Ultrafiltration, fraction concentration

The alternative large scale recovery method to precipitation is ultrafiltration. For concentration of viscous exopolysaccharides, ultrafiltration is only effective for pseudoplastic polymers (shearing reduces effective viscosity see section 7.7). Thus, pseudoplastic xanthan gum can be concentrated to a viscosity of around 30,000 centipoise by ultrafiltration, whereas other polysaccharides which are less pseudoplastic, are concentrated only to a fraction of this viscosity and have proportionally lower flux rates. Xanthan gum is routinely concentrated 5 to 10-fold by ultrafiltration. 

The CCC fractions, HDL-LDL and VLDL-serum proteins, were each separately dialyzed against distilled water until the concentration of the potassium phosphate was decreased to that in the starting buffer used for the hydroxyapatite chromatography. These two fractions were concentrated separately by ultrafiltration. The concentrates of both fractions were chromatographed on the hydroxyapatite column. Fig. 4 shows the elution profile on hydroxyapatite obtained from the HDL-LDL fraction. A 1.4-mL volume of the concentrate was loaded onto a Bio-Gel HTP DNA-grade column (5.0 x 2.5 cm I.D.)... 

Elliott (1978) in a brief communication reported that more than 70% of the aluminium in blood was present in the plasma compartment. In normal subjects, they reported that a very small proportion of the plasma aluminium was ultrafiltrable. These findings were not consistent with thos of Lundin et al. (1978). In the study of (1978) the tendency was for the ultrafiltrable fraction to decrease as the total plasma aluminium concentration fell below 200 fiQlL. At high plasma aluminium levels, studies using polyethene glycol and direct ultrafiltration indicated that 60-70% of the aluminium was bound to high-molecular weight proteins, 10-20% was bound to albumin, and 10-30% was ultrafiltrable. 

The inducible arsenite oxidase from the Eubacterium Alcaligenes faecalis (NCIB 8687) has been purified and characterized (22-24). Anderson et al. (24) isolated the enzyme from a sonicate of washed, lysozyme-treated cells that had been harvested in their late exponential growth phase. The sonicate was fractionated by gel filtration through DEAE-sepharose and active fractions concentrated by ultrafiltration. The purified enzyme was found to be monomeric with a molecular mass of 85 kDa. It consisted of two polypeptide chains in an approximate ratio of 70 30. The enzyme stmcture included one molybdenum, five or six iron atoms, and sulfide. Purification of the oxidase also led to recovery of azurin, a blue protein, which was rapidly reduced by arsenite in the presence of catalytic amounts of Aro, and a red protein. The red protein was a c-type cytochrome, which was reduced by arsenite in the presence of catalytic amounts of Aro and azurin. No reduction of the cytochrome occurred in the absence of Aro, but it did occur in the absence of azurin. Denaturation of Aro led to the release of a pterin cofactor characteristic of molybdenum hydroxylases. In intact cells of A. faecalis, the enzyme resides on the outer surface of the inner (plasma) membrane. The cytochrome and azurin may be part of an electron transfer pathway in the periplasm. 

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. 

Fig. 3. QAE-Sephadex gradient separation of the B fruit extract. An 18 mg (uronic acid equivalents) sample of extract was dissolved in 20 ml of 125 mM imidazole-HCl buffer (pH 7.0) and applied to the column. The column was then eluted with 50 ml 125 mM buffer followed by a 125 mM to 1.5 M buffer gradient (500ml), and, finally, 50 ml of 1.5 M buffer. Fractions of 5 ml were collected and assayed for uronic acids. Groups of fractions (26-41, 45-50, 53-75 and 84-100) were pooled, concentrated by ultrafiltration and analyzed by HPLC.

Influence of U colloidal transport in organic-poor surface waters has been far less studied. Riotte et al. (2003) reported U losses from 0 to 70% during ultrafiltration experiments for surface waters of Mount Cameroon without nearly any DOC. Even in the low concentration waters, U can be significantly fractionated from other soluble elements by the occurrence of a colloidal phase, probably inorganic in origin. However, such fractionations are not systematic because of the occurrence of various colloidal phases, characterised by different physical and chemical properties, and hence different sorption and/or complexation capacities (Section 2.1). 

Presently, the precise determination of the true dissolved Th fraction in water samples remains a challenge. Results from ultrafiltration experiments on organic-rich water samples from the Mengong river tend to demonstrate that Th concentration is less than 15 ng/L in absence of DOC (Table 2 and Viers et al. 1997), and that Th is still controlled by organic carbon in the final filtrate of the ultrafiltration experiments. The latter conclusion is also supported by the results obtained for the Kalix river (Porcelli et al. 2001). These results therefore not only raised the question of the determination of the amount of dissolved Th in water but also of the nature of Th chemical speciation. 

Pool fractions containing PPO and concentrate to 5 mL using YM 10 Amicon ultrafiltration filters. 

Gel filtration may be best used to analyze fractions already separated from a digest supernatant by ultrafiltration, as used in a recent study by Sandstrom, et al. (3.2). A more precise separation of complexes can be obtained with gel filtration, but the size of sample which can be applied is limited. Thus, in many situations, the sample must be concentrated before being applied to the gel column. Either pre-purification or sample concentration could introduce possible shifts in mineral binding which should be understood for proper interpretation of the results (33). 

Amino Acid Content. Amino acid content of field pea products is related to protein level, method of processing, and fraction (starch or protein). The protein fraction contains fewer acidic (glu, asp) amino acids than the starch fraction and more basic (lys, his, arg) amino acids than the starch fraction. Also, there are more aromatic (tyr, phe) amino acids, leu, iso, ser, val, and pro in the protein fraction than in the starch fraction (5). An amino acid profile of pea protein concentrate shows relatively high lysine content (7.77 g aa/16 g N) but low sulfur amino acids (methionine and cystine) (1.08-2.4 g aa/16 g N). Therefore, it is recommended that air classification or ultrafiltration be used because acid precipitation results in a whey fraction which contains high levels of sulfur amino acids (12,23). Also, drum drying sodium proteinates decreases lysine content due to the Maillard reaction (33). 

Cytochrome P-450 fractions were pooled and the free Emulgen 913 removed from the enzyme preparation by stirring with Amberlite XAD-2 beads followed by filtration. The filtrate was concentrated in an Amicon ultrafiltration cell using a YM 10 Diaflo membrane. Dialysis was carried out in 2 liters of Buffer I for 24 hr when required. The fractions containing cytochrome P-450 were stored under nitrogen in 0.5 ml aliquots at -62°. 

The supernatant was first extracted with dichloromethane (2 x 3 L) to eliminate the remaining IMI. The aqueous fraction was then extracted with ethyl acetate (3 L). The ethyl acetate extract, containing 5-hydroxy IMI, wais dried with 30 g anhydrous sodium sulfate and concentrated to about l/20th of the original volume in a vacuum rotary evaporator and then filtered with 0.22 pm pore size ultrafiltration membranes. The filtered solution was evaporated again until white crystals were produced. The crystals were filtered, washed twice with dichloromethane and then dissolved in 10 mL acetonitrile by heating. At 4 °C, the 5-hydroxy IMI crystallized from the above solution and was filtered and dried under vacuum. A total of 413 mg of 5-hydroxy IMI was obtained. 

This simplified calculation is used to illustrate basic computational techniques. It assumes that all of the Fe(OH)3(aq) is a true solute. The quality of this assumption is a matter of debate as at pH 8, Fe(OH)3(aq), tends to form colloids. Thus, laboratory measurements of ferrihydrite solubility yield results highly dependent on the method by which [Fe(lll)]jQ(gj is isolated. Ultrafiltration techniques that exclude colloids from the [Fe(lll)]jQjgj pool produce very low equilibrium solubility concentrations, on the order of 0.01 nM. This is an important issue because a significant fraction of the iron in seawater is likely colloidal, some of which is inorganic and some organic. In oxic... 

Fractionation by Stepwise Elution. Information obtained from the analytical separation was applied for a preparative purification. Lignin peroxidase concentrate was bound to a Q-Sepharose colunm (0= 5 cm, V = 1000 ml) after ultrafiltration and eluted stepwise with 0.08 M, 0.18 M and 0.28 M sodium acetate, pH 6.0. The fraction which was eluted with 0.28 M buffer (V= 3.91, 4400 U/1) was purified further. It was bound to Q-Sepharose and eluted with 0.18 M and 0.3 M sodium acetate. En rnie in the latter fraction was precipitated and dissolved in glycerol as previously described. The volume was 15 ml. 

For all but the recombinant Alg A, extracellular enzymes were obtained from medium concentrated by ultrafiltration. For the E. chrvsanthemi enzymes, a mixture precipitated between 60 and 90% saturated ammonium sulfate was fractionated into individual activities by chromatofocusing. Enzymes from Lachnospira multiparus (strain D15d) and Clostridium populeti cultures wee analyzed directly in concentrated and dialyzed medium. The Alg A enzyme was expressed in E. coli HBlOl transformed with plasmid pAL-A3 (Brown, et al.. University of Florida, unpublished) from the periplasmic fraction. 

Reverse osmosis is applicable for the separation, concentration, and/or fractionation of inorganic or organic substances in aqueous or nonaqueous solutions in the liquid or the gaseous phase, and hence it opens a new and versatile field of separation technology in chemical process engineering. Many reverse osmosis processes are also popularly called "ultrafiltration", and many reverse osmosis membranes are also practically useful as ultrafilters. 

Enzyme Production and Isolation. The production and isolation of veratryl alcohol oxidase (VAO) was described earlier (25). Laccase produced from the same 12-day culture (8 litres) was isolated from the supernatant by precipitation at 0°C with ammonium sulfate (80% saturation). The precipitate was suspended in 0.05 M Na acetate buffer, pH 5.0 and dialysed overnight against 4 litres of buffer. The soluble material was concentrated by ultrafiltration (Amicon PM10) to about 60 mL and applied to a DEAE-Bio-gel A column (2.5 cm x 35 cm). The column was washed with 20 mL of the same buffer, then eluted with a linear gradient from 0 to 0.6 M NaCl (total volume 550 mL). Fractions were monitored for VAO and laccase activity as described below. 

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