CA1214398A - Method for a new type of chromatography and device therefor - Google Patents

Abstract

METHOD FOR A NEW TYPE OF CHROMATOGRAPHY
AND DEVICE THEREFOR
ABSTRACT OF THE DISCLOSURE

The present invention relates to a new type of chromatography technique, referred to as dynamic columm chromatography, for separation of one or more compounds present in a solution, which is characterized by the existence of a moving solid adsorbent bed. According to the invention, the chromatographic system comprises a piston having at its bottom a sealing element and a longitudinal channel containing the adsorbent between two barriers and a test tube having at its bottom a multiple way valve. By pushing the piston into the test tube, the desired eluant which was prior forced through said valve is entering under intrinsic pressure of the closed system through the channel moving the adsorbed compounds to be separated between said barriers, the solution obtained going out through a nozzle located at one of the end parts of the piston. The dynamic column liquid chromatography is applicable to: silica gel chromatography, reversed phase liquid chromatography, affinity chromatography, capillary chromatography, chromato-focusing, gel filtration and ion exchange chromatography.
In the dynamic column liquid chromatography, the equilibrium distribution of the compounds between the adsorbent and liquid is established very rapidly, resulting in sharp and narrow zones of the separated fractions.

Description

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The present invention relates to a new me-thod of chromatography hereafter referred to as Dynamic column Liquid Chromatography (DCLC) for separating two or more components. More particularly the invention relates to a new method for separation of two or more compounds present in a solution using a moving bed chromatographic system.
As known, chromatography is a term that describes a number of physical methods used in chemistry and biology to separate and identify mixtures of chemical compounds.
The principle behind all chromatography variants lies in the repeated subjection of a mixture of chemical compounds to extraction by liquid or adsorption on a solid surface. The mixture is moved physically over a stationary phase (bed or column), which may be either a solid or a liquid immobilized in the pores of a solid (located in said bed or column). The separation of chemical compounds by chromatography may make use of one or more of the following phisico-chemical forces, depending upon the particular chromatographic system:
(a) Differences in adsorption to the porous medium, so called sorbent.
(b) Differences between the relative solubilities of a liquid coating the inert medium (stationary phase) and the liquid, called mobile phase, percolating through the porous column.
(c) Differences in ion exchange with the sorbent.
(d) Differences in molecular size as the solution percol-ates through a gel of very small size.
Chromatography is also named preparative chromatography when it is used for isolation of a fraction from a mixture for further uses such as spectroscopy, identification, synthesis for research or commercial purposes.
The original work on chromatography is based on differences in adsorption over an inert material packed in a column. The separation of components, known also as partition chromatography is based on the relative solubilities in the solvent which is passed over the column. The resolution obtained in this chromatography ..

3~13 depends upon the pH and ionic strength of the solvent - the mobile phase - and the relative solubilities of the constituents in the two phases; the various materials may be eluted with an appropriate solvent and the liquid fractions collec-ted in a series of tubes and subsequently analysed by chemical or physical methods. Thin layer chromatography (TLC) and paper chromatography, are based on differences between the relative adsorption of a component onto an inert medium. In TLC, the stationary phase consists of a thin layer of a finely divided substance applled to a sheet or plastic backing or to a glass plate. Sorbents commonly used, and commercially available as finished plates, include alumina, silica gel and cellulose. In paper chromatography, -the mobile phase may move upwards by capillary action, so called ascending chromatography, or downwards by gravity, so called descending chromatography.
Ion exchange chromatography, involves the separation of molecules based on their ionic charge.
The sorbent or stationary phase, consists of polymers with covalently bound ions. In cation exchange resins, the tightly bound ions are negatively charged and are associated with positive ions that are loosely attached by electrostatic charges. The positively charged substances to be separated from a mixture are first adsorbed to the sorbent, displacing the cations present in the resin.
The solution is buffered at a pH that will facilitate the binding and then eluted with the same buffer to remove the non-binding fractions of the solution. An anion exchanger operates in exactly the same way, except that its covalently bound ions are oppositively charged to attract the anions from the solu-tion.

.,,~,. j , 3~

The separation based on differences in molecular size is encountered in gel filtration also known as molecular sieve chromatography. This method separates molecules according to their size, although the shape of the molecule affects the filtration to some extent. The gels are in the form of beads containing a network of openings of pores in which small molecules may be entrapped. The vast commercial interest in chromatography in general and preparative liquid chromatography in particular, is mani-fested by the large number of publications suggestingvarious microparticulate column packings and prepacked columns claiming to obtain better separation than the known adsorbents used in this field.
The inventors of the present invention have concentra-ted their research in developing a new concept for chrom-atography in which known adsorbents are utilized, but the separation is very fast and more easily conducted. The new concept of the chromatography according to the present invention, is to u-tilize a dynamic column in which the bed with the adsorbent is moving in contrast to the conven-tional chromatography technique wherein the adsorbent material is stationary.
In accordance with one aspect of the present invention, there is provided a method for separating separable compon-ents contained in a liquid sample by a chromatographytechnique referred to as dynamic liquid chromatography, which comprises the steps of (a) absorbing the separable components of the same cnto a movable chromatographic column comparing at least one dense-packed adsorbent bed, the column having an inlet end and an outlet end and being referred to elsewhere herein as a piston having a longitu-dinal channel containing the adsorbent, (b) placing the inlet end of the column into a container defining an elon-gated passage adapted to accept the column in a formfitting manner, the passage containing a predetermined amount of an element, the container being referred to elsewhere as a test tube; and (c) moving the column in the passage through the eluant.

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- 4a -In accordance with another aspect of the present invention, there is provided a device for dynamic liquid chromatography for separating separable components contained in a liquid sample, which comprises (a) a movable chroma-tographic column comprising at least one dense-packed adsorbent bed held between two barriers and a nozzle located at one of the ends of the column, the column being referred to elsewhere as a piston having a longitudinal channel containing the adsorbent; and (b) a container defining an elongated passage having a sealing element and a multiple way valve at its lower end, the elongated passage being adapted to accept the column in a formfitting manner, the container being referred to elsewhere as a test tube.
Thus the invention consists in a method for dynamic column liquid chromatography (DCLC) for separation of one or more compounds present in a solution using a moving solid adsorbent bed. A piston having at its bottom a sealing element and a longitudinal channel containing an adsorbent held betwen two barriers and a test tube having at its bottom a multiple way valve are provided with the piston snugly fitting into the test tube. By pushing the piston into the test tube, the desired eluent which was prior forced through the valve is entering through the channel moving the adsorbed compounds to be separated between the barriers, the solution obtained going out through a nozzle ,. ,~ ., ~2~3~3~

located at one of the ~Id parts of the pistDn. The goneIal case which seems to be more frequently encounter~d is the us~ of a ~olid adso~b~nt ~olu~n or bed, as adsorbent zone, i~ whioh caso th~ ~othod will compete most ~avourably with ~he conY~ntional chr~t~graphy techniques. The method is very accurate and has the following main ad~a~tages ove~ the conventional chromatogxaphy :

(a) In cont~ast to the gravitational flow which exis~s in th0 con~entional chromatography, in the dyna~ic columm liqui~
chro~a~o~raphy some pressure is mherently axe~tod in the adsorbent bed9 which impar*s a better resolution in the separatiOn o the constituents.

~b~ The method is very rapid, also as a ~osult o~ th~ intrmsic pressure exerted in ~he systeDI.

tc) Ihe method recluires less eluent than i~ the con~entio~l ~h~omatogl~aphy.

~d) Ihe presence of intrinsic pressure in the dyna~ic chl~omatogr~hy syste~ enables ~o utilize an adsorbent with smaller p~rticl~s size than in tha conventional chromatography, which enab~s a higher sensitivity.

20 Ihe ~ltiple way valvç is ve~ portant when several consecutivs elu~ntS h~ve to be introduced in the t:est tube, being further p~ssed through the adsorbent bed thus obtaining the desîred ractionati~n.
When the adsorbent bed is utilized mainly for pretreatm3nt o a sE~ple, - or for remo ring of one coD~pound, the valve requirement is not ~dato~y and a simple test tube closed at the bottom~ fitted to the pis*on may be utilized. In this ~ase the eluent ~hould be introduced i~to ~he tsst tube prior to pushing down the pi~t~n. Of course even when no fractionation is required, it s80mS ~hat the test tube with a valve will be more advantageous to be utill~ed, conce~ning washing the adsorbent and introduction of the elu~nt by its aspiration thTough the valve.

The sealing element slides along the inner walls of the t0~t tub~, at the same ti~e staying in good con~act with ssid in~lor w~lls p oxmitting a snugly fitting o the piston in$o the t~st tlibo. l~o sealing element will gerlerally consi~t of a ruWer or other suitablo 5 materi~l O-r~ng having an ori~ice bore and adapted to $1ide along the i~ner walls OI the test tube. I~!hen th~ devico will be ~de cf glass, the polish on the outer wall of the piston ~y b0 accordingly manufactured so that it could replace ths O-ring.

l~e entira mcthod is Yery flexible and c~uld be ~plied in a 10 larga nuJnber of applications with various embsdime~ts~ aEld will b~
thsrefore included in the concspt of th9 pr~sent inv~nt~rl.
A problem which exists in all kinds of chr~matography, îs a~ ~von application of l:he san~ple to the surface of the bed. ~h~ he S a~pl9 is applied directly by ~ravitational flow, an ~v~n ~ppllcation may be ralatively difficult, as the bed has ~ tend0ncy to whirl up when ~he sample is introduced. For this reaso~ it is particularly important to have the surface p~otec~ed sush as a piece of rayon ~ilter paper. One manufacturer of col~ (Phar~acia Pine Chemicals AB) equips some of the columns wi~h a speci~1 devicc called sample applicator, tha~ serves ~o protect th~ bed surface.
In this device, a thin nylon fabric is mounted at the ~d of a short piece of perspex tubing itting inside the chr~m~tographic tube. Such device increases of course the costs of tho ~quipm~nt in addition ~o the disadvantage that its presence causes pr~ssur~
t o ~h~ flow of the sample slowing down its p3rculation thr~ugh the column. In most of the chromatography syst~s utilizing the pr~sent invention, the problem o an even application i~ much all~viated.
In the dynamic flow which exists in the chxoma~ographic bed; the liquid being forced upwards upon pushing the ~tiston into the test ~ube, no whirl tendency will exist due to sample introduction.
- M oreover, the pressure exerted in the syste~ from pushin~ the p iston into the test tube, will accelerate the ~low of the s~pl~.
A nother approach to assist an even application of the sumpie, is to incorporate above the bed another suitable adsorbent different from the adsorbent already present in the bed, having the role of preconcentrating the sample and thus assisting to get a narrow band of resolution. A typical example of such a suitable adsorbent is rough silica, which differs from the active silica with its high adsorbent properties.
The particle size and the particle size dis-tribution must be carefully controlled in most of the conventional chromatography operations. As known, a bed consisting of small particles will generally give good resolution. The reason is that the mechanisms that give rise to zone broadening are amplified, as the particle size is increased. With large particles, diffusion in and out of the particles takes longer. The flow pattern in a bed of large particles is inferior, giving rise to more remixing. On the other hand, the resistance to flow in a bed packed with large particles is lower and the maximum flow rate that can be attained is higher. Thus in the conventional chromatography operations a compromise with respect to particle size should be reached, giving maximum zone resolution under the flow conditions required.
In the new DCLC method according to the present invention, small particle size of adsorbent can be utilized without incurring the disadvantage encountered in the known methods, the small pressure inherently exerted in the system does overcome the resistance to flow raised by the small particle size of the particles. Thus this method can be utilized even for critical fractionation purposes when the use of a finer grade material will be mandatory in order to obtain the desired resolution.
In one of our prior patent applications (Ger.
Offen. 3126926.5) a new method of mass transport and separation through selective barriers was disclosed using a device having similar components as in the present invention. As mentioned therein, the device consists in a mixer-separator which possesses a membrane and a mixing reservoir into which said mixer-separator is pushed in.
On the mixer-separator there ,~`'` 1 3 2~l~3~3 B

are means for accuL~lating of an air pockek 'co decraase the pressure ocerted on the me~rane. During operation of the mixer-s0parator, a dets~rmined amolmt of air is ent~apped in tho air pock~t,which 1lpon coD~preSsion acts as a cushion or shock abso~er to tak0 ~p part of s the pressu~ r~sulting from the membrane re5i5ta~1S:e~ to th~ liquit flow. For the d~namic col~Dn liquid chromatography accor~g to ~o p resent invention, the air pocket requirement ~h~ be con~idered 1 ess mandator~r than in the previous case. Ho~rsYer for coTtain syst~ms wherein relative high pressures will be involved, the air pocket seems to have an important ~ole, the erltrapped ~mount of ~ir foroing back into the ~est tubo any liquid which may h3Ye creeped up in the spaco b~tween the inner walls of the tost ~ube and the outes walls of the terminal end of the piston~ The a~ount of air entrapped by said means on the piston will depend on many actors su~h as the type of barrier, constituents o the ~ixtures to b~ separate~
and the particular conditions exerting in the specific chro~atographic syst~m. A particula~ advantage to utilize such an air pocket is .~ the case whsn it will be required a c~mplete a~oidanco of tho eluent with an 0-ring located at the bottom of the pistoa having tho ~ole of sealing elea~nt.

Il e method according to the present invention can be s ucccssfbllY utilizad in the various areas of chro~atography:
s ilica gel chromatography, re~ersed phase liquid chromatography, c apillary chromatography, affinity chromatography, chromato~ocusing, size-exclusion chromatogTaphy ~also known under ~he naee gel : filtration) and ion exchange t~hromatography A
Silica gel chroma~ography, is one of the most common c hromatography methods. Silica gel being by far one of the best k~own adsorben~ being ralatively in~xp~nsi~e ¢ompared with other 30 ~ ~aterials. The separa~ion results ebtained with silica gel in ~he mathod according to the present invention, are substantially the same 3~

or better conc~rniJIg accu~acy of separatiQn and recovory yields to those obtainad in the conven'cional techniqu~s" but i-~ mor~ CoqlY~Iie~l~
by being faster and requirillg also less sol~ont. It aï30 has a particular ad~rantage that the coïunn can be re-usable. In aWition 5 s maller size silica gel particle5 aJld densor packi~g can be used with the resulting adYalltages o:E higher separation.

Revarsed phase liquid ~omatography is cha~acterizod by the fact that its stationary phase is less polar than t~ nobile phase.
The main drawback with silica ~el is tha~ only a partial recvvc~y of 10 the co~pounds passing through such a bed could be r~coveIed. In view of the a~v~ntages of the DCLC according to th~ prese~c inve~tion, t he re~rersed phase liquid ch~o~atograp~y could be also succ~3ss~1ly utilized in prepara~ive chromatography. Recen~ly, c~pilla~y chromatography received ~ore interest particularly in vi0w of 15 develo~ments in microcolumns for high pe~for~ance liquid cllromatography. The Teason or these devel~p~ents li~s in ~he follow~ng advantages o this type of aromatography:
( a) ~Qt~ntial achie~rement of greater separation eiE~icienees for con~plex D3ixtuTes and hard to resolve solutes.
20. (b~ A substalltial decrease in sonsu~ption of eluent.
The DCLC method arcordin to the present invention could easily bo ap~lied for capillary chromatography providing a nar~ow charmel in ~he piston previo~sly described.

Gel filtration, also bnQwn as size-~xclusio~ chromatography is 25 r~ceiving more and more interest in the purificatio~ of bi410gical s ubstances using an adequa~e adsorbent as sepa~ation media. Good r esults were obtained in t:he separati~n:of labelled I~dine- hCG
from labelled Iodine using a Sephadex G-type (produced by Phar~acia Fine Chemicals, Sweden) adsorben~, using th~-~ynamic chromatcg~aphy according to ~he present invention, ~See Ex~mple 3). ~al ~ ration c hromatography is also considered as a si~ple and ~apid method for desalting or change of buf~er. The gel bed shoul~ ba equilbrated b ef~e the experiment wnth a solution with the ionic compositicn that is desired, for example distilled water is case of desal~ing. The ~ f ~ ~ O ~

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lV

e lution is parformed wi~h the saJna liquid. In Yi~W of 1:he high ~at~s which may be in~rolved, the whole ~peration may be coq~pleted in a sho~t period of time collecting tho desalt~d material in a f~w minutes. Other areas ~or gel filt~ation chroma~ography envisaged 5 with th~ dynamic chromatography ~ill be pretr~a~aeJIt bafo~e HPLC
and concentration of diluted san~ples follo~ed ~y sepa~atiorl.

Chromato-focusing is largely used for separating proteins aceording to their isolcctric points. Sinee chromato-0cusi1lg produces ~xtremely narrow bands of sepa~a~ed ~aterial, a~d reqviros g~erally 10 1 ong narrow columns, it appears tha~ th~ dyn~c col~- liquid chromatography will be ideal ~or this type of chro~at~g~ap1~y9 providing a narrow piston for the device d~scribed befo~e.

The DCLC is also convenient ~r ion exchan~o chrs~atography, w ell known as one of the most popular sepa~ation tec~Qiques.
Se~eral experiments were p~ormed for separati~ copper sulfate and sodi ~ bichra~ate on Dowex 50 WX 8, as adsorb~nt. (See Bxanpl~ 2~.
^ It was ~ound that substantial advantages in texm of tiEe, s~lv~nt Yolumo and convenienco could be aohieved by usi~g th~ DCLC.

Another advantage of the DC~C according to th9 pre~9a~
invention9 is a substantial decrease of the deat Yol~e. As kno~n dead volume i5 de~ined as the volume of the liquid in tho intexstitial space between the grains of the adsor~ent in the b~d. In most of t he conventional chro~atography aperations, the d~ad ~olume c onstitutes a problem which afec s 1~e det~nation of an accurate 25 result. In the DCLC, because dense packing is possi~le, the oquilibrium distribution of the s~bs~ance between the adsorbent and - :1 iquid is established very rapidlr with ve~y low dead ~ol~e.
Aecordingly, it will be possible to obtain ~ p and r~ w z~nes.
This is very important in ~ractionations experimonts wher~in the 30 differenceS in elution lrolume between the s~b5t~ces is genar~lly small. In particular or gel filtration l~ge dead y~ s will impair the ~esolution obtained.

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~ 11 According to another embodiment, the adsorbent is present into a cartridge l~hich is ins~rted into the lon~i$udinal channel o the piston. In this manner, the chromatography device ~ill b e ready for use ~or many purposes only by replacing the cartridge S by one containing the suitable adsorbent. Figur~ 5 attac~ed to ~he specification illustrates this embodiment. The method is very simple and its versatility could be men~ioned a ng its v arious advantages. ~here are many ~mbodiments which can bs envisaged for the device utilizing the method according to the p resent invention. Some of these embodim~nts are presenked hereafter with the attached Figures 1 to 9, being u~erstood that these are given only for a better understanding o the inven~ion without being limited thereto~
In Pigure 1, thc test tube (R) is equipped with a Luer 1 ock ~L) to which a three way valve (LV~ is attached. ~he desired eluent (~) is forced into the test tube ~). The piston (~ has a longitudinal channel into which the adsorbent tP~ is located being held by the two membranes ~l`,F2) at the top and bottom of the piston. Above the upper membrane (Fl~ there is a stopper (S) provided with a nozzle (D) through which the s eparated fraction from the adsorbent bed is collected. At the lower part of the piston there is an 0-ring (O), which has the role of sealing being adapted to slide along the inner walls of the test tube ~R~.

In Fi~ure 2, no valve exists at the bottom of the test tube (R), a limited amount of the chosen eluent ~B) being introduced from the b eginning in the test tube (R). The piston (C) possesses the longitudinal channel in which the adsorbent (P) is located held by the two menbranes (Fl,F2). Above the upper membrane (Fl)there is a stopper (S). The nozzle (D) through which the s~para~ed fraction from the adsorbent bed is collected, is connected to the pist~n (C).

A t the lower part there is ~he 0-ring (0) as a sealing elemsnt.
This device could be utilized when no fractionation is required, ~he operation consisting of only one cycle with a single elusnt.

In Figure 3, the test tube (R) is exactly as in Figure 2, S w ithout a valve at its bottom. The piston (C) p~ssesses the 1 ongitudinal channel into which the adsorbent (P~ is located b eing held by the two membranes ~Fl,F2). Above the upper membrane (F2) there is a stopper (S~ provided, with a nozzle ~D) through which the separated fraction from the adsorbent bed is collected. At the bottom of the piston there is the 0-~ing ~0) as a sealing element.

In Figure 49 there is shown the simplast form of the device also without a valve at the bottom of the test tube and stopper at the top of the piston. A limited amount of the chosen eluent (~ is introduced from ~he beginning in the test tube (R). The p is~on (C) has a longitudinal channel in which the adsorbent (P) is present being held between the two barriers - m~mbranes or filters - (Pl,F2). The 0-ring ~O)~is located at the lower part o the piston and has the role of sealing and being adapted to s lide along the inner walls of the test tube (R). Connected to the channel with the adsorbent (P) there is a nozzle (D) through which ~he separated fraction is collected.

In Figure 5, the method is illustrated wherein a cartridge (CA) - containing the desired adsorbent (P) is introduced into the longit-u dinal channel (I) of the piston (~ . The collection of the eluent can be done through a no~zle as described in the previous Figures.
Tha 0-ring sealing tO) is present at the lower part of the piston ~C).
The mode of operation is very simple as wirl be heraaft~ described in conjunction with Figure 1. ~he piston (C) is pushed down in the t est tube ~R) wllich is filled with the chosen eluent (~)~ This will cause the eluent ~o be ~orced through the lower membrane (Fl) then through the adsorbent ~P) present in the longitudinal channel of the 3~
- ~3 p iston (C) and finally drip khrough ~he nozzle (~ (in Figure 4~ .
lYhen filled with a suitable suppo~t ~aterial ~P~ it will ~ct as a chro~atography col~m. Refilling of the ~est tube (R~ is effected sin~ply by ~locking ~e test tlibe outlet and forcing Dlore 5 eluent through 'ch~ valve (LV3 ~in Figure 1)~

In Figure 6, there is shown an embodiment of the dynamic colwml in which the eolumn piston tC~ moYes ~pwards into th0 test tube (R) and the exit of the eluent is through a ~rerti~al na~row channel tX~ in direct continuation of th~ colu~m chromatog~aphic 10 support material. Pigure 7 is a ~odification of the d~i~c colullm of ~igure 6, whereby a column solvent reservoir is ~oDnect~d (Y?
~o the dynaD~ic col~imn.

Figure 8 illustrates an embod~ent which shows the pisto~
(C~ of the dynamic colwmls consisting of two or more slibunits, eac~
one containing the same or different adsorbents (P~ with the possibility of collectin~ the eluent resu1ting rom cach subunit.

Figure 9 illustrates another cmbodiment which sho~s the versatility of the DCLC whereby the utility can be further extended, according to this embodiment, tha exiting eluent is con~syed to another column whi~h contains tha sa~e or different adso~bent (P).

Figures 10 to 12 represent in a graphic form t~resul~s of the separation for various maxtures-as described in ~xa~ple~
4, 5 and 6. Pigure 13 represents a schema~ic graph or a~fini~y chromatography of IgG isol~tion as dos:cribed in Exan~ple 9.
:;
In principle the dynamic chromatography can be envisa~ed t o be utilized also in liquid ion exchangers.~ Liquid ion exchangers are defined as liquid-liquid extraction sys~ems tha~ operate, a~ :

43~3~
1~ -lczst foxmally, by interchange of ions at the ~nt~r~ac~ between an ~queous solution and an immiscible ~olvant with negligibl~
dist~ibution o the extractant to the aqueous phase. Liqw d a~ion-ex~hangers are used in reversed-pha3e ext~ac~ion chro~atography~
In this technique, ~he support material tsilica gel, c~llulos~
powder etc.~ impregnated with the liquid anion-exchanger, i~ used as the stationary phas~ and an aqueou~ solution of aQ scid or one of its salts is used as the eluent (~obil phase)~ Fo~ the p~sant invention~the membrane should be so sclected to be pe~E~ble ~nlr for the eluent but not o~ the liquid ion exch~ager which should remain in the longitudinal channel of the piston.
, The devics ko be utilized in the DCLC acccrd~ng ~o the p resent inve~tion can be ~ad~ from any inert ~aterial such as glass, polyethyl0ne or any other suitable pl~stic mat~rial and a~en metal c3uld be considered for some special us~s.

' The inventio~ will now be further illustra~od by the following Examples without being limited thereto or to the ombodim0nts described in the speciication. On th~ o~t~ary it is i~nded to cover all alternatives, modifications and 0quivalents as may bo included within the scope of the invention as dafined by the appended Claims.

Example 1: Sep~ration of a mlxturs of Ferocene and Fe~ocene _ - 1.0 g of silica (Mbrk, ~ieselgel H, Type 60) is dispersed in 5 ml of a degassed solution of dichlorometha~e/hexan~
the locked test tube to maks a slurry. The pisto~ ~quippcd with ~he stopper and the upper membra~e~is inserted ~nto the test kube until firm contact between ~0'-ring and the test tube is reached.

, .

lZ1~39B

The whole ~mit is inverted st.~nding vertically on the s topper and the air is removed ~hrough the ou~let in ~he test tubc. The test tube outlet is then locked and packing is effected by moving the test tube down th~ stationary piston a~ a flow rate of about 1 ml~min. When the silica bed is completely settled the test tube outlet is unlocked and the test tube removed-from the pis*on. The Iower membrane is installed and the column is ready for sample application.
(b) Sample A~plication A mixture o Perocene and FerQcene aldehyde is dissolved in 0.2-0.4 ml of dichloromethane and the solution is applied onto the lower membrane of ~he vertically standing column.
The solution penetrates the membrane and the co~ponents are lS adsorbed on silica. This process could be accelerated by applying some air pressure using the locked test tube.

~c) l.lutiGn The packed piston is inserted into the test tube containing 5 ml o$ the chosen eluent. Air is removed as during paaking o f the column and elution is effected by the descent of ~h~
piston into the filled test tube at ~ flow rate of about 1 ml/min.
TABT~ I
1) 1) Separation of a Model ~lixture Con~aining Ferocene (13 mg.) and Ferocene Aldehvde rl9 mE ~
~luent Weigh~ of 0 1 ml l~lexane 0 ; Blank 1 - " - - " - 11.4 mg ~erocene

2 - " - - " - 0.79 Ferocene

3 - " - - " - 0.31 Ferocene .

3~
TABL~ I cont'd.,. 16 Eluent ~eight of F r. No. Volume ~luent Residue Characterisation

4 1 ~1 Dichloromethane Traces Blank S ~ 1.79 Aldehyde 6 - " - - " - 13.36 Aldehyde 7 ~ _ 4,59 Aldehyde 8 - " - - " - 1.69 Aldehyde 9 - " - - " -:TRaces Blank II) Separation of a Model ~ffxture Containing Ferocene ~25.3 mg~
10 and Ferocene Aldehyde (15.6 m~3 _ _ ~luent ~ei~ht of Fr. No. Volume~luent _ Rssidue Characterisa~ion O 1 mlllexane 0.2 mg Forocene l - " -- " - ~ l9.0 mg Ferocene 15 2 ~" - 1.7 mg Ferocene 3 - " -- " - 0.3 mg Perocene 4 - ~ " - 0 Blank - " - Dichloromethane 0 Blank 6 - " - - " - ~ 0.2 mg Aldehyde 20 7 _ ~ 11.5 mg hldehyde 8 - " - - " - ~2.9 mg Aldehyde 9 - " - - " - 0.7 mg Aldehyde - " - - " - 0 Blan]c ~xampl~ 2: Separation of Na2Cr207 ~ 2H20 from Cu SO451-~20 The adsorbent consisted of DOW~X 50 WX8 (200 - 40~ mesh size).
The adsorbent was first washed and after left for ~bout 30 minutes in distilled water acidi~ied with hydroGhloric acid ~2N). ~he . acidity was subsequently removed by washing with distilled water - 30 and the neutral adsorbent introduced into the channel of th~
p iston. The two mer,lbranes which hald the adsor~ent bed consisted o f two discs f porous p~lyethylene filter.

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_ 17 The aqueous solution sample consisted of 359.3 mg of Na2 C~2 7-2H20 and 369.7 mg Cu S04. 5H20 dissolved in 1 cc of water.
The sample was introduced through the adso~bent, *ho amount of s ample taken ~or analysis being 100 ~1. The ions were washed ~rom the column and separated as follows :

- the anions by distilled water;
- the cations by an acidic solution consisting of 2 N hydrochloric acid.
Tho por~ions were collected in test tubes. The end of washing w as determined accordin~ to the colour of the exit solution.
The samples were further quantitatively analysed by drying the v arious portions at 110C and weighing the dry residue.
A blank experiment for tlle residue was perormed wherein 1 00 ~1 of the sample were introduced in a test tube and dried at llO~C. The solid residue weighed 68.5 mg.
The results of the various dried fractions weighed are given in the following Table 2. In ~xpt. 2, the column af~er E~pt. 1 w as washed to neutral and neu~ralized.
TABL~ 2: Separation by ion exchange with the dynamic E No. of the The eluent The wei~lt Total xpot- frac~ionutilized of the dried wei~l~t Remarks ~ fraction~ r~ ~
~ .~, ~, _ ,.
I 1H20 ~ 43.0 2 " ~.2 3 - 0 43.2 *
4HCl(2N) 0.5

5 " , }5,2

6 " 27.5

7 " ~ 1 3 47-7 *

* Fraction without colour.

3~

~pt. No. No. of the The eluenl: The weight of T~tal Remarks fraction utili zed : the dried 2 l ~1 0 - 44 2 " ~l.l 3 ~ ~O.l 4~.3 ~ *
4 HCl~2N) Ø 4 " ~2.3 6 " 34.0 7 " 5.7

8 " ~0.8 43.2 --~ fraction without colour.
From the above results it appears thàt a~ter 8 fractions percolated through the adsor1~ent, substantially all the ~o~polmds were removed and separated.

15 - In order *o poin~ out the efficiency of the DCLC
- according to th0 present invention,;a conparative test was performed using conventional chromatography, by gravitational f low, with the same amount of lO0 lul of sam~le and the same adsorbant. The resul*s are presented in the following 20 T able 3 :
TA5LEi 3: Separation by ion exchange using a conventional chroma*o~p~ column _ _ N o. oflhe eluent lhe weight of the Total Remarks fractionutil i zed ~
H20 0. 8 2 "- 50. 3 3 -"- l.3 ~
4 ~ 0.5 l --"- 0.2 6 -"- 0.3 ... .

~21~39~

19 : -T able 3 cont'd...
No. of ~ e eluentThe weight Or ~he Total 7 ~1 O
8 -"- 0.1

9 -"- 0.3 _"_ 0. 4 1~ -"- ,~00 1 l 2 _~_ 0 3 1 3 _~_ O. l colou~

10 1 4 ilCl ~2N) 0 . 0 1 5 -"- 2.3 1 6 -"- 23. 0 1 7 -"- l2.7 1 8 "
151 9 -"- 1.~1 -~"~ 1.4 44.1 Fraction without colour ~xang?le 3 In this experin~nt a solution of 100 ~1 of ~- hCG containing 30-35%
1 abelled iodine (*I2) was separated by the DCLC ~ ~
using a Sephadex G-lO adsorbent. ~le elution was performed with 10 ml of buffer at pH of about 8. ~ach fraction consisted of about 0.4 ml.
T he resl~lts are present=d in tile fol lowing Table 4:

. .

:~2~3~31~

. _ 20 TABLk 4: Separation of e - h CG containing *I2 on Sephadex C-lt), Fraction No. Blank c p m determined Total 1 29.0 0 2 9348.0 g578.0 3 1946.0 1962.8 ~ 292.0 267,7 130.0 104.8 6 114.0 . 84.5 7 108.0 : 82.0 8 78.0 .48.2 12125 cpm 9 7g.0 49.6 98.0 67.7

11 134.0 102.9 1 2 182.0 152.5 15 1 3 414.0 389.3 14 485.0 464.4 766.0 757.5 1 6 854.0 834.7 1 7 635.0 615.5 20 1 8 450.0 424.6 19 504.0 486.4 378.0 348.5 21 284.0 256.8 2 2 158.0 127.7 25 2 3 131.
- -- . _.
Total 20534.0 20594.4 17294 . . ~
It appears that the recovery is about 85%. This separation llhen p erform0d in a conventional chromatography will require much more e luent and will take also more time or sep~ration.

.

.~

In this e~perim~nt a sol~tion o:~ dyestuf~ ture consisting o~: 35% Ceres red 7B; 28% Nitro fast blue 2B; 25% 2Jit~ fast violet FBL and 12% Ceres yellow R ~all being YoluDIe p~rc~latages3 5 was sepa3~ated with the DCLC method. ~is dyestuf mi~t~re was provided by Merck (oatalogue Number 9354~.

An amount o 30 pl o~ the dyestu~f mixtllre in diehlorome~ e was inj~ct~d in a DCLC containing LI(~HP~PR~P-Si-60 ~Trade Mark produc~d by Merck, cat. No. 9336), a silica-based adsorbent ~AYing particle 10 size of 15-25 pm Silica. me colw0~ sizes were as fvlltP~Irs: 13Dgth 10.6 cm and inte~nal dia~ter 10 mm. The ~ ate was 2 ml/~
and the eluant was dichloTomethane.
The r~sults of the s~paration are presented in Figu~e 10 in th~ or of graphs, op~ical density ~O.D.) at 254 nm versus t~e ractieDs.
As appears from the graphs a ~ast and clean separation was recoived.
:, Exam~le 5. ~.

In this exporiment a mixture o~ polycyclic aro~atics consisti~g of: 50% benzene; 30% naphthalene and 20~ anthracene (volume percentages) in n-heptane solution was separated with the DCLC method.
~ he sample injected consisted of 50 ~11 using a column with the same sizes as in Example 4 containing the same adsorbent.
Ths flow xato was 2 ml/min, the volume of each fraction being 1 ml.
The elusnt was n-hep~ane.
.~ 25 The results of the sepa~ation are pres~nted in Pigur~ n th~ .
f orm of graphs, optical density ~O~D.)~at 254 3~m v~rsus the ~actions.
As appears from said graphs, the componen~s wore separated into three sharp pe~ks. ~ ' .

. _ 2~ -. ~ ', In this e~perimen~ a mixture of alkyl phthal~tos was s ~p~rated with the ~CLC method using a colu~n as in Exæmpl0 4 wi~h ~he sa~e adsorben~.
The alkyl phthalates consisted o~ a ~ixture of dibutylphthalatel diethylphthalate and dime~hylphthala~.in n-heptane / ethyl acetate ~90/10 parts by.vvlu~e). ~he elu~t w~s a mixture of n-heptane/ethyl aceta~e (90/10 ~arts by ~ol~mss). Ths flow ~ate was 3 ml/m~ he volume of each frac~io~ b~i~g l ~1.
The results of the sepaxation are prese~ted in Pigur~ 12 i~:the for~
of gr~phs, optical densi$y (O.D.) at 254 nm versus th~ ~ractions~:
As appears from the graphs a clear sepa~ation was obtained.

Example 7.

Purificatio~ of anti-hCG
.. .
The puriicatio~ of anti-hCG ~as perfo~med with the DCL~
m ethod us m g two different sources of this compounds: a)~SBRON0 and b) MIL~S, the latter being known to be l~ss co w ent~atsd than tho foxmer.
: a) Purifica~ion of anti-hCG ~SBRCN0). ~
One vial o anti-hCG was reconstituted with 1 ml of phosphate b u~fer (pH - 6.3). The so!.ution was applied on a coIumn ~10.6 cm ; length and 10 mm internal diameter3 csntaining 3 grams o~ oellul~e ~DEAE De-52J Txade Mark produced by Wha ~ ) as adsorbent. The :~ colu~n was eluted with phosphate buffer`~pH - 6.3) at a flow rat~ of 2.5 ml/min. Immediately a very high peak of proteins was ~isible in the ~irst fractions t4 to 8~. The column was ~onnect~d to a flON-c ell and recorder for im~ediate detection. A change of buffer to ; pH = 7.1 brought an almost im~ediate appeaTance o~ pro~eins. Tbo other major peaks of proteins were eluted.

The d~te~ation was perono~d by reading on ~b~orl~ e a'c 280 ~p*ical density (O.D.) nn~ ~ch ~ iorl. ThQ ~D~m~lo~ical activity of each fxaction o:f hCG recogniti~n l~ras don~ by RIA
me~od using the followill~ soluti~ns: lûO y} 125I - ~G, lO~
5 serum free o~ hCG ~nd lOû pl o:~ each racti~n. 1~ cubaki~ was for 3 hours at room ~perature. Iho separa~cion wa~ d~ne u8~ a polyethylen0 glycol/do~le antibody (20/1 vslune pa~
The results obtained are prasanted.i~ th~ foll0willg Tablo 5.
TABIE S

P:raction O.D. RIA ~ractio~ O.D. RIA
No. % bi~ldLn~ No. % bind 0.004 0.1 ~ 16 00û13 1~ %
2 O.Oû2 0.1 % 1~ O.û39 ` 16.4 ~
~53 0.00414.1 % ` 18 - 0.133 5.~ %
4 0.006 2~1 % Ig 0.012 3.û 9 0.63 22.8 % 20 ~.019 6.4 %
6 0.87912.3 ~6 21 0.~4 . 10.3 %
7 0.097 2.8 % 22 0.029 4~5 %
208 0.0~ 0.1 % 23 0.013 ~.0 %
9 ~.008 0.1 % 2~, 0.~15 3.0 0.003 0.1 % 25 0.~08. Z.~ %
11 - 0.1% 26 0.~0~ 2.

12 0~002 0.1 ~ 27 0.002 2513 0.0020.1 ~6 28 0,01 0.1 5 ,:: 14 0.003 0.1 % 29~ - 0.1 , 15 0.01 0.1~
., , As appears from the results present~ in Table 5, ,three major p eaks wer0 obtainedJ ~h~ imm~ological~ actiVity ~enained 0xtremly 30 hi~h in con~parison to the pro~eill cwlcentration.

~12~

b ) Purification of an~i-hCG tMIlES~. ~
- 70 111 of antibody ~as rabbit serum) ~ere ~ppl~d to 'chs DEAE D~-52 column, ~s in the previous case) and ~lutod fi~st ~ith phosphate buf~r (pH = 6.3~. Again as in ~he prsviou~; cas~, a 5 fast protein poak was eluted ~n fracti~s 3 a~d 4; by a t~cxe~se in t~e optical density ~O.D.) and cha~l~e o~ bufer to pH ~ 7.1,~
thre~ oth~r major pe~lcs wer~ collect~d.
Ihe rasults are presented în ~che ~ollowing Table 6.
T ABI~; 6 .
10 ~ ,~
Fraction O.D. RIA Protei~l A ~r.No. O.D. RIA Prote~n A

0.01 0.1 % - 21 O.Og4 63.6 % 7.0 %
2 0.102 0.1 ~ 22 ~.042. 64.~ % 7.2 ~6 3 0.656 6~.2% 2.~ %, 23 0,0~1 63.4 %
4 0.4~645. 6% - 2~ 0.:~7 1.0 %
0.091 0.1% - 25 0.518 6~.3 % . 5.g %
. 6 0.055 0.1% - ~ 26 0.51~ 6707 ~ % 5.5 9s: 7 0.04~ 0.~% - 27 O.Z99 55.5 % 4.5 %
8 0,022 0.1% - 2~ 0.161 58.4 % 3.7 9 0.016 0.1% - 29 0.120 66.0 ~6 2.5 0.007 O.lg~ - 30 0,112.. 54 % 1.8 11 0.005 0.1% - 31 O.. Q91 -51.3 % 3.9 $
12 0.003 0.1% - 32 0.07~ 59.? % . 2.4 %
1 3 0.003 0.1~ - 33 0.062 55.7 % unh~ow 1 4 0 . 004. 0 . 1% - 34 0. 062 60 . 9 %
0.001 0.1~ - 35 0.043 59.5 96 . 1.9 %
' ` 1 6 0.004 0. 1% - 36 ~.û34 ~ 1 7 0.011 0.1% - 37 0.~35 30 1~ 0.034 65.4% 7.~ % 38 ~ ~ 0.029 19 0.2~1 55.5% 12.1 ~ 3g . 0.021 0.247 61.4% 11.6 %

4~

~5 As a~pears from the results pr~sent~d in Tabl~ 5, k~
s eparated anti-hCG was collected in th~ee main peak5. hll of tl~em s howed immunological ac~ivity, abso~p~ion at 280 nm a3ld recognition by P~otein A . All peaks were sha~ly Eepara~cd from 5 e a~ other and collected.

~_ .
A separation of }~ n serum was exocuted Usillg ~1~ D~C
method, identic~l to the p~cedur0 ss described in Handb~k o~
10 E~q?erimental Inm~nology (D.M. Weir, Md. Blao1~1ell S~i~ti:~lc p~blications, Ox~o:rd, L~ndon, 1973 2nd ed.).
The chroD~atogr~phy is based on ic~l-exchange o~ ~ cellulose adsorbent and a gradient elution with phosph~t~ bufar tO.02 1 o~pH 5.7.
15 The c~luDm of 10 mm internal diamotex~was pa~k0d with 3 g of DE~
. DE-52 (Trade Mark produced by Whatman~ and washed with tJ18 phosphate bu~er (pH = 8), at ~low ~e of 2.5 ~I/mLn.
The gradie~t was produced with a two chambor system, usl1l~ 40 phosphate bu~fsr pH 8 and 60 ml of phosphate buf~ex ~ 5.7.
20 An amolmt of 3 ~1 of human ser~m~ was sepa~ated; fracti~n~ o~ 1.5 ml each being collected and the p~ot~in content the~in d~texmined by aptical densi~y ~O.D.) at 280 n~, Ths xesul~s are presented in the ~ollowing Table 7.

.. . ... . .... . . . . .. . . . . . . . . . . . . . . . .

3~
~6 _ __ ,_ O. 0017 13 O. ~52 25 O. ~6 2 1 . 3 1~ O. 061 26 ~0 ~26 3 1 . 3 15 0 . 102 27 ~. 0 4 1.3 :16 28 0~0~4 4 . 554 17 O. 187 - 29 O. ~
6 ~. 262 18 O. 355 30 . ~.Q03 7 O. 135 1~ O. 379 - 31 0 . Q~8 8 0 . 082 20 G . 361 ~2 O. ~1 5 9: 0. 067 21 0. 313 33 O. g~6 0 . 064 2~ O. 233 3~ ~. 011 11 0.055 23 0.156 ~ 35 0.055 ~512 0.052 24 0.098 36 O.Q06 As appears from the results present~d in Table 7, the s~aration achieved by DCLC shows th~ t;r~ditional pistur~ of s~paratbd hunan serum wi~h two laajor peaks ~IgG~ Albumins). The~sep~ra*i~n is co~pleted in a short time.

20 ~XA~IE 9.

In this Example th8 DCLC method was a3?plied to afiJ~i~y :i~ chromatography, or isolation of rabbit IgG usin~ as ligand , SEPHAROSE-4B ~Trade Ma:rlc) produced by Pha~macia) - a~tib~dy i' ( 3ntibody = Goa~-an~i rabbi'~) .

~.'2~3~

J~: ~e an~body was coupl~d ~o -the S~PHAaQ6~-4B
(Irade M~rk, p~oduced bq Phax~ci~ bent using the instructions giYen b~ ~:Axel ~Po~a~
et al ~Na~urc 214, 1967~. :
1 g SEPHAROSE-4B~ ~ 50~
30 Dlg Goat-anti rabbit ~ b~din~.
DCLC oolu~n : gl~ss columil os~ 6 mu inte~ dia~eto~ packed with S~pharose-4B - ~ntibod~-.
Dotoceion: was done dir~ctly froqn the colum~ with flo~
cell. With W at 280 mn a~ptioll.
Buffer: 1. Phosphate buffer / N~CI" pH t.8. `
2. Glycine~/ HCl tO.1 M), p~l ~.5.

I`h~ proc~dur~ co~prised the following ste~s :-1. Coupling o~ SEPHAROSB-4B to Goat-all~i rabbit serum.
- 15 2. Pack~ng of DCLC with SEPHAP~SE-4B~ tibody 3 ml g~l" closed both sides with a filter system YY.~N (Trad3 Msrk) app~x~ 4 3. W~shing of the pack~d colu~n with 6 ~l of bu~fer 1.
4 . LDading the column with 0.5 ml N.R.S ~Normal R~bbit Seru~)~
5. Incubation of 2 h at 37C.
. 20. 6. Blution with buffe~ 1 and collection of fraction until th~
optical density at the Photometer is bclow 0.1.
: 7. Elution wi~h bu~er 2 of pH 2.5 and collectio~ o~ ~action$~u~til the optical density is below ~
..
- A schematic graph for the affinity chromatography of this 25 ~xample is presented in Figuris 13.

- '"~ i .

. .

Claims (20)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for separating separable components contained in a liquid sample by chromatography technique hereafter referred to as dynamic column liquid chromatography which comprises the steps of:
(a) adsorbing said separable components of said sample onto a movable chromatographic column comprising at least one dense-packed adsorbent bed, said column having an inlet end and an outlet end;
(b) placing said inlet end of said column into a container defining an elongated passage adapted to accept said column in a formfitting manner, said passage containing a predetermined amount of an eluant; and (c) moving said column in said passage through said eluant, whereby said eluant is forced under an intrinsic pressure developed in said passage through said adsorbent bed to elute at least one of said separable components from said column at said outlet end.

2. A method according to claim 1, wherein an additional suitable adsorbent, different from the adsorbent utilized in the dynamic column liquid chromatography, is incorpora-ted in order to preconcentrate the sample introduced.

3. A method according to claim 1, wherein the container is closed at its bottom, the eluent being introduced into the container, prior to pushing the column downwardly into the container.

4. A method according to any one of claims 1, 2 or 3, wherein the chromatography technique is selected from silica gel chromatography, reversed phase liquid chroma-tography, affinity chromatography, capillary chromatography, chromato-focussing, gel filtration and ion exchange chrom-atography.

5. A method according to claim 1, wherein the solid adsorbent is present in a cartridge located in the column.

6. A device for dynamic column liquid chromatography for separating separable components contained in a liquid sample, which comprises:

(a) a movable chromatographic column comprising at least one dense-packed adsorbent bed held between two barriers and a nozzle located at one of the ends of the column, and (b) a container defining an elongated passage having a sealing element and a multiple way valve at its lower end, said elongated passage being adapted to accept said column in a formfitting manner.

7. A device according to claim 6, wherein the exit of the eluent is through a vertical narrow channel in direct continuation of the column.

8. A device according to claim 6, wherein a sealing element is provided at the base of the column adapted to slide along the inner walls of the container.

9. A device according to claim 8, wherein said sealing element is made of an inert material.

10. A device according to claim 9, wherein said sealing element consists of a rubber-O-ring having an orifice bore connected to the column.

11. A device according to claim 6, wherein the column is constructed of two or more subunits, each one containing the same or different adsorbents.

12. A device according to claim 6, wherein the nozzle is connected to the upper barrier of the column.

13. A device according to claim 6, wherein a stopper is located at the top of the column.

14. A device according to claim 13, wherein the nozzle is connected by a channel passing through the stopper.

15. A device according to claim 6, whereby a column solvent reservoir is connected to the dynamic column.

16. A device according to claim 6, wherein the container tube is closed at its bottom.

17. A device according to claim 6, wherein the column is provided with means for accumulating of gas pocket.

18. A device according to claim 17, wherein said means for accumulating of gas pocket consists of horizontal, vertical or spiral grooves, located on the column.

19. A device according to claim 6, wherein said device is made from an inert material.

20. A device according to claim 19, wherein said inert material is selected from the group consisting of metal, glass, polyethylene or other adequate plastic material.