Lait (1996) 76, 433-443© Elsevier/INRA

Original article

Determination of anions of milkby ion chromatography

F Gaucheron, Y Le Graet, M Piot, E Boyaval

Laboratoire de recherches de technologie laitière,INRA, 65, rue de Saint Brieuc, 35042 Rennes cedex, France

(Received 28 February 1996; accepted 30 May 1996)

Summary - The anions of milk have been successfully analysed by ion chromatography coupledwith suppressed conductivity detection. With an appropriate sample preparation (ultrafiltration alone,or combined with dry mineralization), this method enabled simultaneous identification and quantifica-tion of chloride, phosphate and citrate ions with good repeatability (relative standard deviations ofabout 1%). Moreover, good sensibility (Iess th an 20 Ilglkg) and no interference between ions and othermatrix components were determined. It was also shown that this method olters a very promisingalternative for studying changes in the salt balance of milk du ring some technological treatments(acidification and increase of ionic strength of milk).

ion chromatography / milk / anion

Résumé - Analyse des anions du lait par chromatographie ionique - Les anions du lait ont étéanalysés par chromatographie ionique couplée à une détection par conductimétrie avec autosup-pression. Avec une préparation d'échantillon appropriée (ultrafiltration seule ou combinée avec uneminéralisation par voie sèche), cette méthode a permis l'identification et la quantification simultanéedes ions chlorure, phosphate et citrate avec de bonnes répétabilités (déviations standard relativesd'environ 1 %). De plus, de bonnes sensibilités (inférieures à 20 1l9/kg) et aucune interférence entreles ions à doser et les autres composés du milieu ont été déterminées. Par ailleurs, cette méthodeconstitue un excellent moyen pour étudier des changements d'équilibres salins durant certainstraitements technologiques comme l'acidification ou l'augmentation de force ionique de laits.

chromatographie ionique / lait / anion

INTRODUCTION phosphate associated to small molecules(pentoses, hexoses, glycerol, serine and nu-c1eotides); and 3) covalently bound to peptidechains of caseins; CXsrB, CXs2-A, 13-A2, K-Bcaseins have 8, 11,5 and 1 phosphoserylresidues per molecule respectively (Wal-stra and Jenness, 1984). In casein mi-celles, the MCP (about 50% of the inor-

The principal anions of skim milk are:- phosphate (- 2700-3000 mg/kg);- citrate (- 1500-2200 mg/kg);- and chio ride (- 1000-1100 mg/kg).

Phosphate is present as: 1) inorganicphosphate (soluble phosphate and micellarcalcium phosphate (MCP)); 2) organic

434 F Gaucheron et al

ganic phosphate) plays an important role inmaintaining the structure of casein micellesbecause these micelles are disaggregatedwhen MCP is removed (Walstra andJenness, 1984). Thus, modifications ofphysico-chemical parameters, such as pH,ionie strength and temperature, influencethe dynamic minerai equilibrium of milk andsome changes in minerai balance occur-ring during technological treatments arefundamental to a number of manufacturingprocesses. The phosphate concentrationof milk affects almost ail aspects of cheesemanufacture (Lucey and Fox, 1993). Theeffects of these physico-chemical par-ameter modifications have been investi-gated by several authors (Brulé et al, 1974,1977; Brulé and Fauquant, 1981; Walstraand Jenness, 1984; Grufferty and Fox,1985; Visser et al, 1986; van Hooydonk etal, 1986; Dalgleish and Law, 1988, 1989;Le Graet and Brulé, 1993). Moreover,chloride is totally in the aqueous phase andabout 10% of citrate is electrostaticallylinked to casein micelles.

Different methods to determine the con-centration of milk anions have been de-scribed in the literature. Usually, phos-phate, chloride and citrate ions weredetermined by the following methods:- phosphorus: colorimetrie method (FIL,1987), 31p NMR (Visser et al, 1986), capil-lary ion electrophoresis (Schmitt et al,1993);- chloride: titrimetric (FIL, 1988a (Mohrmethod)), potentiometric (FIL, 1988b) andcolorimetrie (Herrero et al, 1992) methods,capillary ion electrophoresis (Schmitt et al,1993);- citrate: chromatographie (Marsili et al,1981), spectrometrie (Pierre and Brulé,1983) and enzymatic (FIL, 1992a)methods, capillary ion electrophoresis(Schmitt et al, 1993).

Each method has its own merits, but theyare time-consuming and/or require muchmate rial; occasionally they are also sus-ceptible to interference. Moreover, with

each of these methods, except capillary ionelectrophoresis (Schmitt et al, 1993), onlyone element can be determined at a time.Forthe determination of ions, an alternativemethod is the ion chromatography. The ap-plications of this method are in the followingareas (Weiss, 1995): environmental ana-Iysis (water, soil hygiene) (Stahl, 1994),detergent and household product industry(weakly basic c1eansing agent, toothpaste,shampoo, etc), pharmaceutical industry,c1inical chemistry (blood, saliva, urine) andfood (vegetable, baby food) (Ruiz et al,1995) and beverage industry (wine, applejuice, berry juice concentrate).

This article describes the use of ion chro-matography to determine the principal anionsof milk (chloride, phosphate and citrate).Modifications of pH and ionic strength onthe salt balance of milk were also studied.

MATERIALS AND METHODS

Milk

Reconstituted milk was prepared at room tem-perature from INRA low heat skim milk powder ata concentration of 10% (w/w) in water. 0.01 %thiomersal (Sigma Chemical Co, Saint Louis, MO,USA) was added to prevent bacterial growth.

Acidification of milk

Two hours alter the reconstitution of the milk,acidification was realised by addition of 1 mol/LHCI. In order to obtain the same final volumes,the dilution effects were corrected by water ad-dition. Alter overnight storage at room tempera-ture, the pH values were measured. A range ofpH values between 6.73 and 3.93 was obtained.

Increase of ionie strength of milk

0.5, 1 and 2% (w/w) of dry NaCI was added tomilk. Alter overnight storage at room tempera-ture, the pH values were measured.

Milk anion analysis by ionchromatography

Conventional methods for mineraicontent determination

Chio ride concentration was determined bypotentiometry (chloride analyser 926-CibaCorning Diagnostics, Halstead, UK) (Fil,1988a).

Phosphorus concentration was determined ac-cording to Fil method (1987) by molecular ab-sorption spectrometry of phosphomolybdatecompounds. A conversion factor of 95/31 wasused to convert the phosphorus concentrationinto phosphate concentration.

Citric acid concentration was determined outby enzymatic method (Fil, 1992a) using citrateIyase, malate dehydrogenase, lactate dehy-drogenase and NADH,H+ (cat No 139076, Boehr-Inger, Mannheim, ·Germany). Alter enzymaticreactions, the absorbance decrease of NADH,H+which is proportional to citric acid concentration,was determined at a wavelength of 340 nm.

For each sam pie, Iwo independent measure-ments were carried out.

Ion chromatography for mineraicontent determination

A Dionex DX-500 high-performance liquid chro-matographie system (Jouy-en-Josas, France)was used.

lactate, acetate, propionate, butyrate,chloride, nitrate, succinate, carbonate, sulfate,phosphate and citrate were separated on an

435

anion exchange column (AS11 10nPac column,4 x 250 mm) fitted with a AG11 guard column.The AS11 10nPac analytical column stationaryphase was composed of a 13 urn highly cross-1inked polyethylvinylbenzene/d ivi nyl benzenesubstrate agglomerated with anion exchangelatex that had been completely aminated. Theanion trap column (ATC-1) was also used. Thissystem was positioned between the gradientpump pressure transducer and the injectionvalve. Its application was to strip anionic con-taminants such as carbonate from the hydroxideeluent and th us improve the signal to noise ratioof the analysis. Separation under linear gradientelution conditions used NaOH (from a freshbottle of 50% NaOH solution that was low in car-bonate concentration, JT Baker, Deventer, theNetherlands). The gradient elution program isreported in table 1. Solutions A, Band C were2 mmol/l NaOH, 200 mmol/l NaOH and 18-Mnwater respectively. Separations were carried outat 20 "C and at a flow-rate of 2 ml/min. Alterelution and before detection by the ED40 con-ductivity detector maintained at 35 "C, an auto-suppression external water mode was used byan anion self-regenerating suppressor (ASRS-I,4 mm). This system provided high capacity sup-pression of traditional eluents and simplified thedetection of ion chromatography because itmaximised signal to noise ratio for high sensitiv-ity analysis (Weiss, 1995).

Standard solutions of ail the investigated an-ions were prepared from 1000 mgll commercial

Table 1. Gradient elution program used for analysis of anions by ion chromatography. Eluents A, Band C were 2 mmol/l NaOH, 200 mmol/l NaOH and 18-Mn water, respectively. The completechromatographie conditions are described in the legend to figure 1.Programme du gradient d'élution utilisé pour l'analyse des anions par chromatographie ionique.Les éluants A, 8 et C sont respectivement une solution de NaOH à 2 mmollL, une solution de NaOHà 200 mmollL et de l'eau à 18 Mn. Les conditions chromatographiques complètes sont décrites dansla légende de la figure 1.

Time (min) %A %8 %C

0.00 10.0 0 90.00.10 (injection) 10.0 0 90.05.00 50.0 0 50.0

20.00 12.5 11.0 76.522.00 0.0 15.0 85.023.00 10.0 0 90.030.00 10.0 0 90.0

436 F Gaucheron et al

solutions (Merck, Darmstadt, Germany) exceptfor citric acid which was prepared from citric acidmonohydrate commercial salt (Merck). Beforeinjection with an auto-sampling device (251J.1vol-ume), suitable concentrations (0,1,2,5,10 and20 mg!kg for chio ride and citrate, and 0, 2, 5, 10,20 and 40 mg/kg for phosphate) were obtainedby dilution with 18-Mn water.

The detection limits were found by preparingdilute solutions of ions and by identifying the con-centrations of ions that gave a signal calculatedas twice the baseline noise.

ln order to estimate the repeatability (or pre-cision) of this method for each ion, relativestandard deviations (RSD) were calculated asfollows: RSD% = (0 100/m; m and 0 being themean and the standard deviation of series ofdetermination respectively.

Sample preparation

The milk aqueous phase was obtained as fol-lows: 7 ml of milk was placed in an ultrafiltrationmembrane cone (molecular mass cut-off = 25 000 Da, CF 25, Amicon, Epernon,France) and centrifuged at room temperature(1000 g for 30 min). Before ion chromato-graphy analysis, the ultrafiltrate was diluted200-fold (0.1 gin 20 ml) with 18-Mn waterandnot in solution containing NaOH to avoid car-bonation of the sample.

For the determination of total phosphate (or-ganic and inorganic) content in ultrafiltrate or inmilk, a preparation of ashes by dry mineralizationwas carried out. A dry ashing followed by a dis-solving of ashes by HN03 rather than a wet min-eralization by acid such as H2S04 was chosenbecause, in the presence of concentrated acid,the concentration of anions (S042- in this case)would be too high and could induce an overload-ing of the chromatographic column. Therefore,1 9 of sample was mineralised at 550 "C for 4hours. A previous experiment had shown that adry ashing at a temperature higher than 550 "Cinduced a loss of chloride ions probably by vola-tilisation (Fil, 1992b). The ashes obtained weredissolved in 1 ml of 1 N HN03. Then, the vol-ume was adjusted to 200 ml with 18-Mn waterbefore analysis by ion chromatography. Il isnoteworthy that the HN03 solution used does notcontain residual anions as chloride, sulfate,phosphate and citrate. Forsample storage, poly-ethylene vessels were used.

RESULTS AND DISCUSSION

General aspects

A typical chromatogram of standard anions(lactate, acetate, propionate, butyrate,chloride, nitrate, succinate, carbonate, sul-fate, phosphate and citrate) is presented infigure 1. The chromatographie peaks were

12

,20

ChIto

Sul1

Nit LJ1 1 Pho

ÎfPI:~t '. l.ue LI eltILJ'I leo 1, 'LJ' Il: l i.1 1 1 mJ,,.

Minutes

Fig 1. Chromatogram of anions (lactate (lac),acetate (Ace), propionate (Pro), butyrate (But),chloride (Chi), nitrate (Nit), succinate (Suc),carbonate (Car), sulfate (Sul), phosphate (Pho)and citrate (Cit)) of a standard mixture containing5 mg/kg of each ion except for carbonate andphosphate ions which were at concentrations of10 mg/kg. Chromatographic conditions for an-ions were as follows: eluent, NaOH under lineargradient conditions which are indicated in tablel;flow rate, 2.0 ml/min; AS11 column with anAG11 as a guardcolumn; injection volume, 251J.l;suppressed conductivity detection.Chromatogramme des anions lactate (Lac), acé-tate (Ace), propionate (Pro), butyrate (But), chlo-rure (ChI), nitrate (Nit), succinate (Suc), carbo-nate (Car), sulfate (Sul), phosphate (Pho) etcitrate (Cit) d'une solution étalon contenant5 mg/kg de chaque ion, sauf pour les ions car-bonate et phosphate, qui étaient à une concen-tration de 10 mg/kg. Les conditions chromato-graphiques étaient les suivantes: éluant, NaOHutilisé sous les conditions de gradient linéaire quisont indiquées dans le tableau 1; débit, 2,0 mL /min;colonne AS11 avec une précolonne AG Il;volumed'injection: 25 IJ.L; détection conductimétriqueavec autosuppression.

Milk anion analysis by ion chromatography

identified by injecting each ion separately.The organic acids (acetate, lactate, pro-pionate and butyrate) were partially separ-ated. The other peaks were weil resolvedand consequently the quantification stepswere easy. The difference between chro-matographic peak areas with equal molarconcentrations of different ions is related totheirequivalent ionie conductivity which arecharacteristic for each ion (GRG Handbookof chemistry and physics, 1991). Moreover,as the pH value of eluent (containingNaOH) was about 12, ail the anions wereeluted in their fully dissociated forms. It isto be noted that under these chromato-graphic conditions, the citrate moleculesmay be separated from their structuralisomer isocitrate (in milk, 1% of citrate isisocitrate; Walstra and Jenness, 1984), be-cause both molecules have different reten-tion times: 20.2 and 21.0 min respectively.

The resu Iting calibration functions ofchloride, phosphate and citrate ions(table Il) showed that the methods gaveexcellent linearity between injected con-centrations (up to 20 mg/kg for chlorideand citrate, and up to 40 mg/kg for phos-phate) and chromatographic peak areas(conductivity signais). Higher concentra-tions were not tested in order to avoidoverloading of the chromatographic col-umn and because the concentrationrange of the standard solutions usee! in thisstudy corresponded to possible concentra-tions of these ions in milk or in dairy pro-ducts. For ail ions tested, the detectionlimits were lower than 20 llg/kg (table Il). Inthis study, these values were not restrictivebecause the ion concentrations in theaqueous phase of milk are very high. Eachion contributes differently to the currenttransport and the differences in detectionlimits between each ion were due to thedifferences in their equ ivalent ionic conduc-tivity (GRG Handbook of chemistry andphysics, 1991). These detection limits wereapproximately the same as those obtainedby standard methods.

437

Anion determination in the aqueousphase of milk

Unmodified milk

Figure 2 shows anionic chromatographicprofile of diluted milk ultrafiltrate. Additionsof chloride, phosphate and citrate stand-ards (+ 50 and 100% of the initial concen-tration value) to the analysed samples indi-cated that for these samples, there was nosubstantial matrix interference because theadded concentrations were totally re-covered (results not shown).

Ch/aride and citrate concentrations

ln milk ultrafiltrate, concentrations ofchloride and citrate were 1060 and1500 mg/kg respectively. These values ob-tained by ion chromatography are normalfor an unmodified milk and were confirmedby potentiometry for chloride and by enzy-matic method for citric acid (results notshown). The RSD were 0.8% and 1.1% re-spectively (n = 6).

12

Chi1io

~S •

Minute.

Fig 2. Chromatogram of anions in the aqueousphase of milk. The ultrafiltrate was 200-folddiluted with 18-Mn water. Thecomplete chroma-tographieconditionsare described in the legendsto figure 1 and table 1.Chromatogramme des anions de la phaseaqueuse du lait. L'ultrafiltrat était dilué 200 foisavec de l'eau à 18-fvn. Les conditions chroma-tographiques complètes sont décrites dans leslégendes de la figure 1 et du tableau 1.

438 F Gaucheron et al

Table Il. Calibration parameters of chloride, phosphate and citrate ions by ion chromatography.Regression equations (y = ax + b) were calculated from injected concentration (y) as a function ofthe conductimetric signal (x) obtained. The detection Iimit was calculated as being the concentrationwhich gave a signal corresponding to twice the baseline noise. ~ corresponds to the correlationcoefficient obtained by comparison of injected concentrations and conductivity signais obtained. Thechromatographie conditions are described in the legends to figure 1 and table 1.Caractéristiques des droites de calibration des ions chlorure, phosphate et citrate parchromatographie ionique. Les équations de régression (y = ax + b) ont été calculées à partir de laconcentration injectée (y) en fonction du signal conductimétrique obtenu (x). La limite de détectionfut calculée comme étant la concentration qui donna un signal correspondant à deux foix le bruit defond. ; correspond au coefficient de corrélation obtenu par comparaison des concentrationsinjectées et des signaux conductimétriques obtenus. Les conditions chromatographiques complètessont décrites dans les légendes de la figure 1 et du tableau 1.

Time rétention a b ; Détection limit(min) (!!g/kg)

Chloride 7.2 1.01 10-5 - 0.067 0.999 -10Phosphate 17.6 3.0610.5 0.582 0.999 - 10Citrate 20.2 4.2310.5 - 0.316 0.999 - 20

Phosphate concentration

ln milk ultrafiltrate, the phosphate concen-tration was 930 mg/kg and the RSD was0.8% (n = 6). The phosphate concentrationof the same ultrafiltrate determined bycolorimetrie method (Fil, 1987) was1190 mg/kg. In fact, in ail tested samples(results not shown), differences of about250 mg/kg were always observed. An ultra-filtrate of milk contains phosphorus in manyforms. Ali phosphorus is present as ortho-phosphate, but part of it is bound to smallorganic components which are able to passthrough the ultrafiltration membrane.These phosphate groups are either esteri-fied to serine and threonine residues ofpeptides or esterified to several small mole-cules like pentoses, hexoses, glycerol,serine and nucleotides (Walstra andJenness, 1984). The phosphate concentra-tion in milk ultrafiltrate as phosphate estersranges between 150-450 mg/kg (Walstraand Jenness, 1984). This range was inaccordance with the differences found inthis study.

Thus, the organic phosphate concentra-tion of ultrafiltrate was not evaluated by

direct ion chromatography analysis. On thecontrary, the concentration of total phos-phate (inorganic + organic) obtained by dryashing of milk ultrafiltrate and ion chroma-tography (1180 mg/kg) (fig 3) was not sig-nificantly different from the concentrationobtained by Fil method (1987), ie, min-eralization in the presence of H2S04 andmolecular absorption spectrometry(1190 mg/kg). Chloride concentration wasthe same as that found before ashing(1060 mg/kg). On the other hand, in thecase of dry mineralization, citrate was de-stroyed because no chromatographie peakcorresponding to this ion was detected(fig 3). lt is noteworthy that, in the case ofthis mineralization, the small chromato-graphie peaks eluted at 3.8 and 4.6 minwere not identified but can not correspondto the acetate and butyrate ions becausethese ions are also destroyed during themineralization. The high concentration ofnitrate (about 65 000 mg/kg) (fig 3) corre-sponded to the presence of 1 ml of HN03(1 mol/lfjn 200 ml which was necessaryto dissolve the ashes obtained after dryashing and does not affect the chromato-graphie separation.

Milk anion analysis by ion chromatography

Other ions

ln milk ultrafiltrate, other small chromato-graphie peaks eluted at 3.8 and 4.6 min(fig 2) could correspond to acetate and bu-tyrate respectively. Traces of these ions(Iess than 1 mg/kg) were qualitativelydetermined by injection of ultrafiltrateadded with acetic and butyric acid solution-s. Other anionic species such as lactate,propionate, nitrate and succinate were notdetected in these ultrafiltrate samples.

Chromatographie peak eluted at 12.0 min(fig 2) could correspond to carbonate ion. Itwas identified by injection of ultrafiltrateadded with carbonate solution. It should benoted that residual carbonate was also

NI.

121

Chi 1

1 i1

1

1

1

\Pho

Sul 1

i 1 Co, 1

~I~! 1 u0

10

pS •

10 ,.Minutes

Fig 3. Chromatogram of anions in the ashes ofmilk ultrafiltrate. The ashes of ultrafiltrate wereobtained by dry mineralization at 550 "C for 4hours. Then, they were dissolved in 0.5 ml of1 mollL HN03 and the volume adjusted to200 ml, with 18-Mn water. The complete chro-matographie conditions are described in the leg-ends to figure 1 and table 1.Chromatogramme des anions de cendres d'unultrafiltrat de lait. Les cendres de l'ultrafiltrat ontété obtenues par minéralisation sèche à 550 oCpendant 4 heures. Puis elles ont été dissoutesdans 0,5 mL d'HN03 à 1 mollL et le volumeajusté à 200 ml; avec de l'eau à 18 rvn.Les conditions chromatographiques complètessont décrites dans les légendes de la figure 1etdu tableau 1.

439

detected in fresh 18-Mn water used forsample dilution and that the chromato-graphie area of this peak was approxi-mately the same as those found with theultrafiltrate. So, we can conclude that car-bonate was absent in ultrafiltrate.

The chromatographie peak eluted at 13.1min corresponded to sulfate ion (fig 2). Itwas identified by injection of ultrafiltrateadded with sulfate solution. The concentra-tion value of sulfate ions was 140 mg/kg inthe ultrafiltrate and 200 mg/kg after drymineralization followed by analysis by ionchromatography of the same ultrafiltrate(fig 3). This difference in concentrationcould correspond to the presence of sul-phur-containing compounds able to passthrough ultrafiltration membrane (thio-cyanate, amino acids such as methionineand cysteine) but which are not directly de-tectable by ion chromatography as sulfateion. One part of these compounds are prob-ably transformed in sulfate ion du ring dryashing. These values of sulfate concentra-tions are in accordance with the literature(Walstra and Jenness, 1984).

Acidification of milk

Inorganic species in the aqueous phasesof the milk acidified at different pH values(between 6.73 and 3.93) by HCI weredetermined by ion chromatography. For ailsamples and in spite of the high number ofprotons and chloride ions, no change inretention times and in chromatographiepeak resolutions was observed.

The inorganic phosphate concentrationsin the milk aqueous phases increased dur-ing acidification (fig 4). At the pH value of6.73, the inorganic phosphate concentra-tion was 930 mg/kg. Then, at the pH valueof about 5, 1870 mg/kg of inorganic phos-phate were determined and showed a sup-plementary solubilization of 50% of totalinorganic phosphate. The increase in inor-ganic phosphate concentration in theaqueous phase was related to a protona-tion of inorganic phosphate and of acidic

440 F Gaucheron et al

groups of caseins (carboxyls, phosphatesof phosphoseryl residues) and con se-quently corresponded to a direct solubiliza-tion of MCP. At the pH value of 3.94, elec-trostatic binding of inorganic phosphate tocaseins is possible because at this pHvalue, caseins are positively charged andcan bind negative ions such as phosphate.Thus, to determine the concentration oftotal inorganic phosphate, it was necessaryto carry out ultrafiltration of acidified milk ata pH value close to 5. Moreover, at this pHvalue, the ultrafiltration was easier than atother pH values because caseins are pre-cipitated.

ICI! g/kg

3.4

2.9

2.4

1.9

1.4

0.9 0.9U 4 U 5 U 6 U 7

pH

Fig 4. Effect of acidification on the anion concen-trations: chio ride (_), phosphate (+) and citrate(*), in the aqueous phase of milk. A range of pHvalues between 6.73 and 3.93 was obtained byHGI addition. These ion concentrations weredetermined by ion chromatography. The chro-matographic conditions are described in the leg-ends to figure 1 and of table 1.Effet de J'acidification sur la concentration desanions chlorure (II), phosphate (+) et citrate (*)contenue dans la phase aqueuse de lait. Unegamme de pH entre 6,73 et 3,93 a été obtenuepar addition d'HGI. Les concentrations des ionsont été déterminées par chromatographieionique. Les conditions chromatographiquescomplètes sont décrites dans les légendes de lafigure 1 et du tableau 1.

At the same time, in the pH range 6.73-3.93, the citrate concentration increasedslightly. At the pH value of 6.73, the citrateconcentration was 1490 mg/kg (100%). Atthe pH value of 4.71, 1650 mg/kg of citratewere present in the aqueous phase of milk.This value corresponded to a 10% solubili-zation. The significant decrease in the ci-trate concentration between pH 4.41(1650 mg/kg) and pH 3.93 (1590 mg/kg)probably corresponded to the binding ofnegatively charged citrate molecules topositive charges of caseins. Thus, to deter-mine the concentration of total citrate, itwas necessary to carry out ultrafiltration ofacidified milk at a pH value close to 5.

The high and linear increase in chlorideconcentration (from 1052 to 3270 mg/kg)corresponded to the HCI addition necess-ary for the acidification.

These results obtained by ion chromate-graphy were in accordance with thoseobtained by conventional methods used inthis work (results not shown) and agreedweil with the literature (Walstra andJenness, 1984; van Hooydonk et al, 1986;Dalgleish and Law, 1988, 1989; Le Graetand Brulé, 1993).

1.7

1.5

1.3

1.1

Increase of ionic strength of milk

Anionic species in aqueous phase NaCIadded milk were determined by ion chro-matography (fig 5). For ail samples and inspite of the high concentration of sodiumand chloride ions, no change in retentiontimes and in chromatographic peak resolu-tions was observed.

The inorganic phosphate concentrationsof the aqueous phase were slightly in-creased (from 930 to 1000 mg/kg) afterNaCI additions (up to 2%). At the sametime, increases of soluble calcium concen-trations were described and may be due toan exchange of sodium for calcium whichwas attached directly to the phosphoserylresidues of caseins (Brulé et al, 1974;Grufferty and Fox, 1985; Le Graet andBrulé, 1993). These slight increases in in-

Milk anion analysis by ion chromatography

organic phosphate concentrations wereprobably due to slight pH decreases whichoccurred after additions of NaCI. pH valueswere 6.73, 6.68, 6.66 and 6.62 for NaCIaddition of 0, 0.5, 1 and 2% (w/w) respec-tively.

The citrate concentration of the aqueousphase was not affected by the addition ofNaCI.

The high and linear increase of chlorideconcentration (from 1060 to 12 830 mg/kg)corresponds to the NaCI addition (0, 0.5, 1and 2%, w/w).

These results obtained by ion chromato-graphy were in accordance with those

ICII g/kg Icttl, !P04! g/kg13 ;"::"::-~:"'--------- ~1.6

11

9

7

5

3

1 L- __ '-----_-.--J'-_-.--J __ --'-----"0.9

o 0.5 1.5

NaCI % (w/w)

Fig 5. Effect of ionic strength on the anion con-centrations (chio ride (_), phosphate (+) andcitrate (*)) in the aqueous phase of milk. A rangeof added NaCI between 0 to 2% (w/w) wasadded. The ionic concentrations were deter-mined by ion chromatography. The completechromatographie conditions are described in thelegends to figure 1 and table 1.Effet de la force ionique sur les concentrationsdes anions de la phase aqueuse de lait chlorure(II), phosphate (+) et citrate (*). Une gamme deNaCI allant de 0 à 2% (p/p) a été ajoutée au lait.Les concentrations des ions ont été déterminéespar chromatographie ionique. Les conditionschromatographiques complètes sont décritesdans les légendes de la figure 1et du tableau 1.

441

obtained by conventional methods used inthis work. Moreover, these results weresimilar to those obtained by other authors(Grufferty and Fox, 1985; Le Graet andBrulé, 1993).

Anion determination in milk

To determine the total phosphate (inor-ganic + organic) concentration by ion chro-matography, a chromatographie injectionof milk without prior preparation of samplewas not suitable. So, one solution was tocarry out a dry ashing of the milk, as carriedout with the ultrafiltrates, before analysis byion chromatography. Results obtained withthis method (2770 mg/kg) indicated that itwas possible to prepare sample in this waybecause the total phosphate concentrationthus obtained was similar to those obtainedby the FIL method (1987) (2720 mg/kg).The chloride concentration of 1060 mg/kgwas the same as those found in ultrafiltrate.The sulfate concentration was 390 mg/kg(against 140 and 200 mg/kg in an ultrafil-trate and in ashes of ultrafiltrate respec-tively). These differences were probably re-lated to a transformation of sulphurcompounds into sulfate ions du ring dryashing. However, as observed in the caseof dry mineralization of milk ultrafiltrate, nochromatographie peak of citrate was de-tected because it was destroyed du ring thedry mineralization.

1.5

1.4

1.3

1.2

1.1

CONCLUSION

Seve rai methods have been developed fordetermination of anions of milk (Marsili etal, 1981; Pierre and Brulé, 1983; Walstraand Jenness, 1984; Visser et al, 1986; FIL,1987, 1988a, 1988b, 1992a; Herrero et al,1992; Schmitt et al, 1993). In this study, ionchromatography in combination with anadequate sample preparation (table III) hasbeen successfully used for the qualitativeand quantitative determinations of anions(chloride, phosphate and citrate) of milk.

442 F Gaucheron et al

Table III. Sam pie preparations before ion chromatography of milk anions.Préparations d'échantillon avant chromatographie ionique des anions du lait.

Anion determinations

Ultrafiltration of milk

Sample preparations

Chloride, inorganic phosphate", and citrate"concentrations in the aqueous phase

Chloride and total (inorganic and organic)phosphate concentrations in the aqueous phase

Chloride and total (inorganic and organic)phosphate concentrations of milk

Dry mineralization of milk ultrafiltrate''

Dry mineralization of milk sampte?

a The concentrations of total inorganic phosphate and of total citrate can be determined alter ultrafiltration of acidifiedmilk at a pH value close to 5. b Citrate determination can not be determined.a Les concentrations du phosphate inorganique total et du citrate total peuvent être déterminées après ultrafiltrationde lait acidifié à une valeur de pH d'environ 5. b La concentration de citrate ne peut être déterminée.

Also, beside rapid quantification of theseions, ion chromatography provides apowerful method for analysing the mineraitransformations undergone by milk duringtechnological treatments such as acidifica-tion or increase in ionic strength. Evenafter 4000 analyses, no change in retenti ontimes or peak resolutions was observed.

One of the most obvious advantages ofthis technique is that multiple elements canbe determined in one sample with no seriaidilutions and the complete analysis can beperformed by using only one instrument.The total time for each analysis was lessthan 32 min per sample.

Moreover, with the auto-sampling device,about 45 samples can be analysed in asingle day. Conventional methods such aspotentiometric, colorimetrie and enzymaticmethods do not have these advantages.This method can be used for analysis incontrol laboratories in the dairy industryand in other food and drink sectors as de-scribed by Weiss (1995).

The potential of this method for quantita-tive applications appears to be extraordi-nary because it seems possible to charac-terise:

- 1) the minerai composition of differentdairy products (milks, caseinates, wheyproducts, purified milk proteins, yoghurts,cheeses);- 2) the concentrations of the main anionsin the aqueous phase of milk during tech-nological processes such as thermal treat-ments, acidification, membrane separ-ation;- 3) the progress of biochemical reactionsby microorganisms during cheese ripeningby measurement of the organic acid con-tents such as lactate, acetate, propionate,butyrate and succinate. However, furtherwork is necessary to improve the chroma-tographie resolution of lactate, acetate,propionate and butyrate peaks.

ACKNOWLEDGMENTS

We thank JI. Maubois for his help in this workand for the improvement of this manuscript.

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