GB2070964A - Analysis of ionic species using a chromatographic separation medium - Google Patents

Abstract

Method and apparatus for chromatographic separation and quantitative analysis of ions of like charges in a sample, e.g., cations or anions. For the analysis of inorganic anions, the sample and an eluent are directed to a column 13 of hydrophobic porous chromatographic separation medium (organic resin or bonded phase) having high surface area, without permanently attached ion exchange sites. The eluent (a polar mobile liquid) includes an organic cation which reversibly adsorbs to the resin to create ion exchange sites which differentially retard the anions for chromatographic resolution. The eluent also includes a developing reagent of the same charge as the ion to be analyzed. The eluent including the resolved anions is then passed through an ion exchange resin 14 which precludes passage of the counter-ion and its co-ion in ionized form and then through a conductivity cell 15 for quantitative detection. Inorganic cations may be detected in an analogous manner. The system may also be employed to analyze highly organic cations or anions (e.g., surfactants). In this instance, the organic ion of interest is already strongly attracted to the porous chromatographic separation medium, and so the counter-ion may be inorganic. <IMAGE>

Description

SPECIFICATION Analysis of ionic species using a chromatographic separation medium The present invention relates to the quantitative analysis of different anions or cations in a single system. Reversed-phase liquid chromatograph (RPLC) is widely used as a mode of separation in high performance liquid chromatography (H PLC). In RPLC, the mobile phase is more polar than the stationary phase, the reverse being true in conventional chromatography performed prior to development of RPLC. Chemically bonded hydrocarbon chains (alkyl groups) attached to silica substrates are one common form of stationary phase. The mode of formation of such stationary phases and suitable techniques for performing RPLC are well known as set out, for example, in N.H.C. Cooke and K. Olsen, Am. Lab., 45 (August, 1979).One technique of RPLC has gained sufficient popularity to be called Reversed Phase ion Pair Chromatography. In this technique, a salt is added to the mobile phase to improve the chromatographic properties. While there is some conflict in the theory of separation, the experimental techniques described in this paper are commonly employed. Specifically, the sample is directed in an aqueous polar mobile phase, commonly including a lower alcohol, acetonitrile or other water miscible organic solvent, together with a counter-ion, typically tetrabutyl-ammonium ion (TE3A), for anion analysis. In one theory, hydrophobic ion pairs are formed which are relatively nonpolar and so are differentially retarded by the column.In another theory, the counter-ion, e.g., TBA, is adsorbed to the surface to form a reversible ion exchange site on the stationary phase. This technique is employed primarily for the chromatographic separation and analysis of organic acids and bases. Generally the technique has been used with the usual detectors of HPLC such as of the ultraviolet, fluorescence, and refractive index (RI) type. However, these typical detection techniques are unsuitable for the analysis of inorganics separated by RPLC. Also, for non-chromophoric, low pKaorb organic molecules, such as surfactants, these detectors are relatively insensitive.

In Harvey et al patent 4,042,327, a technique for Reversed Phase lon Pair Chromatography is disclosed using a bonded phase packing. The species to be analyzed are generally limited to organic compounds. The packing is unstable at high and low pH levels, leading to limited utility in analyzing inorganic anions and cations, respectively.

A recent paper, F.F. Cantwell and S. Puoh, Anal Chem., 51, No. 6 (May, 1977), 623, has suggested the use of a non-ionic macroporous resin (a copolymer of styrene and divinylbenzene sold under the trademark Amberlite XAD-2) as the stationary phase for RPLC. One significant advantage of such resin is that it is stable at extreme pH ranges in contrast to a silica-based stationary phase. However, the common detection techniques such as described above suffer from the same deficiencies.

Another chromatographic system known as ion chromatography has been utilized in the quantitation of organic and/or inorganic anions and/or cations in aqueous sample solutions. In this technique, chromatographic separation is performed on low capacity ion exchange separating resin column or columns. Then the eluent is directed through a high capacity ion exchange resin suppressor column which converts the eluent frcm a conducting form to a non-conducting form and thereby reduces the background conductivity of the chromatographic system. The ions to be analyzed are eluted from the suppressor column and form highly conductive species which are passed through a conductivity cell and quantitated on the basis of conductivity.This technique is well suited to ionic species eluting from the suppressor column in a form which has a dissociation constant of greater than 1 0 ~~ 7. Molecules with dissociation constants less than this a-a not cletectsble by conductivity at chromatographic concentration levels.

ene limitation of ion chromatography is that the separating resin must be of a conventional permanent ion exchange site containing type. This substantially fixes both the ion exchange capacity and selectivity of the separating column since the ion exchange groups are chemically bonded to the substrate resin. Thus, for a given column and resin, modification of the chromatographic resolution would require chemical modification of the resin, such as by changing of the ion exchange groups by substituting a different type of resin, a time consuming and costly operation. The capacity of the separating resin must be small so that relatively low ionic strength eluents can be used to maximize suppressor column lifetimes.The resolution of highly ionized ionic species in accordance with this technique is set out, e.g., in Small et al.

U.S. Patent 3,920,397.

It is an object of the invention to combine the best features of the aforementioned prior art techniques of (1) reverse phase ion pair chrnmatogrnphy with (2) ion chromatography.

Specifically, the ionic species to be separated are directed in a mobile phase through a first separating column comprising a porous hydrophobic chromatographic bed with essentially no permanently attached ion exchange sites and having a high surface area. A preferably chromatographic bed is formed of an organic resin. The mobile phase also includes an ion exchange site-forming compound with a counter-ion which reversibly adsorbs to the chromatographic substrate to create ion exchange sits and to cause the ionic species to be differentially retarded and chromatographically resolved in the eluent from the bed. The eluent also includes co-ions of the counter-ions. When the eluent is directed through a suppressor column including an ion exchange resin of a type which substantially precludes passage of excess counter-ions and co-ions in ionic form.Finally, the eluent is directed through a conductivity cell having associated readout means to quantitatively detect the resolved ionic species. The mobile phase preferably includes a substantially non-ionic organic polar compound and an inorganic developing ion, both of which can be employed to adjust column selectivity for optimum separation of the ions to be analyzed.

Another aspect of the invention revolves around a different theoretical mechanism. There, instead of the counter-ion of a site-forming compourd forming a reversible adsorbed ion exchange site as set out above, the counter-ion and the ionic species form reversible ion pairs which are reversibly adsorbed onto the chromsatographic bed for differential retardation and chromatographic resolution. Thereafter, the resolved species are directed through the suppressor column and conductivity cell as set forth above. This theory best explains the separation of long chain organic molecules, such as surfactants, which form the primary adsorptive bonds of the ion pairs. In fact, inorganic counter-ions are preferable for use when analyzing such organic ionic species to permit desorption from the column in a reasonable period of time.

It is an object of the invention to provide a technique which combines the best features of reverse phase ion pair chromatography and ion chromatography.

It is a specific object of the invention to provide an ion chromatographic technique in which the parameters of chromatographic selectivity and chromatographic capacity may be varied solely by changes in the eluent composition and concentration.

It is a specific object of this invention to couple this superior chromatographic separating technology with the superior detector system of ion chromatography which facilitates the highly selective and sensitive detection and quantitation of ions with low pKaorb values.

It is a specific object of the invention to provide an optimization of ionic separation over a large range of selectivities by such eluent changes without rapid consumption of the suppressor column.

It is a further object of the invention to provide a technique for separation and detection of large organic ions which are difficult to accomplish by conventional ion chromatography and for which the detection iimits of conventional reverse phase ion pair chromatography using ultravioiet or refractive index detection are inadequate.

It is a particular object of one embodiment of the invention to utilize a porous, hydrophobic chromatographic resin as the stationary phase which is stable to extreme high pH levels and thus capable of extended chromatographic operation of inorganic anions without deterioration.

Further objects and features of the invention will be apparent from the following description taken in conjunction with the accompanying drawings.

Figure 1 is a schematic representation of a simplified apparatus according to the present invention.

Figures 2-6 are chromatograms illustrating the separation of different ions in accordance with the present technique.

The system of the present invention is highly versatile, as it may be employed to determine a large number of strong and/or weak organic and/or inorganic ionic species so long as the species to be determined are solely cationic or anionic. Such ionic species are normally associated with counter-ions but only ionic species of common charge are determinable by the present method. Suitable samples include surface waters, including salt water, and other liquids such as industrial chemical waste streams, body fluids such as serum and urine, beverages such as fruit juices and wines, and drinking water.Covalent molecular compounds, such as amines, which are convertible to ionic form as by forming acids salts, are also analyzable in accordance with the present invention When the term "ionic species" is used herein, it includes species in ionic form and components of molecules whicn are ionizable under the conditions of the present process.

Referring to Fig. 1, a simplified apparatus for performing the method of the present invention is illustrated. Sample is supplied to the system suitably by a syringe (not shown) at sample injection valve 10. The sample is carried through the system by eluent drawn from reservoir 11 by pump 1 2 which thereafter passes into chromatographic separation column 1 3 of a type to be described below. Th eluent from column 1 3 passes through suppressor column 14 in which ions of opposite charge to the ions to be analyzed are substantially precluded from passage in ionic form. Typicaliy. this occurs by stripping of such ions. Then the eluent containing the ionized ionic species flow through a liquid conduit to conductivity cell 1 5. The electrical signal emitted at cell 15, in which the fluctuation in ionic concentration produces an electrical signal proportional to that amount of ionic material registered by conductivity meter 16, is directed to recorder 1 7 which provides a visible readout for the signal from conductivity cell 1 5. After passing through the conductivity cell, the liquid is passed to waste.

The mode of separation in column 1 3 may be explained by two different theorctical mechanisms designated herein as the "ion pair" theory and the "reversible ion exchange" theory. Regardless of the prevailing theory, the system employs a mobile phase more polar in character than the stationary phase, which carries a counter-ion which interacts with the ionic species to be measured. This type of system is commonly referred to as a "reverse phase process." In early reverse phase work, the ion pair theory (as set out in the aforementioned Cooke and Olsen article) was assumed to be applicable, while some recent articles favor the other theory to be described in more detail below.It should be understood that the present invention is applicable to either or both theories, as it is the combination of using the reverse phase process for chromatographic separation together with the ion chromatographic technique for quantitating the separated ionic species that forms a major aspect of the present invention.

The method will first be described in accordance with the reversible ion exchange theory of separation. For simplicity of description, inorganic anions will first be described as the ionic species in the sample to be separated and quantitated in the system. The eluent in reservoir 11 which forms the mobile phase for the sample includes an ion exchange site-forming compound.

This compound is comprised of a counter-ion of opposite charge to the ionic species and a coion of the same charge as the ionic species. (Herein, the term "counter-ion" used alone refers to the last named counter-ion and the term "co-ion" used by itself refers to the co-ion of that counter-ion.) A porous hydrophobic chromatographic bed with essentially no permanently attached ion exchange sites is contained in separation column 1 3. This is to distinguish from conventional ion exchange resin in which the ion exchange sites are permanently attached by covalent bonding to the resin substrate.

In accordance with the reversible ion exchange theory, the counter-ion is of a type which forms reversible adsorptive bonds in situ in the chromatographic bed to create ion exchange sites therein. In this manner, the ionic species are differentially retarded by the thus formed ion exchange sites and are chromatographically resolved in the eluent from this bed. The inorganic anions to which the present technique is applicable includes essentially all types of anionic species from those weakly to strongly retained on the chromatographic bed. For example, the following anions may be separated: fluoride, chloride, nitrite, nitrate, chlorate, perchlorate, bromide, bromate, iodide, iodate, sulphate, thiosulphate, persulphate, pyrosulphate, phosphate, pyrophosphate, azide, cyanide, ferricyanide, and thiocyanate ions.

Prior to the present invention it had not been appreciated that inorganic anions or cations could be separated chromatographically using either of the above two theoretical mechanisms.

A variety of reverse phase chromatographic separation stationary phases may be employed, as of the type illustrated in the aforementioned Cooke and Olsen article. One effective type of chromatographic bed uilized for the stationary phase comprises hydrocarbon chains bonded to a substrate. Such chains are typically 8 to 1 8 carbons in length. Such chemically bonded alkyl phases are commonly produced by the reaction of surface silica silanols with organochlorosilanes. The type of chain may be varied, depending upon the ionic species of interest.

Functionally, the bed provides uniform organic chain surfaces so that the counter ion is readily absorbed onto the surface in a uniform repeatable manner.

A typical bonded-phase porous silica gel packing is silica reacted with an organic material to bear an 1 8-carbon chain aliphatic group thereon. Such packing is sold by Waters Associates, Inc., under the trade designation Bondapak C,8. Other suitable resins are supplied by Altex Coporation and Merck 8 Co., Inc.

A particularly effective stationary phase is a non-ionic porous, hydrophobic organic resin with essentially no ion exchange-sites. One such resin is a copolymer of sytrene and divinylbenzene available under the trademark Amberlite XAD-2 with a surface area of about 300 M2/g. Such resins are stable at pH extremes, e.g., 1-14, in comparison to the bonded phase packings described above which are less stable at pH extremes.

Conventional non-ionic hydrophobic porous organic resins may be utilized for the separation of the present invention if they have sufficient surface area and hydrophobicity. A suitable minimum surface area is from about 10 to 100 M2/g to a maximum of 1000 M2/g or more, with a preferred range ef about a minimum of 280 to 300 M2/g up to 600 M2/g. Typical pore sizes range from 30 to 1 00A to 200A or more. Such porous resins generally are formed by the use of porogens or so-called precipitating agents. Suitable resins and their methods of formation are described in Gustafson patent 3,531,463 and Mindick et al patent 3,549,563.

The method for determining surface area is mercury porosimetry, described in "Advanced Experimental Techniques in Powder Metallurgy", Vol. 5, Plenum Press (1970).

An important characteristic of the chromatographic bed is that, as a result of its large surface area, it has a correspondingly high capacity for forming reversible ion exchange sites with the counter-ions. This high capacity has the benefits of minimizing the required amount of packing.

It is weil-known that the adsorption capacity od a chromatographic packing is proportional to the surface area of the packing.

Another characteristic of the packing is that it is sufficiently hydrophobic so that the organic counter-ion is sorbed onto the surface of the resin and is chromotographically retained thereby.

A suitable hydrophobicity is comparable to a copolymer of styrene and divinyl benzene.

Expressed in a different manner, the preferred packings are cross-linked resins with solubility parameters (expressed in the units

of at least 7.5 up to 1 5, and typically about 9.

Referring to anionic analysis, suitable ion exchange site-forming compounds include: tetrabutylammonium hydroxide, mono-, di- tri- and tetra-alkyl ammonium hydroxide. The counter-ions for inorganic anion analysis must be of opposite charge to the anions and be of a type capable of forming reversible adsorptive bonds with the chromatographic bed. This means that such counter-ions must include organic chains, specifically alkyl chains, of sufficient length for ready adsorption on the column as to be too difficult to remove in a reasonable period of time.

Another parameter of the ion exchange site-forming compound of the present invention is that it must be capable of being substantially precluded from passage through suppressor column 14 in ionic form. As will be explained below, if the ion of interest is an anion, the suppressor column includes a cation exchange resin and the counter-ion is of a type which is removed or stripped by column 14. The co-ion of the counter-ion passes through the column but in substantially unionized form. Such co-ions include carbonate, borate and hydroxide, all of which form weakly ionized acids or water in the suppressor column. However, for those cases where silica comprises the chromatographic bed, hydroxide has limited utility due to dissolution of silica at higher pH levels.

The degree of absorption of counter-ion determines the column capacity which can be tailored to the desired retention time for a particular sample by controlling the amount of organic polar liquid. For example, it has been determined experimentally that the degree of absorption of tetrabutyl ammonium hydroxide (TBAH) increases significantly as the organic polar liquid (e.g., acetonitriie) content decreases. The magnitude of TBAH adsorption is relatively small (e.g., 0.023 meq./ml. for 100 percent water eluent) compared to the exchange capacity of an ion exchange resin (e.g., 0.5 to 1.5 meq./ml.) Thus, the capacity of a reverse phase column in this mode is limited. Typically, the capacity of a system containing 0.004 molar TBAH is limited to about 2 to 4 micrograms of each ionic component per injection.

For the analysis of cations, the ion exchange forming compound must be of a type formed of a counter-ion and co-ion which are cabable of substantially being precluded from passage through the anionic ion exchange resin suppressor column maintained in hydroxide form.

Suitable counter-ions for this purpose include: lauryl sulphuric acid, C,-C20 alkyl sulphuric acid or alkyl suiphonic acid. The counter-ions are retained on the column, while the co-ion, hydrogen, is removed as water molecules from suppressor column 14.

The mobile phase includes the sample and the counter-ion in a polar aqueous liquid. The polar nature of the liquid facilitates ionization and dissolution of the ionic components of the system in the mobile phase.

Another preferable component of the mobile phase is a substantially non-ionic, organic polar compound in an amount which serves to selectively reduce the retardation time of the ionic species in the bed in a controlled manner. This organic polar compound is essentially non-ionic so as not to interfere with the ion conductivity measurement. Viewed one way, the organic compound serves as a mobile attractive force for the counter-ions and, thus, the ionic species of interest to set up an equilibrium which removes these ions from the chromatographic column and passes them selectively into the mobile phase for separation. Viewed another way, the organic polar liquid competes with the organic counter-ion for the available adsorptive binding sites on the stationary phase to cause a reduction in capacity of the same.In either event, a higher concentration of such organic polar compound shortens the retention time. Suitable organic polar compounds include lower alcohols, such as methanol and ethanol, acetonitrile, or any water miscible organic solvent.

The concentration and type of organic polar compound may be varied to a significant extent to modify the desired retention time, depending upon the ionic species to be analyzed. Suitable concentrations of such organic polar liquid can be varied from 0 to 100 percent, with the higher concentrations being employed for the more highly retained counter-ions. At the upper limit there may be solubility problems for the ions of interest and so it is preferable to include water in the mobile phase.

A further component of the mobile liquid phase is a developing reagent which includes an inorganic developing ion of the same charge as the ionic species. Such ion is included in an amount to selectively reduce the retardation time of the ionic species in the chromatographic bed. The developing ion and its co-ion (hereinafter termed the "co-ion of the developing ion") must be of a type which are substantially precluded from passage through suppressor column 1 4. Suitable developing ions include borate and carbonate ions. Both of these ions are converted by a suppressor column in the hydrogen ion form to their respective acids, which are only weakly ionized and so do not provide substantial conductivity cell contaminating interference.Similarly, the co-ion of the developing ion is either stripped by the column or is in the hydrogen ion form, which is the desired form of ionic species for detection in the conductivity cell.

The same principles apply for cation analysis. In this instance, suitable developing reagents include any of a variety of mineral acids, the anions of which are stripped by the suppressor column 14 to form water.

The developing reagent serves a similar function to developing reagents in conventional ion exchange separation in which the ion exchange sites are permanently attached to the resin substrate. That is, the developing reagents provide an equilibrium driving force which thereby displace the ionic species of interest from the stationary phase and, thus, shortens retention time.

The pH level of the eluent solution is another parameter which can affect the chromatographic separation in this technique which can be tailored to the ionic species of interest. The present porous resin is stable at extreme pH levels.

Like the polar organic liquid, the type and concentration of developing reagent may be varied, depending upon the desired retention time. However, at high concentrations, the suppressor column may be rapidly depleted. Although the developing reagent is generally more useful for modifying selectivity and capacity of the separation bed than the polar organic liquid, its type and concentration must be carefully considered to avoid excessive depletion of the suppressor resin.

It is apparent from the foregoing that one of the significant advantages of the system is the ability to vary the developing reagent, polar organic liquid and counter-ion to tune the system resolution to the specific ionic species to be analyzed.

Suppressor column 1 4 is analogous in function to stripper column 11 of Fig. 1 in Small et al.

U.S. Patent 3,920, 297, which relates to ion chromatography. The principles of operation of that column, its detailed description and its relationship and functional characteristics with respect to the separation column are incorporated by reference at this point. Referring to the present system, column 14 is of relatively high specific ion-exchange capacity. This is because the primary function of this suppressor column is to preclude passage of the developing reagent and the ion exchange-site forming compound in highly ionized form while permitting passage of the ionic species resolved on separation column 1 3 without substantial interruption. Suitable ion exchange resins for analysis of anions are polystyrene or modified polystyrene cross-linked with divinylbenzene carrying nuclear groups, the latter providing reactive exchange sites.The strong cation exchange resins typically include nuclear sulphonic acid or sulphonate groups along the polymer chains, while the weak cation exchange resins carry carboxylate groups.

The strong base anion exchange resins carry nuclear chioromethyl groups which have been quarternized. The weak base exchange resins carry nuclear primary, secondary or tertiary amine groups.

The nature of the resin in suppressor column 14 is determined by the ion exchange-site forming compound and developing reagent to be suppressed. For anion analysis, a suitable resin is a high cross-linked polystyrene including sulphonic groups in the hydrogen ion form. The high cross-linking assures that ion exchange effects predominate over chromatographic penetration into the resin. The counter-ion and co-ion of the developing ion are modified by ion-exchange in the suppressor to form products which elute from the column in substantially unionized molecular form and so which do not interfere with detection in the conductivity cell.

The effluence from suppressor column 1 4 is directed through conductivity cell 1 5 and then to a waste. The electrical signal from the conductivity cell is directed to the conductivity meter 1 6 and the output is directed to recorder 1 7.

The mechanism of separation is altered depending upon the nature of the ionic species to be analyzed. Specifically, as the sample ionic species become more hydrophobic (organic) in nature, the predominant mechanism is believed to become one of competitive adsorption between such ionic species and the counter-ion in the eluent at the surface of the stationary phase in column 1 3. For example, alkyl chains of increasing length in the ionic species (e.g., surfactants) enter this competition. This results in unacceptable long retention and poor resolution. This problem can be obviated by changing the counter-ions to a more hydrophilic inorganic ion. In general, as sample ionic species become more hydrophobic, it is preferable to utilize counter-ions which are iess hydrophobic to optimize chromatographic resolution of the ionic species.For example, ammonium ion may be used as the counter-ion for anionic surfactant analysis, while perchlorate ion may be used as the counter-ion for cationic surfactant separations. The suppressor column is still essential to reduce background conductivity of the counter ions.

For analysis of such highly hydrophobic ionic species, the paired ion mechanism is more likely to predominate. In this instance, rather than forming reversible ion exchange sites, the counterion and ionic species form reversible ion pairs which, in turn, form reversible adsorptive bonds with the chromatographic bed for differential retardation of the ionic species on the bed. This is believed to be a major factor in the chromatographic resolution of the ionic species.

By way of emphasis, the choice of counter-ions significantly affects the degree of adsorption of the ion pairs on the stationary phase. Specifically, the more highly organic a counter-ion, i.e., the longer the carbon chain in the motecule, the more firmly retained is the counter-ion and, thus, the ion pair. Thus, for inorganic ionic species, it is preferable to use highly organic counter-ion compounds, e.g.. Ct to C20 carbons long. Conversely, as set out below, for highly organic ionic species such as surfactants, it is preferable to use counter-ion inorganic compounds to avoid excessive retention times.

The steps performed after separation according to the paired ion theory in the process are the same as those described above with respect to the reversible ion exchange theory. That is, after chromatographic separation, the eluent is passed through suppressor column 14 and then through conductivity cell 1 5 for measurement by conductivity meter 1 6 and visible readout on recorder 17.

It is a particular advantage of the present invention to provide a technique for analysis of anionic surfactants. While infrared spectroscopy and nuclear magnetic resonance techniques give some information regarding anionic surfactants, they are of limited value in determining the size and molecular weight distribution. Also, ion chromatography is not capable of analyzing organic surfactants.

Another advantage of the invention is the discovery that ion pair chromatography (or reversible ion exchange chromotography) is effective for inorganic anions or cations.

An overall significant advantage of the above system (in either the reversible ion exchange mode or paired ion mode) is the ability to modify the separation column's capacity (number of counter-ions adsorbed to the column's surface) and selectivity (reiative retention of ionic species retained by such counter-ions on the column) by varying the concentration and type of counterion, developing reagent, and polar organic liquid to accommodate the type of sample to be analyzed. The system is so flexible that the same separation stationary phase may be converted to analyze cations or anions.

The eluent may be fixed for the entire run. In the aiternative, the system is particularly well adapted to the use of continuously changing concentrations of reagents, commonly referred to as a gradient system in the alternative, step changes in concentration may also be employed.

An additional feature of the invention resides in the stability of the disclosed organic resin. It is capable of long-term use without deterioration in the analysis of anions. Also, the co-ions for anion analysis may be strong anions, such as hydroxide, which are readily suppressed in a suppressor column, essential to conductivity detection. This is to be contrasted with the typical co-ions used in bonded-phase packing which would not be suppressible.

A further disclosure of the nature of the present invention is provided by the following specific examples of its practice. It should be understood that the data disclosed serve only as examples and are not intended to limit the scope of the invention.

Example 1 This example illustrates the separation of multiple inorganic anions using a porous chromatographic organic resin packing. A 4 mm X 250 mm stainless steel column packed with a porous resin similar to the XAD-2 Amberlite resin but with 400 M2/g surface area was equilibrated with an eluent composed of 0.002 M tetrabutyiammonium hydroxide, and 0.002 M sodium carbonate dissolved in 15/85 (v/v) acetonitrile/water. The flow rate was 1.5 ml/min.After equilibration the effluent from this column was directed to a 4 mm X 250 mm column of cation exchange resin sold under the trademark Dowex 50WX-16 in the hydrogen form, and then to a conductivity detector supplied by Dionex Corp. 100 y1 of a solution containing 3ppm F-, 4ppm Cl-, lOppm NO2, 50ppm PO4-3, lOppm Br-, 30ppm NO3-, 50 ppm 804- were injected. Fig. 2 shows that all seven anions are separated in less than 1 2 minutes.

Example 2 This example illustrates the separation of inorganic sulfate and organic anionic surfactants. A 4 mm X 250 mm stainless steel column was packed with another batch of the resin of Example 1. The column was equilibrated at a flow rate of 0.5 ml/min with an eluent composed of 0.01 M ammonium hydroxide in 38/62 (v/v) acetonitrile/water. After equilibration the effluent from this column was directed to a 4 mm x 100 mm column of a cation exchange resin, a sulfonated resin (diameter 11 + 1 EL) in the hydrogen form sold by Dionex Corporation under the trademark DC6A, and then to a conductivity detector. A solution containing 35 ppm linear alkyl benzene sulfonates (LAS) of alkyl chain lengths from 9-14 carbon atoms. The chromatogram in Fig. 3 clearly shows that the sample contains at least 10 components.

Example 3 This example illustrates the separation of organic cations. A pumped stream of 5 mM hexanesulfonic acid flowing at 3.0 ml/min. was directed to a 4 X 250 mm column of porous resin described in Example 1.

The effluent from this column was directed to an anion exchange column, a 3 X 250 mm Dowex 1-X10 column in the hydroxide ion form and then to a conductivity cell. Fig. 4 shows results obtained when 20 /ul of a solution containing 25 mg/l each of NH4+, HOCH2CH2NH3+, (CH3)3 NH+ was injected into the column.

Example 4 Example 4 illustrates use of a single column to separate anions or cations by simple eluent changes. A 4 X 250 mm column packed with porous resin of Example 1 was used as described in Example 3 to separate a mixture of 50 mg/l each of NH4+, CH3NH3+, HOCH2CH2NH3 +, (CH3)3 NH2+ and (CH3)3 NH+. The results are shown in Fig. 5. This column was then washed by a pumped stream of water until the pH of the effluent was neutral. The column was then treated with a pumped stream of Ig/l tetrabutylammonium hydroxide in water flowing at 2 ml/min.

Approximately 1 liter was pumped through the column.

Next, a pumped stream, flowing at 0.7 ml/min., of 1 mMtetrabutylammonium hydroxide and 5% (v/v) acetonitrile was directed to the column previously treated with tetrabutylammonium bromide as described above. The effluent from the porous resin column was directed to a stripper column, a 6 X 250 mm Dowex 50W-X16 column in the hydrogen ion form and then to the conductivity flow cell. Twenty microliters of a mixture of 4 mg/l F-, 5 mg/l CL-, 10 mg/l NO2-, 20 mg/l Br-, and 30 mg/l NO3- were injected. The results are shown in Fig. 6.

There was excellent resolution of anions by the column previously used to separate the cations.

Claims (22)

1. The method of chromatographic separation and quantitative analysis of at least a first and second ionic species of ionizable compounds in a polar mobile liquid phase, all of said ionic species being of a positive or negative charge, comprising the steps of: (a) directing said mobile liquid phase, including an ion exchange site-forming compound, through a first column including a porous, hydrophobic chromatographic bed with essentially no permanently attached ion exchange sites, said ion exchange-site forming compound including a counter-ion of opposite charge to said ionic species and also including a co-ion of the same charge as said ionic species, so that said counter-ion forms reversible adsorptive bonds with said chromatographic bed to create ion exchange sites therein, and so that said first and second ionic species are differentially retarded by said ion exchange sites and are chromatographically resolved in the eluent from said first column, said eluent also including counter-ions and co-ions, (b) directing the eluent from said first column through a second column containing an ion exchange resin of a type which substantially precludes passage of said counter-ions and co-ions in ionic form, and (c) directing the elueni mm said second column through a conductivity cell having associated readout means to quantitatively detect said first and second ionic species.

2. The method of Claim 1 in which said mobile liquid phase includes a substantially nonionic, organic polar compound in an amount to reduce the retardation time of said ionic species in said bed in a controlled manner.

3. The method of Claim 1 in which said mobile liquid phase includes a developing reagent, including an inorganic developing ion of the same charge as said ionic species, in an amount to reduce the retardation time of said ionic species in said bed in a controlled manner, the developing ion and its co-ion being of type which are substantially precluded from passage through said second column in ionic form.

4. The method of Claim 1 in which said chromatographic resin is characterized by a surface area of at least 100 M2/gm.

5. The method of Claim 1 in which said chmrnatographic resin is characterized by a solubility parameter in the range between about 7.5 and 1 5.0.

6. The method of Claim 1 in which said ionic species are anions.

7. The method of Claim 6 in which the co-ion of said counter-ion is selected from the group consisting of hydroxide, berate and carbonate.

8. The method of Claim 6 in which said second column is of a hydrogen-form cation exchange type which retains said counter-ions and which passes the co-ions of said counter-ions in hydrogen form.

9. The method of Claim 6 in which said anions are inorganic.

10. The method of Claim 6 in which said counter-ion is an alkyl ammonium ion.

11. The method of Claim 1 in which the ionic species are cations.

1 2. The method of Claim 11 in which the co-ion of said counter-ion is hydrogen.

1 3. The method of Claim 11 in which said second column is of a hydroxide-form anion type which retains said counter-ion and which passes the co-ions of said counter-ion in hydroxide form.

14. The method of Claim 1 in which said chromatographic bed consists essentially of an organic resin.

1 5. The method of Claim 1 in which said chromotographic bed comprises a hydrocarbon chain bonded to a substrate forming a reversed-phase packing.

1 6. The method of Claim 1 in which the substrate of the hydrocarbon chain comprises silica.

1 7. The method of chromatographic separation and quantitative analysis of at least a first and second ionic species of ionizable compounds in a polar mobile liquid phase, all of said ionic species being of a positive or negative charge, comprising the steps of: (a) directing said mobile liquid phase. including a counter-ion of opposite charge to said ionic species and also including a co-ion of the same charge as said ionic species, through a first column inluding a porous, hydrophobic chromatographic bed with essentially no permanently attached ion exchange sites, so that said counter-ion and ionic species form first and second reversible ion pairs which form reversible adsorptive bonds with said chromatographic resin, and so that said ionic species are differentially retarded by said bed and, thus, are chromatographically resolved in the eluent from said first column, said eluent also including a portion of said co-ions, (b) directing the eluent from said first column through a second column containing an ion exchange resin of a type which substantially precludes passage of said counter-ions and co-ions in ionic form, and which converts the ionic species of said first and second ion pairs to a more highly ionized form, and (c) directing the eluent from said second column through a conductivity cell having associated readout means to quantitatively detect said first and second ionic species.

1 8. Apparatus for the chromatographic separation and quantitative analysis of species in a polar mobile liquid phase, including a common counter-ion for said ionic species, all of said ionic species being of a positive or negative charge, said apparatus comprising: (a) a first column containing a porous hydrophobic chromatographic bed with essentially no permanently attached ion exchange sites, said bed including hydrocarbon chains capable of forming reversible adsorptive bonds with organic moieties in said polar mobile liquid phase, (b) means for supplying said polar mobile liquid phase to said first column, (c) a second column containing an ion exchange resin bed of a type and capacity to substantially preclude passage of said counter-ion in ionic form, (d) first conduit means between first and second columns, (e) conductivity measurement means and associated readout means, said measurement means including at least a first flowthrough conductivity cell, and (f) second conduit means between said second column and said conductivity cell.

1 9. The method of Claim 18 in which said chromotographic bed comprises hydrocarbon chains bonded to a substrate forming a reversed-phase packing.

20. The method of Claim 1 8 in which the substrates of the reversed phase comprises silica.

21. The method of Claim 18 in which said chromotographic bed consists essentially of an organic resin.

22. A method of chromatographic separation and quantitative analysis substantially as hereinbefore described with reference to the accompanying drawings.