SEPARATIONS USING. STEP AND LINEAR VOLTAGE

WAVEFORMS AT A CHARGE - CONTROLLABLE

POLYMERIC STATIONARY PHASE

Grant Reference

The United States Government has certain rights in this invention pursuant to Contract No. W-7405 Eng 82 between the Department of Energy and Iowa State University.

Background of the Invention

The invention relates generally to an ion chromatographic system and, more specifically, to an ion chromatographic system using a charge-controllable conductive polymer stationary phase.

Ion exchange chromatography has become a common technique for separating closely similar molecules from complex mixtures. Analyte comprising ionic molecules to be separated is added to a stationary phase having ion exchange sites. The stationary phase adsorbs the ionic molecules by physical and chemical interactions. Ionic species are then selectively displaced or eluted from the stationary phase exchange sites by altering the composition of a mobile phase electrolyte that is passed across the stationary phase. Separation of molecules occurs because different molecules have different affinities for the stationary phase ion exchange sites under different mobile phase electrolyte conditions due to the individual charge characteristics of each ionic molecule.

Conventional ion exchange chromatography employs type and number stationary phases having a fixed composition, i.e. a fixed number of ion exchange sites. The present invention describes a

system in which the stationary phase does not have a fixed composition. The number of ion exchange sites on the stationary phase in the present invention changes as the stationary phase is electrochemically transformed from an ionic to a neutral form. The charge-controllable stationary phase may be manipulated during elution by linear sweep and step voltage waveforms, i.e., sawtooth and square waves, to optimize the efficiency and resolution of the separation of ionic species.

Summary of the Invention

The invention consists of a method and apparatus for ion chromatographic separations using a charge-controllable polymeric stationary phase. The charge-controllable stationary phase is a conductive polymer coated onto a conductive inert substrate. Varying the voltage applied to the stationary phase changes the conductive polymer from a neutral form to a form with a high positive charge density thereby altering the number of charged ion exchange sites available to interact with ionic sample molecules. Ionic sample molecules are separated due to their differing affinities for the ion exchange sites. Separation may be fine tuned by adjusting the voltage applied to the stationary phase in linear sweep or step voltage waveforms.

An object of the invention is to provide a method of ion chromatographic separation that can be controlled using step and linear sweep voltage waveforms applied to a charge-controllable stationary phase.

Another object of the invention is to provide a method of ion chromatographic separation having increased resolution capabilities and separation efficiency.

" Brief Description of the Drawings

Fig. 1 is a schematic representation of the apparatus of the invention.

Fig. 2 is a steady-state cyclic voltammogram obtained using the electrochemical column for a 0.036 urn polypyrrole coating that was electropolymerized onto glassy carbon particles wherein the supporting electrolyte was a 10 mM aqueous solution of LiCIO- and a scan rate of 10 mV/s.

Fig. 3 is a chromatogram showing the separation of adenosine monophosphate (AMP) and adenosine triphosphate (ATP) achieved by applying a voltage step waveform to a charge-controllable stationary phase.

Fig. 4 is a chromatogram showing the separation of AMP and ATP achieved by applying a linear voltage ramp to a charge-controllable stationary phase held at initial voltages: a) - 0.50 Volts (V); b) - 0.70 V; and c) - 0.90 V. The voltages are given with respect to a Ag/AgCl (saturated) electrode.

Fig. 5 is a chromatogram showing the separation of AMP and ATP achieved by applying a linear voltage ramp at varying sweep rates: a) 2 millivolts per second (mV/s); b) 5 mV/s; and c) 20 mV/s to a charge-controllable stationary phase.

Detailed Description of the Preferred Embodiment Figure 1 shows, generally at 10, the apparatus of the invention. The chromatographic column 10 comprises a cation exchange tube 12 containing a gold (Au) mesh strip 14 serving as a high surface area electrode. The cation exchange tube 12 containing the electrode 14 is packed with a conductive inert substrate which is then coated with the conductive polymer. The coated substrate is the charge-controllable stationary phase 16. A platinized platinum (Pt) mesh 18 serves as a high surface area counter electrode and encircles the cation exchange tube 12. The mesh-encased tube is located centrally inside a glass column 20. A side arm 22 of the glass column 20 holds a reference electrode 24. Mobile phase electrolyte occupies the solution contact channel 26 defined by the inner surface of the glass tube 20 and the outer surface of the Pt wrapped cation exchange tube 12. Gold and platinum wires 28, 30 welded to the respective Au and Pt mesh electrodes 14, 18 serve as electrical contacts to a potentiostat/galvanostat and programmer 34. Voltage applied across the stationary phase electrochemically charges the conductive polymer. The chromatographic system is constructed to ensure both effective electrical contact throughout the carbon particulate stationary phase and a reasonably uniform current density across the stationary phase.

Analyte solution is injected into a port 32 near the Au wire electrical contact 28 and flows into the cation exchange tube 12

where it will be in contact with the stationary phase 16. Ionic molecules of the analyte interact and adsorb to the charged stationary phase 16. Adsorbed ionic molecules selectively release from the stationary phase as the voltage across the stationary phase 16 and therefore the charge density of the stationary phase is varied by the potentiostat 34. Ionic molecules eluted from the stationary phase 16 are washed from the column 10 by mobile phase passing through the column driven or pushed by a pump controller 36. Eluted ions are detected by a flow cell positioned in a UV/visible spectrophotometer 38.

The charge density of the stationary phase is electrochemically altered by applying varying voltages across it, therefore, it is possible to use voltage waveforms to optimize and fine tune separations. Voltage waveforms may include simple linear or triangular ramps as well as more complex sine and square functions. Altering the sweep rate and initial and final applied voltages while applying the voltage waveforms to the stationary phase present numerous possibilities for fine tuning the separation of ionic molecules.

In the preferred embodiment the charge-controllable stationary phase 16 is a polypyrrole film electropolymerized onto glassy carbon (GC) particles. Polypyrrole is an attractive candidate for a charge-controllable substrate because it may be electrochemically transformed from a neutral, reduced form to a highly charged cationic, oxidized form. Polypyrrole is also

highly conductive which facilitates propagation of regulating waveforms throughout the stationary phase. A stationary phase of polypyrrole offers additional options for manipulating chromatographic separations. Film porosity may be varied adding size discrimination as a variable to enhance separation. Functional groups may also be substituted onto the pyrrole monomer to modify separation characteristics.

The chromatographic apparatus in Fig. 1 consists of a Nafion #110 cation exchange tube (Perma Pure Products, Inc.) plugged at one end with Teflon ^® wool and containing a Au mesh (Nilaco Corp.) high surface area electrode. The Nafion tube is packed with an inert substrate for the stationary phase and wrapped with Pt mesh (Thomas Scientific) serving as a high surface area counter electrode. The mesh-encased tube is inserted into a glass tube having a side arm holding a silver/silver-chloride (saturated potassium chloride) reference electrode. Gold or platinum wire is attached to each mesh electrode for electrical contact to the Princeton Applied Research Model 173 potentiostat/galvanostat and Model 175 universal programmer. The mobile phase electrolyte occupies the solution contact channel and is driven across the stationary phase by a Dupont Model 870 pump and Model 8800 pump controller maintaining a mobile phase flow rate at 1 milliliter per minute (lml/min.) Analyte is injected onto the stationary phase through a small port near the gold mesh. A Varion DMS 200 UV/Vis Spectrophotometer with an 8 microliter (ul) flow cell

₍ Helma Cells, Inc.) detects ionic molecules displaced from the stationary phase. Chromatograms plotting absorbance vs. time are drawn by a Houston Instrument Omnigraphic 2000 XY recorder.

The conductive polymer stationary phase was a polypyrrole film deposited on the packed glassy carbon (GC) particles within the Nafion tube which, in this arrangement, serves both as a container for the GC particles and as a cation-permeable membrane for electrical contact to reference and counter electrodes. GC particles were prepared by crushing a GC plate (Tokai Carbon) in a diamond mortar and sizing the particles using 200 to 300 mesh sieves (Fisher Scientific). Particle size was determined to be irregular and approximately 70-230 microns (um) by scanning electron microscopy. Particles were activated in an oxygen (0 ₂ ) plasma (Harrick Scientific, Inc.) for 5 minutes at a base pressure of 2x10 torr. The column was packed with a slurry of 0.90 grams (g) of GC particles in 0.1 molar (M) sodium p-toluenesulfonic acid (NaOTs). Fifty milliliters of a deaerated solution of 0.1M pyrrole (Aldrich) and 0.1M NaOTs were pumped through the column at a flow rate of 1 ml/min. Applying a voltage of +1.00 volt (V) for 2 seconds for film nucleation immediately followed by a step to +0.60V for film growth electropolymerized the pyrrole onto the GC particles forming a film. Film growth was allowed to proceed until a thickness of 0.03 um was achieved. Film thickness was estimated from the charge passed during deposition, the density of pyrrole, and an

average geometic surface area of the uncoated GC particles which were assumed to be spheres with an average diameter of 63 um, a value representative of the average size of the meshes used for sizing the particles. After film formation, the column was flushed for 30 minutes with deaerated aqueous 10 millimolar (mM) lithium perchlorate (LiClO.).

Fig. 2 shows the steady-state cyclic voltammetric current-potential (i-E) curve of the polypyrole-coated stationary phase of the preferred embodiment in 10 mM LiCl0 ₄ . The broad shape of the i-E curve reflects a large uncompensated electrolyte resistance and a slow rate of charge transfer due to the low electrolyte concentration of the LiClO. mobile phase. The low concentration of the mobile phase electrolyte sacrifices rapid charge transfer but this is necessary because the LiC10 ₄ ions in the mobile phase compete with the adenosine monophosphate (AMP) and adenosine triphosphate (ATP) ionic species to be separated for ion exchange sites in the polypyrrole film.

Example I

Retention of the monovalent anion adenosine monophosphate (AMP) and the trivalent anion adenosine triphosphate (ATP) in the column is dependent upon the voltage applied across the polypyrrole coated stationary phase. Table I shows the percentage of 1 ul injection volumes of lOmM AMP and ATP solution that is retained on the stationary phase as a function of applied voltage. At a voltage of -0.30 volts (V) (all voltages are given

with respect to the reference electrode) both AMP and ATP are absolutely retained. As the voltage becomes more negative, the retention of the monovalent AMP falls off. Trivalent ATP starts to release from the polypyrrole film at a negative voltage of -0.80V. Neither anion is retained by the column at -1.10V. Elution of the anions were monitored at each voltage for 10 min. A lOmM LiClO- solution was used as the mobile phase electrolyte.

TABLE 1 ^E applied ^(Volts > ^% Retention ³

AMP ATP

-0.30 100 100

-0.40 89.5 100

-0.50 76.2 100

-0.60 39.5 100

-0.70 6.5 100

-0.80 3.8 97.4

-0.85 2.5 94.3

-0.90 0.8 87.0

-1.00 63.5

-1.10 (0) (0)

(a) The percent retention was calculated by integrating the chromatographic peaks obtained at each applied voltage and normalizing to that at -1.10 V. The elution of AMP and ATP was monitored for 10 min.

Example II

Separation of an analyte solution comprising 0.5 ul of lOmM

ATP and 0.5 ul of 10 mM AMP was accomplished by applying a step voltage waveform to the polypyrrole coated stationary phase (see

Fig. 3). An initial voltage of -0.85V was applied to the column.

The applied voltage was stepped to -1.10V 220 sec. after

injection of the 1.Oul aliquot of analyte was added to the stationary phase. AMP is not retained by a column held at -0.85V as illustrated by the first peak on the chromatogram of Fig 3. ATP is released from the column only after the voltage is stepped to -1.10V as shown by the second peak appearing shortly after the 220 sec. mark on the chromatogram. A solution of lOmM LiCl0 ₄ served as the mobile phase electrolyte.

Example III

Fig. 4 illustrates the manipulation of ATP and AMP separation using linear waveforms to control the polypyrrole coated stationary phase. A 2ul sample of 5mM AMP and AMP was injected onto a column which was held at varying initial voltages. A 5 millivolts per second (V/s) cathodic voltage ramp was applied across the stationary phase until a final voltage of -1.10V was reached. Fig. 4(A) shows the chromatogram for a separation with initial voltage -0.50V, (B) initial voltage -0.70V, and (C) initial voltage -0.90V. By manipulating the initial voltages, the retention time of ATP may be altered from 185 sec. in (A) to 105 sec. in (C) . Adjustment of retention time leads to differences in separation efficiency. A solution of lOmM LiC10 ₄ served as the mobile phase electrolyte.

Example IV

Alterations in the slope of the linear voltage ramp provides additional means of manipulating the separation of ATP and AMP. Fig. 4 illustrates a linear sweep from -0.50V to -1.10V applied

across the stationary phase at different sweep rates. Two microliter aliquots of 5mM ATP and AMP were injected onto the column. Fig. 4 (A) illustrates a sweep rate of 2mV/s, (B) a sweep rate of"5mV/s and (C) a sweep rate of 20mV/s. Increased sweep rates markedly decrease the retention of the two anions. At a sweep rate of 2mV/s, AMP is significantly eluted from the stationary phase before ATP elution begins. (B) and (C) illustrate that as sweep rate increases resolution of the eluting species decreases. Varying the sweep rate provides another means to manipulate analyte separation on a charge-controllable stationary phase. A solution of lOmM LiC10 ₄ served as the mobile phase electrolyte.

Although the invention has been described with respect to a preferred embodiment thereof, it is to be understood that it is not to be so limited since changes and modifications can be made therein which are within the full intended scope of the invention as defined by the appended claims.