Question 1: Please describe in details how to measure a sample’s quantum yield on the condition

that the fluorimeter does not have an integral sphere.

Answer:
To measure a sample's quantum yield using a fluorimeter that does not have an integral sphere, you can
follow the steps outlined below:To measure a sample's quantum yield using a fluorimeter that does not
have an integral sphere, you can follow the steps outlined below:
1. Set up the fluorimeter: It also important to make sure that the fluorimeter is standardized, as well as
correctly placed based on the manufacturer’s recommendations. Ensure that the fluorescence wavelengths
are properly chosen from the excitation moiety for the sample being analyzed.
2. Prepare the sample: After dilution, filter the sample and dilute it to the proper ratio in a correct solvent.
In regard to this, it was crucial to confirm that the sample was dissolved in a clear solution and no
precipitation or other interferences that could affect the measurement were present.
3. Measure the absorbance: To determine the absorbance of the sample at the EX, either use an absorption
spectrometer or, if one is available, the absorbance accessory in the fluorimeter. This step is needed to
estimate the number of absorbed photons, Earlier, only diffuse reflectance values were measured by the
radiance probes.
4. Measure the fluorescence intensity: All you need to do is put the sample in a cuvette and then.place it
in the fluorimeter. At the desired emission wavelength, quantify the emission signal coming from the
sample or the fluorescence intensity.
5. Measure the reference intensity: This is done by using a known standard fluorophore where the
fluorophore is diluted with a solvent similar to the sample to produce the reference solution. Prepare
reference solution with a quinine sulfate concentration of 1 µg/mL and measure the fluorescence intensity
under similar conditions with the sample.
6. Calculate the quantum yield: To determine the QY, the following formula can be used: QY = (amount
of fluorescence/ amount of light absorbed) × 100
The equa-tion for QY can be expressed as: QY=(Φs / Φr) * (I_s / I_r) * (n_r^2 / n_s^2)
- Φs: This was done by measuring the quantum yield of the sample.
- Φr: Quantum yield of the reference standard means the first reference value of the quantum yield on
which the calculation of all subsequent values, both on the same and on adjacent scales, is based.
- I_s: Decrease in the fluorescence intensity of the sample
- I_r: relative intensity of the reference standard fluorescence
- n_r: Refractive index of the reference standard (this number is specified by its manufacturer and was
used here to calculate the actual thickness of the reference layer)
- n_s: Refractive index of the sample is refers to the ratio of the speed at which light is moving inside the
sample to the speed of light in vacuum.
In this case, if you replace these values into the formula, you can present the quantum yield of the sample.
7. Repeat the measurements: For the exact and consistent results, I normally suggest taking multiple
measurements of the fluorescence intensity and quantum yield, then take an average to have the most
credible outcome.

Question 2: Please describe in details how to determine a sample’s molar extinction coefficient and
also give the justification that your measurement is accurate.
Answer:
To determine a sample's molar extinction coefficient, you can follow these steps: To determine a sample's
molar extinction coefficient, you can follow these steps:
1. Understand Beer's Law: Beer’s Law has specified that the extent of absorbance is dependent with
sample concentration, and on the path length through which light passes through the sample. The equation
for Beer's Law is A = εbc, where A is the absorbance, ε is the molar extinction coefficient, b is the path
length, and c is the concentration
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2. Prepare a series of standard solutions: Get ready a series of solutions for which the concentration of the
sample is already determined. These solutions should have various concentrations to mark the three points
of the calibration curve.
3. Measure the absorbance: Make an absorbance of the solutions of each standard by using a
spectrophotometer at a particular wavelength. The spectrophotometer is the one which gives the
absorbance values.
4. Plot a calibration curve: Prepare a graph on the absorbance against the concentration of the standard
solutions on the x-axis and the y-axis, respectively. This will give you a calibration curve hence real
accurate point out assessment of someone’s PC-Water relationship.
5. Determine the slope of the calibration curve: Based on the calibration curve analysis, determine the
slope by using the method of first derivative and divide by the change in concentration. The slope of the
plot is the molar extinction coefficient of the compound in the solution, which is denoted as ε.
6. Justification of measurement accuracy: If your measurement is not tight and you want to get accurate
results please consider the following:
a. Precision and accuracy of the spectrophotometer: Ensure that the spectrophotometer used to measure
absorbance is functioning optimally and is standardized. It is also important to regularly do calibration
checks to the equipment and observe rigorous quality check measures.
b. Replicates and averaging: Carry out multiple readings to each standard and arrive at the mean value of
absorbance for each standard solution. This minimizes some of the chances of getting random error and
this is one way of enhancing reliability of the results.
c. Blank correction: The absorbance of each standard solution should of course be corrected for blank
absorbance - which is the absorbance of a solution containing all the reagents used in calibration but not
the analyte. This minimizes on background absorbance so that true absorbance values can be obtained.
d. Quality control standards: Find out ways and means of incorporating quality control measures into your
analyses to ascertain the validity and accuracy of your measurements. Not all standards should be used at
their projected concentrations; the desired and prepared concentrations can also be used for validating
purposes.
Question 3: Why does the emission and excitation spectrum have to be correct? And how to correct
them?
Answer:
The emission and excitation spectra have significance in spectroscopy in that they offer additional data in
ascertaining the actions of molecules and their responses to photons. These spectra allow researchers to
learn about the behaviour of molecules in the context of their absorption and emission, which is important
for processes such as fluorescence microscopy and chemical and materials analysis.
Why do scientists devote considerable effort to experimentally determining both the emission and
excitation spectra of molecular systems?
Determining molecular structure: Spectroscopy is often used to examine the emission and excitation
spectra to determine the potential electronic structures and energy levels of molecules. From these
spectra, one can determine the functional groups of the molecule can be deduced, and certain details
relating to the electron transitions.
Studying fluorescence properties: The process that occurs when a molecule absorbs light at a certain
wavength and re-emits it at a longer wavelength is known as fluorescence. Emission spectrum is
important when the molecule of interest is to emit light at such wavelength and when fluroescent probe or
any fluroescent process has to be studied.
Quantifying analyte concentration: In analytical chemistry, the absorbance and emission are the
parameters to determine the concentration of a particular analyte in the sample. By detecting the
absorbance or fluorescence intensity at these wavelengths, it is possible to calculate the relation between
the intensity and analyte concentration according to the Beer-Lambert Law.
Characterizing materials: Optical properties in the form of emission and excitation spectra are useful for
optoelectronic applications including dyes, nanoparticles and semiconductors. These spectra give details
about energy band structure, electronic transitions as well as optical characteristics which are
constructively used while determining the usage and enhancement of the material.
Emission and excitation spectra correction: Let and be the measured excitation and emission spectra
respectively and be the emission and excitation spectra of the sample respectively.
Calibration: Finally, the following is an important point to note; calibration is crucial in the determination
of both emission and excitation spectra. Depending on this formula, reference material with known
properties is utilized and their spectra are compared to the measured spectra. By correcting the
instrument’s parameters and the spectra themselves by means of correction coefficients, one can
successfully shift them to the reference ones.
Instrument optimization: It is important to properly set up the spectroscopic instrument to balance its
response for improved spectra. This involves fine-tuning some settings like the size of the ablation slit,
the exposure duration, and detector gain to get rid of unwanted noise or distortion on the spectrum.
Background subtraction: Instrumental noise, background from solvent, scattering or any other source
contamination can interfere with accurate spectra. By subtracting the background signal which is
measured simultaneously with the sample one can eliminate these interferences and enhance the quality
of the emission and excitation spectra.
Data analysis and interpretation: Similarly, correcting targets for emission and excitation spectra requires
a comprehensive analysis of the data as well as proper decoding of the results. These are in a way of
excluding all the outliers or artifacts, have to apply the correct mathematical models or functions and
fitting algorithms, to contemplate on the affects such as temperature, pH, solvent etc, upon the spectra.
Question 4: Why do the solvents such as acetone (DMF, DMSO and CH3COCH3) have a long
cutoff at about 270 nm to 330 nm?
The solvents used are acetone, DMF (dimethylformamide), DMSO (dimethyl sulfoxide), and
CH3COCH3 (also known as acetone) and it is to be noted that while all these solvents have a long cutoff
in the wavelength of about 270 nm to 330 nm. This entails that they reflect light in the remaining part of
the electromagnetic spectrum or in this case, they transcribe light in this range of wavelengths. As to the
absorption behavior in these solvents, they might be identified from the electronic structure and molecular
property of the solvents.
Electronic Structure:
Acetone: Combined with the fact that acetone includes a carbonyl group (C=O) in its molecular formula.
The absorption in the UV range is mostly contributed by π→π* transition of carbonyl group The π→π*
transition moves to a lower energy region as the energy gap between the Homodine and LUMO
decreases.
DMF: It also carries a carbonyl group; the fact it shares with acetone. Hence, the absorption in the UV
range can also be due to the π→ π* transition of the carbonyl group of the molecule.
DMSO: It can be seen that DMSO has sulfur atom present in its structure which differentiates it from
both acetone and DMF and has a new electronic transition. The absorption in the UV range is mainly
n→π * transition of the sulphur atom.
CH3COCH3 (acetone): Acetone also has functional groups and one of the functional groups contains a
carbonyl group as seen in the case of DMF above. Also, the absorption in the UV range is contributed to
the π→π* transition of the carbonyl group.
Molecular Properties:
Polarity: DMF and DMSO; the presence of electronegative atoms (oxygen or sulfur) within the molecules
make these solvents polar. This polarity influences the electronic transitions and results in the range of
absorption in the UV region.
Molecular Weight: It is also important to understand that solvents with a higher molecular weight are also
likely to show different levels of absorption. Traditionally, solvents that are characterized with higher
molecular weight have been found to have higher cutoff wavelengths. DMSO is one of the largest
solvents among the mentioned one and s and thus it has longer cutoff wavelength than that of acetone or
DMF. For the solvents acetone, DMF, DMSO, and CH3COCH3, this long cutoff at 270 nm to 330 nm is
attributed to the electronic transitions related to carbonyl group and sulfur atom of these solvent
molecules. The polarity and the molecular weight is also some factors that influence the absorption
characteristic of these solvents in the UV region.