Analysis of Small Molecules

The two major databases containing information obtained from X-ray structure analysis of small molecules are the Cambridge Structural Database (CSD) [25] and the Inorganic Crystal Structure Database (ICSD) [26] both are available as in-house versions. CSD provides access to organic and organometallic structures (mainly X-ray structures, with some structures from neutron diffraction), data which are mostly unpublished. The ICSD contains inorganic structures. 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Pinkerton, T.C. (1991). High-performance liquid chromatography packing materials for the analysis of small molecules in biological matrices by direct injection. J. Chromatogr. 544, 13-23. 

K.D. Altria and D. Elder, Overview of the status and application of capillary electrophoresis to the analysis of small molecules. J. Chromatogr.A 1023 (2004) 1-14. 

A second approach to on-line SPE is to use an SPE extraction column that can be used for hundreds of samples. In the simplest of systems, two pumps (either HPLC systems or stand-alone pumps) are connected to an extraction column and an analytical column via 6 or 10 ports, and these are further linked to an MS system. The pump that is connected in-line with the autosampler loads the sample under high flow rate (3 to 5mL/min). The large molecules from the matrix are not retained by the SPE sorbent and are diverted to waste. The analytes of interest are retained by the sorbent. The valve then switches so that the second pump with the elution solvent is now in-line with the SPE column and elutes the analytes onto the analytical column for HPLC/MS analysis. This type of system has proved useful for the analysis of small molecules in a variety of sample matrices such as plasma and urine. While it is relatively straightforward to plumb this type of system with components already in the laboratory, commercial systems are available from such companies... 

Altria, K. D., Marsh, A., and Sanger-van de Griend, C. (2006). Capillary electrophoresis for the analysis of small-molecule pharmaceuticals. Electrophoresis 27(12), 2263-2282. 

The use of MALDI for the analysis of small molecules was recently reported. Particularly attractive is the coupling of a MALDI source with a triple quadrupole mass analyzer for quantitative analysis in the selected reaction monitoring (SRM) mode due to very high analysis speed. 

Hopfgartner, G. Varesio, E. Tschappat, V. Grivet, C. Emmanuel Bourgogne, E. Leuthold, L. A. Triple quadrupole linear ion trap mass spectrometer for the analysis of small molecules and macromolecules. J. Mass Spectrom. 2004, 39, 845-855. 

Neue, U. D. Fast LC/MS in the analysis of small molecules. Anal Bioanal Chem 2003, 377, 788-802. 

Column Efficiency. The peak capacity (13) for a GPC column used in the analysis of small molecules is related to the number of theoretical plates (N) according to ... 

The number of suppliers of the polymeric gels of the polystyrene-divinyIbenzene type suitable for the analysis of small molecules has been Increasing over the years. Packed columns - usually 30 cm x 8 mm i.d. or gels are currently available from many sources ( ) although some of them sell only the packed columns. 

Monolithic silica Cj3 reversed phase Chromolith RP-18 Merck KgaA 100 X 4.6 mm 50 X 4.6 mm 25x4.6 mm Swift RP analysis of small molecules... 

Monolithic silica Cj reversed phase Chromolith RP-8 Merck KgaA 100x4.6 mm Analysis of small molecules and hydrophobic peptides... 

Shea KJ, Sasaki DY. An analysis of small-molecule binding to functionalized synthetic polymers by 13C CP/MAS NMR and FT-IR spectroscopy. J Am Chem Soc 1991 113 4109-4120. 

Electron ionization (El) was the primary ionization source for mass analysis until the 1980s, limiting the chemist to the analysis of small molecules well below the mass range of common bioorganic compounds. This limitation motivated the development of the techniques commonly known as ESI, 1 MALDI, 2 and fast atom bombardment (FAB) 3,4 (Table 1). These ion sources allow for rapid and easy peptide analyses that previously required laborious sample preparation or were not possible with electron ionization. The mechanism of ionization these ion sources employ, which is somewhat responsible for their ability to generate stable molecular ions, is protonation and/or deprotonation. 

In this chapter we focus primarily on calibration of LC-MS where the mass spectrometer is operating at unit resolution, resolution that is sufficient to separate two peaks one mass unit apart. This kind of low-resolution mass filter covers almost 90 percent of the instruments commonly used for qualitative and/or quantitative analysis of small molecules. Batch-to-batch qualification testing of the instrument is also described. For the calibration of high-resolution mass spectrometers such as magnetic sector, TOF, or FTICR coupled with liquid chromatography, readers are referred to specific publications. 

How often an LC-MS should be calibrated depends on the mass accuracy required. For example, instrument calibration should be verified daily when performing accurate mass measurements of peptides and proteins. However, the quantitative analysis of small molecules requires less frequent calibration. 

In FAB, the sample is usually dispersed in a non-volatile liquid matrix, such as glycerol or diethanolamine, and deposited at the end of a sample probe that can be inserted into the ion source. The sample on the probe is ionised when bombarded by the fast atom beam. However, ionisation of the matrix also occurs, leading to a very large background signal. The technique is thus limited for the analysis of small molecules. Fast-moving ions (Cs+ or Ar+) can be used instead of fast-moving atoms, which is the basis of a technique called liquid secondary ion mass spectrometry (LSIMS). 

This desorption ionisation technique leads to weak fragmentation. The analyte is incorporated into a solid organic matrix (such as hydroxybenzoic acid) and the mixture is placed on a sample holder that is irradiated with UV laser pulses (e.g. N2 laser, A = 337 nm, pulse width = 5 ns). The laser energy is absorbed by the matrix and transferred to the analyte, which becomes desorbed and ionised (Fig. 16.18c). Although MALDI is considered to be a soft ionisation technique, a substantial amount of energy is involved. Because the technique involves pulsed ionisation, it is well suited for time-of-flight mass analysis of biomolecules. The analysis of small molecules (M < 500 Da) is limited because the matrix decomposes upon absorption of the laser radiation. However, solid supports such as silicone can be used as the matrix to overcome this disadvantage. 

However, MIPs offer definitive advantages over the antibodies they display excellent performance in organic samples, their chemical stability is far superior and, in principle, they can be precisely tailored to the target species with low production costs, especially for the analysis of small molecules, for which antibody... 

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