Advanced Analytical Techniques in Dairy Chemistry

Contents
About the Authors
Abbreviations
Chapter 1: Basic Laboratory Skills
1 Laboratory Safety
2 Validation
3 Basic Tools and Operations
3.1 Electronic Weighing Balance
3.2 pH Strips and pH Meter
3.3 Volumetric Laboratory Equipment
3.4 Titration
4 Laboratory Waste Management
References
Chapter 2: Chromatography
1 Chromatographic Parameters
2 Types of Chromatography
2.1 Paper Chromatography
2.2 Thin Layer Chromatography (TLC)
2.3 Column Chromatography
2.4 Adsorption Chromatography
2.5 Size Exclusion Chromatography (SEC)
2.5.1 Principle
2.5.2 Media
2.5.3 Applications
2.6 Ion Exchange Chromatography (IEC)
2.6.1 Principle
2.7 Affinity Chromatography
2.7.1 Introduction
Principle
Advantages of Affinity Chromatography
2.7.2 Types of Ligands
2.7.3 Materials of Affinity Chromatography
2.7.4 Various Matrices Used in Affinity Chromatography
Ligand
Characteristics of Ligand
Spacer arm
2.7.5 Immobilization of Affinity Ligands
Covalent Immobilization
Adsorption
Specific Adsorption
Elution of Analyte
Conditions for the Elution of the Analytes
2.7.6 Modes of Chromatography
Lectin Affinity Chromatography
Immunoaffinity Chromatography
Metal Chelate Chromatography
Psuedoaffinity Chromatography
2.8 Gas Chromatography
2.8.1 GC Columns
Stationary Phases
2.8.2 Sampling Techniques in GC
Sample Introduction
Gas Sampling
Liquid Sampling
Solid Sampling
2.8.3 Selection of Columns
Stationary Phase/Analyte Interactions
Stationary Phase Selection
Column Dimensions
Internal Diameter
Effect of Internal Diameter
Film Thickness (df)
Phase Ratio
Selection of Column
2.8.4 GC Parameters
2.8.5 Head Space GC
Head Space Sample and Solvent Types
Headspace Analysis as an Equilibrium Technique
Headspace Sampling
Equilibration
Analyte Solubility
2.8.6 Detectors Used in GC
Flame Ionisation Detector
Thermal Conductivity Detector (TCD)
Features of TCD
TCD Performance
The Electron Capture Detector (ECD)
Features of ECD
ECD Operation
Performance of ECD
In the Flame Photometric Detector
Nitrogen Phosphorus Detector/Thermionic Detector
Nitrogen Phosphorus Detector
NPD Operation and Optimization
NPD Performance
2.8.7 Quality of Gas Used in GC
Gas Traps/Filters
2.8.8 Split/Splitless Injection for GC
Split Injection
Splitless Injection
Flow Through the Liner Column Flow During Splitless Phase
2.8.9 Temperature Programming in GC
Temperature Control
Isothermal and Temperature Programmed GC
Temperature Programming in GC
Initial Temperature and Hold Time
Optimum Ramp Rate
2.8.10 Selection of GC Column
2.8.11 Derivatization in GC
Purpose of Derivatization in Gas Chromatography
Increase Volatility
Improve Chemical/Thermal Stability
Improve Chromatographic Properties
Improve Sensitivity/Selectivity in Quantitative MS Analysis
Increase Structural Information
Characteristics of Ideal Derivatization Procedure
Silylation
Acylation
Alkylation
Issues with Derivatization
2.8.12 Fast Separations in Capillary GC
2.8.13 Selection of Carrier Gas
Carrier Gasses for GC
Helium
Hydrogen
Nitrogen
Longitudinal Diffusion
Longitudinal Diffusion Can Be Minimized by
Stationary Phase Mass Transfer
Mobile Phase Mass Transfer
2.9 High-Performance Liquid Chromatography (HPLC)
2.9.1 Dimensions of HPLC Columns
2.9.2 HPLC Detectors
2.9.3 Choice of Buffers for HPLC Separations
Buffers Used in MS
2.9.4 HPLC Solvent Pumping Systems
Single Piston Pump
Dual Piston Pump
Binary Pumps
Quaternary Pumps
Ternary Solvents
2.9.5 Preparative HPLC
Objectives of Preparative HPLC
2.9.6 Features of Different Types of Chromatography (Table 11)
2.9.7 Comparison of HPLC, uHPLC, and FPLC (Table 12)
2.9.8 Applications of the Chromatographic Techniques in Dairy Chemistry
Isolation and Detection of Amino Acids by Paper Chromatography
Use of Thin Layer Chromatography to Separate and Identify Amino Acids
Separation of Milk Fat Components by Thin Layer Chromatography (TLC)
Recovery of Proteins from Cheese Whey Using Gel Filtration
Using Sephadex G-25 to Concentrate Dilute Protein Solution
Separation of Bovine Serum Albumin (BSA) and Blue Dextran Using Gel Filtration and Determination of Kav of Bovine Serum Albumin
Fractionation of Casein by Anion Exchange Chromatography
Lactoferrin Isolation from Colostral Whey Using Affinity Chromatography with Immobilized Metal Chelates
Gas-Liquid Chromatography Analysis of the Fatty Acid Content of Milk Fat
Determination of Fatty Acids of Ghee Using GC-MS/MS (ISO 15884:2002/IDF 182:2002, ISO 15885:2002/IDF 184:2002)
References
Chapter 3: Centrifugation
1 Principle
1.1 Buoyant Force
1.2 Frictional Force
1.3 Derivation of Stokes´ Law
1.4 Sedimentation Coefficients
2 Classification of Centrifuges
2.1 Desktop Clinical Centrifuges
2.2 High-Speed Centrifuges
2.3 Microcentrifuge
2.4 Vacuum Centrifuge/Concentrators
2.5 Ultracentrifuge
2.6 Instrumentation of an ultracentrifuge:
2.6.1 Drive and Speed Control
2.6.2 Temperature Control
2.6.3 Vacuum System
2.6.4 Rotors
3 Types of Rotors
3.1 Fixed Angle Rotors
3.2 Vertical Tube Rotors
3.3 Swinging-Bucket Rotors
4 Preparative Centrifugation
4.1 Differential Gradient Centrifugation
4.2 Density Gradient Centrifugation
4.2.1 Rate-Zonal Centrifugation
Application of Rate-Zonal Density Gradient Centrifugation
4.2.2 Isopycnic Centrifugation
Applications
4.3 Analytical Centrifugation
4.3.1 Applications of Analytical Centrifugation Include
5 Applications of Centrifuge
6 Micellar Casein (MC) Preparation by Ultracentrifugation
References
Chapter 4: Polyacrylamide Gel Electrophoresis
1 Introduction: Principle, Components, and Gel Media
2 Sample Preparation and Buffer Systems
3 Types of Gel Electrophoresis Commonly Used for Milk Protein Separation
3.1 Native PAGE
3.2 Urea PAGE
3.3 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS PAGE)
3.4 Tricine PAGE
3.5 Isoelectric Focusing (IEF)
3.6 Two-Dimensional Gel Electrophoresis (2D-GE)
4 Visualization and Detectiondetection
5 Chemistry of Milk Proteins Under Electrophoresis
6 Applications in Dairy Science
7 Tricine-SDS-PAGE Protocol
7.1 Reagents
7.2 Gel Preparation
7.3 Sample Preparation
7.4 Electrophoresis
7.5 Gel Staining
7.5.1 CBB
Reagents
Procedure
7.5.2 Silver Staining
Reagents
Procedure
7.5.3 PAS Staining
Reagents
Procedure
References
Chapter 5: Western Blotting
1 Introduction: Principle and Components
2 Components of Western Blotting
3 Visualization and Detection
3.1 Visualization of Protein Markers
3.2 Visualization of Proteins in the Samples
4 Applications in Dairy Science
References
Chapter 6: Membrane Processes
1 Basic Principle
2 Advantages of Membrane Separation Process
3 Drawbacks of Membrane Technology
4 Principal Types of Membranes
4.1 Isotropic Membrane
4.1.1 Microporous Membranes
4.1.2 Nonporous Dense Membrane
4.1.3 Electrically Charged Membranes
4.2 Anisotropic Membranes
4.3 Inorganic Membranes
4.4 Gas Separation/Permeation
5 Membrane Processes
5.1 Reverse Osmosis
5.2 Nanofiltration
5.3 Ultrafiltration
5.4 Microfiltration
5.5 Dialysis
5.6 Gas Permeation
5.7 Pervaporation
5.8 Electrodialysis
5.9 Liquid Membranes
6 Fractionation of Milk Proteins by Ultrafiltration
6.1 Principle
6.2 Materials
6.3 Procedure
6.3.1 Centrifugal Ultrafiltration
6.3.2 Ultrafiltration Using Stirred Cells
References
Chapter 7: Potentiometry
1 Principle
2 Potentiometric Electrodes
2.1 Metallic Electrodes
2.2 Membrane Electrodes
2.2.1 Glass Membrane Electrodes
2.2.2 Solid State ISEs
2.2.3 Liquid Membrane ISEs
2.2.4 Gas-Sensing Electrodes
2.2.5 Enzyme Electrodes
Application of Ions Selective Electrode
Advantages of Ions Selective Electrode
Disadvantages of Ions Selective Electrode
3 pH Meter and Measurement of pH
4 Buffer Solutions
4.1 pH of a Buffer
5 Measurement of pH of Milk and Whey Sample
5.1 Principle
5.2 Materials and Reagents
5.3 Guidelines to be Followed While Operating the pH/Ion Meter
5.4 Standardization/Calibration of pH Meter
5.5 Measuring pH of the Sample
6 Applicability of ISE for Measuring the Ionic Calcium in Skim Milk
6.1 Principle
6.2 Preparation of Calibration Curve
6.3 Materials and Reagent
6.4 Procedure
References
Chapter 8: Spectroscopy
1 Principle
2 Beer´s Law
3 Limitations to Beer´s Law
3.1 Fundamental Limitations
3.2 Chemical Limitations
3.3 Instrumental Limitations
4 Factors Influencing the Absorption Spectra of Chromophores
5 Energy Levels in Atoms and Molecules
6 Components of UV-Vis Spectrophotometer
6.1 Sources of Electromagnetic Radiation
6.1.1 Wavelength Selector
6.2 Sample Holding Cell
6.3 Detectors
6.4 Signal Processors
7 Single-Beam vs Double-Beam Spectrophotometer
8 Fluorescence Spectroscopy
9 Determination of Absorption Spectrum of Bovine Serum Albumin (BSA)
9.1 Principle
9.2 Materials and Reagents
9.3 Procedure [5]
10 Verification of Beer´s Law Using Bovine Serum Albumin (BSA)
10.1 Principle
10.2 Materials and Reagents
10.3 Procedure
11 Effect of pH on the λmax, Absorbance (A) and Absorbtivity of p-Nitrophenol Solution [5]
11.1 Principle
11.2 Materials and Reagents
11.3 Procedure
11.4 Observations and Calculations
References
Chapter 9: Infrared (IR) Spectroscopy
1 Infrared Region of the Electromagnetic Spectrum
2 Molecular Vibrations
2.1 Infrared Activity
3 Factors Affecting Absorption of Frequency
4 Regions of IR Spectra
4.1 The Fingerprint Region
4.2 Functional Group Region
5 Comparison of Spectra of Alkanes, Alkene, and Alkyne
6 Dispersive vs Fourier Transform Instruments
6.1 Dispersive Instruments
6.2 Fourier Transform Infrared Spectroscopy
6.2.1 What is Fourier Transformation?
6.3 Components of FTIR Instruments
6.3.1 Beam Splitter
6.4 Advantages of FTIR in Food Analysis
6.5 Advantages of FTIR over Dispersive IR
6.6 Sample Preparation and Handling Technique
6.6.1 Gaseous Samples
6.6.2 Liquid Samples
Demountable Liquid Cell
Fixed Thickness Liquid Cell
6.6.3 Solid Samples
6.7 Interpretation of the Spectra
7 Applications of Mid-IR Spectroscopy
7.1 Qualitative Applications
7.2 Quantitative Applications
7.3 Placement of IR Analyzer in a Dairy Industry
8 Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR)
8.1 Basic Principle of ATR Spectroscopy
8.1.1 Configurations of ATR Accessory
8.1.2 Properties of Single-Bounce ATR (SB-ATR) (Fig. 17)
8.1.3 Multiple Bounce ATR (MB-ATR) (Fig. 17)
8.2 Designs of ATR
8.2.1 Traditional Vertical Face ATR
8.2.2 Horizontal ATR (HATR) (Fig. 18)
8.2.3 Cylindrical ATR (Fig. 19)
8.3 ATR Crystals Materials
8.3.1 Diamond
8.3.2 Zinc Selenide (ZnSe)-Easy to Scratch
8.3.3 Germanium (Ge)-Fragile
8.4 Advantages of ATR IR Spectroscopy
8.5 Application of ATR IR Spectroscopy
9 Application of Chemometrics to Develop Spectroscopic Method
9.1 Selection of Variables
9.2 Calibration of Model
9.3 Review of Classification Results
9.4 Validation of the Model
9.5 Selection of Algorithm
9.6 Validation of Calibrated (Prediction) Model
References
Chapter 10: Mass Spectroscopy
1 Principle of Mass Spectroscopy
2 Steps Involved in Mass Spectroscopy
3 Methods of Ionization
3.1 Matrix Assisted Laser Desorption Ionization (MALDI)
3.2 Electrospray Ionization
3.3 Atmospheric Pressure Chemical Ionization
3.4 Atmospheric Pressure Photoionization
4 Mass Analyzers
4.1 Types of Mass Analyzer
4.1.1 Magnetic Sector [5]
4.1.2 Time of Flight (TOF) Mass Analyzer
Principle of Operation
4.1.3 Triple Quadrupole Mass Analyzer
4.1.4 Ion Trap Mass Analyzer
4.1.5 Tandem MS
5 Advantages of MS
6 Acquisition Methods
References
Chapter 11: Atomic Absorption Spectroscopy and Flame Photometry
1 Introduction
2 Basic Principles of AAS
2.1 Sample Atomization Steps [5]
3 Processing of Samples
4 Flame Atomic Absorption Spectroscopy
4.1 Radiation Source
4.2 Atomizers
4.3 Burner
4.4 Monochromator
4.5 Detector
4.6 Readout Devices
4.7 Advantages of Flame AAS
4.8 Disadvantages of Flame AAS
5 Graphite Furnace (Electrothermal) Atomic Absorption Spectroscopy (GFAAS)
5.1 Advantages of Furnace AAS
5.2 Disadvantages of Furnace AAS
5.3 General Practical Considerations of AAS
5.4 Sample Preparation
5.5 Labwares
5.6 Calibration
5.7 Standard Addition Method
6 Interferences in Atomic Absorption Spectroscopy
6.1 Spectral Interference
6.2 Absorption of Source Radiation by Background
6.3 Nonspectral Interference
6.3.1 Transport Interferences
6.3.2 Ionization Interference
6.3.3 Solute Volatilization Interferences
7 Hydride Generation AAS (HGAAS)
8 Mercury Cold Vapor Generation
9 Correction of Drift
9.1 Double-Beam Optics
9.2 Stockdale Optics
10 Correction of Background
10.1 Advantages
10.2 Disadvantages
10.3 Structured Background
10.4 Zeeman Effect
10.5 Disadvantages of Zeeman Correction
11 Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES)
12 Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)
12.1 Introduction of Sample
12.2 Working of RF Generator and ICP Torch
12.3 Reaction
12.4 Monochromator
12.5 Detector and Analysis Through Computer
13 Flame Photometry
13.1 Estimation
13.2 Application
13.3 Disadvantages
13.4 Preparation of Sample
13.5 Preparation of Standard Solution
13.6 Procedure for Preparation of Stock
13.7 Determination of Metals in the Unknown
13.8 Precautions during the Analysis
14 Flame Photometry vs AAS
15 Applications of These Techniques
15.1 Determination of Iron Content in Infant Milk Substitute: Atomic Absorption Spectrophotometric Method
15.2 Determination of Sodium and Potassium Content in Milk Samples by Flame Photometry
References
Chapter 12: Lateral Flow Assay
1 Introduction
2 Components of Lateral Flow Assay
2.1 Sample Pad
2.2 Conjugate Pad
2.3 Membrane
2.4 Adsorbent Pad
2.5 Backing Card
2.6 Position of the Test Line
3 Principles or Formats of Lateral Flow Assay
3.1 Sandwich Format
3.2 Competitive Format
3.3 Multiplex Detection Format
3.4 Adsorption-Desorption Format
4 Biorecognition Molecules
4.1 Antibodies
4.2 Aptamers
4.3 Molecular Beacons
5 Labels for Detection
5.1 Gold Nanoparticles (AuNPs)
5.2 Enzymes
5.3 Colloidal Carbon
5.4 Colored Latex Beads
5.5 Magnetic Particles and Aggregates
5.6 Fluorescent and Luminescent Materials
6 Characterization of Nanoparticles
7 General Steps for Lateral Flow Assay
7.1 Antibody Development against Target Analyte
7.2 Preparation of AuNPs
7.3 Conjugation of AuNPs with Antibody
7.4 Construction of the LFA Strip and Analyte Detection
8 Application of Lateral Flow Assay in Dairy and Food
9 Advantages and Limitations of LFA
10 Conclusion
References