Vol. 29, 2011, No. 6: 634–640 Czech J. Food Sci.

Simultaneous Determination of , and in Foodstuffs by Low Pressure -Exchange Chromatography with Visible Light Detection

Lingyun Yu 1,2, Xinshen Zhang1,3, Jing Jin 2, Shujuan Che 1 and Lan Y u 1

1Light Industry & Textile & Food Engineering, Sichuan University, Chengdu, Sichuan, P.R. China; 2Technology Center of Sichuan Entry-Exit Inspection and Quarantine Bureau, Chengdu, P.R. China; 3State Key Laboratory of Hydraulics and the Exploitation and Protection of Mountain Rivers, Sichuan University, Chengdu, Sichuan, P.R. China


Yu L. Y., Zhang X., Jin J., Che S., Yu L. (2011): Simultaneous determination of chloride, bromide and iodide in foodstuffs by low pressure ion-exchange chromatography with visible light detection. Czech J. Food Sci., 29: 634–640.

An ion-exchange chromatography method with visible light detection was developed for the simultaneous determina- tion of chloride, bromide, and iodide in foodstuffs. They were separated by means of low pressure ion-exchange chro- matography using 4.0mM carbonate solution as the eluent and a low pressure ion-exchange chromatography column as the separation column. The detection limits of chloride, bromide and iodide were 0.011 mg/l, 0.002 mg/l, and 0.023 mg/l, respectively. The relative standard deviations (RSDs) of the peak area were smaller than 2.9%. The recoveries were between 98.61% and 105.65%. Unlike the traditional methods, this validated method is inexpensive and stable.

Keywords: LPIEC; spectrophotometry;

Most analytical approaches reported for chloride, ion-exchange separation with suppressed conduc- bromide, and iodide determination have shortcom- tivity detection (Tucker & Flack 1998; Kapinus ings to some extent. The traditional methods, such et al. 2004; Zhe et al. 2006). IEC with suppressed as titration and colorimetric measurement, with conductivity detection permits the use of higher a relatively low sensitivity and using lots of toxic capacity stationary phases and higher ionic strength reagents (e.g., , mercuric thiocyanate), eluents than those which use conductivity without can determine one substance only. chemical suppression detection methods, and Ion-exchange chromatography (IEC) was first provides a greater variation in ion-exchange selec- introduced about 33 years ago and has become tivity. The other common means of detection are a well-established technique. The simultaneous conductivity without chemical suppression (Zhou analysis of common anions such as chloride (Cl–), & Guo 2000) and direct or indirect UV detection bromide (Br–), iodide (I–), nitrate, phosphate, or (Quattrocchi et al. 2001; Hu et al. 2002). How- sulfate is the most important IEC routine appli- ever, the visible light detection has not been used cation. These solutes are usually determined by to detect Cl–, Br– and I– simultaneously.

Supported by the Hi-tech Research and Development Program of China (863 Program), Project No, 2006AA173Z.

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Commonly, the analyte monitored by optical Table 1. Experimental conditions for Cl−, Br−, and I− de- (visible light) detector are visible light-absorbing termination in LPIEC ions such as Cu2+ or Fe3+. For non-visible light- absorbing ions, the indirect detection and/or post- Analytical separation column derivatisation with subsequent visible light Anion-exchange resin column (0.6 cm × LPIEC column 13 cm, I.D. 30−35 μm), exchange capac- measurement has been considered to be a useful ity 0.001 meq/g detection method. Eluent 4.0mM Na CO This paper describes a low pressure ion-exchange 2 3 Flow rate 0.70 ml/min chromatography (LPIEC) with an optical detector to detect simultaneously Cl–, Br–, and I– by indirect Injection volume 300 µl detection. LPIEC is a type of ion chromatography Chemical reaction variables without the suppressor column and working at 0.15mM Indigo Carmine 5 5 R1 a low pressure of 1.96 × 10 Pa to 2.94 × 10 Pa and 1.1M H2SO4 – – (Jiang et al. 1999; Zhou et al. 2007). Cl , Br , and R1 Flow rate 0.25 ml/min – I are separated by LPIEC column and then each of R2 32.0mM KBrO them catalyses the oxidative discoloration of indigo 3 R2 Flow rate 0.25 ml/min carmine by potassium bromate (KBrO3) in acidic media and heating condition. The spectrophoto- Reaction coil 1.4 m metric measurement can be applied satisfactorily Optical detector – – – to the detection of Cl , Br , and I . Wavelength 610 nm

MATERIAL AND METHODS Sample preparation. All samples including for- tified cookies (No. 1), kelps (No. 2), and radishes All the analyses were conducted on Low Pres- (No. 3) were bought in the morning market of sure Ion Chromatographic Instrument (Jiang et Chengdu city. The fortified cookies were pulverised al. 1999; Zhou et al. 2007) consisting of a low and sieved (80 mesh); fresh kelps and radishes were pressure four-way peristaltic pump (Shanghai carefully rinsed with deionised water, and then the Huxi Analytical Instrument Plant, Shanghai, P.R. deionised water on the surface of the kelps and China), LPIEC column, six-way automatic injec- radishes was removed by a centrifuge, at last the tion valve, optical flow cell, optical detector, and kelps and radishes were separately and completely homothermic heater (installing a reaction coil). A ground by a mill. In the sample preparation, all the HW-2000 Chromatography workstation (Qianpu deionised water was freed from dissolved software Co., Ltd., Zhehang, P.R. China) was used by boiling and was adjusted to pH 10, to prevent for the system control and data collection. The the oxidation of Br– and I–. experimental conditions used for the determina- The processed samples weighing 25.00 g each were tion of Cl–, Br–, and I– are given in Table 1. added to 150 ml flasks with approximately 80 ml Reagents. All the chemicals used were of ana- water, which were subsequently shaken for 30 min lytical grade quality (Chengdu Kelong Chemical in supersonic wave, thereafter, the mixtures were Reagent Factory, Sichuan, P.R. China). The stand- diluted to a final volume of 100 ml with deionised ards were prepared daily by diluting 1000 mg/l ICP water. All samples were filtered before use. standard solutions. The eluent (4.0mM Na2CO3) Procedure. The schematic representation of the was prepared from 1M sodium carbonate aqueous LPIEC setup is shown in Figure 1. The sample was solution. The mixture of Indigo Carmine and sul- loaded via the four-way peristaltic pump and six- furic solution was marked as colour solution way automatic injection valve on a 300 µl sample R1 and was prepared from 1mM Indigo Carmine loop. After injection, the separation of the analyte aqueous solution and 6M sulfuric acid (H2SO4). ions took place in the ion-exchange column by Concentration KBrO3 solution was marked as passing the Na2CO3 eluent (4.0mM Na2CO3). The colour solution R2 and was prepared from 0.1M separated ions with the eluents R1 and R2 mixed

KBrO3 aqueous solution. The above solutions were by mixing coil then came into the reaction coil, in prepared using deionised water with a resistance which the separated ions accelerated the reaction of 18.2 MΩ (Molecular, Shanghai, P.R. China). of R1 and R2 at 85 ± 0.5°C. The reaction solu-

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P the rate of the oxidative, discoloration of indigo S 0.5 carmine. 4 5 The method is based on the observation of dis- 3 Waste Sample loop coloration. Without catalysts, the rate of oxidative C V 6 discoloration of indigo carmine by KBrO3 is very slow 2 1 in acidic media. The rate of discolouring oxidation of – indigo carmine by KBrO3 becoms faster when Cl , – – LPIEC Br , or I is added into the acidic reaction solution. PC – – – 0.25 column The reason is that all of Cl , Br , and I coordinate R1 D with Indigo Carmine and form a six-member ring 0.25 L F R2 M M intermediate, which can weaken the double bond H Waste force of the two 3-sulfo indoles full-ketone. There- (ml/min) fore, the indigo carmine easily oxidises by KBrO3 Valve position Valve position when Cl–, Br–or I– is present in the acidic reaction for sampling step for analysing step solution (Schema 1). In order to find the most appropriate wave-

S – sample; C – Na2CO3 eluent; R1 – Indigo Carmine and length for the analysis on the LPIEC, the effect

H2SO4 solution; R2 – KBrO3 solution; P – four-way peristaltic of wavelength on the peak height was studied in pump; V – six-way automatic injection valve; L – reaction the range from 400 nm to 700 nm using the Spec- coil; M-Mixing coil; H – homothermic heater; F – optical trum UV2800 (You Nike Ltd. Co., Shanghai, P.R. flow cell; D – optical detector China). Figure 2 shows the absorbance spectra of Figure 1. Schematic diagram of the LPIEC system three different solutions. The standard solutions of Cl–, Br–, and I– respectively, accelerated the fading through oxidation reaction of R1 and R2 tion passed through the optical detector, where in the heating condition. The greatest absorbance the peaks of Cl–, Br–, and I– were recognised at a intensity of the solutions was at 610 nm, which wavelength of 610 mm. was selected for further work. Effect of the length of LPIEC column. The rela- tively short LPIEC column (invented by Professor RESULTS AND DISCUSSION Xinshen Zhang) was selected in the study for the following two advantages: the reduced system Detection principle pressure, and improved sensitivity and precision. The column lengths of 11–15 cm were tested, the The catalytic reaction was monitored spectropho- results are shown in Figure 3. When the length of tometrically by measuring the change in absorbance the column was shorter than 13 cm, the separation of the reaction mixture. Only the changes in the of Cl– and Br– was poor. However, if the length of ions (Cl–, Br–, and I–) concentrations influenced the column exceeded 13 cm, the retention time

– O O Cl– (Br– or I–) O H S N – O O O O – – – H O + Cl (Br or I ) S N N O S – O H O O O N S – H O O O

Cl– (Br– or I–)

– O O O H S – O N O O O S O KBrO3 N 2 O O Cl– (Br– or I–) S – + H O N O O H Schema 1

636 Czech J. Food Sci. Vol. 29, 2011, No. 6: 634–640

1.6 (1)

1.4 (2) 1.2


Abs 0.8 (3) 0.6

0.4 (4) 0.2

0.0 400 450 500 550 600 650 700


(1) Mixture solution of 0.15 mM Indigo Carmine solution, 1.1M H2SO4 and 32.0mM KBrO3 − (2) Mixture solution of 0.15mM Indigo Carmine solution, 1.1M H2SO4, 32.0mM KBrO3 and 10.00 mg/l I − (3) Mixture solution of 0.15mM Indigo Carmine solution, 1.1M H2SO4, 32.0m M KBrO3 and 5.00 mg/l Cl − (4) Mixture solution of 0.15mM Indigo Carmine solution, 1.1M H2SO4, 32.0mM KBrO3 and 2.00 mg/l Br Putting the above mixture solution into a temperature of 85 ± 0.5°C constant temperature water bath, it was heated for 2 min, and then quickly taken out and put in the cold water for 3 min to end the reaction. At last, the absorbance spectra were obtained by the Spectrum UV2800 Figure 2. Absorbance spectra increased. Thus, the length of the column of 13 cm tion temperature increased from 65–87°C. When the was chosen for further work. temperature was higher than 85°C, the reproduc- Optimisation of separation conditions. The ibility of the system peak height became poor, and eluent is critical for the analysis by ion-exchange the flow path was prone to bubbles formation, which chromatography. Sodium carbonate (Na2CO3) was impacted on the stability of the system determination. selected as the eluting solution in this study. Differ- To maintain the system stability, the temperature of ent concentrations of Na2CO3 (2.0, 3.0, 4.0, 5.0, and 85 ± 0.5°C was chosen for further study. 6.0mM) were studied to determine the optimum As a consequence, we chose the length 13 cm concentration, with an injection volume of 300 µl for the LPIEC column, 4.0mM Na2CO3 as the and using the visible light detection at 610 nm. eluent with a flow rate of 0.7 ml/min, and 85 ± The results showed that the separation of Cl–, Br–, 0.5°C as the reaction temperature. Under the op- and I– was worse at higher concentrations while timised experimental conditions, the analytes in the low concentrations resulted in a broad peak the solution were well separated within 14 min shape. The separation of these ions with 4.0mM (Figure 3c).

Na2CO3 appeared to be optimum. Under the opti- mum concentration, different flow rates (0.5, 0.6, 0.7, 0.8, and 0.9 ml/min) were set to investigate the Method validation impact of the flow rate on the separation. A faster rate resulted in peaks, overlapping while the lower Linearity was investigated using the stock solu- was the rate, the longer was the overall time, as well tion containing the analytes, which was diluted as the worse was the peak shape. Therefore, it was serially. Eight concentrations of the three analytes suggested to use a flow rate of 0.7 ml/min. solution were injected in triplicate and the calibra- Effect of reaction temperature. To obtain the best tion curves were constructed by plotting the peak overall reaction, the effect of the reaction tempera- height (A, mV) versus the concentration (C, m/l) ture was also investigated in the range of 65–87°C of each analyte. The detection limits were esti- (Figure 4). The peaks became higher with the reac- mated as the mean of the blank sample plus three

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A (mV) (a) Column lenght 11 cm (b) Column lenght 12 cm 450– 400– 350– 300– 250– 200– 150– 100– 50– 0– 450– 400– (c) Column lenght 13 cm (d) Column lenght 14 cm 350– 300– 250– 200– 150– 100– 50– 0– 3 6 9 12 15 3 6 9 12 15 Time (min)

– – – Conditions: Cl 10.0 mg/l, Br 2.0 mg/l, and I 10.0 mg/l; eluent 4.0mM Na2CO3 with a flow rate of 0.7 ml/min; temperature 85 ± 0.5°C Figure 3. Effect of LPIEC column length

A (mV) 450– 450 400 ClȬ 400– BrȬ 350 350– IȬ 300 300– (mV) ȱ 250 250– 200 height 200– ȱ 150 150– Peak 100 100– 50 50– 0 0– 60 65 70 75 80 85 90 3 6 9 12 15 Reationȱtempertureȱ(°C) Time (min) Conditions: Cl– 10.0 mg/l, Br– 2.0 mg/l, and I– 10.0 mg/l; Conditions: Cl– 10.0 mg/l, Br– 2.0 mg/l, and I– 10.0 mg/l; eluent 4.0mM Na2CO3 with a flow rate of 0.7 ml/min; tem- eluent 4.0mM Na2CO3 with a flow rate of 0.7 ml/min perature 85 ± 0.5°C Figure 4. Effect of reaction temperature Figure 5. Chromatogram of water

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Table 2. Analytical characteristic of Cl−, Br−, and I−

Linear range Correlation Detection limit RSD (%) Retention time Analyte Calibration equation (mg/l) coefficient (mg/l) (S/N = 3) (n = 10) (min) Cl− 0.15–35.0 A = 45.51C − 1.45 0.9992 0.011 2.96 6 Br− 0.03–5.0 A = 486.24C − 74.33 0.9991 0.002 2.12 7.5 I− 0.10–25.0 A = 49.71C − 59.64 0.9990 0.023 2.45 14

RSD = relative standard deviation

Table 3. The concentrations and recovery data of Cl–, Br–, and I– in the samples

Concentrations (mg/l) Recovery (%) Analyte No. 1 No. 2 No. 3 No. 1 added (4.0 mg/l) No. 2 added (2.0 mg/l) No. 3 added (1.0 mg/l) Cl– 23.48 20.04 15.93 105.65 103.37 105.21 Br– 0.91 1.69 1.06 101.97 99.47 100.47 I– 4.62 2.42 0.71 98.61 100.12 101.75 times the standard deviation obtained on the blank high accuracy of the results, and it is also easily sample. The precision was evaluated by performing operated in comparison to other methods; it pro- ten replicate analyses of the standard solution at the vides a simple and efficient procedure for analysing concentrations of 10.0 mg/l for Cl–, 2.0 mg/l for B–, the above ions in foodstuffs. The Low Pressure and 10.0 mg/l for I–. The results are illustrated in Table Ion Chromatographic Instrument employed in 2. Under the optimised experimental conditions, all this study not only has a low price ($4000 per in- three analytes showed perfect linearity. strument), but is also portable occupying a small volume (dimension of 25 cm × 20 cm × 20 cm).

Sample determination References To illustrate the application of the method de- veloped, three aquatic samples (No. 1 Fortified Hu W.Z., Yang P.J., Hasebe K., Haddad P.R., Tanaka Cookies, No. 2 kelps, and No. 3 radishes) from K. (2002): Rapid and direct determination of iodide in Chengdu, P.R. China were collected for the de- by electrostatic ion chromatography. Journal of termination. Figure 5 shows the chromatogram of Chromatography A, 956: 103–107. the No. 3 radishes. The contents of the analytes in Jiang X.P., Zhang X.S., Liu M.H. (1999): Ultra-short col- real samples were determined using the external umns for low-pressure ion chromatography. Journal of – – calibration method. Suitable amounts of Cl , Br , Chromatography A, 857: 175–181. and I– standards were added to the real samples of Kapinus E.N., Revelsky I.A., Ulogov V.O., Lyalikov Y.A. known contents and the mixtures were analysed (2004): Simultaneous determination of , chloride, using the proposed procedure. Recovery was ex- nitrite, bromide, nitrate, phosphate and sulfate in aqueous pressed for each component as the mean percent- solutions at 10−9 to 10−8% level by ion chromatography. age ratio between the measured and the added Journal of Chromatography B, 800: 321–323. amounts. The results are shown in Table 3. Quattrocchi O., Frisardi L., Iglesias M., Noya M., Caputto M., Ferraris D.A., Siliprandi D.E., Piccinni E. (2001): Ion exchange chromatographic determina- CONCLUSIONS tion of olpadronate, phosphate, phosphite, chloride and methanesulfonic acid. Journal of Pharmaceutical and This method proves that with the use of visible Biomedical Analysis, 24: 1011–1018. light detection, ion-exchange chromatography Tucker H.L., Flack R.W. (1998): Determination of iodide can detect Cl–, Br–, and I– at the same time; the in ground water and soil by ion chromatography. Journal method has shown good correlation between and of Chromatography A, 804: 131–135.

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Zhe B.H., Zhong Z.X., Yao J. (2006): Ion chromatographic application in the estimation of postmortem interval. determination of trace iodate, , , bromide, Journal of Chromatography B, 852: 278–281. bromate and nitrite in drinking water using suppressed Zhou M.S., Guo D.L. (2000): Simultaneous determination conductivity detection and visible detection. Journal of of chloride, nitrate and sulphate in vegetable samples Chromatography A, 1118: 106–110. by single-column ion chromatography. Microchemical Zhou B., L. Zhang, G.Q. Zhang, X.S. Zhang, X.P. (2007): Journal, 65: 221–226. The determination of potassium concentration in vitre- Received for publication September 2, 2009 ous humor by low pressure ion chromatography and its Accepted after corrections November 15, 2010

Corresponding author: Dr. Zhang Xinshen, Sichuan University, Light Industry & Textile & Food Engineering, Chengdu, 610065, Sichuan, P.R. China tel.: + 86 028 854 075 53, e-mail: [email protected]