Quantitative Spectroscopic Techniques

Several reviews describe various aspects of QTLC [106-114] and three books [115-117] deal with this subject. 

HPTLC on silica gel followed by scanning densitometry is a satisfactory method for the identification and quantitative prior analysis of dye liquors to be used for acrylic fibres and also for dyes extracted from finished products [118]. QTLC has recently been reported for a variety of applications. 

Principles and Characteristics Spectroscopic techniques (mainly UV, F, FTIR, NIRS and NMR) are widely used for quantitation because of speed and flexibility. Ideally, they require pure samples for reliable identification. Before a substance is quantified it needs to be positively identified using spectroscopy or mass spectrometry. 

Quantitation can be carried out using absorption or fluorescence spectroscopy. Measurements can be carried out in transmittance or reflectance mode. The basis of quantitative absorption spectroscopy in transmission mode (UVA IS and FUR) is the usual linear relationship of the Beer-Bouguer-Lambert law, which states that the absorbance A of a solute is directly proportional to its concentration c  

In the simplest type of quantitative analysis, the concentration of a single component is measured in a 

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Another quantitative spectroscopic technique for the analysis of additives in lubricants and hydrocarbons is Raman spectroscopy (Coates, 1975). Theoretically, Raman spectroscopy should be as good as infrared for quantitative analysis. The... 

X-ray photoelectron spectroscopy (XPS) is a quantitative spectroscopic technique that can be used to analyze the surface chemistry of a material, or measure... 

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

Solid state NMR is a relatively recent spectroscopic technique that can be used to uniquely identify and quantitate crystalline phases in bulk materials and at surfaces and interfaces. While NMR resembles X-ray diffraction in this capacity, it has the additional advantage of being element-selective and inherently quantitative. Since the signal observed is a direct reflection of the local environment of the element under smdy, NMR can also provide structural insights on a molecularlevel. Thus, information about coordination numbers, local symmetry, and internuclear bond distances is readily available. This feature is particularly usefrd in the structural analysis of highly disordered, amorphous, and compositionally complex systems, where diffraction techniques and other spectroscopies (IR, Raman, EXAFS) often fail. 

Quantitative information about energies of atomic orbitals is obtained using photoelectron spectroscopy, which applies the principles of the photoelectric effect to gaseous atoms. Our Box (on the next page) explores this powerful spectroscopic technique. 

A chroaatogreuB provides information regarding the complexity (numlser of components), quantity (peak height or area) and identity (retention par uleter) of the components in a mixture. Of these parameters the certainty of identification based solely on retention is considered very suspect, even for simple mixtures. When the identity can be firmly established the quantitative information from the chromatogram is very good. The reverse situation applies to spectroscopic techniques which provide a rich source of qualitative information from which substance Identity may be inferred with a reasonable degree of certainty. Spectroscopic Instruments have, however, two practical limitations it is often difficult to extract quantitative... 

HPLC methods of determining the amounts of different additives in polymeric materials are preceded by an extraction process or dissolution of the polymer matrix. Although extraction-HPLC is often observed to be superior to the traditional spectroscopic techniques (UV and IR) in analysing additives, it is frequently difficult to obtain reproducible results in view of the variability of the extraction yield. On the other hand, it is equally difficult to obtain quantitative data in the dissolution/reprecipitation-HPLC method because of entrapment of analytes in the polymer precipitate and the potential for high absorption of the additives on the polymer surface. 

Each spectroscopic technique (electronic, vibra-tional/rotational, resonance, etc.) has strengths and weaknesses, which determine its utility for studying polymer additives, either as pure materials or in polymers. The applicability depends on a variety of factors the identity of the particular additive(s) (known/unknown) the amount of sample available the analysis time desired the identity of the polymer matrix and the need for quantitation. The most relevant spectroscopic methods commonly used for studying polymers (excluding surfaces) are IR, Raman (vibrational), NMR, ESR (spin resonance), UV/VIS, fluorescence (electronic) and x-ray or electron scattering. 

The amount of information, which can be extracted from a spectrum, depends essentially on the attainable spectral or time resolution and on the detection sensitivity that can be achieved. Derivative spectra can be used to enhance differences among spectra, to resolve overlapping bands in qualitative analysis and, most importantly, to reduce the effects of interference from scattering, matrix, or other absorbing compounds in quantitative analysis. Chemometric techniques make powerful tools for processing the vast amounts of information produced by spectroscopic techniques, as a result of which the performance is significantly... 

Figures 4.31(c), 4.36 and 13.3 from Snyder and Kirkland, Introduction to Modern Liquid Chromatography, 2nd edn., (1979) 9.41(a), (b) and (c) from Cooper, Spectroscopic Techniques for Organic Chemists (1980) 9.46 from Millard, Quantitative Mass Spectrometry (1978) 4.17, 4.18, 4.31 (a), 4.33, 4.34(a), 4.37, 4.38, 4.43 and 4.45 from Smith, Gas and Liquid Chromatography in Analytical Chemistry (1988) figures 4.42 and 13.2 from Berridge, Techniques for the Automated Optimisation of Hplc Separations (1985) reproduced by permission of John Wiley and Sons Limited 11.1, 11.5, 11.6, 11.12, 11.13, 11.14, 11.18 and 11.19 from Wendlandt, Thermal Analysis, 3rd edn., (1986) reprinted by permission of John Wiley and Sons Inc., all rights reserved.

Modern spectroscopy plays an important role in pharmaceutical analysis. Historically, spectroscopic techniques such as infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS) were used primarily for characterization of drug substances and structure elucidation of synthetic impurities and degradation products. Because of the limitation in specificity (spectral and chemical interference) and sensitivity, spectroscopy alone has assumed a much less important role than chromatographic techniques in quantitative analytical applications. However, spectroscopy offers the significant advantages of simple sample preparation and expeditious operation. 

An important tool for the fast characterization of intermediates and products in solution-phase synthesis are vibrational spectroscopic techniques such as Fourier transform infrared (FTIR) or Raman spectroscopy. These concepts have also been successfully applied to solid-phase organic chemistry. A single bead often suffices to acquire vibrational spectra that allow for qualitative and quantitative analysis of reaction products,3 reaction kinetics,4 or for decoding combinatorial libraries.5... 

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