Sample component identification

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

The right chromatography column should separate the sample sufficiently to enable identification or quantitative measurement of the components within a reasonable period of time. The resolution factor (Rs) for two sample components is determined by the width of the two peaks and the distance between the peak maxima. In general, Rs values of 1.0 are required for good qualitative or quantitative work, whereas Rs values >1.5 indicate baseline resolution for two components (3). 

This is an alternative technique to headspace analysis for the identification and determination of volatile organic compounds in water. The sample is purged with an inert gas for a fixed period of time. Volatile compounds are sparged from the sample and collected on a solid sorbent trap—usually activated carbon. The trap is then rapidly heated and the compounds collected and transferred as a plug under a reversed flow of inert gas to an external gas chromatograph. Chromatographic techniques are then used to quantify and identify sample components. 

The response of vertebrates to olfactory stimulation is affected by previous experience but behaviour can be specifically affected by odours (pheromones) (4). The olfactory system has been shown to detect specific components within complex mixtures and analytical chemistry techniques have been used to identify these active components (5). We have assessed the application of these methods to the problems of agricultural odours in an attempt to develop techniques applicable to both slurries and air samples. The identification of the odorous components might allow specific treatment methods to be developed. In addition, the designation of a range of indicator compounds might be useful in practice for monitoring abatement of odour nuisances. 

Coupled columns packed with different stationary phases can be used to optimize the analysis time (71, 75). In this approach the different columns are connected in a series or in parallel. liie sample mixture is first fractioned on a relatively short column. Subsequently the fractions of the partially separated mixture are separated on other columns containing the same or other stationary phases in order to obtain the individual components. Columns differing in length (number of theoretical plates), adsorptive strength or phase ratio (magnitude of specific surface area), and selectivity (nature of the stationary phase) can be employed, whereas, the eluent composition remains unchanged. Identification of the individual sample components via coupled column technique requires a careful optimization of each column and precise control of each switching step. 

The time required by a given analyte to migrate under the sole influence of the applied electric field across the capillary tube from the injection end of the capillary to the detection windows (migration distance) is defined as the migration time (tj and, similarly as the retention time in HPLC, is used for identification of sample components. It is given by... 

Intentionally select or sample components not yet examined. This strategy is frequently applied in art conservation research aimed at the identification of specific alterations or the characterization of the artist s palette. 

In its conventional mode, capillary action TLC is a simple but versatile procedure that does not require expensive equipment. Therefore, TLC has a particular potential as a reliable technique for laboratories with very limited resources for instrumental equipment. The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase. Standards are also applied on the layer to be simultaneously run with the unknown sample for identification purposes. Volume precision and exact positioning are ensured by the use of a suitable instrument. The bottom edge of the plate is placed in a solvent reservoir, and the mobile phase moves up the plate by capillary action for a predetermined distance. In this process, the different components of the sample migrate up the plate at different rates due to differences in their partitioning behavior between... 

Marker. A reference component which is chromatographed with the sample to aid in the identification of sample components. 

There are a variety of techniques currently in use to aid in the identification of sample components. Most techniques, such as spiking and the enzyme peak shift method, are used to confirm the identities of components thought to be in the sample. If, however, the identity of the sample is truly unknown, a combination of techniques is needed to provide a unique fingerprint. ... 

As a first approximation, the most commonly used method of peak identification is that of matching the retention times, or occasionally capacity factors, of the sample components with those of standard reference... 

There are a variety of techniques available to aid in the identification of sample components matching retention times, standard addition, internal standard, isotopic labeling, enzyme peak shift, and UV and mass spectral libraries. 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

Preparative liquid chromatography. Any scale of operation in which the objective is the collection of sample components for subsequent identification or use. Preparative LC may involve submilligram or multigram quantities. 

Techniques in clinical analysis have undergone many advances in the last few decades. The basic needs in clinical chemistry are unambiguous analyte-specific assays that provide both identification of sample components and their concentration levels. The importance of this is self-evident, since most substances analyzed are part of a multicomponent biological fluid. Advances in enzyme and immunochemical assay techniques provide ideal systems for component... 

High performance liquid chromatography (HPLC) has become one of the most powerful tools in contemporary organic analysis as the separation technique which can separate very complex mixtures of compounds and provide qualitative and quantitative information on the sample useful for the identification and determination of sample components. 

The most important peak parameters are the peak area, the elution time of the centre of the peak and the peak variance. The peak area is proportional to the mass of the eluted compound and is usually used as the basis of quantitation. The elution time of the centre of gravity of the chromatographic peak is the elution (retention) time, fR, or the elution (retention) volume, Vr. of the compound. It is controlled by the distribution constant of the compound between the stationary and the mobile pha.ses and can be used for identification of the individual sample components. Finally, the peak variance, o (in time units) or a (in volume units) is a measure of peak broadening and can be used for the evaluation of the efficiency of the chromatographic column. For a truly Gaussian peak, the distance between the two inflection points (at 0.607 peak height) corresponds to 2(7. The peak width, u>, equals 4a and can be determined as the distance between the intersection points of the baseline with tangents drawn to the inflection points of the peak. 

Developing an HPLC method requires a clear specification of the goals of the separation. The primary objective could be (1) resolution, detection and characterisation or quantitation of one or a few substances in a product, so that it is important to separate only a few sample components and complete separation of the sample is not necessary (2) complete resolution, characterisation and quantitation of all sample components (3) isolation of purified sample components for spectral identification or for other assays. Further points that should be considered include the required sensitivity (especially for trace analysis), accuracy, precision, character of sample matrices (which determines sample dissolution, extraction or pretreatment necessary for possible concentration of sample analytes or for removing interference), expected frequency of analyses and the HPLC equipment available. 

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