1

Molecular Luminescence Spectroscopy

Lecture 32

2

The shape of the emission spectrum is expected to be a mirror image of the excitation spectrum since they originate from opposite processes However, instrumental artifacts result in excitation and emission spectra that are not exactly mirror images.

3

3 .Fiber Optic Fluorescence Sensors

These are the same as conventional fluorescence instruments but the beam from the excitation monochromators is guided through a bifurcated optical fiber to the sample container where excitation takes place. The fluorescence at the emission wavelength is then measured and related to concentration of analyte in the sample.

4

5

4 .PhosphorimetersInstruments that can measure phosphorescence are

called phosphorimeters. They are very similar to fluorescence instruments but make use of the fact that phosphorescence has a much longer lifetime than fluorescence and thus can be time resolved from the fluorescence signal. This can be achieved by placing a rotating chamber with a hole directing the beam to the sample. When the hole is aligned so that the incident beam excites the sample, the sample gives both fluorescence and phosphorescence. However, as the chamber rotates the incident beam becomes blocked and the fluorescence ceases. Phosphorescence will continue since it has a much longer lifetime and as the hole faces the detector only phosphorescence will be measured.

6

7

As the chamber rotates, phosphorescence will be detected as the hole in the chamber becomes aligned with the detector slit. No fluorescence interfere as the fluorescence lifetime is much shorter than the time required by the rotating chamber to align its hole with the slit of the detector

8

Applications of Photoluminescence Methods

Fluorescence is the most widely used luminescent technique for determination of many metal ions that react with organic ligands to form fluorescent molecules. On the other hand, although phosphorescence methods were used for analysis of a variety of analytes, they are still rarely used because of lower sensitivity and precision. Furthermore, few chemical systems really show good phosphorescence.

9

10

In addition, too many precautions must be followed for a successful phosphorescent analysis preventing widespread use of these methods. Fluorescence methods are quantitative techniques that are usually highly sensitive. Either the analyte or a reaction product of the analyte must be fluorescent which makes the method highly applicable to many systems that can be made fluorescent.

11

ChemiluminescenceThis luminescence technique emerges in

systems where a chemical reaction produces enough energy to excite an analyte or a reaction product of the analyte. Upon returning to ground state, the excited molecule emits a photon and chemiluminescence is observed. Several systems show the phenomenon of chemiluminescence where the chemiluminescence intensity is proportional to analyte concentration (in the nM to fM range).

12

This can be represented by the general reaction:

A + B = C* + DC* = C + h Analytical Applications of Chemiluminescence Several reactions are known to produce

chemiluminescence under certain conditions, some are described below:

13

Determination of Nitrous Oxide (NO)

Nitrous oxide reacts with electrogenerated ozone to form an excited nitrogen dioxide molecules followed by emission of the excitation energy as photons (chemiluminescence). The intensity of chemiluminescence is proportional to concentration of nitrous oxide.

14

NO + O3 = NO2* + O2

NO2* = NO2 + h A NO concentration down to 1 ppb was

determined using this method. On the other hand, higher nitrogen oxides (NOx) were also determined by this method; by first reducing the oxide to NO followed by reaction with ozone.

15

16

Determination of Sulfur Dioxide Sulfur dioxide reacts with hydrogen to form

an excited sulfur dimer species. The excited sulfur dimer then relaxes to ground state by emission of photons.

4 H2 + 2 SO2 = S2* + 4 H2O

S2* = S2 + h

17

Luminol ChemiluminescenceOne of the most common chemiluminescent

reactions is that of luminal (5-aminophthalhydrazide) with hydrogen peroxide in basic medium.

18

Luminol + H2O2 + OH- = (3-aminophthalate)* + N2 + H2O

(3-aminophthalate)* = 3-aminophthalate + h

This reaction is most important for determination of many bioanalytical substrates which produce hydrogen peroxide. Examples include glucose, cholesterol, alcohol, amino acids, lactate, oxalate, etc… which, in presence of the respective oxidase enzyme, produce hydrogen peroxide. The intensity of chemiluminescence is proportional to substrate concentration

19

In addition, this system can be extended for the analysis of many substrates that can indirectly be made to produce hydrogen peroxide such as cholesterol esters which can be hydrolyzed to cholesterol, using cholesterol estearase, followed by oxidation of generate cholesterol using cholesterol oxidase where hydrogen peroxide is produced.

20

Instrumentation

The instrumentation used in chemiluminescence is rather simple and can be composed of a photomultiplier tube and a readout. However, the PMT should be of very high sensitivity and a very low dark current. A schematic of the instrument can be shown below:

21