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.

Spectrophotometry - IR Spectroscopy - Preparation of Sample

Earlier, we have discussed about theory and concepts, instrumentation of IR spectrophotometer. In this post, we will discuss about another important factor that needs to be considered while performing IR spectroscopy which is the preparation of sample or in other words, sampling techniques.

The sampling technique or the preparation of sample depends on whether the sample is in vapor phase, liquid or solid phase. As there is significant difference in the intermolecular forces in these three phases, hence it is better that the data obtained is specified for the sampling technique used. So, here, we will see about the preparation of samples which are in gas, liquid and solid phases.

Sampling of gases: 

The sample cell is made up of NaCl, KBr etc. and is similar to the liquid sample cell. A sample cell has a series of internal mirrors (multi-pass cells) which reflect the IR beam back and forth lengthening the path-length. A sample cell with a long path length (5 – 10 cm) is needed because the gases show relatively weak absorbance.

Sampling of liquids: 

Liquids are usually observed as a thin film between two IR-transparent windows. Liquid sample cells can be sandwiched using liquid sample cells of highly purified alkali halides, normally NaCl. However, if the sample contains water, they become useless. In such cases, CaF2 flats are used. The sample thickness should be selected so that the transmittance is between 15 – 20%. For most liquids, the sample cell thickness is 0.01 – 0.05 mm. 

Sampling of solids:

Various techniques used for preparing solid samples are as follows:

Mull technique: In this technique, the finely crushed sample is mixed with Nujol or Kaydol (which are mulling agents) in a marble or agate mortar, with a pestle to make a thick paste. A thin film is applied onto the salt plates. This is then mounted in a path of IR beam and the spectrum is recorded.

Deposited films/ Case film technique: If the solid is amorphous in nature, then the sample is deposited on the surface of a KBr or NaCl cell by evaporation of a solution of the solid and ensured that the film is not too thick to pass the radiation.

KBr Disks/ Pressed Pellet technique: In this technique, a small amount of finely ground solid sample is mixed with 100 times its weight of KBr and compressed into a thin transparent pellet using a hydraulic press. These pellets are transparent to IR radiation and it is used for analysis.

So, this was about the various sampling techniques. In the next post, we will have a look at the applications of IR spectrophotometry.