Chromatography compound identification

Chemical separations may first be accomplished by partitioning on the basis of polarity into a series of solvents from non-polar hexane to very polar compounds like methanol. Compounds may also be separated by molecular size, charge, or adsorptive characteristics, etc. Various chromatography methods are utilized, including columns, thin layer (TLC) gas-liquid (GLC), and more recently, high pressure liquid (HPLC) systems. HPLC has proven particularly useful for separations of water soluble compounds from relatively crude plant extracts. Previously, the major effort toward compound identification involved chemical tests to detect specific functional groups, whereas characterization is now usually accomplished by using a... 

TLC plates are of particular interest as substrates for spectroscopy (i) as a storage device for offline spectroscopic analysis (ii) for efficient in situ detection and identification and (iii) for exploitation of spectroscopic techniques that cannot be used in HPLC. Thin-layer chromatography combined with HR MAS (NMR) can be used for compound identification without the need for elution from the stationary phase [413]. Recently also TLC-XRF was found suitable for in situ TLC imaging of elements [414]. The combination... 

In chromatography-FTIR applications, in most instances, IR spectroscopy alone cannot provide unequivocal mixture-component identification. For this reason, chromatography-FTIR results are often combined with retention indices or mass-spectral analysis to improve structure assignments. In GC-FTIR instrumentation the capillary column terminates directly at the light-pipe entrance, and the flow is returned to the GC oven to allow in-line detection by FID or MS. Recently, a multihyphenated system consisting of a GC, combined with a cryostatic interfaced FT1R spectrometer and FID detector, and a mass spectrometer, has been described [197]. Obviously, GC-FTIR-MS is a versatile complex mixture analysis technique that can provide unequivocal and unambiguous compound identification [198,199]. Actually, on-line GC-IR, with... 

Based on a new technology, particle beam enhanced liquid chromatography-mass spectrometry expands a chemist s ability to analyse a vast variety of substances. Electron impact spectra from the system are reproducible and can be searched against standard or custom libraries for positive compound identification. Chemical ionization spectra can also be produced. Simplicity is a key feature. A simple adjustment to the particle beam interface is all it takes. 

Generally we allowed various model compounds to react with alkali for varying lengths of time and then separated the reaction products by paper chromatography. For identification, the products were eluted from the paper, and spectral analyses were made of the eluates. 

Metal complexes are used for compound identification. They can shift or change absorption spectra, change the Rf of compounds in thin layer chromatography, and change visual colors used in chromatography. 

These are detectors that respond to all classes of chemical compounds and at the same time provide definitive compound identification. The only chromatography detector that is universal, i.e. non-selective and selective at the same time, is a mass spectrometer. 

The mass spectrometer provides the most reliable compound identification in chromatography methods. When the concentration levels are sufficiently high and GC/MS or HPLC/MS instruments are available, they should be the preferred confirmation techniques. However, not every detection may be confirmed by the mass spectrometry methods due to the limited sensitivity of the mass spectrometer. 

Compound identifications were made by combined gas chromatography-mass spectrometry (GC-MS) based on relative retention times and mass spectral interpretations. The instrument used was a Finnigan 5100 computerized GC-MS system equipped with a 50 m x 0.32 mm i.d. fused silica capillary coated with CP Sil 8 CB (0.25 jim film thickness). Helium was used as carrier gas and the temperature program was as follows 110°C (2 min)- 3°C/min - 320°C. 

Thompson et al. [113] emphasise that even the use of two dissimilar gas chromatographic columns does not ensure irrefutable compound identification. For example, if the retention characteristics of a given peak obtained from two dissimilar columns suggest the possibility of the presence of a compound which appears wholly out of place in a specific sample, further confirmation is clearly indicated by such techniques as specific detectors, coulometry, p values, or gas chromatography-mass spectrometry or thin layer chromatography. 

Maralikova B, Weinmann W (2004) Confirmatory analysis for drugs of abuse in plasma and urine by high-performance liquid chromatography-tandem mass spectrometry with respect to criteria for compound identification. J Chromatogr B Analyt Technol Biomed Life Sci 811(1) 21—30. doi 10.1016/j.jchromb.2004.04.039 SI570-0232(04)00642-7 [pii]... 

S.E. Stein, An integrated method for spectrum extraction and compound identification from gas chromatography/mass spectrometry data, J. Am. Soc. Mass Spectrom., 10, 770-781 (1999). 

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). 

Process solvents SRC s, and liquefaction products also were examined by column chromatography (11). The sample was dissolved in chloroform or THF, pre-adsorbed on neutral alumina (12), and eluted from neutral alumina to give saturates, aromatics, and three polar fractions. The saturate fraction was analyzed quantitatively by mass spectrometry, and compound identification in distillates and solvents was confirmed by combined GC-MS or high-resolution MS analysis of column chromatography fractions. 

The introduction of GC as an analytical technique has had a profound impact on both qualitative and quantitative analysis of organic compounds. Identification of compounds by GC can be accomplished by their retention times on the column as compared to known reference standards, by inference from sample treatment prior to chromatography, " or by the concept of retention index. " The latter method and tables of retention indices " with associated conditions have been reported. " Although qualitative data and analytical techniques for identification of compounds are well-established " and relative retention data for over 600 substances also have been published, " the main utility of GC undoubtedly lies in its powerful combination of separation and quantitative capabilities. Use in quantitative analysis involves the implementation of two techniques being performed concurrently, i.e., separation of components and subsequent quantitative measurement. 

Wilson, I.D. Spraul, M. Humpfer, E. Thin layer chromatography combined with high resolution solid state NMR for compound identification without substance elution preliminary results. J. Planar Chromatogr.-Mod. TLC 1997, 10, 217-219. 

Identification. The fundamental limitation of gas chromatography in identification studies is that by this method, one can only state nonpresence of a known compound which is available as a standard in a considerably pure form. Thus, gas chromatography is an excellent... 

Selection of the analytical instrumentation for the analysis of the pyrolysate is a very important step for obtaining the appropriate results on a certain practical problem. However, not only technical factors are involved in this selection the availability of a certain instrumentation is most commonly the limiting factor. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS) are, however, the most common techniques utilized for the on-line or off-line analysis of pyrolysates. The clear advantages of these techniques such as sensitivity and capability to identify unknown compounds explain their use. However, the limitations of GC to process non-volatile samples and the fact that larger molecules in a pyrolysate commonly retain more structural information on a polymer would make HPLC or other techniques more appropriate for pyrolysate analysis. However, not many results on HPLC analysis of pyrolysates are reported (see section 5.6). This is probably explained by the limitations in the capability of compound identification of HPLC, even when it is coupled with a mass spectrometric system. Other techniques such as FTIR or NMR can also be utilized for the analysis of pyrolysates, but their lower sensitivity relative to mass spectrometry explains their limited usage. 

Another possible detector for GC is the mass spectrometer. This detector (which was in fact developed independently as a stand-alone instrument) offers the capability of compound identification. Extensive literature is available regarding gas chromatography/mass spectrometry (GC/MS) analysis of organic molecules [e g. 37,... 

Selective reagents form colored or fluorescent compounds on a group- or substance-specific basis and aid in compound identification. They also allow the use of a thin-layer chromatography (TLC) system with less resolution because interfering zones will not be detected. Examples include the formation of red to purple zones by reaction of a-amino acids with ninhy-drin, detection of acidic and/or basic analytes with the... 

Gas chromatography/infrared spectroscopy analysis was also used under differing operating parameters to aid in compound identification. 

The DAD has brought compound identification to HPLC. Previously, mass spectrometry was the sole domain in peak identification for gas or liquid chromatography (GC-MS or LC-MS). This can now be achieved as part of the HPLC analysis, and at a lower cost, because there is the ability to use an HPLC-DAD system as a scouting technique to check the possible identity of an unknown sample. 

Gocan, S. and Cimpan, G. Compound identification in thin layer chromatography using spectrometric methods. Reviews in Analytical Chemistry. 16 1-24, 1997. 

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. ... 

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