Characterization of a Microfabricated Electrochemical Detector and Coupling

UNIVERSITY OF CINCINNATI

Date: 1-Oct-2009

I, Evan T Ogburn , hereby submit this original work as part of the requirements for the degree of: Master of Science in Chemistry It is entitled: Characterization of a Microfabricated Electrochemical Detector and Coupling

with High Performance Liquid Chromatography

Student Signature: Evan T Ogburn

This work and its defense approved by: Committee Chair: William Heineman, PhD William Heineman, PhD

Carl Seliskar, PhD Carl Seliskar, PhD

11/12/2009 288 Characterization of a Microfabricated Electrochemical Detector and Coupling with High Performance Liquid Chromatography

A thesis submitted to the Division of Research & Advanced

Studies of the University of Cincinnati

In partial fulfillment of the requirements for the degree of

Master of Science

In the Department of Chemistry of the College of Arts and Sciences

2009

Evan Ogburn

B.A., Earlham College, 2005

Committee Chair: William R. Heineman, Ph.D.

Abstract

A disposable micro-fabricated electrochemical cell has been developed, characterized with multiple electrochemical systems, and coupled with high performance liquid chromatography to form a high performance liquid chromatography electrochemical detection (HPLC-ED) system.

The detection system consisted of the micro-fabricated electrochemical detector, a flow-cell and a fixture mounted with electrical connections leading from the detector to the potentiostat. The detector is easy to fabricate, inexpensive, and maintains a high performance level which makes it a practical choice for electrochemical detection. The simplicity of the fabrication process for this detector allows it to be used as a disposable device that can be replaced easily if its performance degrades. Parameters for the optimization of the performance were studied in a three-electrode system with a special focus on HPLC-ED, using ascorbic acid, acetaminophen, and potassium ferricyanide as model compounds. A separation of three pharmaceutical compounds, dextrorphan, levallorphan, and acetaminophen was also performed in order to demonstrate the performance of the novel detector in comparison to commercially available macro electrochemical detectors. It was determined that the micro-fabricated detector’s performance was comparable to traditional non-disposable electrodes in these systems, especially HPLC-ED, which was the system of focus. The calibration curves constructed for each model compound showed linearity (R2> 0.99), the limit of detection reached picomolar (< 100) levels, and the peaks generated were more resolved and free from interference when the micro-fabricated detector was used. When this novel detector was used, the limit of detection reached 1.00 nM for acetaminophen, 1.00 nM for ascorbic acid, 50.0 nM for dextrorphan and 80.0 nM for levallorphan. When the nondisposable commercially available detector was used, the limit of detection reached 0.0500 nM for acetaminophen and 1.00 nM for ascorbic acid. Dextrorphan

iii and levallorphan were detected as low as 2.5 nM and 5.00 nM, respectively, with the commercial system when samples were diluted in mobile phase, but the signal reproducibility was not good when tested in human plasma. The results within this thesis demonstrate that a micro-fabricated electrochemical detector can operate at a high level of performance when used in HPLC-ED, while maintaining cost effectiveness.

Acknowledgements

I would like to convey my genuine pleasure and gratitude to my advisor Dr. William R.

Heineman from the Department of Chemistry, University of Cincinnati for giving me the

opportunity to further my education and experience in the field of chemistry.

My thanks are also extended to Dr. Carl J. Seliskar who has served on my committee from the

beginning of my studies here at UC and Dr. Michael Dzwietakowski from Yellow Springs

Instruments, Inc. for offering a great deal of kind help and knowledge throughout my studies.

I would also like to thank all of the members of the Center for Chemical Sensors and Biosensors

at UC as well as the members of Dr. Heineman’s and Dr. Seliskar’s group for support and

guidance. I would like to acknowledge Yellow Springs Instruments, Inc. and the Indiana

University School of Medicine Department of Clinical Pharmacology for providing the detectors

and apparatus necessary to complete my research.

Finally, I would like to thank my girlfriend, family and friends for the love and support they have shown me throughout the duration of my academic career.

Table of Contents

Abstract iii-iv Acknowledgements vi I. Introduction 1-3 Electrochemical Detection as It Occurs in HPLC-ED 3-5 The Basics of Chromatography and Its Use in HPLC-ED 5-7 Characteristics of HPLC-ED Separation and Detection 8-9 Development of a Novel HPLC-ED System 9-10 Background on Microfabricated Electrochemical Detectors Used in HPLC-ED 11-12 Electrochemical Detector Origin 12 Electrochemical Detector Description 12-16 Flow Injection Analysis (FIA) 17-18 Electrochemical Detection as It Occurs in FIA 18-19 Cyclic Voltammetry and How It Is Applied with Microfabricated Electrochemical Detectors 19-22 II. Experimental Chemicals 22 Standard Samples 23 Plating the Reference Electrode Within This System 23-27 HPLC-ED (Commercial System) 28 HPLC-ED (Novel System) 28-29 Flow Injection Analysis (FIA-ED) 29 Cyclic Voltammetry 30 III. Results Cyclic Voltammetry 30-33 Electrochemistry of Ascorbic Acid w/ Conventional and Microfabricated Electrodes 33-34 Flow Injection Analysis 34-38 HPLC-ED 38-40 Degradation of Detector and Signal 41-43 Analyzing Performance when the Degradation of a Novel Microfabricated Electrode Begins 43-44 The Effect of Concentration of Analyte on Electrochemical Detection in HPLC-ED 44-46 Comparing the Novel HPLC-ED System with a Commercialized System 46-47 Limit of Detection 48-50 Band Broadening as a Result of Longitudinal Diffusion 50-54 Chromatographic Properties of Each System 54-56 Separation of Pharmaceutical Compounds in Multiple Matrices 56-58 Chromatographic Attributes 59-67 IV. Conclusion 67-68 References 69

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List of Figures and Tables

Figure 1. L-ascorbic acid (A) and acetaminophen (B) chemical structures. Both compounds were used as model analytes to test the performance of the electrochemical detector. 10 Figure 2. Schematic of the microfabricated electrochemical detector presented within this thesis. 13 Figure 3. (A) A photograph of the entire electrochemical detector presented in this thesis. The circles located on the detector are valve connections allowing liquid to enter and leave the flowcell apparatus. (B) Close up photograph of the electrodes on the detector. The dimensions of these electrodes are listed in Table 1. 15 Table 1. Dimensions of the microfabricated electrochemical detector and coupled microfluidic device. 15 Figure 4. Image of solution fluidic flow channel found in the flowcell used to couple the microfabricated electrochemical detector with FIA and HPLC. 16 Figure 5. A microfluidic-chip that is used to direct fluid flow across the surface of the electrodes on the microfabricated detectors, enabling electrochemical detection. 18 Figure 6. Potential vs. Time, excitation signal for cyclic voltammetry. 20 Figure 7. CV of 4 mM potassium ferricyanide in 1M KNO3 initiated at 0.8 V potential vs. Ag/AgCl in negative direction at 20 mV/s. Gold working electrode used, area = 2.01 mm2. 21 Figure 8. (A) Uniform layer of Ag/AgCl plated on gold to form the reference electrode and (B) a layer that is nonuniform. Environmental scanning-electron microscope (ESEM) images of a uniformly plated (C) and non-uniformly plated (D) reference electrode in which plating was performed simultaneously. 25 Figure 9. CV of 4 mM K3Fe(CN)6 in 1 M KNO3 initiated at 0.8 V in the negative direction at a scan rate of 20 mV/s using (A) the detector pictured in Fig. 8A, uniform plating and with (B) the detector in Fig. 8B, non-uniform plating. 31 Figure 10. (A) CV of a conventional size gold working electrode in a solution of 4 mM K3Fe(CN)6 in 1 M KNO3. (B) CV of the same solution obtained with the microfabricated electrochemical detector presented in this paper. 32 Figure 11. Effect of varying scan rate on cyclic voltammetry of 10 mM ascorbic acid in 10 mM KH2PO4 using microfabricated electrochemical detectors. 34 Figure 12. Electrochemistry, performed with the FIA-ED system, of 2 mM ferricyanide in buffer B at a potential of -75mV. The pressures at which the results were recorded are labeled appropriately. 35 Figure 13. Average peak current (µA) of 2mM potassium ferricyanide analyzed in the FIA-ED system at a working potential of -75 mV. The pressure applied to induce flow was varied between 0.5, 1.2, 0.7, 0.5 and 0.3 psi. The run time for this analysis was 48 h. 36 Figure 14. Peak current (µA) of 2mM potassium ferricyanide analyzed in the FIA-ED system at a working potential of -75 mV. The pressure applied to induce flow was 0.5 psi. The first 17 h of analysis is shown here. 37 Figure 15. Hydrodynamic voltammograms for acetaminophen (50 µM) (A) and ascorbic acid (100 µM) (B). 40

viii

Figure 16. Stability of ascorbic acid (1 µM) and acetaminophen (0.25 µM) over a 10 hour period as shown by peak height. 41 Table 2. Stability of HPLC-ED Response of Acetaminophen (0.25 µM) and Ascorbic Acid (1.0 µM). 42 Figure 17. The response of acetaminophen (50 nM) using 3 different microfabricated electrochemical detectors of different condition, non-working (A), degrading (B) and new (C). 44 Figure 18. Calibration curve of ascorbic acid (A) and the effect of concentration on peak height (B) for acetaminophen detection by HPLC-ED using the microfabricated electrochemical detector. 45 Figure 19. HPLC-ED of acetaminophen (1 µM) examined with the novel microfabricated electrochemical detector (A) and a commercial detector (B). 46 Figure 20. Response for acetaminophen (1.0 x 10-9 M) using the novel HPLC-ED (A) and the response for acetaminophen (5.0 x 10-11 M) in a commerical HPLC-ED system (B). Note the unit increments in each figure in order to depict the relationship between detectors and peak current (nA). 49 Figure 21. Dextrorphan (1 µM) and Levallorphan (1 µM) measured in the commercial (A) and novel (B) HPLC-ED systems. 51 Table 3. Calibration Curve Data For Ascorbic Acid and Acetaminophen. 53 Figure 22. Calibration curves constructed when ascorbic acid was analyzed using commercial HPLC-ED system (Blue) and the novel HPLC-ED system discussed (Red). Acetaminophen was analyzed using a commercial HPLC-ED system (Green) and with the novel HPLC-ED system (Yellow). 54 Figure 23. The capacity factors (k’) for ascorbic acid (Black) and acetaminophen (Red) in the novel HPLC-ED system are compared to the capacity factors for ascorbic acid (Green) and acetaminophen (Blue) in the commercial HPLC-ED system. 55 Figure 24. Chemical structures of the base structure of compounds belonging to the morphinan family (A), levallorphan (B) and dextrorphan (C). Structures were constructed by the author using ChemDraw. 58 Figure 25. Calibration curves of levallorphan and dextrorphan spiked in human plasma and in mobile phase constructed using the novel (A) and commercial (B) HPLC-ED system. These concentrations were plotted versus the ratio of AUC response of drug to the AUC response of the internal standard (acetaminophen, 50 µM). 60 Figure 26. Injections of dextrorphan (500 nM) and levallorphan (500 nM) in mobile phase onto a novel (A) and commercialized (B) HPLC-ED system. Detection occurs at 1.0V for both compounds in the novel system and at 0.75V for both compounds in the commercialized system. 63 Figure 27. Separation of acetaminophen (IS, 50 µM), dextrorphan (0, 0.5, 1, 5, 10, 25, 50, 75, 100 µM) and levallorphan (0, 0.5, 1, 5, 10, 25, 50, 75, 100 µM) in mobile phase detected with the novel HPLC-ED system (A) and the commercial HPLC-ED system (B). 64 Figure 28. Chromatograms of dextrorphan (0, 0.5, 1, 5, 10, 25, 50, 75, 100 µM) and levallorphan (0, 0.5, 1, 5, 10, 25, 50, 75, 100 µM) in spiked human plasma analyzed with a commercial HPLC-ED system (A) and a novel HPLC-ED system (B). 66

I. Introduction

For years there has been an ongoing effort in the field of electroanalytical chemistry to replicate the performance of macroelectrodes in electrodes of smaller size. These electrodes are currently referred to as microelectrodes. According to the International Union of Pure and

Applied Chemistry (IUPAC), “a microelecrtode is defined as any electrode, whose characteristic dimensions, under the given experimental conditions, is comparable to or smaller than the diffusion layer thickness (δ)” (1). Macroelectrodes, on the other hand, “are estimated as an

electrode of infinite dimension”, meaning its specific dimensional definition is rather arbitrary

(1). Most miniature electrode systems today are designed to couple with only one

electroanalytical technique such as cyclic voltammetry (CV) or high performance liquid

chromatography coupled with electrochemical detection (HPLC-ED). The microfabricated electrochemical detector presented in this thesis shows the ability to be coupled with multiple electroanlytical techniques that span all ranges of voltammetry. The versatility shown by this microfabricated electrochemical detector is further demonstrated in these separate electroanalytical systems with the main focus being its performance with HPLC-ED. The comparisons made with conventional size electrochemical detectors demonstrate the practicality and efficacy of this novel detector in the field of electroanalytical chemistry.

High performance liquid chromatography coupled with electrochemical detection

(HPLC-ED) has continued to increase in popularity over the past 30 years. In 1974, Bioanalytical

Systems commercialized the basic fundamental concepts for the HPLC-ED technique, and the technology surrounding this field has continually evolved. The number of HPLC-ED

publications has grown almost 500% since its first introduction into the analytical world (2). The

rise in popularity of this technique can be attributed to its ability to detect certain compounds

with a much better limit of detection than is possible with more commonly used techniques such

as UV-Visible absorption and fluorescence.

Direct current amperometry is the premier technique used in the electrochemical detection of compounds by HPLC-ED. Amperometry has been discovered to be more sensitive

and have lower detection limits for certain compounds than most of the spectroscopic techniques

that are available today. Detection limits as low as 0.1 pmol have been achieved for oxidizable

compounds, although they are found to be about 10 times greater for reducible compounds.

Amperometry works by measuring the current in response to a fixed potential applied at the

electrode. However, in order for HPLC-ED to be performed effectively, the electrochemical

detector must possess some very specific characteristics. The detector must have a rapid response

time, wide dynamic range and low active dead volume (<20 µL) (2). Generally, a transducer

with a small volume (<1 µL) will demonstrate these properties. Electrochemical detectors are

advantageous because they are typically less expensive than spectroscopic detectors and do not

require filters or monochrometers, which are necessary in an HPLC system.

Commercially available detectors, when used in HPLC-ED, require additional maintenance in order to achieve and maintain an optimal level of performance within a three electrode system. The surface of the working electrode needs to be polished frequently in order to remove compound residue deposited from the oxidation or reduction of electroactive species.

This deposition can lead to fouling of the electrode. Polishing electrodes can become tedious and expensive, but is a necessity when non-disposable electrodes are used. The novel detector discussed in this thesis is fabricated to be disposable, which eliminates the required cleaning that commercially available non-disposable electrodes call for. When the electrochemical

performance of these novel micro-fabricated electrochemical detectors begins to degrade, a new detector is installed and the performance is restored. Therefore, the microfabricated electrochemical detector being described provides a more practical and cost-effective method for

electrochemical detection than a typical HPLC-ED system using non-disposable electrodes.

Electrochemical Detection as It Occurs in HPLC-ED. When a conventional working electrode’s dimensions become smaller, the behavior of the electrode then changes. The mode of mass transport of analyte to the electrode surface changes from linear to hemispherical as the electrode area decreases, leading to an increase in the current density produced by the electrode system. The small size of the microelectrode allows for mass transport to take place more efficiently (1).

The electrochemical aspect of HPLC-ED takes place after the eluent has passed through

the analytical column and has entered the flowcell containing the three electrode system. As the

eluent enters the flowcell, it is directed across the surface of a planar detecting electrode via a

precise channel. Electrolysis immediately takes place, considering the fixed potential of the

electrode is greater than the redox potential of the studied compound. If the potential is more

positive than the redox potential then the analyte is oxidized. If the potential is more negative a

reduction occurs. These reactions cause measurable current to arise, which is the basis for

detection of an analyte in amperometric techniques. The current is proportional to the

concentration of the analyte that flows through the channel. HPLC-ED is an example of amperometric detection in electrochemistry, where the potential of the electrode is always set to a fixed value. Using Faraday’s Law we can calculate the value of the measurable current being produced by the redox-reaction taking place at the electrode surface. Faraday’s Law states that the charge produced (Q), in coulombs, is equal to the moles of material electrolyzed (N), the

3 number of moles of electrons gained or lost during the reaction (n), and Faraday’s Constant

(96,500 coulombs/mole of electrons) (F).

Q = nNF (1)

Differentiating this equation with respect to time gives the current, which is equivalent to the rate at which material is being electrolyzed. The equation below shows the relationship between the redox reaction that is taking place at the surface of the electrode and the measurable quantity, which in this case is the current, i.

dQ/dT = i = nF dN/dT (2)

Many factors affect the ability of the redox-reaction to take place, as well as the rate at which it happens in such systems. One very important factor is the composition of the working electrode. In the early days of this technique “carbon paste”, a mixture of graphite and a dielectric material such as paraffin wax was used in most working electrodes (2). This was due to the properties of the material, which were found to be excellent for facilitating redox-reactions of organic molecules. Currently, glassy carbon electrodes are used more frequently than carbon paste electrodes. These electrodes used in traditional voltammetric methods may last up to a few years, but in many cases there are contributing factors that will decrease this lifetime drastically.

If the fixed potential exceeds 1V for an extended period of time or solutions contain chemicals that adsorb on the electrode surface, the performance of the electrode will degrade. Material that is adsorbed into the electrode surface and consequently interferes with electron transfer is

referred to as electrode fouling. Therefore, non-disposable electrodes, used in voltammetric

techniques, can require frequent cleaning and polishing in order to maintain high performance.

Because of the factors mentioned above, the detectors used in HPLC-ED systems require periodic cleaning, which is an unwanted feature. This cleaning is usually done by disassembling the electrochemical cell, polishing the surface of the working electrode, and removing any formed salts present on the reference and auxiliary electrodes. Disposable detectors, such as the novel detector described here, have the advantage of being easily replaced if the performance degrades. Periodic cleaning of the disposable electrodes is still required, however, the time spent cleaning and effort expended is drastically reduced. The necessity of cleaning the electrode surface periodically is marked as a major disadvantage of all HPLC-ED systems.

The HPLC column precedes the electrochemical detector in an HPLC-ED system; therefore, some of the sample components never reach the electrode surface. The analyte being injected into the system will have little or no affect on the surface of the electrode; the adsorption of the analyte or the electrolysis of the product on the electrode surface is minimized due to continuous flow of mobile phase across the electrode surface. Therefore, since the analyte eluent zones are so narrow within HPLC-ED, the analyte will be in contact with the surface of the electrode no longer than a minute’s time. Thus, the performance of electrodes is more conveniently maintained in HPLC-ED systems than in a typical voltammetric experiments.

The Basics of Chromatography and Its Use in HPLC-ED. High-performance liquid chromatography is one of the most popular analytical techniques for chemical separations due to

the fact that most compounds are not sufficiently volatile for gas chromatography and have better

detection properties in liquid form (3). HPLC utilizes high pressure in order to pump solvent

5 through a closed column leading to a high-resolution separation of chemical compounds.

Compounds that have a strong affinity for the stationary phase will remain on the column for longer periods of time than those that are weakly attracted. This phenomenon is the basis for simultaneously separating and detecting multiple compounds using HPLC. Adequate separation is achieved by making modifications to the mobile and stationary phases within a given system.

In order for a useful separation to occur, the compounds must elute the column at different times so that each compound reaches the detector independently, providing the selectivity of the system. The longer the solute spends passing through the column, the broader the peak band becomes, which can affect the extent of separation of compounds when multiple compounds are being detected in a sample. For accurate and precise quantification to take place in HPLC-ED adequate separation between the peaks is necessary. This separation is referred to as resolution, which can be determined by Equation 3:

R = 0.589Δtr / w1/2av (3) where Δtr is the separation between peaks (in units of time) and w1/2av is the peak width at half height (3). A resolution of 2 or greater is desired for a rugged method, allowing slight fluctuations to occur within the conditions without compromising peak resolution.

The column efficiency is ultimately the most important characteristic of an HPLC system, and is defined as the rate at which the solute equilibrates between the stationary and mobile phases. Modifying the stationary-phase particles within the column can result in an increase of efficiency in the column. The smaller the stationary-phase particle size the greater the efficiency of the column. Typically the particle sizes range from 3-10 µm. Increasing column efficiency in

HPLC is also equivalent to decreasing the plate height in the van Deemter equation. The van

Deemter equation is:

H = A + B/ux + C ux (4)

where H is the plate height, ux is the linear flow rate of the solvent, and A, B, and C are constants

that are specific for a given column and stationary phase (3). For a packed HPLC column A, B,

and C are not equal to zero, therefore, all three terms lead to band broadening which decreases

the resolution of the column. The van Deemter equation describes how the column

characteristics and flow rate of solvent are related to one another. The first term, A, is known as the multiple paths term. When applied to packed HPLC columns it is used to analyze the

uniformity of the solvent flow. Smaller particles give a more uniform flow than larger particles.

Small particles decrease the finite equilibration time (C) in an HPLC system along with the plate

height (B). When stationary phase particles are smaller, the distance that the solute must diffuse

to partition in and out of the stationary phase is smaller; ultimately decreasing the time it takes

for the solute to equilibrate between the stationary phase and the mobile phase. The longitudinal

diffusion term, B, explains the broadening of a chromatography band while in the column. When

the band first enters the column it constantly broadens by diffusing away from the center as it

moves throughout the column. As the flow rate of the solvent increases, the time the band spends

on the column decreases, which also decreases the longitudinal diffusion taking place. The van

Deemter equation is used as a critical analysis of a chromatographic system and helps one

improve the performance of the analytical column in order to reach the highest possible

resolution of a given signal.

Characteristics of HPLC-ED Separation and Detection. The performance of an HPLC system

can be described and gauged based on a series of known attributes. The main attributes used to

determine adequate separation are capacity factor, resolution, operating pressure, and asymmetry factor. The capacity factor (k’) measures the retention time of the analyte (tr) compared to the time the mobile phase takes to pass through the column (tm). The equation for this is as follows:

k’ = (tr – tm)/tm (5)

The time the mobile phase takes to pass through the column can be estimated by the equation:

2 Tm ~ Ldc /(2F) (6)

Where L is the column length in centimeters, dc is the column diameter in centimeters, and F is the flow rate at which the solvent is passed through the column in milliliters per minute (3). In order to correctly calculate the retention time and capacity factor for the separation of a compound in the present HPLC-ED, a sample was injected multiple times over the course of an extended period of time. The chromatographic attributes for this system were determined for

each compound detected by analyzing the quality of the separation. For each compound adjusted

retention time, relative retention, capacity factor (k’) and resolution (R) were determined using

this system. The capacity factor was calculated two separate ways, both ways using equation 5,

with tm being the time. The capacity factor was first calculated with the tm value obtained from

the actual chromatograms. A second calculation was then performed using a theoretical value of

tm which was determined from equation 5 above. Resolution was estimated as the change in

retention time (in units of time) per average peak width (in corresponding units) of adjacent

peaks within a separation.

Development of a Novel HPLC-ED System. When performing the method development for the

current system many criteria came into play, criteria that were used in order to optimize the

performance of the analytical column being used, the detection technique (ED) and the available

equipment at hand. The goal when developing this system was to adequately separate chemicals

in a reasonable time period, while achieving an electrochemical detection using a

microfabricated electrochemical detector of high sensitivity. Reversed-phase chromatography

was used to separate the compounds within this novel HPLC-ED system. Reversed-phase

chromatography utilizes a non-polar or weakly polar stationary phase with a more polar mobile

phase to separate chemicals, typically mixtures containing low-molecular mass organic

compounds. Understanding the properties of reversed-phase chromatography allowed model

analytes to be chosen in order to evaluate the performance of the microfabricated

electrochemical detector.

L-ascorbic acid (1A), a water soluble vitamin, is a lactone with a 3,2-enediol group that is extremely effective as a reducing agent and is used in wide applications within analytical chemistry. This compound (Figure 1A) was chosen as one model analyte studied with this novel

HPLC-ED system. Due to ascorbic acid’s use in food chemistry, preventing color and aroma changes in food products and extending storage time, the rapid monitoring of the compound becomes an important step during production and quality control stages of food development (4).

Acetaminophen (1 B), N-acetyl-p-aminophenol, is used extensively as an alternative to aspirin and is important because it does not have an effect on the gastric mucosa within the human body

(5). This compound (Figure 1B) was the second model analyte chosen for evaluating the novel

HPLC-ED system. It was also used as an internal standard for certain pilot experiments throughout this research. These compounds were chosen because they are electrochemically oxidizable and can be separated by reversed-phase HPLC.

A. B.

Figure 1. L-ascorbic acid (A) and acetaminophen (B) chemical structures. Both compounds were used as model analytes to test the performance of the electrochemical detector.

Sample preparation is another important step in HPLC method development and is necessary when trying to produce a reproducible and stable detection signal. Modifications to the sample preparation can drastically improve the reproducibility, sensitivity, selectivity and stability of and HPLC system. Since electrochemical detection was the detection method of choice, the mobile phase as well as the standard samples had to be prepared in a way that would allow for adequate detection of the model analytes. The methods section provides in detail how the samples for this study were prepared.

Background on Microfabricated Electrochemical Detectors Used in HPLC-ED.

Microfabrication of metallic working electrodes has continued to develop since its introduction

during the 1980’s from a thin-film technology. Thin-film technology utilizes the deposition of a

thin layer of metal on a substrate ranging from silicon wafers (6, 7) to glass microscope slides (8,

9). Metallic thin-film electrodes reported thus far have been used as biosensors and microdisk electrodes in which the metal is deposited on a polyimide layer (10-13). This same polyimide layer is used for the novel detector presented here; however, the electrode layers that were chosen for the working electrodes as well as the electroanalytical techniques differed.

There are few reports in the literature that describe thin-film electrodes as used in high performance liquid chromatography. In 2003 Cheng, Jandik and Avdalovic developed and characterized a disposable gold working electrode similar to the detector in this paper, but coupled it with high-performance ion chromatography with integrated pulsed amperometric detection (IPAD) (14). The working electrode was presented in four separate shapes as a circle

(1.00 mm diameter), a square (area = 0.784 mm2) and two rectangular shapes (area= 0.784 mm2)

which compare in area to this microfabricated electrochemical detector (0.178 mm2). When the

gold was deposited on a polyimide layer the signal stability for various amino acids was unstable

and decreased to 50% or less within hours. This prompted Cheng, et al. to perform an analysis of

a large sampling of different polyimide substrates and their effect on the stability of the detector

response. The Kapton layer (used in this novel detector) showed poor signal stability over the

course of 24 hours (signal decreases by 20%) and a limit of detection that was greater than two

times the limit of detection for a non-disposable electrode (>100 ρmol) (14). The limit of

detections from the two systems can not be directly compared since different compounds were

used in the characterization but can be used to show their capabilities. The data generated with

11 the microfabricated electrochemical detector presented in this paper does not reflect the same results that were produced by Cheng, et al. The microfabricated electrochemical detector shows excellent stability in excess of 24 hours of continuous use and has reached a limit of detection in the nM range for multiple compounds.

Electrochemical Detector Origin. Metal electrodes are less common in HPLC-ED but have been increasing in popularity over the past 15 years; they are primarily documented for the detection of inorganic species (2). Developments in microelectronics technology in the 1980 have made it possible to construct very small metal electrodes and measure reliably the very low currents that they produce. These small electrodes began to be studied and applied throughout the electroanalytical field (1). From these studies several important practical advantages associated with the use of microelectrodes were discovered and documented. These advantages include a decrease in the ohmic drop of potential (IR) and a fast establishment of a steady-state signal, increase in current density due to enhanced mass transport giving increased signal to noise ratios (1). The applied potential can be scanned rapidly in microelectrode systems because the charging current is smaller and electrolysis produces lower levels of current. The signal of the electroactive substance is also enhanced when microelectrodes are used in electroanalytical systems with flow, because the substance being detected is constantly replenished within the diffusion layer as the solution passes over the three electrodes. These advantages add to the attractiveness of this novel HPLC-ED system.

Electrochemical Detector Description. In this project the novel microfabricated electrochemical detector contains a three-electrode system, an Au working electrode, Ag/AgCl plated reference electrode and a Pt auxiliary electrode. The schematic layout of this detector is

shown in Figure 2 and the exact dimensions of the detector as well as the electrodes it possesses

are listed in Table 1. A photograph of the microfabricated electrochemical detector after

fabrication at YSI, Inc. (Figure 3A) and an enlarged photograph of its three electrode band

(Figure 3B) show the detector furthermore in its authenticity.

Figure 2. Schematic of the microfabricated electrochemical detector presented within this thesis.

The working and auxiliary electrodes are composed of a bottom substrate of polyimide

on which a layer of chromium is deposited. This polyimide layer is visible in Figure 3A, giving

the detector the brownish color. This chromium layer is used to bind a top layer of metal that will

act as the electrode surface for electrolysis to actually take place. The auxiliary electrode is

comprised of platinum bound to the chromium layer and the working and reference electrodes are made of a gold layer bound to chromium. However, the reference electrode is plated with silver which is the coated with silver chloride and is depicted in Figure 3B. A cover layer of polyimide leaves only the electrical contact pads and the electrode surfaces exposed. The electrodes are connected directly to the electrical connection pads using platinum strips. The

13 connection pads allow for the potential from the potentiostat to be applied to the detector and uniformly reach the surface of each electrode, creating a good cell for an electrochemical reaction to take place.

The method used in plating the reference electrode found on the microfabricated electrochemical detector was developed based on an amperometric biosensor, developed by

Madaras and Buck (11). In order for the reference electrode to maintain a performance similar to conventional electrodes, Madaras and Buck determined that the surface of the electrode had to be modified. There were two modification steps that took place while forming their biosensor; the first being the deposition of a monolayer onto the electrode band surface and the other was the modification of the reference electrode surface by multilayer deposition (11). When YSI Inc. developed the detector presented here multilayer deposition was also used for the modification of the reference electrode. First, a layer of chromium is deposited onto the surface of the electrode band. A layer of gold metal is then deposited on the chromium layer forming the surface of the reference electrode. Multilayer deposition is then used to deposit a layer of silver ions onto the surface of the reference electrode; half of these ions are then converted into silver chloride in the presence of hydrochloric acid. Modifying the electrode surface during micro-fabrication became common-place in the field because it improved electrode performance and increased lifetime.

A. B.

Figure 3. (A) A photograph of the entire electrochemical detector presented in this thesis. The circles located on the detector are valve connections allowing liquid to enter and leave the flowcell apparatus. (B) Close up photograph of the electrodes on the detector. The dimensions of these electrodes are listed in Table 1.

Table 1. Dimensions of the microfabricated electrochemical detector and coupled microfluidic device.

Electrochemical Detector Length (cm) 2.54 Electrochemical Detector Width (cm) 0.508 Three Electrode Band Width (cm) 0.0304 Three Electrode Band Length (cm) 0.147 Reference Electrode Area (cm2) 3.10E-04 Working and Auxiliary Electrode Area (cm2) 1.70E-03 Electrical Connection Pad Area (cm2) 8.13E-03 Diameter of injection valve on syringe pump flowcell (cm) 8.06E-02 Fluidic Channel height (µm) 7.50 Fluidic Channel Width (µm) 25.0

The channel pictured in Figure 4 is a key component in the performance of this microfabricated

electrochemical detector. This channel is used to guide liquid across the surface of the electrodes

and regulates the pressure of the flow. This channel is about 25 µm wide and less than 10 µm

deep. These dimensions dictate the pressure or flow rate in which the mobile phase can be

delivered in this system. If the pressure across the column of this system reaches 3000 psi then

the pressure across the fluidic channel (Figure 4) becomes too great and the flowing liquid can

not be contained and subsequently the system will leak. The optimum operating range for this

system is between 1100 and 2000 psi across the column, translating to a flow rate of 0.25 – 0.50

mL/min in the current system. When operated within this range of flow rates the lifetime of the

detector, its performance, the performance of the fluidic device and column efficiency are at

optimum levels.

Figure 4. Image of solution fluidic flow channel found in the flowcell used to couple the microfabricated electrochemical detector with FIA and HPLC.

Flow Injection Analysis (FIA). Hydrodynamic voltammetry is involved with multiple

electrochemical techniques. The hydrodynamic flow of electrolytic solution across the surface of

an electrode allows for an increase in mass transport enhancing the sensitivity of the system.

These electrodes were used in multiple hydrodynamic systems the first being flow injection

analysis (FIA). In the FIA system a pressure-driven flow injection analysis system (Yellow

Springs Instruments Inc., Yellow Springs, OH) was used to pump the analyte solution across the surface of the electrode. A specifically fabricated microfluidic chip was used to direct the flowing analyte solution in a variety of paths across the surface of the electrodes (Figure 5). The microfabricated electrochemical detector is attached in the top right corner of the microfluidic chip leaving only the electrode surface and contact pads exposed, each are labeled in Figure 5.

Once the detector is attached, the chip is placed inside a specially fabricated fixture that connects

to analyte reservoirs via capillary tubing. There are four main sample inlets within this flow-

injection analysis system, S1, S2, S3 and S4, and they are all used to introduce sample from the

reservoirs into the microfluidic chip (Figure 5). The inlet that is labeled “C” is used to pump the

mobile phase throughout the system, continuously. Once the mobile phase is flowing the analyte

pathway is dictated by opening two of a possible 4 pressure controlled valves that exist in the

FIA system; these valves are labeled 5, 6, 7, 8 (Figure 5). Each valve controls a specific sample

inlet, for instance, valve 5 controls inlet S1 and when valve 5 is opened the sample is introduced

through inlet S1. Two main pathways were used in this research to flow the sample solution from the inlet, throughout the microfluidic chip and eventually across the electrode surfaces. The first pathway flows through valve 5 directly to valve 8. Valve 4, also known as the injection inlet, is then opened simultaneously with valve 8 allowing the analyte to enter the mobile phase and proceed toward the electrode surfaces. Valve 2 is opened next directing the waste is through the

outlet, “W2”, this is specifically used as the large load outlet. This described pathway is the

longer of the two pathways and can pump a maximum of 680 µL of analyte through the system per injection; the smaller sample load pumps 315 µL of analyte per injection. These volumes are based on the volume of the channels being accessed by the pressure control valves opened within each system. The FIA system allowed for a more precise control in solution flow based on the pressures applied to the system which ranged between 0.1 and 10 psi. Pressure control allows for a constant flow of solution making the reproducibility of results much easier to achieve.

Figure 5. A microfluidic-chip that is used to direct fluid flow across the surface of the electrodes on the microfabricated detectors, enabling electrochemical detection.

Electrochemical Detection as It Occurs in FIA. Amperometry coupled with the FIA system measures the electrochemical current of the flowing analyte solution in response to a fixed potential at the electrode surface. Flowcells increase the sensitivity of the system due to the increase in mass transport towards the electrode. In the flow system there are three areas of flow.

The first area is turbulent flow, which occurs throughout the bulk solution of sample. As the

distance between the point in solution and the surface of the electrode decreases, a region of

laminar flow forms. The laminar flow decreases as the electrode surface is approached and

eventually forms a stagnant layer of solution at the electrode. This layer is termed the Nernst

diffusion layer and has a thickness of δ. The reduced species moves away from the surface of the

electrode, pulled back into the bulk solution by the laminar flow region, and is immediately

replaced by new solution ready to undergo the redox process (2). This system was chosen as a

precursor to HPLC-ED detection because it incorporates pressure driven flow of analyte with

amperometric detection.

Cyclic Voltammetry and How It Is Applied with Microfabricated Electrochemical

Detectors. Cyclic voltammetry (CV) has developed into a very practical and perhaps the most

versatile electroanalytical technique (15). It has many advantages and properties that can be used to evaluate microfabricated electrochemical detector designs. In order to evaluate the electrochemical performance of the microfabricated electrochemical detector CV was first performed with these detectors and the results were analyzed and compared to commercial electrodes. The basic principle of CV is that the potential applied to the working electrode is cycled over a certain range and the resulting current is measured, while the electrode is immersed in a stagnant solution. The working electrode potential, as in many electroanalytical techniques, is applied versus a reference electrode whose half-cell potential remains constant (15). The triangular waveform below gives an example of how the potential is scanned in CV. The peaks of the triangles are known as “switching potentials”, the vertical slopes are the forward scans and the ascending slopes are the reverse scans. One cycle includes a forward and reverse scan and multiple scans can be performed. The voltammogram that is produced plots the resulting current

versus the potential applied. When the potential that is applied is sufficiently negative to reduce a

compound the resulting current is known as the cathodic current. When it is positive enough to

oxidize the compound the resulting current is known as the anodic current.

Cycle 1 Cycle 2

-0.2

V 0.2

0.4

0.6

0.8

1 0 10 20 30 40 50 60 70 80

Time, s

Figure 6. Potential vs. Time, excitation signal for cyclic voltammetry.

When interpreting the results of a CV there are important parameters to consider. The anodic

peak current (ipa) and the cathodic peak current (ipc) and the corresponding peak potentials (Epa) and (Epc) (15). These values are generally provided with most software for CV used today but

can be determined from an ideal cyclic voltammogram as in Figure 7.

Figure 7. CV of 4 mM potassium ferricyanide in 1M KNO3 initiated at 0.8 V potential vs. Ag/AgCl in negative direction at 20 mV/s. Gold working electrode used, area = 2.01 mm2.

The formal reduction potential (E°) is centered between Epa and Epc, for an electrochemically reversible couple, a redox process where both species are rapidly exchanging electrons with the working electrode and can be calculated using the following relationship:

E° = (Epa + Epc) / 2 (7)

The number of electrons that are transferred in a reversible process can be determined based on the separation between peak potentials:

ΔEp = Epa - Epc 0.059/n (8)

The peak current for the first scan can be calculated via the Randles-Sevcik equation:

5 3/2 ½ ½ ip = (2.69 x 10 ) n AD Cv (9)

where ip is peak current (A), n is the number of electrons, A is the area of the working electrode

(cm2), D is the diffusion coefficient (cm2/s), C is the concentration of the analyte being tested

(mol/cm3) and υ is the scan rate (V/s) (15). The cathodic and anodic peak currents should be the same for a reversible process. If the peak currents do not show a ratio of one then the process is considered irreversible. Potassium ferricyanide, a model analyte that has been studied thoroughly via CV, was chosen to monitor the performance of the microfabricated electrochemical detector when used in CV. If the detector works correctly in the system a one electron reversible process should be interpreted from the data. The results are presented later in the thesis.

II. Experimental

Chemicals. Potassium phosphate, sodium sulfate, L-ascorbic acid, acetaminophen, o-phosphoric

acid (85%), potassium ferricyanide, sodium hydroxide, potassium nitrate and magnesium

chloride, methanol and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ, USA).

Dextrorphan and levallorphan were obtained from USP References (Rockville, MD, USA).

Potassium phosphate monobasic and cyclohexanone were obtained from J.T. Baker, Inc.

(Phillipsburg, NJ, USA). Potassium carbonate and potassium dicyanoargentate (I) were

purchased from Aldrich Chemical Company (Milwaukee, WI, USA). All chemicals were reagent

grade or higher and solvents were HPLC grade. The deionized water that was used was filtered

using a Mini-Q filter system (Millipore, Bedford, MA, USA).

Standard Samples. Using synthetic powder of acetaminophen, levallorphan, dextrorphan, clopidogrel carboxylic acid and voricanazole, known amounts were dissolved and serially diluted with methanol. In order to create the final concentration of the analytes diluted with methanol,

15.0 µL of a standard solution was added to a clean centrifuge tube and the methanol was evaporated under vacuum. The residual compound was reconstituted with 150.0 µL of mobile phase. For example, to obtain a final concentration of 10.0 µM acetaminophen, 15.0 µL of 100.0

µM acetaminophen was added to a clean eppendorf tube. The methanol was evaporated off and the residue was reconstituted in 150.0 µL of mobile phase. For the ascorbic acid standards they were dissolved in water and diluted further with fresh mobile phase. The final concentration

range differed for each compound, ascorbic acid (10.0 nM – 10.0 µM), acetaminophen (1.0 nM –

50.0 µM), levallorphan (1.0 µM – 1000.0 µM), dextrorphan (500.0 nM – 500.0 µM), clopidogrel carboxylic acid (1.0 µM – 500.0 µM), and voricanazole (5.0 µM – 1.0 mM). The potassium

ferricyanide standards (1.0 mM – 100.0 mM), that were used in the cyclic voltammetry experiments, were made by serially diluting a stock solution (1000mM) further with water as the solvent.

Plating the Reference Electrode Within This System. Plating the reference electrode on the detector is important in order for the system to act as a three-electrode system. The reference

electrode must be able to maintain a constant half-cell potential through the entirety of the

measurement. A common composition for a reference electrode is silver and silver chloride

(Ag/AgCl), which was used for these detectors. In order to achieve maximum performance the

Ag/AgCl had to be plated onto the gold surface to form reference electrode prior to measurements taking place. The plating protocol used was based on a general procedure found in

the literature (11). The protocol began with cleaning the electrodes via sonication in detergent

for three minutes and then rinsing three times each with 2-propanol and deionized water. Once

the electrodes were clean, the plating process was begun. First the silver is plated on the gold

surface of the reference electrode that already exists on the detector. This is achieved by

attaching the detector to electrical connections that lead to the available electrical source,

controlling the current applied to the system. This ensures that the current density that is applied

allows the deposition of Ag to occur at the surface of the electrode, which acts as the working electrode during deposition. The electrodes are then submerged in a solution of 1% potassium dicyanoargentate (I) and 1 % potassium carbonate with deionized water as the solvent; this solution is used to form the initial layer of silver metal on the reference electrode surface. While in the silver formation solution a current density of 5mA/cm2 for 5 minutes is desired for a

uniform layer of silver to be plated. In order for this to be achieved for one reference electrode

(area of 3 x 10-4 cm2) a constant current of 4.5 µA is applied for the 5 minute time period, with

the starting potential being about -1.7 V versus a platinum wire. The next step is the silver/silver

chloride formation, which is done by reversing the current polarity positively from the previous

step until a constant current of 1 µA is achieved (Estart = -0.4V vs. Pt wire). While having the

electrodes submerged in a 0.1M HCl solution the silver chloride formation is allowed to take

place for two and a half minutes. The changes being made between each formation step

theoretically allow about half of the silver atoms to be converted into silver ions and precipitate

as silver chloride on the silver surface.

The plating of the reference electrode is a crucial part of the performance of these

detectors. When plating, one wants a uniform coat of silver/silver chloride to cover the gold

surface to form a reference electrode that should then act in the same manner as a conventional

24 reference electrode in a three electrode system. However, if the layer isn’t uniform then the area of the reference electrode will fluctuate and the composition of the reference will be a combination of silver/silver chloride and exposed gold (Figure 8B). A reference electrode is supposed to have a half-cell potential that is invariant even though the composition of the sample changes, which is normally done by isolating the Ag/AgCl electrode from the sample solution with a barrier. In this case there is no barrier between the reference electrode and the rest of the sample, which makes the stability of the electrode difficult to maintain.

Figure 8. (A) Uniform layer of Ag/AgCl plated on gold to form the reference electrode and (B) a layer that is nonuniform. Environmental scanning-electron microscope (ESEM) images of a uniformly plated (C) and non-uniformly plated (D) reference electrode in which plating was performed simultaneously.

The plating process produces better results when a wafer of detectors are plated simultaneously by connecting the reference electrodes of each one in series using silver epoxy attached to an electrical lead. The lead distributes current to each reference electrode in the series; this method was used while plating the reference pictured in Figures 8A, 8C, and 8D. In this process a hemispherical platinum auxiliary electrode is used in the 3-electrode system. The method used to produce the non-uniform plating observed in Figure 8B plates one reference at a time in solution with a platinum wire used as the auxiliary electrode. An extreme difference in plating uniformity can be observed in the figures as well as a difference in color of the layer deposited on the reference electrode. The brownish color of the reference electrode pictured in

Figure 8A is expected when silver chloride has been plated; notice that this color does not exist on the reference in Figure 8B therefore silver chloride has not been formed. It is not to say that the method used in the plating of Figure 8B will not produce uniform plating because it has been accomplished, however reproducing the plating was much more consistent when the first method was used.

It has been demonstrated that plating of the reference electrodes on these microfabricated electrochemical detectors is much more consistent with the initial method discussed. Obtaining detailed environmental scanning-electron microscope (ESEM) images with a FEI XL30 ESEM

(Royal Philips Electronics, Netherlands) allowed for the plating of these electrodes to be explored in detail at the micron level (Figure 8C, Figure 8D). These images reveal that plating multiple electrodes at the same time does not necessarily mean that the reference electrodes that are plated will have the exact same area; the reference electrodes in Figures 8C and 8D were plated simultaneously in the same “batch”. The reference electrode plating is always going to have variability from one microfabricated electrochemical detector to the next, but if the gold

layer, which is located below the plated Ag/AgCl layer, is not exposed then the affect this has on

the electrochemical performance of the detector is minimal. The areas of the reference electrodes

in Figures 8C and 8D are in fact different, however, the difference between the areas is less than

10 %, 3.1 µm2 (Figure 8C) compared to 3.3 µm2 (Figure 8D). In Figure 8D there are obvious imperfections that are present on the reference electrode that are not present on the

reference electrode in Figure 8C. Theoretically the reference electrode in Figure 8D should be

able to maintain a half-cell potential that is within 10% of the half-cell potential maintained by

the reference electrode in Figure 8C since the difference in reference electrode area is 10%;

assuming the composition of the electrode remains Ag/AgCl. Changes leading to degradation of

the reference electrode performance inside these microfabricated electrochemical detectors will

be analyzed in the Results section.

Initially, the electrodes were stored in deionized water after the plating process was

completed. Conversely it was determined in this study that storing the electrodes like this

weakened the interaction between the silver/silver chloride and the gold surface of the

microelectrode, causing the plating to break away from the detector effortlessly during an

electrochemical application, especially when used with flowing solutions. This bond weakening

is due to the fact that Ag/AgCl is a water soluble salt that will dissolve in DI water. This affected

the results tremendously and is discussed further in the results section. This inconsistency in

plating uniformity is pictured in Figure 8B. The optimum storage conditions for the detectors,

once successfully plated was at room temperature in a sealed container. The detectors were

rinsed with isopropyl alcohol and any remaining liquid was removed with compressed nitrogen

prior to use.

HPLC-ED (Commercial System). An HPLC assay method with electrochemical detection was

developed for the quantification of ascorbic acid, acetaminophen, clopidogrel carboxylic acid,

dextrorphan, levallorphan and voricanazole. The HPLC-ED system was comprised of a Hewlett

Packard 1049A Programmable Electrochemical Detector, coupled with a Waters (Milford, MA) model 515 pump and a model 717 autosampler. The separation system consisted of a Zorbax SB-

C18 column (150 x 2.1 mm, 3.5µM particle size; Phenomenex, Torrance, CA), a Nova-Pak C18

Guard column (4 µM; Waters, Inc., Ireland). An isocratic elution was used to separate the compounds. The mobile phase consisted of 50% methanol and 50% (v/v) 10 mM KH2PO4

buffer (with 1.0 mM EDTA) for ascorbic acid and acetaminophen determination. When

levallorphan and dextrorphan were analyzed, the mobile phase consisted of 70% 10 mM KH2PO4

buffer (with 1.0 mM EDTA) and 30% acetonitrile (v/v). The eluate was introduced, without

splitting, at 0.500 mL/min to the electrochemical detector with a run time of 20 min.

Acetaminophen and L-ascorbic acid were determined at a working potential of 1000 mV, and dextrorphan and levallorphan were determined at a working potential of 0.75mV, respectively.

Each sample was evaporated to dryness and then reconstituted in 150 µL of mobile phase; 100

µL of each sample was injected onto the HPLC-ED system.

HPLC-ED (Novel System). The microfabricated electrochemical detector (Yellow Springs, Inc.,

Yellow Springs, OH) was connected directly with a Hewlett Packard 1049A programmable potentiostat, via custom made electrical pin connections. Data collection was performed using

Empower Pro Software (Waters Inc, Miliford, MA). The solvent delivery and chemical separation systems were identical to those used in the previously mentioned HPLC-ED system.

An isocratic elution was used to separate the compounds. Two different mobile phases were used

in order to test the performance parameters of the detector. The first mobile phase consisted of

50% methanol and 50% (v/v) 10 mM KH2PO4 buffer (with 1.0 mM EDTA) and was used for the

detection of ascorbic acid and acetaminophen. The studies conducted with dextrorphan and

levallorphan used a mobile phase of 70% of 10 mM KH2PO4 (with 1.0 mM EDTA) and 30% acetonitrile (v/v). The eluate was introduced, without splitting, at 0.500 mL/min to the electrochemical detector with a maximum run time of 15 min. The detection potential was set of

1000 mV for all compounds in this system. Each sample was evaporated to dryness and then reconstituted in 150 µL of mobile phase as described earlier; 100 µL of each sample was injected onto the HPLC-ED system.

Flow Injection Analysis (FIA-ED). A flow injection system using the microfabricated electrochemical detector (Yellow Springs, Inc., Yellow Springs, OH) was coupled with a BAS

100B Electrochemical Analyzer in order to perform the FIA-ED analysis in this research. Data was gathered and integrated by Lab View software (National Instruments, Washington, DC,

USA) which was also used to automatically control the flow injection system. The electrochemical detection system was used to monitor the electrical current being produced under a controlled potential of -75mV. FIA-ED was conducted using the pressurized flow injection system (Yellow Springs, Inc., Yellow Springs, OH). The sample reservoirs located on the flow injection system were filled with a 2.0 mM potassium ferricyanide solution in buffer B

(0.0440 M NaH2PO4, 0.0560 M Na2HPO4, 0.10 M KCl, 1g/L of MgCl2-6H2O) and in a second

reservoir a 1.0 M KNO3 carrier buffer. The pressure by which the fluid was forced through the

system was also varied between a range of 0.10 psi and 10.0 psi. The injection time was set at 30

s and the run time was set to 800 s.

Cyclic Voltammetry. A fixture designed by Yellow Springs, Inc. (Yellow Springs, OH) and

modified by the author was used in order to couple the microfabricated electrochemical detectors

to the BAS 100B Electrochemical Analyzer and simultaneously allow for the electrodes to be

submerged in a non-stirred solution of analyte for cyclic voltammetry. Commercially available conventional size gold, glassy-carbon electrodes (Bioanalytical systems, Lafayette, IN) were used as a basis of comparison for the performance parameters of the microfabricated electrochemical detectors in this system. The electrochemical cell was completed with an

Ag/AgCl reference electrode (Bioanalytical Systems, Inc, Lafayette, IN) and a platinum wire for the auxiliary electrode. There were two model analytes used to evaluate the performance of these detectors in this electrochemical technique; potassium ferricyanide and ascorbic acid. The parameters used for detection were as follows: Initial Potential Applied = 0 and –500 mV,

Maximum Potential Reached = 1000 mV, Minimum Potential = -500 mV, Scan Rate = 10, 25,

50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, 1250, 1500 mV/s, Sensitivity = 10.0

µA/V. For the analysis of PFC in the same system the parameters were: Initial Potential Applied

= 800mV, Maximum Potential Applied = 800 mV, Minimum Potential Applied = -200 mV, Scan

Rate = same as AA analysis, Sensitivity = 10.0 µA/V.

III. Results

Cyclic Voltammetry. The initial cyclic voltammetry experiments performed with the novel

detectors evaluated the electrochemistry with both uniformly and non-uniformly plated reference electrodes. A direct correlation was found between the uniformity of the plating and the performance of the detector.

Figure 9. CV of 4 mM K3Fe(CN)6 in 1 M KNO3 initiated at 0.8 V in the negative direction at a scan rate of 20 mV/s using (A) the detector pictured in Fig. 8A, uniform plating and with (B) the detector in Fig. 8B, non-uniform plating.

The voltammograms show well defined, reversible voltammograms when the detector

with the uniformly plated reference electrode (Figure 9A) was used. However, the electrode with the non-uniformly plated reference electrode (Figure 9B) gave a much distorted response. A reduction peak during the forward scan and an oxidation peak during the reverse scan are hard to distinguish in Figure 9B. Therefore, a reversible process is not detected which is what is expected of potassium ferricyanide. These differences show how crucial the plating process of the reference electrode is in the electroanalytical detection performed by these detectors.

The performance parameters of the microfabricated electrochemical detectors were determined in a non-stirred system and were compared with those of a conventional size working electrode (Figure 10).

Figure 10. (A) CV of a conventional size gold working electrode in a solution of 4 mM K3Fe(CN)6 in 1 M KNO3. (B) CV of the same solution obtained with the microfabricated electrochemical detector presented in this paper.

The voltammograms show that both the YSI electrochemical sensor and the conventional size

electrode give a reversible process when used to analyze the potassium ferricyanide solution. The shape of cyclic voltammograms at “microelectrodes” is greatly affected by the scan rate. At slow sweep rates (<10mV/s) a sigmoidal “snake” shape response is expected and as the sweep rate is increased the shape of the response normalizes to that of a CV at conventional sized electrodes

(2). This normalization occurs at high sweep rates as the diffusion shifts from hemispherical to

linear. At low sweep rates steady state mass transport takes place giving a plateau instead of a

peak. This is a phenomenon that takes place near the electrode surface giving the true response

of the sensor. These characteristics are seen in the voltammograms above, which demonstrates

that these detectors perform properly when tested in solution.

The peak currents as well as the peak potentials are different in both sets of data. The

current produced by the detectors is expected to be much less than that of the conventional size

electrode due to the difference in electrode area. The smaller the electrode the less current

produced in a CV according to the Randles-Sevcik equation. This is reflected in the

voltammograms in Figure 9, which show the maximum current achieved by the novel detector at

a scan rate of 200 mV/s to be ~1.6 µA and by the conventional electrode to be ~25 µA at the same scan rate. The performance parameters of the novel detector when used in cyclic voltammetry are comparable to those of conventional commercially available electrodes and show the suitability of these detectors when used for electrochemical detection.

Electrochemistry of Ascorbic Acid w/ Conventional and Microfabricated Electrodes. The microfabricated electrochemical detector was also evaluated on the electrochemistry of ascorbic acid via cyclic voltammetry. Many techniques have been used in the past for quantifying ascorbic acid such as UV/Vis spectroscopy as well as certain titration methods. When ascorbic acid is detected with these methods the selectivity becomes a major issue as ascorbic acid has been found to be interfered with by other components of the sample, which affected the quantification of the nutrient (16). When ascorbic acid is analyzed electrochemically it is oxidized to dehydroascorbic acid and this oxidation produces a current that is proportional to the concentration of ascorbic acid present in the sample (17).

Figure 11. Effect of varying scan rate on cyclic voltammetry of 10 mM ascorbic acid in 10 mM KH2PO4 using microfabricated electrochemical detectors.

The oxidation of ascorbic acid in a non-stirred solution was examined with the microfabricated

electrochemical detector (Figure 11). As the sweep rate increases the resulting current becomes

larger (more negative). The maximum current achieved by the detector at a scan rate of 1.5 V/s

was ~9 µA and the peak potential for the oxidation of ascorbic acid was ~375mV vs. Ag/AgCl.

The absence of a well-defined reduction peak when re-reduction was attempted is also observed,

indicating that the oxidation of ascorbic acid is an irreversible process.

Flow Injection Analysis. The second electroanalytical technique performed with the

microfabricated electrochemical detectors was flow injection analysis coupled with

electrochemical detection (FIA-ED). Flow injection analysis is a rapid technique with good

reproducibility and stability, qualities produced by the microfabricated electrochemical detector

in a FIA-ED system. 34

Figure 12. Electrochemistry, performed with the FIA-ED system, of 2 mM ferricyanide in buffer B at a potential of -75mV. The pressures at which the results were recorded are labeled appropriately.

Attaching the microfabricated electrochemical detector to the chip in Figure 5, allows for a sample solution of analyte to be pumped across the surface of the electrodes. Using potassium ferricyanide (2 mM) as a model analyte, the performance of the microfabricated electrochemical

detector was evaluated in this FIA-ED system at a working potential of -75 mV. The current

peaks detected with this FIA-ED system show good reproducibility for multiple injections at

three flow rate pressures as shown in Figure 12.

Figure 13. Average peak current (µA) of 2mM potassium ferricyanide analyzed in the FIA- ED system at a working potential of -75 mV. The pressure applied to induce flow was varied between 5.0, 1.2, 0.7, 0.5 and 0.3 psi. The run time for this analysis was 48 h.

The pressures and corresponding peak heights are 0.3 psi (0.365 ± 0.004 µA), 0.5 psi

0.377 ± 0.004 µA) , 0.7 psi (0.378 ± 0.016 µA), 1.2 psi (0.376 ± 0.009 µA) and 5 psi (1.100 ±

0.014 µA), respectively. The data in Figure 13 shows good reproducibility at all five pressures tested. An injection of analyte was made at specific time intervals, either 5 or 10 min, over the course of 600 s. The microfabricated electrochemical detector maintains the same electrochemical performance throughout the scan in this FIA-ED system independently of the pressure applied to the mobile phase. The peak width is inversely proportional to the pressure applied to the system because the analyte is in contact with the electrode surface for a shorter time period at higher pressures due to the increase in flow rate. The shorter amount of time it is in contact with the electrodes will result in less time the analyte can be oxidized producing a narrower signal. Consequently, the width of the peak generated by the analyte, which is measured as a unit of time, will decrease as the pressure of the mobile phase increases. The peak

current does not change substantially until the pressure reaches 5psi, where the average peak

current increases from 0.370 µA to 1.100 µA. As the pressure increases the flow rate across the

electrode surface also increases, which enhances mass transport of analyte to the electrode

surface, resulting in an increase in current.

Figure 14. Peak current (µA) of 2mM potassium ferricyanide analyzed in the FIA-ED system at a working potential of -75 mV. The pressure applied to induce flow was 0.5 psi. The first 17 h of analysis is shown here.

The performance of a flow detector can be effectively monitored in a FIA system. With

multiple injections being made in the same run, the reproducibility of the signal is very

dependent upon the detector performance. It was purposely planned to monitor the performance

of the detector in a flow injection analysis system prior to coupling to HPLC. By doing so, the

detector could be characterized in a flow system without having to deal with the

chromatography. The lifetime of the microfabricated electrochemical detector was found to be

about 48 h when used continuously. This was determined by making an injection of 2 mM

potassium ferricyanide onto the FIA-ED system every 5 or 10 minutes for a 48 h time period,

without interruption (Figure 14). As demonstrated in Figure 13, the signal variability when the

FIA-ED was operated at 3 different pressures was 10%. The microfabricated electrochemical

detector demonstrated robust characteristics when coupled with FIA, including reliable working

and reference electrodes, remarkable reproducibility, and durability in this system of flow. The

performance of this microfabricated electrochemical detector within this system granted the

confidence necessary to proceed to an HPLC-ED system.

HPLC-ED. Ascorbic acid and acetaminophen were chosen as model analytes to evaluate the performance of the microfabricated electrochemical detector in HPLC-ED. These compounds were chosen due to the fact that they are electrochemically active and can be separated with ease using reversed-phase HPLC. Both compounds have been detected previously in HPLC-ED systems using conventional size working electrodes (16-18).

The first step in quantitation of any compound via HPLC-ED is determining the optimum working potential for detection. This potential was determined by constructing a hydrodynamic voltammogram of the two model analytes used in this system, acetaminophen and ascorbic acid

(Figure 15). This voltammogram was constructed by injecting the same volume of acetaminophen or ascorbic acid into the system while systematically adjusting the applied potential to cover the potential range of the voltammogram. When interpreting a hydrodynamic voltammogram there are three regions that generate information necessary to determine the optimum potential for detecting an oxidizable compound. The first region is known as the zero current region, where the applied potential is not sufficient enough to force an oxidation (2). This region occurs at potentials for both ascorbic acid and acetaminophen (0 mV – 400 mV). The region located on the slope between 500 mV and 900 mV for ascorbic acid and between 500 mV and 800 mV for acetaminophen is considered the intermediate region. Here the peak height is

rising with increasing potential while the potential controls the kinetics of the electron transfer

between the analyte and the electrode surface. The final region of interest is termed the plateau

region and this region is found between 900 mV and 1500 mV for ascorbic acid and 900 mV-

1200 mV for acetaminophen. Here the current is independent of the applied potential. The diffusion of analyte to the surface of the electrode becomes the rate-determining step. Within this region the optimum working potential is chosen and 1000 mV was selected as the optimum working potential for the HPLC-ED analysis of L-ascorbic acid and acetaminophen in this system using the microfabricated electrochemical detector. At this potential minimal background current was apparent.

2500

2000 A) µ 1500

1000 Peak Current ( Current Peak

500

0 0 200 400 600 800 1000 1200 1400 Detection Potential (mV)

Figure 15. Hydrodynamic voltammograms for acetaminophen (50 µM) (A) and ascorbic acid (100 µM) (B).

Degradation of Detector and Signal. The degradation of the microfabricated electrochemical

detector and the affect on the electrochemical signal was observed and monitored. The length of

time in which the performance of the microfabricated electrochemical detector produces results

that were reproducible and allowed for the limit of detection to be achieved varied based on the plating of the reference electrode. If the reference electrode was comprised of a uniform layer of

Ag/AgCl then the electrode maintained performance in an HPLC-ED system for 12-24 hours of continuous flow of mobile phase. This time range was determined by making 2 injections of ascorbic acid and/or acetaminophen every hour over the course of 10, 12 and 20 hour time periods. An example of the stability test for each compound is shown Figure 16.

Figure 16. Stability of ascorbic acid (1 µM) and acetaminophen (0.25 µM) over a 10 hour period as shown by peak height.

The average response for each compound was compared over a 10 hour, 20 hour, and 40 hour

period (Table 3). It was determined that the percent variability of the signal for each model

analyte, acetaminophen (0.25 µM) and ascorbic acid (1 µM), was around < 1 percent after 10

hours and increased 10-fold over the course of a 30 hour period. For acetaminophen the

variability of response was 0.59% after the first ten hours of testing, 1.30% for hours 11-20 and

the variability increased to 11.3 % the final 20 hours of analysis between hours 21 and 40. For

ascorbic acid the signal varied only 0.75% over the first ten hours, from hours 11-20 the signal

still maintained reproducibility having a % RSD of 1.36%. However, during the last 20 hours

(21-40hr) the signal varied 12.8%.

Table 2. Stability of HPLC-ED Response of Acetaminophen (0.25 µM) and Ascorbic Acid (1.0 µM) Percent Variability Average Signal (µA) Std Dev of Signal of Signal 0-10 hr (Acetaminophen) 185 1.1 0.59 11-20hr (Acetaminophen) 184 2.4 1.3 21-40hr Test (Acetaminophen) 133 15.0 11.3 0-10 hr (Ascorbic Acid) 292 2.2 0.75 11-20hr (Ascorbic Acid) 279 3.8 1.4 21-40hr Test (Ascorbic Acid) 149 19.0 12.8

Table 3 shows that ideal relative standard deviations between multiple injections of

numerous compounds have been observed during the first 10 hours of analysis with these novel

detectors. Between hours eleven and twenty the electrode shows some degradation but the

considerable degradation is observed within the final 20 hours of continuous use. Using this

novel detector that has had 10+ hours of constant flow increases the risk of a decrease in

precision for this system. Based on the results from the stability tests above the detector performs best when it is exposed to the first 10 hours of flowing mobile phase. After 20 hours of flow the

reproducibility of the system becomes >10%, which is greater than is usually desired for an

analytical system.

This stability is not as great as in the FIA-ED system but that is attributed to the greater

flow rate that was used. As the flow rate increases in HPLC-ED the surface of the three

electrodes within the novel detector gradually change their composition due to the solubility of

AgCl in the reference electrode and adsorption on the surface of the gold working electrode. The

silver chloride that is plated on the surface of the reference electrode slowly dissolves during the

continuous flow of mobile phase because of its solubility. Increasing flow rate is one way to increase the sensitivity of a hydrodynamic flow electrochemical system, such as HPLC-ED (19).

Mass transport of analyte to the surface of the electrode is increased and therefore resulting in a

higher response from oxidized and reduces species in HPLC-ED when compared to FIA-ED

(19).

Analyzing Performance when the Degradation of a Novel Microfabricated Electrode

Begins. The degradation of the novel detector performance becomes evident in the signal of a model analyte, as shown in Figure 17. In this case, before each sample set was analyzed, acetaminophen (50 µM) was injected onto the system and a peak current at or around 25.0 µA

was expected for acetaminophen. If the signal varied more than 10%, then the detector was

immediately replaced with a new detector. When the detector performance degrades, the noise

level increases and it becomes difficult to form a stable baseline for detection. The performance

of a new detector was compared to that of a degraded detector and one that had considerable or

all AgCl removed from the surface of the reference electrode.

Figure 17. The response of acetaminophen (50 nM) using 3 different microfabricated electrochemical detectors of different condition, non-working (A), degrading (B) and new (C).

The Effect of Concentration of Analyte on Electrochemical Detection in HPLC-ED. The effect of analyte concentration on the electrochemical detector response produced with this

HPLC-ED system was studied using ascorbic acid as a model analyte. Standard samples of ascorbic acid and acetaminophen were prepared and injected onto the HPLC-ED system containing the novel detectors. In most analytical systems the response is directly proportional to analyte concentration as shown in Figure 17B for acetaminophen. This is due to the increase in analyte concentration at the surface of the working electrode, increasing the yield of oxidative product which increases the current that is translated into the measurable signal. A calibration

44 curve was formed using ascorbic acid and is used to depict this relationship of analyte concentration to response, in Figure 18.

A. B.

3500

y = 312.1x R² = 0.9997 3000

2500

2000 µA 1500

1000

500

0 0 5 10 15 Ascorbic Acid (µM)

Figure 18. Calibration curve of ascorbic acid (A) and the effect of concentration on peak height (B) for acetaminophen detection by HPLC-ED using the microfabricated electrochemical detector.

The formation of calibration curves is a useful tool when characterizing the performance of an analytical system. The calibration curve shows a linear response when ascorbic acid (1nM-

10µM) was determined with this system, confirming the hypothesis that the response in this system should be proportional to analyte concentration. The equation of the line, when 0 µM

45 ascorbic acid is included in the sample set, was y = 312.1x and the r2 value was 0.999, further confirming the expected linear relationship between analyte concentration and detector response.

The standard deviation for 3 injections for each concentration of ascorbic acid tested, is minimal and the error bars may be difficult to distinguish in Figure 16 (A.).

Comparing the Novel HPLC-ED System with a Commercialized System. The main purpose in this research was to compare the performance of the microfabricated electrochemical detectors to a commercial electrochemical detector used within the analytical world today. A Hewlett

Packard model HP1049A programmable electrochemical detector was chosen for comparison because it was readily available and easily accessible; the data produced for this detector is described as system 2.

Figure 19. HPLC-ED of acetaminophen (1 µM) examined with the novel microfabricated electrochemical detector (A) and a commercial detector (B).

There are noticeable differences between the two detectors chosen for comparison in this

paper. The first difference is the area of the working electrodes that are contained in each

detector. The area of the working electrode on the microfabricated electrochemical detectors

(system 1) is 17.0 µm2, while the area of the working electrode for the HP1049A is 5020 µm2.

This difference in working electrode size will result in substantial difference in the

electrochemical signal that is produced, since the current produced by a redox reaction of an electroactive species is directly proportional to the area of the working electrode (19). This difference in resulting current between two different working electrodes is evident from the

results in Figure 19 where acetaminophen (1 µM) was injected onto both system 1 (Fig. 19A)

and system 2 (Figure 19B). The working electrode area contained in system 2 is approximately

295 times larger than the working electrode found on the microfabricated electrochemical

detector (system 1) and as expected the resulting peak current for 1 µM acetaminophen in system

2 is approximately 300 times greater than the current produced in system 1. This is due to the amount of analyte that can be oxidized or reduced on the working electrode surface, the larger the area the more analyte that can be converted. The increase in working electrode surface area also increases the background current and thereby the noise associated with the detection. As the noise increases, the signal-to-noise ratio produced within a given electrochemical detection system decreases. Thereby, the limit of detection of the system ultimately is affected. The detectors used in this comparison both scale linearly and therefore do not benefit from hemispherical diffusion. If an electrode is small enough in size and can benefit from hemispherical diffusion a real difference in noise levels can be observed, however this is not the case for this comparison as shown in Figure 19.

Limit of Detection. The limit of detection was determined for an HPLC-ED system using both microelectrodes (system 1) and macroelectrodes (system 2) size electrode. The limit of detection is variously defined in the field of electroanalytical chemistry, under conditions of flow, as a concentration, or quantity, and derived from the smallest measurable net signal, that can be determined with reasonable certainty based on a statistical basis (19). The limit of detection is typically referred to in the general analytical community (American Chemical Society, IUPAC) as the minimum concentration of a given analyte required to produce a response that maintains a signal-to-noise ratio that is greater than 3 to 1 (20, 21). When comparing the limits of detection of acetaminophen in each system it was observed that when the microfabricated electrochemical detector was used the limit of detection (1 x 10-9 M, 1 nM) was higher in value than when the

commercial detector was used (5 x 10-11 M, 0.05 nM) (Figure 20). The limit of detection was determined for each system by serially diluting acetaminophen to known concentrations in mobile phase (150 µL). Once the dilutions were prepared 100 µL was injected onto the chosen

HPLC-ED system. When the limit of detection was being approached the dilutions were decreased by 10 nM until the signal to noise ratio became less than 3 at which the limit of detection is surpassed. The noise level at low concentrations is much less in system 1, confirming that the noise level is proportional to working electrode size in HPLC-ED. The limit of detection was determined to be lower with the commercial detector than the microfabricated electrochemical detector. Even though the noise level is greater (Figure 20), its signal is also substantially greater for a given concentration allowing for a higher signal to noise ratio and lower limits of detection.

Figure 20. Response for acetaminophen (1.0 x 10-9 M) using the novel HPLC-ED (A) and the response for acetaminophen (5.0 x 10-11 M) in a commerical HPLC-ED system (B). Note the unit increments in each figure in order to depict the relationship between detectors and peak current (nA).

The importance of a small noise level at low concentrations is reflected in the quantitation of the

peak response. If high noise levels interfere with the symmetry of the analytical peak then the

accuracy for quantitating and the ability to consistently reproduce the same signal are

jeporadized. In this instance the microfabricated electrochemical detector shows a small noise

level (Figure 20A) and a peak having an asymmetry factor (A/B) of 1.0, meaning the area of the

first half (A) of the peak with respect to time is equal to the second half (B). When the detection

limit is approached with sytem 2 (Figure 20B) the noise level is observed within the peak itself

causing the asymmetry factor to decrease to 0.6 which is slightly less than the accepted value of

a good separation by IUPAC standards (0.9 ≤ A/B ≤ 1.5). When the area of an electrode

decreases certain changes take place that can make them advantageous; conditions are changed

leading to a decrease in ohmic drop, an increase in current due to enhancement of mass transport

at the boundary of the electrode and as well as an increase in the signal to noise ratio (1). The microfabricated electrochemical detector possesses these properties.

Band Broadening as a Result of Longitudinal Diffusion. As solute moves through an HPLC column it broadens. Harris states “ideally an infinitely narrow band applied to the inlet of the column emerges with a Gaussian shape,” (3). A main cause of band broadening is diffusion, the movement of a substance from a region of high concentration to low concentration as it passes through the column and reaches the detector. This diffusion when applied to HPLC is called longitudinal diffusion because it takes place along the axis of the column. The term B/ux , in

Equation 3, occurs due to longitudinal diffusion. In HPLC, the faster the linear flow of solvent

the less time the band of substance remains on the column, decreasing the amount of band

broadening that takes place as a result of longitudinal diffusion (3). When comparing the results

from the novel HPLC-ED system to those of the commercial system, they are similar. Therefore

it is understood that there is little to no effect of the detector, for either system, on band

broadening because the same analytical column was used for separation with each system. The

volume of the device that the novel detector uses to connect to the HPLC plumbing has a much

smaller volume (<100 uL) than the commercial system (> 1mL). Typically in order to

accommodate for this decrease in volume within the system the linear flow of solvent must be

decreased as well in order to maintain a working pressure. However, in this instance both

systems were operated at the same flow rate, 0.500 mL/min.

Figure 21. Dextrorphan (1 µM) and Levallorphan (1 µM) measured in the commercial (A) and novel (B) HPLC-ED systems.

In Figure 21, dextrorphan and levallorphan are shown to be determined with both systems

and the difference between systems seems minimal. The peak width at the base for dextrorphan

averages 1.52 ± 0.10 min and for levallorphan 1.9 ± 0.2 min when examined in the novel HPLC-

ED system. In the commercial system the dextrorphan peak (1.27 ± 0.08 min) and the leavallorphan peak (1.3 ± 0.1 min) both are slightly narrower but the effect is not substantial.

Band spreading does take place in the microfabricated electrochemical detector; however, the effect on the chromatographic properties of the system is not significant. This demonstrates that the microfabricated electrochemical detector is suitable for coupling to an HPLC system while having minimal effect on the chromatography due to a decrease in detector volume.

Ascorbic acid and acetaminophen were quantified by using the peak area from the direct

injection of the compound onto the HPLC-ED system. The other compounds, dextrorphan and

levallorphan, were quantified by using the ratio of peak area of the compound to peak area of internal standard (acetaminophen, 50 µM) because the samples containing these compounds were extracted before injection onto the HPLC-ED system. By adding an internal standard to these samples eliminated unnecessary variability caused by the extraction and sample preparation. Calibration curves of dextrorphan and levallorphan were constructed using known concentrations of each compound. Ascorbic acid and acetaminophen were analyzed separately diluted only in mobile phase. Spiked samples of levallorphan and dextrorphan, in mobile phase and human plasma samples were analyzed simultaneously to show a practical application of the novel HPLC-ED system. Calibration curves were constructed for each compound in each matrix; attributes from standard curves constructed with multiple concentrations of ascorbic acid and acetaminophen are listed in Table 4.

Table 3. Calibration Curve Data for Ascorbic Acid and Acetaminophen Ascorbic Acid Ascorbic Acid Acetaminophen Acetaminophen

HPLC-ED Novel Commercial Novel Commercial

System

Slope (µA / 3100 1 x 106 5000 2 x 106

µM)

R2 0.9998 0.9991 0.9993 0.9995

Linear 0.01µM – 0.01µM – 100 0.001µM – 0.0008µM –

Range 100µM µM 100µM 100µM

The calibration graphs constructed using both the novel and commercial HPLC-ED systems demonstrate the expected linear relationship between analyte concentration and electrochemical response. The percent relative standard deviations for all compounds at each concentration tested within the linear range was less than 2% after triplicate injections. The capacity of the novel

HPLC-ED system to perform adequate detection is realized when analyzing the calibration curves displayed in Figure 22. The noticeable differences when comparing the data produced by both systems is that that slope of the line is much greater when ascorbic acid and acetaminophen are analyzed with the commercial HPLC-ED system. This is a direct reflection of the increase in signal for this system due to the size of the working electrode used. The area of the working electrode in the commercial HPLC-ED system is about 300 times greater which means more electroactive compound can be electrolyzed at once, resulting in more current being produced.

Since peak height and background current scale linearly with electrode area the higher current

produced by the commercial system is expected. The linear range determined for ascorbic acid is equal in both systems, but for acetaminophen the linear range is lower for the commercial detector than the microfabricated electrochemical detector; R = 0.999 in all experiments (Table

4).

y = 2E+06x 100000000 R² = 0.9995

10000000 y = 1E+06x 1000000 R² = 0.9991

100000 y = 4972.5x R² = 0.9993 (µA) 10000 10 y = 3121x 1000

log R² = 0.9998 100

1 0.001 0.01 0.1 1 10

log10 [µM]

Figure 22. Calibration curves constructed when ascorbic acid was analyzed using commercial HPLC-ED system (Blue) and the novel HPLC-ED system discussed (Red). Acetaminophen was analyzed using a commercial HPLC-ED system (Green) and with the novel HPLC-ED system (Yellow).

Chromatographic Properties of Each System. For further analysis and comparison of each

system chromatographic attributes were calculated and used for the basis of comparison. These

attributes are universally used to determine the quality of an analytical separation. For each

compound the capacity factor (k’) was calculated in two separate ways, both ways using

Equation 5 where tm, is the time in which the first baseline disturbance occurs in minutes. The

54 first capacity factor value was calculated when tm was determined from the actual chromatograms. The second capacity factor value used a theoretical value of tm which was calculated using Equation 6. For ascorbic acid the capacity factor was calculated when tm was determined from actual chromatograms for the commercial system (1.600 ± 0.002 min) and for the novel HPLC-ED system (2.1 ± 0.1 min). When tm was theoretically determined the capacity factor for ascorbic acid in the commercial HPLC-ED system (2.65 ± 0.02 min) remains shorter than when the novel HPLC-ED is used (3.7 ± 0.2 min). For acetaminophen, the average capacity factor value was 3.99 ± 0.03 min when tm was determined from an actual chromatogram in the commercialized system and (4.53 ± 0.01) min when analyzed in the novel HPLC-ED system.

When a theoretical value is calculated for tm the capacity factor for acetaminophen was 6.06 ±

0.02 (commercial HPLC-ED) and 6.37 ± 0.02 min (novel HPLC-ED).

Figure 23. The capacity factors (k’) for ascorbic acid (Black) and acetaminophen (Red) in the novel HPLC-ED system are compared to the capacity factors for ascorbic acid (Green) and acetaminophen (Blue) in the commercial HPLC-ED system.

Due to the decrease in flow rate of the mobile phase for the novel HPLC-ED system the

capacity factors are higher for ascorbic acid and acetaminophen when compared to the capacity

factors for both when analyzed in the commercial system. The same conditions used in the novel

system were tested within the commercialized system but due to a significant decrease in peak

resolution those conditions were modified to the current conditions used in the commercialized

system. An increase in flow is known to increase the sensitivity of the electrochemical detection

due to the increase in mass transport of electroactive substance at the electrode surface (19).

However, this increase in signal becomes negligible when comparing these two HPLC-ED systems based on the difference in electrode size. The electroactive species is continually replenished in the diffusion layer as the eluate passes when microelectrodes are used. This phenomenon allows for microelectrodes to be used in systems of lower flow than systems typically coupled with macroelectrodes without sacrificing performance. For both systems, all peaks and injections of ascorbic acid and acetaminophen expressed a capacity factor value within the accepted range (0.5 < k’ < 20) and the resolution was greater than the accepted value of 2 (3).

The operating pressure is approximately 1300 psi for the commercial system and 2000

psi for the novel HPLC-ED system, which is within the accepted standard of less than or equal

to, 2200 psi, described by Harris (3). A system with a pressure within this range allows for the

minimum damage to occur to the analytical column. The novel HPLC-ED system showed

remarkable reproducibility and sensitivity and seems a good choice to separate and detect

multiple compounds, simultaneously, in various matrices.

Separation of Pharmaceutical Compounds in Multiple Matrices. The ability to accurately

detect chemical compounds and their metabolites is essential in understanding the metabolic

pathways taken by drugs in the human body. If the metabolic pathway can be characterized for a

select drug, the knowledge gained can help to optimize the drug’s efficacy and limit its toxicity

in the human body. Dextromethorphan (DXM), a synthetic analog of codeine belonging to the

morphinan family of drugs, is an antitussive and is used to treat various neurological disorders

(22). Dextromethorphan is known to metabolize to dextrorphan (DXO, Figure 24C) through O-

demethylation which is mediated by polymorphic CYP2D6 (23). The ability to detect

dextrorphan allows for dextromethorphan to be used as a probe for human CYP2D6 enzymatic

activity both in vitro and in vivo. Levallorphan (Figure 24B) also belongs to the morphinan

family, and was the second model analyte chosen to characterize this microfabricated

electrochemical detector when coupled with high performance liquid chromatography. Drugs belonging to the morphinan family are characterized by the base structure, morphinan (Figure

20A) and their capabilities as NMDA antagonists (22,23). Compounds that are similar in structure present a difficult challenge when trying to adequately separate and quantify multiple compounds simultaneously. However, when compounds have similarities within their chemical structure they can usually be detected at similar working electrode potentials, as is the case for both dextrorphan and levallorphan in this system.

Figure 24. Chemical structures of the base structure of compounds belonging to the morphinan family (A), levallorphan (B) and dextrorphan (C). Structures were constructed by the author using ChemDraw.

In this study, dextrorphan and levallorphan were separated and simultaneously detected

with a high degree of accuracy and precision in two HPLC-ED systems (novel and commercial)

and in two different sample matrices, mobile phase and human plasma. The human plasma

samples were prepared differently than samples run in mobile phase, as described in the Methods

section. Levallorphan and dextrorphan are known to be detected using electrochemical detection

at a potential of +1.0 and +1.2 V and were found in this system to have the highest signal at 1.0

V. Acetaminophen was shown in the novel system to have an optimum detection potential

between +0.9 and +1.2 mV (Figure 15A). The same separation system was used when both the

novel and commercial systems were tested.

Chromatographic Attributes and HPLC-ED Analysis. The average capacity factor value for dextrorphan in the commercial HPLC-ED system was determined to be 9.72 ± 0.07 min when tm

is theoretically determined and 9.76 ± 0.09 for the novel HPLC-ED system. The average

capacity factor value (tm theoretical) for levallorphan was determined to be 12.73 ± 0.04 min for the novel system and 13.11 ± 0.05 for the commercial system. For all peaks and injections, for each compound, the capacity factor value fell within the accepted range (0.5 < k’ < 20) and the resolution was greater than the accepted value of 2 (3).

Calibration curves were constructed when samples in mobile phase and drug free human plasma were spiked with known concentrations of dextrorphan and levallorphan. These curves were compared within both systems and matrices based on reproducibility and accuracy. The

standard curve for dextrorphan in mobile phase (y = 0.013x, R2 = 0.993) and human plasma (y =

0.014x, R2 = 0.991) showed linearity and reproducibility when the microfabricated

electrochemical detector was used in the HPLC-ED system (Figure 25A). When levallorphan

was determined using the microfabricated electrochemical detector the samples in each matrix

also demonstrate a linear relationship between drug concentration and AUC response, as expected (Figure 25A).

The same linear response was shown in the commercial system for both dextrorphan and levallorphan when diluted in mobile phase. However, when the drugs are spiked in human plasma the relationship becomes less linear (Figure 25B). When samples were prepared in mobile phase the response for both systems was clean and precise and reproducible across the entire linear range for dextrorphan and levallorphan (500 nM-100 µM). The limit of detection for dextrorphan (2.5 nM) and levallorphan (5.0 nM) in mobile phase is different than in human plasma, 10.0 nM for dextrorphan and 15.0 nM for levallorphan, when analyzed with the

59 commercial HPLC-ED system. When the novel HPLC-ED system is used this is not the case, as the limit of detection was found to be 50.0 nM for dextrorphan and 80.0 nM for levallorphan in both mobile phase and human plasma.

2.5 A y = 0.019x R² = 0.9906 2 Dextrorphan in Human Plasma y = 0.0142x Dextrorphan in Mobile Phase R² = 0.9919 1.5 y = 0.0131x Levallorphan in Mobile Phase R² = 0.9936 1 Levallorphan in Human Plasma y = 0.0105x R² = 0.9914

0.5 Avg AUC Drug/ISAcetaminophen AUC Avg 0 0 20 40 60 80 100 120 Drug [µM]

450 y = 3.7639x B R² = 0.9908 400 Dextrorphan in Human Plasma 350 y = 3.6543x Dextrorphan in Mobile Phase R² = 0.9901 300 Levallorphan in Mobile Phase y = 2.4231x 250 R² = 0.9621 200

150 y = 1.4833x 100 R² = 0.6469

50 Avg AUC Drug/IS AUC Acetaminophen Avg 0 0 20 40 60 80 100 120 Drug [µM]

Figure 25. Calibration curves of levallorphan and dextrorphan spiked in human plasma and in mobile phase constructed using the novel (A) and commercial (B) HPLC-ED system. These concentrations were plotted versus the ratio of AUC response of drug to the AUC response of the internal standard (acetaminophen, 50 µM).

The calibration plots constructed in Figure 25 can be used to compare the performance of the disposable microfabricated electrochemical detector, present in the novel HPLC-ED system, to the non-disposable conventional electrochemical detector that is found in the commercial system. The ratio of the area of the working electrode found on the conventional electrochemical detector to that of the microfabricated electrochemical detector is approximately 295, as previously stated. This difference in electrode area is further indicative of the ratio of the slopes that are expected for dextrorphan and levallorphan within the two HPLC-ED systems. The ratio of the slopes should be similar in value to the ratio of working electrode areas when comparing the two systems because it is known that electrochemical response is directly proportional to working electrode area. The slope of dextrorphan, when diluted in mobile phase and detected with the commercial electrochemical detector (m = 3.76), is greater than that determined by microfabricated electrochemical detector (m = 0.013), by a factor of 290. When dextrorphan is spiked in human plasma the ratio of the slopes decreases to approximately 100 amid the commercial (m = 1.4833) and microfabricated detectors (m = 0.0142) used in the individual

HPLC-ED systems. Levallorphan, when diluted in mobile phase and detected with the commercial detector, has a slope of 3.65 which is almost 200 times greater than when it is detected using the microfabricated electrochemical detector, m = 0.019. This difference in slope is not observed when levallorphan is spiked in human plasma, as the ratio of the slopes for levallorphan decreases from 200 to 160.

This decrease in slope for levallorphan and dextrorphan observed within the two matrices can be associated with a number of variables that are present in these analytical systems. These can include the extraction procedure used with the human plasma samples, fouling of the working electrode within either system due to residual remains on the electrode surface, or

contaminant peaks within the chromatography which leads to incorrect analyte peak integrations.

As this section progresses, related variables discovered within these two HPLC-ED systems along with the consequential affects they have on the final results, will be investigated.

The non-disposable working electrode found in the commercial HPLC-ED system requires a polishing regiment in order for optimum performance to be achieved. When working with biological samples such as human plasma, cleaning of the working electrode becomes a vital part in the performance of the detector and can drastically affect the results if it is not often maintained. The benefit that the microfabricated electrochemical detector presents is the potential to eliminate the cleaning and maintenance that is necessary for a non-disposable working electrode to perform optimally, yet another advantage that this microfabricated electrochemical detector exhibits. However, not all facets of the microfabricated electrochemical

detector are advantageous. Due to the small working electrode area it possesses, the

amplification of electrochemical signal is much less for the microfabricated electrochemical

detector than a conventional size working electrode, thus current signals for lower concentrations

sometimes become problematic. As a result, the limit of detections for the model analytes tested

here are considerably higher for the novel HPLC-ED system as compared to the commercial

system, as previously stated.

The difference in the current produced in each system is evident in the chromatograms

shown in Figure 26 where dextrorphan (500 nM) and levallorphan (500 nM) were analyzed with

the novel (Figure 26A) and commercial (Figure 26B) detectors. The signal produced for

dextrorphan and levallorphan, when analyzed in the commercial system, has a strength of

approxiamately 100 µA and when the microfabricated electrochemical detector is used the

signals are close to 0.3 -0.5 µA. This difference in signal is expected due to the difference in

working electrode area between the commercial and novel systems, which is about 300 fold.

Figure 26. Injections of dextrorphan (500 nM) and levallorphan (500 nM) in mobile phase onto a novel (A) and commercialized (B) HPLC-ED system. Detection occurs at 1.0V for both compounds in the novel system and at 0.75V for both compounds in the commercialized system.

When dextrorphan and levallorphan are extracted and reconstituted with mobile phase

and directly injected onto the commercial (Figure 27B) and novel (Figure 27A) HPLC-ED systems, the response is very clean and resolute for both compounds, across the entire linear range (500 nM – 100 µM). The sample matrix, mobile phase, and the sample preparation ensure that the samples do not contain contaminants that can affect the peak shape of dextrorphan or levallorphan. This is evident in the chromatograms in Figure 27. When comparing the response of dextrorphan and levallorphan with both detectors, the previously mentioned trends apply to this data as they did for the data obtained when acetaminophen and ascorbic acid were analyzed.

The commercial detector (Figure 27B), when used, produces a higher current for both

compounds than the microfabricated electrochemical detector (Figure 27A).

Figure 27. Separation of acetaminophen (IS, 50 µM), dextrorphan (0, 0.5, 1, 5, 10, 25, 50, 75, 100 µM) and levallorphan (0, 0.5, 1, 5, 10, 25, 50, 75, 100 µM) in mobile phase detected with the novel HPLC-ED system (A) and the commercial HPLC-ED system (B).

The peak shape for dextrorphan and levallorphan, when evaluated in the system containing the microfabricated electrochemical detector (Figure 27A), is slightly broader than when detected with the commercial system (Figure 27B), both trends are expected. Conversely, when the sample matrix is changed from mobile phase to human plasma the expected trends do not hold true and the results are negatively affected.

The lower end of the linear range (i.e. 50 nM-1 µM) for both dextrorphan and levallorphan are affected by contaminating peaks when prepared in human plasma and analyzed with the commercial system (Figure 28A). Since the samples were extracted prior to injection the plasma and other biological components should be completely removed, resulting in a cleaner response, yet, this does not transpire. For the dextrorphan peak there is a visible contamination peak that in some instances, results in peak splitting (Figure 28A). Near the end of the levallorphan peak a second peak occurs having an effect on the area and height of the model analyte peak itself (Figure 28A). These interferences, especially for lower concentrations, become extremely problematic and have a considerable affect on quantification. Since the commercialized system is one containing a non-disposable working electrode the cleaning performed on this system may be considered to have not been adequate enough to remove all contaminants that could cause an interfering current. However, even after thorough cleaning of the working electrode, the interference appears within the first 10 injections of samples prepared in human plasma made (Figure 28B). These aforementioned interferences are not present when the microfabricated electrochemical detector is used since the signal is not amplified to the level of the commercial detector. This is apparent when comparing signals for 1 uM dextrorphan and

1uM levallorphan prepared in human plasma in the commercial system (Figure 28A) and in the novel HPLC-ED system (Figure 28B) where the signal is barely visible.

Figure 28. Chromatograms of dextrorphan (0, 0.5, 1, 5, 10, 25, 50, 75, 100 µM) and levallorphan (0, 0.5, 1, 5, 10, 25, 50, 75, 100 µM) in spiked human plasma analyzed with a commercial HPLC-ED system (A) and a novel HPLC-ED system (B).

The microfabricated electrochemical detector shows a performance level that strongly

compares to the macroelectrode in this HPLC-ED system and has the ability to eliminate interference from electronics as well as the environment. The results above show that when this disposable microfabricated three-electrode detector is used in HPLC-ED systems it maintains

stability (less than 15% variability) and reproducibility over the course of 10-24 hours of

continuous use. A high performance level coupled with a low level of maintenance makes the

microfabricated electrochemical detector a practical choice for use in various electroanalytical

techniques.

IV. Conclusion

The microfabricated electrochemical detector was found to perform adequately in multiple

electroanalytical systems in comparison with a commercially available detector, especially high-

performance liquid chromatography coupled with electrochemical detection. The deciding factor

in the performance of the microfabricated electrochemical detector, independent of the

electrochemical technique, is the plating of the reference electrode. If the reference electrode is

uniformly plated then the microfabricated elelctrochemical detector demonstrates that it can

peform at a satisfactory level in multiple electrochemical techniques. When used in cyclic

voltammetry (CV), the detector is able to produce normal response for the oxidation of

electroactive compounds when tested against conventional size electrodes (Figure 10) and maintains normal response when the scan rate is varied (Figure 11). The microfabricated electrochemical detector displayed robust characteristics when coupled with flow-injection

analysis (FIA). The reference and working electrodes were found to be stable for 48 h when the

detection is uninterrupted. Throughout this 48 h period the analyte signal varied less than 10 % at three different flow rate pressures (Figure 13). The performance of the microfabricated

electrochemical detector in the FIA system, along with the stability exhibited by the reference and working electrodes, confirmed the microfabricated electrochemical detector could operate in

a system of flow, which led to the coupling of the detector with HPLC. When used in HPLC-

ED, the stability of signal produced by the microfabricated electrochemical detector was shown

to decrease over the first 10 h of run time with a considerable fluctuation in signal being

observed between hours 20 and 40 (Table 3). Due to the size of the working electrode the

detector was less sensitive, for each compound that was measured, than its commercial

counterpart used for comparison in HPLC-ED, but the limits of detection were comparable.

When the microfabricated electrochemical detector was used for detection in HPLC-ED the

noise and contamination levels were minimized and the reproducibility was good. The ability of

the microfabriacted electrochemical detector to examine compounds at slower flow rates than

commercial systems operate minimizes mobile phase consumption without terminating the

analytical peak shape. The microfabricated electrochemical detector has shown the capability to

act as a versatile electroanalytical instrument while maintaining cost effectiveness and

convenience via its disposability, making it a sensible choice when selecting an electroanalytical

detector.

References

1.) K. Stulik, C. Amatore, K. Holub, V. Marecek, W. Kutner, Pure Appl. Chem., 2000, 72, 1483-1492. 2.) P.T. Kissinger, W.R. Heineman (Editors), “Laboratory Techniques in Electroanalytical Chemistry,” Dekker, New York, in press. 3.) D. Harris, “Quantitative Chemical Analysis,” 2003, Freeman, USA, in press. 4.) I. Casella, Electroanalysis, 1996, 8, 128-134. 5.) C. Li, G. Zhan, Q. Yang, J. Lu, Bull. Korean Chem. Soc., 2006, 27, 1854-1860. 6.) M.D. Porter, T. Bright, D. L. Allara, C.E.D. Chidsey, J. Amer. Chem. Soc., 1987, 109, 3559-3368. 7.) D.N. Batchelder, S.D. Evans, T.L. Freeman, L. Haussling, H. Ringsdorf, H. Wolf, J. Amer. Chem Soc. 1994, 116, 1050-1053. 8.) K. Sinniah, J. Cheng, S. Terrattaz, J. Reutt-Robey, C.J. Miller, J. Phys. Chem., 1995, 99, 14500-14507. 9.) J. Cheng, G. Saghi-Szabo, J. Tossell, C.J. Miller. J. Amer. Chem. Soc., 1996, 118, 680-684. 10.) V.V. Cosofret, M. Erdosy, T.A. Johnson, R.P. Buck, R.B. Ash, M.R. Neuman. Anal Chem., 1995, 67, 1647-1653. 11.) M.B. Madaras and R.P. Buck, Anal. Chem., 1996, 68, 3832-3839. 12.) C.S. Henry, I. Fritsch, Anal. Chem., 1999, 71, 550-556. 13.) E. Lindner, R.P. Buck, Anal. Chem., 2000, 336A-345A. 14.) J. Cheng, P. Jandik, N. Avdalovic, Anal. Chem., 2003, 75, 572-579. 15.) P.T. Kissinger, W.R. Heineman, J. Chem. Educ., 1983, 60, 702-706. 16.) H. Iwase, I. Ono, J. Agric. Food Chem., 1997, 45, 4664-4667. 17.) P.W. Washko, W.O. Hartzell, M. Levine, Analytical Biochemistry, 1989, 181, 276-282. 18.) J.-P. Courade, D. Besse, C. Delchambre, N. Hanoun, M. Hamon, A. Eshalier, F. Caussade, A. Cloarec, Life Sciences, 2001, 69, 1455 – 1464. 19.) K. Toth, K. Stulik, W. Kutner, Z. Feher, E. Linder, Pure Appl. Chem. 2004, 76, 1119-1138. 20.) J.N. Miller, J.C. Miller, Statistics and Chemometrics for Analytical Chemistry, Prentice Hall, Harlow. 21.) IUPAC, Analytical Chemistry Division, Spectrochem. Acta, 1978, 33B, 241-245. 22.) R. Pechnick, R. Poland, J. of Pharmacology and Experimental Therapeutics, 2004, 309, 515-522. 23.) A. Yu, R. Haining, Am. Soc. for Pharm. and Experimental Therapeutics, 2001, 29, 1514-1520.