Lecture 6 : Molecular Spectrometry

Question 1

6.1. . Basic principles of spectroscopy

What are the 3 main ways that matter can interact with light?

Answer 1

1. Absorption
2. Emission – material glows and emits light when light is shined on it
3. Diffraction – when light interacts with matter, it gets split and spread out (e.g. prism split 1 ray of white light into 7 different coloured rays)

Question 2

6.1. . Basic principles of spectroscopy

What are the 3 energy states that molecules can possess when interacting with light?

Answer 2

1. Electronic energy
2. Vibrational energy
3. Rotational energy

Question 3

6.1. . Basic principles of spectroscopy

What is electronic energy? What does it mean when a molecule has higher electronic energy?

Answer 3

It is the energy stored as potential energy in excited electron configurations.

• Higher electronic energy : when the electron in a molecule jumps to a higher valence shell that is further away from the nucleus, causing the electronic energy of the molecule to be higher

Question 4

6.1. . Basic principles of spectroscopy

What is vibrational energy? What is meant by higher vibrational energy?

Answer 4

Vibrational energy is the back and forth movement in molecules, causing it to stretch and bend along covalent bonds (e.g. C=C stretch, O-H bend)

• Higher vibrational energy = molecule can stretch and bend more

Question 5

6.1. . Basic principles of spectroscopy

What is rotational energy? What is meant by higher rotational energy?

Answer 5

Rotational energy is amount of energy a molecule possesses due to it spinning around an axis.

• Higher rotational energy = molecule can spin faster

Question 6

6.1. . Basic principles of spectroscopy

What is the relationship between energy and wavelength?

Answer 6

Energy and wavelength is inversely proportional.
- At shorter wavelength, frequency is higher, thus energy level is higher

Question 7

6.1. . Basic principles of spectroscopy

What are the 3 main steps for a molecule to emit fluorescence when it interacts with light?

Answer 7

1. Absorption of light (excitation) : electrons in molecules get excited to a higher electronic state
2. Relaxation : Non-radiative loss of energy as the electron relaxes to the lowest vibrational level of the excited state
   - Non-radiative loss of energy means that energy is lost without light being emitted = no fluorescence
3. Emission : Fluorescence is emitted as the electron returns to the ground state.

Question 8

6.1. . Basic principles of spectroscopy

In fluorescence spectroscopy, why is the wavelength of the emitted radiation longer than the wavelength of radiation used for excitation of analyte?

Answer 8

Energy is inversely proportional to wavelength. Excitation = energy gained = shorter wavelength ;; emission of light = energy lost = longer wavelength
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More detailed explanation :
- When excited to higher energy state, e- will not stay in excited state for too long and quickly relaxes to lowest vibrational state. Thus some energy is lost due to non-radiative process
- When e- returns to ground state, energy is emitted in the form of light, but the energy emitted is less than the energy of absorbed light because some energy was already lost due to the non-radiative process

Question 9

6.2. . UV-Vis spectroscopy in food analysis

What are the 2 equations for Beer Lambert’s law in calculating absorbance?

Answer 9

1. A = acL ; where a = absorptivity, c = concentration, L = pathlength

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2. A = εcL, ε = molar extinction coefficient, c = concentration, L = pathlength

Question 10

6.2. . UV-Vis spectroscopy in food analysis

What is transmittance?
What is the equation for calculating transmittance?

Answer 10

Transmittance measures the amount of light that passes through a material (unabsorbed).

• Transmittance = P/P_0, where P = power of exit beam (light going out) , P_0 = power of incident beam (light coming in)

Question 11

6.2. . UV-Vis spectroscopy in food analysis

How do I convert transmittance (fraction form) into absorbance?

Answer 11

Absorbance = -log(transmittance)

Question 12

6.2. . UV-Vis spectroscopy in food analysis

How do I convert transmittance (%) into absorbance?

Answer 12

Transmittance = 2-log (T%)

Question 13

6.2. . UV-Vis spectroscopy in food analysis

In experiments, why can’t absorbance be more than 1?

Answer 13

If absorbance is more than 1: - the curve becomes non linear and there is high relative error (because the spectrophotometer cannot detect the transmitted light accurately since 90% of light is being absorbed)
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- Beer Lambert’s law will not hold true, thus cannot calculate conc

Question 14

6.2. . UV-Vis spectroscopy in food analysis

What does it mean when my sample gives me an absorbance of more than 1? What can I do to solve this?

Answer 14

It means the sample is too concentrated. I can dilute my sample to fit the linear range of the absorbance against concentration curve.

Question 15

6.3. Procedural considerations for UV-Vis abs> sample prep

What are two important procedural considerations when preparing a sample for quantitative analysis by UV-Vis absorbance?

Answer 15

1) Dissolve sample in a solvent that is UV-transparent (does not absorb UV light)
- This ensures that absorbance readings is only due to analyte absorbing the UV light, and not due to other intefering compounds
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2) Filter and remove a particulate matter, which can block path of light and absorb the light, affecting absorbance readings.

Question 16

6.3. Procedural considerations for UV-Vis abs> ref solution

What is a reference solution? [2]

Answer 16

A reference solution is a solution that :
1. Is same as the solvent used to dissolve the sample
2. The solvent, but without target analyte in it (to set a baseline absorbance value)

Question 17

6.3. Procedural considerations for UV-Vis abs> cuvette solution

What 3 cuvette materials are commonly used, and which is the most common?

What property must cuvettes possess?

Answer 17

1. Quartz
2. Fused silica
3. Plastic (most common)

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• Cuvettes must be UV-transparent so that they dont intefere with absorbance readings

Question 18

6.3. Procedural considerations for UV-Vis abs> wavelength

Which wavelength should be chosen to measure absorbance and quantify concentration of an analyte? [2]

Answer 18

1. Wavelength with the highest peak/absorbance = maximum sensitivity
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2. Wavelength at which the difference between absorbance between target analyte and interfering compounds are the largest

Question 19

6.3. Procedural considerations for UV-Vis abs> cal curve

When costructing a calibration curve, you must have at least how many serial dilutions?

Answer 19

5

Question 20

6.3. Procedural considerations for UV-Vis abs> cal curve

Where should the expected concentration of the unknown sample fall in relation to the calibration curve?

Answer 20

The expected concentration of the unknown sample must fall within the range covered by the calibration curve.

Question 21

6.3. Procedural considerations for UV-Vis abs> cal curve

Why is it ideal to prepare standard solutions at the same time as the unknown sample?

Answer 21

To ensure consistency in reagent preparation and minimize variation (=higher precision) between the standard and the unknown sample.

Question 22

6.3. Procedural considerations for UV-Vis abs> intefering compound

What are the 3 types of intefering compounds in UV-Vis?

Answer 22

1. Compounds that absorb at same wavelength as target analyte
2. Compounds that intefere with absorbance of analyte (by chemically / physically reacting with analyte and enhance / reduce absorbance of analyte)
3. Compounds that react with modifying agents that are supposed to be specific to the target analyte.

Question 23

6.3. Procedural considerations for UV-Vis abs> intefering compound

What is the purpose of adding modifying agents to sample in UV-Vis?

Answer 23

Modifying agents are added to specifically react with analytes to produce a measurable change (e.g. colour change)

Question 24

6.3. Procedural considerations for UV-Vis abs> std addition

How to conduct a standard addition protocol? (e.g. target analyte : urea in urine)

Answer 24

1. Take your urine sample with unknown urea concentration and measure absorbance
2. To this sample, add a known concentration of urea (target analyte) and measure absorbance.
3. Keep adding the same known concentration of urea to sample, until a minimum of 5 data points are obtained
4. Plot standard curve of absorbance against added conc of urea, and extrapolate until where y-axis = 0 (absorbance = 0) and get x-intercept . This gives you the original concentration of target analyte in clean matrix
   -bc tho u measure abs of yr sample, the conc of urea calc frm abs of sample cld b higheror lower than actual conc, due to interference. Thus std addition helps to account for matrix effect.

Question 25

6.3. Procedural considerations for UV-Vis abs> std addition

What is the main purpose of using a standard addition protocol, rather than a external calibration method?

Answer 25

It helps to correct for matrix effects.
- More accurate because the known analyte is added to sample matrix (which may interfere with signal).

Question 26

6.3. Procedural considerations for UV-Vis abs> std addition

From standard additional protocol, absorbance against concentration of analyte is plotted. How to find the concentration of analyte in the absence of matrix?

Answer 26

Back-extrapolate the graph, and find the x-intercept. The absolute value is the actual concentration of analyte found in the sample, in the absence of matrix.
- because when measuring analyte concentration in a matrix, the absorbance could be higher due to inteference.
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- by adding known amounts of the analyte and plotting the results, you can isolate and correct for these matrix effects. The x-intercept represents the analyte concentration as if the matrix was not present.

Question 27

6.3. Procedural considerations for UV-Vis abs> instrument

What is indiscriminate insturment error and when can it be observed?

Answer 27

It is instrument noise, where there is random fluctations in the UV-Vis spectrophotometer.
- It can be observed when the absorbance of the same sample is measured a few times, a different absorbance value is obtained everytime due to random fluctuations. (Or when u open and close the door of the machine, the abs machine fluctuate etc)

Question 28

6.3. Procedural considerations for UV-Vis abs> instrument

Why is it important to ensure that absorbance does not exceed 1? (relative error & transmittance)

Answer 28

When absorbance does not exceeds 1, there is low relative error since there is intermediate transmittance that can be accurately detected by the UV-Vis spectrometer.
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- At too high transmittance values (concentration too low = absorbs less = transmits a lot) OR too low transmittance values (concentration too high = absorbs more = transmits less), the detector in the uv-vis spectrometer cannot accurately detect absorbance

Question 29

6.3. Procedural considerations for UV-Vis abs> instrument

What does it mean by the UV-Vis spectrometer uses monochromatic light?

Answer 29

It means that the light only has 1 specific wavelength.

Question 30

6.3. Procedural considerations for UV-Vis abs> instrument

What is the difference between a single beam and double beam UV-Vis spectrometer ?

Answer 30

• Single beam : light passes through as 1 beam, can only measure absorbance of samples 1 by 1
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• Double beam : there is a light splitter that splits the monochromatic light into 2 beams, thus absorbance of 2 samples can be measured at the same time

Question 31

6.3. Procedural considerations for UV-Vis abs> instrument

In a double beam uv-vis spectrometer, the light gets split into 2 beams of different wavelengths. True or false?

Answer 31

False, the 2 beams of light are still at the same wavelength as UV-Vis spectrometers utilises monochromatic light

Question 32

6.3. Procedural considerations for UV-Vis abs> instrument

What is the main benefit of double beam uv-vis spectrometer over the single beam uv-vis spectrometer?

(Hint : random error)

Answer 32

Double beam less susceptible to random fluctations (random error) and drifts, leading to more reliable results.
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- Because in double beam, can measure absorbance of 2 samples at once, won’t need to keep opening and closing the machine

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- in single beam, assuming there is 1 compartment only (in lab, the UV-Vis spectrophotometer has like 6 compartments), samples have to put in 1 by 1 and measure absorbance 1 by 1. Thus, since machine is opened and closed more often, there will be more random fluctutations

Question 33

6.3. Procedural considerations for UV-Vis abs> instrument

What is a disadvantage of double beam spectrometer over a single beam spectrometer?

Answer 33

Double beam spectrometer requires regular calibration and alignment, more costly. Also more complex.

Question 34

6.4. Infrared spectroscopy

What is the difference between Near Infra-red (NIR), Mid Infra-red (MIR) and Far Infra Red (FIR)?

State any values if needed.

Answer 34

• NIR : lowest wavelengths (highest energy) –> 780-2500 nm. Measures energy states of covalent bonds.
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• MIR : intermediate wavelengths –> 2.5 to 25µm. Measures energy states of different functional groups
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• FIR : Highest wavelengths –> 25µm

Question 35

6.4. Infrared spectroscopy

IR spectroscopy involves using light at only 1 specific wavelength. True or False?

Answer 35

False

Question 36

6.4. Infrared spectroscopy

What is the idea behind Fourier Transformation?

Answer 36

• Light of all wavelengths arrive at inteferometer (intefere) (detector) simultaenously
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• The in teferometer consists of beam splitters and moving mirrors (left-right / up-down) to split light into different wavelengths –> the output is initially a complex inteferogram

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- Fourier transformation (mathematical transformation) converts the results into an FTIR spectrum (transmittance against wavenumber, absorbance against wavenumber)
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- At certain wavelengths, specific functional groups (found in carbs/proteins/fat/water) will absorb light. Thus, at these wavelengths, transmittance is low and absorbance is high (peaks). From the wavelengths where the absorbance peaks, you can tell what functional groups are there, and their vibrational and rotational states.

Question 37

6.4. Infrared spectroscopy > Transmittance / reflection modes

Transmission mode is used primarily for what kinds of samples?
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How does transmission mode work?

Answer 37

• Liquid samples

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- the sample is dispersed and mixed with chemically inert halide salt (to disperse the liquid bc liquid particles v closely packed tgt, resulting in v high absorbance and high relative errir)
- An IR beam (light) is passed through sample that is placed in between 2 IR transparent windows
- It measures transmittance

Question 38

6.4. Infrared spectroscopy > Transmittance / reflection modes

Why are cells of pathlength 0.1-1mm used in tranmission mode?

Answer 38

Absorptivity coefficients are high.
- Since A = acL, when a is high, L should be reduced to ensure absorbance does not exceed 1.

Question 39

6.4. Infrared spectroscopy > Transmittance / reflection modes

What kind of samples is Attenuated Total Reflectance (ATR) used for?

Answer 39

All kinds of samples

Question 40

6.4. Infrared spectroscopy > Transmittance modes

How does ATR work?

Answer 40

• Samples are placed onto crystals with high refractive index.
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• Incident ray is passed through the crystals with high refractive index. As the ray passes through to the interface between sample and crystal, certain functional groups in food sample interacts with ray and absorbs certain wavelengths, and evanscent / standing waves are formed.
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• The attenuated ray then leaves the crystal, and attenuated ray gives unique info of functional groups present in sample. (degree of attenuation, i.e. reduction in energy of light** at specific wavelengths corresponds to the presence of those functional groups.)

Question 41

6.4. Infrared spectroscopy > Transmittance / reflection modes

What is the main benefit of Attenuated Total Reflectance (ATR)?u

Answer 41

Minimal sample prep required. Since radiation is not passed through samples :
- dont need to dilute samples / samples dont have to be thin

Question 42

6.4. Infrared spectroscopy > Transmittance / reflection modes

What kind of food products is diffused reflection used for?

Answer 42

Powdered products, where there are many spaces in between and light gets reflected in all different directions.

Question 43

6.4. Infrared spectroscopy > Transmittance / reflection modes

What is meant by :
1. Specular reflected light
2. Diffuse reflected light

Answer 43

• Specular reflected light : light that exits at the same angle as incident ray
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• Diffuse reflected light : light that exits at different angle as incident ray

Question 44

6.4. Infrared spectroscopy > Transmittance / reflection modes

How does diffused reflection mode work?

Answer 44

• Sample is placed in an integrating sphere, and light shines through the sphere. Due to the many spaces within the powdered products, light is reflected in all directions

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- Detectors are put along the circumference of the integrating sphere to detect the diffuse reflected light (no detector put at the opening because not interested in measuring the specular reflected light).
- reflectance spectra obtained, convert into abs spectra, calc conc

Question 45

6.5. Infrared spectroscopy in food analysis

In infrared spectroscopy, which wavelengths should I look at if I want to quantify :
1. Moisture content
2. Protein content
3. Fat content
4. Carbohydrate content

Answer 45

Wavelengths to look at :
1) Moisture content –> wavelengths where –OH absorb
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2)** Protein content** –> wavelength where –NH absorb (because only protein has N)
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3) Fat content –> wavelength where –CH absorb (fat is in triglyceride form, esters of alcohol + FA, no –OH. Even if there is –OH in fatty acid chain, inteference is negligible.

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4) Carbohydrate content –> wavelength where –CH & –OH absorb

Question 46

6.5. Infrared spectroscopy in food analysis

What are the general steps in quantifying (e.g.protein) using FTIR spectroscopy?

Answer 46

1) Put in standards with known protein concentrations through the FTIR machine → obtain spectral data [At least 5 different concentrations for calibration].

• Narrow down the spectral data to wavelengths where amide bonds primarily absorb light, so that it is more specific to protein (i.e. wavenumber of 1750 cm-1)
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  2) Use a statistical methods (by software) to correlate spectral data to concentration of the sample as a calibration curve.
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  3) Validate the model : test calibration model with additional samples to ensure accuracy
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  4) Adjust and finetune the model : add necessary adjustments to improve model’s accuracy and reliability
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  5) Put in your milk sample with unknown protein concentration. Obtain the spectral data
  Spectral data of milk sample with unknown protein concentration is compared to the spectral data of milk sample with known protein concentration, and concentration of protein in milk sample is obtained through calibration curve.

Question 47

6.5. Infrared spectroscopy in food analysis

In NIR, the beam of light is split into different wavelengths, and measurements are made across the whole NIR spetrum (many wavelengths). Why is it necessary to make measurements at more than 1 wavelength? [3]

Answer 47

1) Improved accuracy : diff wavelengths provide information on diff components of the sample, enhancing overall accuracy
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2) Noise reduction : measuring at multiple wavelengths help average out noise, improve S/N ratio
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3) **Component differentation **: - different components in a sample can absorb at the same wavelength (water and carbohydrates contain –OH groups, which can absorb light at similar wavelengths, leading to interference). By measuring at multiple wavelengths, it is possible to differentiate between overlapping signals and more accurately quantify specific components in the presence of others. This approach allows for better separation of signals and reduces the impact of interferences from other food components.

Question 48

IMPT QN

What is the units for molar extinction coefficient, ε?

Answer 48

M^-1.cm^-1
ε = A/cL
- absorbance has no units
- Usually concentration,c, is given in mM so need to convert to M by x10^-3!!