Table Of ContentKamal Gandhi · Neelima Sharma
Priyae Brath Gautam · Rajan Sharma
Bimlesh Mann · Vanita Pandey
Advanced Analytical
Techniques in
Dairy Chemistry
S P H
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Advanced Analytical Techniques
in Dairy Chemistry
Kamal Gandhi
Dairy Chemistry Division, National Dairy Research Institute, Karnal, India
Neelima Sharma
National Referral Center for Milk Quality and Safety, National Dairy Research Institute, Karnal, Haryana,
India
Priyae Brath Gautam
Dairy Chemistry Division, National Dairy Research Institute, Karnal, Haryana, India
Rajan Sharma
Dairy Chemistry Division, National Dairy Research Institute, Karnal, Haryana, India
Bimlesh Mann
Dairy Chemistry Division, National Dairy Research Institute, Karnal, Haryana, India
Vanita Pandey
Quality and Basic Sciences, Indian Institute of Wheat and Barley Research, Karnal, Haryana, India
KamalGandhi NeelimaSharma
DairyChemistryDivision NationalReferralCenterforMilkQualityandSafety
NationalDairyResearchInstitute NationalDairyResearchInstitute
Karnal,India Karnal,Haryana,India
PriyaeBrathGautam RajanSharma
DairyChemistryDivision DairyChemistryDivision
NationalDairyResearchInstitute NationalDairyResearchInstitute
Karnal,Haryana,India Karnal,Haryana,India
BimleshMann VanitaPandey
DairyChemistryDivision QualityandBasicSciences
NationalDairyResearchInstitute IndianInstituteofWheatandBarleyResearch
Karnal,Haryana,India Karnal,Haryana,India
ISSN1949-2448 ISSN1949-2456 (electronic)
SpringerProtocolsHandbooks
ISBN978-1-0716-1939-1 ISBN978-1-0716-1940-7 (eBook)
https://doi.org/10.1007/978-1-0716-1940-7
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Contents
AbouttheAuthors ........................................................... xiii
Abbreviations ............................................................... xv
1 BasicLaboratorySkills................................................... 1
1 LaboratorySafety....................................................... 1
2 Validation.............................................................. 2
3 BasicToolsandOperations.............................................. 4
3.1 ElectronicWeighingBalance.......................................... 4
3.2 pHStripsandpHMeter ............................................. 6
3.3 VolumetricLaboratoryEquipment.................................... 7
3.4 Titration ............................................................ 8
4 LaboratoryWasteManagement.......................................... 8
References ............................................................... 9
2 Chromatography........................................................ 11
1 ChromatographicParameters............................................ 11
2 TypesofChromatography............................................... 15
2.1 PaperChromatography............................................... 16
2.2 ThinLayerChromatography(TLC)................................... 16
2.3 ColumnChromatography............................................ 17
2.4 AdsorptionChromatography ......................................... 18
2.5 SizeExclusionChromatography(SEC)................................ 18
2.6 IonExchangeChromatography(IEC) ................................ 21
2.7 AffinityChromatography............................................. 22
2.8 GasChromatography ................................................ 34
2.9 High-PerformanceLiquidChromatography(HPLC) .................. 57
References ............................................................... 83
3 Centrifugation.......................................................... 85
1 Principle............................................................... 85
1.1 BuoyantForce....................................................... 87
1.2 FrictionalForce...................................................... 87
1.3 DerivationofStokes’Law............................................ 87
1.4 SedimentationCoefficients ........................................... 88
2 ClassificationofCentrifuges............................................. 88
2.1 DesktopClinicalCentrifuges ......................................... 88
2.2 High-SpeedCentrifuges.............................................. 89
2.3 Microcentrifuge...................................................... 90
2.4 VacuumCentrifuge/Concentrators ................................... 90
2.5 Ultracentrifuge ...................................................... 90
2.6 Instrumentationofanultracentrifuge:................................. 92
3 TypesofRotors........................................................ 93
3.1 FixedAngleRotors .................................................. 93
3.2 VerticalTubeRotors................................................. 93
v
vi Contents
3.3 Swinging-BucketRotors.............................................. 94
4 PreparativeCentrifugation .............................................. 94
4.1 DifferentialGradientCentrifugation .................................. 95
4.2 DensityGradientCentrifugation...................................... 95
4.3 AnalyticalCentrifugation............................................. 98
5 ApplicationsofCentrifuge............................................... 100
6 MicellarCasein(MC)PreparationbyUltracentrifugation .................. 101
References ............................................................... 102
4 PolyacrylamideGelElectrophoresis ....................................... 103
1 Introduction:Principle,Components,andGelMedia...................... 103
2 SamplePreparationandBufferSystems................................... 105
3 TypesofGelElectrophoresisCommonlyUsedforMilkProteinSeparation... 106
3.1 NativePAGE........................................................ 106
3.2 UreaPAGE.......................................................... 108
3.3 SodiumDodecylSulfate–PolyacrylamideGelElectrophoresis
(SDSPAGE) ........................................................ 108
3.4 TricinePAGE........................................................ 110
3.5 IsoelectricFocusing(IEF)............................................ 111
3.6 Two-DimensionalGelElectrophoresis(2D-GE)....................... 112
4 VisualizationandDetectiondetection..................................... 113
5 ChemistryofMilkProteinsUnderElectrophoresis ........................ 114
6 ApplicationsinDairyScience............................................ 115
7 Tricine-SDS-PAGEProtocol ............................................ 116
7.1 Reagents ............................................................ 116
7.2 GelPreparation...................................................... 117
7.3 SamplePreparation .................................................. 117
7.4 Electrophoresis ...................................................... 117
7.5 GelStaining......................................................... 118
References ............................................................... 120
5 WesternBlotting........................................................ 121
1 Introduction:PrincipleandComponents................................. 121
2 ComponentsofWesternBlotting........................................ 123
3 VisualizationandDetection ............................................. 125
3.1 VisualizationofProteinMarkers...................................... 125
3.2 VisualizationofProteinsintheSamples............................... 125
4 ApplicationsinDairyScience............................................ 129
References ............................................................... 129
6 MembraneProcesses .................................................... 131
1 BasicPrinciple.......................................................... 131
2 AdvantagesofMembraneSeparationProcess.............................. 133
3 DrawbacksofMembraneTechnology .................................... 133
4 PrincipalTypesofMembranes........................................... 134
4.1 IsotropicMembrane ................................................. 134
4.2 AnisotropicMembranes.............................................. 135
4.3 InorganicMembranes................................................ 136
Contents vii
4.4 GasSeparation/Permeation .......................................... 136
5 MembraneProcesses.................................................... 136
5.1 ReverseOsmosis..................................................... 137
5.2 Nanofiltration ....................................................... 138
5.3 Ultrafiltration........................................................ 138
5.4 Microfiltration....................................................... 138
5.5 Dialysis.............................................................. 139
5.6 GasPermeation...................................................... 139
5.7 Pervaporation........................................................ 140
5.8 Electrodialysis ....................................................... 141
5.9 LiquidMembranes................................................... 141
6 FractionationofMilkProteinsbyUltrafiltration........................... 143
6.1 Principle............................................................. 143
6.2 Materials ............................................................ 144
6.3 Procedure........................................................... 144
References ............................................................... 145
7 Potentiometry.......................................................... 147
1 Principle............................................................... 148
2 PotentiometricElectrodes............................................... 149
2.1 MetallicElectrodes................................................... 149
2.2 MembraneElectrodes................................................ 149
3 pHMeterandMeasurementofpH ...................................... 154
4 BufferSolutions........................................................ 157
4.1 pHofaBuffer....................................................... 157
5 MeasurementofpHofMilkandWheySample............................ 157
5.1 Principle............................................................. 157
5.2 MaterialsandReagents............................................... 157
5.3 GuidelinestobeFollowedWhileOperatingthepH/IonMeter......... 157
5.4 Standardization/CalibrationofpHMeter............................. 158
5.5 MeasuringpHoftheSample ......................................... 158
6 ApplicabilityofISEforMeasuringtheIonicCalciuminSkimMilk.......... 158
6.1 Principle............................................................. 158
6.2 PreparationofCalibrationCurve...................................... 159
6.3 MaterialsandReagent................................................ 159
6.4 Procedure........................................................... 159
References ............................................................... 160
8 Spectroscopy ........................................................... 161
1 Principle............................................................... 163
2 Beer’sLaw............................................................. 165
3 LimitationstoBeer’sLaw............................................... 165
3.1 FundamentalLimitations............................................ 166
3.2 ChemicalLimitations ............................................... 166
3.3 InstrumentalLimitations............................................ 166
4 FactorsInfluencingtheAbsorptionSpectraofChromophores.............. 166
5 EnergyLevelsinAtomsandMolecules................................... 167
6 ComponentsofUV-VisSpectrophotometer .............................. 168
6.1 SourcesofElectromagneticRadiation................................ 168
viii Contents
6.2 SampleHoldingCell................................................ 170
6.3 Detectors........................................................... 170
6.4 SignalProcessors ................................................... 171
7 Single-BeamvsDouble-BeamSpectrophotometer......................... 172
8 FluorescenceSpectroscopy .............................................. 173
9 DeterminationofAbsorptionSpectrumofBovineSerumAlbumin(BSA) ... 174
9.1 Principle ........................................................... 174
9.2 MaterialsandReagents.............................................. 174
9.3 Procedure[5] ...................................................... 174
10 VerificationofBeer’sLawUsingBovineSerumAlbumin(BSA) ........... 174
10.1 Principle.......................................................... 174
10.2 MaterialsandReagents............................................. 174
10.3 Procedure......................................................... 175
11 EffectofpHontheλ ,Absorbance(A)andAbsorbtivity
max
ofp-NitrophenolSolution[5].......................................... 175
11.1 Principle.......................................................... 175
11.2 MaterialsandReagents............................................. 175
11.3 Procedure......................................................... 175
11.4 ObservationsandCalculations...................................... 176
References ............................................................... 176
9 Infrared(IR)Spectroscopy............................................... 177
1 InfraredRegionoftheElectromagneticSpectrum......................... 177
2 MolecularVibrations ................................................... 179
2.1 InfraredActivity .................................................... 179
3 FactorsAffectingAbsorptionofFrequency ............................... 180
4 RegionsofIRSpectra................................................... 181
4.1 TheFingerprintRegion............................................. 181
4.2 FunctionalGroupRegion........................................... 181
5 ComparisonofSpectraofAlkanes,Alkene,andAlkyne..................... 182
6 DispersivevsFourierTransformInstruments.............................. 183
6.1 DispersiveInstruments.............................................. 183
6.2 FourierTransformInfraredSpectroscopy............................. 184
6.3 ComponentsofFTIRInstruments................................... 185
6.4 AdvantagesofFTIRinFoodAnalysis ................................ 186
6.5 AdvantagesofFTIRoverDispersiveIR .............................. 186
6.6 SamplePreparationandHandlingTechnique......................... 186
6.7 InterpretationoftheSpectra......................................... 188
7 ApplicationsofMid-IRSpectroscopy..................................... 189
7.1 QualitativeApplications............................................. 189
7.2 QuantitativeApplications............................................ 189
7.3 PlacementofIRAnalyzerinaDairyIndustry......................... 191
8 AttenuatedTotalReflectance-FourierTransformInfraredSpectroscopy
(ATR-FTIR)........................................................... 192
8.1 BasicPrincipleofATRSpectroscopy ................................. 192
8.2 DesignsofATR..................................................... 194
8.3 ATRCrystalsMaterials.............................................. 194
8.4 AdvantagesofATRIRSpectroscopy................................. 195
8.5 ApplicationofATRIRSpectroscopy................................. 196
Contents ix
9 ApplicationofChemometricstoDevelopSpectroscopicMethod............ 196
9.1 SelectionofVariables................................................ 197
9.2 CalibrationofModel................................................ 197
9.3 ReviewofClassificationResults...................................... 197
9.4 ValidationoftheModel............................................. 197
9.5 SelectionofAlgorithm.............................................. 197
9.6 ValidationofCalibrated(Prediction)Model.......................... 197
References ............................................................... 198
10 MassSpectroscopy...................................................... 199
1 PrincipleofMassSpectroscopy .......................................... 200
2 StepsInvolvedinMassSpectroscopy..................................... 200
3 MethodsofIonization.................................................. 201
3.1 MatrixAssistedLaserDesorptionIonization(MALDI)................ 201
3.2 ElectrosprayIonization.............................................. 203
3.3 AtmosphericPressureChemicalIonization........................... 204
3.4 AtmosphericPressurePhotoionization............................... 205
4 MassAnalyzers......................................................... 206
4.1 TypesofMassAnalyzer ............................................. 207
5 AdvantagesofMS...................................................... 215
6 AcquisitionMethods.................................................... 215
References ............................................................... 217
11 AtomicAbsorptionSpectroscopyandFlamePhotometry.................... 219
1 Introduction........................................................... 219
2 BasicPrinciplesofAAS.................................................. 220
2.1 SampleAtomizationSteps........................................... 221
3 ProcessingofSamples................................................... 222
4 FlameAtomicAbsorptionSpectroscopy .................................. 224
4.1 RadiationSource ................................................... 226
4.2 Atomizers.......................................................... 226
4.3 Burner............................................................. 227
4.4 Monochromator.................................................... 227
4.5 Detector ........................................................... 227
4.6 ReadoutDevices.................................................... 227
4.7 AdvantagesofFlameAAS ........................................... 227
4.8 DisadvantagesofFlameAAS......................................... 228
5 GraphiteFurnace(Electrothermal)AtomicAbsorptionSpectroscopy
(GFAAS) .............................................................. 228
5.1 AdvantagesofFurnaceAAS ......................................... 229
5.2 DisadvantagesofFurnaceAAS....................................... 229
5.3 GeneralPracticalConsiderationsofAAS ............................. 229
5.4 SamplePreparation ................................................. 229
5.5 Labwares........................................................... 229
5.6 Calibration......................................................... 230
5.7 StandardAdditionMethod.......................................... 230
6 InterferencesinAtomicAbsorptionSpectroscopy ......................... 230
6.1 SpectralInterference................................................ 230
6.2 AbsorptionofSourceRadiationbyBackground ...................... 230
6.3 NonspectralInterference............................................ 230
