(8) Spectrometry Flashcards
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
Mechanism of mass spec
MS measures the mass to charge ratio of a substance
- mass spectrometer bombards a substance under investigation with an electron beam and quantitatively records the results as a spectrum of positive ion fragments (the record = mass spectrum)
- A mass spectrum is a presentation of the masses of the positively charged fragments vs their relative concentration
- Separation of the positive charge ion fragment is the basis of mass (mass/charge)
mechanism + application of atomic absorption spec
- Uses the absorption of light to measure the concentration of gas-phase atoms
- since samples are usually liquids or solids, the analytes or ions must be vaporised in a flame or graphite furnace
- the atoms absorb uv or vis light and make transitions to higher electronic energy level
- the analyte concentration is determined from the amount of absorption
applications: identification and quantitation of trace elements
Why is it difficult to apply the beer lambert law to AA spec? (3)
- variation in the atomization efficiency from the sample matrix
- non-uniformity of concentration and path length of analyte atoms (in graphite furnace AA)
- so concentration measurements are usually determined from a standard curve (calibrate the instrument with standards of known conc)
transmittance vs absorption
- transmission does not represent the interaction between incident light and the sample inside the cuvette unlike absorbance (%T is not directly proportional to the concentration of the solute)
- absorbance is directly proportional to the concentration of the absorbing species in the solute (not transmittance)
fluorescence
- when sample is hit by a specific EM transmission, electron will be excited from low to high energy level
- excited form is not stable so will go back to ground state and emit the energy that it has absorbed (fluorescence)
transmittance
Transmittance (T) of a solution is defined as the ratio of transmitted intensity, I to incident intensity, I。
- expresses the fraction of incident light absorbed by the solution
- not directly proportional to the concentration of the solute
- can be expressed as a percentage as well
Reasons for failure to obey the Beer-Lambert Law: stray light
always keep the lid of the spectrometer closed when taking readings; light from the room can affect the measurement
Reasons for failure to obey the Beer-Lambert Law: poor wavelength choice
perform wavelength scan to choose a wavelength that corresponds to a peak (optimal wavelength), to avoid problems from sloping regions of the absorption spectrum of the substance
Reasons for failure to obey the Beer-Lambert Law: changes in pH or temperature
alters solubility properties of the substance
- samples should all be at the same pH and temp
Reasons for failure to obey the Beer-Lambert Law: light scattering
from particles inside the cuvette such as cells
Reasons for failure to obey the Beer-Lambert Law: molecules associate in a concentration dependant manner
try different solvent if see particles form suspension/cloudy appearance
Reasons for failure to obey the Beer-Lambert Law: solvent-substance interactions
if not appropriate, sample will precipitate; try a different solvent
Reasons for failure to obey the Beer-Lambert Law: fluorescence of the substance
choose alternative wavelength, away from the excitation wavelength
what is measured in fluorescence spectroscopy?
the EM radiation that is emitted from the analyte when it relaxes from an excited electronic level to its corresponding ground state
- process of activation and deactivation occur simultaneously during fluorescence measurement
excitation and emission wavelengths
- for each unique molecular system, there will be an optimum radiation wavelength for sample excitation and another (longer wavelength) for monitoring fluorescence emission
- respective wavelengths depend on the chemistry of the system
what is quenching + types? (4)
reduction of the fluorescence intensity by the presence of substances in the sample other than the fluorescent analyte
- Inner fluorescence effect
- concentration self effect
- collisional quenching
- static quenching
Quenching: inner fluorescence effect
- absorption of incident light or emitted fluorescent by primary and secondary filters leads to decrease in fluorimeter intensity
Quenching: self quenching
at low concentration, linearity is observed while at larger concentration of the same substance, increase in fluorimetry is less detected (less sensitive)
Quenching: collisional quenching
between the fluorescent substances and halide ions (as they collide, they quench some of the fluorescent energy)
Quenching: static quenching
complex formation between the fluorescent molecules and other molecules
Food sample: NMR
- can be anything; syrup, beverage, meat, oil
- sample size is limited by the magnet-coil combination (sample usually 1-50cm^3)
absorbance, A
A of the solution states how much of the light is absorbed by the sample
