SE442591B - SET UP CHROMATOGRAPHIC SEPARATION AND CHROMATOGRAPHY FOR THIS SEPARATION - Google Patents

Description

7-13892--3 2 dynamic size of the components. The molecular weight (MW) of the components can be calculated as a function of the hydrodynamic size. A curve with the molecular weight fraction eluted at a given retention volume (VR) for a particular packing material shows that for a given pore volume in the packing material, certain molecular weight fractions are completely excluded due to their large size and certain molecular weight fractions penetrate * completely due to their small size. size. Between these two extreme cases is a range of molecular weight fractions, which are preferentially retarded by contact with the porous particles, and materials containing these molecular weight fractions can be fractionated by this particular packing material.

The actual functional relationship in size exclusion chromatography is the molecular weight calibration curve, which is usually a logarithmic curve of the molecular weight plotted against the retention volume.

Molecular weight calibration curves generally have a substantially linear portion, so that the molecular weight of a retained fraction in a sample can be determined exactly if the retention volume of this particular molecular weight fraction is in the linear portion of the molecular weight calibration curve and less precise, if it is outside the linear range. Molecular weight calibration curves generally have linear portions, which comprise approximately two decades in the molecular weight logarithmic scale for a single pore size. To obtain a calibration curve with a linear part comprising more than two decades of the molecular weight scale, a chromatographic resolution zone composed of many columns, each with different pore sizes, was used.

As stated in "Know More About Your Polymer", published in 1976 by Waters Associates, Hilford, Hass., Is specifically combined, for expansion of the linear range of the molecular weight calibration curve, so that materials containing a wide range of molecular weight fractions can be separated and detected. , four or five columns, each with molecular weight calibration curves, the linear parts of which overlap.

Unfortunately, this does not result in the range of expected linear molecular weight calibration. The linear portion of the molecular weight calibration curve for the combined particles can be increased in this way, but the maximum appears to be only about three decades, which is often less than the molecular weight range found in normal sample compositions. The invention relates to a chromatograph having a resolution zone which provides a wider range of linearity in its molecular weight calibration curve and avoids the problem of using multiple columns with overlapping linear calibration. This dissolution zone according to the invention comprises a plurality of macroparticles, selected so as to provide a dissolution zone with a pore size distribution divided into at least two intervals for the pores within the macroparticles, the average pore size within each interval being such that the linear the portions of the molecular weight calibration curve for each pore size in the distribution are substantially non-overlapping and the pore volume within each range is such that the linear portions are substantially parallel. Interval means the same as the English word "mode". As used herein, the term "average pore size" refers to the volume average pore size. To achieve a maximum area for the linear part of the molecular weight calibration curve, the pore size within the intervals in the distribution should be approximately one order of magnitude apart. The use of more than two ranges of particle size distribution is not excluded, as long as two adjacent ranges have substantially parallel linear non-overlapping portions in their calibration curves, the average pore volume within each of the two adjacent ranges being separated by approximately an order of magnitude.

The macroparticles suitable for this invention can be constituted by indigestible particles, such as e.g. silica or alumina, or they may be non-digestible, such as crosslinked polymer gels. In the preferred embodiment, the macroparticles consist of difficult-to-digest macroparticles, composed primarily of silica and the component in the pore size distribution which exhibits the smaller average pore size constitutes about 30-60%, preferably about 40-60%, especially 40-55%. , and in particular about 45-55%, of the total pore volume of the macroparticles in the dissolution zone, the rest of the total pore volume being constituted by the component in the pore size distribution which has the larger average pore size. The dissolution zone may be composed of a plurality of necroparticles, each with a pore size distribution divided into two intervals, or it may consist of a plurality of macroparticles, each with a pore size distribution, combined with a plurality of macroparticles, with a different pore size distribution.

The invention also relates to an improved method of performing chromatographic separation, comprising the steps of: (a) placing the material to be separated in a carrier liquid, I (b) contacting the carrier liquid with a dissolution zone and (c) the degree of retention of the materials in the dissolution zone is determined, the method being characterized by using a chromatograph according to the invention.

The present invention is described below with reference to the following figures, in which Fig. 1 is a representative calibration curve for molecular weight, plotted on a log scale, against retention volume, Fig. 2 is a calibration curve for a dissolution zone composed of six different particles with different pore sizes, Fig. 3 is a schematic view of a liquid chromatograph, showing the insertion point 12 of the carrier liquid, dissolution zone 13 and detector 14, and showing in particular that the dissolution zones consist of 1 and 2 separate columns, Fig. 4 Fig. 5 is a representative calibration curve for a dissolution zone provided with a pore size distribution divided into two or more intervals, Fig. 5 is a schematic view with a partial section through an embodiment of a completely porous macroparticle with a pore size distribution divided into two intervals, 6 is a schematic view with a partial section through a second embodiment of a completely porous macroparticle with a pore size distribution divided into two intervals Fig. 7 is a schematic view with a partial section through an embodiment of a surface porous macroparticle with a pore size distribution divided into two intervals, Fig. 8 is a mercury penetration curve for particles, shown in Fig. 6, Fig. 9 is a comparison between a calibration curve obtained with two of the particles exhibiting non-overlapping particle size distribution, as shown in Fig. 2, compared with a calibration curve obtained using five of the particles, all having overlapping particle size distributions, as shown in Figs. Fig. 2, and Fig. 10 is a calibration curve obtained using the particles shown in Fig. 6.

In size exclusion chromatography, such a chromatograph is used, which is shown schematically in Fig. 3. A material to be separated is introduced into a carrier liquid stream at an insertion point 12 and forced under pressure through a chromatographic dissolution zone 13 to a detector. 14. As they pass through the dissolution zone, the materials in the carrier liquid are brought into contact with the packing material in the dissolution zone and are retained for a time characteristic of their molecular weight (MW). As a larger volume of carrier liquid passes through the chromatographic column, over time the material temporarily retained by the packing material elutes from the column. The detector then determines each component of the material leaving the dissolution zone. The information obtained from the detector typically consists of a peak, as shown schematically at the bottom of Fig. 4.

The dissolution zones used in size exclusion chromatography generally consist of columns packed with porous particles, e.g. such as those described in U.S. Pat. Nos. 3,505,785 and 3,855,172, or more recently the macroporous microspheroids described in U.S. Pat. No. 4,070,286.

Using such packing materials, a typical relationship between log MW (a function of the hydrodynamic radius of solute) and the retention volume (VR) is shown by the simple solid line on the left in Fig. 1. The limiting retention volume at a point A is known as the total exclusion volume, which is determined by the maximum pore size available for permeation of the dissolved materials, which is completely rejected from the material retained in the inner pores at this retention volume, and dissolved material corresponding to this molecular weight and larger is not fractionated by the system. Point B represents the volume associated with substances that completely penetrate the inner pores of the packing material, and is called the total penetration volume. Thus, materials corresponding to this MW and less can not be fractionated to any significant degree by this separation system. The difference between retention volumes A and B represents partial penetration of dissolved material, and it is within this volume range that separation occurs. The difference between the retention volume at B and the retention volume at A is a function of the total inner pore volume in the packing material. Between the retention volumes at A and B is an approximately linear range of log MW / retention volume curve (points C to D), which is described by the following equations: 7713892-3 6 ll vB 0.1 _ G2 logmw (1) and Mw =: belt-eva (2) G2 is the slope of the linear part of the calibration curve (si ml / decad-MW) and G4 is the cut-off of this linear part. Experimental chromatograms and Equation 2 are used to obtain molecular weight information from this calibration curve.

D1 refers to the intersection of this linear part of the calibration curve and D2 refers to its slope. -These equations are well known to those skilled in the art and were generally used to characterize macromolecules.

The additional properties of two identical columns used in size exclusion chromatography are well known.

As stated in rig. 1, interconnection of two identical columns (same particles, same length) with identical calibration curves, shown by the two solid lines, is equivalent to doubling the length of a single column. It follows that interconnection of these two columns increases the total available pore volume, thus increasing the retention volume range between total penetration and total exclusion, while maintaining the same molecular weight fraction range. As shown in Fig. 1, when two columns are interconnected, the molecular weight reaction range remains about 5000 - 80,000, even when the retention volume is doubled. The calibration curve for the combination of the two columns is shown by the dashed line.

The additional function which describes this relationship is: 02 = §L (0231, or (5) 132 * l / š (4) To date, general polymer fractionation has been carried out with packing material with the widest possible pore size distribution, this is usually achieved by interconnecting several columns with different Pore size to obtain a separation system covering the molecular weight range of interest Fig. 2 shows a series of molecular weight calibration curves for six different chromatographic resolution zones, each filled with porous silicon dioxide particles with different pore sizes.Designs and average pore volumes for table I below: 1 7713892-3 7 Table I Designation Pore size (AO) 1 PMS-50 60 2 PMS-300 125 3 PMS ~ 600 195 4 PMs-aoo _ 300 5 PMS-1500 '750 6 PMS-4000 3500 These particles was prepared as described in U.S. Patent Nos. 4,070,286 and 3,782,075.

The bar graphs to the right of Fig. 2 show the linear range for each calibration curve. To achieve a linear combined calibration curve, which includes a molecular weight range of 103 - 106, a combination of six columns would traditionally be used; each composed of the individual particles, which correspond to the six curves.

In this invention, the relationship set forth in Equation 3 has been utilized to improve accuracy, flexibility and convenience in the size exclusion process. This relationship predicts a previously unrecognized phenomenon, namely that to obtain a broad linear log MW / retention volume relationship, a series of columns with substantially overlapping linear molecular weight fractionation ranges or ranges (ie, linear parts) should not be used.

Instead, use columns with only two pore sizes, chosen so that the linear parts of the molecular weight / retention volume curves do not overlap. This gives a much wider linear range in the calibration curve. As shown, for example, in Fig. 4, a pore distribution divided into several areas in the dissolution zone gives a narrow linear part on the molecular weight calibration curve, and a two-partition distribution gives a much wider linear area. The calibration curve for the distribution in several areas does not include the entire molecular weight distribution of the sample within its linear range, which is the case for the calibration curve for the distribution in two areas.

The advantage of using chromatographic columns with a pore size distribution divided into two intervals, whether it is the interconnection of columns with individual pore size or the use of columns containing a physical mixture of particles having two pore sizes within the particles, can be thus seen in Fig. 4. Molecular weight calibration curves of the type shown with pore size distribution divided into two intervals are highly preferred, as the intention is to characterize a polymer with the type of molecular weight distribution illustrated at the bottom of the figure.

A quantitative comparison of a pore size distribution divided into several intervals compared with a pore size distribution divided into two intervals is given in Fig. 9. Fig. 9 shows a polystyrene calibration curve for a multi-area and for a two-area resolution zone. The set of columns used to produce the distribution divided into several intervals are filled with a packing material marked 1, 2, 4, 5 and 6 in Fig. 2. The individual columns show substantially overlapping calibration curves as in the traditional process. The approximately linear calibration range (dashed line shows the exact linearity) for this combined wide pore size distribution set is only about 2.5 decades in molecular weight. On the other hand, the division divided into two intervals, shown in Fig. 9, obtained by interconnecting columns with only two pore sizes (namely of particles 1 and 5 in Fig. 2), results in a linear molecular weight calibration curve, which more than four decades of molecular weight.

To obtain these unexpected and improved results, the individual calibration curves for the two pore sizes used in the two-interval distribution must not overlap. This is achieved by selecting particles with a suitable pore size. The average pore sizes for the two-interval distribution should be about an order of magnitude apart. In this way, linear calibration curves with up to five ccades in the molecular weight range are obtained.

A three-range device of a similar type can result in up to seven decades of linearity in the molecular weight range. In addition to non-overlapping molecular weight calibration weight curves, the internal pore volume of the two regions should be such that the linear portions of the calibration curves are substantially parallel. The term "substantially parallel" means that the shape of the linear portions of the calibration curves need not be exactly parallel, provided that some deviation from linearity can be accepted. For example, Fig. 9 shows that the overlapping multi-area calibration curve, which represents prior art, is far from linear over the expected range. When about 30-60%, preferably 40-60%, especially about 40-55%, and especially about 45-55%, of the total pore volume of the macroparticles 7715892-3 in each dissolution zone is obtained by the component in it at intervals divided pore distribution, which has the lower average pore size, the remainder being the component with the larger average pore size, the linear parts of the individual calibration curves are substantially parallel. Fig. 10 shows the deviation from linearity in the calibration curve, when the pore volume ratio is 40:60. In the most preferred embodiment, however, the pores within each range of the pore distribution should constitute about 50% of the total pore volume of the macroparticle in the dissolution zone to reduce the deviation from linearity in the calibration curve.

Best results are obtained using packing material which exhibits a very narrow pore size distribution. Pore size distributions for each of the ranges should be 1.0 or less (2o§, as shown in conventional log-normal curves for mercury porosimetry measurements. Areas of 0.5 (26) are preferred. Within these ranges, the pore size distribution is a insignificant factor in determining D2 for the calibration curve, and the internal volume of the particles is dominant in determining D2 for the calibration curve. If the pore size distribution is greater than the values given above, the relationship between the two pore size distributions plus internal volume determines the gene for the calibration curve.

The pore size distribution used in the present invention can be achieved in two different ways. The pore distribution divided into two intervals can be achieved by a plurality of macroparticles, each with such a divided pore size distribution. In this case, a single column, e.g. as shown in the upper part of Fig. 3, is used. Alternatively, the pore size distribution divided into two intervals can be achieved by using a plurality of macroparticles with one pore size distribution and a plurality of macroparticles with a different pore size distribution. Although particles with different pore size distributions can be mixed in a single column, the packing of such columns is less convenient and it is best to use two or more columns, each packed with particles of a single type.

Individual particles of the desired pore size to form the divided pore size distribution can be prepared by the methods described in the above-mentioned patents. Polymer gels, alumina and the large range of refractory particles mentioned in these documents can be used, but silica is the preferred material, especially for chromatographic separations. Particles with a pore size distribution divided into two intervals can either be completely porous or on the surface porous macroparticles. The term "macroparticle" as used herein refers to the composite macroparticle (either completely or on the surface porous) having an average diameter in the range of about 0.5 to 500 microns. The totally porous embodiment of this particle is shown in Fig. 5, the macroparticle having a average diameter of about 0.5 - 500 nm.-Preferred macroparticles have average diameters of about 5 - 50 μm. The macroparticle is composed of a plurality of microparticles 16, each having an average diameter in the range of about 0.005 - 1.0, preferably 0.005 - 0.5 μm The individual microparticles are in turn composed of a plurality of ultramicroparticles 17 having an average diameter in the range of about 1.0 - 30.0 nanometers, with 2-20 nm being preferred. 18, and between each ultramicroparticle there is a micropore 19. Although these particles, in general, can have any shape, it is preferred that they be of spherical shape, so that the macroparticles are in fact macrospheres, the microparticles are micro spheres and the ultramicroparticles are ultramicrospheres. The spherical nature of these materials enhances their properties in chromatographic columns.

Alternatively, as shown in Fig. 6, the completely porous macroparticle 15 may be composed of a core 20, comprising a plurality of ultramicroparticles 21 having an average diameter in the range of about 1-30 nanometers, and a coating composed of a plurality of microparticles 22, each with a diameter in the range of about 0.1 - 1.0 μm, or usually 0.1 - 0.5 μm. The completely porous macroparticle, prepared by currently known methods, preferably has a diameter in the range of about 0.5-50 μm.

An embodiment of a surface porous macroparticle is shown in Fig. 7. This macroparticle 22 has a diameter in the range of about 0.5 - 500, or preferably 5 - 50 μm, and comprises an impermeable macro core 24 and a coating of a variety of similar monolayers of the same colloidal inorganic microparticles joined to and surrounding the core. Each microparticle has an average diameter in the range of about 0.005 - 1.0 μm, preferably 0.1 - 0.5 μm, and constitutes about 0.2 - 25% of the total volume of the macroparticle. The microparticle may be the same as shown in Fig. 5, composed entirely of ultramicroparticles, or it may be similar to the microparticle shown in Fig. 7, composed of an impermeable micronucleus 27 and ...._..._... .í ._.._ .. .. _ ... ... ._ .. 7713892-3 11 a coating of a plurality of identical monolayers of the same colloidal inorganic ultramicroparticles 28, joined to and surrounding the core. In both cases, for the completely porous or surface porous particle, the pores between the individual microparticles in the macroparticle should be referred to as the macropore 30 and they form an interval in the two-interval pore size distribution, and the pores between the the individual ultramicroparticles should be called the micropore 31 and form the second range in the divided pore size distribution. Recently used terminology sometimes defines pores of the size referred to herein as "micropores", now as "mesopores".

Example I The preparation of scaled particles with a pore size distribution divided into two intervals is described below. Such a particle is shown in Fig. 7. 75 g of Zipax <š) (du Pont, chromatographic support) with controlled porosity, (<37 μm) was gently stirred with 800 ml of 0.5% "Lakeseal" laboratory cleaning solution for 30 minutes. the excess solution was removed by decantation and washed with distilled water.

This was repeated seven times and the resulting powder was filtered on a coarse sintered glass filter and dried in air. The dry powder was then placed in a 7 cm diameter funnel with coarse sintered glass and treated with 100 ml of 0.5% Zelec (š> DX (du Pont antistatic agent and release agent) in solution for 5 minutes with stirring. the treated beads were filtered, then washed twice with 200 ml of distilled water and dried in the funnel with vacuum.

The beads were then treated with 100 ml of 10% silica sol, produced by Ludox <É) AS (colloidal silica from du Pont, ~ / 140 Å silica particles) consisting of 125 g of 30% by weight silica in Ludox (ï) AS diluted to 400 g with distilled water. The mixture of beads and silica was allowed to stand for 15 minutes in the funnel with occasional gentle stirring. Excess Ludox was then filtered off and the resulting wet cake was washed four times by gentle slurry with about 400 ml of tap water and filtration. The cake was then allowed to air dry in the filter under vacuum. This material was then dried at 150 ° C for one hour in a circulating air oven and a small sample was removed for filtration.

The above-described Zelec R DX silica sol treatment 73777113 8192-.3 12 was repeated three more times to build up a shell of the silica ultramicroparticles having a diameter of 140 Å on the surface of the silica microparticles having a diameter of 2000 Å, which originally constituted the shell of the Zipax R particles. The finished particles were dried and heated at 650 DEG C. for two hours to burn off the organic intermediate layer and sinter the particles to a mechanically stable state. This sintered sample was then allowed to stand for two hours in a large excess of 0.001 M ammonium hydroxide with occasional stirring. The particles were then washed twice with a large excess of distilled water by decantation, filtered on a coarse sintered glass funnel, air dried and heated at 150 ° C for two hours in a circulating air bath. The finished material was sieved dry with stainless steel sieves to obtain a fraction <38 μm weighing 45 g.

The surface area of the products obtained during the manufacturing steps was obtained by the nitrogen flow method with the following results: 2 _ Sample Surface area / q (I) Starting material Zipax 0.89, 0.99 First treatment with 7 Lua0x®1is _ 2.03, 2.09 Second treatment with ® in Ludox AS 2.35 2.46 Tred] e.behÉïdling with R) Ludox AS 3.07 3.01 Fourth treatment with Ludoxßbxs _ 3.33 3.50 Sintered at 650 ° C for _ two hours 2, 67 2.67 Final rehydrated material 2.85 2.36 'A mercury porosimetry measurement of this sample showed three interruptions in the mercury penetration curve, one at about 10lpm, representing the penetration of mercury between the individual particles, an interruption at about 0lpm. 07 μm (700 Å), which represents the macropores between the solar microparticles in the shell of the original Zipaxíg) structure, and a break at about 0.006 μm (60 Å), which represents the pores between the 140 Å solar ultramicroparticles, 7713892- 3 13 which are present as several layers on the original Zipax <š> structure through the n the procedure set forth herein. The volumes associated with the pore size distribution divided into two intervals were: Macropores - (700 Å pores) - 0.011 cm3 / g Micropores - (60 Å pores) - 0.014 cm3 / g These data show that the finished particles contained the desired pore configuration, with pores about a decade in size difference, and about equal pore volumes for each pore size.

Example II Particles of the type shown in Fig. 6 can be prepared as follows: 15 g of porous silica microspheres (PSM-40, 47 Å pores) prepared according to U.S. Patents 3,782,075 and 3,855,172 were treated. with 200 ml of 0.001 M ammonium hydroxide, allowed to stand for 10 minutes with occasional stirring and centrifuged in a 250 ml polyethylene flask for two minutes (from the start) at about 2000 rpm. The clear supernatant was decanted off and to the wet cake was added 100 ml of 0.5% solution of Selec DX (from du Pont de Nemours and Co.), which had been adjusted to pH 7 with ammonium hydroxide. The system was carefully slurried and then allowed to stand for 10 minutes with gentle stirring occasionally. The resulting mixture was centrifuged for one minute using the above method and the excess Zelec DX solution was decanted off. The wet cake was washed twice with 200 ml of distilled water (adjusted to pH 7 with ammonium hydroxide) by careful slurrying, centrifugation for one minute and decantation.

To these treated particles was added 50 ml of 5% by weight 2000 Å silica sol mixture adjusted to pH 8 (the sol can be prepared by procedures described in W. Stober, A. Fink and E. Bohn, J. Colloid, Inter. Sci., Gg, 62 (1968)) and the mixture was carefully slurried and sometimes stirred for 10 minutes. This mixture was centrifuged as above, decanted and excess sun was retained. The coated beads were then washed twice with 200 ml of distilled water (pH 7) by slurry, centrifugation and decantation. The second wash water from the decantation was ready.

The wet cake was then filtered on a 3 μm "Nuclepore" filter and dried in a circulating air oven at 150 ° C for two hours.

The sample was then annealed at 700 ° C for one hour in a muffle furnace. 77130892-3 14 A small part of this material was subjected to scanning electron microanalysis, which showed an excellent coverage of the surface of the original beads with the 2000 Å silica sol. No bare spots could be seen on the particles. A second layer of 2000 Å of silica sol was placed on the PSM particles using the technique described above, after the annealed silica particles were first hydrolyzed in 0.01 M hydrochloric acid overnight and elimination of the acid by washing with distilled water. The material was again treated with Zelec R DX, followed by 2000 Å of sol (25 ml of new substance plus the recovered sol excess from the first treatment) as described above. Examination of this material by scanning electron microscopy showed that the second layer was coated as desired. Very few stains could be seen on the beads, and a very small amount of particle bridging could be noted.

A third, fourth, fifth and sixth treatment of the beads was performed in substantially the same manner as above to build up the desired shell of 2000 Å of silica sol particles on the particles.

These treated beads were annealed at 75,000 for one hour and hydrolyzed again by treatment with dilute acid, as above. SEM examination of the finished beads showed good coverage, but it was not possible to observe the exact thickness of the desired, surface-porous shell.

Mercury penetration measurements (see curve in Fig. 8) show that the pore volume of the larger pores for these particles is about 40% of the total pore volume, and the pore volume of the smaller pores is about 60% of the total volume. Log molecular weight / retention volume calibration curve for a 25 x 0.62 cm id column of these particles is shown in Fig. 10. Due to the differences in internal volumes associated with the two regions, a certain deviation from linearity.

Claims (26)

7713892-3 Patent claims A method of chromatographic separation, wherein (a) a material to be separated is placed in a carrier liquid, (b) the carrier liquid is contacted with a dissolution zone. and (c) the degree of retention of the material in the zone is determined, characterized in that a dissolution zone is used, which comprises a plurality of macroparticles. which macroparticles are selected so that the dissolution zone obtains a pore size distribution divided into at least two intervals for the pores within the macroparticles, the average pore size within each interval being such that the linear portions of the molecular weight calibration curve for each pore size are within the two interval sizes substantially non-overlapping and the pore volume for each interval is such that the linear portions are substantially parallel. 2. A method according to claim 1, characterized in that the pores in the pore size distribution are divided into two intervals. which have a smaller average pore size. constitutes about 30 - 60 t of the total pore volume, and that the pores with a larger average pore size constitute about 70 - 40 t of the total pore volume of the macroparticles in the dissolution zone. 3. A method according to claim 2, characterized in that the pores within each of the two ranges within the pore size distribution constitute about 40 - 60 2 of the total pore volume of the macroparticles in the dissolution zone and that the average pore sizes within the two intervals differ by about an order of magnitude. 4. A method according to claim 3, characterized in that the pores in the pore size distribution divided into two parts are divided. which have the smaller average pore size. constitutes about 40 - 55% of the total pore volume and that the pores with the larger average pore size constitute about 45 - 60 t of the total pore volume for the macroparticles in the dissolution zone. 5. A method according to claim 4, characterized in that the pores within each of the two intervals within the pore size distribution constitute about 45 - 5% of the total pore volume of the macroparticles in the dissolution zone. 6. A method according to claim 4, characterized in that the macroparticles have an average diameter of about 0.5 - 5007 μm and are composed of a plurality of microparticles with a diameter of cafO; 005 - 1; 0, and a plurality of ultramicroparticles having a diameter of about 1 to 30 nm. each macroparticle exhibiting a pore size distribution. divided into two intervals. 7. a method according to claim 4, wherein the macroparticles have an average diameter of about 0.5 - 500 .mu.m and: are composed of a plurality of microparticles with a diameter of about 0.005 to 10 .mu.m). The pore size distribution in the zone divided into two intervals is achieved by a plurality of macroparticles having an average pore size within a range of the distribution and a plurality of macroparticles having an average pore size within the second range of the distribution. 8. A method according to claim 6, characterized in that the macroparticles are completely porous macroparticles with an average diameter of about 0.5 - 50 .mu.m and are either composed of a plurality of microparticles with an average diameter of 0.005 .mu.m. 0.5 / microparticles with a diameter of about 1 - 30 nm. or is upp- um. which in turn are composed of ultra-built of a porous core composed of ultramicroparticles with an average diameter of about 1 - 30 nm surrounded by a porous shell of microparticles with an average diameter of about 0.005 - 0.5 / um. 9. A method according to claim 6, characterized in that the macroparticles are porous on the surface and have an average diameter of about 5 to 50 microns and are composed of an impermeable macronucleus, surrounded by microparticles having a diameter of 0. , - 1 - - 0.5 / um, which in turn are composed of ultramicroparticles with a diameter of about 1 - 30 nm 7713892-3 17 10. A method according to claim 7, characterized in that the macroparticles consist of completely porous macroparticles with an average diameter of about 0.5 - 50 / um and are composed of a plurality of microparticles with an average diameter of about 0.005 - - 0.5 / um. 11. ll. A method according to claim 7, characterized in that the macroparticles are porous on the surface and have an average diameter of about 5 - 50 .mu.m and are composed of a macro core. surrounded by microparticles with a diameter of about 0.1 - 0.5 / um. 12. A chromatograph for chromatographic separation, characterized in that its dissolution zone comprises a plurality of macroparticles. which macroparticles are selected so that the dissolution zone obtains a pore size distribution divided into the pores within the macroparticles divided into at least two intervals, the average pore size within each range being such that the linear portions of the molecular weight calibration curve for each pore size are non-distributive. and the pore volume for each range is such. that the linear parts are substantially parallel. 13. A chromatograph as claimed in Claim 12, characterized in that the pores in the pore size distribution divided into two intervals, which have a smaller average pore size. constitutes about 30 - 60 t of the total pore volume. and that the pores with a larger average pore size constitute about 70 - 40 l of the total pore volume of the macroparticles in the dissolution zone. Chromatograph according to claim 13, characterized in that the pores within each of the two ranges within the pore size distribution constitute about 40 - 60 3 of the total pore volume of the macroparticles in the dissolution zone and that the average pore sizes of the pores within the two ranges differ from each other by about an order of magnitude. 15. A chromatograph according to claim 14, characterized in that the pores in the pore size distribution divided into two intervals 7713-892-3 18. which show the smaller average pore size. constitutes about 40 - 55 2 of the total pore volume and that the pores. which has the larger average pore size, »constitutes about 45 - 60-% of the total pore volume of the macroparticles in the dissolution zone. A chromatograph according to claim 15, characterized in that the pores within each of the two ranges within the pore size distribution constitute about A5 - 55 2 of the total pore volume of the macroparticles in the dissolution zone. Chromatograph according to one of Claims 12 to 16, characterized in that the macroparticles have an average diameter of about 0.5 - 500 .mu.m and are composed of a plurality of microparticles with a diameter of about 0.005 to 1.0 .mu.m. and a plurality of ultramicroparticles having an average diameter of about 1 - - 30 nm. each macroparticle having a pore size and distribution divided into two intervals. 18., l8. A chromatograph according to claim 15, characterized in that the macroparticles have an average diameter of about 0.5 - - 500 / um and are composed of a plurality of microparticles with a diameter of about 0.005 - 1.0 / um. wherein the pore size distribution in the zone divided into two intervals is achieved by a plurality of macroparticles with an average pore size within one range and a plurality of macroparticles forging an average pore size within the second range. 19. A chromatograph according to claim 17, characterized in that the macroparticles are completely porous macroparticles with an average diameter of about 0.5 - 50 / um and that they are either composed of a plurality of microparticles with an average diameter of 0.005 - 0.5 / um. which in turn are composed of ultra-microparticles with a diameter of about 1 - 30 nm. or are composed of a porous core of ultramicroparticles with an average diameter of about 1 - 30 nm and a porous shell of microparticles with an average diameter of 0.005 - 0.5 / um. 7713892-3 19 Chromatograph according to claim 17, characterized in that the macroparticles are porous on the surface, have an average diameter of about 5 - 50 μm and are composed of an impermeable macronucleus, surrounded by microparticles with a diameter of 0.1 - 0, 5 μm, which in turn are composed of ultramicroparticles with a diameter of about 1 - 30 nm. 21. A chromatograph according to claim 18, characterized in that the macroparticles consist of completely porous macroparticles with an average diameter of about 0.5 - 50, um and are composed of a plurality of microparticles with an average diameter of about 0.005 - 0, 5 fun. Chromatograph according to claim 18, characterized in that the macroparticles are porous on the surface with an average diameter of about 5 - 50 μm and are composed of a macro nucleus, surrounded by microparticles with a diameter of about 0.1 - 0.5 nm . Chromatograph according to one of Claims 19 to 22, characterized in that the macroparticles are composed mainly of silica. A chromatograph according to claim 17, characterized in that the macroparticles are on the surface porous macroparticles with an impermeable core and a coating of a plurality of similar monolayers of colloidal microparticles joined to and surrounding the core, the microparticles having a diameter of about 0.00 1.0 μm and constitutes about 0.2 - 25% of the total volume of the macroparticle. Chromatograph according to claim 17, characterized in that the macroparticles are completely porous with a core of a plurality of ultramicroparticles with a diameter of about 1 -30 nm and a coating of a plurality of microparticles with a diameter of about 0.1 - 1.0 Fm. Chromatograph according to claim 24 or 25, characterized in that the macroparticles have a diameter of 5 - 50 μm and the microparticles have a diameter of 0.1 - 0.5 μm.