Determination of Molecular Mass

In a modem laboratory, the molecular mass is determined through the use of mass spectrometry. The details of this method and the means of determining molecular mass can be found in Section 1.6 and Chapter 8, Section 8.4. This section reviews some classical methods of obtaining the same information. 

An old method that is used occasionally is the vapor density method. In this method, a known volume of gas is weighed at a known temperature. After converting the volume of the gas to standard temperature and pressure, we can determine what fraction of a mole that volume represents. From that fraction, we can easily calculate the molecular mass of the substance. 

One of the most important pieces of information required to elucidate the molecular structure of an unknown organic compound is its molecular mass, which provides a window within which the elemental composition and the final structure of the compound must fit. Therefore, the first essential step to identifying a compound is to measure its molecular mass by determining the m/z value of the molecular ion. Molecular mass measurements can be performed at either low or high resolution. A low-resolution measmement provides information about the nominal mass of the analyte, and its elemental composition can be also determined for low-molecular-weight compounds from the isotopic pattern. From a high-resolution mass spectrum, the accurate molecular mass can be determined, from which it is also feasible to deduce the elemental composition. Chemists who work with synthetic compounds and natural products rely heavily on the exact mass measurement data for structmal assignment. This value is acceptable in lieu of the combustion or other elemental analysis data. An acceptable value of the measured mass should be within 5 ppm of the accmate mass [1]. As shown below, the mass measurement error is reported either in parts per million (ppm) or in millimass units (mmu). 

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Rothe, GM, Determination of Molecular Mass, Stoke radius. Frictional Coefficient and Isomer-Type of Non-denatured Proteins by Time-Dependent Pore Gradient Gel Electrophoresis, Electrophoresis 9, 307, 1988. 

Determination of molecular mass of pectic enzymes The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with Tris-HCl 50 mM, pH 7,5 buffer, plus 100 mM KCl. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were tyroglobulin (660 kDa) apoferritin (440 kDa) P-amylase (200 kDa) alcohol dehydrogenase (150 kDa) bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were myosin (205 kDa) p-galactosidase (116 kDa) phosphorylase b (97 kDa) bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). 

Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers - tyroglobulin, 2- apoferritin, 3- p-amylase, 4-alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers 1- myosin, 2- p-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.

The approximate determination of molecular masses of polygalacturonases present in the medium (Fig. 5) showed two activity peaks corresponding the values of about 40 kDa for polygalacturonase and 50 kDa for exopolygalacturonase. 

A measure of the breadth of the molecular mass distribution is given by the ratios of molecular mass averages. The most commonly used ratio Mw/Mn — H, is called the polydispersity index. Wiegand and Kohler discuss the determination of molecular masses (weights) and their distributions in Chapter 6. 

Experiment 4 Determination of Molecular Mass by Freezing-Point Depression ... 

It is necessary to notify, that the critical analysis of the Flory theory application for the determination of molecular mass and the crossing density of the coal structure has been done in the Painter s works [16], Authors assert, that the possible formation of hydrogen bonds between the hydroxy groups of low-metamorphized coal has an important role here that is why, even a lot of empirical amendments introduction into calculations leads to obtaining the understated values of molecular masses of clusters. 

Protonated and cationized species are the most commonly detected ions using the ES process if a positive ionization mode is selected. Protonation is a result of the addition of proton(s) to a neutral molecule for every proton added, a net charge of +1 will result. Similarly, canonization is due to the addition of cation(s) to a neutral molecule. Detection of cationized ions can be useful in the determination of molecular mass of unknown analytes.If ESI is operated under negative ion mode, deprotonated ions will usually be the most dominant ions. Eor either operation mode, solvent adducts of the protonated/deprotonated ions are frequently detected in ESI/MS mass spectra. 

Budzikiewicz H, Schafer M, Meyer JM (2007) Siderotyping of Fluorescent Pseudomonads -Problems in the Determination of Molecular Masses by Mass Spectrometry. Mini-Rev Org... 

Determination of molecular mass by freezing-point depression... 

The independent determination of molecular masses by SDS-PAGE is impossible. To estimate the molecular mass of a protein, measure the path of that protein or calculate its Rf value (distance of the protein from origin/distance of electrophoresis front from origin) and compare these values with that of marker proteins, i.e., proteins with independently determined molecular masses. This method is successful only if the protein of interest behaves regularly in SDS-PAGE, i.e., it is totally unfolded by SDS, has a rod-like shape, and the SDS/protein ratio is the same for the unknown and the marker protein. Especially highly hydrophobic proteins and glycoproteins often deviate from these assumptions. 

DETERMINATION OF MOLECULAR MASS OP A VOLATILE LIQUID BY MEYER S METHOD... 

Figure 7.5—Determination of molecular mass. The use of a calibration curve made with standards of known molecular masses. It should be noted that the calibration curve is linear over a wide range of masses due to the use of a mixture of stationary phases. (Reproduced by permission of Polymer Lab.) The bottom right figure shows the geometry assumed by a linear polymer in solution (figure from PSS).

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

Colligative properties have many practical uses, including the melting of snow by salt, the desalination of seawater by reverse osmosis, the separation and purification of volatile liquids by fractional distillation, and the determination of molecular mass by osmotic pressure measurement. 

The ylide was identified by its 1H and 13C NMR and mass spectra, by chemical analysis, and by a eryoscopic determination of molecular mass in solution. It could also be obtained, in lower yields, from TaCle and 5 equivalents of the neopentyl Grignard reagent. It has a melting point of 71°C and can be distilled ( ) at 75°C under vacuum. The compound is very sensitive to oxygen and moisture, but may be stored indefinitely at room temperature in an inert atmosphere. 

NICI mass spectra do not contain isomer-specific information. Therefore, this technique is most suitable for the determination of molecular mass information and quantification (see Sect. 3.3.1.1). 

Determination of Molecular Masses of Volatile Liquids from... 

Particle beam LC/FT-IR and LC/MS spectrometries provide both different and complementary information. LC/MS measurements provide fragment identity and sequence information through an accurate determination of molecular mass. PB LC/FT-IR spectrometry measurements provide information on the solution structure of the peptide that includes residual or chromatographically induced secondary stmcture and aggregation, as well as information on the presence of some amino acid functionalities. The PB technique is especially useful with larger peptides which posses formal secondary structure. 

Calculation of reagents introduced into the reaction for determination of molecular mass was conducted by the formula = 3 /m = (A/m-219)/272, where m = number of diphenylsilandiol (DPhSDO) moles n = degree of polymerization = required molecular mass 219 = molecular mass of hexamethyldisilazane, 272 = molecular mass ofDPhSDO. 

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