GB2172812A

GB2172812A - Chromatographic separation medium for rapid analysis of small samples

Info

Publication number: GB2172812A
Application number: GB08607241A
Authority: GB
Inventors: David J Burke; Gary S Ott
Original assignee: Bio Rad Laboratories Inc
Current assignee: Bio Rad Laboratories Inc
Priority date: 1985-03-25
Filing date: 1986-03-24
Publication date: 1986-10-01
Also published as: GB2172812B; DE3609021A1; GB8607241D0; JPS61257236A; CA1265116A

Abstract

Chromatographic separation of very small volume samples, typically 500 micrograms or less, of proteins (including blood haemoglobin), nucleic acids, or other species in short time periods, typically 15 minutes or less, are achieved by the use of a separation medium comprising a solid, substantially nonporous inert support having polymeric chains with multiple functional groups coupled to the surface thereof. Preferred polymeric chains are selected from polyethyleneimine, polylysine, aspartic acid polymers, and derivatives thereof.

Description

SPECIFICATION Chrornaitographic separation medium for rapid analysis of small samples BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to chromatographic separations, and particularly to the separation of components in a liquid sample by passage through a packed bed of solid separation medium. This invention is primarily directed to rapid analytical separations of protein mixtures of small sample size.

2. Description of the Prior Art The separation of protein mixtures by ion exchange chromatography is well known. Existing media, however, represent comprise designs which allow for preparative separations containing milligram amounts of protein, and require twenty minutes or more to elute all components in a typical mixture.

There is an increasing need for the separation of smaller size samples in shorter periods of time. This is particularly true in clinical applications requiring the analysis of the large number of samples in a rapid quantitative manner. In many cases, available sample quantities are very small, and a high performance liquid chromatography (HPLC) column of small volume and rapid separation time would be appropriate.

Existing materials used as the stationary phase in such columns generally consist of porous supports with ion exchange functional groups coupled to the support surfaces, the major fraction of which are inside the pores. Both isolated point charges and short polycationic or -anionic chains have been used as the functional groups. Although suitable for large columns, these media fail to provide clean separations when rapid separations are required.

SUMMARYOFTHE INVENTION It has now been discovered that small volume samples of protein mixtures, particularly those on the order of about 500 micrograms of protein or less, and more particularly about 200 micrograms or less of total protein, are capable of being separated on small columns with high resolution in an unusually short period of time by using a separation medium comprised of a substantially non-porous inert solid support, the surface of which has been coupled to polymeric chains, each containing multiple binding sites. Even without the greatly increased surface area provided by the pores in the materials of the prior art, the media of the present invention are capable of separating the proteins into discrete bands which, upon detection by conventional methods, provide a highly accurate analysis in an unusually short period of time.The present invention is useful for a wide variety of separation mechanisms and mixtures.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1a through le are hemoglobin separation chromatograms using a series of separation media within the scope of the invention, the media differing in the number of binding sites per unit volume, Figures 2a and 2b show the same type of separation shown in Figures large, except that the media differ in column length.

Figures 3a through 3f show the same type of separation shown in Figures ia-1e,comparing non-porous media within the scope of the invention to porous media at several carrier fluid flow rates.

Figures 4a and 4b are mouse ascites fluid separation chromatograms, comparing a separation medium within the scope of the invention to a preexisting commercial medium.

Figures 5a through 5d are chromatograms showing the separation of ovalbumin from bovine serum albumin, using a separation medium within the scope of the invention with different sample sizes.

Figure 6 is a chromatogram of the same mixture shown in Figures la-le, using a further separation medium within the scope of the invention.

Figure 7 is a chromatogram of a red cell hemolysate, using a still further separation medium within the scope of the invention.

Figures 8a and 8b are graphical representations of quantitative analyses using a separation medium within the scope of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The separation media of the present invention are solid phase media, preferably in the form of beaded materials suitable for use in HPLC. Unlike the macroporous and mesoporous media of the prior art, the base materials which support the functional groups in the media of the present invention are substantially non-porous, which term is used herein to include microporous media, i.e., media which are characterized by small particle diameter, low pore volume, and small surface area. Thus, virtually all of the chromatographic interaction between solutes in the mobile phase and the stationary phase during passage of the mobile phase through the bed occurs on the outer surface of the solid phase rather than in the pores.The base materials of the present invention have particle diameters between 0.5 and 30 microns, preferably between 1.0 and 10 microns.

When analyzed by nitrogen adsorption-desorption (B-E-T) analysis, the base materials of the present invention have less than 100 and preferably less than 25 microliters of pore volume per gram of dry material. For beads of the present invention with a mean particle diameter of 7 microns, B-E-T analysis will show less than 5.0 and preferably less than 1.5 m2/g of surface area. Beads with smaller diameters will have proportionately larger surface areas.

A mixture of components designed as a standard for gel filtration experiments may be used to demonstrate the microporous nature of the support base. The components range in molecular weight from 1350 (cyanocobalamin) to 670,000 (thyroglobulin). When passed through a typical gel filtration column such as Bio-Sil TSK#250TM (Bio Rad Laboratories, Richmond, California), the proteins separate and emerge as discrete peaks due to the variation in molecular size. However, when the mixture is passed through a packed bed of comparable dimensions consisting of the support base used in the present invention (prior to derivatization), no separation occurs and all species emerge as a single peak. This indicates that species having molecular weights of 1350 and above do not penetrate the support base.

For the purposes of the present invention, the surface of the support must be capable of covalent coupling to the polymeric chains referred to above, or capable of chemical derivatization to provide active sites capable of covalent coupling. Thus, the bare support will conveniently be one which has reactive groups such as, for example, hydroxyl groups, epoxide groups, carboxyl groups, acyl halide side chains or aldehydes, exposed on the surface.

The bead itself may be of an organic or inorganic material. Examples of organic materials are methacrylate resins such as, for example, poly (hydroxypropyl methacrylate), poiy(glycidyl methacrylate), and various copolymers thereof, including cross-linking agents such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate. Examples of inorganic materials are glass and silica.

It is preferred that the support base have a rigid surface which is inert to the materials which come in contact with it, and remains so over a wide pH range. It is further preferred that the support base itself have substantially no binding affinity for any of the substances in the mixture being separated, thus avoiding non-specific binding on any portion of the bead other than the functional groups in the polymeric chains coupled to the bead surface. Thus, for protein and nucleic acid separations, the support base is preferably hydrophilic in character, such that after it is derivatized, the free surface, i.e., thb surface which is accessible to the carrier fluid during analytical separations, is hydrophilic.

As used in HPLC, the support base is preferably comprised of substantially spherical beads. The particle diameter of the beads may be varied widely subject to considerations of back pressure and flow rate in the HPLC system. In most applications, beads having a mean particle diameter of about 0.5 to about 30 microns, preferably about 1 to about 10 microns, will provide the best results.

The polymeric chain which is coupled to the bead may be any polymer whose units contain functional groups capable of interaction with the species in the sample being analyzed. Examples of such functionalities are ion exchange sites including both anionic and cationic sites, and any other sites having a characteristic binding affinity, such as, for example, hydrophobic sites which interact with corresponding hydrophobic sites on the sample species.

Ion exchange interactions are preferred. Any of the wide variety of polymeric chains currently known which contain anionic or cationic sites may be used. Examples are polyethyleneimine and polylysine. The active sites on these chains may be further derivatized for conversion from anionic to cationic character, and vice versa, or for increasing or decreasing the binding strength. A preferred chain is polyethyleneimine, the active sites of which may be derivatized in a variety of ways including, for example, addition of carboxyl groups or sulfonic acid derivatives, or fully methylating the nitrogens to form quaternary amines.

In preferred embodiments, the polymer chains are substantially branched and are formed of units containing approximately one binding site each.

The binding affinity and capacity of the chains may be further varied by appropriate selection of the molecular weight of the chains or their concentration along the support surface. In general, polymeric chains having an average molecular weight of from about 100 to about 100,000, preferably from about 250 to about 5,000, will provide the best results. The concentration of binding sites is conveniently characterized by the overall binding capacity of the separation medium, in terms of equivalents or microequivalents per unit volume. Effective separations may be obtained over a wide range of binding capacities.Expressed in terms of chloride binding capacity, best results are generally achieved within the range of about 10 to about 1,000 microequivalents per milliliter, with about 30 to about 350 microequivalents per milliliter preferred, and about 70 to about 130 microequivalents per milliliter particularly preferred.

Although applicable to a wide variety of sample mixtures, the present invention is particularly useful in the separation of proteins, peptides and nucleic acids. Very small sample sizes, particularly those on the order of about 500 micrograms or less, preferably about 200 micrograms or less, may be separated in the present system in an unusually short period of time while still obtaining high resolution among the components. The preferred form of the separation medium is a packed bed in the form of a column having a volume of about 0.1 to about 2.0 cubic centimeters, preferably about 0.2 to about 1.5 cubic centimeters. The length of the column is preferably about 20 cm maximum, with about 1.0 to about 10 cm more preferred, and about 2.0 to 7.5 cm the most preferred.

The separation is generally achieved with the aid of a carrier fluid passing through the bed at a flow rate of about 0.5 to about 10 ml/min, preferably about 1.0 to 5.0 ml/min.

In preferred embodiments, particularly for protein separations, separation of the species is enhanced by the use of either a pH gradient of a salt gradient during the elution procedure. The gradient value may vary widely depending on the species to be separated. A typical pH gradient may range from about 4 to about 10 over the course of the separation, while a typical salt gradient may entail a net increase of from about 0.03 to about 1.0 molar concentration, preferably from about 0.05 to about 0.5 molar. In most applications, the gradient will be sustained over a time period ranging from about 0.5 to about 20 minutes, preferably from about 1 to about 5 minutes, with the pH and/or salt concentration changing as the separation proceeds.

Gradual or stepwise changes in salt composition, such as the substitution of one anion for another, may also be used in some cases to beneficial effect.

The following examples are offered for illustrative purposes only, and are intended neither to define nor limit the invention in any manner.

EXAMPLE 1 A. Bead Preparation Microporous polymethacrylate beads were prepared from glycidyl methacrylate monomer with diethylene glycol dimethacrylate cross-linking agent bythefollowing procedure.

A reaction flask was charged with 2 liters of distilled deionized water and 30.0 grams (1.5%) poly(vinyl alcohol) of average molecular weight approximately 1800. The flask was heated to 40 C and maintained at that temperature until all ofthe polymer had dissolved. Sodium dodecyl sulfate (36.0 grams, 1.5%) and Dow Corning 544 defoaming agent (2.0 grams dissolved in 18 ml water) were added to the solution.

A mixture was prepared by combining 204.0 grams (1.44 moles) glycidyl methacrylate with 136.0 grams (0.56 moles) diethylene glycol dimethacrylate, and filtering through a column of basic alumina (Bio-Rad At~10 resin 100-200 mesh) to remove inhibitors present in the monomers. After filtration, 2.0 grams of free radical initiator was added and the entire mixture added to the poly(vinyl alcohol) solution.

The resulting mixture was stirred for 30 minutes at 400C and 800 rpm. The temperature was then increased over a two-hour period to 780C and maintained there for ten hours. The mixture was then allowed to cool to room temperature and was diluted with deionized water. The resulting polymer beads were collected by centrifugation, resuspended in water, stirred for a half hour at room temperature and centrifuged, then resuspended, stirred and centrifuged again. The resulting water wet cake was suspended in methanol, stirred for a half hour, and centrifuged, and the latter sequence repeated twice. The resulting methanol wet cake was then washed twice with acetone (2 volumes each time), and twice with 500 ml dry ether.The filter cake was finally air dried for one hour and then transferred to a vacuum drying oven overnight at 40 C. The resulting beads were sized to a mean particle diameter of 7.5 microns. B-E-T analysis showed a surface area of 1.00 m2/g and a pore volume of 10.5 microliters/g.

B. Derivatization Polyethyleneimine was coupled to the beads prepared above as follows.

A methanol wet cake of 100 g of the beads was suspended in 400 ml of a solution of polyethyleneimine (100 g of polyethyleneimine of average molecular weight of approximately 1800 dissolved in 300 ml of methanol). The suspension was shaken at 40 C for sixteen hours, then diluted with methanol, centrifuged and decanted. The beads were then resuspended in methanol, centrifuged and decanted again. The supernatant was discarded and the beads were washed twice with 0.5N HCI by suspension, centrifugation and decanting. Following a similar procedure, the beads were then washed four times with filtered deionized water.

The binding capacities were determined using chloride ion as the binding agent. The determination consisted of saturating the sites with chloride ion by equilibration with hydrochloric acid followed by an aqueous wash, then displacing the chloride ion with 0.5N nitrate ion using concentrated silver nitrate, followed by titration of the displaced chloride ion with ARNO, to a K2CrO4 end point.

The above-described procedure produced beads with a chloride-binding capacity of 90 1 10 microequivalents per milliliter of beads. For the following tests, however, a series of five batches of varying chloride-binding capacities were prepared by varying the concentration of polyethyleneimine in the reaction mixture. The resulting capacities were 36,74,95, 133 and 203 microequivalents per milliliter.

C. Chromatographic Separations A mixture of hemoglobin A, F, S and C was prepared for separation on the various derivatized beads, with the following proportions: A--42+3%; F-23+2%; S~20+1%; C-1612%.

The five batches of beads were individually packed into cylindrical columns measuring 4.6 mm in diameter (inner) and 30 mm in length. Mixing apparatus was assembled for feeding buffer solution to a column with a linearly increasing salt gradient. The buffers used were both 1 ohm tris chloride at pH 8.5, the NaCI content rising from zero to 0.1 M over five minutes. The separation was performed using a sample size of 20 microliters (40 micrograms) and a buffer flow rate of 1.0 ml/min.

The results are shown in Figs. la through 1 e, which represent traces recording the response of an ultraviolet detector having an 8-microliter cell. The chloride-binding capacities of the packing were as follows: Fig. la-36 microeq/ml; Fig. 1b74 microeq/ml; Fig. 1c-95 microeq/ml; Fig. 1d-133 microeq/ml; Fig. 1#203 microeq/ml.

EXAMPLE 2 This example compares columns of different length in terms oftheirelution time and separation capability.

A single column identical to that prepared in connection with Example 1 with a chloride binding capacity of 74 microeqlml and a pair of such columns coupled in series by a low dead volume connector were used to separate the same hemoglobin mixture (sample size 20 microliters) used in Example 1. The salt gradient in the carrier fluid was established by use of two buffer solutions as follows: Buffer A: 16mM tris chloride, pH 8.5; Buffer B: 20mM tris chloride plus 0.2M NaCI, pH 8.5.

The gradient program consisted of S15% B during the period e9 minutes after injection, followed by 1540% B during the period W15 minutes after injection.

Detection was accomplished in the same manner as Example 1, and the results are shown in Figs. 2a (single column) and 2b (double column). The hemoglobin peaks and their retention times are given for each run. The traces indicate that the longer column gave better peak resolution, although atthe expense of analysis time and band width.

EXAMPLE 3 This example compares a separation medium of the present invention with a pre-existing separation medium, to demonstrate the superiority of the former. The former was identical to that prepared in connection with Example 2, while the latter was SynChropak AX~300, a product obtained from Brownlse Labs Inc., Santa Clara, California, consisting of a porous silica support having polyethyleneimine coupled to its surfaces, having an average particle diameter of approximately 10 microns, an average pore diameter of approximately 300 A and a picric acid binding capacity of 656 micromoles/g (equivalent to a chloride binding capacity of about 850 microeq/ml).

Columns measuring 4.6 mm by 30 mm and sample volumes of 20 microliters were used in each case, at three differentcarrierfluid flow rates. The results are shown in Figs. 3a through Sf, Figs. 3a-3c representing the derivatized beads of the present invention at carrier fluid flow rates of 0.5, 1.0 and 2.0 ml/min, respectively, and Figs. 393f representing the A)(-300 beads at the same carrier fluid flow rates. The buffer solutions of Example 2 were used, with gradient programs as follows: Figs. 3a and 3d: S70% B over seven minutes; Figs. 3b and 3e: 0~50% B over five minutes; and Figs. 3e and 3f: 0#0% B over three minutes.

The results indicate thatthe non-porous beads are superior in resolution, band width and sensitivity at all flow rates, particularly the higher flow rates.

EXAMPLE 4 This example compares a separation medium of the present invention with a further pre-existing separation medium in the analysis of lgG present in mouse ascites fluid, again demonstrating the superiority of the former. The separation medium of the present invention was the same as that used in Example 2. The comparison medium was Bakerbond, a product of J. T. Baker, Phillipsburg, New Jersey, consisting of a porous silica support having anion exchange groups coupled to its surfaces, with average particle diameter of approximately 5 microns.

A column measuring 4.6 mm by 30 mm was used for the medium of the present invention. The Bakerbond column was a 4.6 mm by 250 mm analytical column packed by the manufacturer.

Sample volumes of 20 microliters were used in each case. In the non-porous bead test, buffer A was 16 mM tris at pH 7.5, while buffer B was 20 mM tris plus 0.5M NaCI at pH 7.5; with a gradient program of 0~100% B over twenty minutes at a flow rate of 1.0 ml/min. In the Bakerbondtest, buffer Awas 10 mM potassium phosphate at pH 6.8, while buffer B was 500 mM potassium phosphate at pH 6.4; with a gradient program of 0~25% B over sixty minutes at a flow rate of 1.0 ml/min.

The detector trace for the non-porous bead test is shown in Fig. 4a (with retention times shown for each peak) while thatforthe Bakerbond comparison test is shown in Fig. 4b. It is clear that the nonporous bead provides an analysis of similar quality in a much shorter period of time.

EXAMPLE 5 This example demonstrates the separation of a mixture of ovalbumin and bovine serum albumin using the separation medium of Example 2.

A 4.6 by 30 mm column was used, with four sample sizes ranging from 65 to 650 micrograms of total protein. Buffer A was 10 mM tris at pH 8.0, and Buffer B was the same plus 0.5M NaCI. The gradient program was S100% B over fifteen minutes at a flow rate of 1.0 ml/min.

The results are shown in Figs. 5a through 5d where the sample sizes were as follows: Fig. 5a-65 micrograms; Fig.59130 micrograms; Fig. 5c-260 micrograms; and Fig. 5d-650 micrograms; with salt gradients and retention times shown. It is clear that effective separations are achieved in under fifteen minutes, with peak broadening occurring above about 250 micrograms.

EXAMPLE 6 This example demonstrates the preparation and use of poly(hydroxypropyl methacrylate) beads derivatized with polyethyleneimine.

A. Bead Preparation A reaction flask was charged with 2 liters of distilled deionized water and 30.0 g (1.5%) of Gelvatol# 20--30, a poly(vinyl alcohol) (Monsanto Industrial Chemicals Co., St. Louis, MO). The flask was heated to 400C and maintained at that temperature until all of the polymer had dissolved.

Sodium pyrophosphate (4.0 g, 0.2%) was then added.

A mixture was prepared by combining 150.0 g (1.04 moles) of hydroxypropyl methacrylate and 50.0 g (0.25 mole) of ethylene glycol dimethacrylate.

The former was a mixture of 72% 2-hydroxypropyl methacrylate and 28% l-methyi-Zhyd 1-methyi-2-hydroxyethyl methacrylate. The mixture was filtered through a column of basic alumina (Bio-RadAG-10 resin 100~200 mesh), and 0.8 g of lauroyl peroxide dissolved in 80 g cyciohexanol was added. This mixture was then combined with the poly(vinyl alcohol) mixture prepared above.

The resulting mixture was stirred for 30 minutes at 400C and 800 rpm. The temperature was then increased over a two-hour period to 78 C and maintained at that level forten hours. The mixture was then allowed to cool to room temperature, then diluted with deionized water. The resulting beads were collected by centrifugation, resuspended in water, stirred, centrifuged, then resuspended, stirred and centrifuged again. The resulting cake was suspended in methanol,-stirred, centrifuged, resuspended, stirred and centrifuged again, then washed twice with methanol and twice with dry ether.The filter cake was finally air dried, then vacuum dried overnight at 40 C. The resulting beads were sized to a mean particle diameter of 10 microns. B--EE-T analysis showed a surface area of 1.16 m2/g and a pore volume of 5.0 microliters/g.

B. Derivatization A 40 g quantity of the beads was washed with dry acetone and suspended in acetone to a volume of 100 ml. Recrystallized p-toluenesulfonyl chloride (50 9) and dry, distilled pyridine (50 ml) were added.

The reaction flask was then sealed and shaken at 600C overnight. The beads were centrifuged out, washed twice with acetone to remove excess p toluenesulfonyl chloride and pyridine, then resuspended in 100 ml of water. A 175 ml solution containing 82 g of polyethyleneimine (average molecular weight 1800) dissolved in water was added, and the reaction flask was'shaken at400Cfor two days. The resulting beads had a chloride binding capacity of 62 microequivalents per milliliter.

C. Chromatographic Separation The mixture of hemoglobin A, F, S and C of Example 1 was used, with a sample size of 10 microliters.

The beads were packed into a column measuring 0.4 cm in diameter by 25 cm in length, and a buffer mixing apparatus was set up as in Example 1 to provide a flow of buffer through the column at 1.0 ml/min, the buffer containing 0.01 or tris chloride at pH 8.0 and a NaCI content rising from zero to 0.1 so over the time period extending from two to twenty minutes from the start of the separation. The results are shown in Fig. 6, which shows absorbance at 415 nm in the detector as a function of time. The major peaks as they appear from left to right were identified as follows, with retention times in parentheses: C(7.5 min.), S(8.6 min), A (10.2 min), F (13.3 min).

EXAMPLE 7 The example demonstrates the preparation and use of a cation exchange support by succinylation of the support prepared in Example 1.

Poly(glycidyl methacrylate) beads derivatized with polyethyleneimine were prepared according to the procedure described in Example 1, sections A and B, to provide a chloride binding capacity of 90 microequivalents per milliliter. The beads were then washed twice with 0.5N HCI, then several times with water. The beads were then resuspended in water and the pH adjusted to 6.0.

A ten-fold molar excess of succinic anhydride (relative to the chloride binding capacity) was then added in three equal portions with stirring, keeping the pH at 6.0 by addition of NaOH. The reaction was permitted to proceed for two hours. The degree of succinylation was checked by measuring the chloride binding capacity. If the latter was above 15 microeqiml, the succinylation was repeated.

The resulting beads were used to resolve hemoglobin Air from a red cell hemolysate containing 11.9% of the Aic. A column measuring 4.6 mm in diameter and 3.0 cm in length was used, with a 0.010M bis-tris buffer at pH 6.3 flowing at 1.5 ml/min, with NaCI increasing from zero to 0.05M over ten minutes. The sample size was 20 microliters.

Detection was achieved by absorption measurements at 415 nm, and the results are shown in Fig. 7. The two peaks were identified as hemoglobin A, (retention time 3.36 min) and hemoglobin Ao (4.40 min), respectively.

EXAMPLE 8 This example demonstrates the use of a separation medium of the present invention in quantitative analysis.

A sample containing 60% hemoglobin A and 40% hemoglobin C was prepared, then serially diluted, resulting in a series of solutions differing in the concentrations of these two proteins. A fixed volume (20 microliters) of each solution was then repeatedly injected onto columns identical to those described in Example 2 (single column). Peaks eluting from the columns were detected by absorbance at 415 nm and the peak areas were determined by electronic integration of the detector output.

Figs. 8a and 8b show the peak areas generated by the integrator plotted against the amounts of protein injected for hemoglobin A and hemoglobin C individually, each figure representing a separate series of dilutions. The linearity of the results indicates that recovery of these proteins in quantitative peaks is excellent over the entire range tested, i.e., 2 to 100 micrograms of input protein.

The foregoing description is offered for illustrative purposes only. Numerous modifications and variations beyond those mentioned herein but still falling within the spirit and scope of the invention will be readily apparent to those skilled in the art.

Claims

1. Achromatographic separation medium comprising a solid, substantially non-porous inert support to the surface of which are covalently coupled polymeric chains, each said chain containing multiple binding sites.

2. A chromatographic separation medium in accordance with claim 1 in which said binding sites are selected from the group consisting of anionic sites, cationic sites, and hydrophobic sites.

3. A chromatographic separation medium in accordance with claim 1 in which the free surface of said support is hydrophilic.

4. A chromatographic separation medium in accordance with claim 1 in which said support is comprised of a member selected from the group consisting of poly(hydroxypropyl methacrylate), poly(hydroxyethyl methacrylate) and poly(glycidyl methacrylate) and derivatives thereof including a cross-linking agent selected from the group consisting of diethylene glycol dimethacrylate and ethylene glycol dimethacrylate.

5. A chromatographic separation medium in accordance with claim 1 in which said support is comprised of substantially spherical beads having a mean diameter of about 0.5 to about 30 microns.

6. A chromatographic separation medium in accordance with claim 1 in which said support is comprised of substantially spherical beads having a mean diameter of about 1.0 to about 10 microns.

7. A chromatographic separation medium in accordance with claim 1 in which the average molecular weight of said polymeric chains is about 100 to about 100,000.

8. A chromatographic separation medium in accordance with claim 1 in which the average molecular weight of said polymeric chains is about 250 to about 5000.

9. A chromatographic separation medium in accordance with claim 1 in which said polymeric chains are substantially branched and each unit thereof contains approximately one binding site.

10. A chromatographic separation medium in accordance with claim 1 in which said binding sites are anion exchange sites and said polymeric chains have a chloride binding capacity of about 10 to about 1000 microequivalents per milliliter, based on total chromatographic separation medium.

11. A chromatographic separation medium in accordance with claim 1 in which said binding sites are anion exchange sites and said polymeric chains have a chloride binding capacity of about 30 to about 350 microequivalents per milliliter, based on total chromatographic separation medium.

12. A chromatographic separation medium in accordance with claim 1 in which said binding sites are anion exchange sites and said polymeric chains have a chloride binding capacity of about 70 to about 130 microequivalents per milliliter, based on total chromatographic separation medium.

13. A chromatographic separation medium in accordance with claim 1 in which said polymeric chains are selected from the group consisting of polyethyleneimine, polylysine, polymers of aspartic acid, and derivatives thereof.

14. A chromatographic separation medium in accordance with claim 1 in which said polymeric chains are selected from the group consisting of polyethyleneimine and polyethyleneimine having active sites converted to a member selected from the group consisting of carboxyl moieties, sulfonate moieties and quaternary ammonium moieties.

15. A chromatographic separation medium comprising a solid, substantially non-porous inert support selected from the group consisting of crosslinked poly(hydroxypropyl methacrylate) and crosslinked poly(glycidyl methacrylate), to the surface of which are coupled polyethyleneimine chains having an average molecular weight per chain of about 250 to about 5000 in an amount providing said medium with a chloride binding capacity of about 70 to about 130 microequivalents per milliliter, said support being in the form of substantially spherical beads having a mean diameter of about 1 to about 10 microns.

16. A chromatographic separation column containing a packed bed of chromatographic separation medium in accordance with claim 1, said column being a maximum of about 20 cm in length.

17. A chromatographic separation column containing a packed bed of chromatographic separation medium in accordance with claim 1, said column having a length of from about 1.0 to about 10cm.

18. A chromatographic separation column containing a packed bed of a chromatographic separation medium in accordance with claim 1, said column having a length of from about 2.0 to about 7.5 cm.

19. A chromatographic separation column containing a packed bed comprising a solid, substantially non-porous inert support selected from the group consisting of cross-linked poly(hydroxypropyl methacrylate) and cross-linked poly(glycidyl methacrylate), to the surface of which are coupled polyethyleneimine chains having an average molecular weight per chain of about 250 to about 5000 in an amount providing said medium with a chloride binding capacity of about 70 to about 130 microequivalents per milliliter, said support being in the form of substantially spherical beads having a mean diameter of about 1 to about 10 microns, said column having a length of from about 2.0 to about 5.0 cm.

20. A method for detecting species in a mixture thereof, comprising: (a) passing said mixture through a packed bed comprised of a solid, substantially non-porous inert support to the surface of which are bonded polymeric chains, each said chain containing multiple binding sites, to separate said species into substantially discrete bands; and (b) detecting said bands as they emerge from said bed.

21. A method in accordance with claim 20 in which said packed bed is a column having a volume of about 0.1 to about 2.0 cubic centimeters, said mixture is less than about 500 micrograms in size, and said species are selected from the group consisting of proteins, peptides and nucleic acids.

22. A method in accordance with claim 20 in which said packed bed is a column having a volume of about 0.2 to about 1.5 cubic centimeters, said mixture is less than about 200 micrograms in size, and said species are selected from the group consisting of proteins, peptides and nucleic acids.

23. A method in accordance with claim 20 in which said packed bed is a column of a maximum of about 20 cm in length, and said mixture is less than about 500 micrograms in size.

24. A method in accordance with claim 20 in which said packed bed is a column of about 1.0 to about 10 cm in length, and said mixture is less than about 500 micrograms in size.

25. A method in accordance with claim 20 in which said packed bed is a column of about 2.0 to about 7.5 cm in length, and said mixture is less than about 200 micrograms in size.

26. A method in accordance with claim 20 in which step (a) is accomplished by flowing an inert carrier fluid through said bed at a flow rate of about 0.5 to about 10 mi/min.

27. A method in accordance with claim 20 in which step (a) is accomplished by flowing an inert carrier fluid through said bed at a flow rate of about 1.0 to about 5 ml/min.

28. A method in accordance with claim 20 in which a parameter of said carrier fluid selected from the group consisting of salt concentration, salt composition and pH is continuously varied during step (a).

29. A method in accordance with claim 20 in which said carrier fluid contains a salt dissolved therein at a concentration continuously increasing over the course of step (a) with a net increase in molarity of about 0.03 to about 1.0.

30. A method in accordance with claim 20 in which said carrier fluid contains a salt dissolved therein at a concentration continuously increasing over the course of step (a) with a net increase in molarity of about 0.05 to about 0.5.

31. A method for detecting proteins in a mixture thereof in a sample size of about 200 micrograms or less, comprising: (a) passing said mixture through a packed bed of about 0.2 to about 1.5 cubic centimeter in volume and about 1.0 to about 10 cm in length, said bed being comprised of a solid, substantially nonporous inert support to the surface of which are bonded polymeric chains, each said chain containing multiple binding sites, by flowing through said packed bed an inert carrier fluid ata flow rate of about 1 to about 5 ml/min, said carrier fluid containing a salt dissolved therein at a concentration continuously increasing with a net increase in molarity of about 0.1 to about 0.5 over a period of about 1 to about 5 minutes, to separate said proteins into substantially discrete bands; and (b) detecting said bands as they emerge from said bed.

32. A method in accordance with claim 20 in which said inert support is comprised of substantially spherical beads having a mean diameter of about 0.5 to about 50 microns.

33. A method in accordance with claim 20 in which said polymeric chains are selected from the group consisting of polyethyleneimine and polyethyleneimine having active sites converted to a member selected from the group consisting of carboxyl moieties, sulfonate moieties, and quaternary ammonium moieties.

34. A method in accordance with claim 20 in which said binding sites are anion exchange sites and said polymeric chains have a chloride binding capacity of about 10 to about 1000 microequivalents per milliliter, based on total chromatographic separation medium.

35. A method for detecting proteins in a mixture thereof in a sample size of about 200 micrograms or less, comprising: (a) passing said mixture through a packed bed of about 0.2 to about 1.5 cubic centimeter in volume and about 2.0 to about 7.5 cm in length, comprised of a solid, substantially non-porous inert support in the form of substantially spherical beads having a mean diameter of about 1.0 to about 10 microns, to the surface of which are coupled polyethyleneimine chains having an average molecular weight per chain of about 250 to about 500 in an amount providing said medium with a chloride binding capacity of about 70 to about 130 micro quivalents per milliliter, by flowing through said packed bed an inert carrier fluid at a flow rate of about 1.0 to about 5 ml/min, said carrier fluid containing a salt dissolved therein at a continuously increasing concentration with a net increase in molarity of about 0.05 to about 0.5 over a period of about 1 to about 5 minutes, to separate said proteins into discrete bands; and (b) detecting said bands as they emerge from said bed.

36. A chromatographic separation medium as claimed in Claim 1 substantially as herein described with reference to any one of the Examples.

37. A method for detecting species in a mixture thereof as claimed in Claim 20 substantially as herein described with reference to any one of the Examples.

38. A method for detecting species in a mixture thereof as claimed in Claim 20 substantially as herein described with reference to the accompanying drawings.