Molecular weight ultrafiltration

Fig. 29. Rejection of test proteins as a function of molecular weight, in a series of ultrafiltration membranes with different weight cut-offs (69).

Membrane Sep r tion. The separation of components ofhquid milk products can be accompHshed with semipermeable membranes by either ultrafiltration (qv) or hyperfiltration, also called reverse osmosis (qv) (30). With ultrafiltration (UF) the membrane selectively prevents the passage of large molecules such as protein. In reverse osmosis (RO) different small, low molecular weight molecules are separated. Both procedures require that pressure be maintained and that the energy needed is a cost item. The materials from which the membranes are made are similar for both processes and include cellulose acetate, poly(vinyl chloride), poly(vinyHdene diduoride), nylon, and polyamide (see AFembrane technology). Membranes are commonly used for the concentration of whey and milk for cheesemaking (31). For example, membranes with 100 and 200 p.m are used to obtain a 4 1 reduction of skimmed milk. 

Reverse Osmosis and Ultrafiltration. Reverse osmosis (qv) (or hyperfiltration) and ultrafilttation (qv) ate pressure driven membrane processes that have become well estabUshed ia pollution control (89—94). There is no sharp distinction between the two both processes remove solutes from solution. Whereas ultrafiltration usually implies the separation of macromolecules from relatively low molecular-weight solvent, reverse osmosis normally refers to the separation of the solute and solvent molecules within the same order of magnitude in molecular weight (95) (see also Membrane technology). 

Ultrafiltration (qv) (uf) is increasingly used to remove water, salts, and other low molecular-weight impurities (21) water may be added to wash out impurities, ie, diafiltration. Ultrafiltration is rarely used to fractionate the proteins because the capacity and yield are too low when significant protein separation is achieved. Various vacuum evaporators are used to remove water to 20—40% dry matter. Spray drying is used if a powdery intermediate product is desired. Tyophilization (freeze-drying) is only used for heat-sensitive and highly priced enzymes. 

Sodium poly(a-L-glutamate). It was washed with acetone, dried, dissolved in water and ppted with isopropanol at 5°. Impurities and low molecular weight fractions were removed by dialysis of the aqueous solution for 50h, followed by ultrafiltration through a filter impermeable to polymers of molecular weights greater the 10. The polymer was recovered by freeze-drying. [Mori et al. J Chem Soc, Faraday Trans I 2583 1978.]... 

Like normal filtration, with ultrafiltration (UF), a feed emulsion is introduced into and pumped through a membrane unit water and some dissolved low molecular weight materials pass through the membrane under an applied hydrostatic pressure. In contrast to ordinary filtration however, there is no build-up of retained materials on the membrane filter. 

Ultrafiltration Solution or colloidal suspension of high molecular weight organics One stream concentrated in high molecular weight organics one containing dissolved ions... 

Tangential crossflow filtration Process where the feed stream sweeps the membrane surface and the particulate debris is expelled, thus extending filter life. The filtrate flows through the membrane. Most commonly used in the separation of high-and-low-molecular weight matter such as in ultrapure reverse osmosis, ultrafiltration, and submicron microfiltration processes. 

Ultrafiltration of micellar solutions combines the high permeate flows commonly found in ultrafiltration systems with the possibility of removing molecules independent of their size, since micelles can specifically solubilize or bind low molecular weight components. Characteristics of this separation technique, known as micellar-enhanced ultrafiltration (MEUF), are that micelles bind specific compounds and subsequent ultrafiltration separates the surrounding aqueous phase from the micelles [70]. The pore size of the UF membrane must be chosen such, that the micelles are retained but the unbound components can pass the membrane freely. Alternatively, proteins such as BSA have been used in stead of micelles to obtain similar enan-tioselective aggregates [71]. 

Purification of poloxamers has been extensively investigated due to their use in medical applications, the intention often being to remove potentially toxic components. Supercritical fluid fractionation and liquid fractionation have been used successfully to remove low-molecular weight impurities and antioxidants from poloxamers. Gel filtration, high-performance liquid chromatography (HPLC), and ultrafiltration through membranes are among the other techniques examined [5]. 

Commercially available plate- and frame- type ultrafiltration equipment are used for exopolysaccharide concentration. The membranes are polysulphone or polyvinylidine fluoride with molecular weight cut-off between 20-60,000. There is a relatively low eneigy requirement (1-2 kWh m 3) for pumping the fluid through the filtration unit at the desired pressure. Pressure difference across the membrane is of the order 2-14 atmospheres. 

Ultrafiltration has the advantage that there is removal of low molecular weight fermentation products and medium components during concentration of the exopolysaccharide. In addition, biological degradation is minimised because fluid is held only for a short time during the filtration process. Other advantages lie in file fact that there is no requirement for solvent recovery and the process is carried out at ambient (not elevated) temperature. 

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. 

Eig. 16.9. Dependence of rejection coefficient on molecular weight for ultrafiltration membranes. 

A limitation to the more widespread use of membrane separation processes is membrane fouling, as would be expected in the industrial application of such finely porous materials. Fouling results in a continuous decline in membrane penneation rate, an increased rejection of low molecular weight solutes and eventually blocking of flow channels. On start-up of a process, a reduction in membrane permeation rate to 30-10% of the pure water permeation rate after a few minutes of operation is common for ultrafiltration. Such a rapid decrease may be even more extreme for microfiltration. This is often followed by a more gradual... 

Fig. 17.9. Purity comparison (SDS-PAGE) of the conventional purification process and integrated cell disrupt tion/fluidised bed adsorption.The numbers given in the flow sheet indicate the origin of samples and correspond to their respective lane numbers. Lanes M, low molecular weight markers 1, Erwinia disruptate, 15% biomass ww/v 2, eluate CM HyperD LS, fluidised bed 3, desalted eluate (after dia/ultrafiltration, 30 K MWCO membrane) 4, flow-through, DEAE fixed bed 5, elution, DEAE fixed bed 6, eluate CM HyperD LS 7, CM cellulose eluate 8, CM cellulose eluate, final 9, final commercial product.

Kj is the distribution ratio of uncomplexed ions and all low molecular weight inorganic and organic complexes (ultrafiltration cutoff was at a molecular weight of 1000). 

The polyethersulfone capillary ultrafiltration membranes (Daicel Chemical Industries, Ltd., inner diameter 0.8 mm, outer diameter 1.3 mm, length 40 cm, molecular weight cutoff 150 000, water permeability 3 x 10 m m s kPa at 298 K) were used. The length and area of membrane consisting of seven hollow fibers are 40 cm and 70 cm. ... 

Ultrafiltration of heterogenous colloidal suspensions such as citrus juice is complex and many factors other than molecular weight contribute to fouling and permeation. For example, low MW aroma compounds were unevenly distributed in the permeate and retentate in UF in 500 kd MWCO system (10). The authors observed that the 500 kd MWCO UF removed all suspended solids, including pectin and PE. If PE is complexed to pectate in an inactive complex, then it is conceivable that release of PE from pectin with cations will enhance permeation in UF. At optimum salt concentration, less PE activation was observed at lower pH values than at higher pH (15). In juice systems, it is difficult to separate the effect of juice particulates on PE activity. Model studies with PE extracts allows UF in the absence of large or insoluble particulates and control of composition of the ultrafilter. In... 

Until this point, the sample preparation techniques under discussion have relied upon differences in polarity to separate the analyte and the sample matrix in contrast, ultraflltration and on-line dialysis rely upon differences in molecular size between the analyte and matrix components to effect a separation. In ultrafiltration, a centrifugal force is applied across a membrane filter which has a molecular weight cut-off intended to isolate the analyte from larger matrix components. Furusawa incorporated an ultrafiltration step into his separation of sulfadimethoxine from chicken tissue extracts. Some cleanup of the sample extract may be necessary prior to ultrafiltration, or the ultrafiltration membranes can become clogged and ineffective. Also, one must ensure that the choice of membrane filter for ultrafiltration is appropriate in terms of both the molecular weight cut-off and compatibility with the extraction solvent used. 

On-line dialysis also separates the analyte from tissue matrix based upon molecular size, but in this case, the sample extract is passed over a membrane filter through which the analyte (and other low molecular weight compounds) is diffused into a second solvent on the other side of the membrane filter. Usually, the second solvent is then concentrated on to an SPE column to minimize the dilution effect that is caused by the dialysis process. Agasoester used on-line dialysis to separate oxytetracycline from muscle, liver, milk, and egg tissue matrix components. A problem encountered with on-line dialysis is the inability of analyte molecules that are bound to proteins in the sample extract to pass through the membrane filter. Problems with membrane clogging are reduced with on-line dialysis compared with ultrafiltration because no external force is being applied to bring the analyte across the membrane filter. 

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