Fluorescence - Pre-Laboratory Questions Explain why a UV-vis spectrophotometer c
ID: 1036349 • Letter: F
Question
Fluorescence - Pre-Laboratory Questions Explain why a UV-vis spectrophotometer could not be used to measure the amount of quinine in tonic water. (Hint: look at the ingredients in tonic water.) The quinine standards and unknown were made in an acidic solution. Explain why this is important. What errors occur when fluorescence measurements are made using solutions that are too concentrated? What physically causes this? What are two major differences between a UV-vis spectrophotometer and a fluorimeter?Explanation / Answer
uv visible spectrophotometer could be used to measure the amount of quonine in tonic water, but that depends on the ingredients in tonic water. As the ingredients aren't given we can't tell why we can't use uv visible spectrophotometer for this specific experiment.
Difference between UV - vis spectrophotometer and a fluorimeter.
Microplate readers can be either fluorometer or spectrometer or both, as is more common today. If you have something with emission/excitation values then, yes it is a fluorescent marker, so use a fluorometer.
Fluorometers work by shining a light of shorter frequency (higher energy) onto the marker, which then emits light at a longer frequency (lower energy)... e.g. blue excitation will give green fluorescence for an alexafluor 488 dye (488=excitation frequency). The machine uses a filter between light source and the sample to provide the excitation light and between the sensor and the sample to block out light from the excitation, so that only emission light gets through.
Absorbance kind of works in reverse to this. A sample of colour yellow, is not yellow itself, it is reflecting light that is yellow and absorbing the rest, so you could shine a light at it and measure in any colour other than yellow, how much light is getting through the sample on the other side. In this case you are looking for a loss of light or absorbance of the sample. This can be done for the whole spectrum or only at a particular wavelength(s) (e.g. DNA and RNA are measured based on absorbance at 260 and 280 nm). The light for specific wavelengths can be filtered before or after the sample, but usually before.
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It is often simpler to think of it like this: absorbance passes through but fluorescence bounces back.