1 Preparation of Solutions ..................................... 1
1.1 Types of Solutions ...................................... 1
1.1.1 Standard Solutions ............................... 1
1.1.2 Stock Solution ................................... 3
1.1.3 Saturated Solution ............................... 4
1.1.4 Solution of Acids ................................ 4
1.1.5 General Precautions .............................. 5
2 Expression of Concentration .................................. 9
2.1 Molarity (M) ............................................ 9
2.2 Molality (m) ........................................... 10
2.3 Normality (N) .......................................... 10
2.4 Mass Concentration ..................................... 11
2.5 Mass Fraction .......................................... 12
2.6 Mass Percent % (w/w) ................................... 12
2.7 Percentage by Volume or % (v/v) ........................ 12
2.8 Parts per Million (ppm) ................................ 13
2.8.1 Preparation of 1,000 ppm Solutions .............. 13
2.9 Parts per Billion (ppb) ................................ 14
2.10 Preparation of 10-1-10-10 M Solutions from 1 M Stock
Solution by Serial Dilutions ........................... 14
2.11 Conversion of Molarities into ppm ...................... 15
2.12 Conversion of ppm to Parts per Hundred (%) (ppm ×
10-6 × 100) ............................................ 15
2.13 Preparation of One Molar (1 M) Solutions of
Concentrated Acids ..................................... 15
2.14 Formula to Calculate the Volume of Stock Solution
Required to Prepare Solution of Desired Normality ...... 16
2.15 Formula to Calculate Volume of Stock Solution
Required to Prepare Solution of Desired Percentage ..... 16
2.16 Formula to Calculate the Dilution Factor Used
in Spectroscopic Estimations ........................... 17
2.17 Preparation of Standard Solution of Acids and Bases .... 17
2.17.1 Normal Solutions ................................ 18
2.18 Percentage Solutions ................................... 24
2.18.1 Reagents ........................................ 24
2.18.2 Preparation ..................................... 25
2.19 Ratio Solutions ........................................ 25
2.20 Titration .............................................. 26
3 Buffers and Their Preparation ............................... 29
3.1 Selection of Buffers ................................... 29
3.2 Acids and Bases ........................................ 30
3.3 Concept of pH .......................................... 30
3.4 Buffer System .......................................... 33
3.5 Preparation of Buffers ................................. 34
3.6 Preparation of Some Commonly Used Buffers .............. 34
4 Techniques in Biochemical Evaluation ........................ 39
4.1 Spectrophotometry ...................................... 39
4.2 Measurement of pH with Glass Electrode ................. 41
4.3 Chromatography ......................................... 42
4.3.1 Types of Chromatographic Techniques ............. 42
4.4 Centrifugation ......................................... 62
4.5 Ultracentrifugation .................................... 63
4.6 Isotopic Methods ....................................... 64
5 Carbohydrate Estimations .................................... 67
5.1 Classification of Carbohydrates ........................ 67
5.2 Qualitative Tests for Carbohydrates .................... 68
5.2.1 Molisch'sTest ................................... 68
5.2.2 Anthrone Test ................................... 69
5.2.3 Iodine Test ..................................... 70
5.2.4 Barfoed's Test .................................. 70
5.2.5 Seliwanoff s Test ............................... 71
5.2.6 Fehling's Test .................................. 72
5.2.7 Benedict's Test ................................. 73
5.2.8 Picric Acid Test ................................ 73
5.2.9 Mucic Acid Test ................................. 74
5.2.10 Bial's Test ..................................... 75
5.2.11 Quantitative Determination of Carbohydrates ..... 75
6 Estimation of Lipids ........................................ 77
6.1 Extraction, Separation and Estimation of Lipids from
Oil Seed ............................................... 77
6.2 Determination of Saponification Value of Fats and
Oils ................................................... 79
6.3 Determination of Acid Value of Fats and Oils ........... 80
6.4 Determination of Iodine Number of Fat .................. 81
6.5 Solubility Test for Lipids ............................. 83
6.6 Acrolein Test for Glycerol ............................. 84
6.7 Qualitative Test for the Presence of Fatty Acids
by Titrimetric Method .................................. 84
6.8 Test for Unsaturation of Fatty Acids in Lipid Sample ... 85
6.9 Separation of Different Lipid Fractions by Thin Layer
Chromatography (TLC) ................................... 86
6.10 Separation and Identification of Lipids by Column
Chromatography ......................................... 87
6.11 Estimation of Fatty Acids by Gas-Liquid
Chromatography ......................................... 89
7 Qualitative and Quantitative Estimations of Amino
Acids and Proteins .......................................... 93
7.1 Qualitative Tests for Proteins ......................... 93
7.2 Nitrogen Estimation by Micro-Kjeldahl Method (FAO
1970) .................................................. 93
7.3 Sample Preparation for Amino Acid Estimation ........... 98
7.4 Estimation of Total Free Amino Acids .................. 100
7.5 Estimation of Proline ................................. 101
7.6 Lysine Estimation in Grains ........................... 102
7.7 Estimation of Lysine in Cereal Grains ................. 103
7.8 Estimation of Methionine in Food Grains ............... 105
7.9 Estimation of Tryptophan .............................. 106
7.10 In Vitro Protein Digestibility ........................ 107
7.11 Protein Efficiency and Net Protein Ratios ............. 108
7.12 Determination of Net Protein Utilization,
Digestibility and Biological Value .................... 110
7.13 Protein Estimation by Lowry's Method .................. 113
7.14 Polycrylamide-Sodium Dodecyl Sulphate Slab Gel
Electrophoresis (SDS-PAGE) of Proteins ................ 115
7.15 Fluorography of Polyacrylamide Gels ................... 119
7.16 Quantification of Protein in Polyacrylamide Gels ...... 122
7.17 Protein Electrophoresis Using Starch Gel .............. 124
7.18 Isoelectric Focusing of Proteins ...................... 126
7.19 Western Blotting ...................................... 128
7.20 Production of Antiserum ............................... 131
7.21 Immunodiffusion in Agarose Gels ....................... 133
7.22 Enzyme-Linked Immunosorbant Assay ..................... 136
7.23 Preparations of S-30 Extract for Protein Synthesis
In Vitro .............................................. 139
7.24 In Vitro Translation Assay ............................ 140
7.25 Ammonium Sulphate Fractionation of Proteins ........... 142
7.26 Methods for Determining Amino Acid Sequences of
Protein ............................................... 142
7.26.1 Identification of the NH2-Terminal Amino Acid .. 144
7.26.2 Identification of the COOH-Terminal Amino
Acid ........................................... 144
7.26.3 Cleavage of Protein into Smaller Units ......... 146
8 Protein Purification Techniques ............................ 149
8.1 Protein Extraction Procedure .......................... 150
8.2 Ammonium Sulphate Fractionation ....................... 151
8.3 Ion-Exchange Chromatography ........................... 153
8.3.1 Selection of Ion-Exchange Matrix ............... 153
8.4 Gel Filtration ........................................ 157
8.5 Characterization of Proteins by SDS-PAGE .............. 161
8.6 Protein Analysis by Reversed Phase HPLC ............... 165
8.6.1 Separation of Proteins on C-18 Column .......... 168
9 Cell Disruption and Fractionation .......................... 169
9.1 Homogenization Media .................................. 169
9.2 Cell Disruption ....................................... 169
9.3 Fractionation of Tissue Homogenate .................... 170
9.4 Isolation of Mitochondria ............................. 170
9.5 Isolation of Chloroplasts ............................. 173
10 Enzymes in Metabolism ...................................... 175
10.1 Main Steps During Enzyme Purification ................. 175
10.2 Preparation of Acetone Powder ......................... 177
10.3 Estimation of Activities of Some Important Enzymes .... 177
10.3.1 Malate Dehydrogenase (L-Malate: NAD+
Oxidoreductase EC 1.1.1.37) .................... 177
10.3.2 Glutamate Dehydrogenase (L-Glutamate: NAD
Oxidoreductase (Deaminating) EC 1.4.1.2)
(L-Glutamate: NADP Oxidoreductase
(Deaminating) EC 1.4.1.4) ...................... 178
10.3.3 Glutamate Synthase (L-Glutamate: NADP+
Oxidoreductase (Transaminating) EC 1.4.1.13) ... 180
10.3.4 Ascorbic Acid Oxidase (L-Ascorbate: Oxygen
Oxidoreductase EC 1.10.3.3) .................... 181
10.3.5 Catalase (Hydrogen Peroxide: Hydrogen
Peroxide Oxidoreductase EC 1.11.1.6) ........... 182
10.3.6 Nitrate Reductase (NADH: Nitrate
Oxidoreductase EC 1.6.6.1 (NADH-Dependent))
(NAD(P)H: Nitrate Oxidoreductase EC 1.6.6.2
(NAD(P)H-dependent)) (NADPH: Nitrate
Oxidoreductase EC 1.6.6.3 (NADPH-Dependent)) ... 184
10.3.7 Nitrite Reductase (NAD(P)H: Nitrite
Oxidoreductase EC 1.6.6.4) ..................... 185
10.3.8 Peroxidase (Donor: H2O2 Oxidoreductase
E.C. 1.11.1.17) ................................ 186
10.3.9 Polyphenol Oxidase (Monophenol,
Dihydroxyphenylalanine: Oxygen Oxidoreductase
EC 1.14.18.1) .................................. 188
10.3.10 Indole Acetic Acid Oxidase .................... 190
10.3.11 Lipase (Triacylglycerol Acylhydrolase EC
3.1.1.3) ...................................... 191
10.3.12 Acetylcholine Esterase (Acetylcholine
Hydrolase EC 3.1.1.7) ......................... 193
10.3.13 Pectin Methylesterase (Pectin Pectyl
Hydrolase EC 3.1.1.11) ........................ 195
10.3.14 Aspartate Aminotransferase (Glutamate:
Oxaloacetate Aminotransferase EC 2.6.1.1) ..... 196
10.3.15 Alanine Aminotransferase (Glutamate:
Pyruvate Aminotransferase EC 2.6.1.2) ......... 198
10.3.16 Phosphatases (Orthophosphoric-Monoester
Phosphohydrolase, Alkaline Medium 3.1.3.1)
(Orthophosphoric-Monoester Phosphohydrolase,
Acid Medium 3.1.3.2) .......................... 199
10.3.17 Amylases (α-1,4 Glucan 4-Glucanohydrolase
EC 3.2.1.1) and (α-1,4 Glucan
Maltohydrolase EC 3.2.1.2) .................... 201
10.3.18 Cellulases (1,4-(1,3:1,4): β-D-Glucan
4-glucanohydrolase EC 3.2.1.4) ................ 203
10.3.19 Polygalacturonase Activity (Poly 1,4-α-D-
Galacturonide Glycanohydrolase EC 3.2.1.15) ... 205
10.3.20 Estimation of Papain (Papainase EC 3.4.22.2) .. 208
10.3.21 Phosphoenol Pyruvate Carboxylase
(Orthophosphate: Oxaloacetate Carboxylase
Phosphorylative EC4.1.1.31) ................... 210
10.3.22 Ribulose Biphosphate Carboxylase (3-Phospho
D-Glycerate Carboxylyase EC 4.1.1.39) ......... 211
10.3.23 Phenylalanine Ammonia Lyase (L-Phenylalanine
Ammonia Lyase EC 4.3.1.5) ..................... 212
10.3.24 Glutamine Synthetase (L-Glutamate: Ammonia
Ligase EC 6.3.1.2) ............................ 213
10.3.25 Asparagine Synthetase (L-Aspartate; Ammonia
Ligase EC 6.3.1.4) ............................ 215
10.3.26 Lactate Dehydrogenase (L-Lactate: NAD
Oxidoreductase, EC 1.1.1.27) .................. 216
10.3.26 Glutamate-Oxaloacetate Transaminase ........... 218
10.3.28 Glutamate-Pyruvate Transaminase ............... 221
10.3.29 Phosphatases .................................. 223
11 Isoenzyme Analysis ......................................... 227
11.1 General Methodology for Isozyme Analysis .............. 228
11.2 Isozyme Analysis of Esterase .......................... 228
11.3 Peroxidase ............................................ 229
11.4 Polyphenol Oxidase (PPO) .............................. 229
11.5 Acid Phosphatase ...................................... 229
11.6 Ghitamine Synthetase .................................. 230
11.7 Phosphoenol Pyruvate Carboxylase ...................... 230
11.8 Glutamate Dehydrogenase ............................... 230
11.9 Indolacetic Acid Oxidase .............................. 231
11.10 Malate Dehydrogenase ................................. 231
12 Chromatographic Separations ................................ 233
12.1 Separation and Identification of Amino Acids
by Descending Paper Chromatography .................... 233
12.1.1 Principle ...................................... 233
12.1.2 Reagents ....................................... 233
12.1.3 Procedure ...................................... 234
12.1.4 Observations and Calculations .................. 235
12.1.5 Note ........................................... 235
12.2 Separation and Identification of Amino Acids
by Ascending Paper Chromatography ..................... 236
12.2.1 Reagents and Materials ......................... 236
12.2.2 Procedure ...................................... 236
12.3 Separation and Identification of the Amino Acids
by Two-Dimensional Paper Chromatography ............... 237
12.3.1 Principle ...................................... 237
12.3.2 Reagents ....................................... 237
12.3.3 Procedure ...................................... 238
12.4 Identification of Lipids by TLC ....................... 238
12.4.1 Principle ...................................... 238
12.4.2 Reagents and Materials ......................... 239
12.4.3 Procedure ...................................... 239
12.5 Separation of Pigments by Adsorption Column
Chromatography ........................................ 240
12.5.1 Principle ...................................... 240
12.5.2 Materials and Reagents ......................... 240
12.5.3 Procedure ...................................... 240
12.6 Separation and Identification of Sugars by
Adsorption TLC ........................................ 241
12.6.1 Principle ...................................... 241
12.6.2 Reagents and Materials ......................... 242
12.6.3 Procedure ...................................... 242
12.6.4 Note ........................................... 243
12.7 Separation of Amino Acids by Ion Exchange Column
Chromatography ........................................ 244
12.7.1 Principle ...................................... 244
12.7.2 Materials and Reagents ......................... 244
12.7.3 Procedure ...................................... 244
12.8 Concentration of Dilute Protein Solutions
with Sephadex G-25 .................................... 245
12.8.1 Principle ...................................... 245
12.8.2 Materials and Reagents ......................... 245
12.8.3 Procedure ...................................... 246
12.9 Concentration of Dilute Protein by Column
Chromatography using Sephadex G-25 .................... 247
12.9.1 Principle ...................................... 247
12.9.2 Reagents ....................................... 247
12.9.3 Procedure ...................................... 247
12.10 Determination of Molecular Weight of Protein
by Gel Filtration ..................................... 248
12.10.1 Principle ..................................... 248
12.10.2 Materials and Reagents ........................ 248
12.10.3 Procedure ..................................... 249
13 Methods for Nutritional Quality Evaluation of Food
Materials .................................................. 251
13.1 Determination of Iron ................................. 251
13.2 Estimation of Protein Content by Micro-Kjeldahl
Method ................................................ 253
13.3 Estimation of Tryptophan .............................. 255
13.4 Estimation of Methionine .............................. 256
13.5 Estimation of Starch .................................. 257
13.6 Estimation of Amylose ................................. 258
13.7 Estimation of Gluten in Wheat ......................... 259
13.8 Estimation of Lysine .................................. 260
13.9 Protein Fractionation in Cereals ...................... 262
13.10 Estimation of Crude Fibre ............................ 264
13.11 Estimation of Dietary Fibre .......................... 265
13.12 Estimation of Oil Content in Oilseeds ................ 266
13.13 Estimation of Fatty Acids by Gas-Liquid
Chromatography (GLC) ................................. 268
13.14 Determination of Lipase and Lipoxygenase Activity .... 270
13.15 Estimation of Carotenoid Pigments .................... 272
13.16 Estimation of Lycopene ............................... 276
13.17 Estimation of Chlorophylls ........................... 277
13.14 Estimation of Curcumin ............................... 279
13.19 Estimation of Thiamine ............................... 279
13.20 Estimation of Riboflavin ............................. 282
13.21 Estimation of Niacin ................................. 283
13.22 Estimation of Ascorbic Acid .......................... 284
13.23 Biological Evaluation of Protein Quality by Rat
Feeding Experiments .................................. 287
13.24 Determination of In Vitro Protein Digestibility
in Food Grains ....................................... 292
13.25 Estimation of Blood Glucose and Cholesterol .......... 294
13.26 Estimation of High Density Lipoprotein (HDL), Low
Density Lipoprotein (LDL) and Very Low Density
Lipoprotein (VLDL) Cholesterol in Serum .............. 297
13.27 Estimation of Phenols ................................ 299
13.28 Chlorogenic Acid ..................................... 300
13.29 Estimation of Tannins ................................ 301
13.30 Estimation of Lignin ................................. 303
13.31 Estimation of Capsaicin in Chillies .................. 305
13.32 Estimation of Glucosinolates Content in Oil Seeds .... 306
13.33 Determination of Polyphenols in Pulse Grains ......... 309
13.34 Estimation of Aldehydes in Food Stuffs ............... 310
13.35 Estimation of ODAP ................................... 311
13.36 Assessment of Rancidity of Oil and Fats .............. 312
13.37 Determination of Phytin Phosphorus ................... 315
13.38 Estimation of Trypsin Inhibitor in Legumes ........... 317
13.39 Estimation of Cyanogens .............................. 319
13.40 Estimation of Indoleactic Acid ....................... 320
13.41 Estimation of Ethylene ............................... 321
14 Nutritional Evaluation of Forages .......................... 323
14.1 Preparation of Plant Extract for Analysis ............. 323
14.2 Proximate Analysis of Forages ......................... 324
14.3 Estimation of Dry Matter (DM) ......................... 327
14.3.1 DM in Bulk Samples ............................. 327
14.3.2 Dry Matter in Laboratory Samples ............... 328
14.3.3 DM in Silage, Haylage and Molasses ............. 329
14.4 Crude Protein (CP) .................................... 330
14.5 Determination of True Protein and Non-Protein
Nitrogen .............................................. 332
14.6 Estimation of Ammonia Nitrogen ........................ 333
14.7 Estimation of Urea Nitrogen ........................... 334
14.8 Estimation of Ether Extract (Crude Fat) ............... 335
14.8.1 Preparation of Thimble ......................... 335
14.9 Determination of Crude Fibre (CF) ..................... 336
14.10 Estimation of Total Ash .............................. 338
14.11 Estimation of Acid Insoluble Ash ..................... 339
14.12 Nitrogen-Free Extract ................................ 341
14.13 Determination of Free Fatty Acids .................... 341
14.14 Estimation of Fibre Fractions ........................ 342
14.14.1 Estimation of Neutral-Detergent Fibre
(Determination of Cell Contents and Cell
Wall Constituents) ........................... 343
14.14.2 Determination of Acid-Detergent Fibre ........ 344
14.15 Determination of Acid-Detergent Lignin ............... 346
14.16 Permanganate Lignin, Cellulose and Silica ............ 347
14.17 Estimation of Non-Starch Polysaccharides ............. 350
14.17.1 Determination of Starch ...................... 350
14.18 Determination of Digestibility by In Vitro and In
Sacco Techniques ..................................... 351
14.18.1 In Vitro Dry Matter Digestibility ............ 352
14.18.2 Modified Method of In Vitro Dry Matter
Digestibility ................................ 353
14.19 Quantification of Tanins in Foliage .................. 355
14.20 Method for Extraction of Tannins ..................... 356
14.21 Measurement of Total Phenolics and Tannins Using
Folin-Ciocalteu Method ............................... 357
14.22 Determination of Condensed Tannins
(Proanthocyanidins) .................................. 358
14.23 Gallotannin Determination by Rhodanine Assay ......... 359
14.24 Gallotannin Determination Using HPLC ................. 361
14.25 Determination of Protein-Precipitable Phenolics ...... 362
14.26 Protein-Binding Capacity by Filter Paper Assay ....... 364
14.27 Characterization of Phenolic Compounds by Thin
Layer Chromatography (TLC) ........................... 365
14.28 Estimation of Nitrates and Nitrites .................. 368
14.29 Estimation of Total Glucosinolates ................... 370
14.30 Estimation of Oxalic Acid ............................ 371
14.31 Estimation of Trypsin Inhibitor in Forages ........... 372
14.32 Estimation of Cyanogenic Glycosides .................. 373
14.32.1 Qualitative Test ............................. 373
14.32.2 Titrimetric Method for Quantitative Test ..... 373
14.33 Qualitative Estimation of Ricin ...................... 375
15 Techniques in Molecular Biology ............................ 377
15.1 Polymerase Chain Reaction (PCR) ....................... 377
15.2 Isolation of Plant DNA ................................ 380
15.3 Isolation of RNA ...................................... 383
15.4 Quantitative Estimation of DNA ........................ 385
15.5 Quantitative Estimation of RNA ........................ 386
15.6 Southern Blot Analysis of Plant DNA ................... 387
15.7 Isolation of Plasmids ................................. 394
15.8 Rapid Isolation of Plasmid DNA ........................ 397
15.9 Miniprep of Plasmid DNA ............................... 399
15.10 Cultivation of Lambda (λ) Phage ...................... 401
15.11 Extraction Lambda DNA ................................ 403
15.12 Restriction (Digestion) of DNA ....................... 405
15.13 DNA Electrophoresis in Agarose Gel ................... 407
15.14 Recovery of DNA Fragments from Agarose Gels .......... 409
15.15 Isolation of mRNA by Affinity Chromatography ......... 410
Appendix A Glossary ........................................... 417
Appendix В Abbreviations ...................................... 419
Appendix С Reference Tables ................................... 421
References .................................................... 425
Index ......................................................... 431
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