UV-Vis and fluorescence spectroscopy

Question 1

What are the four main parts inside a spectrophotometer?

Answer 1

Lamps
Monochromator
Optics
Detector

Question 2

The bigger the path length, the bigger the absorbance. True or false?

Answer 2

True

Question 3

Why can’t normal glass be used for cuvette?

Answer 3

Because it has an absorbance of its own

Question 4

Absorbance of the sample should be >1.5 otherwise dilution is recommended. True or false?

Answer 4

False <1.5

Question 5

Why are samples with an absorbance of >1.5 diluted?

Answer 5

Because molecules can pack together and cause deviations

Question 6

Cuvettes should be handled from the clear side, not frosted side to prevent transfer of proteins from the hand. True or false?

Answer 6

False - hold from the frosted side

Question 7

What is derivative spectrophotometry and how does it differ from normal?

Answer 7

The normal plot of absorbance against wavelength is zero order.
In derivative spectrophotometry, the absorbance is differentiated with respect to wavelength and as a result, sharp waves become amplified

Question 8

Derivates help to identify peaks, they do not increase data. True or false?

Answer 8

True

Question 9

What is jablonski’s diagram?

Answer 9

An energy diagram that describes the process of photon emission

Question 10

What is the only way in which fluorescence can be generated?

Answer 10

Electrons going from S1 to S0

Question 11

Can electrons go from S2 to S0 directly?

Answer 11

No, have to go to S1 before S0

Question 12

Phosphorescence is produced when electrons pass from S1 to ___

Answer 12

T1

Question 13

The return of an electron from excited singlet state to ground state requires change in spin orientation. True or false?

Answer 13

False - doesn’t require

Question 14

Phosphorescence has a longer lifetime than fluorescence. True or false?

Answer 14

True

Question 15

Intersystem crossing requires spin orientation to change. True or false?

Answer 15

True

Question 16

What is an internal conversion?

Answer 16

Radiationless transition, but vibrational levels need to match

Question 17

What is intersystem crossing?

Answer 17

Molecules relax via a non-radiative transition to the T1. Requires spin orientation to change

Question 18

Phosphorescence takes place when electrons return from a triplet excited state to ground state. True or false?

Answer 18

True

Question 19

Why does phosphorescence occur at longer wavelengths than fluorescence?

Answer 19

Because the energy difference between S0 and T1 is lower

Question 20

A chromophore that can emit fluorescence is called a _____

Answer 20

fluorophore

Question 21

Describe the fluorescence process

Answer 21

Absorption of light leads to excitation to a higher vibrational state (either within S0 or to S1 or S2
Vibrational relaxation takes place, till the lowest vibrational state in S1 is achieved - the molecule may undergo conformational change to achieve this
Molecule relaxes from the lowest vibrational energy level of the excited state to one of the vibrational energy levels in S0 = fluorescence

Question 22

What is stokes shift?

Answer 22

The difference in wavelength between absorption and emission

Question 23

Why is the wavelength of light emitted higher than that of light absorbed?

Answer 23

Because so of the absorbed energy is lost due to process that happen in the excited state lifetime e.g. vibrational relaxation etc.

Question 24

The excited state of electron lifetime is short. True or false?

Answer 24

True

Question 25

What is on the x and y axis for an emission spectrum?

Answer 25

Fluorescence intensity on y | wavelength on x

Question 26

Fluorescence is less sensitive than UV-visible light for detection of analytes. True or false?

Answer 26

False - more sensitive

Question 27

Why is fluorescence more sensitive to UV-visible light for detection of analytes?

Answer 27

Because it can be repeated more than once - some molecules can be excited repeatedly to produce more photons Emission is at a higher wavelength than absorption so background signal can be quite low at detection wavelength

Question 28

Instruments that measure fluorescence are called?

Answer 28

Spectrofluorometers

Question 29

What is a key difference in the instrumentation between spectrophotometers and spectrofluorometers?

Answer 29

Spectrofluorometers have two monochromators

Question 30

The cell holder in fluorescence needs to be clear on all four sides. True or false?

Answer 30

True

Question 31

What are the advantages of using fluorescence?

Answer 31

Higher selectivity than UV-vis Inexpensive High sensitivity Can be applied in many cases without a separation step

Question 32

What are the limitations of using fluorescence?

Answer 32

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

Question 33

What are the main factors that affect fluorescence intensity?

Answer 33

``` Inner filter effect pH Temp Viscosity Presence of oxygen Photobleaching ```

Question 34

How does the inner filter effect affect fluorescence?

Answer 34

At high drug concentration, fluorescence intensity reaches a plateau. At further increase in concentration, intensity decreases because of inner-filter effects

Question 35

Barbituates only fluoresce in the di-anionic form. True or false?

Answer 35

True

Question 36

Phenol fluoresces at pH __ anion

Answer 36

7

Question 37

How do viscosity and temperature have an effect on fluorescence intensity?

Answer 37

They have opposite effects - as molecules move more freely (low viscosity, high temp) more collisions take place and so they lose more energy leading to lower fluorescence

Question 38

A high oxygen concentration quenches fluorescence through collisions. True or false?

Answer 38

True

Question 39

Structural rigidity increases fluorescence. True or false?

Answer 39

True

Question 40

Electron withdrawing groups lower fluorescence, while electron donating groups increase fluorescence. True or false?

Answer 40

True

Question 41

What are the analytical problems that digoxin and digitoxin have?

Answer 41

They have weak absorbance at 220nm They are potent drugs so only a small quantity is available The excipients of the tablet also absorb at 220nm

Question 42

The dehydration of the steroid molecule in digoxin and digitoxin with strong acids or oxidising agents generates analytically useful fluorphore form. True or false?

Answer 42

True