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Fluorescence Instrumentation 2

fluorescence for analytical chemistry

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fatimazafaraziz2526
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18 views16 pages

Fluorescence Instrumentation 2

fluorescence for analytical chemistry

Uploaded by

fatimazafaraziz2526
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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Fluorescence

Instrumentat
ion
By Sadaf Batool
Light sources

A
Excitation filters
fluorescence
instrument
Sample holder
consists of
the following
Emission
basicfilters
components:
Detectors
Diagram
The basic instrument is a
spectrofluorometer

• It contains a light source, two monochromators, a sample holder and a detector.

• There are two monochromators, one for selection of the excitation wavelength, another for
analysis of the emitted light.

• The detector is at 90 degrees to the excitation beam.


• Upon excitation of the sample molecules, the fluorescence is emitted in all directions and
is detected by photocell at right angles to the excitation light beam.
The basic instrument is a
spectrofluorometer

• The lamp source used is a xenon arc lamp that emits radiation in the UV, visible and near-
infrared regions.
• The light is directed by an optical system to the excitation monochromator, which allows
either preselection of wavelength or scanning of certain wavelength range.
• The exciting light then passes into the sample chamber which contains fluorescence
cuvette
Light sources
Almost any type of light source can be used for
fluorescence spectroscopy so the best choice
depends on the actual requirements to spectral
wavelength coverage such as
 intensity,
size,
cost,
efficiency
Excitation
filters
• The excitation filters is used
to select wavelengths that
efficiently excite the analyte
fluorescence. This filter is
typically a short-pass or
band pass filter.
Sample holder

• The choice of sample holder really depends on


the application.
• The main thing to consider is that in the case a
transmissive cuvette or flow cell you must
ensure that the sample holder material is
transparent for both your excitation and
emission wavelength.
• This is especially important for UV
wavelengths where most glasses absorb light so
special types of materials needs to be used.
Emission filters
Emission filter
Exciter filters permit only selected
wavelengths from the illuminator to
pass through on the way toward the
detector.
Detectors
• The detector can be either single- or
multi-channeled.
• The single-channel detector can only
detect one wavelength’s intensity at a
time, while the multi-channel detector
simultaneously detects the intensity at all
wavelengths, rendering the
monochromator or filter of the emission
unnecessary.
Detectors
• The most flexible fluorimetry can record
both an excitation spectrum and a
fluorescence spectrum with dual
monochromators and a continuous excitation
light source.
• When measuring fluorescence spectra, the
excitation light wavelength is kept constant,
ideally at a high absorption wavelength, and
the emission monochromator scans the
spectrum.
Photodetectors
Photomultiplier tube (PMT),Chargecoupled detector (CCD) PMT
commonly used detector in spectroflorometers
Important features of the PMT for florescence measurements consist
of :
 nanosecond photon response time,
 sensitivity.
Sensitivity is due to the possible gain of 106 electrons at the anode of
the PMT for each incident photon hitting the photo cathode
How it works
1. Electron is released by the photocathode
2. Electron is then multiplied by the electrodes
3.At the end of the chain is the collection electrode
4.The current flowing from the anode to ground is directly proportional to the
photoelectron flux generated by the photocathode
The Variables

• Photocathode thickness
• Too thick and more photons will be absorbed, less electrons will be
emitted
• Too thin and too many photons will pass through without being
absorbed
• Semitransparent Photocathode
• Multiplies the electrons to up to 100 million
Questions ?

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