Question

1. What is the effect of slit width variation in Fluorescenece Spectrum?

2. Why is the fluoresence measured 90 degree from the exiciting light source?

3. Why must the quinine solutions be acidified?

Answer 1

• In Fluorescence spectroscopy, we can measure the concentration of a sample based on their property of fluorescence.
• Molecules have various enery levels and each of these electronic energy leveles are associated with a number of vibrational energy levels.
• A light source ( usually UV light) of a apecific wavelength is passed through a sample. The sample absorbs a photon and an electro from the ground state enery level gets excited to one of the vibrational energy levels in the excited states. Due to number of collisions, the electron drops down to the lowest vibrational state of the excited state. Eventually, the electron drops down to the vibrational states of the ground state.
• In this process it emits a photon. This gives rise to both absorption and emission spectra.

1)

• Basically, slit width is the rectangular spaces through which light enters and exits a monochromator in a spectrometer.
• It helps in effeciently separating close wavelengths of a light source.
• Slit width will alter the fluoresence of a sample. When we increase the slit width, the intensity of fluorescence increases as more exciting light sources passes through the sample. But, this decreases the resolution of wavelength. The monochromator will not effectively separate so many wavelengths from the broad light beam.
• When we decrease the slit width, the opposite happens. The fluorescence intensity decreases but the targeted wavelength can be obtained easily by the monochromator.

2)

• Monochromators are used in the instrument.
• The monochromator helps to transmit light of the targeted wavelength to the sample, among the different wavalengths that come from the exciting light source.
• As mentioned in the question, fluorescence is measured 90 degrees from the light source. This position is preferred over placing the sample in line of the excitation source.
• This is to avoid the interference of the unwanted wavelengths with the detected fluorescence light.
• The monochromator will transmit this unwanted wavelength in the form of stray light. So that this light does not interfere in the measurement of fluoresence, sample is placed at 90 degrees to the light source.

3)

• Acidified quinine solution is an ideal standard for fluorescence spectroscopy
• This solution has optimum quantum yield. Quantum yield is the ration of photons absorbed to the photons emitted by a sample.
• Also, optimum absorbance occurs for quinine in acidified solution. And, quinine decomposes in water. Hence, it is necessary to acidify the solution.