INSTRUMENTAL METHODS OF ANALYSIS, B.PHARM 7TH SEM. AND FOR BSC,MSC CHEMISTRY. This is Geeta prasad kashyap (Asst. Professor), SVITS, Bilaspur (C.G) 495001

1. Introduction to Instrumentation
• The basic components of fluorescence
spectrometers are a white light source, excitation
monochromator, sample chamber, emission
monochromator, and detector.
• Advanced instruments can be configured with
multiple sources, detectors and sample holders.

2. Cont….
• Steady state fluorescence measurementsrequire a continuous
excitation source.
• This is usually a continuous xenon lamp which generatesintense
white light, from 230 nm up to the near-infrared range.
• The excitation monochromator selects the desired wavelength of
excitation light, which is focused at the sample position.
• The monochomator utilises diffraction gratings and slits to select
light if a particular wavelength and reject the rest of the excitation
lamp spectrum: as the gratings rotate, the centre wavelength varies.
• The slits control the bandpass, or the width of the wavelength
interval that passes through the monochromator such as the one in
the figure. Double monochromators provide better stray light
rejection and therefore a higher signal-to-noise ratio for the
instrument.

3. Cont…
• The emission light, which is of longer wavelength than the
excitation, is filtered by the emission monochromator.
• This selects the wavelength of the light that reaches the
detector which is typically a photon-counting photomultiplier
tube (PMT). PMT detectors are capable of detecting single
photons so it is important that any unwanted light is effectively
rejected by the monochromator, to avoid any background on
the emission spectrum.
• The excitation and emission paths are set at an angle of 90∘ to
each other to minimise the fraction of excitation light entering
the emission arm.

4. Excitation and Emission Spectra
• Two types of fluorescence spectra may be measured: excitation and
emission.
• An excitation spectrum is obtained by scanning the excitation
wavelength and keeping the emission wavelength constant, and
recording the photons reaching the detector.
• An emission spectrum is recorded in the same fashion, but scanning
the emission wavelength at a constant excitation wavelength. It is
also possible to acquire synchronous spectra by simultaneously
scanning the excitation and emission wavelengths.
• The spectra provide information on the absorption and emission
properties of the sample, and are typically plotted as a function of
counts (number of detected photons) against wavelength.