Involatile compounds

Problems may be encountered in the analysis of thermally labile compounds, as heat is required for mobile-phase removal and for the transfer of analyte from the belt into the source of the mass spectrometer, and highly involatile compounds which cannot be desorbed from the belt, unless FAB is used for ionization. 

Involatile compounds are not usually ionized with good efficiency. 

A number of thermally labile and relatively involatile compounds which do not yield El spectra when using more conventional inlet methods do so when introduced via the particle-beam interface. 

Neither extremely volatile or extremely involatile compounds are ideal for investigation using the particle-beam interface. 

Principles and Characteristics In the early mass-spectrometric ionisation techniques, such as El and Cl, the sample needs to be present in the ionisation source in its gaseous phase. Volatilisation by applying heat renders more difficult the analysis of thermally labile and involatile compounds, including highly polar samples and those of very high molecular mass. Although chemical derivatisation may be used to improve volatility and thermal stability, many compounds have eluded mass-spectrometric analysis until the emergence of fast atom bombardment (FAB) [72]. 

Table 7.40 summarises the general characteristics of on-line SFC-MS. The method is potentially most useful for thermally labile and involatile compounds that are unsuitable for GC-MS. Because the MS instrument is the main source of information, the reproducibility of the retention and the separation selectivity are much less important than for other SFC applications. As a result, mass spectroscopists do not feel restrained by the limits on reproducibility, which slowed the uptake of SFC by chromatographers. Method development should not be underestimated. Practical problems are associated with interfacing and the effect of the expanding... 

The use of ionisation techniques such as El and Cl for TLC stationary phases has generally been limited to relatively nonpolar and thermally stable molecules. Polar involatile compounds, separated on silica gel, generally strongly adsorb on to the matrix, and decompose when heat is applied for volatilisation [817]. Use of less-adsorbent phases, such as polyamide, is particularly useful for TLC-EIMS work, because the analytes are not as strongly adsorbed to this phase and do not require high probe temperatures [818,819]. For compounds that are not suitable candidates for TLC-EIMS, FAB can be employed. Chemical ionisation, although suitable for TLC-MS, appears to have been little used. 

One of the major problems has been to determine the site of attachment of the PAH to the base. Some information may be obtained directly from the nmr spectra eliminating certain points of attachment. As mentioned above, if the C-8 proton of guanine or adenine can be identified, then this cannot be the point of attachment of the carcinogen. Estimation of the pKa s of the adducts either by titration (108) or partition (110) has, however, provided additional valuable information. Mass spectral fragmentation patterns can be of help in determining the site of substitution as well as in determining which bases are involved in binding (108.111-113). Substantial advances have been made in recent years on the mass spectral analysis of involatile compounds and derivatization is not always essential (114-118). X-ray analysis of DNA adducts has, to date, only been applied to model systems (119-121). 

Gas chromatography is one of the most powerful analytical techniques available. Its only major limitation is that it can not analyse involatile compounds such as fats. The solution in this case is to make a volatile derivative, e.g. the use of fatty acid methyl esters to analyse triglycerides. 

The sample must be in the gas phase. Microwave absorption lines are considerably broadened by molecular collisions at intermediate and high pressures at atmospheric pressure, microwave absorption lines are tens of thousands of megacycles wide. Hence the gas is kept at low pressure, typically 0.01 to 0.1 torr under these conditions, line widths run about MHz. The compound studied need not be a gas at room temperature, but it must have sufficient vapor pressure to give detectable absorption. To study involatile compounds such as the alkali halides, the waveguide must be heated to 500-1000°C high-temperature microwave spectroscopy presents great experimental difficulties, but it has been used to study most of the alkali halides. 

A graphite quartz tube for decomposing involatile compounds of metals, with extremely high sensitivity. 

For ionic and involatile compounds, data must be obtained in the solid state. This has disadvantages in that bands tend to be broader, calibration of samples presents difficulties, and only surface layers of the sample are probed. These may well not be characteristic of the bulk. Also surfaces are very sensitive to contamination, and effective sample cleaning is needed rigorous vacuum conditions must prevail during the experiment to prevent recontamination. 

Inverse Gas Chromatography (IGC) has been used to measure solubility parameters for three polymers at 25° C using the method of Guillet and DiPaola-Baranyi. The linear relationship noted with other polymers was found and the results add further credance to the method. Solubility parameters have also been calculated for six small molecule involatile compounds of the type use as plasticizers. The original method did not yield values in good agreement with literature results but estimation of the different contributions to the solution interactions allowed calculation of more meaningful values. 

Desorption/chemical ionization mass spectrometry is a useful technique for the investigation of underivatized polar and involatile compounds such as triterpenoid saponins.162 The cleavage of saponins by anodic oxidation has been investigated,163 as has the glycosidation of triterpenoids.164... 

As reported in the structural determination of BL, CS, DL, and typhasterol, MS is an essential technique for BRs isolated in pure form. However, in most cases, isolation of BRs in pure form is time-consuming and tedious work because of their very low concentration in plant materials. BRs are highly polar and involatile compounds. Therefore, conversion of BRs into volatile derivatives in gas phase makes it easy to characterize BRs in a partially purified bioactive fraction by GC/MS or GC/selected ion monitoring (SIM), which are analytical techniques most frequently used in natural products chemistry. The desired derivatives of BRs are BMBs or MB-TMSs. Another convenient and useful technique is HPLC. HPLC has now been routinely and effectively employed in the purification of natural BRs. Microanalysis of BRs by HPLC has recently been developed, which involves transformation of BRs into derivatives with a fluorophore or an electrophore by use of pre-labeling reagents. Immunoassay techniques to analyze plant hormones have recently advanced and are readily accessible by plant physiologists. RIA for BRs has also been developed. In this section, micro-analytical methods of BRs using GC/MS (SIM), HPLC, and RIA are described. 

For a molecular compound, the full molecular formula can be established from the empirical formula and the molecular mass (RMM). Various physical properties, including the vapour density of a gas, and so-called colligative properties (such as freezing point depression) in solution, can be used to determine the RMM. However the most important technique in modem research is mass spectrometry (MS) where molecular ions are accelerated in an electric field, and then pass through a magnetic field where their paths are bent to an extent that depends on the mass/charge ratio. The traditional MS method requires a volatile sample, ionized by electron bombardment, but methods are now available that overcome the limitations of that method. Direct desorption from solids by a laser beam or by fast atom bombardment (FAB) allow measurement of involatile compounds. Solutions may also be sprayed directly into the spectrometer inlet and the spectrum measured after the solvent has evaporated. 

Using the CFD methods one can analyse polymeric and other involatile compounds by converting them, prior to chromatographic separation, into characteristically volatile products. These volatile products can be obtained either by pyrolysis or by using more selective chemical conversions. It is customary to regard the application of pyrolysis reaction to identify and analyse quantitatively involatile samples as an independent part... 

Derivatives of this type are widely used in gas chromatography to obtain volatile derivatives of involatile compounds. Silyl derivatives, e.g., for the case of the TMS group donor, can be obtained in accordance with the following scheme ... 

Analytical pyrolysis is one of the most important methods in analytical chemistry, known for many years. Thermal degradation and subsequent analysis of the degradation products have long been used for the qualitative and quantitative analysis of involatile compounds and for determining their structures [1—6]. The use of GC analysis of pyrolysis products has increased the practical value of the method because only certain of the products contained in the complex mixture formed are characteristic of a particular sample. 

The use of the formation of involatile compounds in the GC analysis of hydrocarbons has also been described by other workers [61,62]. The subtraction method is also used successfully in the analysis of organic compounds of other classes. 

The formation of involatile compounds has also been used for the selective removal of alcohols from a mixture of organic compounds. Ykeda et al. [63] used a reactor... 

It is extremely advantageous to use for subtraction purposes inorganic compounds that form strong complexes or involatile compounds with particular organic components of the test mixture. An example (see below) is the use of copper(II) salts as reagents for the removal of amines [88]. 

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