Retention time chemical identification

The quantitation of products that form in low yields requires special care with HPLC analyses. In cases where the product yield is <1%, it is generally not feasible to obtain sufficient material for a detailed physical characterization of the product. Therefore, the product identification is restricted to a comparison of the UV-vis spectrum and HPLC retention time with those for an authentic standard. However, if a minor reaction product forms with a UV spectrum and HPLC chromatographic properties similar to those for the putative substitution or elimination reaction, this may lead to errors in structural assignments. Our practice is to treat rate constant ratios determined from very low product yields as limits, until additional evidence can be obtained that our experimental value for this ratio provides a chemically reasonable description of the partitioning of the carbocation intermediate. For example, verification of the structure of an alkene that is proposed to form in low yields by deprotonation of the carbocation by solvent can be obtained from a detailed analysis of the increase in the yield of this product due to general base catalysis of carbocation deprotonation.14,16... 

Chromatographic methods are used to separate the components in a mixture, but in a complex mixture, a single chromatographic method or step many not separate all components. In these cases, using simple retention time to identify the components will not suffice and the identification of components in the mixture will be incorrect. Thus, the addition of a method of identification such as mass spectrometry (MS) or Fourier transform infrared (FTIR) is essential. In some cases, it may even be necessary to confirm either an FTIR or MS identification by the same method applied in a different way. For example, FTIR may be followed by MS, or electron ionization (El) MS followed by chemical ionization (Cl) MS or by an entirely different method. 

It is a great deal of work to actually determine a true equilibrium constant and most chemical separation methods speak in terms of values which are proportional to the actual equilibrium constant. At constant flow, the time that a given type of molecule is retained is related to the time for the void volume to pass after the sample is placed in a column or on a plate with the addition of the time for the net retention volume. If the flow remains constant, the temperature of the separation remains constant and no stationary phase is gained or lost, one can attempt qualitative identification using retention times. It is more reasonable to calculate the ratio of net retention volume to the void volume and call the result partition factor or capacity factor, k. ... 

MS, chemical ionization-MS, and sometimes GC/infrared spectroscopy (IR) have been used with GC/MS to obtain structural information. Examples of the use of GC/ MS for identifying new DBFs include the recent identification of iodo-acids. The iodo-acids were discovered in drinking water treated with chloramination through the use of full-scan GC/MS on the methylated extracts. Empirical formula information for both the molecular ions and the fragment ions was obtained by high-resolution electron ionization (EI)-MS, and the spectra were interpreted to yield tentative identifications of five new iodo-acids (iodoacetic acid, bromoiodoacetic acid, ( )-3-bromo-3-iodopropenoic acid, (Z)-3-bromo-3-iodopropenoic acid, and )-2-iodo-3-methylbutenedioic acid). Structural assignments were then confirmed by the match of mass spectra and GC retention times to authentic chemical standards, several of which had to be synthesized. 

Identification of compounds in the river water extracts was based on the coincidence of gas chromatographic retention times and on the equivalence of electron impact and chemical ionization mass spectra with those of authentic compounds. Quantitation was based on standard curves generated for selected compounds. 

A combination of (.Z)-8-dodecenyl acetate and ( )-8-dodecenol was identified from the sex pheromones glands of the carambola fruit borer, Eucosma notanthes Meyrick (Lepidoptera Tortricidae). The ratio of the alcohol to the ester in the extracts was 2 7. GC, GC-MS, chemical deriva-tization, and comparison of retention times with authentic standards were used for identification. ... 

Solvent extraction of the sample is also frequently used in the analysis of particulate matter. Through the appropriate choice of solvents, the organics can be separated into acid, base, and neutral fractions, polar and nonpolar fractions, and so on. This grouping of compounds according to their chemical properties using extraction techniques simplifies the subsequent analysis. Each fraction can then be analyzed by GC-MS, with the GC retention time and the mass spectrum used for identification and measurement. 

Unlike with GC-MS, quality criteria for identification of drug residues by LC-MS have not been yet defined within the European Union, but this is currently under review. Criteria for GC-MS stipulate the measurement of preferably at least four diagnostic ions. However, this is not always possible with LC-MS because most compounds will only produce an M ion in positive mode or a M ion in negative mode, with little fragmentation when using thermospray (TSP), electrospray (ESP), or atmospheric pressure chemical ionization (APCI). Even where the ions and ratios are in agreement, there will be still possibility of misidentification. For this reason, mass spectra data are often interpreted with additional supporting data such as the LC retention times, as, for example, in the LC-MS analysis of sulfadimethoxine and sulfadoxine that present identical mass spectra (24). 

Chemical Analysis of Extracts. The extracts were analyzed by capillary column GC-MS for OCs, TAAPs, and PAHs (see the list on page 313). The GC-MS parameters used at the two laboratories are shown in Table II. The identification and quantitation were all done by using automatic routines based on a mass spectra library created from authentic standards of the selected compounds. Compounds were located by searching the reconstructed ion chromatogram for each library entry within a narrow retention time window relative to the internal standard (anthracene-dio or phenanthrene-dio). Quantitation was achieved by comparison of characteristic ion areas in the field samples with ion areas of the internal standard. These ion areas were normalized by response factors established by comparison of ion ratios of a standard mixture of all 66 analytes at a concentration of 2.5 ng//zL. 

Identification of unknown compounds by retention times or peak enhancement is not conclusive or absolute proof of identity. It is possible for two different substances to have identical retention times under the same experimental conditions. For positive identification, the sample must be collected at the exit port and characterized by mass spectrometry, infrared, nuclear magnetic resonance, or chemical analysis. 

The method is based on the principle of chromatographic separation of components of a mixture on a GC column, followed by their identification from their mass spectra. The compounds are separated on a suitable GC column, following which, the components eluted from the column are subjected to electron-impact or chemical ionization. The fragmented and molecular ions are identified from their characteristic mass spectra. Thus, the substances present in the sample are determined from their characteristic primary and secondary ions and also from their retention times. 

Individual compound identification in all GC methods with the exception of GC/MS relies on the compound retention time and the response from a selective or non-selective detector. There is always a degree of uncertainty in a compound s identity and quantity, particularly when non-selective detectors are used or when the sample matrix contains interfering chemicals. To reduce this uncertainty, confirmation with a second column or a second detector is necessary. Analyses conducted with universal detectors (mass spectrometer or diode array) do not require confirmation, as they provide highly reliable compound identification. 

When a new impurity is encountered during chemical process research, retention time and molecular weight information are compared to the database for rapid identification. This approach is similar to the procedure described for natural product dereplication. If the compound is not contained in the structure database, then the corresponding LC/MS/MS analysis is performed to obtain substructural detail and the proposal of a new structure. 

In most cases, absolute retention times are used for the identification. Modem GCs have high precision and accuracy of retention times within 0.05 %, or better can be obtained in subsequent runs during the same day. When the analysis is repeated by another instrument, the deviation may be of several percent. The reproducibility of absolute retention times is strongly dependent on the proper adjustment of all chromatographic parameters. In addition, the column properties are not exactly the same even when similar columns from the same manufacturer are used. On the other hand, absolute retention times are the most useful identification parameters if a few chemicals are to be monitored and the background is low. This method requires frequent calibration because even small changes in chromatographic conditions will influence the absolute retention times. 

The OPCW requires two independent techniques for identification. In the case of GC/MS, El, and Cl spectra, acquired from separate chromatographic runs, are regarded as independent techniques. A combination of the two is accepted as unequivocal identification - one as a fingerprint, the other to confirm molecular mass. LC/MS provides an alternative to GC/CI/MS for confirmation of the molecular mass and LC/MS/MS provides a partial fingerprint. However, LC/MS/MS spectra, which are normally acquired under identical chromatographic conditions as LC/MS spectra, are not currently considered as a second independent identification. Further development of these criteria may be required with instrumental development. LC retention time is accepted as a second technique if the retention time falls within a window of 0.2 min of the retention time for an authentic chemical, with a signal-to-noise ratio of at least 5 1. However, great care has to be exercised if identification is based solely on LC/MS. 

Database, which contains as much data on CWC-related chemicals as possible. Since on-site analysis in particular by MS is planned for future verification activities, library of mass spectra is mandatory. Retention times of chemicals are collected for chromatography analyses. Although NMR spectroscopy is not suitable for on-site analysis, it is nevertheless considered an essential technique (as may be also IR) for laboratories specialized in the detection and identification of CW agents and related chemicals. Qualified laboratories from all parts of the world were therefore asked to submit their mass, NMR, and IR spectra and retention time data to be included in the OCAD. 

Each cycle creates additional chemical noise. In TLC or GC, this noise build-up creates a substantial problem. In LC, however, the very polar, noise-causing compounds elute before the PTH-amino acids and, therefore, do not interfere with the analysis. The sequencing of a protein or peptide can be done manually or on a commercial device called a sequencer. The HPLC is the analysis tool which aids in the identification of the amino acid by matching the retention time of the unknown to a known, specific amino acid. An example of the separation and identification of the output of a sequencer is shown in Figure 2-11. 

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