 25

When recording fluorescence spectrum, what wavelengths are used?

Answer 25

When recording a fluorescence spectrum the sample may be excited at single wavelength (excitation wavelength) and the emission recorded at several wavelengths. The plot is displayed as fluoresce intensity Vs wavelength.

Question 26

What are the advantages of using fluorescence

Answer 26

o Very sensitive (µg to ng , depending on quantum yield) o Higher selectivity than UV-Vis o Relatively inexpensive (more expensive than UV-Vis but less than MS, NMR) o Can be applied in many cases without separation step

Question 27

What are the limitations of fluorescence?

Answer 27

 Not all drugs are fluorescent  Use of standards normally required in pharmaceutical analysis  Changes in conditions can affect fluorescence properties

Question 28

Describe the BP method using fluorescent light to calculate the concentration of the substance in the solution

Answer 28

Fluorescent light emitted by the substance is examined in relation to that emitted by a given standard. Dissolve substance in solvent. Set excitation wavelength Measure the intensity of the emitted light at an angle of 90° to the excitant beam. Calculate the concentration cx of the substance in the solution to be examined, using the formula: Cx = (IxCs)/Is ``` cx = concentration of the solution to be examined, cs = concentration of the standard solution, Ix = intensity of the light emitted by the solution to be examined, Is = intensity of the light emitted by the standard solution. ```

Question 29

What are mixed fluorophores?

Answer 29

When there are two molecules with different absorption spectra, it is important to consider where a fixed wavelength excitation should be placed. This method can also be important to minimise fluorescence from impurities by selecting an Ex λ where impurity can not absorb light

Question 30

What are the main factors of fluorescence?

Answer 30

1) Inner filter effect 2) pH 3) Temperature 4) Viscosity 5) Presence of oxygen 6) Photobleaching 7) Structural 8) Solvent

Question 31

What is the inner filter effect?

Answer 31

At high [drug] fluorescence intensity reaches a plateau. As concentration increases further the intensity decreases because of inner-filter effects, in which ground-state molecules absorb (present in abundance) the fluorescence emitted by excited molecules.

Question 32

What is the effect of pH?

Answer 32

o Fluorescent drugs with ionisable groups can be sensitive to pH. o Barbiturates only fluoresce in the di-anionic form o Phenol fluoresces at pH 7 (neutral or slightly acidic). anion.

Question 33

What are the effects of viscosity and temperature?

Answer 33

They have opposite effects. As molecules move more freely (low viscosity or high temp) there is more chance of intermolecular collisions (including solvent molecules) happening and this causes loss energy or deactivation of excited state by non-radiative means and thus lower fluorescence

Question 34

What is the effect of O2?

Answer 34

High concentration of oxygen quenches fluoresence through collisions. Copper (Cu2+) ions quench fluorescein by complexation-static quenching

Question 35

What are the effects of photobleaching?

Answer 35

too high incident radiation can irreversible degrade the fluorophore and cause loss of fluorescence

Question 36

What are the effects of the solvents?

Answer 36

polar fluorophores are especially sensitive. Interaction with solvent molecules can offer non-radiative pathway for energy loss.

Question 37

What effect does rigidity have?

Answer 37

Rigidity increases fluorescence's. E.g.. formation of a chelae (BP limit test for aluminium)

Question 38

What functional groups have affects?

Answer 38

Substituents that affect resonance stability changes can alter fluorescence Electron withdrawing groups (COOH, Nitro, halide ions) lowers fluorescence while electron donating (NH2, OH) have the opposite effect

Question 39

In the assay of ethinylestradiol what can be added to ionise the OH group to make it non-fluorescent?

Answer 39

NaOH

Question 40

What may peptide/protein drugs contain that are fluorescent?

Answer 40

Tyrosine and/or tryptophan. The fluoresence of such drugs often changes on folding/defolding (denaturation) – result of solvent access and rigidity change.

Question 41

How can some other drugs be made to be fluorescent?

Answer 41

Some drugs can be made to become fluorescent by chemical treatment. Digitoxin treated with H2SO4 shows strong fluorescence. Drugs can also be labeled with specific fluorophores that are reactive towards certain functional group (generally amines, COOH or SH).

Question 42

Digitoxin and digoxin examples:

Answer 42

Digitoxin and digoxin are glycosides commonly used for treatment of heart disease. Analytical Problems : o Weak absorbance at 220nm o Small quantities- potent drug so only 0.2mg/tablet o The excipients also absorb at 220nm. Thus method needs both : selectivity and sensitivity in this case. Fluorimetric assay would be an advantage to solve the selectivity and sensitivity problem. BP recommends fluorescence method for assay or dissolution test. But if absorption is weak then fluorescence may also be weak. Digitoxin or digoxin can be treated chemically to produce products that are strongly absorbing across UV-Vis but can also be made to fluoresce selectively. The dehydration of the steroid molecule with strong acids or oxidizing agents generate an analytically useful fluorophore from. BP uses methanol, ascorbic acid, hydrogen peroxide and concentrated hydrochloric acid to do this.