Difference Between UV Spectrophotometer and Fluorescence Spectrophotometer

The primary components such as cuvettes, detectors, and recorders found in UV spectrometers are identical to those in a fluorescence spectrophotometer. The light emitted from the xenon arc lamp of the spectrophotometer is converted into intermittent light. The monochromator in the instrument converts the excitation light into monochromatic fluorescence, which is later irradiated on the photomultiplier tube used for the test sample.

UV spectrophotometer:

A UV-spectrophotometer is used for the quantitative monitoring of colored substances. The instrument employs hydrogen or deuterium lamps in the UV regions and deuterium or bromine tungsten lamps in the visible region. As far as a fluorescence spectrophotometer is concerned, it is used to test the emission and excitation spectrum and time scan of luminescent materials and does not require a display. The sensitivity is 2–3 folds higher than that of a UV-spectrophotometer.

How does a Fluorescence Spectrophotometer Work?

The light irradiated by the xenon lamp is converted into intermittent light with the help of the light cutter. The excitation light is modified to monochromatic fluorescence with the help of a monochromator and later irradiated on the photomultiplier tube used for sample measurement. The cam-driven, with the motor’s help, regulates the gratings present on the monochromator. It’s essential to fix the grating of the excitation light monochromator to an appropriate excitation length when depicting the fluorescence emission spectrum. Allow the monochromator to rotate and yield the fluorescence intensity of the signal of each wavelength to the recorder. The recorded spectrum is the fluorescence spectrum.

During the depiction of the fluorescence excitation spectrum, the fluorescence spectrophotometer is supposed to adjust and fix the monochromator’s grating to an appropriate fluorescence wavelength. The cam of the excitation light monochromatic port should be rotated to produce the intensity signal of excitation light of each wavelength to the recorder. For quantitative sample solution analysis, the excitation light monochromatic is affixed at the pre-specified excitation light wavelength, and the monochromator is reset to the selected fluorescence wavelength. The signal is recorded, which gives the fluorescence intensity.

How to use a fluorescence spectrophotometer?

Step 1: Press the power button present on the left side of the instrument. It will turn on the running indicator and xenon lamp indicator light on the right side.

  1. Leave the instrument switched on for 15 minutes and then turn on the computer and the fluorescence spectrophotometer. A green light indicates that the computer is connected to the instruments, and the work plan can be initiated. On the right side of the workstation, a vertical line of instructions appears; click “method” to develop a new measuring method. It can be utilized if it has been previously set.

Four different options are given in the general section:

  • Wavelength scan
  • Time scan
  • Photometric method
  • 3D scan.

An operator option is also available where it’s vital to enter the operator’s name. Soon, the instruments will present the model; if the experiments include the determination of sample group and allotment of the sample data, then it should be set in the space below comments. However, no sample data is required if a blank sample is used.

Operations in the Quantitation screen

The various quantitative types that can be chosen in quantitation are:

  • Peak area
  • Peak height
  • Derivative method
  • Ratio

Select the wavelength option.

The number of options includes single wavelength, dal wavelength, and three-wavelength calculations. The single-wavelength calculation should be selected. For the collaboration type, the options are none, first-order straight line, second-order curve, third-order curve, and segmented. The first order straight one should be selected.

If neither is selected, up to 6 wavelengths can be selected in the wavelength setting. If other options are selected, the maximum value is 3 wavelengths. In concentration, set the data unit as a percentage. Not many options are available for manual calibration or force curve through zero. The standard operation is necessary.

Fill in the digits after the decimal point as needed in the Digit after decimal option, and the input range is 0–3 is set to 0. The lower and upper limit concentrations are set to 0–1000. When the determined concentration exceeds the upper concentration limit, an “H” will be displayed on the average concentration array, and if the value is below the upper concentration limit, an “L” will be displayed.

Actions in the Instrument interface: 

There are three data modes are

  • Fluorescence intensity,
  • Luminescence intensity,
  • Phosphorescence intensity.

Fluorescence intensity should be selected.

Select the wavelength type in Wavelength: EX WL excitation light wavelength and EM WL emission light wavelength.  The experimenter will select both WL Fixed upon request. The desired wavelength is entered for the experiment.

EX: Excitation light slit width, EM: Emission light slit width. All choose 5nm.

PMT Voltage: It is used to regulate the voltage of the photomultiplier tube detector, selecting a value from 400–700V: 400V.

Auto statistic calculations: Calculate mathematical statistics in an automated manner. Set once you’ve entered your value. The number of samples, the mean, standard deviation, and coefficient of variation can be calculated. Set the device to 2.

Replicates: The number of replicate measurements can be between 1 and 20. It is set to 1 in this case.

Integration time: Average data values are recorded in a specified interval which provides stable data.

Setting time: 0.1s.

Scan delay time: The default delay scan time pre-specified in the instrument is in the input range of 0–9999s. The speed can be regulated manually without involving the scan delay time. The number of standards should be chosen in the standard interface and set the standard concentration. The Y-axis’s minimum (0) and maximum (100) values are selected.

Select the number of standards in the Standards interface, select the number in the space below, set the standard concentration, etc. Select the option to open a data processing window after the data collection. Finally, print the data by selecting the print report option in the report interface.

3) Press “OK” to feed the measurement interface once the method selection is complete.

Determine as per the specifications of the workstation. No method is set in the front. First, the reference sample is measured, followed by the unrelated sample. Soon after putting the blank sample securely in the detection chamber, the blank measurement option should be selected in the lower-left corner. Once the blank is measured, it’s time to put the sample in the detection chamber for measurement. The samples were measured twice to obtain an average value. The instrument produces a report on the test details. For inconsistent results, the samples can be re-measured.

4) To finish the measurement process, click the End button. Reports can be printed directly. The files can be saved for accessing later. If samples need to be measured, the monitor window can be closed, but the lamp should be left open. If no samples are left, turn off the lamp as well. The fluorescent light is switched off, and once the instrument cools down, the power is also turned off. Place the dust cover.


Why do two wavelengths need to be set when measuring fluorescence intensity with a spectrofluorophotometer?

The fluorescence spectrophotometer has two modes:

  • Scan Em with fixed Ex.
  • Scan Ex with fixed Em.

Ex is the excitation from the light source. Adjusting the excitation allows only a specified wavelength (254nm) to be used for exciting the sample. The fluorescence intensity displayed at various wavelengths is observed. Em is the excited light that is received by the receiver.

What steps should be taken if the excitation and emission wavelength are unknown?

The empirical wavelength is measured before the emission wavelength as it fluctuates slightly. Excitation wavelength cannot be determined without emission wavelength. The fluorescence samples have excitation wavelengths within 300–410nm. The fluorescent intensity pattern is mapped under the excitation wavelength. The wavelength of emission that corresponds to the spot with the highest fluorescence intensity is the value on the abscissa and is known as emission wavelength. To acquire a spectrum again, the fluorescence spectrophotometer places the emission wavelength above the excitation wavelength. The spectrogram shows maximum fluorescence intensity and the emission wavelength corresponding to the given intensity, which is the optimal excitation wavelength.

What is the difference between Atomic Absorption Spectrophotometer and Atomic Fluorescence Spectrophotometer?

The Atomic absorption spectrophotometer measured the amount of characteristic radiation absorbed and evaluated the measured element’s content. The working principle is based on the emission of characteristic spectral radiation from the light source.

The emitted light passes through the atomizer chambers, and the monochromatic light is received by the spectroscopic system passing through the photomultiplier tube and finally reaching the detector. The terminal computer changes the signal into processed data. If the atomizer is left uninjected, the passing light fails to get absorbed, and light transmittance appears to be 100%. In an opposite-case scenario, the light transmittance decreases. The Lamber-Beer Law states, “The absorbance is proportional to the concentration of the sample. Thus, the reference to the standard can help determine the sample concentration according to the absorbance.”