Molecular mass, determination examples

Aluminum, gallium, indium, and thallium have been successfully incorporated in cyclic systems, although little structural information has been obtained (75). Hoberg (34) has prepared many such ring compounds, including, for example, 8a and 8b, and has demonstrated the former to be dimers from molecular mass determinations and from mass spectra. 

A study of the fast atom bombardment mass spectra and low-energy collision activation tandem mass spectra of a series of monoquatemary and bisquatemary bisbenzylisoquinoline Thalictrum alkaloids was undertaken [143]. It was determined that the relative molecular mass of the free base alkaloid and its bisquatemary salt can be obtained from FAB data, but that the monoquatemary ammonium salt derivatives produce only a (M-X)+ ion, thus precluding the determination of the relative molecular mass. For example, the bisquatemary iodide ammonium salts produced ions (M-I)+, (M-I-HI)+, and (M-I-CH3I)+. The relative molecular mass of the counter anion may be determined with facility, thereby affording the relative molecular mass of the alkaloid. However, with a monoquatemary ammonium salt such as a monochloride, only a (M-X)+ ion is produced, and there is insufficient mass spectral data to determine its counter anion from positive-ion FAB. Negative-ion FAB was unable to be employed to determine absolutely the counter anion of the alkaloid, since chloride anions occur as common background anions. 

Intact protein mass spectrometry allows the molecular mass determination of either proteins or complexes of proteins and covalently bound ligands/other proteins. In a first step, the sample is desalted to detach from buffer components and small ions that would interfere through noncovalent complexes in the gas phase. Next, the isolated protein is ionized, for example, by electrospray ionization (ESI). The acid in the eluent causes protonation of the protein at basic sites, particularly lysine and arginine residues, so that m/z values of multiple species with different charges can be measured in a mass spectrometer. These data are then combined during the deconvolution process to yield the mass of the protein or complex. 

Molecular mass determination by MS is also useful for analysis of substrate specificities and cleavage patterns of enzymes, including chitinases, chitosanases, lysozymes, and chitin deacety-lases, as summarized with a few representative examples in Table 11.4. Continuous infusion of reaction mixtures into an ESI mass spectrometer (so called real time monitoring) was nsed to analyze the hydrolysis of D (z = 4 - 6) by several chitosanases and some mutants (Dennhart et al. 2008). ESl-MS is also snitable for simnltaneous measurement of individual kinetic constants of enzymes in mixtures of substrates, as reported for a bacterial sulfotransferase with A (z = 2 - 5) (Pi and Leary 2004). 

Historically, chemists have used the group of colligative properties— vapor pressure lowering, freezing-point depression, boiling-point elevation, and osmotic pressure—for molecular mass determinations. In Example 14-9, we showed how this could be accomplished with osmotic pressure. Example 14-10 shows how freezing-point depression can be used to determine a molar mass and, with other information, a molecular formula. To help you understand how this is done, we present a three-step procedure in the form of answers to three separate questions. In other cases, you should be prepared to work out your own procedure. 

The broad pore size distribution of Sepharose makes it well apt for the analysis of broad molecular mass distributions of large molecules. One example is given by the method for determination of MWD of clinical dextran suggested in the Nordic Pharmacopea (Nilsson and Nilsson, 1974). Because Superose 6 has the same type of pore size distributions as Sepharose 6, many analytical applications performed earlier on Sepharose have been transformed to Superose in order to decrease analysis time. However, Sepharose is suitable as a first try out when no information about the composition of the sample, in terms of size, is available. 

The dashed line in Fig. 3.2 corresponds to a linear regression calculation yielding Me = 8 kg/mol for the average molecular mass between entanglements if no chemical crosslinks are present (MR - oo). This result agrees reasonably with values for various thermoplastics as determined from elasticity measurements on melts [30, 31, 32], Examples are given in Table 3.2. 

Data from f.a.b.-m.s., and also f.d.-m.s., revealed the existence of naturally occurring, large cyclic polysaccharides. The first indication that a molecule may be cyclic comes from its precise molecular-weight determination. Cyclic molecules are 18 mass units less than their linear counterparts. Loss of water may, of course, occur in a number of ways, for example, by dehydration or lactonization, and conclusive evidence for the presence of a cyclic molecule can only be obtained from f.a.b.-m.s. of suitable derivatives, such as the permethyl derivative. Cyclic and dehydrated linear polymers are distinguishable after permethylation, as the cyclic polymer will incorporate one methyl group less than the linear molecule. 

Complex polymers are distributed in more than one molecular property, for example, comonomer composition, functionality, molecular topology, or molar mass. Liquid chromatographic techniques can be used to determine these properties. However, one single technique cannot provide information on the correlation of different properties. A useful approach for determining correlated properties is to combine a selective separation technique with an information-rich detector or a second selective separation technique. 

If we know the formula of a compound, it is a simple task to determine the percent composition of each element present. For example, suppose you wanted the percentage carbon and hydrogen in methane, CH4. First, calculate the molecular mass of methane ... 

We can use Graham s law to determine the rate of effusion of an unknown gas knowing the rate of a known one or we can use it to determine the molecular mass of an unknown gas. For example, suppose you wanted to find the molar mass of an unknown gas. You measure its rate of effusion versus a known gas, H2. The rate of hydrogen effusion was 3.728 mL/s, while the rate of the unknown gas was 1.000 mL/s. The molar mass of H2 is 2.016 g/mol. Substituting into the Graham s law equation gives ... 

A second example of a membrane-bound arsenate reductase was isolated from Sulfurospirillum barnesii and was determined to be a aiPiyi-heterotrimic enzyme complex (Newman et al. 1998). The enzyme has a composite molecular mass of 100kDa, and a-, P-, and y-subunits have masses of 65, 31, and 22, respectively. This enzyme couples the reduction of As(V) to As(III) by oxidation of methyl viologen, with an apparent Kra of 0.2 mM. Preliminary compositional analysis suggests that iron-sulfur and molybdenum prosthetic groups are present. Associated with the membrane of S. barnesii is a h-type cytochrome, and the arsenate reductase is proposed to be linked to the electron-transport system of the plasma membrane. 

Calculation of the molecular mass of an unknown protein follows the same procedure as, for example, quantitative protein determination plotting of the Rf of the calibration proteins against their molecular mass computing of a standard curve and estimation of the MW of the unknown protein and using the regression functions of the standard curve (c.f Fig. 2.1). 

Yield stress values can depend strongly on filler concentration, the size and shape of the particles and the nature of the polymer medium. However, in filled polymer melts yield stress is generally considered to be independent of temperature and polymer molecular mass [1]. The method of determining yield stress from flow curves, for example from dynamic characterization undertaken at low frequency, or extrapolation of shear viscosity measurements to zero shear rate, may lead to differences in the magnitude of yield stress determined [35]. 

Ribosomes are large complexes of protein and rRNA (Figure 31.8). They consist of two subunits—one large and one small—whose relative sizes are generally given in terms of their sedimentation coefficients, or S (Svedberg) values. [Note Because the S values are determined both by shape as well as molecular mass, their numeric values are not strictly additive. For example, the prokaryotic 50S and 30S ribosomal subunits together form a ribosome with an S value of 70. The eukaryotic 60S and 40S subunits form an 80S ribosome.] Prokaryotic and eukaryotic ribosomes are similar in structure, and serve the same function, namely, as the "factories" in which the synthesis of proteins occurs. 

Although a typical mass spectrum contains ions of many different masses, the heaviest ion is generally due to the ionized molecule itself. By measuring the mass of this ion, the molecular mass of the molecule can be determined. The naphthalene sample discussed in the previous section, for example, gives rise to an intense peak at mass 128 amu in its spectrum, consistent with its molecular formula of Ci0H8 (Figure 3.10b). 

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