Dextrans, linear calibration

Dextrans were employed to evaluate the linear calibration method for utility in aqueous SEC. The dextrans were obtained from Pharmacia Chemical Co. (Upsala, Sweden) of the following molecular weights ... 

The blend of T-70 and T-40 dextran materials was utilized as a polydisperse calibration standard for the linear calibration method and the T-40 dextran standard was used as a sample for evaluation. Concentrations of 0.15% W/V were injected for each dextran material chromatographed. 

Dextran polymers were used to evaluate the utility of the linear, polydisperse calibration method for water-soluble polymer characterization. A blend of T-40 and T-70 dextran standards was used as a polydisperse calibration standard. Table VIII displays the report from the linear calibration method using this standard. Nine Iterations of the search algorithm were required for convergence to the true and Mn values of the standard. As can be seen in the report, the elution volume profile of the standard contained 72 area/time slices upon which calibration calculations were based. The slice width was set at 10 seconds/siIce. Figure 5 shows a plot of the calibration curve generated from the linear calibration method utilizing the dextran standard,... 

The amount of active urease immobilized into the polyelectrolyte microcapsules was determined under assumption that the enzyme retains its activity while encapsulated. Free urease had 100 U/mg according to the data sheets. The activities of free and encapsulated enzyme were determined from the decomposition of urea into two ammonia molecules and CO using a pH-sensitive d e Bromocresol purple [24], The urease aliquot solutions were added to a reaction mixture contained a necessary amount of urea and 0.015 mM Bromocresol, whose pH was aprioiy brought up to 6.2. The reaction kinetics was recorded as a change in the optical absorption of the dye at 588 run to obtain the linear calibration plot. Then, the known number of microcapsules containing urease and SNARF-1 dextran was added to the reaction solution. The revealed activity of enzyme was compared with amount of free urease. 

FIGURE 16.5 Broad standard calibration (linear mode) of a semipreparative Sephacryl S-IOOO system (95 x 1.6 cm) with an aqueous mixture of Blue Dextran, Dextran T-SOO, and glucose eluent 0.005 M NaOH V, i = 75 ml. = 162 ml. 

Figure 5 Calibration curve for polydisperse dextran standard using a linear method. Plot of log (MW) vs. elution volume. Horizontally, each - represents 0.U8 units.

Each sample to be analyzed was dissolved in tris(ethylenediamine)cadmium dihydroxide (1 mL) by stirring overnight, and then water (1 mL) was added. A 1-mL aliquot (concentration < 1.0%) was applied to the column, and elution proceeded with a pressure head of 100 cm and flow rate of 10 mL/h. A Turner 111 fluorometer (excitation filter 2A plus 47B and emission filter 8 plus 65A) fitted with a flow-through door allowed for automatic continuous monitoring of carbohydrates as they were eluted. Relative fluorescence was automatically recorded on a linear strip recorder. Fractions of 3 mL were collected on a FC-80K Gilson microfractionator. Typically, each sample was analyzed several times, usually at different concentrations, to ensure the reproducibility and accuracy of the data. A calibration run using the labeled dextrans was performed a minimum of one time per week. 

There were also attempts to calibrate the SEC columns with help of broad molar mass dispersity poplymers but this is less lehable. The most common and well credible SEC cahbration standards are linear polystyrenes, PS, which are prepared by the anionic polymerizatioa As indicated in section 11.7, according to lUPAC, the molar mass values determined by means of SEC based on PS calibration standards are to be designated polystyrene equivalent molar masses . Other common SEC calibrants are poly(methyl methaciylate)s, which are important for eluents that do not dissolve polystyrenes, such as hexafluoroisopropanol, further poly(ethylene oxide)s, poly(vinyl acetate)s, polyolefins, dextrans, pullulans, some proteins and few others. The situation is much more complicated with complex polymers such as copolymers. For example, block copolymers often contain their parent homopolymers (see sections 11.8.3, 11.8.6 and 11.9). The latter are hardly detectable by SEC, which is often apphed for copolymer characterization by the suppliers (compare Figure 16). Therefore, it is hardly appropriate to consider them standards. Molecules of statistical copolymers of the same both molar mass and overall chemical composition may well differ in their blockiness and therefore their coils may assume distinct size in solution. In the case of complex polymers and complex polymer systems, the researchers often seek support in other characterization methods such as nuclear magnetic resonance, matrix assisted desorption ionization mass spectrometry and like. 

V = elution volume of small molecule. The standards used to derive these calibra- 0 tion curves are frequently dextrans or sulphonated polystyrenes, although this tends Z to (incorrectly) assume a linear structure for the biological macromolecule. The m behaviour of proteins and peptides on SEC columns is more accurately represented by the use of globular proteins as calibration standards. Anderson and Stoddart observed (3) that in the middle of the Kj range (/fp = 0.15—0.80) these theoretical plots are y essentially linear (see Figure 2), and they can therefore assist in the choice of SEC CO column. It is consequently graerally best (4) to choose a column on which the molecules of interest elute near the middle of the calibration curve, where this linearity occurs. Occasionally, two or more columns may be used in series. When connected in series, columns of the same pore size will increase resolution, whilst columns of different pore size will broaden the range of molecidar sizes separated. 

Fig. 4-1). This explains why some polypeptides may fall outside calibration curves prepared with standard globular proteins. Theoretically, the steepest selectivity curve and thus the highest separation power is obtained with the idealized gel medium in which all the pores are of identical size, giving a separation range per pore volume of approximately one decade [7]. Such gels do not exist but are approached by gels made of point cross-linked linear poljmiers such as dextran and polyacrylamide. 

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