Lab Project

Exploration of the Voltammetry of Glucose

Objectives

• To examine the electrochemistry of glucose at a gold electrode in alkalinesolution.

• To examine the electrochemistry of a gold electrode in alkaline solution.

• To become familiar with the operation of a rotating disk electrode (RDE).

• To gain enough information regarding the electrochemistry of both glucoseand a gold electrode in alkaline solution to be able to apply this information tothe electrochemical detection of sugars following their separation by HPLC.

Introduction

The voltammetry that we've done in the lab to this point has all been performed inunstirred solutions. However, as we will discuss in class, the shape of the voltammogramchanges significantly when the solution is stirred. One of the most straightforwardmethods of generating a constant rate of stirring is by simply rotating the electrode. Thedynamics of the rotating disk electrode (RDE) have been thoroughly examined. Thelimiting current (i

�) at an RDE is described by the Levich equation:

i� = 0.62nFAD2/3ν−1/6ω1/2Cb

where A is the electrode area, D is the diffusion coefficient of the analyte, ν is thekinematic viscocity of the solution, ω is the rotatiopn rate, and Cb is the bulkconcentration of analyte. In this experiment we are going to examine the voltammetry ofglucose at a gold rotating disk electrode (RDE).

Electrochemistry of Glucose in Alkaline Solution

Glucose shows some very interesting behavior at a gold RDE. The oxidation in alkalinesolution occurs in two steps. The first step, signified by an increase in the anodic(positive) electrode current, is believed to correspond to the oxidation of the aldehydegroup of the glucose molecule. The second step is signified by another large increase incurrent. This step shows some very interesting behavior: the current peaks and then shutsoff. It is believed that this second step involves some sort of C−C bond breaking. Theshutting down of the process coincides with the beginning of oxide formation on the goldelectrode. This oxide apparently passivates the surface toward further glucose oxidation.

Mass Transport-Controlled Processes vs. Surface Adsorption-Controlled Processes

Often times we are interested in determining whether an electrochemical reaction arisesfrom analyte being brought to the electrode surface from the bulk of the solution (a masstransport-controlled process), or from the reaction of species already adsorbed on theelectrode surface (an adsorption-controlled process). One way to tell the difference is by

varying the stirring rate of the solution. If an increased stirring rate increases the current,then we know that the process is mass transport-controlled. However, if stirring has noeffect on the current, then we can usually assume that the process is due to some speciesalready adsorbed on the electrode surface.

We can also distinguish between adsorption-controlled and mass transport-controlledprocesses by varying the scan rate of the potential waveform. A mass transport-controlled process should be independent of the scan rate. An adsorption-controlledprocess, however, will show a larger current at faster scan rates.

Procedure

A. Solution Preparation. Prepare 100 mL of a 0.1 M solution of glucose in water.

B. Electrode Preparation. Polish the gold RDE with diamond paste and alumina asoutlined in the polishing manual.

C. Examination of the Electrochemistry of the Gold Electrode.

1) Fill the cell approximately 1/3 full with 0.25 M NaOH. (Know the volume ofsolution you added.) Use a disposable pipet to fill the arms of the cell to the samelevel as the main body.

2) Set up the 3-electrode cell and begin cycling the potential between −1.0 V and+0.6 V while rotating the electrode at 1600 rpm. Run the cyclic voltammetryLabVIEW VI to see the voltammetry that's occurring.

3) Cover the cell with the Teflon lid and begin purging the solution with N2. Noticehow the oxygen wave disappears on the voltammogram. Play with the upper andlower scan limits. Be able to identify both the oxidation and the reduction ofwater, along with the oxidation and reduction of the gold itself. Record and savea CV that shows all of these features. Then return the scan limits to their initialvalues. When the oxygen has been completely purged, switch the stopcockposition on the N2 line to begin blowing the N2 over the solution.

4) Record and save voltammograms of the Au electrode in 0.25 M NaOH at scanrates of 1, 2, 3, 4, 5, and 6 V/min at a constant rotation speed of 1600 rpm.

5) Record and save voltammograms of the Au electrode in 0.25 M NaOH at rotationrates of 100, 200, 400, 900, 1600, and 2500 rpm at a constant scan rate of yourchoice.

D. Electrochemistry of Glucose.

1) Calculate the amount of glucose stock solution you'll need to add to bring theconcentration of glucose in the cell to 2 mM. Add this amount.

2) Record and save voltammograms for glucose concentrations of 2, 4, 6, 8, and 10mM at a rotation rate of 1600 rpm.

3) Record and save voltammograms for a 10 mM glucose solution at rotation rates of100, 200, 400, 900, 1600, and 2500 rpm.

Report

THIS REPORT MUST BE COMPLETED BEFORE THE NEXT LABORATORYPERIOD.

Tell the story of the electrochemistry of glucose on gold in 0.25 M NaOH. Begin bytalking about the electrochemistry of the gold electrode in the absence of glucose. Youshould show:

• a single CV of a gold electrode in 0.25 M NaOH indicating the major features ofthe voltammogram such as Au oxidation, AuO stripping, oxygen evolution, andhydrogen evolution.

• a single graph showing all the CV's taken as a function of scan rate.

• another graph showing all the CV's taken as a function of rotation rate.

What's the main difference between these last two graphs? How do you account for thisdifference?

Now begin talking about the electrochemistry of glucose. You should show:

• a single graph showing all the CV's taken as a function of concentration.

• a plot of i� vs. glucose concentration for both the wave and the peak. Is this plot

consistent with Levich?

• a single graph showing all the CV's taken as a function of rotation rate.

• a plot of i� vs. ω1/2 for both the wave and the peak. Is this plot consistent with

Levich?

Lab Project

Pulsed Amperometric Detection of Glucose

Introduction

There are a number of effective detection methods routinely employed in HPLC. Whilethe most common method is UV absorbance, it is not always the best. Many importantclasses of compounds lack a natural "chromophore", i.e. they possess no functional groupthat will absorb light to any appreciable extent. In such cases, electrochemical detectioncan offer a powerful alternative to UV absorbance.

The most straightforward way of performing electrochemical detection is to simply placean electrode in the flow path of the eluant, then apply a potential to the electrode that willeither oxidize or reduce the analytes as they flow by. Plotting the electrode currentversus time generates the chromatogram. Unfortunately, we're going to see that it's notalways this easy. Your assignment today is to develop an electrochemical detectionassay for glucose in fruit juice. In this set of experiments we're going to see:

1) how a constant electrode potential will NOT work for the detection of carbohydratesfollowing their separation by HPLC

2) how we can use what we've learned about the oxidation/reduction behavior of glucoseand the gold electrode to develop an electrochemical detection method forcarbohydrates

HPLC Separation of Carbohydrates

Simple carbohydrates (sugars, sugar alcohols, etc…) are weakly acidic (pKa ~ 12-14),therefore they are at least partially ionized in solutions of high pH. This suggests that wemay be able to use an anion-exchange column along with a strongly basic mobile phaseto separate a mixture of carbohydrates. This works out beautifully if we intend to useelectrochemical detection since we saw that glucose (and other carbohydrates) gives astrong oxidation signal at high pH.

To accomplish the separation we'll use the Dionex CarboPac PA-1 column. The PA-1 isan anion-exchange column specifically designed to separate carbohydrates. By the way,this column costs $800 to replace, so be careful with it.

Detection of Carbohydrates at Constant Potential

You've already looked at the voltammetry of glucose in alkaline solution. Mostcarbohydrates will generate voltammograms quite similar to glucose. Therefore,examination of your glucose voltammetry data should give you a pretty good idea as towhat electrode potential will give a strong oxidation signal for all carbohydrates. So ifyou're interested in developing an LC-EC (liquid chromatography - electrochemicaldetection) method for carbohydrates, you may naively conclude that all you need to do isset your working electrode at this potential, then sit back and let the peaks roll on by.Unfortunately, it isn't quite this easy.

When glucose is oxidized at a gold electrode, some of the products of the oxidationreactions tend to stick to the electrode. Over time these products build up on theelectrode surface and decrease the area available for further oxidation to occur. We oftensay that these oxidation products poison the electrode.

Pulsed Amperometric Detection of Carbohydrates

We can cleverly use what we've learned about gold oxide formation and stripping to getaround this problem of poison formation. If we increase the electrode potential into theregion of gold oxide formation, this oxide will "push" away all the poisons that formedduring the carbohydrate reaction. So this rids the surface of the poison. But now we'vegot a new problem. We've got an electrode that is completely oxidized, and we saw fromour glucose voltammetry experiments that oxide formation passivates the electrodetoward further glucose oxidation. So now we need to decrease the electrode potentialinto the region where the gold oxide is stripped away. This should leave us with a cleangold surface that is once again ready to oxidize any carbohydrates that flow by.

What we've just developed is a 3-step waveform that we'll apply to our workingelectrode. In other words, we'll be pulsing the electrode potential. Let's review each stepof the waveform:

a) Edet (detection potential): This is the potential at which the carbohydrates areoxidized. We choose this potential based on the results of our glucosevoltammetry experiments.

b) Eox (oxidation potential): At this potential the gold electrode is oxidized. Thisoxide formation rids the surface of the poisons that formed during thecarbohydrate oxidation. We want to be sure that this potential is high enoughto form gold oxide, but not high enough to evolve O2 gas.

c) Ered (reduction potential): The gold oxide formed at Eox is stripped at thispotential, once again restoring our clean gold electrode. This potential mustbe low enough to strip away the gold, but not so low that H2 gas is evolved.

The graph on the next page depicts this 3-step waveform. The times chosen for each ofthe potential steps are critical. The variables tox and tred are easy to understand. They aresimply the times needed for oxidation and reduction, respectively. But notice that theelectrode is held at Edet for a time of tdel + tint. The delay time (tdel) is a waiting periodduring which the background charging (non-faradaic) current is allowed to decay to zero.(Remember that anytime a potential step is applied to any electrode, a large spike in thecharging current results.) Following this delay time the current at the electrode ismeasured for a time period known as the integration time (tint). Any non-zero currentmeasured during this time should be the result of a faradaic process. Remember thatcurrent integrated over time results in charge. Therefore, the units of our detector outputwill be in coulombs (C).

The total time period for the waveform is therefore tdel + tint + tox + tred. It is importantthat this time period be long enough to result in appreciable current measurement,complete oxide formation, and complete stripping of the gold oxide. But this time periodmust be short enough to sample the eluent stream at a high enough frequency to properlydepict any peaks that may elute. The total time period of the waveform is usually ~1 s.This means that our sampling frequency is 1 Hz, or data point on the chromatogram isaccumulated every second.

The method we've just developed is called pulsed amperometric detection (PAD). It isnow the most popular analytical method for determining carbohydrates in a mixture. Ifyou were to walk into the analytical lab at American Crystal Sugar, you wouldimmediately see several commercial PAD systems.

The Experiment

Provided Chemicals

• glucose (dextrose) • sucrose

• fructose

Procedure

A. Solution Preparation. Prepare 100.0 mL of 0.10 M stock solutions of glucose,fructose, and sucrose.

time

Ele

ctro

de

Po

ten

tial

Eox

Ered

Edet

tdel tint tox tred

B. Detection of Glucose at Constant Potential.

1. Dilute 0.500 mL of your glucose stock solution to 100.0 mL. What is theresulting concentration?

2. Use your voltammograms of glucose to select a detection potential for theworking electrode in the flow-through cell of the chromatography system.

3. Use the LC-EC program to detect glucose following its separation on the PA-1column. Do this with multiple injections (~10) of the solution created above. Irecommend just starting the LC-EC program and making a new glucose injectionevery minute.

C. Pulsed Amperometric Detection of Glucose.

1. Open the LC-PED program. Use what you know about the oxidation of glucose,and the oxidation/reduction of the gold surface, to choose values for Edet, Eox, andEred.

2. We want the waveform to cycle once a second. Use the following values for thetime parameters:

tdel 200 mstint 200 mstox 200 mstred 400 ms

1000 ms3. Apply the waveform to the working electrode. Let this run for ~5 minutes to

condition the electrode. Then perform the multiple injection experiment again.Hopefully you'll see a big difference.

4. Generate calibration data for glucose and make a calibration plot of peak heightversus glucose concentration (in mM). See how low you can make theconcentration before you lose the signal. Also try to find the upper limit oflinearity of the method.

D. Determination of Retention Times for Fructose and Sucrose. Make separate solutionsof fructose and sucrose by diluting 1.00 mL of each stock solution to 100.0 mL.Inject a sample of each solution to determine the retention times of these compounds.

E. Determination of Glucose in Fruit Juice. Our goal here is to dilute our juice sampleuntil the glucose peak is in the range of our calibration data. It's better to make thesample too dilute than too concentrated. I recommend beginning with a 1:1000dilution in water. Filter the sample with a syringe-filter before injecting it into thechromatograph.

Report

Trace your steps through each of these experiments, showing the relevant data whereappropriate. Make sure that you calculate the glucose concentration in the fruit juice.Also point out the fructose and sucrose peaks if they are present.

Instructor Notes

I. Exploration of the Voltammetry of Glucose

Sample data:

A. Voltammetric features of Au in alkaline solution.

-0.14

-0.1

-0.06

-0.02

0.02

0.06

-1100 -900 -700 -500 -300 -100 100 300 500 700

Potential (mV vs SCE)

Cu

rren

t (m

A)

with oxygen

without oxygen

• Au RDE in 100 mL 0.25 M NaOH before and after purgingwith N2 to eliminate O2.

• Scan rate: 6 V/min; Rotation rate: 1600 rpm

• Upper potential limit could be raised slightly to see moreobvious oxidation of H2O to O2. Lower potential limitcould be dropped to see reduction of H2O to H2.

B. Scan rate dependence.

-0.1

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

-1100 -900 -700 -500 -300 -100 100 300 500 700

Potential (mV vs. SCE)

Cu

rren

t (m

A)

• Scan rates of 1 (smallest curve), 2, 3, 4, 5, and 6 V/min.

• Rotation rate: 1600 rpm.

C. Rotation rate dependence.

• Changing rotation rate has no effect on voltammogram.

• Gold oxidation is adsorption controlled and not masstransport controlled.

D. Concentration dependence of glucose.

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

-1100 -900 -700 -500 -300 -100 100 300 500 700

Potential (mV vs SCE)

Cu

rren

t (m

A)

• Concentrations of 2 (smallest curve), 4, 6, 8, and 10 mMglucose in 0.25 M NaOH.

• Rotation rate: 1600 rpm; Scan rate: 6 V/min.

E. Levich plot for glucose concentration dependence.

R2 = 0.9993

R2 = 0.9999

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.0 2.0 4.0 6.0 8.0 10.0 12.0

Glucose Concentration (mM)

Cu

rren

t (m

A)

• Peak current (■ ) measured at approximately +160 mV;Wave current (◆ ) measured at –300 mV.

F. Rotation rate dependence of glucose.

-0.05

0.05

0.15

0.25

0.35

0.45

0.55

0.65

-1100 -900 -700 -500 -300 -100 100 300 500 700

Potential (mV vs SCE)

Cu

rren

t (m

A)

• For clarity, only the forward (anodic) scan is shown.

• 10 mM glucose; Scan rate: 6 V/min.

• Rotation rates: 100 (smallest curve), 200, 400, 900, 1600,and 2500 rpm.

G. Levich plot for rotation rate dependence.

R2 = 0.9994

R2 = 0.9914

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.0 10.0 20.0 30.0 40.0 50.0 60.0

(rotation rate)1/2

Cu

rren

t (m

A)

• Peak current (■ ) measured at approximately +160 mV;Wave current (◆ ) measured at –300 mV.

II. Pulsed Amperometric Detection of Glucose

A. Experimental Set-Up:

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B. Notes.

• The entire procedure can be completed in one 4-hour lab period. Studentsshould work in groups of 3-4, dividing the tasks among themselves so thatthere is very little downtime.

• The LabVIEW VI's are compatible with either LabVIEW 5.1 (or higher)or the Student Edition of LabVIEW 6i.

• Our voltammetry data was acquired using a saturated calomel referenceelectrode, however the BAS LC44 uses a silver/silver chloride reference.We are now consistent and use a silver/silver chloride for each, howeverthe potential difference between the two reference electrodes is smallenough (~44 mV) that the potentials determined from the SCEvoltammetry data work well in the silver/silver chloride PAD cell.

• We use a Pine AFRDE5 bi-potentiostat, but a BAS CV-27 has also beensuccessfully tested.

• Our experimental configuration gives a large level of background noise, sowe are unable to detect low concentrations of the carbohydrates.Commercial LC-PAD instruments yield limits of detection several ordersof magnitude below what we are able to see.

• Since we are only able to detect relatively high carbohydrateconcentrations we must be aware of the possibility of overloading theHPLC column. A mobile phase of 0.25 M NaOH is strong enough to keepthe column clean, yet weak enough to still give an adequate separation ofglucose and fructose. (More complicated LC-PAD applications call formobile phase concentrations in the 20–100 mM range.) It may benecessary, however, to briefly clean the column with 1.0 M NaOH ifretention times begin to decrease.