Properties of the analyte

The solubility of a solute in a supercritical fluid is dictated by its polarity, volatility and molecular weight. Thus, the less volatile and more polar a given analyte is, the less readily soluble in a non-polar extractant such as supercritical CO, it will be. 

The highest possible solubility is achieved when the solubility parameter for the extracting SF is similar to that for the solute. The extraction conditions must be chosen in such a way that the solute solubility in the fluid will be maximal whenever a large amount of solute (a major constituent) is to be extracted. The influence on the extraction efficiency of the analyte concentration in the target sample increases as the analyte solubility decreases. 

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An analysis of a sample to find the identity, concentration, or properties of the analyte. 

The first important distinction we will make is among the terms analysis, determination, and measurement. An analysis provides chemical or physical information about a sample. The components of interest in the sample are called analytes, and the remainder of the sample is the matrix. In an analysis we determine the identity, concentration, or properties of the analytes. To make this determination we measure one or more of the analyte s chemical or physical properties. 

The goal of an analytical separation is to remove either the analyte or the interferent from the sample matrix. To achieve a separation there must be at least one significant difference between the chemical or physical properties of the analyte and interferent. Relying on chemical or physical properties, however, presents a fundamental problem—a separation also requires selectivity. A separation that completely removes an interferent may result in the partial loss of analyte. Altering the separation to minimize the loss of analyte, however, may leave behind some of the interferent. 

Secondly, the intensity of response for a certain compound from one type of detector is not necessarily the same as that obtained from the other detector. This should not be unexpected, since the two detectors are measuring quite different properties of the analyte, in this case UV absorption at a particular wavelength and how readily it is ionized and fragmented under the conditions employed. These properties are urn-elated. 

Solute-property detector A detector which monitors a property of the analyte, e.g. the UV detector. 

Confirmatory techniques must be submitted if the analytical method is not highly specific. A confirmatory method will not be required if the original method uses GC/MS, provided that at least three fragment ions with an m z ratio of >100 are used for identification/quantitation. The rationale for the selection of the ions monitored should also be provided. When a confirmatory method/technique is required to demonstrate specificity, the properties of the analyte should be considered when deciding on an appropriate method/technique. In SANCO/825/00 acceptable confirmatory techniques are specified as follows ... 

The following physico-chemical properties of the analyte(s) are important in method development considerations vapor pressure, ultraviolet (UV) absorption spectrum, solubility in water and in solvents, dissociation constant(s), n-octanol/water partition coefficient, stability vs hydrolysis and possible thermal, photo- or chemical degradation. These valuable data enable the analytical chemist to develop the most promising analytical approach, drawing from the literature and from his or her experience with related analytical problems, as exemplified below. Gas chromatography (GC) methods, for example, require a measurable vapor pressure and a certain thermal stability as the analytes move as vaporized molecules within the mobile phase. On the other hand, compounds that have a high vapor pressure will require careful extract concentration by evaporation of volatile solvents. 

Another equally important consideration before development of a determinative or confirmatory method is an understanding of the chemical properties of the analyte. Such an understanding becomes the cornerstone of a successful method since the unique chemical properties of each analyte provide the basis for isolation and detection schemes. Table 1 lists some of the important chemical properties that could be considered. For example, knowing the or p/fb of an analyte could influence the choice of a liquid-liquid extraction scheme, solid-phase extraction (SPE) cartridge, mobile phase pH, or mass spectrometric ionization. Knowing the overall polarity of the analyte can be very helpful in the evaluation of an extraction or separation. Currently, computational methods are available to obtain an estimate of the logP... 

Once the determinative or confirmatory method has been developed to take full advantage of the chemical properties of the analyte molecule, a study is necessary to prove that the method is valid. Criteria for method validation are outlined in guidelines from the US FDA, US EPA, and EU. A summary of the differences in regulatory requirements for method validation is provided in Table 3. The parameters addressed by all of the regulatory guidelines include accuracy, precision, sensitivity, specificity, and practicability. 

The separation of analytes from undesirable matrix components, or cleanup , of sample extracts can be accomplished through a variety of techniques that take advantage of differences in the physicochemical properties of the analytes from co-extracted matrix components. 

The IMS response for a compound is strongly dependent on temperature, pressure, analyte concen-tration/vapour pressure, and proton affinity (or elec-tron/reagent affinity). Pressure mainly affects the drift time, and spectral profiles are governed by concentration and ionisation properties of the analyte. Complex interactions among analytes in a mixture can yield an ambiguous number of peaks (less, equal to, or greater than the number of analytes) with unpredictable relative intensities. IMS is vulnerable to either matrix or sample complexity. 

Now returning to the coulometric analysis proper we can. say that any determination that can be carried out by voltammetry is also possible by coulometry whether it should be done by means of the controlled-potential or the titration (constant-current) method much depends on the electrochemical properties of the analyte itself and on additional circumstances both methods, because they are based on bulk electrolysis, require continuous stirring. 

Optical sensors rely on optical detection of a chemical species. Two basic operation principles are known for optically sensing chemical species intrinsic optical property of the analyte is utilized for its detection indicator lor label) based sensing is used when the analyte has no intrinsic optical property. For example, pH is measured optically by immobilizing a pH indicator on a solid support and observing changes in the absorption or fluorescence of the indicator as the pH of the sample varies with time1 20. 

A wide variety of ionization techniques is nowadays available and one can choose the most suitable on the basis of different factors, such as the chemico-physical properties of the analyte under investigation, its molecular weight, polarity, etc. 

Generally, there are a number of ways in which the adsorption and binding of charged macromolecules (in particular, DNA immobilization and hybridization) can affect the electrochemical properties of the analyte-FED interface. In the case of field-effect devices, two basic effects are usually considered ... 

Thermal analysis methods are defined as those techniques in which a property of the analyte is determined as a function of an externally applied temperature... 

Properties of the analyte, such as volatility, sensitivity to light, thermal stability and chemical reactivity, all have to be considered when designing a sampling strategy. These factors need to be taken into account to ensure the quality of the sample does not degrade before the measurements are made. 

A more recent development is a technique known as flow injection analysis, in which a discrete volume of a liquid sample is injected into a carrier stream. Reagents required for the development of the analytical property of the analyte, e g. colour developing reagents for spectrophotometry, are already present in the stream. The stream then flows straight to the detector and the technique depends upon the controlled and reproducible dispersion of the sample as it passes through the reaction zone. Thus the reaction does not necessarily need to develop to completion,... 

LOD is defined as the lowest concentration of an analyte that produces a signal above the background signal. LOQ is defined as the minimum amount of analyte that can be reported through quantitation. For these evaluations, a 3 x signal-to-noise ratio (S/N) value was employed for the LOD and a 10 x S/N was used to evaluate LOQ. The %RSD for the LOD had to be less than 20% and for LOQ had to be less than 10%. Table 6.2 lists the parameters for the LOD and LOQ for methyl paraben and rhodamine 110 chloride under the conditions employed. It is important to note that the LOD and LOQ values were dependent upon the physicochemical properties of the analytes (molar absorptivity, quantum yield, etc.), methods employed (wavelengths employed for detection, mobile phases, etc.), and instrumental parameters. For example, the molar absorptivity of methyl paraben at 254 nm was determined to be approximately 9000 mol/L/cm and a similar result could be expected for analytes with similar molar absorptivity values when the exact methods and instrumental parameters were used. In the case of fluorescence detection, for most applications in which the analytes of interest have been tagged with tetramethylrhodamine (TAMRA), the LOD is usually about 1 nM. 

The dynamic range of protein expression represents a main obstacle since abundant proteins are seldom of interest and others such as transcription factors are only present in a few copies. There is no detector that is able to visualize all proteins at the same time so that prefractionation and the investigation of subproteomes is required. In fact, pre-MS sample preparation techniques exploiting electrophoretic, chromatographic, or chemical properties of the analyte are often the bottleneck of proteomics. 

Retention time is the basic measure used in GC to identify compounds. It is a physical property of the analyte and is dependant on the separation conditions such as temperature, flow rate and chemical composition of the stationary phase. Solubility of the analyte in the stationary phase, which is based on the energy of intermolecular interactions between the analyte and stationary phase, is the most important factor in determining retention time. In Fig. 14.1, the retention... 

Many compounds exhibit near-IR and mid-IR absorption. By using IR transparent optical fibers, detection of an absorption band in the IR region is possible for optical sensing. Both direct sensing using the absorption property of the analyte or indicator sensing are widely exploited. Most mid-IR sensing schemes are based on the principles of internal reflection spectroscopy, or the attenuated total reflection (ATR) [3,14-21],... 

The text develops an understanding of the relevance of four fundamental properties of the analyte shape, polarity, charge and size, to the three key types of analysis separation, identification and quantification.The third edition of Analytical Biochemistry has been fully updated in content and format, making it even more accessible to students learning how to select analytical techniques and recognise their scope and limitations. 

The physical and chemical properties of stationary phase materials are described in Chapter 3 (including methods for their synthesis) to clarify the differences in similar stationary phase materials supplied from different manufacturers. A detailed selection guide to solvents is given in Chapter 4. The unlimited selection of eluent components and their concentrations is a powerful force in developing separations in liquid chromatography. Although this area seems rather complicated, it is easy to understand the selection of a suitable eluent when you first identify the molecular properties of the analytes and solvents. 

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