Saturday, May 17, 2014

Spectrophotometry - Luminometry

Till now, we have seen various posts on UV-visible spectrophotometry, IR-spectrophotometry and spectrofluorimetry. Now, we will have a look at the next type of spectrophotometry which is luminometry.
As the name suggests, this type of spectrophotometry is associated with the phenomenon of luminescence. Now, the question is “what is luminescence?” Luminescence can be described as the emission of light by certain materials which do NOT result from heating (that is, the emission of light is when the temperature is below that of incandescence). Luminescence is the basic principle behind the working of luminometers. This phenomenon is usually ascribed to oxidative reactions which take place in solution producing molecules in an excited state. Some of these reactions release energy in the form of heat while others release in the form of photons.
Examples of luminescent compounds are luciferin (light emitting compound found in organisms), luminol (chemical exhibiting luminescence).

There are two major categories of luminescence as chemiluminescence and bioluminescence. It is easy to understand them as the name itself suggests the meaning. So, chemiluminescence is the luminescence produced by some chemical means. For example, luminol when oxidized with hydrogen peroxide (H2O2) in the presence of a catalyst produces luminescence which is called the chemiluminescence. On the other hand, luminescence which is produced by the interference of an enzyme is referred to as bioluminescence. 

Advantages of luminometry 
There are various advantages of luminometry over spectrophotometry. Firstly, luminometry is more sensitive as around femtomole quantities can be measured. Next advantage is that of a simple instrumentation (as we will see below). In luminometers, wavelength selectors are not required. This is so because the luminescent light is monochromatic as a result of its emission from a specific reaction.

The basic components of luminometers are:
a. A light-tight chamber in which the cuvette containing the sample can be kept

b. A facility for the addition of luminescent reagents in light-tight fashion
c. A detector (which is generally a photomultiplier)
d. An amplifier
e. A recorder

The light which is emitted by the reaction taking place in the cuvette is measured either as a peak value (which generally measures the concentration of compound of interest) or the rate of change of intensity (which is generally while measuring enzyme intensities). 


We will discuss here three main systems which are of frequent use as firefly, bacterial luminescence and luminol chemiluminescence. The principle and applications of each of these are described below:

a. Firefly luminescence and ATP measurement:

Luciferase enzyme catalyses the following reaction in the presence of magnesium:

Here, for each molecule of ATP reacting, one photon of intensity of 562nm is produced. This system is highly specific for ATP if all the reagents are pure. By linking this reaction with various other reactions, it can be used to assay a number of ATP-specific enzymes and their substrates such as creatine kinase, creatine phosphate etc.

b. Bacterial luminescence and coenzymes measurement:

The coenzymes that can be measured by this method are the NADH and NADPH. This system utilizes a purified oxidoreductase obtained from the bacterium Benecka harveyi. The reaction can be then coupled to bacterial luciferase as follows:

Here, bacterial luciferase catalyzes the oxidation of aldehyde by oxygen in the presence of FMNH2 during which a photon of maximum intensity at 495nm is produced.
c. Luminol based chemiluminescent assays:
Luminol is oxidized by hydrogen peroxide at pH 10-11 if chromium, copper or iron compounds are used as catalysts. Photons with maximum intensity at 430nm are produced.

Tuesday, March 18, 2014

Spectrophotometry - Spectrofluorimetry Part 3

Till now we have seen about the principle, theory and concepts and instrumentation of spectrofluorimetry; advantages and disadvantages of spectrofluorimetry and various factors giving rise to fluorescence. Now, coming to the last topic in spectrofluorimetry which are its applications. Following are some of the applications of spectrofluorimetry:
Studies on Protein Structure:
A lot of information regarding the structure of the protein can be determined with the help of fluorescent studies. The fluorescence spectra may change depending on the position of amino acids in the protein, the composition of active site, protein denaturation etc.

Qualitative Analysis:
The identity of the compound can be determined by comparing the fluorescence spectra and the absorption spectra of the particular compound.
Compounds which are fluorescent are readily determined with simple instruments and here the solution for examination is normally obtained by dissolving the sample in a suitable solvent.
Certain substances which are in themselves non-fluorescent may be determined as a result of a chemical change. This method can be used for both inorganic and organic compounds. For example, determination of primary and secondary aliphatic amines through the reaction with NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-I,3-diazole which gives yellow fluorescence.

Quantitative Analysis:
This includes the determination of the concentration of various vitamins, hormones, drugs etc. For example, the quantitative analysis includes assay of vitamins like thiamine, riboflavin; hormones such as cortisol, estrogen, serotonin, dopamine and drugs such as lysergic acid and barbiturates.

Intracellular Free Calcium Concentration Assay:
The three probes which allow us to perform the assay for free calcium concentrations are Quin-2, Quin-2 AM and Flura-2. These probes are permeable and they enter into the plasma membrane.

Fluorescent Microscopy:
When spectrofluorimeter is combined with a microscope, it allows the determination of subcellular location of fluorescent compounds or of materials which can bind to the fluorescent dyes. This technique is important especially in the field of pharmacology and immunology. Thus, an antibody can detect the fluorescent labelled antigen present on the surface of the cell. For example, this technique allows the visualization of nucleic acids within subcellular organelles with the help of acridine orange dye.

Assay of Membrane Potential:
There occur changes in membrane potential which regulate entry of ion into the cells. These membrane potential changes can be monitored by using some of the fluorescent probes.

Studies on Membrane Structure:
There are various fluorescent probes such as ANS (anilinonapthalene 8-sulphonate) and N-methyl-2-anilino-6-naphthalene sulphonate (MNS). They both contain charged and hydrophobic areas and therefore are situated at the water-lipid interface of the membrane. The fluorescent properties of the molecule vary with its mobility and also with the polarity of the environment. Studies with the ANS probe has shown that structural changes occur in mitochondrial membrane during oxidative phosphorylation. These probes have also helped in giving information about the structural features of the plasma membrane. 

Thus, by this post, we complete the topic of spectrofluorimetry. In the next post, we will start with another type of spectrophotometry.

Tuesday, February 25, 2014

Spectrophotometry - Spectrofluorimetry Part 2

In the last post, we have discussed about the principle, theory and instrumentation of spectrofluorimetry. In this post, we will have a look at various advantages, disadvantages of spectrofluorimetry and also various factors which give rise to fluorescence.

Advantages of Spectrofluorimetry:
High Sensitivity: Spectrofluorimetry gives extremely accurate results even when samples of very low concentrations are used; even in ppm. Substances can be determined at concentrations up to 1000times lower than those required for absorption spectrophotometry. The concentrations which are as low as μg/ml or ng/ml can be determined using spectrofluorimetry. Precision upto 1% can be achieved easily using spectrofluorimeter.

Spectral Selectivity:  As we have seen in the instrumentation that two monochromators are used in spectrofluorimetry where one monochromator selects the activating wavelength while the other one selects the fluorescent wavelength. This arrangement gives great spectral selectivity to spectrofluorimeter.

Disadvantage of Spectrofluorimetry:
Quenching: What is quenching? So, quenching refers to any process which decreases the fluorescence intensity of a given substance.  There is high degree of absorption of fluorescent radiation by the emitting sample itself i.e.; there is quenching by the sample itself. Quenching can also occur because of impurities. Dissolved oxygen is a very effective quencher. And, if the sample contains dissolved oxygen, then nitrogen is bubbled through the sample to remove oxygen.  Thus, this quenching is a major drawback of spectrofluorimetry.

Factors Affecting Fluorescence Intensity:
There are certain factors that give rise or inhibits fluorescence thereby affecting fluorescence. Some of them are as follows:

Conjugation: Aromatic molecules or the molecules having multiple conjugated double bonds with a high degree of resonance stability generally fluoresce. Molecules must have π electrons. Both the groups, (i.e.; the aromatic molecules and the molecules with conjugated double bonds) possess delocalized π electrons and the higher the number of π electrons, the higher will be the fluorescence. For this reason, the polycyclic compounds are more fluorescent than the benzene derivatives.
The substituent groups do affect fluorescence by either increasing it or decreasing it. For example, the electron-donating groups like –NH2, -OH, etc. enhance fluorescence while the electron-withdrawing groups which delocalize π electron like –NO2, -COOH enhance the fluorescence.

Rigidity: The more rigid the structure of compound, the more will be the intensity of fluorescence. Also, the more the sterically uncrowded, the more will be the fluorescence. Also, chelation of aromatic compounds with metal ions promotes rigidity and reduces internal vibrations. Thus, chelation promotes fluorescence. 

Viscosity: If there is an increase in viscosity, there will be decreased collision of molecules thereby increasing fluorescent intensity. 

Temperature: Increase in temperature leads to increased collision between molecules thereby decreasing fluorescent intensity.

I hope these are clear to you. In the next post, we will have a look at the various applications of spectrofluorimetry.

Sunday, February 16, 2014

Spectrophotometry - Spectrofluorimetry Part 1

In this post, we will start with the next kind of spectrophotometry which is spectrofluorimetry. Under spectrofluorimetry, we will discuss about the principle, theory and instrumentation for spectrofluorimetry, various factors which give rise to fluorescence, advantages and disadvantages of spectrofluorimetry and lastly the applications.
In this post, we will have a look at the basic principle, the theory and instrumentation for spectrofluorimeter.

Spectrofluorimetry, as the name suggests takes the advantage of the fluorescent properties. So, before understanding about spectrofluorimetry, it is necessary to know what is fluorescence. When a molecule after absorbing radiations, emits radiation of a longer wavelength, then this phenomenon is referred to as “fluorescence.” Because of this, the compound absorbing in ultraviolet range might emit radiation in visible range. This is called Stoke’s shift wherein the shift is towards a longer wavelength. Fluorescence is an extremely short-lived phenomenon which lasts for about 10-7 seconds or less and thus can provide information about events which take less than 10-7seconds to occur.

After understanding the basic principle of fluorescence, we will now come to the main principle of spectrofluorimetry. As we have learnt in Chemistry, when an atom or molecule absorbs radiation, the energy of the photon absorbed lifts an electron to a higher orbital. Now, the electron needs to come down back to its ground state. It can do so in two different ways. In one way, the electron can directly return to its ground state in a single step where it will emit radiation of the same wavelength that it has absorbed. In another case, the electron can do so in a step-wise manner through intermediate energy levels and in this process, it will obviously emit quanta of radiation to each energy step. Since, each quantum will have a smaller amount of energy; the radiation emitted will have a longer wavelength than the original exciting radiation (since we know that energy is indirectly proportional to wavelength). If this happens, then the emitted light will have many different wavelengths which will correspond to each of the intermediate level which the electron will adopt on its way back to the ground state. Thus, fluorescence spectra are band spectra and they are independent of the wavelength of the radiation absorbed.

Fluorimetry can be used as a tool for the determination of very small concentration of substances which exhibit fluorescence. Beer-Lambert law (discussed previously) can also be applied in this case of fluorimetry as:
where εis the absorptivity of the fluorescent material. C is the concentration of the substance and b is the path length, Isolvent and Isample represents the values of intensities of the incident radiant energy and transmitted energy respectively.  The intensity of the radiation absorbed can thus be given by Isolvent - Isample. The intensity of fluorescence is thus given by:

The major instrumentation of spectrofluorimeter differs from the spectrophotometer in two major aspects as follows:
Firstly, there are two monochromators (instead of one as is the case of spectrophotometer). These two monochromators are placed before and after the sample holder respectively.
Secondly, the sample-holder has a device to maintain the temperature as the fluorescence is maximum between 25oC - 30oC.

The following are the different components of spectrofluorimeter:
a. A continuous source of radiant energy (mercury lamp or xenon arc or tungsten lamp)
b. A monochromator usually a prism (P1), to choose the wavelength with which the sample is to be irradiated. 
c. Sample cell: Sample cells are cylindrical or polyhedral made up of color corrected fused glass and path length normally 10mm to 1cm.
d. A second monochromator (P2) which, placed after the sample, enables the determination of fluorescent spectrum of the sample.
e. A detector which is usually a photomultiplier or photo-voltaic cell or photo-tubes suited for wavelengths greater than 500nm and lastly
f. An amplifier

The fluorescent radiation is emitted in all directions by the sample but in most of the instruments the sample is viewed at the right angles (90o) to the incident beam as can be seen in the diagram.

This was about the theory, concepts and instrumentation of spectrofluorimeter. In the next post, we will have a look at the various factors giving rise to fluorescence and its advantages and disadvantages.