Gilchrist and Reynolds
180
plastic that is often polycarbonate and disposable. Of course, it is essential that the cuvette
chosen is suitable for the application and experiment and that it is clean, and handled with
maximum care.
Checking the transmission of the cuvette and solvent in the wavelength range of excitation and emission is critical. This should be done using a quality absorption spectrophotometer. After all, if there is no transmission of light to the sample or the emission signal
is blocked to the analyzing channel of the fluorimeter then there will be no fluorescence
measurement. Plastic cuvettes are useful because they are inexpensive, disposable, do not
require cleaning and are robust. Even so, such cuvettes are unsuitable for some applications
such as:
They demonstrate strong polarization effects and are therefore not suitable for any meas•
urement involving polarizers.
They can be dissolved by many organic solvents commonly used for fluorescence
•
measurements.
In most cases, cuvettes should be used with stoppers, especially if the solvent is corrosive or volatile. This will help to minimize the possibility of spills and to maintain sample
concentration. The optical windows that form the cuvette should not be touched with bare
hands (powder-free gloves are often used), as human fingerprints exhibit fluorescence. In
addition, careful and thorough cleaning of fused silica and glass cuvettes is fundamental
to accurate measurements. For this, it is common to soak cuvettes for several hours in a
nitric acid solution followed by a thorough rinse with deionized water. Before use, cuvettes
should be rinsed several times with the solvent to be used in the experiment. An alternate
method of cleaning glassware is to use a detergent solution but this has the risk that the
detergent may have some fluorophore in it. Therefore, it is sometimes appropriate to make
a fluorescence measurement on a dilute solution of any detergent that is used for cleaning purposes. At the same time, please note that there are whitening agents in many filter
papers, clothes, and tissues and these can introduce contaminant fluorophores.
5.4.15 Solvents and Contaminants
The fluorescence emission from a fluorophore can be strongly dependent on its local
environment. As such, choice of solvent and solvent purity are very important. Incorrect
choice of solvent can cause spectral shifts and changes in the peak shapes and reduce samples emission. As a matter of good practice, high-purity solvents should be used and then
checked before use by checking their absorption and fluorescence properties. Handling
techniques should be employed to minimize the possibility of stock solutions becoming
contaminated – even very small levels of contaminants may cause appreciable background
signals. As such, solvents should not be stored in plastic containers and should be regularly
screened for contaminations before use. Regular screening of the solvent using a fluorimeter will also indicate the position and magnitude of any Raman signal from the solvent or
contaminants in the analysis spectral range. Contamination opportunities exist at each stage
180
plastic that is often polycarbonate and disposable. Of course, it is essential that the cuvette
chosen is suitable for the application and experiment and that it is clean, and handled with
maximum care.
Checking the transmission of the cuvette and solvent in the wavelength range of excitation and emission is critical. This should be done using a quality absorption spectrophotometer. After all, if there is no transmission of light to the sample or the emission signal
is blocked to the analyzing channel of the fluorimeter then there will be no fluorescence
measurement. Plastic cuvettes are useful because they are inexpensive, disposable, do not
require cleaning and are robust. Even so, such cuvettes are unsuitable for some applications
such as:
They demonstrate strong polarization effects and are therefore not suitable for any meas•
urement involving polarizers.
They can be dissolved by many organic solvents commonly used for fluorescence
•
measurements.
In most cases, cuvettes should be used with stoppers, especially if the solvent is corrosive or volatile. This will help to minimize the possibility of spills and to maintain sample
concentration. The optical windows that form the cuvette should not be touched with bare
hands (powder-free gloves are often used), as human fingerprints exhibit fluorescence. In
addition, careful and thorough cleaning of fused silica and glass cuvettes is fundamental
to accurate measurements. For this, it is common to soak cuvettes for several hours in a
nitric acid solution followed by a thorough rinse with deionized water. Before use, cuvettes
should be rinsed several times with the solvent to be used in the experiment. An alternate
method of cleaning glassware is to use a detergent solution but this has the risk that the
detergent may have some fluorophore in it. Therefore, it is sometimes appropriate to make
a fluorescence measurement on a dilute solution of any detergent that is used for cleaning purposes. At the same time, please note that there are whitening agents in many filter
papers, clothes, and tissues and these can introduce contaminant fluorophores.
5.4.15 Solvents and Contaminants
The fluorescence emission from a fluorophore can be strongly dependent on its local
environment. As such, choice of solvent and solvent purity are very important. Incorrect
choice of solvent can cause spectral shifts and changes in the peak shapes and reduce samples emission. As a matter of good practice, high-purity solvents should be used and then
checked before use by checking their absorption and fluorescence properties. Handling
techniques should be employed to minimize the possibility of stock solutions becoming
contaminated – even very small levels of contaminants may cause appreciable background
signals. As such, solvents should not be stored in plastic containers and should be regularly
screened for contaminations before use. Regular screening of the solvent using a fluorimeter will also indicate the position and magnitude of any Raman signal from the solvent or
contaminants in the analysis spectral range. Contamination opportunities exist at each stage