Feature Article

Determination of Carrageenan in Food Products UsingPotentiometric Polyion SensorsSaad S. M. Hassan,*a M. E. Meyerhoff,b I. H. A. Badr,a and H. S. M. Abd-Rabboha

a Department of Chemistry, Faculty of Science, Ain Shams University, Cairo, Egypt; e-mail saadsmhassan@yahoo.comb Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

Received: March 28, 2001Final version: July 6, 2001

AbstractPolymeric matrix tubular membrane sensors incorporating poly(vinyl chloride) matrix, o-nitrophenyloctylether (o-NPOE) plasticizer and either dinonylnaphthalene sulfonate (DNNS) or tridodecylammonium chloride (TDMAC)ionophore are prepared and electrochemically evaluated for determination of carrageenan polyion. These sensors areused for monitoring potentiometric titration of carrageenan with protamine titrant. Carrageenan at the concentrationlevels of 0.2 ± 1.5% (m/m) in a variety of food products (e.g., cream, chocolate, caramel, ice cream, and salad dressing)are satisfactory determined. Results agree fairly well with nominal values are obtained and confirmed by recoveryexperiments of spiked samples. The method has several advantages over all previously reported methods in beingmore simple, accurate and applicable for routine analysis of real samples without prior treatment.

Keywords: Polyion potentiometric sensors, Carrageenan in food products, Protamine titrant, Potentiometrictitrimetry, PVC membrane

1. Introduction

It has been reported that specially formulated polymericmembranes doped with suitable electroactive materialsexhibit high and reproducible potentiometric responsetowards a variety of polyanions (e.g., heparin, DNA) [1, 2]and polycations (e.g., protamine, arginine- and lysine-richsynthetic polypeptides) [3, 4]. The potentiometric responseof these sensors was explained by the development of a non-equilibrium steady-state electrochemical phase-boundarypotential on the membrane/sample interface [5]. Heparin-sensitive membrane electrode based on tridodicylmethy-lammonium chloride (TDMAC) dispersed in poly(vinylchloride) PVC matrix has been described and utilized forthe determination of heparin levels in whole blood [2] andheparin binding to various biological macromolecules [6]via simple potentiometric titrations. Further, heparin sensorhas been shown to be respond to carrageenan [7]. Ananalogous protamine-sensitive membrane electrode em-ploying tetra-p-chlorophenylborate in a polymeric film hasalso been applied for the determination of the bindingconstants of protamine with various macromolecules, aswell as for the assay of proteinases that cleave polycationicsubstrates (e.g., protamine, arginine- or lysine-rich syntheticpolypeptides) into much smaller fragments that yield littleor no potentiometric response [4, 8].The present work is dealing with the determination of

carrageenan; a naturally occurring, highly sulfated poly-saccharides (average molecular mass of ca. 4500) extractedfrom different seaweed algae, such as E. cotonii, E.spinosum, G. pistillata, G. aciculaire, and C. crispus [9].There are three main types of carrageenan differ from each

other in their sulfate contents: Lambda, Kappa and Iotacarrageenan (Fig. 1).Carrageenan is used in food industry asfood additive [10] stabilizer, homogenizer, thickening agentor gel-forming agent. The content of the three types indifferent food products ranges from 0.03 to 3.0% (m/m)according to the purpose of their addition [10]. In the

Fig. 1. Chemical structures of carrageenan types III (Kappa), IV(Lambda), and V (Iota) (Average charges are: �� 600; �� 400;and �� 470, respectively).

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therapeutic research field, carrageenan was introduced asan inhibitor for the replication of hepatitis A virus in cellculture [11] and was examined as a natural product in thefight against AIDS [12].Different methods and techniques are currently used for

the assay of carrageenan. Gas and liquid chromatography,which require a tedious acid hydrolysis of samples beforeanalysis have been suggested [13]. Precipitationwith bariumchloride, barium chloranilate, cetylpyridinium chloride,alkyldimethyl-benzylammonium chloride, and cationicdyes followed by direct gravimetric or spectrophotometricassay of the reaction products after dissolution or indirectlyby measuring the absorbance of the excess unreactedreagents have been described [14 ± 23]. The precipitationmethods involve centrifugation steps, long incubation times(12 ± 60 h), and are applicable to large quantities of carra-geenan (� 40 mg).Colorimetric methods have also been reported for

carrageenan assay [9, 18, 24, 25]. Complexation of carra-geenan with carbocyanine dye produces a soluble complexand results in a hypsochromic shift in the absorptionmaximaof the dye. Themethod, however, requires fresh preparationof the dye reagent for each assay. A colorimetric methodusing 2-thiobarbituric acid has been suggested to form ayellow coloured complex. Despite the sensitivity of thismethod, it requires a long reaction time (ca. one hour) forfull color development. Reaction with methylene blue dyegives a complex with absorption maximum at a shorterwavelength (559 nm) compared with those of the free dye(610 and 660 nm). The disadvantage of this method is thatthe formed complex is sparingly soluble in water and causesan error in absorbance measurements. Spectrofluorimetrywas also utilized for carrageenan analysis. A loss in thefluorescence intensity of acridine orange upon binding tocarrageenan was used for carrageenan assay [26]. Themethod, however, requires a prior tedious enzyme digestionof the samples.In this work an electrochemical method for simple

potentiometric determination of carrageenan in differentfood products utilizing polyion sensors is described. Themethod has many advantages over spectrophotometry andfluorimetry methods as being more simple, rapid, sensitive,accurate, time resuming and not affected by sampleturbidity and matrix color.

2. Experimental

2.1. Apparatus

An Orion digital pH/mV meter (Model SA 720) was usedfor pH and mV measurements with polyion sensors inconjunction with an Orion Ag/AgCl single-junction refer-ence electrode (Model 90-01) filled with 10% m/v KCl. Acombination Ross glass-pH electrode (Orion 81-02) wasused for all pHmeasurements. Calibrated automatic micro-pipette (0.1 �L devisions) (Brand transferpette) was usedfor the titrations.

2.2. Reagents

Analytical grade carrageenan types III, IVandV, protaminesulfate (average molecular mass �4500) and poly-�-argi-nine (molecular mass 10000 ± 25000) were obtained fromSigma (Buch, Switzerland). Tridodecylmethylammoniumchloride (TDMAC), potassium tetrakis(p-chlorophenyl)-borate, and high molecular weight poly(vinylchloride)(PVC) powder, (molecular mass of 100000), were obtainedfrom Aldrich Chemical Co. (Milwaukee, WI). Tetrahydro-furan (THF), dioctylsebacate (DOS), dioctylphthalate(DOP) and o-nitrophenyloctylether (o-NPOE), were pur-chased from Fluka Chemika-Biochemika (Ronkonkoma,NY). Dinonylnaphthalene sulfonate (DNNS) was obtainedas a gift from King Industries (Norwalk, CT). All otherchemicals were analytical reagent grade unless otherwisestated, and deionized doubly distilled water was usedthroughout. Food products containing carageenan wereobtained from the local market.

2.3. Membrane Preparation and Sensor Construction

Two polyion membrane sensors were prepared, the poly-anion carrageenan and thepolycation protaminemembranesensors. The polyanion carrageenan membrane sensor(tubular type) was prepared as previously described [27]by dissolving the membrane components [33% (m/m) o-NPOE plasticizer, 66% (m/m) PVC, and 1% (m/m)TDMAC] in 9 mL of freshly distilled THF. The membranecocktail solution was then dip coated (12 times at 20-minintervals) over glass rods protruding from a narrow boreTygon tube (i.d. ca. 1.3 ± 1.5 mm) and then dried overnight.After soaking in 15 mM NaCl for about 3 h, the glass rodswere carefully removed. The working sensors were assem-bledby fillingwith 0.12 MNaCl solution and then inserting aAg/AgCl reference electrode wire into the bore of the tube.Prior to use, the tubular membrane sensors were condi-tioned by soaking in 0.12 M NaCl solution for at least 5 h.The polycation protamine membrane sensor was preparedas previously described [28] by dissolving the membranecomponents [49.5% (m/m) dioctylsebacate plasticizer,DOS, 49.5% (m/m) PVC, and 1% (m/m) dinonylnaphtha-lene sulfonate, DNNS] in 9 mL of freshly distilled THFandthe electrode is prepared as described above.Calibration of polyion sensors towards carrageenan was

examined in a saline background solution (0.12 M NaCl).The polyion sensor in conjunction with the referenceelectrode was introduced in 10 mL of the saline solutionand the potential reading was recorded after stabilization.Then, 5 ± 300 �L aliquots of carrageenan calibrants (1 mg/mL) were added. Potential readings were recorded after5 min of each addition.

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2.4. Potentiometric Titration of Carrageenan

Thepolycationic sensor in conjunctionwith a single junctionAg/AgCl reference electrode was immersed in a 10 mL of0.12 M NaCl solution. After potential stabilization, a fixedvolume (10 ± 30 �L) of a standard solution of carrageenan(10 mg/mL) was added and the potential reading wasrecorded after 5 minutes of the addition. The solution waspotentiometrically titratedwith either a standard protamineor poly-�-arginine (Fig. 2) solution as a titrant (1 ± 10 mg/mL). The solution was stirred 5 min after addition of eachincrement of the titrant. Precipitation of carrageenanyielded a potential change with a sigmoidal curve. The endpoints were calculated from first derivative titration curves.The stoichiometry for the titration of different standardcarrageenan types with protamine are 1 :0.6, 1 :0.9, and1 :0.7 (m/m) carrageenan: protamine for types III, IV, andV,respectively, and those for the titrations against poly-�-arginine are: 1 : 0.5, 1 : 0.9, and 1 :1 (m/m) carrageenan :poly-L-arginine for types III, IV, and V, respectively.

2.5. Determination of Carrageenan in Food Products

Known weights of the food product samples (100 ± 400 mg)were dissolved in a 10- mL portion of 0.12 MNaCl solution,shaked well and titrated with a standard protamine solution(10 mg/mL). A polycation protamine sensitive membraneelectrode based on DNNS was used to monitor the titrationend point. Carrageenan concentrations were calculatedfrom the end point break of the titration curves. The resultsare expressed as carrageenan type IV (1.0 mL of protaminetitrant, 10 mg/mL�9.0 mg of carrageenan type IV).

3. Results and Discussion

3.1. Effect of Sensor Configuration

A comparison was made between the responses of bothtubular and conventional [27, 29] sensors based on TDMACwith o-NPOE plasticized membranes towards type IVcarrageenan and the results are shown in Table 1. Tubularmembrane sensors offered lower limit of detection, wider

working concentration range and higher calibration slope.This behavior is expected based on the responsemechanismand the response function for polyion. The measuredpotential reaches a steady-state value after about 5 minand then continues to slowly decrease to a minimum valueover amuch longer time period (10 ± 20 h).At the endof thisperiod the EMF begins to drift back to the original baselinepotential value. This reversal in EMF response is due to thetest polyanion eventually diffusing through the polymermembrane and reaching the inner interface at membrane/internal solution of the electrode, thereby changing theinner phase boundary potential of themembrane [3] (i.e., nopolyanion is originally present within the membrane andinternal solution). It is apparent that the potentiometricresponseof a polyion sensor is truly dependent on reaching aquasi steady state of polyion flux up to and into the bulk ofthe organic membrane phase [2]. Changes in electrodegeometry influence the EMF response function of thesensor.With a tubular sensor configuration, the steady-stateaccumulation of polyionic species in the surface layer of themembrane phase is expected to be larger than that forconventional sensor configuration [27,29] resulting in en-hancement of themembrane sensitivity. Tubular membranesensors were used for subsequent experiments.

3.2. Effect of Membrane Composition

Three different tubular PVC membrane sensors wereprepared using DOP, DOS and o-NPOE plasticizers.Calibration experiments for type IV carrageenan werecarried out using these sensors. As depicted in Table 2, o-NPOE-based membrane sensor exhibited the best poten-tiometric response characteristics; a detection limit of0.7 �g/mL, a linear range of 1.9 ± 19.9 �g/mL, and a slopeof 41.5 mV/decade.

Fig. 2. Subunit structures of protamine (molecular mass �4500)and poly-�-arginine (molecular mass �10,000 ± 25,000) titrants.

Table 1. The response characteristics of conventional and tubularPVC based membrane sensors plasticized with o-NPOE towardtype IV carrageenan.

Parameter Conventional type Tubular type

Limit of detection (�g/mL) 0.05 0.70Linear range (�g/mL) 1.9 ± 6.3 1.9 ± 19.9Nernstian response (mV) 39.2 41.5Slope (mV/decade) 14.8 18.8

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Poly(vinylchloride)membrane sensors, plasticizedwitho-NPOE and doped with 1% (m/m) TDMAC, were preparedwith different mole ratios of the additive potassiumtetrakis(p-chlorophenyl)borate (10 ± 30 mol% relative toTDMAC) and examined. It was reported that the presenceof additional lipophilic ionic additives in the membrane ofpotentiometric sensors enhances its response characteristics[30]. However, the present results revealed a decrease in thelinear range and the slope of the calibration plot, by theincrease of the borate ratio indicating no enhancement inthemembrane response towards carrageenanwithin the lowcarrageenan levels normally found in food samples. It isconcluded that the best membrane composition for thecarrageenan assay consisted of 66% (m/m) PVC as amembrane matrix, 33% (m/m) o-NPOE as a plasticizerand 1% (m/m) TDMAC as the electroactive species.

3.3. Potentiometric Titration of Carrageenan UsingPolyanion Carrageenan Sensor and Protamine Titrant

Potentiometric titration technique was found to be moreefficient than the direct potentiometry using calibrationtechnique for polyion sensing. There are some factorsinfluencing the stability and reproducibility of data obtainedby direct potentiometry. Of these, changing the stirring rateduring the analysis significantly affects the flux of thepolyionic species from the bulk of the sample to the

membrane. This change influences the steady-state poten-tial of the membrane. Sample volume, sample fluidity, andelectrode geometry all affect the accuracy of direct potenti-ometry of carrageenan [3, 6, 7]. For these reasons, carra-geenan levels in solutions were measured by means ofpotentiometric titration using either polyanion carrageenanor polycation protamine sensors, and utilizing protamine asa titrant (Fig. 2). Standard solutions (10 ± 30 �g/mL) ofvarious carrageenan, (types III, IV and V), were preparedand titrated in replicates (n� 5) with a protamine titrant(1.0 mg/mL).The inflection breaks of the potentiometric titration

curves were 3.7 ± 5.6 mV, 7.3 ± 10.6 mV, and 8.9 ± 9.6 mVfor types III, IVand V-carrageenan, respectively. Figure 3adepicts some representative typical potentiometric titrationcurves of type IV carrageenan. Other carrageenan typesdisplay similar titration curves. As shown from these data,polyanion carrageenan sensitive membrane sensor showinflection breaks with small potential change. The smallinflection break is probably due to the limited reversibilityof the sensor after prolonged contact with carrageenan.However, sharp end points are obtained from the firstderivative titration curves. It is well known that polyionsensors do not offer complete signal reversibility. Toenhance the potential change at the inflection break, apolycation protamine sensor was used instead.

3.4. Potentiometric Titration of Carrageenan UsingPolycation Protamine Sensor and Protamine or Poly-�-arginine Titrant

Aprotamine PVCmembrane sensor based on dinonylnaph-thalene sulfonate was used for the end point detection ofcarrageenan titration [8]. Inflection breaks 4 ± 10 timesmuch greater than those obtained with carrageenan sensorwere obtained. The inflection breaks were 35.4 ± 40.2 mV,26.3 ± 29.0 mV, and 30.1 ± 32.5 mV for types III, IV, and Vpolyanion carrageenan, respectively. Figure 3b presents

Table 2. Response characteristics of polyanion (TDMAC tubularPVC-based) membrane sensors with DOP, DOS, and o-NPOEplasticizers toward type IV carrageenan.

Parameter DOP DOS o-NPOE

Limit of detection (�g/mL) 0.05 2.81 0.70Linear range (�g/mL) 0.2 ± 2.2 4.2 ± 22.3 1.9 ± 19.9Slope (mV/decade) 21.0 35.1 41.5Correlation coeff. (r) 0.9956 0.9996 0.9966Intercept (mV) �16.1 8.6 �11.7

Fig. 3. Typical potentiometric titration curves of (�) 10, (�) 20, and (�) 30 �g/mL of type IV carrageenan with 1.0 mg/mL protamine orpoly-�-arginine titrant using: a) polyanion carrageenan sensor, b) and c) polycation protamine sensor.

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typical titration curves of type IV carrageenan. The titrantused was 1.0 mg/mL protamine solution. Similar titrationcurves were obtained with types III, and V carrageens. Theprotamine sensorwas used for all subsequentmeasurementsof real samples.Poly-�-arginine (Fig. 2) was also tested as a titrant for

carrageenan. Since this titrant has a higher charge densitycompared to protamine, this leads to a higher potentiomet-ric response, and larger inflection breaks, compared toprotamine when used as a titrant for carrageenan. Theinflection breaks for the titration curves of various carra-geenan types with poly-�-arginine range from 79 ± 81.5 mV,52 ± 76 mV, and 31.7 ± 66 mV for types III, IV, and Vcarrageenan, respectively. Figure 3c represents typical titra-tion curves of type IV carrageenan. Types III and V displaysimilar titration curves. Although, poly-�-arginine titrantdisplays inflection breaks much greater than those obtainedusing protamine titrant, it is more expensive.

3.5. Determination of Carrageenan in Some FoodProducts Using Polycation Protamine Sensor andProtamine Titrant

Total carrageenan expressed as carrageenan type V atconcentration levels of 0.2 ± 1.5% (m/m)weremeasured in avariety of food samples with different matrix compositions.Five food samples were tested: tart creams (Dr.Oetker:vanilla and chocolate), cream caramel, ice cream (Mˆve-npick: vanilla), and a vinaigrette salad dressing. Totalcarrageenan levels in these samples were measured bypotentiometric titration technique (Fig. 4) using 10 mg/mLprotamine as a titrant and polycation protamine sensor forthe end point detection. Since carrageenan in real samples isnot a single compound but may comprise any or all of theother types, the results obtained are expressed as carra-geenan type IVand are shown in Table 3. The data obtainedare found within the expected ranges of the nominal valuesfor these types of food products.A comparison of the results with data obtained using the

standard colorimetric methods was next tried. Two pub-lished colorimetric methods were tested as referencemethods [9, 25].The first involved theuseof 2-thiobarbituricacid as a chromogenic reagent for the residue 3,6-anhydro-�-galactose, which is produced by the acid digestion ofcarrageenan. The secondmethod is based on a reaction withmethylene blue dye, which binds carrageenan yielding asparingly soluble complex with a hypsochromic shift in theabsorption maxima of the methylene blue dye solution (at610 and660 nm).Themajor disadvantage of bothmethods istheir need for clear samples for the colorimetric measure-ments, which can×t be obtained for the tested food sampleseven by ultrafiltration. In addition, the detection limit of thefirst method is relatively higher than the expected carra-geenan levels in the tested dilute food samples (Table 3).Onthe other hand, the complex formed between methyleneblue and carrageenan is partially insoluble at the concen-tration level of carrageenan in food samples. To the best of

our knowledge, all of the reportedmethods for carrageenanassay were not applied for real samples, probably due to theabove reasons, except for onemethod,whichwas applied forcarrageenan assay in clear samples (jelly and Italian saladdressing) [22].The validity of the proposed potentiometric method was

checked, however, using recovery measurements of spikedreal food samples. The same weights of each sample of thefive test samples were spiked with various concentrations oftype IV carrageenan and the titrations of these spikedsamples were carried out using protamine titrant and apolycation protamine sensors. The results obtained showaverage carrageenan recoveries of 103.4, 102.3, 98.5, 101.2,and 102.2%with relative standard deviations of 1.2, 1.1, 1.3,1.3 and 0.9% for tart cream (vanilla), salad dressing,caramel, ice cream (vanilla) and tart cream (chocolate)

Fig. 4. Typical potentiometric titration curves of some realcarrageenan-containing food products using 10 mg/mL protaminetitrant and polycation protamine sensor: (�) salad dressing(Vinaigrette); (�) ice cream vanilla (Mˆvenpick); (�) creamcaramel (Dream); (�) tart cream chocolate (Dr. Oetker); and (X)tart cream vanilla (Dr. Oetker).

Table 3. Carrageenan concentration levels measured in some foodsamples using polycation protamine membrane sensor andprotamine titrant.

Sample types Carrageenan (% m/m)

Nominal Found [a]

Tart cream, vanilla (Dr. Oetker) 0.2 ± 0.4 0.220� 0.004Tart cream, chocolate (Dr. Oetker) 1.5 ± 2.5 2.793� 0.003Cream caramel (Dream) 0.2 ± 0.6 0.376� 0.005Ice cream, vanilla (Mˆvenpick) 0.5 ± 0.8 0.813� 0.005Salad dressing (Vinaigrette) 0.2 ± 0.5 0.403� 0.004[a] Average of 5 measurements

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samples, respectively, indicating negligiblematrix effect. AnF-test revealed that there was no significant differencebetween the means and variances of the two sets of results.These data render the proposed method applicable forquality control of food products.

4. Conclusions

Fast and simple assay method for carrageenan polyanion infood products is presented. The method involves potentio-metric titration with protamine or poly-�-arginine titrantusing a PVC matrix tubular membrane protamine sensorincorporating dinonylnaphthalene sulfonate as ionophore,PVC as amatrix andDOS as solventmediator in the ratio of1 :49.5 :49.5% (m/m), respectively. Compared to otherknown methods, the proposed technique is less timeconsuming, does not need prior enzymatic or acid hydrol-ysis, and directly applicable for real samples. The resultsobtained are in good agreement with the nominal levels andthe method accuracy is confirmed by recovery experimentsof spiked samples. The outlined procedure presents a simplesolution for quantification of a group of compounds noteasily assayed by most instrumental methods.

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