An obvious difference between certain compounds is their color. Thus, quinone is yellow; chlorophyll is green; the 2,4-dinitrophenylhydrazone derivatives of aldehydes and ketones range in color from bright yellow to deep red, depending on double bond conjugation; and aspirin is colorless. In this respect the human eye is functioning as a spectrometer analyzing the light reflected from the surface of a solid or passing through a liquid. Although we see sunlight(or white light)as uniform or homogeneous in color, it is actually composed of a broad range of radiation wavelengths in the ultraviolet (UV), visible and infrared (IR) portions of the spectrum. As shown on the right, the component colors of the visible portion can be separated by passing sunlight through a prism, which acts to bend the light in differing degrees according to wavelength. Electromagnetic radiation such as visible light is commonly treated as a wave phenomenon, characterized by a wavelength or frequency. Wavelength is defined on the left below, as the distance between adjacent peaks (or troughs), and may be designated in meters, centimeters or nanometers (10-9 meters). Frequency is the number of wave cycles that travel past a fixed point per unit of time, and is usually given in cycles per second, or hertz (Hz). Visible wavelengths cover a range from approximately 400 to 800 nm. The longest visible wavelength is red and the shortest is violet. Other common colors of the spectrum, in order of decreasing wavelength, may be remembered by the mnemonic: ROY G BIV. The wavelengths of what we perceive as particular colors in the visible portion of the spectrum are displayed and listed below. In horizontal diagrams, such as the one on the bottom left, wavelength will increase on moving from left to right.
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The discovery of near-infrared energy is ascribed to Herschel in the 19th century, but the first industrial application began in the 1950s. In the first applications, NIRS was used only as an add-on unit to other optical devices that used other wavelengths such as ultraviolet (UV), visible (Vis), or mid-infrared (MIR) spectrometers. In the 1980s, a single unit, stand-alone NIRS system was made available, but the application of NIRS was focused more on chemical analysis. With the introduction of light-fiber optics in the mid 80s and the monochromator-detector developments in early nineties, NIRS became a more powerful tool for scientific research.
This optical method can be used in a number of fields of science including physics, physiology, or medicine. It was only in the last few decades that NIRS began to be used as a medical tool for monitoring patients.
The polypyrrol film was coated on an ITO glass using cyclic voltammetry (20 cycles). The ITO glass was bulked in an acetonitril solution (Bu4NPF6 0.2 M) containing 1-methylpyrrol monomer in concentration 10-2 M. The potentialsweep was used instead of a polymerization inpotentiostatic mode in order to obtain asmoothed film. The potential sweep wasperformed from -0.2 V to 1.015 V/AgCl with 100mV/s scan rate. We have used a three electrodecell with an ITO glass as working electrode, aplatinum wire as counter electrode and aAg/AgCl electrode as reference electrode.
The spectro-electrochemical measurements were performed in the Visible wavelengths range (300 to 750 nm) with an homemade spectroelectrochemical cell. The spectrometer used was a diode array with 256 diodes (MOS-DA) allowing 256 simultaneous absorbance
controlled by the potentiostat and swept from
-0.2 to 1 V/AgCl (and back to -0.2 V) with 100
mV/s scan rate. The description of the spectro-electrochemical assembly can be seen on figure 1. The computer is used to manage the diode array with BioKine software and to store and plot data points. It is also used to manage the potentiostat with ECLab software and store electrochemical data points. Both instruments are triggered together
with EC-Lab in order to start at the same time the potential sweep and spectroscopic measurement.
Biochemical Analysis :-
Simplicity and speed are the key benefits of using UV-VIS spectroscopy for the determination of protein content. A reagent is chosen that will bind to specific groups or bonds in the protein to form a colour product. The Agilent 8453 UV-Visible Spectrophotometer and ChemStation software is used to measure different dilutions of a protein standard. Measurements of absorbance at specified wavelengths plotted against concentration giving a calibration curve fitted using a least-squares method. Unknown samples are quantified by comparing their measurements with those in the calibration curve. Quantification is performed automatically after the measurement with protein results displayed almost instantaneously. Since the 8453 stores all spectra data, the measurements can be compared to multiple calibration methods without measuring the sample again.
Chemical Analysis :-
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The UV-VIS spectrophotometer is a key component in every laboratory. The Agilent 8453 provides fast, reliable data capture of the entire UV-visible spectrum in less than one second.
R&D: In research and development labs the spectrophotometer is used to quickly analyze a new formulation. The Agilent 8453 captures the complete picture including the wavelengths of interest as well as any unexpected results that may occur. Multiple spectra can be captured quickly to understand even the most fast-acting chemical reactions. Autosamplers and multi-cell transports make it possible to study multiple formulations. UV-VIS ChemStation software and its add-on modules provide a complete set of analysis routines for thorough understanding of the data and rapid reporting of the results. Data can be archived locally or in a central database.
Dissolution Test :-
Dissolution testing of a finished drug is critical to ensure product quality. Efficient testing and rapid feedback is a challenge that can be easily met with Agilent's UV-VIS spectroscopy solution. Using the Agilent 8453 UV-Visible Spectrophotometer and ChemStation software makes it possible to analyze the release of multiple ingredients. The key benefit to this approach is simultaneous sample analysis as opposed to a sequential and time-consuming approach using HPLC. Entire release profiles are possible with automated sampling and online analysis.
Environmental Samples :-
Environmental regulations call for routine samples of soil and sludge. During routine preparation background contaminants may be extracted along with the target analytes. The contaminants can invalidate analyses and potentially cause instrument damage for hours or even days. To avoid this contamination laboratories introduce cleanup steps. However, some complex matrices require additional cleanup. UV-VIS spectroscopy can provide a quick, non-interfering analysis of the samples to detect hydrocarbons and PCBs before they interfere with GC inlets and columns and cause a shutdown of the lab. In seconds the Agilent 8453 UV-Visible Spectrophotometer and ChemStation software can detect the harmful contaminants.
Multicomponent Analysis :-
Agilent chemists have found the results from an Agilent UV-VIS spectrophotometer and an Agilent LC for a single component formulation to be identical. However a multicomponent formulation poses more of a challenge if only using the UV-VIS absorbance spectra. Using a first-order derivative (rate of change of absorbance with respect to wavelength) ensures correction for minor background scattering or absorbance. Applying derivative spectroscopy across a range of wavelengths results in higher spectral differences between components. With the Agilent 8453 UV-Visible Spectrophotometer and UV-VIS ChemStation software a complete spectra can be captured in less than a second resulting in a fast and accurate multicomponent analysis.
Sunscreens are designed to absorb ranges of wavelengths in the ultraviolet spectrum. For rapid analysis UV-VIS spectroscopy can be used. If the components in the sunscreen the multi-component analysis is fast and accurate. However, if some of the components have virtually identical spectra it is difficult to distinguish individual components. In this case it is best to look at the quantification of their sum. In either case, it's relatively straightforward to compare the spectra of the sample to that of the standard to determine the validity of the product. Using the Agilent 8453 UV-Visible Spectrophotometer and UV-VIS Advanced ChemStation software one can measure a specific range of wavelengths and do the comparison.
Absorption :- Absorption spectroscopy is a technique in which the power of a beam of light measured before and after interaction with a sample is compared. Specific absorption techniques tend to be referred to by the wavelength of radiation measured such as ultraviolet, infrared or microwave absorption spectroscopy. Absorption occurs when the energy of the photons matches the energy difference between two states of the material.
Fluorescence spectroscopy uses higher energy photons to excite a sample, which will then emit lower energy photons. This technique has become popular for its biochemical and medical applications, and can be used for confocal microscopy, fluorescence resonance energy transfer, and fluorescence lifetime imaging.
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When X-rays of sufficient frequency (energy) interact with a substance, inner shell electrons in the atom are excited to outer empty orbitals, or they may be removed completely, ionizing the atom. The inner shell "hole" will then be filled by electrons from outer orbitals. The energy available in this de-excitation process is emitted as radiation (fluorescence) or will remove other less-bound electrons from the atom (Auger effect). The absorption or emission frequencies (energies) are characteristic of the specific atom. In addition, for a specific atom small frequency (energy) variations occur which are characteristic of the chemical bonding. With a suitable apparatus, these characteristic X-ray frequencies or Auger electron energies can be measured. X-ray absorption and emission spectroscopy is used in chemistry and material sciences to determine elemental composition and chemical bonding.
Liquid solution samples are aspirated into a burner or nebuliser/burner combination, desolvated, atomized, and sometimes excited to a higher energy electronic state. The use of a flame during analysis requires fuel and oxidant, typically in the form of gases. Common fuel gases used are acetylene (ethyne) or hydrogen. Common oxidant gases used are oxygen, air, or nitrous oxide. These methods are often capable of analyzing metallic element analytes in the part per million, billion, or possibly lower concentration ranges. Light detectors are needed to detect light with the analysis information coming from the flame.
Many atoms emit or absorb visible light. In order to obtain a fine line spectrum, the atoms must be in a gas phase. This means that the substance has to be vaporised. The spectrum is studied in absorption or emission. Visible absorption spectroscopy is often combined with UV absorption spectroscopy in UV/Vis spectroscopy. Although this form may be uncommon as the human eye is a similar indicator, it still proves useful when distinguishing colours.
All atoms absorb in the Ultraviolet (UV) region because these photons are energetic enough to excite outer electrons. If the frequency is high enough, photo ionization takes place. UV spectroscopy is also used in quantifying protein and DNA concentration as well as the ratio of protein to DNA concentration in a solution. Several amino acids usually found in protein, such as tryptophan, absorb light in the 280nm range and DNA absorbs light in the 260nm range. For this reason, the ratio of 260/280nm absorbance is a good general indicator of the relative purity of a solution in terms of these two macromolecules. Reasonable estimates of protein or DNA concentration can also be made this way using Beer's law.
Infrared spectroscopy offers the possibility to measure different types of inters atomic bond vibrations at different frequencies. Especially in organic chemistry the analysis of IR absorption spectra shows what type of bonds is present in the sample. It is also an important method for analysing polymers and constituents like fillers, pigments and plasticizers.
Near Infrared (NIR) :-
The near infrared NIR range, immediately beyond the visible wavelength range, is especially important for practical applications because of the much greater penetration depth of NIR radiation into the sample than in the case of mid IR spectroscopy range. This allows also large samples to be measured in each scan by NIR spectroscopy, and is currently employed for many practical applications such as: rapid grain analysis, medical diagnosis pharmaceuticals/medicines, biotechnology, genomics analysis, proteomic analysis, interactomics research, inline textile monitoring, food analysis and chemical imaging/ hyper spectral imaging of intact organisms, plastics, textiles, insect detection, forensic lab application, crime detection, various military applications, and so on.
Raman spectroscopy uses the inelastic scattering of light to analyse vibrational and rotational modes of molecules. The resulting 'fingerprints' are an aid to analysis.
Coherent anti-Stokes Raman spectroscopy :-
CARS is a recent technique that has high sensitivity and powerful applications for in vivo spectroscopy and imaging.
Nuclear magnetic resonance spectroscopy analyzes the magnetic properties of certain atomic nuclei to determine different electronic local environments of hydrogen, carbon, or other atoms in an organic compound or other compound. This is used to help determine the structure of the compound.
Ultraviolet light is shone at the sample using a Helium lamp emitting at 21.2 eV (He I radiation) or 40.8 eV (He II radiation), although synchrotron radiation can provide photon energies from approx. 10 eV up to the XPS region. The low photon energy in UPS means that deep core electron levels cannot be excited and only photoelectrons emitted from the valence band or shallow core levels are accessible. Angle Resolved UPS (ARUPS) can be used to determine the band structure of the material under investigation. UPS can also be used to identify molecular species on surfaces by identifying characteristic electron energies associated with the bonds of the molecules.
Transmission or conversion-electron (CEMS) modes of Mossbauer spectroscopy probe the properties of specific isotope nuclei in different atomic environments by analyzing the resonant absorption of characteristic energy gamma-rays known as the Mossbauer effect.
Ohno , Yoshi "CIE Fundamentals for UV-Visible spectroscopy
Oxford encyclopedia 2003