Types of spectrometry (6)
- UV, Visible
- Fluorescence
- Infrared
- Nuclear magnetic resonance
- Mass
- Atomic absorption
Discuss the meaning of energy absorption
- energy required for the transition from a state of lower energy to a state of higher energy by components of a compound
- directly related to the frequency of EM radiation that causes the transition **mechanism of absorbing energy is different in each case
define Spectrometric analysis
the study of how a compound interacts with different wavelengths in a given region of EM radiation
Application of UV-Vis (3)
- industrial
- amino acids: aromatics
- vitamin: Pantothenic acid
- Glucose determination - research
- enzyme activities
- identification and characterisation of organic compounds and proteins that contains organic chromophores
power of the incident and exiting beams
- relationship expressed in terms of transmittance or absorbance of the solution
What is energy transition?
- the absorption of light energy by organic compounds in the visible and UV region involves the promotion of electrons in the sigma, pi and n- orbitals from the ground state to a higher energy state
- these higher energy states are molecular orbitals called antibonding orbitals
Discuss electron excitation
- energy transitions in light absorptions
- the higher energy transitions (from sigma to sigma) occur at a shorter wavelength and the low energy transitions (pi to pi or n to pi) occur at a longer wavelength
What are chromophores?
functional group of a compound that absorbs a characteristic UV or visible region
Beer-Lambert Law
amount of light absorbed by any solute is directly proportional to the concentration of the absorbing species in the solution
* If the assumptions of the beer lambert law are violated, then absorption may no longer be proportional to concentration
Reasons for failure to obey the Beer-Lambert Law (7)
- Stray light
- Poor wavelength choice
- Change in pH or temp
- Light scattering: From particles like cells
- molecules associate in a concentration dependent manner
- Solvent substance interaction
- Fluorescence of the substance
- Always use standards to calibrate concentration measurements. Then, only measure in the straight line area
- dilute samples as necessary
What is molecular fluorescence
the optical emission from molecules that have been excited to higher levels by absorption of electromagnetic radiation
Advantage of fluorescence detection compared to UV-VIs
- greater sensitivity because the fluorescence signal (in principle) has zero background
- 1 to 3 orders of magnitude more sensitive than corresponding absorption spec methods
Analytical application of fluorescence spec
Quantitative measurement of molecules in solution and fluorescence detection in liquid chromatography
Industrial
- Vitamin assay (quantitation)
Research
- identification and characterization tool
Limitations of quantitative fluorimetry
- many limitations of the beer lambert law also affect quantitative fluorimetry
- measurements also susceptible to quenching effects
Practical application of fluorescence: ATP bioluminescence
- ATP bioluminescence is used to measure the cleanliness of a surface
- The ATP collected from a surface reacts with Luciferin and Luciferase compounds to create bioluminescence light.
- amount of bioluminescence light is measured by the Luminometer and is expressed in Relative Light Units (RLU).
-RLU numbers are directly proportional to the amount of ATP, and therefore the
amount of organic/food residue or microbial biomass on the sampled surface.
<100 RLU = clean
100 -150 = suspect
>150 = unclean
Advantages of ATP bioluminescence (3)
- Rapid result generation
- detects ATP from microbial and food residue
- it gives an accurate appraisal of the overall surface cleanliness
Disadvantages of ATP bioluminescence (6)
- does not easily distinguish between microbial and non-microbial surface contaminants
- less sensitive than traditional microbial test when low level of ATP is present
- at least 10^3 CFU (colony forming unit) is needed to obtain a reliable ATP value
- It is not very sensitive for spore detection since the level of ATP is very low in spores
- Affected by pH and temp
- chemical contaminants interfere with enzyme reaction
What is infrared spectrometry? (3)
> used to identify FUNCTIONAL GROUPS in foods
- Samples are irradiated with infrared light which is absorbed by the functional groups in a food sample and converted into energy of molecular vibration
- the energy absorption pattern thus obtained is commonly referred to as infrared spectrum
- this consists of a a plot of the intensity of radiation absorption vs wavelength of absorption
Principles of Infrared spectroscopy
Different functional groups in a food sample absorb different frequencies of radiation
Application of IR spec
Widely used in milk for quantitative determination of compounds containing C-H, O-H or N-H groups
(MilkoScan)
What is MilkoScan + advantages (4)
- Equipment developed and modified to scan the full IR spectrum
- no filters have to be changed (usually need to change filter for different types of food like carbs, protein)
- analyses milk and dairy products without dilution
- solids and semi-solids have to be diluted (liquid phase)
Advantages:
(1) Quick
(2) minimal sample prep
(3) decrease reagent consumption
(4) less hazard or pollution to environment (bc doesnt involve sample prep)
Nuclear magnetic resonance (NMR) spectrometry mechanism
NMR spectrum reflects the chemical environment around the protons of the compound thus, aids STRUCTURAL determination of the compound
allows the molecular structure of a material to be analyzed by observing and measuring the interaction of nuclear spins when placed in a powerful magnetic field.
When a nucleus that possesses a magnetic moment (such as a hydrogen nucleus 1H, or carbon nucleus 13C) is placed in a strong magnetic field, it will begin to precess, like a spinning top.
In the absence of an external magnetic field the direction of the spin of the nuclei will be randomly oriented. However, when a sample of these nuclei is place in an external magnetic field, the nuclear spins will align or oppose the field.
If the ordered nuclei are now subjected to EM radiation of the proper frequency the nuclei aligned with the field will absorb energy and “spin-flip” to align themselves against the field, a higher energy state. When this spin-flip occurs the nuclei are said to be in “resonance” with the field.
As the strength of the magnetic field increases the energy difference between the two spin states increases and a higher frequency (more energy) EM radiation needs to be applied to achieve a spin-flip .
Explain the magnetic properties of nuclei (4)
- Nuclei of certain atoms possess a mechanical spin/angular momentum.
- total angular momentum depends on the nuclear spin or spin number
- the numerical value of the spin number (I) is related to the mass number and the atomic number
- each proton and neutron has its own spin and (I) is a result of these spins
Discuss the chemical effect on proton response (chemical shift)
- difference in the absorption frequency of a particular proton of the sample from the absorption frequency of a reference proton
- When the sample is immersed in an external magnetic field, the electron density begins to fluctuate as to induce its own magnetic field that opposes the external magnetic field. Therefore, the net magnetic field will be less than the external magnetic field as a result of this induced magnetic field. This is called the shielding effect because the electron density is said to shield the proton in the nucleus from the external magnetic field by decreasing the net magnetic field that is experiences.
- The greater the electron density is around the particular proton, the greater the strength of the induced magnetic field created by the electron density. Thus, the smaller the net magnetic field that the proton feels. (stronger EM frequency required to resonate proton)
- suppose that you attached the hydrogen to something more electronegative. The electrons in the bond would be further away from the hydrogen nucleus, and so would have less effect on the magnetic field around the hydrogen
- external magnetic field needed to bring the hydrogen into resonance will be smaller if it is attached to a more electronegative element, because the hydrogen nucleus feels more of the field
