Small-pore Membranes Research Articles

Precolumn phenylisothiocyanate derivatization and liquid chromatography of free amino acids in biological samples

A method for the analysis of free amino acids using precolumn phenylisothiocyanate derivatization and liquid chromatography was tested on a range of sample matrices: bovine plasma, dry cat food, dry infant milk formula and potatoes. The chromatographic resolution of the phenylthiocarbamyl derivatives of free amino acids was satisfactory, with few unidentified compounds present. Standard deviations for triplicate analyses were generally less than 10% of their means. 5-Sulfosalicylic acid (SSA) and a small pore membrane ultrafilter were tested for the deproteinization of bovine plasma. Recoveries of standards were highest when 1% and 4% SSA were used for deproteinization (98% average recovery), and lower (91%) when the ultrafilter was used. The 4% SSA treatment gave higher average free amino acid contents and protein removal than the other deproteinization techniques. These higher values were not due to protein hydrolysis by SSA. Interaction of free amino acids with plasma protein remaining after deproteinization may account for the lower free amino acid contents measured after 1% SSA and ultrafilter treatment. Deproteinization of plasma with 4% (w/v) (final concentration after mixing with the sample) SSA is recommended.

A widely applicable automated sampling apparatus for dissolution testing

An automated sampling apparatus for drug release testing is described that is chemically inert and can filter particulates larger than 0.2 μm. Samples are pushed through filters with inert piston pumps capable of delivering up to 100 psi, so that small-pore membrane filters can be reliably used. These filters are now available in a variety of materials and pore sizes so that in every case a filter was identified that could remove undissolved drug and excipients while avoiding excessive adsorption. A PC-based controller provides flexibility, ease of use, and operation of multiple units. The system proved applicable with heavy particulate loading and for several hydrophobic drugs. Important considerations in designing an automated system are discussed.

Hindered diffusion of synthetic polyelectrolytes in charged microporous membranes

To examine electrostatic effects on the diffusion of macromolecules in membranes, diffusivities of narrow molecular-size fractions of the polyelectrolytes ficoll sulfate and dextran sulfate were measured in polycarbonate track-etch membranes. Radius, number density, and surface charge density of membrane pores were determined from a combination of hydraulic permeability, glucose diffusion, and streaming potential measurements. Molecular charge and Stokes—Einstein radius for each macromolecule fraction were determined from free-solution electrophoretic mobility and diffusivity in a large pore radius membrane, D∞, respectively. As ionic strength was increased from 0.005 to 0.1 M, D∞ for ficoll sulfate remained constant while D∞ for dextran sulfate increased slightly (15-18%). Macromolecule diffusivities in small pore membranes, D, were much more sensitive to ionic strength. For membranes where the ratio of Stokes—Einstein radius to pore radius ranged from 0.08 to 0.29, D/D∞ for ficoll sulfate and dextran sulfate increased by factors ranging from 2.5 to 14 for the same increase in ionic strength. Recent theoretical results for electrostatic double layer interactions in hindered diffusion are in good quantitative agreement with these findings.

Interstitial fluid oncotic pressures in rabbit subcutaneous tissue.

Interstitial fluid oncotic pressures in rabbit subcutaneous tissues were measured by chronically implanted membrane osmometers. Pairs of osmometers were used, one using a membrane permeable and one impermeable to plasma proteins. Measurements from the small-pore membranes averaged 10.2 mmHg while pressures measured from the large-pore membrane osmometers averaged -1.2 mmHg, indicating that the measured pressures were osmotic rather than hydrostatic in nature. These values are in agreement with previously published values from this laboratory on oncotic pressures of excised rabbit skin and with a computer-simulation study of capillary fluid balance. The oncotic pressures described in this study are of the s-me general magnitude as those of lymph from most organs in the body.

Charcoal assay for intrinsic factor using membrane filtration

A modification of the albumin-coated charcoal method for the assay of intrinsic factor and other vitamin B 12 binders is described. It replaces centrifugation with ultrafiltration under reduced pressure through small pore membranes. The resulting method is quicker, and more accurate. In addition, it makes it possible to study the kinetic of agents thought to split vitamin B 12 macromolecular complexes by separating the free from the bound vitamin in seconds.

The behaviour of nucleic acids in membrane chromatography and electrophoresis on nitrocellulose filters

Nitrocellulose membrane filters of various types, both intact or impregnated with Tween or serum albumin, were used as carrier material for small-scale chromatography and electrophoresis of native and denatured DNA, RNA and proteins. DNA isolated from calf thymus, and from B. subtilis, total RNA from chick myeloblasts and human serum albumin and their mixtures, in various binary combinations, served as model samples in the experiments. Horizontal chromatography and electrophoresis of 0.5–2 μg/μl samples was completed within 1–30 min on 3–4 cm long strips in citrate, veronal or acetate buffers pH 7, 8.6 or 4.5. Nucleic acids were stained with toluidine blue, proteins with nigrosin. Characteristic behaviour of different samples under different conditions is described and optimal conditions are found for the separation of most substances under study. In general the model substances to be separated either remained at or moved from the start in single spots. However, the sieving effect of small-pore membranes permitted the convenient detection of the presence of three components of total RNA by electrophoresis. The micromethod also appears to extend the applicability of membrane chromatography and electrophoresis on nitrocellulose filters into the field of nucleic acids, especially where rapid information and simple characterization of minute samples is needed.