Unit 4 - UV-Visible Spectrophotometry

Question 1

The nature of light (5 properties)

Answer 1

1) Light has a wavelength and frequency
2) Light has a polarization, defined by the direction of its electric field
3) Light has a direction and travels with speed c = 3 x 10^8 m/s in vacuum
4) Light has perpendicular electric and magnetic field components
5) Light has wave-particle duality

Question 2

Why do dark lines appear in the spectrum of white light shone through a cloud of hydrogen gas?

Answer 2

Atoms absorb the light as electrons move from a lower energy level to a higher energy level

Question 3

What is resonance?

Answer 3

It is the condition that the absorption of electromagnetic (EM) radiation by matter must have the same energy as a transition between 2 states. The length of the line is proportional to the energy of photon

Question 4

What type of transition occurs in UV-visible spectroscopy?

Answer 4

Transition of valence electrons

Question 5

3 Properties of Light Absorption

Answer 5

1) Light is energy. Energy is conserved
2) The light energy is converted into electronic potential energy
3) Electrons move from a ground state orbital to an excited state orbital

Question 6

2 Properties of Atomic Absorption

Answer 6

1) Multiple absorption lines (different transitions)
2) Narrow spectral width (0.001-0.05). It is narrow because it only represents the transition from the lower electronic state to a higher electronic state. The energy gap can be easily calculated

(view slide 16 of lecture 1 of UV-VIS)

Question 7

What forms of energy can be released?

Answer 7

Energy can be released as heat or as photons

Question 8

List 3 ways an atom or molecule can be promoted to an excited state

Answer 8

1) Thermal energy
2) Absorption of a photon
3) Chemical reaction

Question 9

Difference between absorption and emission. What are its implications?

Answer 9

Absorption is when a molecule or atom absorbs energy for the electron to be promoted to an excited state. Emission is the process of the same electron being relaxed back into its ground state, which then emits energy (heat or photons). This implies that the emission spectrum is a visual of how much E was emitted when the electron relaxed. It can be used to differentiate elements

Question 10

What is the most common valence electron transition?

Answer 10

HOMO to LUMO. It is an energetically favoured excitation. The jumps take a certain wavelength

Question 11

Ground state singlet state (S_0)

Answer 11

The total spin quantum number is 0. Two electrons of opposite spin are in the HOMO

Question 12

1st excited singlet state (S_1)

Answer 12

The total spin quantum number is 0. One electron is located in the HOMO and a spin paired electron is located in the LUMO

Question 13

Describe the conservation of energy in terms of how a molecule/atom returns to its ground state

Answer 13

The molecule/atom returns to its ground state via the production of heat, light, or energy transfer to another molecule

NOTE: ground state-to-excited state transition energies depend on molecular structure, intermolecular interactions, and the local environment

Question 14

Why does the HOMO to LUMO gap have the highest valence electron transition?

Answer 14

Because it has the lowest energy gap

Question 15

Draw a Jablonski diagram

Answer 15

(think about it)

Question 16

How different phases affect spectral widths

Answer 16

Vapour phase: molecules can rotate and vibrate freely. Vibrational and rotational fine structure observed

• Dissolved in hexane: rotational freedom is lost and collisions with solvent broaden the vibrational transitions
• Dissolved in water: Stronger interactions with solvent broaden the transitions further and obliterate all fine structure

NOTE: (*) - more interaction with the solvent means broader transitions

Question 17

De-excitation pathways of Absorption, Fluorescence, and Phosphorescence

Answer 17

Absorption has normal de-excitations (aka, electrons just go back to the ground state). Fluorescence is normal and undergoes internal conversion. Phosphorescence takes a long time because its electrons cannot go directly down (Pauli exclusion principle) and undergoes intersystem crossing, therefore, it takes a longer time. Fluorescence in nature is rare because most animals emit heat when the electrons transition back down to the ground state.

Question 18

Chromophores

Answer 18

Functional groups that can absorb visible light. They are responsible for an absorption band and the approximate location of the corresponding electron transition (ex. aromatics, alkenes, alkynes, carbonyls (unsaturated bonds)

Question 19

How can you decrease transition energies?

Answer 19

Conjugation of unsaturated bonds. More conjugation, longer wavelength, less E gap from HOMO to LUMO. More conjugation allows absorption strength to increase (ie. it shifts the absorption maximum to longer wavelengths)

***just because you see a double bond, doesn’t mean the system is conjugated. They can to be close together (aka they have to be at most, one sigma bond away so resonance can occur)

Question 20

Hyperchromic shift

Answer 20

The strength of the absorption (called absorptivity) roughly doubles with each new conjugated double bond

Question 21

Bathochromic shift

Answer 21

Each additional double bond in the conjugated pi-electron system shifts the absorption maximum by about 30nm to the red

Question 22

Auxochrome

Answer 22

Chemical group that is attached to a chromophore and modifies its light absorption properties (by altering the energies of the MOs). Different configurations of the functional groups can modify the light absorption property

ex. pH indicators are made of up chromophores attached to auxochromes

Question 23

The absorbance of bromocresol purple is affected by pH because the deprotonated form (present at higher pH) causes what?

Answer 23

A decrease in the HOMO-LUMO energy gap

Question 24

Transmittance

Answer 24

The ratio of the power (P) of the transmitted beam to the incident beam (P_0). Often expressed as a percentage

Question 25

Absorbance

Answer 25

The negative logarithm of transmittance. Effectively the number of orders of magnitude by which transmittance is decreased

Question 26

Blank measurement

Answer 26

Account for non-absorption losses of light. Measured using only the solvent and the same/matched cell. It is used to compensate for reflection and absorbance of the solvent

Question 27

Single beam instrument

Answer 27

P_solution and P_solvent must be measured sequentially rather than simultaneously

Question 28

Beer-Lambert Law

Answer 28

A, absorbance = the amount of light absorbed. Wavelength dependent

ε, molar absorption coefficient (M-1cm-1) = measure of how strongly a molecule absorbs light. Wavelength-dependent. Proportional to the absorption cross-section of a molecule (cross-section of photon capture). Generally, the more conjugated double bonds, the larger the value of ε

b, pathlength (cm) = the distance light travels through the sample

c, concentration (M) = the amount of chromophore per unit volume

Question 29

Graph of absorbance vs concentration

Answer 29

ε (molar absorption coefficient) and path length (b) determine slope of the calibration plot, and thus the sensitivity of the method. Larger slope means the method is more sensitive, and sensitivity is determined by ε*b (they are constants)

Therefore, we choose the wavelength with the highest ε when doing absorption measurements

Question 30

Does ε change if the species are the same?

Answer 30

No, ε is the same for the same species. We can use this knowledge when comparing graphs. If one line is steeper and the species are the same, then we know that pathlength between the two measurements are different

Question 31

Name 2 factors the does not limit the sensitivity of a UV-visible absorption spectrophotometric method?

Answer 31

1) The intensity/power of the light source
2) the efficiency of the photodetector

We know that sensitivity is only related to the ε value and the b value

Question 32

Deviations from the Beer-Lambert Law

Answer 32

Real deviations:
At high concentrations (>0.01 M), each chromophore affects the charge distribution of nearby chromophores (changes ε)
The value of ε depends on refractive index (dielectric constant), which can change as concentration increases

Apparent deviations:
Spectrophotometer radiation is not perfectly monochromatic
The chemistry is not suitably controlled (ex. the chromophore reacts with solvent or solutes). That is, the concentration of chromophore does not vary linearly with the amount added

Question 33

Spectroscopy

Answer 33

The study of how physical systems interact with or produce electromagnetic radiation (EM radiation)

NOTE: mass spectroscopy is the exception

Question 34

Spectrometry

Answer 34

the measurement of EM radiation to obtain information about systems and their components

NOTE: Mass spectrometry is the exception

Question 35

Spectrophotometry

Answer 35

Describes the measurements made with ultraviolet (UV), visible, and infrared (IR) light

Question 36

Molecular spectrophotometry

Answer 36

A method of molecular analysis. Most samples comprise a collection of molecules

Question 37

Atomic absorption spectrophotometry

Answer 37

A method of elemental analysis. Isolated atoms do not usually exist in samples. It must generate an atomic population with energy input

Question 38

Tungsten-halogen lamp (light source)

Answer 38

Quartz envelope containing tungsten filament, inert gas, and small amount of iodine. Gives off visible and near infrared (NIR) radiation, and almost no UV radiation. Iodine extends the lifetime of the lamp by re-depositing sublimated tungsten

Question 39

Deuterium lamp (light source)

Answer 39

Mostly UV radiation and has a quartz envelope. Deuterium gas is excited by electrical arc and dissociates. Can dissociate to emit photons

Question 40

Reflection gratings (wavelength selector)

Answer 40

Most commonly used for spectrophotometric components for variable wavelength selection. Less expensive and more compact than prisms. It is made up of a reflective substrate with a series of highly parallel, closely spaced grooves

Question 41

Diffraction orders

Answer 41

Different orders always show up together, so adding another diffraction grating will not remove unwanted wavelengths. To remove unwanted orders, use secondary filtering (ex. long-pass filter)

Wavelength multiples constructively interfere at the same angles. There are multiple angles that support constructive interference

Question 42

Implications of the size of the slits in a monochromator

Answer 42

Entrance and exit slit width determine the monochromator bandwidth and limit the spectral resolution. The smaller the bandwidth, the greater the spectral resolution

We want the slit as narrow as possible, however, this would mean less light is entering, therefore, decreased light intensity (exiting the monochromator) and decreased S/N. But if we open the slit wider, we get increased light intensity and results in a broad wavelength distribution, meaning it will not be monochromatic anymore.

Question 43

Dispersion

Answer 43

The angular spread of diffracted wavelengths increases as the groove density (lines per millimeter) increases. Resolution increases, working range decreases

Question 44

Double monochromator configuration

Answer 44

Reduces stray light by orders of magnitude. The light throughput is reduced by the second monochromator, but does NOT sort out diffraction orders. Double monochromators have additional components (ex. slit 2 of 3 is used as both an exit and entrance slit)

Question 45

Cuvettes and Spectrophotometer cells

Answer 45

Must provide a defined and reproducible path length and must be transparent to wavelength of interest. Some examples:

Plastic: visible wavelengths only
Silicate glass: approx. 400-2000 nm
Corex glass: approx. 350-2000 nm
Quartz: approx. 200-3000 nm (used to measure absorbance in the UV)

Question 46

Microtiter plates

Answer 46

Fast, high-throughput analysis and consists of a 2D array of “sample cells” or microwells. They have small volumes and come in different sizes. Some plates are UV transparent and are measured with a plate reader (instrument)

Question 47

Photoelectric effect

Answer 47

Photon incident on alkali metal or metal oxide photocathode ejects an electron

Question 48

Photodetector: Photomultiplier Tube (PMT)

Answer 48

Signal amplification through an electron cascade across a series of dynodes
Electrons are collected at the anode producing an amplified photocurrent (depends on wavelength). The voltage calculated is from the first dynode to the last dynode. The higher the voltage, the easier it is for the electron to move from one dynode to another, but the lifetime might be shorter

Main points:
PMTs are more sensitive with more dynodes and the more voltage is applied
PMTs are not equally sensitive to all wavelengths of light

Question 49

Efficiency

Answer 49

Gratings are not equally efficient across all wavelengths. They are blazed to optimize efficiency for a certain wavelength region

Question 50

Does PMT of efficiency affect absorbance?

Answer 50

No. Absorbance is A=εbc. PMT is not a part of Beer-Lambert Law, so A is not affected by PMT of efficiency

Question 51

Photodetector: Silicon Photodiode

Answer 51

A silicon photodiode is a zero, or reverse biased P-N semiconductor junction
Absorbed photons generate mobile charge carriers (electrons and holes) in the junction resulting in a photocurrent under reverse bias or a photovoltage at zero bias
The magnitude of the current is proportional to the incident light intensity

NOTE: photodiodes are not equally sensitive to all wavelengths

Question 52

Photodiode array (PDA) chips

Answer 52

It consists of a series of P-N photodiodes arranged in a line. The advantage of a PDA is that an entire spectrum can be measured simultaneously. They do this by removing the exit slit of a monochromator, which allows the light to be dispersed onto the PDA

Question 53

UV-Visible Plate Reader

Answer 53

Monochromator output coupled to optic fiber
Optical fiber delivers light to individual well
Uses silicon photodiode detector
Translation system moves fiber and detector from well to well

Question 54

Does adding water to both wells (sample and blank) affect the absorbance value?

Answer 54

No, the absorbance stays the same. Only way we can change absorbance is to pour a small amount of sample solution out, which affects pathlength, but concentration stays the same; therefore, absorbance will decrease

Question 55

UV-Visible spectrophotometer design

Answer 55

Dual lamp design
Wavelength selection prior to sample
Wavelength scanning
Double beam for sample and reference (blank)
More complicated, more precise, more expensive

Question 56

Atomization process

Answer 56

Allows Atomic Absorption Spectrometry (AAS) an elemental analysis technique, approx. independent of the molecular or solid state of the element in the original sample

Question 57

Modulation

Answer 57

Turn the source on and off to identify contributions to the observed signal from emission processes

Question 58

Light source requirements for Atomic Absorption Spectrophotometer (AAS)

Answer 58

For good S/N, the line width of the source should be more narrow than that of the absorption band
The spectral position of the light source should overlap with the absorption line of the element of interest

Question 59

Hollow cathode lamp

Answer 59

Glass envelope filled with Ar or Ne (inert gases). It generates a current when the filler gas is ionized and an electric potential is applied across the anode and cathode.

Electrons will be transferred to metal vapour and converts metal from ground to excited state. Metal atoms return to ground state by the emission of a photon

Question 60

Premix burner for AAS

Answer 60

It prepares atomic population by burning material (using nebulizer) and generates vapour with atoms, which requires a large amount of energy. The flame temperatures are low enough that most atoms are in the ground state. Long burner path length to improve sensitivity

Question 61

Electrothermal Atomizer/Graphite Furnace for AAS advantages (3) and disadvantages (2)

Answer 61

Advantages:

1) Good for small samples
2) Minimal sample preparation (solids and liquids suitable)
3) 10-100x more sensitive than flame methods

Disadvantages:

1) Poor precision
2) Compatible with fewer elements

Question 62

Interferences in AAS and how to fix them

Answer 62

Spectral interferences:

1) Unwanted lines from light sources
- Choose analytical wavelengths to minimize effects
2) Absorption lines of other atoms or ions
- Adjust flame temperature or optical height in the flame (high T can yield ions and low T can yield molecules)
3) Absorption bands of metal oxides or hydroxides, other molecules

Chemical interferences:

1) Chemical reactions that produce low volatility species (introduce negative bias)
2) Ionization of analyte (ions have different absorption lines than their parent atoms)
- Use an ionization buffer (cesium salts) to suppress ionization of analyte