Week 6 (spectroscopic methods)

Question 1

What are the different spectroscopic methods?

Answer 1

• untraviolet-visible spectroscopy (UV-VIS)
• fluorescence spectrophotometry
• atomic absorption spectroscopy (AAS)
• Nuclear Magnetic spectroscopy (AAS)

Question 2

Explain ultraviolet visible spectroscopy (UV-VIS)

Answer 2

• All organic compounds absorb UV light, sometimes at very short wavelength
• UV energy is similar to the bonding energy in organic molecules
• The UV-Vis light is passed through a sample and the transmittance of light by a sample is measured
• An absorbance spectrum is obtained that shows the absorbance of a compound at different wavelengths. The amount of absorbance at any wavelength is due to the chemical structure of the molecule

Each wavelength of light has a particular energy associated with it. If that particular amount of energy is just right for making one of these energy jumps, then that wavelength will be absorbed - its energy will have been used in promoting an electron to higher energy (empty molecular orbitals)

The larger the energy jump, the lower the wavelength of the light absorbed.

UV-Vis can be used in a qualitative manner, to identify functional groups or confirm the identity of a compound by matching the absorbance spectrum. It can also be used in a quantitative manner, as concentration of the analyte is related to the absorbance using Beer’s Law

ΔE= E₂-E₁=hv

E= energy absorbed or emitted

h= Planck’s constant

v = frequency of radiation

Question 3

What is Beers law?

Answer 3

1. Light absorption is proportional to concentration of absorbing species

Question 4

What is Lamberts law?

Answer 4

Light absorption is proportional to path length

Question 5

What is the Beer-Lambert law?

Answer 5

A= log₁₀(I_o/I)= εcl

A= absorbency

Io= Incidence light intensity

I= transmitted light intensity

ε_h= molar absorbance coefficient at wavelength h

c= concentration of sample

l= path length (cuvette size)

Units are often: dm³mol^-1cm^{<span>-1</span>}

Question 6

Explain the components of the Beer-Lambert law equation: A=εcl

Answer 6

A=εcl

where A is absorbance, ε is the molar extinction coefficient (which depends on the nature of the chemical and the wavelength of the light used), l is the length of the path light must travel in the solution in centimetres, and c is the concentration of a given solution

Question 7

What are some clauses for deviation from the Beer-Lambert law?

Answer 7

Sample

• contamination (with material that’s absorbs in the same region of the UV spectrum)
• precipitation
• degradation (photolysis)
• fluorescence
• tautomerisation
• pH effects
• temperature

Question 8

What are some other causes for deviation from the Beer-Lambert law?

Answer 8

• stray light
• non- monochromatic light source
• mismatched cells
• sensitivity A< 0.002
• solvent absorption

Question 9

What must happen for each sample under the measurement conditions to be used over an appropriate concentration range?

Answer 9

Must establish the validity of the Beer-Lambert law

Question 10

Calibration curves:

Answer 10

• use at least 5 standard solutions spanning the working concentration range
• Measure in duplicate in a matched pair of cells against the solvent as reference

Question 11

Multicomponent systems:

Answer 11

• the absorption spectra often overlap
• if the components obey the Beer-Lambert law AND the law of additivity of absorbance applies then the observed spectrum is simply the sum of the components
• simultaneous equations (one per component)

Question 12

What do multicomponent systems need?

Answer 12

• accurate absorptivity values
• non- overlapping h_max regions for components
• the errors are very great for similar components
• make derivatives for colourmetric analysis?

Question 13

Explain fluorescence spectrophotometry

Answer 13

It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light.

• very sensitive
• better than absorption spectrophotometry (where I_o/I is difficult at low concentration)
• measured against a “dark” background (Incident light is at a different wavelength to what is measured- result light)
• selective - fluorescent drugs or biomolecules and their metabolites May be analysed more readily than by conventions, spectroscopy

Question 14

Using the diagram how does it work?

What are the applications of fluorescence spectrophotometry?

Answer 14

There is a light source and a monochromator (something that allows selection for the wavelength of light). The light goes in and it meets molecules in the cuvette and excites some of them, flurescence energy comes out in all directions. Another monochromater and detector is organised perpendicular to the initial light source (avoid the light passed directly through arriving on the detector)

• enzymes assays and kinetic analysis
• protein structure
• membrane structure
• fluorescence bleaching recovery
• energy transfer studies - fluorescence depolarisation
• microspectrofluorimetry
• fluorescence immunoassay
• Fluorescence activated cell sorting
• multicomponent analysis by synchronous luminescence spectrophotometry

Question 15

Fluorescence spectrophotometry: What is signal intensity affected by?

Answer 15

• generally more sensitive to environment than absorbance measurements
• the signal intensity may be affected by
• pH (any ionisable groups)
• temperature (T^, increasedcollisions, quenching - use a thermostat for precise work)
• quenching (formation of complex between the sample and another species-photons absorbed by molecules)
• interfering substances
• often limiting in the analysis of biological samples; can be reduced by pre treatment of the sample, use of pure solvents, clean glassware)
• solvent
• interference from Rayleigh and Raman scattering

Question 16

Explain the Jablonski diagram

Answer 16

• In molecular spectroscopy, a Jablonski diagram is a diagram that illustrates the electronic states of a molecule and the transitions between them. The states are arranged vertically by energy and grouped horizontally by spin multiplicity
• Electronic ground state with several vibrational energy levels to create a vibrational energy ladder and electronic excited state also with several vibrational energy levels
• Radiative transitions involve the absorption of a photon, if the transition occurs to a higher energy level, or the emission of a photon, for a transition to a lower level.
• For flourescent comounds, the emission wavelengths is longer than the excitaion wavelength

Question 17

Fluorescence spectrophotometry: Quantitative applications

Answer 17

In dilute solution fluorescence intensity I_f = KQI_oebc

I_f proportional to c

• k= instrumental constant
• Q= quantum efficiency of fluorescence
• I_o= incident light intensity
• e_h= molar absorbance coefficient at wavelength h
• c= concentration of sample
• b= path length

Question 18

Explain Fluorescence spectrophotometry

Answer 18

• If an atom or molecule first absorbs energy – for instance a photon -, this is called excitation. Very shortly (in the order of nanoseconds) after excitation it emits a photon of a longer wavelength. We call that fluorescence
• In fluorescence spectroscopy, a beam with a wavelength varying between 180 and ∼800 nm passes through a solution in a cuvette. We then measure – from an angle - the light that is emitted by the sample.
• In fluorescence spectrometry both an excitation spectrum (the light that is absorbed by the sample) and/or an emission spectrum (the light emitted by the sample) can be measured. The concentration of the analyte is directly proportional with the intensity of the emission.

Question 19

What must the total absorbance of the system (εcl) not exceed?

Answer 19

0.005 absorbance units

Question 20

What may happen at high drug concentrations?

Answer 20

Ground-state molecules may absorb the fluorescence emitted by excited molecules

>negative deviations from linearity

Question 21

What must the range of linearity of the calibration curve (I_f vs c) use?

Answer 21

at least 5 standard solutions

Question 22

Unlike absorption spectrophotometry:

Answer 22

Spectrofluorimetry does not give an absolute scale of values.

> MUST USE A REFERENCE STANDARD for quantitative work

Question 23

What is fluorescence derivatisation?

Answer 23

Some weakly fluorescent or non- fluorescent molecules may be derivatised with a fluorescent reagent e.g:

• Dansyl chloride Derivatives primary and secondary amines and phenols
• Fluorescamine Derivatives primary aliphatic amines including peptides and proteins
• Isatonic anhydride Derivatives primary and secondary amines and alcohols
• o-Phthaldehyde Derivatives primary amines

Question 24

What are some considerations of fluorescence derivatisation?

Answer 24

• fluorescent contaminants
• extent derivatisation
• reliability of the derivatisation reaction

Question 25

Explain Atomic absorbance spectroscopy (AAS)

Answer 25

• quantitative determination of chemical elements using the absorption of optical radiation (light) by free metallic atoms in the gaseous state.

specific light source

• Hollow cathode filled with e.g Fe
• Emits light at frequencies characteristic of Fe that produce the right mix of wavelengths to be absorbed by any Fe atoms from the sample
• In AAS, the sample is atomised – ie converted into ground state free atoms in the vapour state – and a beam of electromagnetic radiation emitted from excited Fe atoms is passed through the vaporised sample. Some of the radiation is absorbed by the Fe atoms in the sample.

The greater the number of atoms there is in the vapour, the more radiation is absorbed. The amount of light absorbed is proportional to the number of Fe atoms. A calibration curve is constructed by running several samples of known Fe concentration under the same conditions as the unknown. The amount the standard absorbs is compared with the calibration curve and this enables the calculation of the Fe concentration in the unknown sample.

• One element at a time - Compare I_o and I (Beer-Lambert law)

Question 26

What are the advantages and disadvantages of AAS?

Answer 26

• high sensitivity (ppm, sometimes ppt)
• sharp lines
• little overlap between different elements in the same sample
• SELECTIVE
• Not as accurate as some wet methods
• precision rarely better than 1- 2%
• equipment expensive but widely available
• need calibration curve- standard solutions

Question 27

describe nuclear magnetic resonance spectroscopy

Answer 27

• radiation in the radiofrequency region is used to excite the nuclei of atoms( ¹H, ¹³C, ¹⁵N, ³¹P) so that their spins switch from being aligned with to being aligned against an applied magnetic field

The energy transfer takes place at a wavelength that corresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to yield an NMR spectrum for the nucleus concerned

Question 28

What are the advantages of nuclear resonance spectroscopy?

Answer 28

• the magnetic field experienced by a nucleus is a summation of the external (applied field and the local environment as determined by molecular structure
• the range of frequencies required for excitation and the complex splitting patterns produced are very characteristic of the chemical structure of the molecule
• a powerful technique for the characterisation of the exact structure of molecules
• provides much more information about molecular structure than any other technique
• a relatively insensitive technique requiring > 5mg of sample for proton nuclear magnetic resonance (NMR) and > 20 mg for carbon-13 NMR
• molecular behaviour can be studied in solution e.g protein folding
• ³¹P has been used extensively to study phosphate metabolism

Question 29

Magnetic resonance imaging (MRI)

Answer 29

• in MRI, owing to sensitivity issues, the hydrogen nucleus is used predominantly
• materials such as urine, blood, cerebrospinal fluid and tissue biopsy samples may be studied directly
• the measurement of metabolic concentrations at specific sites in tissues is possible
• whole animals may be studied for:

ATP metabolism in humans

Distribution of water constant in tissues

Lipid distribution

Question 30

Explain MRI

Answer 30

1. To perform a study, the person is positioned within an MRI scanner that forms a strong magnetic field around the area to be imaged.
2. First, energy from an oscillating magnetic field is temporarily applied to the patient at the appropriate resonance frequency.
3. When a radiofrequency current is then pulsed through the patient, the protons are stimulated, and spin out of equilibrium, straining against the pull of the magnetic field.
4. When the radiofrequency field is turned off, the MRI sensors are able to detect the energy released as the protons realign with the magnetic field as they produce a radio signal that can be measured by receivers in the scanner and made into an image
5. The time it takes for the protons to realign with the magnetic field, as well as the amount of energy released, changes depending on the environment and the chemical nature of the molecules a
6. Protons in different body tissues return to their normal spins at different rates, so the scanner can distinguish among various types of tissue

Question 31

Describe MRI

Answer 31

In most medical applications, hydrogen nuclei, which consist solely of a proton, that are in tissues create a signal that is processed to form an image of the body in terms of the density of those nuclei in a specific region. Given that the protons are affected by fields from other atoms to which they are bonded, it is possible to separate responses from hydrogen in specific compounds.

Question 32

Describe what would be seen in an MRI scan of a transverse section though the abdomen of a fat person?

Answer 32

• the fatty deposits are indicated in white
• this resonance arises from the methylene protons if long fatty acid chains

Question 33

What allows white and grey matter to be distinguished and most tumors differentiated from normal tissue?

Answer 33

Differences in water content

Question 34

X-Ray

Answer 34

• X-rays interact with heavy elements best and least well with hydrogen and carbon > soft tissue not detected well
• clearly shows the contrast between soft tissue and bone density
• often used to examine broken bones
• a few seconds
• exposure to ionising radiation
• cheaper

Question 35

MRI

Answer 35

• MRI scanning detects hydrogens and discriminates between different types of hydrogens
• shows a better contrast beween different kinds of soft tissue
• produces detailed images of the brain and other tissues
• ability to change the imaging plane without moving the patient
• scanning typically run for about 30 minutes
• no exposure to ionising radiation
• more expensive

Question 36

What Sensitive analytical techniques useful for quantitative measurements?

Answer 36

• Ultraviolet
• Visible Spectroscopy (UV - VIS) Fluorescence Spectrophotometry
• Atomic Absorption Spectroscopy (AAS)

Question 37

What spectroscopic method is:

• Less sensitive than the above but far richer in information
• Used for identification and imaging purposes

Answer 37

Nuclear Magnetic Resonance Spectroscopy (NMR / MR