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.

 

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.

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.

Principle:

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.

Theory:

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 ε_f  is the absorptivity of the fluorescent material. C is the concentration of the substance and b is the path length, I_solvent and I_sample 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 I_solvent - I_sample. The intensity of fluorescence is thus given by:

Instrumentation:

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 25^oC - 30^oC.

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 (90^o) 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.

Spectrophotometry - IR Spectroscopy - Applications

After discussing about the theory and concepts, instrumentation and sampling technique for infra-red (IR) spectrophotometer, we will now discuss the last part of IR spectroscopy which are its applications.

IR spectroscopy is a simple and reliable technique. IR spectroscopy is widely used in industry as well as in research. Here are some of the applications:

Determination of functional group in an organic material: 

By IR spectroscopy, information about various functional groups of an unknown compound can be obtained by having a look at the regions in which the absorption band appears. As we have seen in earlier post that each compound has its own signature or fingerprint. Each and every peak obtained (in the IR spectrum) corresponds to different functional groups of the compound. So, as per the corresponding peak, functional group can be determined.

Determination of substances/Identification of compounds:

Another application is the determination of substances or identification of the compounds. Also, IR spectroscopy can used to establish whether two given compounds are identical or not. As we know, maximum number of absorption bands is observed in the IR spectra of organic molecules and there is almost a zero possibility that the two compounds have the identical IR spectra (as each compound has its own fingerprint or characteristic peak). If we suspect that some the compound is identical with the sample, then all we have to do is take the IR spectra of both. If the IR spectra match, then the compounds are identical.

Lets take an example of benzaldehyde. The IR spectrum of benzaldehyde will be as follows:

C-H stretching aromatic ring – 3080 cm^-1

C-H of aldehyde – 2860 cm^-1 and 2775 cm^-1

C=O stretching of aromatic aldehyde – 1700 cm^-1

C=C stretching of an aromatic ring – 1595 cm^-1

C-H bending – 745 cm^-1 and 685 cm^-1

No other compound apart from benzaldehyde will show this IR spectrum.

Assaying the progress of reaction/Determination of rate of reaction:

As we have seen that the IR spectra can used to determine the functional groups. Hence, any enzymatic reaction involving these functional groups – which are either consumed in the reaction or are produced in the reaction – can be determined with the help of IR spectra. For example if a reactant  contains carbonyl group and the product does not contain this group, so the progress of reaction can be determined by measuring the rate of disappearance of carbonyl (-C=O) stretching vibration. Thus, the rate of disappearance of a characteristic absorption band (of the reactant group) and/or the rate of appearance of the characteristic absorption band (of the product group) due to formation of the product is observed from time to time. In this way, the progress of the enzymatic reaction can be assayed.

Detection of impurities:

If we suspect that the compound is not pure, then IR spectroscopy can be used. The IR spectrum of the sample can be compared with the standard compound. If we see any changes in the spectrum like an additional band, then we can say that it is due to the impurities present in the compound.

Quantitative determination of compounds in mixtures:

The base line technique is used for quantitative determination of compound. The quantity of the compound or a mixture of two or more compounds can be determined. In this, the characteristic peak corresponding to the compound is chosen and log Io/It of peaks for the standard and the test sample is compared. 

By this, another kind of spectrophotometry, i.e.; the IR spectroscopy is completed.