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The Medicine (Y2) > UV-Vis and fluorescence spectroscopy > Flashcards

UV-Vis and fluorescence spectroscopy Flashcards

1
Q

What are the four main parts inside a spectrophotometer?

A

Lamps
Monochromator
Optics
Detector

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2
Q

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

A

True

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3
Q

Why can’t normal glass be used for cuvette?

A

Because it has an absorbance of its own

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4
Q

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

A

False <1.5

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5
Q

Why are samples with an absorbance of >1.5 diluted?

A

Because molecules can pack together and cause deviations

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6
Q

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

A

False - hold from the frosted side

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7
Q

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

A

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

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8
Q

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

A

True

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9
Q

What is jablonski’s diagram?

A

An energy diagram that describes the process of photon emission

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10
Q

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

A

Electrons going from S1 to S0

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11
Q

Can electrons go from S2 to S0 directly?

A

No, have to go to S1 before S0

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12
Q

Phosphorescence is produced when electrons pass from S1 to ___

A

T1

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13
Q

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

A

False - doesn’t require

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14
Q

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

A

True

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15
Q

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

A

True

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16
Q

What is an internal conversion?

A

Radiationless transition, but vibrational levels need to match

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17
Q

What is intersystem crossing?

A

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

18
Q

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

A

True

19
Q

Why does phosphorescence occur at longer wavelengths than fluorescence?

A

Because the energy difference between S0 and T1 is lower

20
Q

A chromophore that can emit fluorescence is called a _____

A

fluorophore

21
Q

Describe the fluorescence process

A

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

22
Q

What is stokes shift?

A

The difference in wavelength between absorption and emission

23
Q

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

A

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

24
Q

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

A

True

25
Q

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

A

Fluorescence intensity on y

wavelength on x

26
Q

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

A

False - more sensitive

27
Q

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

A

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

28
Q

Instruments that measure fluorescence are called?

A

Spectrofluorometers

29
Q

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

A

Spectrofluorometers have two monochromators

30
Q

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

A

True

31
Q

What are the advantages of using fluorescence?

A

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

32
Q

What are the limitations of using fluorescence?

A

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

33
Q

What are the main factors that affect fluorescence intensity?

A 
Inner filter effect
pH
Temp
Viscosity 
Presence of oxygen
Photobleaching
34
Q

How does the inner filter effect affect fluorescence?

A

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

35
Q

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

A

True

36
Q

Phenol fluoresces at pH __ anion

A

7

37
Q

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

A

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

38
Q

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

A

True

39
Q

Structural rigidity increases fluorescence. True or false?

A

True

40
Q

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

A

True

41
Q

What are the analytical problems that digoxin and digitoxin have?

A

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

42
Q

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

A

True

The Medicine (Y2) (34 decks)

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