Lecture 6 : Molecular Spectrometry Flashcards
(48 cards)
6.1. . Basic principles of spectroscopy
What are the 3 main ways that matter can interact with light?
- Absorption
- Emission – material glows and emits light when light is shined on it
- 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)
6.1. . Basic principles of spectroscopy
What are the 3 energy states that molecules can possess when interacting with light?
- Electronic energy
- Vibrational energy
- Rotational energy
6.1. . Basic principles of spectroscopy
What is electronic energy? What does it mean when a molecule has higher electronic energy?
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
6.1. . Basic principles of spectroscopy
What is vibrational energy? What is meant by higher vibrational energy?
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
6.1. . Basic principles of spectroscopy
What is rotational energy? What is meant by higher rotational energy?
Rotational energy is amount of energy a molecule possesses due to it spinning around an axis.
- Higher rotational energy = molecule can spin faster
6.1. . Basic principles of spectroscopy
What is the relationship between energy and wavelength?
Energy and wavelength is inversely proportional.
- At shorter wavelength, frequency is higher, thus energy level is higher
6.1. . Basic principles of spectroscopy
What are the 3 main steps for a molecule to emit fluorescence when it interacts with light?
- Absorption of light (excitation) : electrons in molecules get excited to a higher electronic state
- 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 - Emission : Fluorescence is emitted as the electron returns to the ground state.
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?
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
6.2. . UV-Vis spectroscopy in food analysis
What are the 2 equations for Beer Lambert’s law in calculating absorbance?
- A = acL ; where a = absorptivity, c = concentration, L = pathlength
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- A = εcL, ε = molar extinction coefficient, c = concentration, L = pathlength
6.2. . UV-Vis spectroscopy in food analysis
What is transmittance?
What is the equation for calculating transmittance?
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)
6.2. . UV-Vis spectroscopy in food analysis
How do I convert transmittance (fraction form) into absorbance?
Absorbance = -log(transmittance)
6.2. . UV-Vis spectroscopy in food analysis
How do I convert transmittance (%) into absorbance?
Transmittance = 2-log (T%)
6.2. . UV-Vis spectroscopy in food analysis
In experiments, why can’t absorbance be more than 1?
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
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?
It means the sample is too concentrated. I can dilute my sample to fit the linear range of the absorbance against concentration curve.
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?
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.
6.3. Procedural considerations for UV-Vis abs> ref solution
What is a reference solution? [2]
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)
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?
- Quartz
- Fused silica
- Plastic (most common)
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- Cuvettes must be UV-transparent so that they dont intefere with absorbance readings
6.3. Procedural considerations for UV-Vis abs> wavelength
Which wavelength should be chosen to measure absorbance and quantify concentration of an analyte? [2]
- Wavelength with the highest peak/absorbance = maximum sensitivity
<br></br> - Wavelength at which the difference between absorbance between target analyte and interfering compounds are the largest
6.3. Procedural considerations for UV-Vis abs> cal curve
When costructing a calibration curve, you must have at least how many serial dilutions?
5
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?
The expected concentration of the unknown sample must fall within the range covered by the calibration curve.
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?
To ensure consistency in reagent preparation and minimize variation (=higher precision) between the standard and the unknown sample.
6.3. Procedural considerations for UV-Vis abs> intefering compound
What are the 3 types of intefering compounds in UV-Vis?
- Compounds that absorb at same wavelength as target analyte
- Compounds that intefere with absorbance of analyte (by chemically / physically reacting with analyte and enhance / reduce absorbance of analyte)
- Compounds that react with modifying agents that are supposed to be specific to the target analyte.
6.3. Procedural considerations for UV-Vis abs> intefering compound
What is the purpose of adding modifying agents to sample in UV-Vis?
Modifying agents are added to specifically react with analytes to produce a measurable change (e.g. colour change)
6.3. Procedural considerations for UV-Vis abs> std addition
How to conduct a standard addition protocol? (e.g. target analyte : urea in urine)
- Take your urine sample with unknown urea concentration and measure absorbance
- To this sample, add a known concentration of urea (target analyte) and measure absorbance.
- Keep adding the same known concentration of urea to sample, until a minimum of 5 data points are obtained
- 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.
- 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.
- 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
- 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
- Because in double beam, can measure absorbance of 2 samples at once, won't need to keep opening and closing the machine
- 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
- **MIR** : intermediate wavelengths --> **2.5 to 25µm**. Measures energy states of different functional groups
- **FIR** : Highest wavelengths --> **25µm**
- 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
- Fourier transformation (mathematical transformation) converts the results into an FTIR spectrum (transmittance against wavenumber, absorbance against wavenumber)
- 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.
How does transmission mode work?
- 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
- 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.
- 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.)
- Diffuse reflected light : light that exits at different angle as incident ray
- 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
2)** Protein content** --> wavelength where **--NH** absorb (because only protein has N)
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.
4) **Carbohydrate content** --> wavelength where **--CH & --OH** absorb
2) Use a statistical methods (by software) to correlate spectral data to concentration of the sample as a calibration curve.
3) Validate the model : test calibration model with additional samples to ensure accuracy
4) Adjust and finetune the model : add necessary adjustments to improve model's accuracy and reliability
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.
2) **Noise reduction** : measuring at multiple wavelengths help average out noise, improve S/N ratio
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.
