Determination of inorganic anions in red algae (Eucheuma cottonii ...

R. Suhaili et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 406-410)ISBN 978-979-18962-0-7Jatinangor, 30-31 October 2008Determination of inorganic anions in red algae (Eucheuma cottonii) andcarrageenan by suppressor system of ion chromatographyRefilda Suhaili 1 *, Edison Munaf 1 , Rahmiana Zein 1 , Abdi Dharma 1 ,Anang Sedyohutomo 2 , Lim Lee Wah 2 , Toyohide Takeuchi 21 Department of Chemistry, Faculty of Mathematics and Natural Sciences,Andalas University, Padang 25163, Indonesia2 Department of Chemistry, Faculty of Engineering, Gifu University,1-1 Yanagido, Gifu, 501-1193, Japan*e-mail: refilda_59@yahoo.com; Phone: +81-26-628-806AbstractDetermination of inorganic anions in red algae (Eucheuma cottonii) and carrageenan by using two 6-port switching valves and two packed column suppressors system of ion chromatography wasinvestigated. Sodium carbonate-bicarbonate as a mobile phase to reduce the background conductivityand to increase the analyte signal was maintained at low detection limits. The relative standarddeviations (RSDs) for the retention time, peak area and peak height of eight common inorganicanions (0.3 mM each of F - , Cl - , NO 2 - , Br - , NO 3 - , HPO 4 2- , SO 42-and I - ) were between 0.0-0.2, 0.1-0.4and 0.1-0.3%, respectively. Inorganic anions contained in red algae (Eucheuma cottonii) andcarrageenan shown in table.Table Inorganic anions contained in red algae (Eucheuma cottonii) and carrageenanSampleF - Cl - NO 2-Anion contained mg/100gBr - NO 3-HPO 42-SO 42-PureCarrageenan 1.75 16.60 1.28 5.22 445.47 8.64 19.16 11.66ExtractedCarrageenan 1.80 17.79 0.89 5.10 442.45 8.02 25.14 10.13Eucheumacottonii 2.33 10.02 1.71 7.77 448.94 7.99 22.14 15.12I -Keywords: Common inorganic anions, ion chromatography, packed column suppressor, red algae(Eucheuma cottonii), carrageenan, six-port switching valveIntroductionThe oceans cover 71% of the earth’s surface andinclude an abundance of fauna and flora. Thechemical composition of sea water is quite constant.but the nutrient levels for the plants in sea water areextremely variable and concentrations for some traceelements and vitamins are more variable.Red algae (Eucheuma) is harvested throughout theworld as a food sources as well as an exportcommodity for the production of agar andcarrageenan products). F - , Cl - , NO 2 - , Br - , NO 3 - ,HPO 4 2- , SO 42-and I - are minor essential nutrients infood that can lead to health dieses. The ions are anindicator of the food quality [1]Since its introduction in 1975, high performanceliquid chromatography (HPLC) is a main tool toseparate and determine inorganic cations and anionsin food. Many advances in ion chromatographic (IC)separation and detection have been reported. Themain goal of ion chromatography (IC) as an analyticaltechnique is to provide complete information aboutthe ionic composition of the analyzed sample. Forobvious reasons, it is often necessary to use twodifferent sets of IC conditions for the separation anddetermination of cationic and anionic species.Some of these are described in several books andpapers [2-6]. Some of the most important advancesrelate to suppressor development [4-8]. Recentdevelopments in conductivity suppressor technologyhave been reported for inorganic ion analysis by ionchromatography [5-10]. Packed-column suppressorssuffers from a number of disadvantages, involving theneed for off-line regeneration, band-broadeningoccurring in the suppressor resulting in loss ofchromatographic efficiency, and variable retention inthe suppressor column due to ion-exclusion effectsIn IC, a ‘‘suppressor system’’ is commonly usedfor a sensitive detection of ions on the basis of theirelectrical conductance. Its function is to reduce406

R. Suhaili et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 406-410)Jatinangor, 30-31 October 2008chemically the background conductivity of theelectrolyte of an eluent before it enters theconductivity cell [11-12]. Suppressors are used inconjunction with conductivity detectors to improvedetection sensitivity, especially for anion analysisTwo suppressor types are commonly used foranion analysis. They are continuously regeneratedmembrane suppressors and intermittently regeneratedpacked-bed suppressors. Electrochemical andchemical regeneration have been used with both.Packed-bed suppressors are more rugged and reliablethan membrane suppressors [5-13], but intermittentregeneration limits run time and requires mechanicalswitching mechanisms. While more fragile,membrane suppressors offer continuous operation andregeneration. In addition to suppressing the mobilephase and enhancing the analyte signal through acid–base neutralization reactions like traditional ICsuppressors, this new suppressor also removescarbonic acid as CO (g) from the analyte stream,reducing background conductivity with carbonate–hydrogencarbonate mobile phases to near zero. Thisimproves the signal-to-noise ratio, reduces the waterdip that often interferes with early eluting peaks, andenables gradient separations with carbonate–hydrogencarbonate mobile phases with minimalbaseline shift.The propose of this research is to determinationof eight inorganic anions (F - , Cl -, NO 2 - , Br - , NO 3 - ,HPO 4 2- , SO 4 2- , I - ) by using two 6-port switchingvalves and two packed column suppressors tomaintain low conductivity of mobile phase in redalgae (Eucheuma cottonii) and carrageenan withsuppressed ion chromatography for achieving higherselectivity, shorter analysis time, lower quantitationand detection limits via optimization ofchromatographic parameters for the routine analysis.Materials and MethodsChemicalsAnalytical reagent grade chemicals were purchasedfrom Nacalai Tesque (Kyoto, Japan). Standardsolutions (0.3mM for each anion) were prepared bydissolving NaF, NaHPO 4 , NaNO 2 , NaCl, NaBr, NaI,NaNO 3 , and Na 2 SO 4 by using deionized water. Allstandard solutions were stored in polyethylenecontainers and kept under refrigeration at 4 ◦ C. wereprepared. The deionized water used throughout thisstudy was prepared in the laboratory using a GS-590water distillation system (Advantec, Tokyo, Japan).The stock standard solutions for anions contained ionsof interest. The standard solution of anions Na 2 CO 3and NaHCO 3 were obtained from Wako (Osaka,Japan), and used for the eluent. The eluent wasprepared daily before use.he pH of the carbonat eluentwas measured with an IM-20E ion meter (ToaElectronics, Tokyo, Japan). A 0.45 µm membranefilter for IC obtained from GL Sciences (Tokyo,Japan) and stored in polyethylene containers then keptunder refrigeration at 4 ◦ C. The samples were injectedto the chromatographic system without dilution.SamplesRed algae Eucheuma cottonii which was 40 daysages, harvested from, big Aceh sub-province ofNangro Aceh Darussalam, Indonesian. The algaewere extensively washed with distilled water toremove the particulate material from their surface andwere dried under the sunlight Dried biomass of redalgae was cut, ground and then screened to particlesizes of 150-425 µm. Carrageenan was extracted fromred algae by alkaline solution. Dried red algae andcarrageenan were digested with nitric acid andhydrogen peroxide, the solution was filtered with a0.45 µm membrane filter for IC, stored inpolyethylene containers and kept under refrigerationat 4 ◦ C.IC systemThe ion chromatograph consisted of a PU-2080i plusHPLC pump (Jasco, Tokyo, Japan), a Rheodyne 5095injector equipped with a 20- µl sample loop (Cotati,CA, USA), a CM-8020 conductivity detector (Tosoh,Tokyo, Japan), two Model 7610-600-6 port switchingvalve (Rheodyne), and a Computer AidedChromatography data processor (Nippon Filcon,Tokyo, Japan). The column employed was a TSKgelIC-Anion-PW XL column (50 mm×4.6 mm i.d.) andtwo suppressor columns obtained from Tosoh (Tokyo,Japan). One suppressor was being used forchromatographic run, and the other suppressor wasregenerated with 0.5 M sulfuric acid (Wako PureChemical Industries, Osaka, Japan) at a flow rate of2.0µl/min by using pump. The suppressor column wasthen rinsed with deionized water for futurechromatographic run.Operating conditionsThe mixture of (Na 2 CO 3 : NaHCO 3 ) was used as theeluent for the determination of anions. The systemwas operated under isocratic mode. Since the pressurelimit of the columns is 8MPa. To find the optimumcondition of anion separation, the effect of eluentflow-rate, eluent concentration and anionconcentration were studied. The optimum conditionwas applied to determine anion in red algae(Eucheuma cottonii) and carrageenanResults and discussionEffect of sodium carbonate and sodium bicarbonateconcentration on anions separationSodium bicarbonate and sodium carbonate solutionconcentration ratio were evaluated for the efficient407

R. Suhaili et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 406-410)Jatinangor, 30-31 October 2008separation of anions on the TSKgel Super IC-Anioncolumn. Fig. 1 presents ionic chromatograms showingthe anion separation of a standard mixture sample.The retention order at the optimum concentration wasF - , Cl - , NO 2 - , Br - , NO 3 - , HPO 4 2- , SO 42-and I - .The retention times and resolutions of analyte ionswere strongly dependent on the concentration of theeluent. Carbonate eluents were investigated forevaluating the efficient separation of anions on theIC-Anion-PW XL column. For investigation of theoptimum concentration of the eluent, the sodiumcarbonate and sodium bicarbonate concentration ratiowere varied from 1.0:1.0mM; 1.2:1.4 mM and1.5:1.75 mM. Considering the retention times, peakshapes and peak resolutions of the analyte anions, theoptimum ratio concentration of the eluent was found[bicarbonate : carbonate] = [1.2 : 1.4] witch gave thegood resolution.. When the concentration ratio ofbicarbonate and carbonate less than 1.2:1.4 mM, theresolution was not good. When the eluentconcentration was higher than 1.2:1.4 mM, theresolution of anions were good but the inlet pumppressure higher than 8MPa. It was not satisfactory andcan cause the instrument damage.×10 5 ] 3Conductivity (µS/cm)21Figure 1 Effect of Na 2 CO 3 :NaHCO 3 concentrationRepeatability11222331 60a0 5 10 15on anion separation. 0.7Flow ratemL/min. a. 1.0:1.0 mM; b. 1.2:1.4 mM; c.1.5:1.75 mM. Columns TSKgel IC-AnionPW XL (50x4.6 mm i.d). Columntemperature: room temperature. Injectionvolume: 20 µL. Peak: 1. F - ; 2. Cl - -; 3. NO 2; 4. Br - ; 5. NO 3 -; 6. HPO4 = ; 7. SO = 4 ; 8. I -Table 1 shows the repeatability of the signal for fivesuccessive measurements under the conditions. Allthe relative standard deviation (RSD) values forretention time, peak area and peak height weresmaller than 1% except for F-. For this system F- wasearly eluting analyte and interfered with water dip.Calibration curves and detection limits43 4 54 5576Time (min)The linearity and detection limits data are summarizedin Table 3. All the calibration curves of the anions and677888cbthe cations showed good linear correlations. Thelinear relationships between the peak height and thesample concentration of anions with concentrationranged from 0.1 to 0.5 mM showed good linearrelationships with the correlation coefficients ofanions varied between 0.9958 and 1.0000, as shownin Table 3. From the table, the correlation coefficientsr2 > 0.999 proved good linearity of the method..These values were sufficiently low as the suppressedconductivity IC. Separation time in this method wasless than 15 min, while conventional nonsuppressorpacked-bed column, inorganic anions separation timewas 24 min.Table 1 Relative standard deviations (RSDs) of theretention time and the peak signals of theinorganic anions under the optimumchromatographic conditions with Na 2 CO 3 :NaHCO 3 1.2 : 1.4 mM , flow rate of 0.7mL/min, Columns TSKgel IC- AnionPW XL (50x4.6 mm i.d).AnionRSD (%), n=5Ret. Time Peak Height Peak AreaF- 0.00 0.12 0.38Cl- 0.21 0.17 0.32NO2- 0.16 0.28 0.34Br- 0.13 0.22 0.39NO3- 0.17 0.25 0.24HPO4= 0.13 0.15 0.44SO4= 0.20 0.28 0.14I- 0.10 0.27 0.31Table 2 Summarized data for the standard calibrationgraphs, coefficient correlation (r 2 ) of anions,and retention time were obtained under theoptimum operating conditionIon [anion] mM r 2 t r minF- 0.1-0.5 0.9958 1.84Cl- 0.1-0.5 0.9999 2.80NO2- 0.1-0.5 0.9987 3.80Br- 0.1-0.5 0.9995 4.60NO3- 0.1-0.5 0.9995 5.61HPO42- 0.1-0.5 0.9997 6.70SO42- 0.1-0.5 0.9997 8.00I- 0.1-0.5 1.0000 13.70Determination inorganic anions in red algae(Eucheuma cottonii) and carrageenanThe present suppressed ion chromatography systemwas applied to the determinae inorganic anions in redalgae (Eucheuma cottonii) and carrageenan. Eachsample was injected after filtration through a 0.45-µmmembrane filter.408

R. Suhaili et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 406-410)Jatinangor, 30-31 October 2008Conductivity (µS/cm)600004000020000123 4 5651 2 3 4 6 7 800 5 10Figure 2 Chromatogram of anion separation a. Eucheuma cottonii, b. Anion Standard solution 0.3 mM. Flowrate of Na 2 CO 3 : NaHCO 3 [1.2:1.4 mM] 0.7 mL/min, Columns TSKgel IC-Anion PW XL (50x4.6 mmi.d). Column temperature: room temperature, Injection volume: 20 µL. Peak: 1. F - , 2. Cl - , 3. NO 2 - , 4.Br - , 5. NO 3 - , 6. HPO 4 - , 7. SO 4 = , 8. I -7Retention Time (min)8baTable 3 Anion contained in red algae (Eucheuma cottonii) and carragenanSamplePureCarrageenanExtractedCarrageenanEucheumacottoniiAnion contained mg/100gF - Cl - -NO 2 Br - -NO 32-HPO 42-SO 4 I -1.75 16.60 1.28 5.22 445.47 8.64 19.16 11.661.80 17.79 0.89 5.10 442.45 8.02 25.14 10.132.33 10.02 1.71 7.77 448.94 7.99 22.14 15.12Good separations for common inorganic anions wereachieved. Ion contain in sample is shown in Table 3.-Table 3 shown that the sample contains NO 3 as amain component in both red algae (Eucheumacottonii) and carragenan. The heigh concentration ofnitrate in samples is caused by red algae consumednitrate very rapidly due to light conditions [1].ConclusionsIn conclusion, an ion chromatographic system usingtwo 6-port switching valves and two packed columnsuppressors to maintain the suppressed conductivity,were used for continuous suppression to obtain bettersensitivity. Eight common inorganic anions (F - , Cl -,NO 2 - , Br - , NO 3 - , HPO 4 2- , SO 4 2- , I - ) could bedetermined by contactless conductivity detector. Themixture of 1.4 mM sodium bicarbonate and 1.2 mMsodium carbonate with flow rate 0.7 mL/min wassuitable for the determination of the above inorganicanions. The system was satisfactorily applied to thedetermination of common anions in red algae(Eucheuma cottonii) and carrageenan could be carriedout within 15 min. The relative standard deviations(RSDs) for the retention time, peak area and peakheight of eight common inorganic anions (0.3 mMeach of F - , Cl - , NO 2 - , Br - , NO 3 - , HPO 4 2- , SO 42-and I - )were between 0.0-0.2, 0.1-0.4 and 0.1-0.3%.References1. Anna. C.U, M. B´athorib, G. Blundenc, 2000,Determination of elements in algae by differentatomic spectroscopic methods, MicrochemicalJournal 67,.39-422. Saari-Nordhaus R., J.M. Anderson Jr. 1997,Recent advances in ion chromatographysuppressor improve anion separation anddetectionJ. Chromatogr. A 782, 75.3. Shinji S ,, Y. Ogura, A. Miyanaga, T. Sugimoto,2002 Ion chromatographic system with a novelswitching suppression device, Journal ofChromatography A, 956 53–584. Chen. Y, L. Jing, X. Li, Y. Zhu, 2006 Suppressedanion chromatography using mixed zwitter-ionicand carbonate eluents, Journal ofChromatography A, 1118, 3–115. Toshimitsu. O, A. Isozakib, H. Nagashima, 1998,Studies on elution conditions for thedetermination of anions by supressed ioninteractionchromatography using a graphitizedcarbon column, Journal of Chromatography A,800, 239–245409

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