Anal. Chem. 1980, 52, 1542-1544
1542
Optically Transparent Thin-Layer Electrochemical Flow Cell for Liquid Chromatography Thomas C. Pinkerton,' Kiamars Hajizadeh, Edward Deutsch, and William R. Heineman" Department of Chemistty, University of Cincinnati, Cincinnati, Ohio 4522 1
Since t h e optically transparent thin-layer electrode (OTT L E ) was first reported in 1967 ( I ) , the basic microscope slide-gold minigrid cell design has been modified to accommodate a wide variety of spectroelectrochemical research interests ( 2 ) . Cells have been fabricated with quartz plates for measurements in t h e UV ( 3 ) ,KC1 plates for measurements in the infrared ( 4 ) ,and multiple grids for increasing the optical path length ( 4 ) . Also included in the design have been oxygen removal by vacuum-nitrogen cycling ( 5 ) ,optical measurement capability a t cryogenic temperatures (6),very small cell volume (7, B ) , and temperature control capability for measuring temperature dependence (9). Optically transparent electrodes other than the gold minigrid have been used in the OTTLE: mercury-coated gold (IO) and nickel (11) minigrids, vapordeposited platinum film (12, 13),reticulated vitreous carbon (14), and platinum grid (15). Described here is a n O T T L E flow cell which has been developed for use with liquid chromatography. This cell is particularly advantageous for thin-layer spectroelectrochemical studies of mixtures of electroactive species which are sufficiently complicated to require chromatographic separation so t h a t each component can be examined separately. T h e chromatographic eluent flows directly into the OTTLE, thereby enabling individual components to be examined by spectroelectrochemistry as they elute from the chromatograph. Several advantages are gained by performing spectroelectrochemical characterization on-line with the chromatograph. A higher concentration of the electroactive species is contained in the OTTLE when t h e peak of a chromatographic band is trapped in a small volume cell as opposed to collection of the entire fraction with subsequent injection into the cell. An unknown separated component can be evaluated immediately after chromatographic purification, thereby minimizing interference from possible reactions that could occur in the time between collection of the fraction and experimentation. On-line experimentation decreases the amount of sample handling. This latter advantage becomes important when dealing with components that may be toxic or radioactive. T h e O T T L E flow cell has been demonstrated with a radiopharmaceutical analogue consisting of a technetium-99hydroxyethylidene diphosphonate (HEDP) complex mixture which was prepared by the reduction of pertechnetate with hydroxylamine hydrochloride in the presence of HEDP. This analogue is hereafter referred to as Tc(NH20H)-HEDP. The resulting Tc-HEDP complexes are radioactive, inorganic complexes which have not been obtained in crystalline form. Little is known about these complexes except t h a t they are negatively charged. When prepared with the y-emitting isotope *Tc, these complexes function as efficacious skeletal imaging agents. T h e Tc(NH,OH)-HEDP reaction mixture was separated by anion-exchange liquid chromatography with on-line spectroelectrochemical characterization of a separated yellow component. T h e techniques described here have proved useful in the characterization of radiopharmaceutical analogues of skeletal imaging agents.
EXPERIMENTAL Cell Construction. The OTTLE flow cell shown in Figure 1 was machined from a 11/4X 11/4X l / , in. Plexiglas block. A Present address: Department of Chemistry, Purdue University, West Lafayette, Ind. 47907.
li4-in.diameter hole was drilled in the block, and a x 3 / s in. diameter inset was machined to accommodate a quartz disk (Esco Products, Oak Ridge, N.J.) for the light path. The inlet and outlet ports were drilled and tapped to accommodate 'i4-28 Teflon fittings. A 5/32-in.diameter channel was drilled at an angle for the NaSCE reference electrode which was fitted with a modified 'i4-28 fitting. The inlet and outlet ports were equipped with Solder Seal R-201 rubber washers. The reference electrode was fitted with a rubber washer fashioned from the plunger tip of a 1-mL Tuberculin disposable syringe by clipping off the solid end. A 2-mil Teflon spacer (Dilectrix Corp., Farmingdale, N.Y.) was placed on the surface of the Plexiglas block upon which a 1 X 1 X in. quartz plate rested. A 120-line per inch gold minigrid working electrode (WE) and parallel auxiliary electrode (Aux 2) were sandwiched between the quartz plate and Teflon spacer, and sealed to the Plexiglas by means of epoxy. Another auxiliary electrode (Aux 1)consisting of 22-gauge platinum wire was sealed with epoxy into the outlet channel just above the reference port. Contact wires were soldered to the gold minigrids as previously described (7). Instrumentation. The OTTLE flow cell was positioned in a Harrick Rapid Scan Spectrometer (Harrick Scientific Corp., Ossining, N.Y.) as shown in Figure 2. A similar OTTLE flow cell was positioned in the reference beam of the spectrophotometer. Spectral data collection was accomplished by means of an interfaced 8080 microprocessor. The potential of the working electrode was controlled by a PAR 173 potentiostat (Princeton Applied Research). During chromatographic elution, spectra were continuously monitored on a Tektronix storage oscilloscope. Chromatographic Column. The anion-exchange column consisted of a macroporous polystyrene/quarternary ammonium resin (AG-MP1, 100-200 mesh, chloride form, Bio-Rad Laboratories) slurry packed with a 0.1 M LiC104/0.1hl HEDP solution (pH -5) into a 20 cm X 8 mm Kontes Chromaflex disposable column. A small amount of glass wool was placed at the base of the column to prevent resin passage. The column tip was fitted into a plastic stopcock type switching valve (Ace Glass Co.) which was attached to a piece of Plexiglas machined to accommodate a 1/4-28Teflon fitting. The column was then attached to the OTTLE flow cell by 3 in. X 0.8 mm Teflon tubing with 1/4-28 fittings. Sample Preparation. The Tc-HEDP mixture was prepared by first dissolving 56 mg of NH,OH.HCl (Fisher Scientific Co.) in triply distilled water with 400 mg Na,HEDP (Procter & Gamble, Cincinnati, Ohio). To this solution was added 2 mL of a 50 mM N H 4 Y c O 4solution. The total volume of the solution was 10 mL, producing concentrations of 8 mM, 80 mM, 160 mM, for Tc04-, ",OH, and HEDP, respectively. The sample was then heated at 90 O C for 20 min resulting in a final volume of approximately 5 mL. The reaction mixture was allowed to cool before introduction to the chromatographic column. Procedure. Prior to positioning of the OTTLE flow cell into the spectrophotometer, the cell was washed with triply distilled water, HPLC grade methanol, dried in an oven, and then subjected to radiofrequency plasma cleaning for 10 min. This cleaning procedure minimized the adherence of air bubbles to the cell walls, thus allowing the cell to be easily flushed with eluent from a 5-mL syringe attached to the switching valve (Figure 2). Approximately 0.5 mL of the Tc(NH,OH)-HEDP reaction mixture was placed at the top of the anion-exchange column and eluted with a 0.1 M LiC104/0.1 M HEDP solution (pH -5). The eluting complex components were visually monitored on a storage oscilloscope (10 scans/s, 250 to 550 nm), and the gravity flow elution was stopped by means of the switching valve (Figure 2) when the absorbance at 306 nm, corresponding to a yellow TcHEDP component, had reached its maximum. With the separated component trapped in the OTTLE cell, thin-layer cyclic voltammetry or spectropotentiostatic characterization was performed.
0003-2700/80/0352-1542$01.00/00 1980
American Chemical
Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980
1543
A Tc ( “ZOH yellow
I
\PLEXIGLAS SIDE
FRONT
Flgure 1.
Diagram of OTTLE flow cell. WE represents the working
0
) H E DP fraction
-200
-400
E r n v vs
NaSCE
-600
electrode
a=n -w a
CHROC MO A LTUOMGNR A P H I C
SWITCHING
7R I N G S T A N D
B Tc(NH2OH) H E D P yellow
fraction
m / % N G
VALVE
E L U A N T SYRINGE PMT (REF
P M T (SAMPLE) O T T L E ” FLOW C E L L
,
I
Arrangement of OlTLE flow cell and chromatograph in Rapid Scan Spectrometer
Figure 2.
