Chromatographic fractions, molecular weights

Bis(4-imino-2-pentanonato)nickel(II) crystallizes from a benzene-petroleum ether mixture as dark red needles or as fine red-orange needles. The two forms have identical melting points. The compound is very soluble in chloroform, but less soluble in benzene, pyridine, and carbon tetrachloride, and very insoluble in water. The compound crystallizes from pyridine without adduct formation. The compound is diamagnetic and apparently has the trans configuration. Partial resolution in optically active fractions has been achieved by means of a chromatographic technique. Molecular weight determinations indicate that the compound is monomeric in chloroform and benzene solution. The visible absorption spectrum of this compound in chloroform is characterized by a band centered at 552 m/i (e = 43). The ultraviolet absorption maxima for solutions in 1 1 benzene-petroleum ether occur at 298, 348, and 364 m x (e = 4150, 4760, and 4460, respectively). ... 

Both preparative and analytical GPC were employed to analyze a standard (NBS 706) polystyrene sample. Fractions were collected from the preparative column, the solvent was evaporated away, and the weight of each polymer fraction was obtained. The molecular weights of each fraction were obtained usmg an analytical gel permeation chromatograph calibrated in terms of both and M. The following data were obtained ... 

Analytical Approaches. Different analytical techniques have been appHed to each fraction to determine its molecular composition. As the molecular weight increases, complexity increasingly shifts the level of analytical detail from quantification of most individual species in the naphtha to average molecular descriptions in the vacuum residuum. For the naphtha, classical techniques allow the isolation and identification of individual compounds by physical properties. Gas chromatographic (gc) resolution allows almost every compound having less than eight carbon atoms to be measured separately. The combination of gc with mass spectrometry (gc/ms) can be used for quantitation purposes when compounds are not well-resolved by gc. 

The eluted luciferase is precipitated with ammonium sulfate. The precipitate is dissolved in 1 mM Tris-HCl, pH 8, containing 0.1 mM EDTA, 3 mM DTT and 0.1 M NaCl, and chromatographed on a column of Sephacryl S-300 (2.6 x 97 cm) using the same buffer. Luciferase is eluted in two peaks, corresponding to the molecular weights of about 420,000 (an aggregate) and 130,000, in a ratio of about 8 1. The fractions of these two peaks are pooled separately the Mr 420,000 luciferase is concentrated by either ultrafiltration or precipitation with ammonium sulfate. 

With notable exceptions, the application of HPLC to clinical chemistry has not as yet been extensive. This is somewhat surprising in view of the potential the method has for this area. This potential arises, in part, from the fact that HPLC is well suited to the types of substances that must be analyzed in the biomedical field. Ionic, relatively polar species can be directly chromatographed, without the need to make volatile derivatives as in gas chromatography. Typically, columns are operated at room temperature so that thermally labile substances can be separated. Finally, certain modes of HPLC allow fractionation of high molecular weight species, such as biopolymers. 

Because of chromatographic dispersion, the sample fraction in the detector cell is polydisperse. The weight-average and number-average molecular weights of the polydisperse fraction are calculated as... 

Figure 9 shows the result of injecting 10 gA of the total low molecular weight fraction from GPC 1 (Column Code A2) into GPC 2 (Column Code Bl). With this column code, GPC 2 is performing as a High Performance Liquid Chromatograph (HPLC). Separation is based upon solubility (i.e. composition differences) rather than upon molecular size. Methyl methacrylate monomer was used as a reference and added to the solution injected into GPC 1. Concentrations of n-butyl methacrylate, styrene and conversion are readily calculated from the peak areas and initial concentrations. 

In the absence of adsorption, inclusion, or exclusion, a polymer is fractionated on a GPC column according to the hydrodynamic volume.40138 The hydrodynamic volume is a function of monomer identity, as well as polymer molecular weight, branching, and cross-linking. The polymer chains in any given chromatographic fraction have roughly the same hydrodynamic volume. 

The brownish, oily nonpolar fractions was chromatographed on the same column as was the polar leachate. One distinct peak, labelled peak A, was observed (Figure 4). An additional peak comprised of several smaller peaks was collected and labelled peak B. Estimates of molecular weight suggest that peak A and B are also between 600 and 1000. 

In conclusion one can say that SEC is a very powerful method for polymer characterization, especially in combination with other composition sensitive or absolute calibration methods. A big advantage is also that the sample amount is fairly small, typically 10 mg. For more complex polymers, such as polyelectrolytes, enthalpic effects often become dominant and also for rather high molecular weight polymers chromatographic methods such as field-flow fraction (FFF) techniques might be more suitable. For fast routine measurements linear columns are often used. 

A later study (45) indicates that cucurbitin from pumpkin has a molecular weight of TT2,000 dal tons that can be electrophoretically separated into subunits of 63,000 and 56,000 dal tons. Reduction of disulfides produces polypeptides of 36,000 and 22,000 dal tons. Globulins from six cucurbits examined chromatographically (46) have molecular weights of 220,000 to 260,000 dal tons that exhibit predominantly 10.4 - 11.2 S values (about 95% of the three globulin fractions). Cucurbitin from Cucumis sativus appears a tetramer of... 

Procedure The chromatographic procedure may be carried out at room temperature using (a) a column (1 M x 25 mm) packed with a cross-linked dextran suitable for fractionation of globular proteins in the range of molecular weights from 5,000 to 350,000 (Sephadex G-150 is suitable), (b) mixed phosphate buffer pH 7.0 with azide as the mobile-phase with a flow rate of about 20 ml (4 ml per square centimetre) of column cross-sectional area) per hour, and (c) a detection wavelength of 280 nm. 

Fig. 2.2 Comparative chromatographic profiles of low molecular weight fraction isolated from rat brain...

Chromatographic purification of cross-links. The initial steps of the purification were to separate the cross-links from monomeric amino acids, followed by the separation of individual cross-links. The acid hydrolyzate of demineralized roof powder (13.0 g collagen) was separafed on a size exclusion column fo yield the high molecular weight fractions. 

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