Determination of calcium and magnesium in milk
Determination of calcium and magnesium in milk
Determination of calcium and magnesium in milk
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Thermo. Titr. Application Note No.Title:H-103<strong>Determ<strong>in</strong>ation</strong> <strong>of</strong> Calcium <strong>and</strong> Magnesium <strong>in</strong>MilkScope:<strong>Determ<strong>in</strong>ation</strong> <strong>of</strong> <strong>calcium</strong> <strong>and</strong> <strong>magnesium</strong> <strong>in</strong> <strong>milk</strong>.Pr<strong>in</strong>ciple:A measured amount <strong>of</strong> <strong>milk</strong> is treated with trichloroaceticacid to coagulate <strong>milk</strong> solids <strong>and</strong> liberate <strong>calcium</strong> <strong>and</strong><strong>magnesium</strong> as dissociated ion. The coagulated <strong>milk</strong> isfiltered or centrifuged, <strong>and</strong> an aliquot <strong>of</strong> the clear serum istitrated with a st<strong>and</strong>ard solution <strong>of</strong> 1mol/L tetra-sodiumEDTA to thermometrically determ<strong>in</strong>ed endpo<strong>in</strong>ts for Ca<strong>and</strong> Mg. Acetylacetone is added to alter the Ca- <strong>and</strong> Mg-EDTA stability constants for better endpo<strong>in</strong>t sharpness.Ca 2+ + Y 4- CaY 2- (Y = EDTA)Mg 2+ + Y 4- MgY 2-Thermodynamic Constants:Heat <strong>of</strong> chelation, Ca 2+ with EDTA: H r -23.4 KJ/molHeat <strong>of</strong> chelation, Mg 2+ with EDTA: H r +20.1 KJ/molThe reaction with Ca 2+ is thus exothermic, <strong>and</strong> that <strong>of</strong> Mg 2+endothermic.Stability Constants <strong>of</strong> EDTA complexes (as log K):Ca 2+ = 10.7, Mg 2+ = 8.7Reagents:3.1. c(Na 4 EDTA)=1mol/L. Prepare from A.R. tetrasodiumEDTA. Alternatively, weigh 372.24 g A.R. Na 2 H 2 EDTA <strong>and</strong>quantitatively transfer to a 1000mL volumetric flask. 80gA.R. NaOH is carefully dissolved <strong>in</strong> 500mL D.I. water,cooled, <strong>and</strong> added to the same flask. When all solids havedissolved (with the addition <strong>of</strong> more water), make tovolume <strong>and</strong> mix well. The EDTA solution may best<strong>and</strong>ardized aga<strong>in</strong>st a c(Mg 2+ ) = 0.2 mol/Lsolution,prepared from Mg metal accord<strong>in</strong>g to AN H-075.3.2. NH 3 /NH 4 Cl buffer. Dissolve 17.5 g A.R. NH 4 Cl <strong>in</strong> 172mL A.R. conc. (28%) NH 3 soln. <strong>and</strong> make to 250 mL withdeionised water.3.3. Acetylacetone (2, 4-Pentanedione). CAS 123-54-6,Aldrich cat. no. P7754.3.4. Trichloroacetic acid, 25% w/v <strong>in</strong> D.I. water1
Method:Basic Experimental Parameters:Titrant delivery rate (mL/m<strong>in</strong>.) 4ERC Ca EP -15ERC Mg EP +5Data smooth<strong>in</strong>g factor (DSF) 15Stirr<strong>in</strong>g speed (802 stirrer) 15Delay before start <strong>of</strong> titration (secs.) 10Prepare an aliquot <strong>of</strong> serum <strong>of</strong> the <strong>milk</strong> product bypipett<strong>in</strong>g 100 mL <strong>of</strong> <strong>milk</strong> <strong>in</strong>to a 250 mL beaker equippedwith a large magnetic sp<strong>in</strong> bar. Set on a magnetic stirrer,<strong>and</strong> slowly add by bulb pipette 10 mL 25% w/vtrichloroacetic acid soluton. Allow to stir for 10 m<strong>in</strong>utes.Separate clarified <strong>milk</strong> serum either by filtration through aWhatman no. 4 filter paper or by centrifugation. Pipette 50mL <strong>of</strong> <strong>milk</strong> serum <strong>in</strong>to a titration vessel <strong>and</strong> add 800 µLacetylacetone. Prior to the titration with Na 4 EDTA, 5 mL <strong>of</strong>NH 3 /NH 4 Cl buffer is added automatically by Dos<strong>in</strong>o. Thevolume <strong>of</strong> orig<strong>in</strong>al <strong>milk</strong> conta<strong>in</strong>ed <strong>in</strong> a 50 mL aliquotdelivered for titration is 100*50/(100+10)=45.4545 mLFull cream<strong>milk</strong>Low fat <strong>milk</strong>Skim <strong>milk</strong>with <strong>milk</strong>solids <strong>and</strong><strong>milk</strong> <strong>calcium</strong>NutritionInformation onlabelCa mg/100 mLTitrotherm Camg/100 mLTitrotherm Mgmg/100 mL117 145 175125.9±0.50(n=5)13.2±0.41(n=5)147.0±0.44(n=9)14.8±0.59(n=9)209.7±0.45(n=8)14.5±0.33(n=9)Calculations:Ca mg/100 mL =((EP1-Ca blank)*M EDTA*40.078*100)/sample vol., mLMg mg/100 mL = ((EP2-EP1-Mg blank)*M EDTA*24.305*100)/sample vol,mL2
<strong>Determ<strong>in</strong>ation</strong> <strong>of</strong> Ca <strong>and</strong> Mg blanksThe method blank is determ<strong>in</strong>ed by titrat<strong>in</strong>g differentamounts <strong>of</strong> a representative sample <strong>of</strong> the product <strong>and</strong>plott<strong>in</strong>g the sample amount aga<strong>in</strong>st the titrantconsumption. The method blank is determ<strong>in</strong>ed as the y-<strong>in</strong>tercept from a l<strong>in</strong>ear regression <strong>of</strong> the titration data.Changes <strong>in</strong> titrant dose rate or filter factor will require anew determ<strong>in</strong>ation <strong>of</strong> the method blank.In the case <strong>of</strong> this determ<strong>in</strong>ation, it is necessary tocompute two method blanks: one for the Ca endpo<strong>in</strong>t(EP1) <strong>and</strong> one for the Mg endpo<strong>in</strong>t (EP2). There ismutual <strong>in</strong>terference between Ca <strong>and</strong> Mg dur<strong>in</strong>g thetitration, <strong>and</strong> this must be compensated correctly.To obta<strong>in</strong> an accurate estimate for the Mg blank,separate regression analyses are performed on EP1<strong>and</strong> EP2, with the <strong>in</strong>tercept for EP1 be<strong>in</strong>g subtractedfrom that for EP2.Examples <strong>of</strong> Ca <strong>and</strong> Mg blank estimations:Full cream<strong>milk</strong>Low fat <strong>milk</strong>Skim <strong>milk</strong>with <strong>milk</strong>solids <strong>and</strong><strong>milk</strong> <strong>calcium</strong>Ca <strong>in</strong>tercept(blank), mL(1)Mg <strong>in</strong>tercept,mL (2)CorrectedMg blank(2)-(1)0.0623 0.0547 0.03530.1320 0.1329 0.16260.0697 0.0782 0.12743
Examples <strong>of</strong> titration plotsBlue curve = solutiontemperatureBlack curve = secondderivative <strong>of</strong> solutiontemperatureEP1 = <strong>calcium</strong> endpo<strong>in</strong>t(exothermic reaction,negative endpo<strong>in</strong>t“peak”)EP2 = <strong>magnesium</strong>endpo<strong>in</strong>t (endothermicreaction, positiveendpo<strong>in</strong>t “peak”)Fig. 1. Ca <strong>and</strong> Mg <strong>in</strong> full cream <strong>milk</strong>Fig. 2. Ca <strong>and</strong> Mg <strong>in</strong> low fat <strong>milk</strong>Fig. 3. Ca <strong>and</strong> Mg <strong>in</strong> skim <strong>milk</strong> with added <strong>milk</strong> solids <strong>and</strong> <strong>milk</strong> <strong>calcium</strong>Notes on safe usage <strong>and</strong> disposal <strong>of</strong> trichloroacetic acid.CCl 3 COOH (trichloroacetic acid) <strong>and</strong> its solutions are toxic <strong>and</strong> corrosive. Wearappropriate protective cloth<strong>in</strong>g. Adhere to recommendations <strong>in</strong> MSDSdocumentation. Disposal <strong>of</strong> CCl 3 COOH solutions <strong>and</strong> residues conta<strong>in</strong><strong>in</strong>g CCl 3 COOHshould be <strong>in</strong> accordance with local regulations.4