Molecular weight analysis method

Molecular weight analysis by MS is an essential analytical tool for the determination of disulfide connectivities of multiple-cystine peptides. MS analysis is applied in all the methods described above. Three types of MS analyses are presently used, FAB-MS, MALDI-MS, and ES-MS. The related methodology is developing rapidly and thus continuously increasing the analytical power of these techniques. 

The technique described here is finding increasingly widespread use for the resolution and molecular weight estimation of naturally occurring polypeptides. Its intended function is to supplement the classical exact techniques of molecular weight analysis and is not intended as an alternative procedure. Although we are aware of no instance in which invalid data have been obtained, the method can only be considered an approximate one since measurement involves calibration with secondary standards, and at present it is not suitable for precise theoretical analysis. 

I. Jardine. 1990. Molecular weight analysis of xotQms, Methods Enzymol. 193 441-455. (PubMed)... 

The fact that a humic substance is not a pure compound, but is a heterogenous mixture of many compounds with generally similar chemical properties, places an important constraint on all these characterization methods. Examples of the multicomponent mixture problem are presented in the chapters discussing interpretation of elemental analysis, determination of molecular weight, analysis of potentiometric data, and interpretation of infrared and other spectroscopic data. 

The methods used can be conveniently arranged into a number of categories (a) fractionation by precipitation (b) fractionation by distillation (c) separation by chromatographic techniques (d) chemical analysis by spectrophotometric techniques (infrared, ultraviolet, nuclear magnetic resource. X-ray fluorescence, emission, neutron activation), titrimetric and gravimetric techniques, and elemental analysis and (e) molecular weight analysis by mass spectrometry, vapor pressure osmometry, and size exclusion chromatography. 

Molecular weight Absolute methods end group analysis, membrane osmometry, vapor pressure osmometry, static light scattering, mass spectrometry, sedimentation measurements... 

Hydrocarbons from waxes are usually removed by dissolution in a nonpolar solvent such as purified hexane and analyzed by capillary gas chromatography. Most common hydrocarbons can be identified readily by mass spectrometry (Bagneres and Morgan, 1990). Analysis of wax esters is more difficult, as several different compounds in the rather complex mixtures obtained possess the same molecular weight. Analysis of these mixtures has been effected by tandem mass spectrometry (MS-MS) methods (Spencer and Plattner, 1984). 

We have used the tandem ADMET/hydrogenation method to produce a series of model EVA copolymers with pendant acetate functionality on every 19, 2V 23, and 2T carbon atom along the polyethylene backbone Figure 2, polymer 3, n = 18, 20, 22, and 26) [23]. Molecular weight analysis by GPC indicates that all of these polymers are in the range of 30,000 to 70,000 g/mol and have typical ADMET molecular weight distributions (PDI 2). Due to its low (23 °C), the copolymer with n = 18 lacks form stability at room temperature. However, the copolymers with n = 20, 22, and 26 all yield tough films and fibers from solution or the melt, and transparent or- translucent materials can be obtained from the melt. 

Jardine, I. Molecular weight analysis of proteins. Methods in Enzymol. 1990, 193, 441—455. 

The major molecular weight determination methods include number average molecular weights determined by end-group analysis. 

Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

This method is applicable for mineral oil fractions whose molecular weight is between 290 and 500 and for < 60% and 40% < Cp< 70%. The analysis is fast, approximately 10 minutes, and the correlation with other methods is satisfactory. 

Scale of Operation Coulometric methods of analysis can be used to analyze small absolute amounts of analyte. In controlled-current coulometry, for example, the moles of analyte consumed during an exhaustive electrolysis is given by equation 11.32. An electrolysis carried out with a constant current of 100 pA for 100 s, therefore, consumes only 1 X 10 mol of analyte if = 1. For an analyte with a molecular weight of 100 g/mol, 1 X 10 mol corresponds to only 10 pg. The concentration of analyte in the electrochemical cell, however, must be sufficient to allow an accurate determination of the end point. When using visual end points, coulometric titrations require solution concentrations greater than 10 M and, as with conventional titrations, are limited to major and minor analytes. A coulometric titration to a preset potentiometric end point is feasible even with solution concentrations of 10 M, making possible the analysis of trace analytes. 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

The method of end group analysis for molecular weight determination is not only simple to understand, but can also be done with ordinary laboratory equipment in many instances. 

Note that the method of end group analysis is inapplicable to copolymers, since the presence of more than one repeat unit adds extra uncertainty as to the nature of chain ends. The above example included the remark that the molecular weights calculated in the example were average values. In the next section we shall examine this point in greater detail. 

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