Relative molecular masses, determination

The relative molecular mass determination of an unknown protein is generally performed automatically using various deconvolution algorithms, but the procedure is limited to relatively simple mixtures. 

At low compressions, up to c. 1 mN m i, protein films tend to be gaseous, thus permitting relative molecular mass determination. 

In view of experimental simplicity and accuracy, viscosity measurements are extremely useful for routine relative molecular mass determinations on a particular polymer-solvent system. K and a for the system are determined by measuring the intrinsic viscosities of polymer fractions for which the relative molecular masses have been determined independently - e.g. by osmotic pressure, sedimentation or light scattering. 

Based on elemental composition and relative molecular mass determinations, the formula of benzene was found to be The saturated... 

Based on elemental composition and relative molecular mass determinations, the formula of benzene was found to be C6H6. The saturated hydrocarbon hexane has the molecular formula C6H14 and therefore it was concluded that benzene was unsaturated. Kekule in 1865 proposed the cyclic structure 4 for benzene in which the carbon atoms were joined by alternate single and double bonds. Certain reactions of benzene, such as the catalytic hydrogenation to cyclohexane, which involves the addition of six hydrogen atoms, confirmed that benzene was a ring compound and that it contained three double bonds. However, since benzene did not undergo addition reactions with HC1 and HBr, it was concluded that these double bonds were different from those in ethene and other unsaturated aliphatic compounds. 

The observation of molecular size or polydispersity and the subsequent determination of relative molecular mass, (MJ or molecular mass (weight) distribution (MWD), is the most common analytical application of SEC. The goal of these types of experiments is to either observe the solvated size of one or more molecular species or to observe the distribution of sizes present in a mixture... 

The molecular weight (mean relative molecular mass) was obtained by determination of density but, in order to determine that the gas was monatomic and its atomic and molecular weights identical, it was necessary to measure the velocity of sound in the gas and to derive from this the ratio of its specific heats kinetic theory predicts that Cp/C = 1.67 for a monatomic and 1.40 for a diatomic gas. 

The term titrimetric analysis refers to quantitative chemical analysis carried out by determining the volume of a solution of accurately known concentration which is required to react quantitatively with a measured volume of a solution of the substance to be determined. The solution of accurately known strength is called the standard solution, see Section 10.3. The weight of the substance to be determined is calculated from the volume of the standard solution used and the chemical equation and relative molecular masses of the reacting compounds. 

DETERMINATION DF THE RELATIVE MOLECULAR MASS OF AN ORGANIC ACID 10.39... 

The theory of titrations between weak acids and strong bases is dealt with in Section 10.13, and is usually applicable to both monoprotic and polyprotic acids (Section 10.16). But for determinations carried out in aqueous solutions it is not normally possible to differentiate easily between the end points for the individual carboxylic acid groups in diprotic acids, such as succinic acid, as the dissociation constants are too close together. In these cases the end points for titrations with sodium hydroxide correspond to neutralisation of all the acidic groups. As some organic acids can be obtained in very high states of purity, sufficiently sharp end points can be obtained to justify their use as standards, e.g. benzoic acid and succinic acid (Section 10.28). The titration procedure described in this section can be used to determine the relative molecular mass (R.M.M.) of a pure carboxylic acid (if the number of acidic groups is known) or the purity of an acid of known R.M.M. 

Discussion. Very pure silver can be obtained commercially, and a standard solution can be prepared by dissolving a known weight (say, 10.787 g) in nitric acid in a conical flask having a funnel in the neck to prevent mechanical loss, and making up to a known volume (say, 1 L for a 0.1 M solution). The presence of acid must, however, be avoided in determinations with potassium chromate as indicator or in determinations employing adsorption indicators. It is therefore preferable to employ a neutral solution prepared by dissolving silver nitrate (relative molecular mass, 169.87) in water. 

Throughout the main text of this book standard solutions and quantities have all been expressed in terms of molarities, moles and relative molecular masses. However, there are still many chemists who have traditionally used what are known as normal solutions and equivalents as the basis for calculations, especially in titrimetry. Because of this it has been considered desirable to include this appendix defining the terms used and illustrating how they are employed in the various types of determinations. 

The complete analysis of alcohol sulfates is described in the Standard Methods of the International Organization of Standards (ISO) [200] and of the American Society for Testing and Materials (ASTM) [201]. These methods describe the analysis of inorganic sulfate content, chloride content, unsulfated matter, and water as well as other analytical values. Other ISO standards describe the analysis of sodium secondary alkyl sulfates [202], determination of pH [203], determination of water content [204,205], chlorides [206], total active matter in sul fated ethoxylated alcohols and alkylphenols [207], mean relative molecular mass in sulfated ethoxylated alcohols and alkylphenols [208], sulfate content... 

The third described enzyme form with pH optimum about 4.7 [11, 4], we found in Fraction C - the fraction from carrot roots pulp (Fig. 2). We supposed that this form of exopolygalacturonase is relatively strongly bound on carrot cell walls and so it can be released only by higher salt concentrations. The approximative molecular mass determination on Superose 12 (Fig. 3c) showed the molecular mass about 50 000 for this form and the second, with more acidic pH optimum, form present in the fraction. The further characterization of these enzymes showed the exopolygalacturonase with pH optimum 4.7 to be identical with enzyme described sooner by Pressey and Avants [4] and exopolygalacturonase with pH optimum 3.8 to be identical with the enzyme from Fraction A. In conclusion, the exopolygalacturonase form with pH optimum 3.8 can be considered to be the main enzyme form present in carrot roots. 

In Fig. 3.4d the relative molecular mass of the solute, Mr, is plotted on a log scale against the retention volume. The interstitial volume, which represents the volume range within which separations occur, and the size range of solutes that are eluted in this volume range, depend on the sort of material that is used for the stationary phase. Because for a given separation, F0 and V are constant, we can reliably predict the total volume of solvent or the time taken for a particular analysis. The calibration curve is established by determining the retention volume for standards of known Mr. 

Relative molecular mass distributions for components of biochemical and polymer systems can be determined with a 10% accuracy using standards. With biochemical materials, where both simple and macro-molecules may be present in an electrolyte solution, desalting is commonly employed to isolate the macromolecules. Inorganic salts and small molecules are eluted well after such materials as peptides, proteins, enzymes and viruses. Desalting is most efficient if gels with relatively small pores are used, the process being more rapid than dialysis. Dilute solutions of macro-molecules can be concentrated and isolated by adding dry gel beads to absorb the solvent and low RMM solutes. 

An aid to identification is the ability to determine the relative molecular mass (RMM) with a high degree of accuracy and to establish the empirical molecular formula. The former depends on recognition of the parent or molecular ion peak M produced when an electron is ejected from the molecule... 

Used in conjunction with infrared, NMR, UV and visible spectral data, mass spectrometry is an extremely valuable aid in the identification and structural analysis of organic compounds, and, independently, as a method of determining relative molecular mass (RMM). The analysis of mixtures can be accomplished by coupling the technique to GC (p. 114). This was formerly done by using sets of simultaneous equations and matrix calculations based on mass spectra of the pure components. It is well suited to gas... 

Prior to its cloning, GLT-1 was reconstituted and purihed and shown to have a relative molecular mass of 64 kDa, which agrees well both with the value of 65 kDa of the purihed and deglycosylated transporter (28,37) and its 573 amino acids determined from its nucleotide sequence (25). Reconstituted GLT-1 has been shown to form dimers, trimers, and higher molecular-weight homomers in the absence of reducing agents (38). The possibility of homomeric assemblies is corroborated by a recent freeze fracture, which shows pentameric assemblies (39). 

The movement of the analyte is an essential feature of separation techniques and it is possible to define in general terms the forces that cause such movement (Figure 3.1). If a force is applied to a molecule, its movement will be impeded by a retarding force of some sort. This may be as simple as the frictional effect of moving past the solvent molecules or it may be the effect of adsorption to a solid phase. In many methods the strength of the force used is not important but the variations in the resulting net force for different molecules provide the basis for the separation. In some cases, however, the intensity of the force applied is important and in ultracentrifugal techniques not only can separation be achieved but various physical constants for the molecule can also be determined, e.g. relative molecular mass or diffusion coefficient. 

Cellophane is frequently used for dialysis and it has a pore size of approximately 4—8 gm, which makes it impermeable to molecules with a relative molecular mass in excess of about 10 000. The development of a variety of membrane materials in which the pore size is much more rigorously controlled, has led to wider applications of ultrafiltration (Table 3.11). Various cellulose and polycarbonate membranes are available with pore sizes down to 5 nm which are capable of excluding molecules with a relative molecular mass of about 50. The internal structure of such membranes, as well as the pore size, determines their exclusion range and as a result precise specifications of membranes vary from one manufacturer to another. 

The elution volume of a solute is determined mainly by its relative molecular mass and it has been shown that the elution volume is approximately a linear function of the logarithm of the relative molecular mass. It is possible to determine the relative molecular mass of a test molecule using a calibration curve prepared from the elution volumes of several reference substances of known relative molecular mass. This should be done using the same column and conditions (Figure 3.37) and in practice it may be possible to calibrate the column and separate the test substance at the same time by incorporating the reference compounds in the sample. Such a method is rapid and inexpensive and does not demand a highly purified sample, provided that there is a specific method for detecting the molecule in the eluate. 

Figure 3.37 Determination of relative molecular mass by gel permeation chromatography. A mixture of proteins (approximately 10 /ug of each) was separated on an UltroPak TSK SW column and the elution volume for each protein plotted against the logarithm of its relative molecular mass (RMM). 

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