zhang 2008

Original article

A novel method for the determination of sodium chloride in

salted fish

Yinliang Zhang1,2 & Wenshui Xia1*

1 School of Food Science and Technology, Southern Yangtze University, Wuxi 214036, Jiangsu Province, China

2 School of Food and Biological Engineering, ZhengZhou University of Light Industry, Zhengzhou 450002, Henan Province, China

(Received 30 March 2006; Accepted in revised form 4 December 2006)

Summary A novel, simple and reliable spectrophotometric method to determine sodium chloride in salted sh was

developed and validated. The method was based on the reaction of chloride with silver nitrate and the

determination of turbidity of silver chloride formed. The absorbance was measured at 385 nm. The method

was tested under various conditions, and we conrmed an optimal operation with 2 mL 1:4 (v v) nitric acid,2 mL 1.5 g L)1 gelatin, 5 mL 0.5% silver nitrate in a total volume of 50 mL, mixed and heated at 60 C for10 min. The range of linearity and the detection limit were 0.424 mg L)1 and 0.2 mg L)1, respectively. The

relative standard deviations and the recovery were 0.83% to 2.87% and 93.74% to 103.6%, respectively.

The accuracy of this method was comparable with Volhard method, and it was cheap and successfully

applied to determine sodium chloride in salted sh.

Keywords Gelatin, salted sh, sodium chloride, spectrophotometry, Volhard method.

Introduction

Salting sh is one of the oldest treatments in foodpreservation and is still popular even in developedcountries for its low processing costs and satisfyingconsumers habits (Zugarramurdi & Lupin, 1980). Ahigh level of salt decreases water activity and thushinders germ development and proliferation of fungiand yeast. It also contributes to developing desirablecharacteristic avour to the products (Ismail & Woot-ton, 1982). A high level of salt results in consumerrejection whereas a low level of salt brings about shelf-life problem. Therefore, simple, sensitive and reliableassay of salt content becomes more important for saltedfoods.There are several methods to detect sodium chloride.

First is the volumetric method (AOAC, 1997; Yu,(1991)). This method is based on Mohr method andVolhard method, but they are time-consuming anddicult for routine analysis of a large number ofsamples. This method consumes much of silver reagentand there may be serious interference from other samplecomponents. Second is the potentiometry method (Lapaet al., 1995; Perez-Olmos et al., 1997). It is simple,economical and has higher sampling rates. The most

serious problem with this method is its lack of accuracy.An error of 0.5 mV in the electromotive force cancause a 2% error in the concentration calculated for theelectrode sensitive to single charge species. The third isthe ion chromatography method (Zhou & Guo, 2000;Alcazar et al., 2003), which needs high investment onequipment and consumes more reagent. Another disad-vantage in this method is that it is less selective. Thereare other methods such as ow injection analysis(Taylor & Grate, 1995; Chalk & Tyson, 1998; Coutoet al., 1998; Silva et al., 1999) and robotic method(Velasco-Arjona et al., 1998). These methods need highinvestment on equipment. Until now, no publishedliterature is available on the determination of sodiumchloride in food by measuring turbidity with spectro-photometer (Xian & Chen, 2003).The aim of this study was to set up a new method with

turbidity measurement and to compare this methodwith Volhard method.

Materials and methods

Reagents

All solutions were prepared from analytical grade orguarantee reagents (Sinopharm Group ChemicalReagent Co. Ltd, Shanghai, China). Solutions of gelatinwere freshly prepared and stored no longer than 1 day.

*Correspondent: Fax: +86-510-85869455;

e-mail: [email protected]

International Journal of Food Science and Technology 2008, 43, 927932 927

doi:10.1111/j.1365-2621.2007.01544.x

2007 The Authors. Journal compilation 2007 Institute of Food Science and Technology Trust Fund

Salting process

Pike eel (Muraenesox cinereus) was obtained from alocal supermarket and processed in llets within 24 hafter the catch. The dorsal muscle was manually lletedand then soaked in 25%NaCl solutions at 20 C. Filletsamples were removed from the solution at regularintervals and then drained and gently dried with papertissue, and stored for 12 h at 4 C. Then, the llets wereminced and aliquots were taken for the determination ofsalt content.

Sample preparation

Minced sample (20 g) was mixed with 100 mL hot water(70 C). The mixture was then oscillated in boiling waterbath for 15 min. followed by rapid cooling to roomtemperature. Finally, 4 mL potassium hexacyanoferrateand 4 mL zinc acetate were added, followed by dilutingthe mixture to 250 mL. The solution was ltered and thesupernatant was used for determination.

Principle of determination

The principle of turbidity spectrophotometry is thereaction between chloride and silver nitrate at acidiccondition. The resulted precipitate, which was protectedwith colloid, had absorption at a given wavelength.

Maximum absorption wavelength

Three millilitre of 0.1 mg mL)1 standard sodium chlor-ide was put in a 50 mL volumetric ask. Then, 4 mL(1:4) nitric acid, 5 mL gelatin colloid (5 g L)1), 5 mLsilver nitrate (0.5%) and distilled-deionized water weresuccessively added and mixed thoroughly. Distilled-deionized water was used as reference and samples werescanned from 300 to 900 nm with a UV-1900 UV-Vis double-beam spectrophotometer (Beijing PurkjinjeGeneral Instrument Ltd, Beijing, China). A UV-2100UV-Vis spectrophotometer (Unico Shanghai AnalysisInstrument Ltd, Shanghai, China) was used for validation.

Optimization determination condition

The mixture, which consists of 3 mL standard sodiumchloride (0.1 mg mL)1), 4 mL (1:4) nitric acid, 5 mLgelatin colloid (5 g L)1), and 5 mL silver nitrate (0.5%),was heated to 30, 40 and 60 C, respectively, andabsorbance at 385 nm was determined with distilled-deionized water as reference.Gelatin concentration and volume were changed while

other conditions were xed to optimize gelatin concen-tration and volume. Similarly, nitric acid concentrationand volume were changed while other conditions werexed to optimize nitric acid concentration and volume.

Interference study

Four millilitre of 0.1 mg mL)1 standard sodium chlor-ide was put into a 50 mL volumetric ask and dierentamount of sodium bromide and bovine serum albumin(BSA) was added followed by 2 mL (1:4) nitric acid,2 mL gelatin colloid (1.5 g L)1), 5 mL silver nitrate(0.5%) and distilled-deionized water to a nal volume of50 mL. After being mixed thoroughly, the mixture washeated and kept at 60 C for 10 min before absorbancewas measured at 385 nm.

Range of linearity

Standard sodium chloride (0.1 mg mL)1) of 0.0, 0.2, 0.4,0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10 and 12 mL was, respect-ively, put into 50 mL volumetric ask, followed by2 mL (1:4) nitric acid, 2 mL gelatin colloid (1.5 g L)1),5 mL silver nitrate (0.5%) and distilled-deionized waterto a nal volume of 50 mL. After mixing thoroughly,the mixture was heated and kept at 60 C for 10 minbefore absorbance was measured at 385 nm.

Precision test

According to the above detection procedure, ninesamples were determined, and each sample was meas-ured six times.

Recovery test

To dierent sample solution series, standard sodiumchloride was added. According to the above detectionprocedure, recovery was calculated as follows:

Recovery% Ma MbMs

100%

where Ma was total chloride content after addingstandard solution, Mb was original chloride content insamples, andMs was standard chloride content added insample.

Statistical analysis

For uniform design, Mathematics 4.0 software anovaanalysis was applied (Wolfram Research Inc., USA).Statistical signicance was determined by t criterion.

Results and discussion

The UV-visible spectrum of silver chloride had twoabsorption peaks at 320 and 385 nm as shown inFig. 1a, and Fig. 1b shows the validation that maximumabsorption wavelength was 385 nm. This was slightlydierent from the results reported by Zhang & Xu

A reliable method to determine sodium chloride in salted fish Y. Zhang and W. Xia928

International Journal of Food Science and Technology 2008 2007 The Authors. Journal compilation 2007 Institute of Food Science and Technology Trust Fund

(2003). Meanwhile, the results of series concentration ofsodium chloride solution were the same. Therefore,detection wavelength for turbidity of silver chloride wasselected at 385 nm.Reaction time and temperature are very important

parameters in turbidity spectrophotometry. Figures 2aand b show the changes of absorbance with time at 30and 40 C, respectively. The absorbance remainedalmost constant after 150 min (Fig. 2a) and after70 min (Fig. 2b) at respective temperatures. The higherthe temperature, the faster the absorbance reachedconstant. The result of absorbance changes at 60 C isshown in Table 1, where the absorbance did not increasealong with time between 10 and 30 min. Therefore, theoptimal reaction conditions were set at 60 C and10 min.

Two kinds of colloids (gum Arabic and gelatin) weretested for the protection of stability of turbidity of silverchloride. The trials showed that they had no signicantdierence (data are not shown here). Considering thesolution preparation and cost, we chose gelatin.The optimal gelatin concentration (x1) and volume

(x2) were examined with Uniform Experimental DesignU*6(6

4) (Fang, 1994) (Table 2). The analysis of variance(anova) and P-values were used to check the signi-cance of the eects on absorbance. The main eects and

340 360 380 400 420 440 4600.16

0.17

0.18

0.19

0.20

0.21

Abso

rban

ce

Wavelength (nm)

(b)

(a)

Wavelength (nm)

Abso

rban

ce

300.00.0000

0.0750

0.1500

0.2250

0.3000

400.0 600.0 800.0 900.0

Figure 1 UV-Vis double-beam curve with (a) and without

(b) scanning. (a) UV-Vis double-beam scanning from 300 to 900 nm.

(b) UV-Vis validation from 340 to 460 nm.

0.10

0.15

0.20

0.25

0.30

0.35

Time (min)

Abso

rban

ce

30 C

0 50 100 150 200 250 300 350 400

10 20 30 40 50 60 700.10

0.12

0.14

0.16

0.18

0.20

Abso

rban

ce

Time (min)

40 C

(b)

(a)

Figure 2 Changes of absorbance with time at 30 and 40 C.(a) At 30 C, turbidity absorbance of silver chloride changes with time.(b) At 40 C, turbidity absorbance of silver chloride changes with time.

Table 1 Changes of absorbance with reaction time at 60 C

Reaction time (min) 10 20 30

Absorbancea 0.171 0.009 0.172 0.011 0.176 0.008

aValues of mean (4 replication) SE.

A reliable method to determine sodium chloride in salted fish Y. Zhang and W. Xia 929

2007 The Authors. Journal compilation 2007 Institute of Food Science and Technology Trust Fund International Journal of Food Science and Technology 2008

their interactions may be observed in the equation givenbelow:

y 0:4899 0:05979x1 0:06243x2 0:01013x1x2The factors of gelatin concentration (P = 0.0225) and

volume (P = 0.0251) were signicant and they hadinteractive inuence. The result of single factor is listedin Table 3. When the total gelatin quantity was between0.5 and 3.0 mg, the absorbance remained almost con-stant. Considering results in Table 2, we have chosen touse 2 mL 1.5 g L)1 gelatin.The eect of nitric acid was also studied. The

concentration (x1) and volume (x2) were optimised byUniform Experimental Design U*6(6

4) (Table 4). UsingMathematics 4.0 software analysis, a linear regressionequation was obtained as follows:

y 0:2872 0:007176x1 0:002945x2The negative values obtained in this equation

()0.007176; )0.002945) indicate that, the absorbance willdecrease by increasing these factors. Therefore, we chose2 mL 1:4 (v v) nitric acid for further measurement.Interference must be considered for every new method

(McMahon et al., 2006). The eect of sodium bromideand protein (BSA) as interfering agents on standardsolution was studied and the results are shown inTables 5 and 6. It revealed (Table 5) that bromine did

not interfere with the determination of chloride whenbromine content was less than 0.5% of chloride. Table 6shows that protein interfered with the detection ofchloride. Therefore, protein must be completely separ-ated with a precipitator.The calibration curves were evaluated by plotting

absorbance against the concentrations of sodium chlor-ide. The representative regression equation for thecalibration curve (Fig. 3) was Y = 0.0071 + 0.77343X(R = 0.9997, n = 9) over the concentration range from0.4 to 24 mg L)1 for sodium chloride. Based on a signalvs. noise ratio of 3, the limit of detection for thedetermination of sodium chloride was 0.2 mg L)1.The precision was determined by performing six

replicate analyses of the same solution and evaluatedby RSD of the content of sodium chloride (Table 7).Nine samples were prepared and analysed in one day orsix dierent days in order to evaluate intra-day or inter-day variations, respectively. The RSD of responses wascalculated in each case. The method was found precise

Table 2 Uniform experiment design of gelatin

Experiment

number

Concentration

(g L)1, x1)

Volume

(mL, x2) Absorbancea

1 3 3 0.211 0.008

2 3 5 0.148 0.009

3 5 7 0.113 0.010

4 5 2 0.175 0.011

5 7 4 0.099 0.006

6 7 6 0.123 0.008


Table 3 Optimization condition of gelatin for single factor trial

Gelatin concentration

3 g L)1 1.5 g L)1

Volume (mL) Absorbancea Volume (mL) Absorbancea

0.5 0.221 0.008 0.1 0.221 0.008

1 0.215 0.009 0.3 0.242 0.011

2 0.209 0.010 0.5 0.246 0.010

3 0.208 0.011 1 0.243 0.009

4 0.203 0.008 2 0.254 0.012

5 0.178 0.007 4 0.234 0.011


Table 4 Uniform experiment design of nitric acid

Number Concentration (v:v)x1 Volume (mL,x2) Absorbancea

1 (1:3) 3 0.248 0.011

2 (1:3) 5 0.247 0.010

3 (1:4) 7 0.240 0.009

4 (1:4) 2 0.258 0.012

5 (1:5) 4 0.256 0.012

6 (1:5) 6 0.253 0.011


Table 5 Effect on the determination of chloride of sodium bromide

Number

Standard

chloride (mg)

Add sodium bromide

(%Cl)) Absorbancea

1 0.4 0 0.317 0.010

2 0.4 0.01 0.312 0.009

3 0.4 0.05 0.312 0.008

4 0.4 0.1 0.315 0.011

5 0.4 0.5 0.309 0.008

6 0.4 1 0.304 0.003

7 0.4 3 0.297 0.009


Table 6 Effect on the determination of chloride of BSA

Number Standard chloride (mg) Add BSA (mg) Absorbancea

1 0.4 0 0.316 0.006

2 0.4 0.1 0.295 0.007

3 0.4 0.2 0.302 0.009

4 0.4 0.3 0.302 0.008

5 0.4 0.4 0.304 0.006




with the RSD values from 0.83% to 1.08% for intra-dayassays and 1.00% to 2.87% for inter-day assays,respectively.The mean values of the recovery percentage for each

concentration of sodium chloride are shown in Table 8.The mean recovery of spiked samples was 98.0%

(RSD = 3.11%, n = 35). These results indicate theaccuracy of the proposed method for the determinationof sodium chloride in salted sh.The accuracy of the new method as compared with

Volhard method is summarized in Table 9. A t-test wascarried out to determine the dierence at 95% cond-ence level; the calculated t (1.15) was less than criticalt0.05,8 (2.30), and so there was no signicant dierence.

Conclusions

This assay fulls the requirements of a reliable andfeasible method, including selectivity, accuracy, stabilityand limit of detection (LOD). It is a precise analyticalprocedure to analyse a large number of samples in ashort period. In addition, this method is rapid, conveni-ent and inexpensive, and it may also be suitable fordetermination of sodium chloride in other salted foodproducts.

Acknowledgment

The authors express gratitude to Nantong Lu XianAquatic Products Ltd. of China for providing support.

References

Alcazar, A., Fernandez-Caceres, P.L., Martin, M.J., Pablos, F. &Gonnzalez, A.G. (2003). Ion chromatographic determination ofsome organic acid, chloride and phosphate in coee and tea.Talanta, 61, 95101.

AOAC (1997). Ofcial method 937.09 Salt in seafood - volumetricmethod, 16th edn, p. 7. Chapter 35. Washington, DC: Association ofOcial Analytical Chemists.

Chalk, S.J. & Tyson, J.F. (1998). Determination of chloride by owinjection spectrophotometry with membrane reagent introduction.Analytica Chimica Acta, 366, 147153.

Couto, C.M.C.M., Lima, J.L.F.C. & Montenegroo, M.C.B.S.M.(1998). Construction and evaluation of a crystalline silver double-membrane tubular potentiometric detector for ow injectionanalysis. Application to chloride determination in wine. Analysis, 26,182186.

Fang, K.T. (1994). Uniform Design and Uniform Design Table. Pp. 6970. Beijing: Scientic Press.

Ismail, N. & Wootton, M. (1982). Fish salting and drying: a review.ASEAN Food Journal, 7, 175183.

Lapa, R.A.S., Lima, J.L.F.C., Perez-Olmos, R. & Ruiz, M.P. (1995).Simultaneous automatic potentiometric determination of acidity,chloride and uoride in vinegar. Food Control, 6, 155159.

McMahon, M., Regan, F. & Hughes, H. (2006). The determination oftotal germanium in real food samples including Chinese herbal

0.0

0.2

0.4

0.6

0.8

1.0

Y = 0.0071 + 0.77343X

R = 0.99971

Abso

rban

ce

NaCl (mg)0.0 0.2 0.4 0.6 0.8 1.0 1.2

Figure 3 The standard curve. Turbidity absorbance of silver chloride

of standard NaCl solution at 385 nm.

Table 7 The precision of determination sample

Sample

Number of determinationMean

(%) SD

RSD

(%)1 2 3 4 5 6

1 15.30 15.24 15.11 15.30 15.14 14.94 15.17 0.141 0.93

2 15.81 15.46 15.71 15.66 15.76 15.56 15.66 0.130 0.83

3 13.28 13.19 12.92 13.14 13.10 13.32 13.16 0.142 1.08

4 8.99 9.11 9.15 9.22 8.99 9.02 9.06 0.090 0.99

5 9.65 9.52 9.55 9.47 9.68 9.44 9.55 0.096 1.00

6 9.97 9.83 9.78 9.67 9.61 9.58 9.74 0.148 1.52

7 8.55 8.55 8.77 8.71 8.52 8.52 8.60 0.101 1.24

8 11.87 11.78 11.63 11.57 11.78 11.94 11.76 0.140 1.19

9 7.73 7.73 7.65 7.22 7.36 7.45 7.52 0.216 2.87

Table 8 Recovery of determination

Number sample 1 2 3 4 Mean (%)

1 94.70 92.78 95.68 91.80 93.74

2 96.00 93.74 96.33 94.06 95.03

3 98.91 97.94 92.45 104.40 98.43

4 100.85 102.14 100.53 101.82 101.34

5 98.26 100.20 98.59 100.53 99.40

6 96.00 98.27 94.06 96.33 96.17

7 104.41 102.47 104.73 102.79 103.60

8 95.68 96.65 98.91 99.88 97.78

9 94.71 95.68 97.29 98.26 96.49

Table 9 Comparison with Volhard method

Sample 1 2 3 4 5 6 7 8 9

Volhard method 14.96 15.89 13.16 9.20 9.72 10.69 8.28 11.85 7.71

Turbid absorbance

method

15.17 15.66 13.16 9.06 9.55 9.74 8.60 11.76 7.52

RSD% 1.39 1.46 0.00 1.53 1.76 9.30 3.79 0.76 2.49

A reliable method to determine sodium chloride in salted fish Y. Zhang and W. Xia 931

2007 The Authors. Journal compilation 2007 Institute of Food Science and Technology Trust Fund International Journal of Food Science and Technology 2008

remedies using graphite furnace atomic absorption spectroscopy.Food Chemistry, 97, 411417.

Perez-Olmos, R., Herrero, R., Lima, J.L.F.C. & Montenegro,M.C.B.S.M. (1997). Sequential potentiometric determination ofchloride and nitrate meat product. Food Chemistry, 59, 305311.

Silva, F.V., Souza, G.B., Ferraz, L.F.M. & Nogueira, A.R.A. (1999).Determination of chloride in milk using sequential injectionautomated conductimetry. Food Chemistry, 67, 317322.

Taylor, R.H. & Grate, T.W. (1995). A ow injection analysis techniquefor the determination chloride using reectance detection. Talanta,42, 257261.

Velasco-Arjona, A., Garcia-Garrido, J.A., Quiles-Zafra, R. & Luquede Castro, M.D. (1998). Full automated robotic method for thedetermination of chloride, nitrite and nitrate in curing meat product.Talanta, 46, 969976.

Xian, X.Q. & Chen, Z.H. (2003). Study on determination tracechloride in boiler water of power plant according to turbidity. NorthChina Electric Power, 2, 78. In Chinese.

Yu, Q.Q. (1991). China National Standard GB/T 12457-90. Method fordetermination of chloride sodium in foods. Beijing: Standard Press ofChina.

Zhang, G.X. & Xu, B.L. (2003). Determination of total chlorinecontent in industrial salt by miscellaneous spectrophotometry.Sea-lake Salt and Chemical Industry, 32, 2022. In Chinese.

Zhou, M.S. & Guo, D.L. (2000). Simultaneous determination ofchloride, nitrate and sulphate in vegetable samples by single-columnion chromatography. Microchemical Journal, 65, 221226.

Zugarramurdi, A. & Lupin, H.M. (1980). A model to explainobserved behaviour on sh salting. Journal of Food Science, 45,13051311.



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