AustinPeayStateUniversityDepartmentofChemistry

CHEM1021

ISOLATIONOFCASEINFROMMILK
Purpose:Inthislabyouwillbeisolatingtheproteinscaseinandlactalbuminfromasampleofmilk.You
willusethesevaluestodeterminethepercentproteininmilkcomparedtothelistedvalueonthebox.
Caution:Weareyeprotectionbecauseofthepossibleshatteringofdroppedglasswareandbecause
10%aceticacidisusedinthisexperiment.Ifanyofthisacidcomesincontactwithyourskin,washitoff
immediatelywithplentyofwater.Also,avoidbreathingvaporsofthissolutionandofcourse,donot
drinkanyofthesolutionsinthisexperiment.
Introduction:Milkisacomplexbiologicalmixtureofchemicalsthatservesastheprimaryfoodsource
forinfantmammals.Milkcontainsmostofthebiologicalmoleculesnecessarytosustainlifeincluding
water, a variety of vitamins, minerals, proteins, sugars, and lipids (DNA, although necessary for life, is
made by your body and therefore is not needed on a dietary level). Although most mammals stop
drinkingmilkuponmaturity,many(butnotall)humanculturescontinuedrinkingmilkandconsuming
milkproducts(forexample,cheese,butter,andcream)throughouttheirentirelife.

Itwouldbequitechallengingtoisolateeverycomponentofmilkbecausesomeofthechemicals
are present in very small quantities. You can very easily, however, isolate the most abundant
componentsfromeachother.Acommonexampleofthis
Table1.Milkcomponents(%composition)
isremovingthefatfromcowsmilktomakeskimmilkor

Cow
Human
Goat
removingthesugarfrommilktomakelactosefreemilk.
Water
87.8
87.4
87.0
Table 1 shows the average composition of milk from
Protein
3.0
1.4
3.3
Lipids
3.9
4.0
4.2
several mammals consumed by humans. A small
Sugars
4.6
7.0
4.8
discussion of the components of milk is warranted and
Minerals
0.7
0.2
0.7
describedbelow.
Lipids:

If you were asked if fat mixes with water, chances are you would say no. However, milk
contains around 4% fat in water. This means that in 100 mL of water, about 4 mL of fats would be
present. This is possible because milk contains, in addition to fats, a number of lipids called
phospholipids.Thesemoleculesaresimilartothefattyacidsyoustudiedinclass,buthaveanegatively
chargedphosphategroupattachedtooneend(Figure1).

Figure 1. Example phospholipid

O
P

hydrophobic ( water hating) area

hydrophilic ( water loving) area

The negatively charged phosphate group, like other charged, polar groups, is quite soluble in
water. The nonpolar, uncharged hydrocarbon area is completely insoluble in water. The dual

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AustinPeayStateUniversityDepartmentofChemistry

CHEM1021

ISOLATIONOFCASEINFROMMILK
personality of the phospholipid
(chemists would call it
amphipathic)letsthemilkfatbe
soluble in the milk water by
aligning a number of its
hydrophobic areas with the
other fats, with the phosphates
facing out toward the water
(Figure2).Thismakesamixture
called an emulsion, meaning a
fat dissolved in water by means
of an amphipathic molecule, called an emulsifying agent (soaps are emulsifying agents that work to
dissolvegrease).Inthislab,youwillbeusingpowderedmilkthathasalreadyhadthefatremoved.
Proteins:
There are three main proteins in milk; casein (casein), lactalbumin (lactalbumin), and
lactoglobulin (lactoglobulin). In this lab you will be isolating the casein, and a mixture of the
lactalbumin and lactogobulin proteins (they isolate under the same conditions and are difficult to
separatefromeachother).

Chancesareyouhaveconsumedcaseinwithoutknowingit.Caseincanprecipitatefromwater
whenitsnormalstructureisdestroyed.Wecallthisprocessproteindenaturation.Thecurdsincottage
cheeseareprecipitatedcaseinprotein(theenzymerenninisusedinthiscasetoprecipitatetheprotein)
andtheIndiancheesepaneerismadebyaddinganacidtomilktoprecipitatethecurds.Manycheeses
aremadebyprecipitatingcaseinandremovingitfromtheleftoverliquid,thewhey.Thelactalbumin
proteins are easily precipitated by heating. After casein proteins are removed, heating the whey
solutioncanprovidethesolidprotein.Althoughaminorcomponent,thelactoglobulinproteinsarethe
immuneproteinspresentinmilkthatprotectababyfromillnessuntilitcandevelopitsownimmune
system.
Sugars:

Themaincarbohydratepresentinmilkisthesugarlactose.Lactoseisadisaccharidecontaining
the monosaccharides galactose and glucose. Mammals produce an enzyme called lactase that breaks
the disaccharide into its monomers during digestion. Many mammals stop producing lactase after
maturity leading them to be lactose intolerantthey lack the ability to digest the sugar in milk.
Althoughmostpeoplecandigestlactoseuponmaturity,therearemanywhocant.Thesepeoplecan
buylactosefreeproducts,orbuytheenzymesupplementlactasetoaidindigestionofthesugar.

Onapracticalnote,whenbacteriagetintomilk,theydigestthelactoseandformtheacidlactic
acid.Thiscausesaprecipitationoftheproteincasein.Thisiswhathappenswhenoldmilksours.

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CHEM1021

ISOLATIONOFCASEINFROMMILK
Equipment:
10%aceticacidsolution

spatula

filterflask
10mLGraduatedcylinder

disposablepipettes

filterpaper
Powderedmilk

nylonhose

Buchnerfunnel

Procedure:
Caseinisolation
1. Placeabout4gofpowderednonfatmilkand42mLofwaterintoabeakerandrecordthemass.
Heatthemilkto40oC.
2. Whenthemilkhasreached40 oC,startaddingtheaceticacidsolution5dropsatatime.After
every5drops,stirthesolutionwitha spatula.Youwillseeawhitesolidforming.Thisisthe
casein.Pushthecaseintothesideofthebeakerabovethesolutionandpushtheliquidoutand
tocompresstheprotein.Transfertoawatchglassasyougo.Stopafter20dropsoftheacetic
acid(1mL).Ifyouseeanyliquidonthewatchglass,pipetteitbackintothebeaker.
3. Add0.2gofcalciumcarbonatetothemilksolutioninthebeakertoneutralizethepH.
4. Transferthecaseintoasectionofnylonhose.Wraptheproteinandgentlysqueezeoverthe
topofthebeakertoremoveasmuchofthemilksolutionaspossible.
5. Weighaplasticboatandrecord.Addthecaseinandrecordthemassonthedatasheet.
Lactalbuminisolation
6. Heatthemixtureto75 oCfor5minutes.Thisprecipitatesthelactalbuminprotein(andanyof
thelactoglobulinproteininthemilk).
7. Useafilterfunnel,flask,andfilterpapertoseparatethesolidprecipitatefromthehotsolution.
8. Transferthelactalbumintoaweighboatandrecordmassondatasheet.

WasteDisposalandCleanup:Properlydisposeofallsolutionsandsolidsaccordingtoyourlab
instructor.Thoroughlywashandrinseallglasswareandreturntotheiroriginalplaces.

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CHEM1021

ISOLATIONOFCASEINFROMMILK

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CHEM1021

ISOLATIONOFCASEINFROMMILK

DATASHEET

Name:______________________________________
Partner:_____________________________________

1. Exactmassofdriedmilk

_________________g

2. Massofisolatedcasein

_________________g

3. Massoflactalbumin

_________________g

Usingtheboxofmilktofindnumberswecanworkwithtocheckqualitycontrol:
4. Massofproteinlistedperservingonmilkbox

_________________g/serving

5. Volumeofreconstitutedmilkperserving(inmL)

_________________mL/serving

6. Convertthevolumeofmilk(in#5)tothemassofreconstitutedmilk.
Assumethedensityofmilkis~1g/mL.

7. Percentproteininareconstitutedmilksample

#
#

100

_________________g

_________________%

Compareyourdatatothelistednutritionalinformationyoudeterminedabove:
8. Totalmassofproteininyoursample(#2+#3)

_________________g

9. Findthepercentoftotalproteininyoursample

_________________%

100
10. Findthepercentofcaseininyoursample

_________________%

11. Findthepercentoflactalbumininyoursample

_________________%

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CHEM1021

ISOLATIONOFCASEINFROMMILK

POSTLABQUESTIONS
Name:__________________________________

Partner:________________________________

1.Percenterror:Determinetheerrorinthisexperimentbyusingthefollowingequation:

100%

_________________%

2.Assumingyourexperimenthadsomeleveloferror(inotherwords,youhaveanonzero
answerfor#1),whatdoyouthinkcouldbeareasonforyourerror?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

3.Whatdoestheaceticaciddothestructureofcaseinthatcausesittoprecipitate?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

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CHEM1021

ISOLATIONOFCASEINFROMMILK

PRELABASSIGNMENT

Name:_________________________________

1. Whatiscasein?
__________________________________________________________________________________
__________________________________________________________________________________
2. Whatisthepurposeoflactoglobulin?
__________________________________________________________________________________
__________________________________________________________________________________
3. Whatisanemulsion?
__________________________________________________________________________________
__________________________________________________________________________________
4. Whatisanexampleofanemulsifyingagent,otherthanphospholipids?___________________

5. Ifyouarelactoseintolerant,whatenzymeareyoulacking?
__________________________________________________________________________________
__________________________________________________________________________________
6. Whatarethemonomersthatmakeuplactose?_______________________________________

7. Whydoesmilkspoil?Beasspecificaspossible.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________

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