electrochemical determination of caffeine content in ethiopian coffee samples ...€¦ ·...

Research ArticleElectrochemical Determination of Caffeine Content in EthiopianCoffee Samples Using Lignin Modified Glassy Carbon Electrode

Meareg Amare and Senait Aklog

Bahir Dar University PO Box 79 Bahir Dar Ethiopia

Correspondence should be addressed to Meareg Amare amaremearegyahoocom

Received 20 January 2017 Accepted 5 March 2017 Published 23 April 2017

Academic Editor Sibel A Ozkan

Copyright copy 2017 Meareg Amare and Senait Aklog This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Lignin film was deposited at the surface of glassy carbon electrode potentiostatically In contrast to the unmodified glassy carbonelectrode an oxidative peak with an improved current and overpotential for caffeine at modified electrode showed catalyticactivity of the modifier towards oxidation of caffeine Linear dependence of peak current on caffeine concentration in the range6 times 10minus6 to 100 times 10minus6mol Lminus1 with determination coefficient and method detection limit (LoD = 3 sslope) of 099925 and837 times 10minus7mol Lminus1 respectively supplemented by recovery results of 9379ndash10217 validated the developed method An attemptwasmade to determine the caffeine content of aqueous coffee extracts of Ethiopian coffees grown in four coffee cultivating localities(WonberaWolega Finoteselam and Zegie) and hence to evaluate the correlation between users preference and caffeine content Inagreement with reported works caffeine contents (ww) of 0164 inWonbera coffee 0134 inWolega coffee 0097 in Finoteselamcoffee and 0089 in Zegie coffee were detected confirming the applicability of the developed method for determination of caffeinein a complex matrix environment The result indicated that usersrsquo highest preference for Wonbera and least preference for Zegiecultivated coffees are in agreement with the caffeine content

1 Introduction

Alkaloids are broad category of nitrogen containing organicmetabolites produced by plants the plants that produce thesealkaloids make their leaves unattractive to eating by insectsand higher animals [1] The awfully known toxic compoundsmorphine quinine cocaine and codeine belong to thisgroup of compounds Caffeine (37-dihydro-137-trimethyl-1H-purine-26-dione) is one of the naturally occurring alka-loids that is widely contained in plant products and beveragesCaffeine is a natural stimulant contained in many sourceslike coffee tea chocolate soft drinks and tablets for thetreatment of many diseases such as asthma nasal congestionand headache and even for improving athletic endurance andfacilitating weight loss [1] Many of caffeine consumers getcaffeine from multiple sources the caffeine content of whichvaries with the type of source and its amount [2ndash5]

However reports on human and animal studies showedthat caffeine produces mental and behavioral effects that aresimilar to those of typical psychomotor stimulant drugs such

as amphetamine and cocaine [6] Stimulation of the centralnervous system diuresis gastric acid secretion and increasedblood pressure are among the reported physiological effectsassociated with caffeine [7] Among the different possiblesources coffee is known to have the highest caffeine concen-tration and yet the most utilized source of caffeine [5]

The word coffee is believed to be derived from Keffawhich is the name of the locality in AbyssiniaEthiopia wherecoffee beans were first discovered by Sheferds in the 6thcentury [8] Since then coffee has become one of the mostwidely consumed beverages throughout the world [9]

Coffee beans which are seeds of Rubiaceae botanic familyand Coffea genus [10] have two common species Coffeaarabica andCoffea canephora [11 12]Whilemost of the coffeeplants cultivated in Ethiopia are Coffee arabica there arehowever wide ranges of variabilities including decaffeinatedvarieties in the country [2 13 14] which might be attributedto variation in the soil altitude and climate of the localitieswhere they are grown [4 15]

HindawiJournal of Analytical Methods in ChemistryVolume 2017 Article ID 3979068 8 pageshttpsdoiorg10115520173979068

2 Journal of Analytical Methods in Chemistry

Among the coffee varieties in the country coffees cul-tivated at four localities of Ethiopia Finoteselam WolegaZegie and Wonbera are commonly consumed by the con-sumers in Bahir Dar City North West Ethiopia Of these thecoffee cultivated in Wonbera locality is the most expensiveand preferred while the Zegie coffee is the cheapest and leastpreferred the basis for which might be psychological aromaotherwise the caffeine content Thus the aim of this researchwas to check whether the usersrsquo preference can be related tothe caffeine content or not which needs determination of thecaffeine content of the four coffee varieties using a sensitiveselective fast and environmentally friendly method

High performance liquid chromatography [16 17] cap-illary chromatography [18 19] spectroscopy [20 21] andelectrochemical [22 23] methods are among the reportedmethods for determination of caffeine in coffee tea and colabeverage samples Most of the reported methods require car-cinogenic organic solvents for extraction and hence are envi-ronmentally nonfriendly Moreover using time-consumingsample preparation procedures such as liquid-liquid extrac-tion and solid-phase extraction or the use of more thanone chromatographic step costly instrumentation and highskilled technician [12 23] makes them inconvenient

In contrast to the conventional analytical methods elec-trochemical methods are powerful and versatile analyticaltechniques that offer high sensitivity accuracy and preci-sion relatively wider linear dynamic range and low-costinstrumentation specificity for a particular oxidation stateof an element relative inexpensiveness and being usuallyenvironmentally friendly (use no or minimum amount oforganic solvent) [23 24]

In recent years working electrodes including borondoped diamond [25ndash28] carbon paste and carbon nanotubes[29 30] and glassy carbon [22 31 32] based electrodes havebeen reported for determination of caffeine in real samples

Carbon materials have broad potential window lowbackground current rich surface chemistry comparativechemical inertness relatively easy electrode preparation andlow cost making them electrodes of choice [33] Deliberateand controlled modification of the electrode surface canproduce electrodes with new and interesting properties thatmay form the basis of new applications of electrochemistry[34] Polymer-modified electrodes (PMEs) have receivedconsiderable attention in recent years due to their goodstability reproducibility increased active sites homogeneityin electrochemical deposition and strong adherence to theelectrode surface [35 36]

Lignin is a natural polymer contained in most woodytrees and shrubs [37] Approximately 2of the lignin releasedduring chemical pulping in pulp and paper industries is usedfor chemical and material applications and the remainder isburned as boiler fuel showing its availability Although theelectrochemical behavior of lignin at the surface of glassycarbon electrode has been reported [38] to the best of ourknowledge its application for electrochemical determinationof caffeine in aqueous extracts of coffee samples has notbeen reported This paper reports a simple cheap and envi-ronmentally friendly lignin based electrochemical methodfor determination of caffeine in aqueous extracts of coffee

samples cultivated in different localities of Ethiopia theorigin of coffee

2 Experimental

21 Chemicals and Instruments Caffeine (Fischer Scientific)H3PO4

(Cipla Ltd India) K2HPO4

(Wagtech Interna-tional Ltd UK) CH

3COOH and NaOH (Landmark chem-

icals PVT India) HCl (Riedel-De Haen Germany) lignin(Riedel-De Haen Germany) HNO

3(65ndash70) and H

2SO4

(99) (both from Loba chemie Pvt Ltd) were used Allreagents were of analytical grade and were used directlywithout further purification Glassware was cleaned usingchromic solution prepared by dissolving 1 g of potassiumdichromate in 1 L of H

2SO4followed by rinsing with distilled

water Distilled water was used for the preparation of allsolutions

Voltammetric experiments were carried out usingCHI760D Electrochemical Workstation (Austin TexasUSA) connected to a personal computer All electrochemicalexperiments were performed employing a conventionalthree-electrode system with a glassy carbon electrode (3mmin diameter) or a Lignin modified glassy carbon electrode astheworking electrode platinum coil as an auxiliary electrodeand AgAgCl as a reference electrode All experiments werecarried out at 20 plusmn 2∘C

22 Real Sample Preparation Coffee samples were purchasedfrom the respective local markets (Wolega FinoteselamZegie andWonbera) Aqueous extracts of the coffee sampleswere prepared following the procedure outlined elsewhere[22] Briefly exactly 50 g of coffee from each sample wasroasted by using conventional coffee roasting machine Eachroasted coffee sample was ground using a mortar and pestleand screened through 250 120583M sieve to get a uniform textureAn accurately weighed 4 g of sieved coffee from each samplewas then boiled in 200mL of distilled water in the tempera-ture range 80ndash90∘C for 30min while stirring using magneticstirrer The water extracted caffeine was finally separatedfrom the residue by decantation and was ready for the DPVanalysis

23 Preparation of Lignin Modified Glassy Carbon ElectrodeThe lignin modified glassy carbon electrode (LGCE) wasprepared following a procedure reported elsewhere [38]Briefly a glassy carbon electrode (3mm diameter) wasfirst rinsed with distilled water and polished carefully withalumina powder having different particle size (10 03 and005 120583m) to a mirror finish surface The residual polishingmaterial was removed by repetitive rinsing of the surfacewith distilled water The resulting electrode was immersedin pH 7 Phosphate buffer solution (PBS) and was activatedpotentiodynamically by scanning the potential between minus02and+15 V for five cycles at a scan rate of 100mV sminus1Then theactivated electrode was transferred to a cell containing ligninin a mixture of 05M H

2SO4and 01M HNO

3prepared by

dissolving 10mgof lignin in 50mLof themixture (1 1 volumeratio)

Journal of Analytical Methods in Chemistry 3

Lignin polymer was then deposited at the electrodesurface at a potential of +09V for 2 minutes The ligninmodified electrode was then rinsed with distilled water toremove physically adsorbed and unreacted species from theelectrode surface Subsequently the modified electrode wasstabilized in pH 7 PBS by scanning the potential betweenminus02 V and +10V until a steady cyclic voltammogram wasobtained Finally the modified electrode was dried in air andmade ready for use

24 Preparation of Standard Solutions For cyclic voltammet-ric studies 10mM stock solution of caffeine was preparedby dissolving 01941 g of caffeine in 100mL of 01M pH 5Acetate buffer solution (ABS) [22] From the stock solutionwhile 1mMsolution of caffeinewas used for the voltammetricinvestigation of caffeine at both the unmodified andmodifiedelectrodes working solutions of different concentrations ofcaffeine (6 8 10 20 30 50 60 80 and 100 120583M) in pH 5 ABSwere prepared from 500 120583M intermediate solution throughserial dilution

3 Results and Discussion

Cyclic voltammetric and differential pulse voltammetrictechniques were used to investigate the electrochemicalbehavior of caffeine and determine caffeine content in coffeesamples respectively

31 Cyclic Voltammetric Behavior of Caffeine at LGCE Fig-ure 1 presents the cyclic voltammograms of unmodifiedand modified glassy carbon electrodes in the absence andpresence of caffeine

As can be seen from the figure no peak was observed atboth the unmodified and modified glassy carbon electrodesin caffeine-free buffer solution On the contrary well resolvedoxidative peak was recorded at the unmodified GCE andLGCE at a potential of 1578 and 1510V respectively Absenceof a reductive peak indicated that caffeine undergoes anirreversible oxidation at both the modified and unmodifiedelectrodes which is in agreement with previously reportedworks [22 36]

Appearance of an oxidative peak with a relatively largerpeak current and improved peak potential at the ligninmodified electrode compared with the unmodified GCE alsoshowed the catalytic activity of the lignin modified electrodewhich might be due to an increased electrode surface areaandor improved electron exchange at the electrode surface[22 38] making it suitable for caffeine analysis

32 Effect of Scan Rate To investigate the type of reactionkinetics the caffeine follows at the surface of lignin modifiedglassy carbon electrode the determination coefficients (1198772)for the plots of peak current as a function of scan rate andsquare root of scan rate were compared in the scan rate range10ndash400mV sminus1 (Figure 2) As can be seen from the figurethe oxidative peak current shifted to a higher positive valuewith increasing scan rate confirming the irreversibility ofthe oxidation reaction of caffeine at the modified electrode

(a)

(b)

minus50

minus40

minus30

minus20

minus10

0

Curr

ent (

휇A

)

16 14 12 10 0818E (V versus AgAgCl)

(a㰀)

(b㰀)

Figure 1 CVs of unmodified GCE (a and a1015840) and LGCE (b and b1015840)in pH 5 ABS containing no (a and b) and 1mM (a1015840 and b1015840) caffeine

(a)

(m)minus50

minus40

minus30

minus20

minus10

0Cu

rren

t (휇

A)

16 14 12 10 08 0618E (V versus AgAgCl)

Ipa (휇A) = minus565 minus 211 V sminus1)12

R2 = 099915

minus50minus45minus40minus35minus30minus25minus20minus15minus10

Ipa (

휇A)

4 6 8 10 12 14 16 18 20 222(Scan rate)12

]12 (

Figure 2 Cyclic voltammograms of lignin modified GCE at pH 5ABS containing 1mMcaffeine at different scan rates ((a)ndash(m) 10 2040 60 80 100 125 150 200 250 300 350 and 400mV sminus1 resp)Inset plot of oxidative peak current as a function of the square rootof scan rate

Furthermore a higher determination coefficient (1198772 = 0999)for the dependence of peak current on the square root ofscan rate (inset of Figure 2) than for the dependence ofpeak current on the scan rate indicated that the oxidation ofcaffeine at themodified electrode follows diffusion controlledkinetics which is in agreement with reported works [33]

33 Effect of Solution pHon the PeakCurrent Theeffect of pHon the electrochemical response of lignin modified electrodefor caffeine was studied in the pH range of 3 to 6 Figure 3presents the cyclic voltammograms of 1mM caffeine in 01MABS of various pHs at LGCE As can be observed from thefigure the oxidative peak current increased with pH 3 to4 which then decreased in pH values beyond 4 Thus pH4 was selected as the optimum pH which was used in thesubsequent experiments


335445

5556

minus60

minus50

minus40

minus30

minus20

minus10

0

10

Curr

ent (

휇A

)

16 14 12 10 08 0618E (V versus AgAgCl)

4 62pH

minus26minus28minus30minus32minus34minus36minus38minus40

Ipa (

휇A)

Figure 3 CVs of LGCE in ABS of different pH values (30ndash60)containing 10 times 10minus3mol Lminus1 of caffeine Inset plot of Ipa versus pHin pH range 30ndash60 at a scan rate of 100mV sminus1

(a)

(b)

minus18

minus15

minus12

minus9

minus6

minus3

0

Cor

rect

edI a

(휇A)

16 14 12 1018A

Figure 4 Differential pulse voltammograms (corrected for back-ground) of unmodified (a) and modified (b) GCE in pH 40 ABScontaining 1mM caffeine

34Differential PulseVoltammetric Studies of Caffeine at LGCESince it is an effective voltammetric method with estab-lished advantages including good discrimination againstbackground current and low detection limit differentialpulse voltammetry (DPV) was used for the quantificationof caffeine content of coffee samples cultivated in differentlocalities of Ethiopia Figure 4 presents differential pulsevoltammograms of unmodified and lignin modified glassycarbon electrodes in pH 4 ABS containing 1mM caffeineAn oxidative peak with an improved peak current and peakpotential at the modified electrode (curve (b) of Figure 4)relative to at the unmodified electrode (curve (a) of Figure 4)confirmed the catalytic effect of the lignin modified glassycarbon electrode towards oxidation of caffeine

35 Optimization of Method Parameters The effect of dif-ferential pulse voltammetric parameters such as the steppotential and pulse amplitude on the peak current responseof lignin modified glassy carbon electrode for 1mM caffeineunder the optimized pH 4 ABS was investigated (Figure 5)Although it is customary that the peak current increaseswith step potential and pulse amplitude it is necessary tooptimize these parameters by compromising the peak cur-rent enhancement with the accompanied peak broadening(capacitive peak current) Taking both the peak currentenhancement and peak shape broadening into consideration8 and 75mV were taken as the optimum pulse step potentialand differential pulse amplitude respectively

After optimizing the method and solution parametersDPV voltammograms were obtained for the LGCE in pH 40ABS containing no (a) 1mM caffeine (b) and the correctedvoltammogram for the blank (c) (Figure 6)

As can be observed from the figure closeness of thepeak current of the subtracted (c) and unsubtracted (b)voltammograms of the LGCE showed low capacitive currentof the modified electrode and hence fitness of the electrodefor caffeine determination

36 Calibration Curve and Method Detection and Quantifi-cation Limits Under the optimized solution and methodparameters anodic peak current of caffeine at ligninmodifiedGCE was linearly proportional to the caffeine concentrationin the range of 6 to 100 times 10minus6mol Lminus1 (Figure 7) with alinear regression equation and determination coefficient of119868119886120583A = minus0060 + 0034 [Caffeine] 120583M and 1198772 = 099900

respectively Method limit of detection (LoD = 3 sm) andlimit of quantification (LoQ = 10 sm where s is blankstandard deviation for 119899 = 7) were calculated to be 837 times10minus7 and 279 times 10minus6M respectively making the developedmethod comparable with themethod usingNafion [31] whichis expensive electrode modifier

37 Application of theMethod for theDetermination of Caffeinein Coffee Samples Aqueous coffee extract samples were pre-pared as described in the procedure under the experimentalpart To determine the concentration of caffeine in real coffeesamples cultivated in four different localities of Ethiopiathe differential pulse voltammetric peak current for eachextract sample was recorded which then was converted toconcentration units using the regression equation of thecalibration curve Figure 8 presents the DPVs for the coffeeextracts of the analyzed four coffee types Table 1 presentsthe summary of the amount of caffeine in the analyzed coffeesamples expressed in different units As can be seen from thetable the caffeine content of the four coffee samples in anincreasing order is Zegie FinoteselamWolega andWonberawhich is in agreement with the reported trend [39 40] Theobserved difference in caffeine content could be attributedto the difference in the agronomical differences between thelocalities where they were cultivated This also indicated thatthe highest and least preference of the people around thelocalities for Wonbera cultivated coffee and Zegie cultivatedcoffee respectively might be associated with the caffeinecontent of the coffees


minus20

minus15

minus10

minus5

0

Ipa (

휇A)

16 14 12 081018E (V versus AgAgCl)

(a)

(c)

(a)

16 14 12 10 0818

minus30

minus25

minus20

minus15

minus10

minus5

0

Ipa (

휇A)

(a)

(d)

E (V versus AgAgCl)

(b)

Figure 5 DPVs of lignin modified GCE in pH 4 ABS containing 1mM caffeine at (a) different step potentials ((a)ndash(c) 4 8 and 12mV resp)and pulse amplitude of 50mV and (b) different amplitudes ((a)ndash(d) 25 50 75 and 100mV resp) and step potential of 8mV

(a)

(b)

(c)

minus35

minus30

minus25

minus20

minus15

minus10

minus5

0

I a(휇

A)

16 14 12 1018Ea (V versus AgAgCl)

Figure 6 DPVs of LGCE in pH 4 ABS containing (a) no (b)1mM caffeine and (c) 1mM caffeine corrected for the blank underoptimized conditions

38 Recovery Study of the Developed Method To evaluatethe accuracy of the developed DPV method using ligninmodified GCE the recovery of spiked standard caffeinein aqueous extracts of Wolega coffee which showed anintermediate amount of caffeine among the studied coffeesamples was checked Figure 9 presents the voltammogramsof an aqueous extracts of Wolega coffee detected to contain2631 120583M caffeine spiked with different volumes of 60120583Mstandard caffeine

As can be seen from the figure the DPV anodic peakcurrent increased with increasing volume of the spiked

(a)

(j)

minus30

minus25

minus20

minus15

minus10

minus05

00

05

I a(휇

A)

14 12 10 08 0616Ea (V versus AgAgCl)

0

2

4I a

(휇A)

R2 = 099900

Ia (휇A) = minus0060 + 0034C (휇M)

20 40 60 80 1000conc (휇M)

Figure 7 DPVs of LGCE in pH 40 ABS containing variousconcentrations of caffeine ((a)ndash(j) 6 8 10 20 30 40 50 6080 and 100 120583M resp) Inset plot of anodic peak current versusconcentration of caffeine

standard indicating the sensitivity of the method devel-oped Excellent recovery results in the range 9379ndash10217(Table 2) were obtained indicating the applicability of thedeveloped method for the determination of caffeine in acomplex aqueous coffee extracts

4 Conclusion

This work presents the differential pulse voltammetric deter-mination of water extracted caffeine content of coffee samples


Table 1 Summary of concentration of caffeine in aqueous coffee extract and corresponding amount of caffeine per gram of roasted coffee ofcoffee samples from different localities of Ethiopia

Locality of coffee sample DPV oxidative peak current (120583A) Caffeine concentration (120583A) Caffeine (mggminus1)lowast

Wonbera 145 4441 1078Wolega 117 3618 878Finoteselam 083 2618 635Zegie 076 2412 585lowastCaffeine content per gram mass of powdered coffee sample

Table 2 Summary of percentage recoveries of spiked standard caffeine in aqueous coffee extract

Sample Initial120583M Spiked standard (120583M) Expected

120583M recovery

Coffee extract (a) 2631 mdash mdash mdashCoffee extract (b) 2631 543 545 9963Coffee extract (c) 2631 800 783 10217Coffee extract (d) 2631 1299 1385 9379Coffee extract (e) 2631 1533 1556 9852

(a)

(d)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00

I pa

(휇A)

10 11 12 13 14 15 1609E (V versus AgAgCl)

Figure 8 DPVs of LGCE in pH 4 ABS containing aqueous ex-tracts of coffee samples cultivated in different localities of Ethiopia((a)ndash(d) Zegie Finoteselam Wolga and Wonbera resp)

cultivated in different localities of Ethiopia using a ligninmodified glassy carbon electrode The electrode modifierreported is relatively cheap environmentally friendly easilydeposited at the electrode surface exhibiting low capacitivecurrent and easy to clean

Differential pulse voltammetric current response showedlinear dependence on the concentration in the range6ndash100 120583M with a linear regression equation LoD and LoQof 119868119886120583A = minus0060 + 0034 [Caffeine] 120583M 837 times 10minus7M

and 279 times 10minus6M respectively Low detection limit highprecision and excellent recovery results indicated the appli-cability of the developed method for the electrochemicaldetermination of caffeine in water extracts of real samplesof coffee without any interference Caffeine contents ofthe coffee samples from four localities (Wonbera WolegaFinoteselam and Zegie) of the origin of coffee Ethiopia were

(a)

(e)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00I p

a(휇

A)

105 120 135 150 165090E (V versus AgAgCl)

Figure 9 DPVs of aqueous Wolega coffee extract samples spikedwith different volumes of 60120583M of standard caffeine ((andashe) 0 2 36 and 7mL resp)

found in the order of (mg gminus1) 1078 878 635 and 585respectively From the result it seems that the highest andlowest preference of the coffee consumers around the studyarea for Wonbera and Zegie cultivated coffee respectively isassociated with the caffeine content of the coffee varieties

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

References

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[2] J J Barone and H R Roberts ldquoCaffeine consumptionrdquo Foodand Chemical Toxicology vol 34 no 1 pp 119ndash129 1996

[3] M S Christian and R L Brent ldquoTeratogen update evaluationof the reproductive and developmental risks of caffeinerdquo Tera-tology vol 64 no 1 pp 51ndash78 2001

[4] C D Frary R K Johnson andM Q Wang ldquoFood sources andintakes of caffeine in the diets of persons in the United StatesrdquoJournal of the American Dietetic Association vol 105 no 1 pp110ndash113 2005

[5] C A Knight I Knight D CMitchell and J E Zepp ldquoBeveragecaffeine intake in US consumers and subpopulations of interestestimates from the Share of Intake Panel surveyrdquo Food andChemical Toxicology vol 42 no 12 pp 1923ndash1930 2004

[6] B E Garrett and R R Griffiths ldquoThe role of dopamine inthe behavioral effects of caffeine in animals and humansrdquoPharmacology Biochemistry and Behavior vol 57 no 3 pp 533ndash541 1997

[7] R P Heaney ldquoEffects of caffeine on bone and the calciumeconomyrdquo Food and Chemical Toxicology vol 40 no 9 pp1263ndash1270 2002

[8] T Ranheim and BHalvorsen ldquoCoffee consumption and humanhealthmdashbeneficial or detrimentalmdashMechanisms for effects ofcoffee consumption on different risk factors for cardiovasculardisease and type 2 diabetes mellitusrdquo Molecular Nutrition andFood Research vol 49 no 3 pp 274ndash284 2005

[9] M C R Camargo ldquoCaffeine daily intake from dietary sourcesin Brazilrdquo Food Additives and Contaminants vol 16 no 2 pp79ndash87 1999

[10] M N Clifford C L Gibson J-J R Rakotomalala E Crosand A Charrier ldquoCaffeine from green beans ofMascarocoffeardquoPhytochemistry vol 30 no 12 pp 4039ndash4040 1991

[11] J V Higdon and B Frei ldquoCoffee and health a review of recenthuman researchrdquoCritical Reviews in Food Science andNutritionvol 46 no 2 pp 101ndash123 2006

[12] M J Martin F Pablos and A G Gonzalez ldquoCharacterizationof green coffee varieties according to their metal contentrdquoAnalytica Chimica Acta vol 358 no 3 pp 177ndash183 1998

[13] M B Silvarolla PMazzafera and L C Fazuoli ldquoPlant biochem-istry a naturally decaffeinated arabica coffeerdquo Nature vol 429no 6994 p 826 2004

[14] A Belay K Ture M Redi and A Asfaw ldquoMeasurement ofcaffeine in coffee beans with UVvis spectrometerrdquo Food Chem-istry vol 108 no 1 pp 310ndash315 2008

[15] Y Dessalegn M T Labuscagne G Osthoff and L HerselmanldquoVariation of green bean caffeine chlorogenic acids sucroseand trigolline contents among Ethiopian Arabica coffee acces-sionsrdquo SINET Ethiopian Journal of Science vol 30 no 1 pp77ndash82 2008

[16] P L Fernandez M J Martın A G Gonzalez and F PablosldquoHPLC determination of catechins and caffeine in tea differen-tiation of green black and instant teasrdquo Analyst vol 125 no 3pp 421ndash425 2000

[17] V Sharma A Gulati S D Ravindranath and V Kumar ldquoAsimple and convenient method for analysis of tea biochemicalsby reverse phase HPLCrdquo Journal of Food Composition andAnalysis vol 18 no 6 pp 583ndash594 2005

[18] F Regan and Y Shakalisava ldquoRapid simultaneous determina-tion of alkylxanthines by CZE and its application in analysisof pharmaceuticals and food samplesrdquo Analytica Chimica Actavol 540 no 1 pp 103ndash110 2005

[19] A Wang L Li F Zang and Y Fang ldquoAmperometric detectionof three purine alkaloids following their separation by micellarelectrokinetic capillary chromatographyrdquo Analytica ChimicaActa vol 419 no 2 pp 235ndash242 2000

[20] A R Khanchi M Khayatzadeh Mahani M Hajihosseini MG Maragheh M Chaloosi and F Bani ldquoSimultaneous spectro-photometric determination of caffeine and theobromine inIranian tea by artificial neural networks and its comparisonwithPLSrdquo Food Chemistry vol 103 no 3 pp 1062ndash1068 2007

[21] L Lopez-Martınez P L Lopez-de-Alba R Garcıa-Camposand L M De Leon-Rodrıguez ldquoSimultaneous determination ofmethylxanthines in coffees and teas by UV-Vis spectrophotom-etry and partial least squaresrdquo Analytica Chimica Acta vol 493no 1 pp 83ndash94 2003

[22] M Amare and S Admassie ldquoPolymer modified glassy carbonelectrode for the electrochemical determination of caffeine incoffeerdquo Talanta vol 93 pp 122ndash128 2012

[23] A Carolina Torres M M Barsan and C M A Brett ldquoSim-ple electrochemical sensor for caffeine based on carbon andNafion-modified carbon electrodesrdquo Food Chemistry vol 149pp 215ndash220 2014

[24] O A Farghaly R S Abdel Hameed and A-A H Abu-Nawwas ldquoAnalytical application using modern electrochemicaltechniquesrdquo International Journal of Electrochemical Sciencevol 9 no 6 pp 3287ndash3318 2014

[25] B C Lourencao R AMedeiros R C Rocha-Filho L HMazoand O Fatibello-Filho ldquoSimultaneous voltammetric determi-nation of paracetamol and caffeine in pharmaceutical formu-lations using a boron-doped diamond electroderdquo Talanta vol78 no 3 pp 748ndash752 2009

[26] B C Lourencao R A Medeiros R C Rocha-Filho and OFatibello-Filhoa ldquoSimultaneous differential pulse voltammetricdetermination of ascorbic acid and caffeine in pharmaceuticalformulations using a boron-doped diamond electroderdquo Electro-analysis vol 22 no 15 pp 1717ndash1723 2010

[27] C A Martınez-Huitle N Suely Fernandes S Ferro A DeBattisti and M A Quiroz ldquoFabrication and application ofNafion-modified boron-doped diamond electrode as sensorfor detecting caffeinerdquo Diamond and Related Materials vol 19no 10 pp 1188ndash1193 2010

[28] L Svorc P Tomcık J Svıtkova M Rievaj and D BustinldquoVoltammetric determination of caffeine in beverage sampleson bare boron-doped diamond electroderdquo Food Chemistry vol135 no 3 pp 1198ndash1204 2012

[29] Z M Khoshhesab ldquoSimultaneous electrochemical determina-tion of acetaminophen caffeine and ascorbic acid using a newelectrochemical sensor based on CuO-graphene nanocompos-iterdquo RSC Advances vol 5 no 115 pp 95140ndash95148 2015

[30] J Zhang L P Wang W Guo X D Peng M Li and Z BYuan ldquoSensitive differential pulse stripping voltammetry ofcaffeine in medicines and Cola using a sensor based on multi-walled carbon nanotubes and Nafionrdquo International Journal ofElectrochemical Science vol 6 no 4 pp 997ndash1006 2011

[31] B Brunetti E Desimoni and P Casati ldquoDetermination ofcaffeine at a nation-covered glassy carbon electroderdquo Electro-analysis vol 19 no 2-3 pp 385ndash388 2007

[32] S Yang R Yang G Li L Qu J Li and L Yu ldquoNafionmulti-wall carbon nanotubes composite film coated glassy carbonelectrode for sensitive determination of caffeinerdquo Journal ofElectroanalytical Chemistry vol 639 no 1-2 pp 77ndash82 2010


[33] B Uslu and S A Ozkan ldquoElectroanalytical application ofcarbon based electrodes to the pharmaceuticalsrdquo AnalyticalLetters vol 40 no 5 pp 817ndash853 2007

[34] J A Young X Jiang and J R Kirchhoff ldquoElectrochemicaldetection of histamine with a pyrroloquinoline quinone modi-fied electroderdquoElectroanalysis vol 25 no 7 pp 1589ndash1593 2013

[35] A Galal ldquoElectrochemistry and characterization of someorganic molecules at ldquoMicrosizerdquo conducting polymer elec-trodesrdquo Electroanalysis vol 10 no 2 pp 121ndash126 1998

[36] U Lange N V Roznyatovskaya andVMMirsky ldquoConductingpolymers in chemical sensors and arraysrdquo Analytica ChimicaActa vol 614 no 1 pp 1ndash26 2008

[37] R J A Gosselink E de Jong B Guran and A Abacherli ldquoCo-ordination network for ligninmdashstandardisation production andapplications adapted tomarket requirements (EUROLIGNIN)rdquoIndustrial Crops and Products vol 20 no 2 pp 121ndash129 2004

[38] H Degefu M Amare M Tessema and S Admassie ldquoLigninmodified glassy carbon electrode for the electrochemical deter-mination of histamine in human urine and wine samplesrdquoElectrochimica Acta vol 121 pp 307ndash314 2014

[39] B Tewabe Gebeyehu and S L Bikilla ldquoDetermination ofcaffeine content and antioxidant activity of coffeerdquo AmericanJournal of Applied Chemistry vol 3 no 2 pp 69ndash76 2015

[40] B R Singh M A Wechter Y Hu and C Lafontaine ldquoDeter-mination of caffeine content in coffee using fourier transforminfra-red spectroscopy in combination with attenuated totalreflectance technique a bioanalytical chemistry experiment forbiochemistsrdquo Biochemical Education vol 26 no 3 pp 243ndash2471998


Among the coffee varieties in the country coffees cul-tivated at four localities of Ethiopia Finoteselam WolegaZegie and Wonbera are commonly consumed by the con-sumers in Bahir Dar City North West Ethiopia Of these thecoffee cultivated in Wonbera locality is the most expensiveand preferred while the Zegie coffee is the cheapest and leastpreferred the basis for which might be psychological aromaotherwise the caffeine content Thus the aim of this researchwas to check whether the usersrsquo preference can be related tothe caffeine content or not which needs determination of thecaffeine content of the four coffee varieties using a sensitiveselective fast and environmentally friendly method

High performance liquid chromatography [16 17] cap-illary chromatography [18 19] spectroscopy [20 21] andelectrochemical [22 23] methods are among the reportedmethods for determination of caffeine in coffee tea and colabeverage samples Most of the reported methods require car-cinogenic organic solvents for extraction and hence are envi-ronmentally nonfriendly Moreover using time-consumingsample preparation procedures such as liquid-liquid extrac-tion and solid-phase extraction or the use of more thanone chromatographic step costly instrumentation and highskilled technician [12 23] makes them inconvenient

In contrast to the conventional analytical methods elec-trochemical methods are powerful and versatile analyticaltechniques that offer high sensitivity accuracy and preci-sion relatively wider linear dynamic range and low-costinstrumentation specificity for a particular oxidation stateof an element relative inexpensiveness and being usuallyenvironmentally friendly (use no or minimum amount oforganic solvent) [23 24]

In recent years working electrodes including borondoped diamond [25ndash28] carbon paste and carbon nanotubes[29 30] and glassy carbon [22 31 32] based electrodes havebeen reported for determination of caffeine in real samples

Carbon materials have broad potential window lowbackground current rich surface chemistry comparativechemical inertness relatively easy electrode preparation andlow cost making them electrodes of choice [33] Deliberateand controlled modification of the electrode surface canproduce electrodes with new and interesting properties thatmay form the basis of new applications of electrochemistry[34] Polymer-modified electrodes (PMEs) have receivedconsiderable attention in recent years due to their goodstability reproducibility increased active sites homogeneityin electrochemical deposition and strong adherence to theelectrode surface [35 36]

Lignin is a natural polymer contained in most woodytrees and shrubs [37] Approximately 2of the lignin releasedduring chemical pulping in pulp and paper industries is usedfor chemical and material applications and the remainder isburned as boiler fuel showing its availability Although theelectrochemical behavior of lignin at the surface of glassycarbon electrode has been reported [38] to the best of ourknowledge its application for electrochemical determinationof caffeine in aqueous extracts of coffee samples has notbeen reported This paper reports a simple cheap and envi-ronmentally friendly lignin based electrochemical methodfor determination of caffeine in aqueous extracts of coffee

samples cultivated in different localities of Ethiopia theorigin of coffee

2 Experimental

21 Chemicals and Instruments Caffeine (Fischer Scientific)H3PO4

(Cipla Ltd India) K2HPO4

(Wagtech Interna-tional Ltd UK) CH

3COOH and NaOH (Landmark chem-

icals PVT India) HCl (Riedel-De Haen Germany) lignin(Riedel-De Haen Germany) HNO

3(65ndash70) and H

2SO4

(99) (both from Loba chemie Pvt Ltd) were used Allreagents were of analytical grade and were used directlywithout further purification Glassware was cleaned usingchromic solution prepared by dissolving 1 g of potassiumdichromate in 1 L of H

2SO4followed by rinsing with distilled

water Distilled water was used for the preparation of allsolutions

Voltammetric experiments were carried out usingCHI760D Electrochemical Workstation (Austin TexasUSA) connected to a personal computer All electrochemicalexperiments were performed employing a conventionalthree-electrode system with a glassy carbon electrode (3mmin diameter) or a Lignin modified glassy carbon electrode astheworking electrode platinum coil as an auxiliary electrodeand AgAgCl as a reference electrode All experiments werecarried out at 20 plusmn 2∘C

22 Real Sample Preparation Coffee samples were purchasedfrom the respective local markets (Wolega FinoteselamZegie andWonbera) Aqueous extracts of the coffee sampleswere prepared following the procedure outlined elsewhere[22] Briefly exactly 50 g of coffee from each sample wasroasted by using conventional coffee roasting machine Eachroasted coffee sample was ground using a mortar and pestleand screened through 250 120583M sieve to get a uniform textureAn accurately weighed 4 g of sieved coffee from each samplewas then boiled in 200mL of distilled water in the tempera-ture range 80ndash90∘C for 30min while stirring using magneticstirrer The water extracted caffeine was finally separatedfrom the residue by decantation and was ready for the DPVanalysis

23 Preparation of Lignin Modified Glassy Carbon ElectrodeThe lignin modified glassy carbon electrode (LGCE) wasprepared following a procedure reported elsewhere [38]Briefly a glassy carbon electrode (3mm diameter) wasfirst rinsed with distilled water and polished carefully withalumina powder having different particle size (10 03 and005 120583m) to a mirror finish surface The residual polishingmaterial was removed by repetitive rinsing of the surfacewith distilled water The resulting electrode was immersedin pH 7 Phosphate buffer solution (PBS) and was activatedpotentiodynamically by scanning the potential between minus02and+15 V for five cycles at a scan rate of 100mV sminus1Then theactivated electrode was transferred to a cell containing ligninin a mixture of 05M H

2SO4and 01M HNO

3prepared by

dissolving 10mgof lignin in 50mLof themixture (1 1 volumeratio)










(a)

(b)

minus50

minus40

minus30

minus20

minus10

0

Curr

ent (

휇A

)


(a㰀)

(b㰀)


(a)

(m)minus50

minus40

minus30

minus20

minus10

0Cu

rren

t (휇

A)

16 14 12 10 08 0618E (V versus AgAgCl)


R2 = 099915


Ipa (

휇A)

4 6 8 10 12 14 16 18 20 222(Scan rate)12

]12 (





335445

5556

minus60

minus50

minus40

minus30

minus20

minus10

0

10

Curr

ent (

휇A

)

16 14 12 10 08 0618E (V versus AgAgCl)

4 62pH


Ipa (

휇A)


(a)

(b)

minus18

minus15

minus12

minus9

minus6

minus3

0

Cor

rect

edI a

(휇A)

16 14 12 1018A










minus20

minus15

minus10

minus5

0

Ipa (

휇A)


(a)

(c)

(a)

16 14 12 10 0818

minus30

minus25

minus20

minus15

minus10

minus5

0

Ipa (

휇A)

(a)

(d)

E (V versus AgAgCl)

(b)


(a)

(b)

(c)

minus35

minus30

minus25

minus20

minus15

minus10

minus5

0

I a(휇

A)





(a)

(j)

minus30

minus25

minus20

minus15

minus10

minus05

00

05

I a(휇

A)


0

2

4I a

(휇A)

R2 = 099900

Ia (휇A) = minus0060 + 0034C (휇M)

20 40 60 80 1000conc (휇M)



4 Conclusion








120583M recovery


(a)

(d)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00

I pa

(휇A)

10 11 12 13 14 15 1609E (V versus AgAgCl)





(a)

(e)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00I p

a(휇

A)

105 120 135 150 165090E (V versus AgAgCl)





References




















































(a)

(b)

minus50

minus40

minus30

minus20

minus10

0

Curr

ent (

휇A

)


(a㰀)

(b㰀)


(a)

(m)minus50

minus40

minus30

minus20

minus10

0Cu

rren

t (휇

A)

16 14 12 10 08 0618E (V versus AgAgCl)


R2 = 099915


Ipa (

휇A)

4 6 8 10 12 14 16 18 20 222(Scan rate)12

]12 (





335445

5556

minus60

minus50

minus40

minus30

minus20

minus10

0

10

Curr

ent (

휇A

)

16 14 12 10 08 0618E (V versus AgAgCl)

4 62pH


Ipa (

휇A)


(a)

(b)

minus18

minus15

minus12

minus9

minus6

minus3

0

Cor

rect

edI a

(휇A)

16 14 12 1018A










minus20

minus15

minus10

minus5

0

Ipa (

휇A)


(a)

(c)

(a)

16 14 12 10 0818

minus30

minus25

minus20

minus15

minus10

minus5

0

Ipa (

휇A)

(a)

(d)

E (V versus AgAgCl)

(b)


(a)

(b)

(c)

minus35

minus30

minus25

minus20

minus15

minus10

minus5

0

I a(휇

A)





(a)

(j)

minus30

minus25

minus20

minus15

minus10

minus05

00

05

I a(휇

A)


0

2

4I a

(휇A)

R2 = 099900

Ia (휇A) = minus0060 + 0034C (휇M)

20 40 60 80 1000conc (휇M)



4 Conclusion








120583M recovery


(a)

(d)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00

I pa

(휇A)

10 11 12 13 14 15 1609E (V versus AgAgCl)





(a)

(e)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00I p

a(휇

A)

105 120 135 150 165090E (V versus AgAgCl)





References












































335445

5556

minus60

minus50

minus40

minus30

minus20

minus10

0

10

Curr

ent (

휇A

)

16 14 12 10 08 0618E (V versus AgAgCl)

4 62pH


Ipa (

휇A)


(a)

(b)

minus18

minus15

minus12

minus9

minus6

minus3

0

Cor

rect

edI a

(휇A)

16 14 12 1018A










minus20

minus15

minus10

minus5

0

Ipa (

휇A)


(a)

(c)

(a)

16 14 12 10 0818

minus30

minus25

minus20

minus15

minus10

minus5

0

Ipa (

휇A)

(a)

(d)

E (V versus AgAgCl)

(b)


(a)

(b)

(c)

minus35

minus30

minus25

minus20

minus15

minus10

minus5

0

I a(휇

A)





(a)

(j)

minus30

minus25

minus20

minus15

minus10

minus05

00

05

I a(휇

A)


0

2

4I a

(휇A)

R2 = 099900

Ia (휇A) = minus0060 + 0034C (휇M)

20 40 60 80 1000conc (휇M)



4 Conclusion








120583M recovery


(a)

(d)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00

I pa

(휇A)

10 11 12 13 14 15 1609E (V versus AgAgCl)





(a)

(e)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00I p

a(휇

A)

105 120 135 150 165090E (V versus AgAgCl)





References












































minus20

minus15

minus10

minus5

0

Ipa (

휇A)


(a)

(c)

(a)

16 14 12 10 0818

minus30

minus25

minus20

minus15

minus10

minus5

0

Ipa (

휇A)

(a)

(d)

E (V versus AgAgCl)

(b)


(a)

(b)

(c)

minus35

minus30

minus25

minus20

minus15

minus10

minus5

0

I a(휇

A)





(a)

(j)

minus30

minus25

minus20

minus15

minus10

minus05

00

05

I a(휇

A)


0

2

4I a

(휇A)

R2 = 099900

Ia (휇A) = minus0060 + 0034C (휇M)

20 40 60 80 1000conc (휇M)



4 Conclusion








120583M recovery


(a)

(d)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00

I pa

(휇A)

10 11 12 13 14 15 1609E (V versus AgAgCl)





(a)

(e)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00I p

a(휇

A)

105 120 135 150 165090E (V versus AgAgCl)





References

















































120583M recovery


(a)

(d)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00

I pa

(휇A)

10 11 12 13 14 15 1609E (V versus AgAgCl)





(a)

(e)

minus14

minus12

minus10

minus08

minus06

minus04

minus02

00I p

a(휇

A)

105 120 135 150 165090E (V versus AgAgCl)





References











































electrochemical determination of caffeine content in ethiopian coffee samples ...€¦ ·...

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