Analyte identification

The retention time or volume at which an unknown solute elutes from a column or the distance traveled on a plate is often matched to that of a reference compound. The appearance of a solute peak, band, or spot at the same time as that of a reference compound is consistent with the two compounds being the same. The simultaneous appearance does not prove identity, however, because other compounds can have the same retention time as the reference compound. 

In planar chromatography, reference compounds are chromatographed with the unloiown sample. Tentative identification is made by comparison of the migration distances and detection characteristics of the reference compounds with those of the unknown analytes. If the Rf of the unicnown analyte and the Rf of the reference compound do not match, the compounds are judged to be different. If they match, the compounds are presumed to be identical. However, as more than one compound can have the same Rf in a particular chromatographic system, the presumptive identification has to be confirmed by the use of specific spray reagents, antibody complexation, or isolation of the compound followed by chemical and/or instrumental analysis. Software is now available for compound identification by library searching of UV spectra based on corrected Rf values.  

With capillary GC and LC columns, it is possible to simultaneously introduce the components of a single injection into two columns made of dissimilar stationary phases. The columns are connected to separate detectors of the same or a different type. Matching the retention properties of a single analyte with a reference compound on two columns of dissimilar phases enhances the chance for correct identification of the analyte. The most reliable analyte identification, however, is provided by a detector that features structural information, such as a mass spectrometer (see Chapter 7). 

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Sjogren. A. and Dasgupta, P. K., Two-dimensional conductimetric detection in ion chromatography. Analyte identification, quantitation of very weak acid anions, and universal calibration, Anal. Chem., 67, 2110, 1995. 

Tandem mass spectrometry provides the highest degree of certainty in analyte identification and may be employed in accordance with recent EU guidelines to obtain data with relevant unambiguity (EC Council Directive SANCO/1805/2000). The sensitivity of MS/MS is typically in the sub-pg range. 

Quite often a normal electron ionization mass spectrum appears insufficient for reliable analyte identification. In this case additional mass spectral possibilities may be engaged. For example, the absence of the molecular ion peak in the electron ionization spectrum may require recording another type of mass spectrum of this analyte by means of soft ionization (chemical ionization, field ionization). The problem of impurities interfering with the spectra recorded via a direct inlet system may be resolved using GC/MS techniques. The value of high resolution mass spectrometry is obvious as the information on the elemental composition of the molecular and fragment ions is of primary importance. 

Analytical identification of monoazo colorants and the other decomposition products requires effective (analytical) methods of concentration, which is made possible by high performance liquid chromatography (HPLC). Prior to HPLC analysis, the pigmented medium was extracted for 20 hours with toluene in a soxhlet extractor. These analytical methods also showed that above 240°C, especially after prolonged exposure of the pigmented polymer material to heat, dichlorobenzidine (DCB) is also formed. 

Retention time the time it takes for an elnate to move through a chromatographic system and reach the detector. Retention times are reprodncible and can therefore be compared to a standard for analyte identification. 

Combinatorial chemistry techniques produce hundreds, if not thousands, of samples that need to be analyzed in a relatively short period of time. While fast HPLC analyses with short columns and fast gradients have increased throughput, there are still limits on the number of samples that can be processed. Some systems have evolved that incorporate multiple injectors, pumps, and UV detectors, but are still limited by having only one mass spectrometer available for detection and analyte identification. 

Drifts of migration times can partly be compensated by calculating the mobility for analyte identification and using corrected PAs or internal standards for quantitation. 

MS sensitivity depends on both the type of instrumentation and the nature of the analytes, but, typically, a minimum sample size of 5-10 ng is in most cases sufficient. Limited sensitivity in a certain application is often not due to the inherent sensitivity of the MS but rather the level of background impurities that are in the isolate. It is not always appreciated that very slowly eluting LC solutes from previous separations can create substantial MS background peaks that obscure analyte identification. Thus, it is important to use a blank sample to ensure that the background of the trapped fraction is adequately free of possible interferences to the desired identification. 

Recognizing the fact that shifts in retention time of individual compounds may cause false negative results, laboratories use retention time windows for target analyte identification. Retention time windows are experimentally determined retention time ranges for each target analyte. To minimize the risk of false positive results, EPA methods require that chromatography analysis be performed on two columns with dissimilar polarities. This technique, called second column confirmation, is described in Chapter 4.4.3. It reduces the risk of false positive results, but does not eliminate them completely. 

In individual compound analysis with the FID, analyte identification relies solely on retention time. As a result, single compound determinations (for example, individual phenols or PAHs) are usually gravely affected by interferences that often render the results unreliable or unusable. Because the FID methods for individual compound analysis are so susceptible to false positive results, they should be used with caution and only for interference-free matrices. 

Compound(s)1 Source of PGS Prior sample cleanup Column Analytical identification Ref. 

Analytical HPLC of PGS. A number of reports are currently available on the use of HPLC for the analytical identification of native PGS (Table 5). These techniques have primarily relied on purification by prior partitioning and chromatographic separation... 

Analytical methods for PGS research have been greatly improved during this past decade. GLC-MS analysis has proven to be the method of choice, particularly when appropriate internal standards are used for accurate assessment of PGS recovery. HPLC, the most rapidly developing form of separation science, should substantially enhance present PGS analytical efforts. One advantage of HPLC is the substantial purification obtained for PGS compounds from crude plant extracts. For analytical identification by instrumentation, scrupulous purification is required, along with selective identification of the PGS. Preferably, two different analytical procedures should be utilized for positive identification of a given compound. 

For the purpose of analyte identification, the response of the detector array to exposure to the analyte vapor must be recorded, and a unique pattern or chemical signature for each analyte must be extracted from the data. A good signature pattern highlights the characteristic features of the data that yield high differentiation between patterns and eliminates those features that provide little or no differentiation information. 

As noted in the preceding table, elution of PAHs is detected by UV absorbance at two different wavelengths 280 nm and 365 nm. Fluorescence detectors are also applicable to the HPLC analysis of PAHs (9, 19). The UV detector monitors the sample simultaneously at two wavelengths, aiding in compound identification. For a specific compound, the ratio of absorbances at two different wavelengths is an intrinsic physical characteristic. Therefore, it is possible, in principle, to identify a sample analyte by this characteristic ratio. The chromatographic retention time of each of the specific peaks observed in the sample eluate is compared with those of known standard compounds for tentative analyte identification. For quantitation, peak areas of each standard, at each of six... 

Selectivity is a qualitative estimate of how well the analyte identification procedure is able to distinguish an analyte in a sample from one or many similar analytes with similar, or even some of the same, physical or chemical properties. 

While the structure determination methods seen in the previous section are based on the analytical identification of the positive library individuals, other methods exist to perform the so-called deconvolution of the library complexity and allow the eventual identification of one or more positives from the library without determining their structure by analytical methods. The most common approach is based on iterative synthesis cycles of less complex pools deriving from the original active pool(s) until single compounds are prepared and tested iterative deconvolution) (3). 

Inorganic and bioinorganic applications of IR and Raman spectroscopies are covered in some detail in subsequent sections. General types of information that can be obtained include analytical identification, stracture and symmetry, ligand and functional group identification, metal-ligand and metal-metal bonding potentials and force constants, structural kinetics and dynamics, excited-state properties, vibronic... 

Fourier transform-infrared spectroscopy analysis of collected peaks used to confirm analyte identification. 

Scheidegger, A., Borkovec, M. and Sticher, H. (1993) Coating of silica sand with goethite Preparation and analytical identification. Geoderma, 58 43-65. 

Added to these constraints are the issues raised by the certainty of a result in terms of both analyte identification and quantitation. The uncertainty of a result is dependent on the analyte concentration. In trace analysis it might be argued that the traceability of a method in identifying a substance would have a traceability chain completely different from that of a method which quantitates the same substance. The traceability chain for a method which both quantitates and identifies a substance could be different again. This differentiation is important in many regulatory analyses in which a zero tolerance for a substance has been set. Under these circumstances, only the presence of the substance has to be established. This problem is discussed in more detail later in this paper. 

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