Filtration

Type of physical science: Chemistry

Field of study: Chemical methods

Filtration is the passage of a solution or suspension of molecules through a medium such as a gel that allows solvent molecules to pass but retards passage of one or more of the molecules in solution or suspension. Because the degree of retardation depends primarily on molecular size, the method may be used for the separation, purification, and analysis of mixtures of molecules.

Overview

Filtration involves the passage of a solution or suspension of molecules through a medium that allows solvent molecules to pass but retards passage of one or more of the molecules in solution or suspension in the solvent. The filtering medium may consist of materials such as a sheet of porous paper or plastic, or a layer of gel or plastic beads. Retardation of filtered molecules may be complete, so that none of the molecules succeeds in passing through the filter, or partial, so that molecules are slowed to a greater or lesser extent in passage but are potentially capable of movement through the filtering medium. For molecules that are retarded, the degree of retardation depends on such factors as molecular size, shape, or charge.

Complete stoppage of molecules by a filtering medium is of relatively little use as an analytical method; however, partial retardation, because it depends on molecular size, shape, or charge, can provide information on one or more of these molecular characteristics and can therefore be analytical. In addition, because the degree of retardation depends on these factors, filtration can be used to separate molecules, often to a very high level of purity. For most of the analytical methods based on filtration, molecular size is the primary characteristic of importance in the filtering mechanism.

The most common applications of filtration as an analytical technique use gels of various kinds as the filtering medium. In a gel, long, chainlike molecules are tied together by cross-links into a more or less open network. The gel molecules may be naturally occurring biological substances or synthetic plastics. Water and other smaller molecules can penetrate readily into the open spaces of the gel network, but, depending on the dimensions of the spaces in the network, larger molecules may be excluded entirely or delayed in their entry into the network. The filtering gel may take the form of small beads packed into a glass or plastic cylindrical column, or may be cast into a solid tube or slab.

For many filtering gels, particularly the synthetic ones, the dimensions of the spaces in the gel network may be varied by increasing or reducing the number of cross-links between the gel molecules. In this way, a filtering gel may be adjusted to retard molecules extending over a desired size range and to exclude others above this range. The gel material may be neutral, or carry a positive or negative charge. If charged, the gel will retard molecules of opposite charge and allow molecules with the same charge to pass through more rapidly. In some applications, reactive molecules or chemical groups may be attached to the gel molecules to bind desired molecules selectively from the solution being analyzed.

In practice, the solution to be analyzed is layered carefully at one end of a column containing gel beads, or along the top of a gel slab. Flow through the gel depends on gravity or hydrostatic pressure applied by means of a pump; no electrical currents are used. The solvent (usually water) and excluded molecules pass rapidly through the gel; other molecules with size, shape, or charge allowing them to enter the gel network are retarded to a greater or lesser degree.

Because the forces moving solutions through filtering gels are minimal and the gels are relatively chemically inert, most of the retarded and excluded molecules are undisturbed by the technique and remain in their native form. The method is particularly valuable for the analysis of biological molecules such as proteins and nucleic acids, which may be separated by gel filtration with little or no disturbance of their biological activity.

The analytical separation of molecules by filtration is often termed chromatography, derived from the Greek words chroma, "color," and graphein, "to write." The term was first used by Mikhail Tsvett in 1906 to describe the separation of leaf pigments into a series of bands of different color by a filtering column. Chromatography is now used as a term to describe any separation of molecules by filtration, whether the molecules are pigments or colorless. Three different applications of the technique are commonly used in analytical studies: gel filtration, affinity chromatography, and ion exchange.

Applications

Gel filtration is highly effective in separating mixtures of molecules according to molecular size. The gels used for this purpose usually take the form of small beads a millimeter or so in diameter, packed into a cylindrical glass or plastic column that is held vertically. The beads used in this technique are available with networks that exclude molecules with molecular weights ranging from as low as 500 to more than 1 million. Thus, if a group of molecules of interest have molecular weights less than 50,000, for example, a filtering gel can be chosen that excludes all molecules with weights above this value. The excluded molecules, because they do not enter the gel network, quickly pass through the column, and if desired for other analyses, can be collected immediately in the solution running off the bottom of the gel.

Retardation of molecules entering the gel according to size occurs through a process that might at first seem opposite to the expected effect. The largest nonexcluded molecules pass through the gel most rapidly, and the smaller ones pass through at successively slower rates, with the smallest ones exiting last. Passage in this sequence evidently occurs because the smallest molecules enter the spaces in the gel network most easily, and are retarded to the greatest extent because they pass through more of the network. The larger nonexcluded molecules undergo collisions with parts of the gel molecules surrounding openings in the network, and thus enter the network less frequently. As a result, larger molecules tend to pass around the gel beads more often than smaller molecules, and therefore pass through the entire gel more rapidly. The effect is proportional to size, so that each successively larger molecular type passes through the gel more rapidly than the next smaller type.

In more technical terms, the smallest molecules in a sample, because they can pass through all parts of the gel, have access to, and must pass through, the entire volume of fluid in the column, both inside and outside the gel. Larger molecules, depending on the degree to which they are excluded by the framework of the gel, have access to less of the volume of the fluid in the column, and therefore pass through less of the fluid on their way to the bottom of the column.

As a result, the larger molecules encounter less delay, and pass more rapidly through the column, than the smaller types.

If all the molecules have approximately the same overall shape and charge but differ in size, the largest molecular type, passing through the gel most rapidly, exits from the column as a group. After an interval that depends on the molecular size difference and the length of the gel, the next smaller molecular type exits as a second group. Each type of molecule will follow, with molecules of successively smaller size exiting the gel as separate groups. The longer the gel, the greater the interval between the appearance of each successive molecular type.

The filtering gel thus separates the molecules of the mixture according to size, often allowing separate samples of great purity to be collected after only a single pass through the gel.

Molecules that are too large to enter the spaces in the gel network are excluded, as has been noted, and pass through the gel as a mixed group well in advance of any of the retarded molecules.

Separation by a filtering gel according to size is often precise enough to allow estimation of molecular weights, particularly in situations in which the molecules in a sample have equivalent shapes. In order to carry out this estimation, a series of molecules with known molecular weights and easily identifiable characteristics (such as color) is added to a sample containing the unknowns. The known molecules are selected so that their molecular weights bracket the unknowns. The entire mixture is then run through the filtering gel. The rate at which the solution runs through is kept as constant as possible, usually by means of a pump that feeds the solution at a precisely constant rate, and the volume of fluid running through the gel is monitored.

The knowns and unknowns pass through the gel in separate groups that depend on their molecular sizes. The total volume of fluid that has passed through the gel as an unknown exits the column is compared with the volumes that have passed through as the bracketing knowns exit; comparison of the volumes, which are proportional to molecular weight, allows the molecular weight of the unknowns to be estimated. In practice, a graph is set up with the vertical axis as the logarithm as the base 10 logarithm of the molecular weight, and the horizontal axis as the volume of fluid passing through the gel. For each known, the total volume of fluid having passed through gel as it exits is plotted against its known molecular weight. The plot produces a straight line. The total volume of fluid having passed through the column as each unknown exits is also plotted on the same straight line. The molecular weight for an unknown can then be read directly from the graph by extending a horizontal line from the point taken by the unknown on the plotted line to the vertical axis. Because molecular weights determined by this method are estimated by relating unknowns to knowns, the values obtained are relative rather than absolute.

The method has proved to be especially valuable for the determination of the molecular weights of proteins.

The filtering situation becomes more complex when a mixture of molecules that vary in both shape and size is poured on one end of the gel. Departures from a spherical form affect both the ease by which molecules enter gel networks and, once inside, how rapidly they thread through the spaces in the network. As a result, each molecular type passes through a filtering gel at a rate that represents a compromise between the effects of molecular size and departures from spherical shape.

Because departures from an ideal spherical form affect the rate at which molecules pass through a filtering gel, determination of molecular weight is considerably less accurate if molecules vary greatly in shape. Fortunately for the analysis of protein, most soluble proteins approximate a spherical shape and pass through filtering gels at rates that are determined primarily by molecular size.

Many natural and artificial substances form gels that can be used for analytical filtration. Three materials, dextran, polyacrylamide, and agarose, are most often used to make filtering gels, particularly for work with biological molecules. Dextran, a long-chain carbohydrate assembled from glucose units, is a natural product made by certain bacteria. This highly stable, uncharged substance can be cross-linked to a greater or lesser extent, forming gels that exclude molecules of greater or lesser sizes. The most open networks obtainable with dextran filtering gels exclude molecules with molecular weights of more than 800,000 or so, and therefore allow analysis of molecules below this size. Polyacrylamide is an entirely synthetic plastic with chemical properties similar to dextran. The maximum molecular weight excluded by the most open polyacrylamide gel networks is about 500,000, allowing molecules below this size to be analyzed. Agarose is derived from agar, a long-chain carbohydrate mixture extracted from certain marine algae. Agarose, assembled from galactose subunits, forms a more open network than dextran or polyacrylamide. As a consequence, it can retard and allow analysis of molecules of higher molecular weight than either dextran or polyacrylamide, easily up to molecular weights of 10 million. It is more reactive chemically, however, and carries the additional disadvantage of being a strongly charged substance. Both of these characteristics can lead to unwanted interactions with molecules under analysis. Polyacrylamide and agarose are also used in gel electrophoresis, another technique widely employed for analytical separation and purification of molecules and determination of relative molecular weights.

Affinity chromatography is a method related to gel filtration that is much used in biochemical studies to separate proteins that can recognize and bind specific molecules. In this technique, a molecule recognized and bound by the protein of interest is attached chemically to the beads in a column. A mixture of proteins, which may include the complete protein complement of an organism under study, is then passed through the column. Among the varied proteins in the mixture, only the protein recognizing and binding the substance attaches to the beads; the remainder pass through without hindrance. After the unbound proteins are washed from the column, the protein of interest can be removed from the beads by the addition of a solution in which the substance bound by the protein is suspended in free, unattached form. As the solution passes through the column, the freely suspended molecules of the substance compete with the molecules attached to the beads for the binding site on the protein. The competition eventually releases most of or all the protein molecules from the beads for collection as a highly purified sample at the bottom of the column.

Affinity chromatography can rapidly separate a single protein from crude extracts containing hundreds or thousands of proteins. For example, if one wishes to isolate and identify a protein acting as a cellular receptor for a hormone such as insulin, the hormone is the molecule attached to the beads in the column. The receptor for the hormone, if present in a crude preparation of cellular proteins, will attach specifically to the bound hormone when the mixture is passed through the column. All other cellular proteins exit the column without delay. Adding a solution containing the hormone in free, unbound form to the column then removes the receptor from the beads.

This method has proved to be especially useful as a means to identify proteins that control gene activity in living organisms. In this application, the nucleic acid molecule of a gene is linked to the beads in an affinity column, and a mixture of proteins extracted from cells is poured through the column. The control proteins recognizing and binding the gene become attached to the beads, and can be separated for analysis and identification.

Context

Gel filtration was developed partly through a chance observation made by a vacationing scientist hiking some years ago in the mountains of Europe. His guide was a local who brought along a light repast for the hike, including an elongated loaf of bread and a rather crude homemade wine. When the pair stopped for lunch, the guide prepared the wine for drinking by what was evidently a local method. He cut both ends from the loaf and poured the wine through the bread. The wine exiting the bread was much clearer and more drinkable than the original. The guide then split the loaf lengthwise and gave half to the scientist to eat. To the scientist's surprise the opened bread revealed that some of the materials filtered from the wine were layered, producing transverse bands of differing tint along the loaf. According to the story, the layering gave the scientist the idea for filtration as a method for the analytical separation of molecules.

It has been said that the history of chemistry is to a large extent the history of the development of techniques allowing substances to be separated and purified. Gel filtration, developed most extensively in the early 1960's, represented a major technical breakthrough that offered a relatively inexpensive and uncomplicated technique for separating molecules, particularly biological molecules of relatively high molecular weight. Until the advent of gel filtration, the only workable method for separating and purifying molecules with molecular weights on the order of proteins was ultracentrifugation, in which a solution of molecules is spun at very high speeds in an electrically driven rotor. Depending on factors such as molecular weight and shape, molecules descend at different rates in a centrifuge, and can be separated and purified by this means. The method is time-consuming, however (it may take as much as a week or more to centrifuge down some biological molecules), and ultracentrifuges are highly expensive and complex machines.

A gel column capable of equivalent or even better analytical separations can be assembled or purchased relatively cheaply, and a run can be completed in a few hours. In addition, very large solution volumes can be analyzed by gel filtration; most ultracentrifuges are limited to a few milliliters of sample. For these reasons, gel filtration has become one of the most widely applied and successful analytical separation methods used in analytical chemistry today, particularly in biochemistry.

Principal terms

AGAROSE: a long-chain carbohydrate commonly used for filtering gels; part of agar, extracted from marine algae

CHROMATOGRAPHY: a general term applied to filtration methods that separate a mixture of molecules into distinct groups

DEXTRAN: a long-chain carbohydrate commonly used for filtering gels; a product of certain bacteria

EXCLUDED MOLECULES: molecules that are too large to enter a gel network, and pass through a filtration column or slab gel without retardation

GEL: a material consisting of long-chain molecules crosslinked into the network, with water molecules surrounding and penetrating the network

POLYACRYLAMIDE: a synthetic plastic commonly used as a filtering gel

Bibliography

Alberts, Bruce, et al. MOLECULAR BIOLOGY OF THE CELL. 2d ed. New York: Garland, 1989. Chapter 4, "How Cells Are Studied," describes gel electrophoresis along with other analytical methods used in biochemical and molecular analysis of proteins and nucleic acid. The text, intended for the college level, is clearly written and includes many excellent illustrations.

Fischer, Lave. AN INTRODUCTION TO GEL CHROMATOGRAPHY. New York: North-Holland, 1968. Part of a series titled Laboratory Techniques in Biochemistry and Molecular Biology, this book describes the theory and practice of gel filtration in some detail. Although intended for practicing scientists, it explains the basics of the technique, and much of the book is accessible to the layperson.

Giddings, J.C. DYNAMICS OF CHROMATOGRAPHY. Part 1, PRINCIPLES AND THEORY. New York: Marcel Dekker, 1965. Written at a more advanced technical level, this work provides a complete description of the theory underlying gel filtration and other chromatographic methods.

Schleif, Robert F., and Pieter C. Wensink. PRACTICAL METHODS IN MOLECULAR BIOLOGY. New York: Springer-Verlag, 1981. This book, a laboratory manual intended for the college level, explains gel filtration in step-by-step terms along with the other commonly used methods for separating and purifying biological molecules.

Streyer, Lubert. BIOCHEMISTRY. 3d ed. New York: W. H. Freeman, 1988. Chapter 3, "Exploring Proteins," presents a brief and lucid explanation of gel filtration and its use in purifying proteins, along with many other methods used in the biochemical analysis of these molecules. Although intended for use at the college level, the text is clearly and simply written, and should be understandable to readers with a background in high school chemistry.