Spectrophotometry is the quantifiable study of interaction of electromagnetic radiations with the matter. Electromagnetic radiations do not require any medium for its transmission. It consists of two components, electric and magnetic field. Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color (or more specifically the wavelength) of light. Spectrophotometry is the spectroscopic technique used to assess the concentration or amount of a given species. Spectrophotometer makes use of the transmission of light through a solution to determine the concentration of a solute within the solution. It is often used in physical and analytical chemistry for the identification/characterization of substances through the spectrum emitted from or absorbed by them. It is also used to examine the behavior of chemical substances after electromagnetic irradiation such as gamma rays, X-rays, ultra violet rays, infrared rays, radio waves and microwaves. It gives detailed information about inter-molecular bonding types or molecular changes occurring during enzymatic reactions and mitochondrial electron transport chain. Qualitative and quantitative measurement of biomolecules even in impure samples can be done rapidly and conveniently.
- To determine the molecular structure
- To estimate the energy levels of the ions and complexes in a chemical system along with the compositions.
- To get an idea regarding absorption and emission details of the specimen
- To understand the intrinsic configuration and relative association and chemical shifts
- Determine the wavelength of maximum absorbance.
UV-visible spectroscopy investigates the interactions between ultraviolet or visible electromagnetic radiation and matter. Ultraviolet and visible spectroscopy (UV-vis) is a reliable and accurate analytical laboratory assessment procedure that allows for the analysis of a substance. Specifically, ultraviolet and visible spectroscopy measures the absorption, transmission and emission of ultraviolet and visible light wavelengths by matter.
UV-visible spectroscopic measurements provide precise information about atomic and molecular structure. It consists of light of several colors ranging from violet to red. This is now termed the UV-visible electromagnetic spectrum. The ultraviolet and visible regions of the electromagnetic spectrum are linked in UV-vis spectroscopy because similarities between the two regions allow many of the same research techniques and tools to be used for both regions. The ultraviolet region (about 450-200 nm) is particularly important for the qualitative and quantitative determination of many organic compounds. In the visible region (about 450-700 nm), spectrophotometric methods are widely used for the quantitative determination of many trace substances, especially inorganic species.
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Special instrumentation is used in UV-vis spectroscopy. Hydrogen or deuterium lights provide the source of light for ultraviolet measurements. Tungsten lamps provide the light for visible measurements. These light sources generate light at specific wavelengths. Deuterium lamps generate light in the UV range (190 to 380nm). Tungsten-halogen lamps generate light in the visible spectrum (380 to about 800 nm).Xenon lamps which can produce light in the UV and visible portions of the spectrum are used to measure both UV and visible spectra.
Uv/Vis Spectrophotometry is used to determine the absorption or transmission of Uv/Vis light (180 to 820 nm) by a sample. It can also be used to measure concentrations of absorbing materials based on developed calibration curves of the material. It is routinely used in the quantitative determination of solutions of transition metal ions and highly conjugated organic compounds. Its main applications are;
- Quantitative determination of chromophores concentrations in solution
- Impurity determination by spectrum subtraction
- Determination of reaction kinetics
Fluorescence spectroscopy, fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample.Fluorescence occurs when a molecule absorbs photons from the U.V.-visible light spectrum (200-900 nm), causing transition to a high-energy electronic state and then emits photons as it returns to its initial state, in less than 10-9 sec. Fluorimetry characterizes the relationship between absorbed and emitted photons at specified wavelengths. It is a precise quantitative analytical technique that is inexpensive and easily mastered. Fluorescence spectroscopy is an important investigational tool in many areas of analytical science, due to its extremely high sensitivity and selectivity. With many uses across a broad range of chemical, biochemical and medical research, it has become an essential investigational technique allowing detailed, real-time observation of the structure and dynamics of intact biological systems with extremely high resolution. It is particularly heavily used in the pharmaceutical industry where it has almost completely replaced radiochemical labelling. Fluorescent compounds or fluorophors can be identified and quantified on the basis of their excitation and emission properties. The excitation and emission properties of a compound are fixed, for a given instrument and environmental condition, and can be used for identification and quantification. The principal advantage of fluorescence over radioactivity and absorption spectroscopy is the ability to separate compounds on the basis of either their excitation or emission spectra, as opposed to a single spectra. This advantage is further enhanced by commercial fluorescent dyes that have narrow and distinctly separated excitation and emission spectra. The sensitivity of fluorescence is approximately 1,000 times greater than absorption spectrophotometric methods.
Fluorescence spectroscopy is used in, among others, biochemical, medical, and chemical research fields for analyzing organic compounds. There has also been a report of its use in differentiating malignant, bashful skin tumors from benign.In particular, the measurements of fluorescence spectrum, lifetime and polarization are powerful methods of studying biological structure and function. The fluorescence spectrum is highly sensitive to the biochemical environment of the fluorophor. Fluorophors have been designed such that their spectra change as a function of the concentration of metabolites, such as pH and calcium. A major disadvantage of fluorescence is the sensitivity of fluorescence intensity to fluctuations in pH and temperature.
Flame photometry (more accurately called flame atomic emission spectrometry) is a branch of atomic spectroscopy in which the species examined in the spectrometer are in the form of atoms. Flame photometry is suitable for qualitative and quantitative determination of several cations in biological specimens, especially for metals that are easily excited to higher energy levels at a relatively low flame temperature (mainly Na, K, Rb, Cs, Ca, Ba, and Cu). This technique uses a flame that evaporates the solvent and also sublimates and atomizes the metal and then excites a valence electron to an upper energy state. Light is emitted at characteristic wavelengths for each metal as the electron returns to the ground state that makes qualitative determination possible. Flame photometers use optical filters to monitor for the selected emission wavelength produced by the analyte species. Comparison of emission intensities of unknowns to either that of standard solutions or to those of an internal standard allows quantitative analysis of the analyte metal in the sample solution. Because of the very narrow and characteristic emission lines from the gas-phase atoms in the flame plasma, the method is relatively free of interferences from other elements. Flame photometry has many advantages. It is a simple, relatively inexpensive, high sample throughput method used for clinical, biological, and environmental analysis.
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The flame photometers are relatively simply instruments. There is no need for source of light, since it is the measured constituent of the sample that is emitting the light. The energy that is needed for the excitation is provided by the temperature of the flame (2000-3000 Â°C), produced by the burning of acetylene or natural gas (or propane-butane gas) in the presence of air or oxygen. By the heat of the flame and the effect of the reducing gas (fuel), molecules and ions of the sample species are decomposed and reduced to give atoms, e.g.: Na+ + e- ïƒ Na. Atoms in the vapour state give line spectra. (Not band spectra, because there are no covalent bonds hence there are not any vibrational sub-levels to cause broadening). The mono chromator selects the suitable (characteristic) wavelength of the emitted light. The emitted light reaches the detector. This is a photomultiplier producing an electric signal proportional to the intensity of emitted light.
Atomic Absorption Spectrometry:
In analytical chemistry, atomic absorption spectroscopy is a technique for determining the concentration of a particular metal element in a sample. The technique can be used to analyze the concentration of over 70 different metals in a solution. The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. Atomic absorption spectroscopy (AAS) determines the presence of metals in liquid samples. Metals include Fe, Cu, Al, Pb, Ca, Zn, Cd and many more. It also measures the concentrations of metals in the samples. Typical concentrations range in the low mg/L range. The electrons of the atoms in the atomizer can be promoted to higher orbitals for a short amount of time by absorbing a light of a given wavelength. This amount of energy (or wavelength) is specific to a particular electron transition in a particular element, and in general, each wavelength corresponds to only one element. This gives the technique its elemental selectivity.
In order to analyze a sample for its atomic constituents, it has to be atomized. The sample should then be illuminated by light. The light transmitted is finally measured by a detector. The light source is usually a hollow-cathode lamp of the element that is being measured. Lasers are also used in research instruments. Since lasers are intense enough to excite atoms to higher energy levels. The disadvantage of these narrow-band light sources is that only one element is measurable at a time. AA spectroscopy requires that the analyte atoms be in the gas phase. Ions or atoms in a sample must undergo desolvation and vaporization in a high-temperature source such as a flame or graphite furnace. Flame AA can only analyze solutions, while graphite furnace AA can accept solutions, slurries, or solid samples. The graphite furnace has several advantages over a flame. It is a much more efficient atomizer than a flame and it can directly accept very small absolute quantities of sample. It also provides a reducing environment for easily oxidized elements. Samples are placed directly in the graphite furnace and the furnace is electrically heated in several steps to dry the sample, ash organic matter, and vaporize the analyte atoms.
AA spectrometers use monochromators and detectors for uv and visible light. The main purpose of the monochromator is to isolate the absorption line from background light due to interferences. Simple dedicated AA instruments often replace the monochromator with a bandpass interference filter.
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