Midterm Material Flashcards

Question

Describe the purpose of grinding in sample preparation, and considerations regarding the sample characteristics and sources of error.

Answer 1

* Results in size reduction & homogenization * **Moist samples** → bowl cutters, meat mincers, blenders * **Dry samples** → mortars and pestles, mills * Grinding can cause (1) moisture loss, (2) chemical changes, and (3) metal contamination. * Reduce undesirable changes by grinding frozen samples. * Determine and control particle size through sieving or dynamic light scattering for particles \<50um

Answer 2

* Denature/unfold by: * Heating * Changing pH * (high pressure) * Inactivate by: * Changing pH * Adding salt * Adding reducing agents * Adding inhibitors

Answer 3

* Remove reactants, slow the reaction * Limit oxygen exposure (store under nitrogen, or in a vacuum) * Lower temperature * Limit light * Add antioxidants

Answer 4

* Low temperature * Drying * Freeze-drying * Add preservatives

Answer 5

* **Objective of the analysis** → e.g., quality control/monitoring, nutritional labelling, health claim, detecting food fraud * **Properties of the sample** → consideration of food composition and characteristics → e.g., major chemical components, liquid or solid/dry powder, stable or reactive, amount available, preparation required * **Characteristics of the method** * _Inherent properties_ → specificity; precision; accuracy; sensitivity * _Applicability of method to laboratory_ → sample size, reagents, equipment, cost * _Usefulness_ → time required, reliability, need * _Personnel_ → safety, training, competent * **Validity of the method** → representative sample and number of samples; proper calibration; use of standard reference materials or check samples; adequate procedure; repeatable results

Answer 6

To determine the number of significant figures in a number use the following 3 rules: 1. Non-zero digits are always significant 2. Any zeros between two significant digits are significant 3. A final zero or trailing zeros in the decimal portion ONLY are significant Example: .5**_00_** or .632**_000_** the zeros are significant .**_00_**6 or .**_000_**968 the zeros are NOT significant

Answer 7

For addition and subtraction use the following rules: 1. Count the number of significant figures in the decimal portion ONLY of each number in the problem 2. Add or subtract in the normal fashion 3. Your final answer may have no more significant figures **to the right of the decimal** than the LEAST number of significant figures in any number in the problem.

Answer 8

For multiplication and division use the following rule: 1. The LEAST number of significant figures in any number of the problem determines the number of significant figures in the answer. (You are now looking at **the entire number**, not just the decimal portion) \*This means you have to be able to recognize significant figures in order to use this rule\* Example: 5.26 has 3 significant figures 6.1 has 2 significant figures

Answer 9

* (1) sample is heated in an immiscible liquid like toluene and co-distilled, distillate is collected and volume of water removed is measured. * (2) * (3) clinging water droplets; water-solvent emulsions * (4) spices, cheese, animal feed, nuts, oils; not amenable to routine testing of many samples; less thermal degradation than oven drying * (5) AOAC method for spices

Answer 10

* (1) K-F titration is based on a stoichiometric reaction involving reduction of iodine by sulfur dioxide in the presence of water; titration of sample with K-F reagent (contains I₂); reach endpoint of titration when excess I₂ has no water to react with (visually = red/brown, or based on conductance); volume of titrant is used to determine % moisture. * (2) * (3) atmospheric moisture, moisture adhering to apparatus, interferences (e.g., ascorbic acid, carbonyl compounds, unsaturated fatty acids) * (4) _low-moisture foods,_ especially those high in sugar, protein, or unstable to heat or vacuum e.g., dried fruits & veg, candy, chocolates, coffee, oils & fats; may need pre-extraction of water (e.g., boiling with methanol) * (5) Production, quality control, product development, basic research

Answer 11

* (1) Compare relative density (specific gravity) of sample to than of water at same temperature; **hydrometer** → based on Archimedes' principle that a solid suspended in a liquid will be buoyed by a force qual to the weight of the liquid displaced; **pycnometer** → standardized glassware used to compare weights of equal volumes of a liquid and water * (2) * (3) * (4) Total solute in concentration e.g., brine, sugar in juice, % alcohol, milk solids; limited applications but fast, simple and cheap * (5) Quality control

Answer 12

* (1) Based on bending of light; the ratio of sines of angle of incidence and refraction; RI determines [compound of interest] * (2) temperature and wavelength of light is held constant * (3) * (4) % alcohol, milk solids, % sucrose in beverages, syrups; limited applications * (5) Quality control, product development, basic research

Answer 13

* ADVANTAGES * Relatively safe and inexpensive * Requires no acids, other reagents, or blanks * Can analyze many samples at one time * Requires little technician time or attention * Resultant ash can be used for **some** other analyses * DISADVANTAGES * Long time required to get results (12-18h) * Some minerals can be volatilized * Some minerals can be hard to resolubilize * More expensive than wet ashing

Answer 14

When sample is heated to high temperatures, all organic matter is incinerated, leaving inorganic material to be quantified gravimetrically.

Answer 15

* **Sample preparation & contamination** → losses during pre-drying; inorganic contamination * **Crucibles must be inert & resistant to high temperatures** → no binding of minerals; heat-stable e.g., quartz fibre, porcelain, steel, quartz, platinum * **The ash residue is fluffy and hygroscopic** → easy to spill; picks up moisture * **Volitalization of elements** → Fe, Se, Pb, Hg, Cu, Zn, P * **Incomplete combustion**

Answer 16

Organic matter is oxidized using acids and oxidizing agents, leaving inorganic matter (solubilized) * **Advantages** * Minerals stay in solution * Less volitilization * Short time = few hours * Requires only hood, hot plate, long tongs, and safety equipment * **Disadvantages** * Requires constant operator attention * Can be dangerous (due to corrosive and/or explosive reagents) * Handles small numbers of samples

Answer 17

* **Advantages** → more rapid than conventional wet or dry ashing; can process many samples at once (though, generally less than with conventional methods) * **Disadvantages** → requires specialized instrumentation = more expensive

Answer 18

True. This is because we are reporting the amount of ash either in relation to the total sample amount including water, or to just the total solids part of the sample.

Answer 19

False. This is because we are reporting the amount of ash either in relation to the total sample amount including water, or to just the total solids part of the sample.

Answer 20

Sample is heated in oven to evaporate water. Weight loss equals moisture content.

Answer 21

Sample is heated in oven under reduced pressure, so water evaporates at a lower temperature. Weight loss equals moisture content.

Answer 22

Sample is heated with microwave energy to evaporate water. Weight loss equals moisture content.

Answer 23

Infrared lamp supplies heat that penetrates sample to evaporate water. Weight loss equals moisture content.

Answer 24

Sample is heated with heating elements to evaporate water. Weight loss equals moisture content.

Answer 25

When sample is heated to toluene (an immiscible liquid), the toluene and water are co-distilled. Collected moisture distills off, is condensed and collected, and volume of water is measured.

Answer 26

In titration of sample with Karl Fischer reagent, water in sample reacts with sulfur dioxide to cause reduction of iodine. Endpoint of titration is detected when excess iodine cannot react with water. Volume of titrant (i.e., volume of Karl Fischer reagent titrated) is used to calculate % moisture.

Answer 27

Archimedes' principle. Compare relative density (specific gravity) of sample to that of water at same temperature.

Answer 28

Weight change is measured. Heat evaporates water when it boils at 100 degrees Celsius.

Answer 29

Heat sample under reduced pressure to evaporate water at ~70 degrees Celsius. Weight change is measured.

Answer 30

Heat from microwave energy causes water evaporation. Weight change is measured.

Answer 31

Heat from infrared lamp evaporates water. Weight change is measured.

Answer 32

Heat evaporates water when it boils at 100 degrees Celsius. Weight change is measured.

Answer 33

Co-distill water from sample with toluene. Measure the volume of water from sample collected after distillation and condensation.

Answer 34

Water in sample reacts with iodine and sulfur dioxide to cause reduction of iodine. Volume of Karl Fishcer Reagent titrated is measured.

Answer 35

Based on solids content of the solution, to determine specific gravity compared to pure water. Volume displaced by hydrometer is measured. Read specific gravity directly from hydrometer. **Measuring solids content.**

Answer 36

Control time, temperature; control sample particle size. Must pre-dry some samples to avoid splattering.

Answer 37

Control time and temperature. Pull and release vacuum slowly.

Answer 38

Control power and time to prevent sample decomposition. Spread sample evenly. Check calibration and analytical balance.

Answer 39

Control time and temperature. Spread sample evenly.

Answer 40

Control time and temperature. Spread sample evenly. Regular calibration of analytical balance.

Answer 41

Any emulsion formed must break to read volume of water. Need very clean glassware with no water. Use caution with solvents (fire hazards; toxic).

Answer 42

Control particle size and humidity of room. Prevent any water in glassware. Must standardize KRF. Choose another method if interferences from certain food constituents (e.g., ascorbic acid, carbonyl compounds, unsaturated fatty acids).

Answer 43

Control temperature. Need clean hydrometer.

Answer 44

_Advantages_ * Easy to handle many samples at one time. _Disadvantages_ * Takes long time to get results. * High temperature can cause loss of volatiles, lipid oxidation, Maillard browning, sucrose hydrolysis (not suitable for some foods).

Answer 45

_Advantages_ * Easy to handle many samples at one time. * Lower temperature for evaporating water reduces problems associated with high-sugar products. _Disadvantages_ * Takes long time to get results (though usually less time than with a forced draft oven). * More expensive than forced draft oven.

Answer 46

_Advantages_ * Rapid. _Disadvantages_ * More expensive than other drying methods. * Can only run one sample at a time.

Answer 47

_Advantages_ * Rapid. _Disadvantages_ * Expensive. * Can only run one sample at a time.

Answer 48

_Advantages_ * Rapid _Disadvantages_ * Expensive. * Can only run one sample at a time.

Answer 49

_Advantages_ * Causes less thermal decomposition in some foods than oven drying. * Solvent protects sample from losing volatiles and minimizes oxidation. * Water is measured directly. _Disadvantages_ * Can only run one sample at a time. * Solvent is likely flammable and toxic. * Reading volume of water in receiving tube may be less accurate than gravimetric methods.

Answer 50

_Advantages_ * No heat, so no thermal decomposition. * Rapid. * Higher accuracy than many other methods for low-moisture foods. _Disadvantages_ * Can only run one sample at a time. * Expensive, if using automated unit.

Answer 51

_Advantages_ * Rapid. * Easy. * Inexpensive. _Disadvantages_ * Limited applications. * Measures only solids content.

Answer 52

Not suitable for rapid quality control results. Not suitable for samples subject to loss of volatiles, lipid oxidation, Maillard browning, or sucrose hydrolysis.

Answer 53

An official method for many types of products. Not suitable for rapid quality control. Not suitable for powdered products, since they can blow around when vacuum is pulled and released.

Answer 54

Suitable for rapid quality control, especially for liquid products, since use of pads avoids splattering.

Answer 55

Suitable for rapid quality control, but not for high moisture products (would get splattering).

Answer 56

Suitable for rapid quality control, but not for high moisture products (would get splattering).

Answer 57

AOAC method for spices.

Answer 58

Method of choice of many low-moisture foods (e.g., dried fruits and vegetables, candies, chocolate, roasted coffee, oils and fats, and many low-moisture foods that are high in sugar or protein). Good method to try if method with heating and/or vacuum gives erratic results.

Answer 59

Commonly used as rapid method to measure solids content of beverages, salt brines, and sugar solutions. Best applied to solutions with only one solute in a medium of water.

Answer 60

* **Nutrition** * Major source of energy. * Carrier of vitamins (A, D, E, K) * Source of essential fatty acids. * **Physical properties** * Texture * Mouthfeel * Flavour * Heat transfer / cooking medium * **Stability/shelf-life** * Oxidation; rancidity

Answer 61

* **Simple lipids** → fatty acid esters of alcohols (TAG, DAG, MAG, waxes) * **Compound lipids** → fatty acid esters of alcohols + other groups (e.g., phospholipids) * **Derived lipids** → fatty acids, long-chain alcohols, sterols, fat-soluble vitamins

Answer 62

Insoluble in water & soluble in organic solvents Lipids = in general, are soluble in ether, chloroform, acetone, ethanol, benzene, hexane, etc., but are sparingly soluble in water. FDA's regulatory definition of ‘total fat’ for nutrition labelling purposes = sum of fatty acids from C4 to C24, calculated as triglycerides (e.g., include weight of glycerol per 3 FA)

Answer 63

* **Principle** → fat is extracted with solvent; fat content is measured by weight loss of sample or by weight of fat removed * **Considerations** * What type of solvent should be used? * Sample type (requires dry samples) * Bound fat (lipoproteins, liposaccharides)

Answer 64

**Nonpolar solvents cannot penetrate wet samples.** * Require dry samples for solvent extraction * Drying conditions are important * Particle size reduction may also be required. * The polarity of the solvent used for extraction influences the type of fat extracted. * Alternatively, use non-solvent methods for wet samples.

Answer 65

* Pre-drying sample: * Makes sample easier to grind * Breaks fat-water emulsion to more easily dissolve fats in organic solvent * Helps release lipids from food tissue. * Solvent extraction requires dry samples because nonpolar solvents. cannot penetrate wet samples.

Answer 66

* Organic nonpolar solvent. * BP = 34.6 degrees Celsius * Generally a better solvent for fat * More expensive * Greater danger of explosion and fire hazard * Hygroscopic * Forms peroxides

Answer 67

* Not actually an ether! * BP ~30-80 degrees Celsius * Selective for more hydrophobic lipids * Cheaper * Less hydroscopic * Less flammable

Answer 68

Acid and/or alkaline hydrolysis to break interactions between proteins &/or carbohydrates

Answer 69

Increases extraction efficiency

Answer 70

**Continuous solvent extraction** * Procedure * Dry sample in porous thimble; solvent in pyrex beaker * Extract with solvent * Mass loss = fat content &/or * Collect solvent * Evaporate solvent * Weigh the fat

Answer 71

_Advantages_ * Efficient, fast (4 hours or so) * Multiple samples _Issues_ * Channeling

Answer 72

**Semi-continous solvent extraction** * Procedure * Extract with solvent * Mass loss = fat content &/or * Collect solvent * Evaporate solvent * Weigh the fat * Difference from Goldfisch → solvent pools around the sample, then siphons back to boiling flask

Answer 73

_Advantages_ * More complete extraction * Less chance of ‘channeling’ than continuous solvent extraction _Disadvantage_ * Slower (16 hours or so)

Answer 74

**Discontinuous solvent extraction** * Procedure * Treat sample, in series, with: * Ammonium hydroxide → **precipitate proteins** * 95% ethanol → **prevents gelation** * Ethyl ether, then petroleum ether * Decant ether layer → **contains fat** * Repeat extraction series * Evaporate solvent * Weigh fat

Answer 75

* Simple/low cost * Moisture removal from sample is not necessary * Used mainly for dairy products (proteins surround milk globules → precipitate proteins = easier to extract fat)

Answer 76

**Discontinuous solvent extraction** * Procedure * Treat sample with HCl → **free lipids from protein or carbohydrate** * Extract with ethyl ether then petroleum ether * Decant ether layer → **contains fat** * Repeat extraction series * Evaporate solvent * Weigh fat

Answer 77

* Moisture removal from sample not necessary. * Simple/low cost * Used for many products.

Answer 78

**GC analysis** → solvent extraction + quantify lipids using gas chromatography = official method for nutrition labelling. * Procedure * Add internal standard (e.g., known amount of specific fatty acid) * Add antioxidant (to limit lipid oxidation) * Acid/alkaline hydrolysis procedure * Extract lipids with ethyl ether, petroleum ether * Derivatize fatty acids (add methyl group) = fatty acid methyl esters (FAMEs) → makes them volatile * Separate and quantify using gas chromatography (i.e, areas under peaks) * Total fat = sum of individual FAMEs * Area under the curve is proportional to the amount of fatty acid present in sample.

Answer 79

Ascorbic acid reduces the Karl Fischer reagent. Ascorbic acid undergoes a redox reaction with the KFR. Ascorbic acid reduces I₂ → 2I^- (in turn, ascorbic acid is oxidized). During a KF titration, ascorbic acid uses up I_2,effectively prolonging the end point leading to the overestimation of the water content.

Answer 80

**Discontinuous solvent extraction** * Procedure * Sample is mixed/homogenized in chloroform-methanol solution, then filtered. * Potassium chloride is added to break solution into two phases: aqueous and chloroform (contains fat) * Isolate chloroform layer (separatory funnel; centrifuge) * Evaporate chloroform * Weigh fat.

Answer 81

* Moisture removal from sample not necessary * Well suited to samples of low fat content * Simple/low cost

Answer 82

**Non-solvent wet extraction** * Procedure * Sulfuric acid digests protein, generates heat, and releases fat * Add water * Centrifugre samples → fat separates to top * Fat content is measured volumetrically

Answer 83

* Moisture removal from sample not necessary * Mainly used for determining milk fat; also fat in seafood. * Simple method.

Answer 84

**Non-solvent wet extraction** * Procedure * Sulfuric acid and amyl alcohol digests protein, generates heat, and releases fat. * Centrifuge samples * Fat content is measured volumetrically.

Answer 85

* Moisture removal from sample not necessary * Mainly used for determining milk fat * Simple method

Answer 86

**Soxhlet** - but need to dry samples.

Answer 87

Gas chromatography.

Answer 88

* Nutrition labelling (total protein content) * Quality control & product development * Pricing (e.g., cereal grains, milk products) → fat & protein = high value commodities * Functional property investigation * Biological activity determination (e.g., enzymes, enzyme inhibitors, allergens → so can express as % of total protein)

Answer 89

* **Physical, chemical properties** * Provide structure (bulk, texture, viscosity) * Stabilize gels, foams, emulsions * Add flavour, aroma (amino acids) * **Other important functions/features:** * Nutrition * Bioactivity (hormones, allergens) * Enzyme activity (spoilage, processing, assays for safety/quality)

Answer 90

* Primary, secondary, tertiary, quaternary structures * Polypeptide → linear change of L-amino acids connected via amide/peptide bonds * Peptide backbone = relatively hydrophilic

Answer 91

* **Nitrogen content** = unique among major food components (water, CHO, fat, ash) * **Dye binding capacity** = charged groups, hydrophobic pockets, peptide backbone * **Peptide bonds** = amide groups = characteristic IR and UV absorption * **Aromatic amino acids** = characteristic UV absorption

Answer 92

1. **Total nitrogen** → Kjeldahl (acid digestion); Dumas (nitrogen combustion) 2. **Spectroscopic methods** → Infrared spectroscopy (peptide bonds)

Answer 93

1. **Colorimetric methods** → anionic dye binding (dye binding to basic a.a.); Bradford assay (dye-binding by amino acid side chains); Biuret, Lowry, & bicinchoninic acid (BCA) assays (based on reaction of peptide bonds with copper) 2. **Spectroscopic methods** → UV absorbance (peptide bond, aromatic residues)

Answer 94

Determine the amount of nitrogen in a sample by **(1)** acid digestion to release N as NH₄⁺, **(2)** neutralization to convert NH₄⁺ → NH₃, simultaneous with distillation to isolate NH₃, **(3)** titration to quantify the NH_3, and **(4)** calculate the %N and relate to %protein using a conversion factor. Catalyst = CuSO_4,or others

Answer 95

1. Digestion with acid → H₂SO₄, catalyst, heat (\>370 degrees Celsius), releases N as NH₄⁺ 2. Neutralization & distillation → base added = generates NH₃, distill & collect in solution of boric acid 3. Titration → NH₃ + boric acid → borate ion (**blue/red indicator**); titrate borate ion + HCl → boric acid (**pale grey**) 4. Calculation → moles HCl required to titrate borate ion = moles N Assumptions → All N derived from protein (includes all organic N) The amount of borate produced is equivalent to the amount of ammonia that is derived from the protein in the sample. Borate is titrated with HCl back to boric acid in order to quantify (blue-red → pale grey). Measurable is how much HCl is needed to titrate the borate. **The method consists essentially of transforming all nitrogen in a weighed sample into ammonium sulfate by digestion with** [**sulfuric acid**](https://www.britannica.com/science/sulfuric-acid)**, alkalizing the solution, and determining the resulting** [**ammonia**](https://www.britannica.com/science/ammonia) **by distilling it into a measured volume of standard acid, the excess of which is determined by** [**titration**](https://www.britannica.com/science/titration)**.**

Answer 96

Conversion factors vary between different proteins, due to **differences in amino acid composition**.

Answer 97

Convert sample N to N₂ by combustion, then measure using gas chromatography. Assumption = All N is derived from protein (includes organics & inorganics/nitrates)

Answer 98

1. Convert protein nitrogen to N₂ (&NOx) by flash combustion at 700-1000 degrees Celsius → NOx compounds are converted to N₂ by combustion 2. Determine N₂ by gas chromatography & thermal conductivity detector → integrate area under the curve to tell you then amount of N 3. Convert N content to protein content using a conversion factor. 4. Results in only a few minutes.

Answer 99

* _Both_: * Official methods for crude protein * Applicable to all types of food * Measure total nitrogen (not only protein N) = possible to spike foods with N-rich chemicals (e.g., Melamine adulturation) * Requires correct %N → %protein conversion factor * _Differences_ * **Kjeldahl** = corrosive reagents, environmental concerns, time-consuming, technical expertise required * **Dumas** = fast, little attention required if using automated instrument, but expensive!

Answer 100

**Isolate proteins by precipitation (e.g.,using TCA) & perform analysis on remaining non-protein nitrogen** * Procedure * To sample, add TCA (trichloroacetic acid) to 10% m/v final concentration [or, dissolve dry sample into 10% TCA solution] * Proteins denature & precipitate * Filter or centrifuge sample to remove protein pellet * Proceed with analysis (Kjeldahl, Dumas) of supernatant * Any nitrogen = non-protein nitrogen

Answer 101

Dye-binding induces protein precipitation; remove protein precipitate and measure [dye] in the supernatant. The [unbound dye] is inversely proportional to [protein]

Answer 102

1. Dye binds protein basic amino acids (His, Arg, Lys) and free terminal amino group of protein = causes protein to precipitate 2. Remove protein-dye precipitate (spin, or filter) 3. Quantify the unbound [dye] 4. Calculate [dye]-bound to protein 5. Relate [dye]_{protein-bound} to [protein] using a **calibration curve** → Kjeldahl or Dumas analysis

Answer 103

1. Dye binds protein basic amino acids (His, Arg, Lys) and free terminal amino group of protein = causes protein to precipitate 2. Remove protein-dye precipitate (spin, or filter) 3. Quantify the unbound [dye] 4. Calculate [dye]-bound to protein 5. Relate [dye]_{protein-bound} to [protein] using a **calibration curve** → Kjeldahl or Dumas analysis

Answer 104

* Dye (Coomassie Brilliant Blue G-250) binds to proteins via basic and aromatic side chains, particularly Arginine (and Phe, Trp, Tyr) * Max wavelength shifts from 465nm to 595nm upon binding protein * Measure absorbance at 595nm and relate to [protein] using a **calibration curve**.

Answer 105

* Prepare a series of standards of increasing concentration e.g., BSA solutions from 0.1 → 1.0 mg/mL * Mix the standards with dye solution (acidic pH) * Mix the sample with dye solution (acidic pH) * Measure absorbance at 595nm * Calibration curve: plot A_595nm vs [BSA] * Calculate [sample] using the calibration curve

Answer 106

Variability in how dye binds to different proteins. BSA most often used as a standard. Ideally = use purified form of protein of interest e.g., if we want to quantify [ovalbumin], use ovalbumin as standard.

Answer 107

* Under alkaline conditions, Cu²⁺ binds to proteins giving rise to a colour change * Increase in VIS absorbance proportional to [protein] * Use calibration curve (e.g., BSA) * Absorbance measured at * Biuret - 540nm * Lowry - 650-750nm * BCA - 562nm

Answer 108

Vibrational transitions

Answer 109

_Advantages_ * Accurate if extinction coefficient is accurate (experimental, calculate) * Non-destructive * Rapid _Disadvantages_ * Requires pure and clear solution

Answer 110

* **Principle** * Trp and Tyr absorb UV light at ~280nm * [protein] proportional to absorbance at 280nm * **Procedure** * Measure A₂₈₀ of blank (e.g., buffer) * Measure A₂₈₀ of sample; subtract blank * Calculate [protein] using Beer-Lambart law

Answer 111

* **Principle** * Different functional groups give characteristic absorption of IR light * Peptide bond gives absorption bands in the mid (6500nm) and near-IR (3300, 2200, 1600nm) regions * Requires a calibration curve (i.e., relies on other methods) * **Application**: * Rapid, on-line analysis

Answer 112

Nitrogen (total organic) Determine N by method that involves digestion, neutralization, distillation, and titration. Use N content to calculate protein content.

Answer 113

Nitrogen (total organic and inorganic) N is released upon combustion of sample at very high temperature. N gas is quantitated by gas chromatography using a thermal conductivity detector. Use N content to calculate protein content.

Answer 114

Peptide bond Presence of peptide bond in protein molecules causes absorption of radiation at specific wavelength in mid-or near-IR region

Answer 115

Basic amino acid residues (of histidine, arginine, and lysine), and N-terminus of protein molecule. Residues identified react with anionic sulfonic acid dye to form an insoluble complex. Unbound soluble dye is measured by absorbance and related to protein concentration.

Answer 116

Peptide bond and specific amino acids (cystein, cystine, tryptophan, and tyrosine) Peptide bond is complexed with cupric ions under alkaline conditions. Cuprous ions are chelated by BCA reagent to give colour measured by spectroscopy.

Answer 117

Peptide bond Peptide bond is complexed with cupric ions under alkaline conditions to give colour that is quantitated by spectroscopy.

Answer 118

Tyrosine and tryptophan Aromatic amino acids, tryptophan and tyrosine, cause proteins to absorb at 280nm. Absorbance can be used to estimate protein content.

Answer 119

Peptide bond Peptide bonds cause proteins to absorb at 220nm. Absorbance can be used to estimate protein content.

Answer 120

_Advantages_ * Inexpensive (if not automated system) * Widely used and accepted method for over a century. _Disadvantages_ * Measures total organic nitrogen and not just protein N. * Uses corrosive reagents. * Lower precision than some other methods.

Answer 121

_Advantages_ * Inexpensive (if not automated system) * Widely used and accepted method for over a century. _Disadvantages_ * Measures total organic nitrogen and not just protein N. * Uses corrosive reagents. * Lower precision than some other methods.

Answer 122

_Advantages_ * Requires no hazardous chemicals. * Rapid (few minutes). * Automates instruments allow for analyzing many samples without attention. _Disadvantages_ * Expensive equipment. * Measures total organic and inorganic N, and not just protein N.

Answer 123

_Advantages_ * Rapid way to estimate protein content. * Requires minimal training. _Disadvantages_ * Expensive equipment. * Only provides an estimate of protein content. * Instrument must be calibrated against results of official methods.

Answer 124

_Advantages_ * Rapid (15 mins or less) * Relatively accurate. * No corrosive reagents. * Does not measure non-protein N. * More precise than Kjeldahl method. * Can be used to estimate changes in available lysine content, since the dye does not bind altered, unavailable lysine. _Disadvantages_ * Not as sensitive as some other colorimetric methods. * Requires a calibration curve for a given food commodity, since foods differ in basic amino acid content and so differ in dye-binding capacity. * Not suitable for hydrolyzed proteins due to dye binding to N-terminal amino acids. * Some non-protein components bind dye or protein, to cause error.

Answer 125

_Advantages_ * Good sensitivity, and micro-BCA method is even better. * Nonionic detergents and buffer salts do not interfere with the reaction, nor do medium concentrations of denaturing reagents. _Disadvantages_ * Colour is not stable with time. * Any compound capable of reducing Cu²⁺ to Cu⁺ will lead to colour formation. * Reducing sugars and high concentrations of ammonium sulfate interfere. * Get colour variation among proteins.

Answer 126

_Advantages_ * Rapid * Relatively sensitive. * No interference from ammonium sulfate and other buffer salts. * Nondestructive (So samples can be used after protein determination) _Disadvantages_ * Nucleic acids can absorb at 280nm. * Aromatic amino acid contents in proteins vary between food sources, so results are qualitative. * Requires relatively pure, clear, and colourless samples.

Answer 127

_Advantages_ * Rapid * Nondestructive (so samples can be used after protein determination) _Disadvantages_ * Many things other than peptide bonds absorb at 220nm. * Requires relatively pure, clear, and colourless samples.

Answer 128

_Advantages_ Less expensive, faster, and simpler than Kjeldahl method Does not detect non-peptide sources Few interferences.

Answer 129

Applicable to all foods. Little use now, due to availability of automated Dumas systems.

Answer 130

Applicable to all foods. Widely used now, compared to Kjeldahl method, for both official and quality control purposes.

Answer 131

Applicable to wide range of food products (grains, cereal, meat, dairy). Used as a rapid, quality control method.

Answer 132

Automated version used for quality-control purposes, especially as a method to compare results against a nitrogen-based method (to check for economic adulturation).

Answer 133

Widely used method for protein isolation and purification. Has largely replaced other quantitative research colorimetric methods.

Answer 134

Best used in purified protein systems (e.g., postcolumn detection of intact proteins)

Answer 135

Best used with purified hydrolyzed protein systems (e.g., postcolumn detection of hydrolyzed proteins)

Answer 136

* **Validity of the method** e.g., representative sample? reproducible results? * **Applicability to the lab** e.g., cost of assay, equipment, reagents required * **Inherent properties** e.g., specificity, precision, accuracy, sensitivity, sample size required * **Usefulness** e.g., time required, speed, reliability, necessity * **Personnel** e.g., safety, training, competency, procedures

Answer 137

Method A is more precise because the data has a smaller standard deviation from the mean compared to Method B. This implies that method A has a greater replicability than method B. This is because the smaller the standard deviation, the more likely the result is to match the target value. Method B is more accurate because its mean is closer to the true value than Method A's mean.

Answer 138

Relative error is more useful → the absolute error relative to the true value Absolute error → the difference between the experimental mean and the true value; typically, not as informative as relative error

Answer 139

Systematic error Account for the systematic error by calibrating the pipettor

Answer 140

Blunder Redo assay, be more careful.

Answer 141

**Sensitivity** → magnitude of change in measurement with change in concentration of compound; a more sensitive instrument requires a smaller change in concentration of the compound to have a large response; a less sensitive instrument requires a larger change in concentration of the compound to have a small response. Lower sensitivity method may be preferred in certain scenarios, however. **Limit of detection** → lowest amount to detect with statistical significance; the mean of the blank sample plus three times its standard deviation.

Answer 142

* What is measured? * How is the water removed/reacted/identified? * Assumptions? * Sources of error? * Applications? * Expected moisture content * Nature of other food constituents * Equipment available * Speed necessary * Intended purpose

Answer 143

Drying under lower pressure allows a lower temperature and a faster process. The lower temperature helps to mitigate issues associated with decomposition of the sample. By drying under reduced pressure, more complete removal of water and volatiles can be achieved at a lower temperature and without decomposition in a shorter period than with a forced draft oven.

Answer 144

Underestimate; potentially water is entrapped within the food particle and the sample is not entirely dry; water in interior of particles will not be volatized

Answer 145

Overestimate; some of the weigh difference between the wet and dry samples will be due to the loss of the volatile flavour compounds; weigh loss due to volatiles will be assumed as water

Answer 146

Underestimate; sample has weight gain due to peroxide formation

Answer 147

Overestimate; if the sample is retaining or acquiring water at any point during the measurements then error could be introduced; if moisture absorbed before initial weighing; measure water absorbed as moisture in product Underestimate; if moisture absorbed after drying and before final weighing

Answer 148

Overestimate; a reducing sugar dehydrates an amino group (which usually comes from protein) and along the biochemical pathway water molecules are generated; the chemical generation of ‘new water’ means an overestimation

Answer 149

Underestimate; water is a reactant and is incorporated into the sucrose hydrolysis products, so for every molecule of sucrose hydrolyzed a molecule of water is lost that cannot be measured; this water will not be removed by drying as it has become incorporated into the monosaccharides; water is being used in the chemical reaction

Answer 150

Underestimate; water becomes trapped within interior of the sample because the crust prevents its evaporation; cannot remove residual water from under crust

Answer 151

Overestimate; loss of sample due to splattering treated as water loss

Answer 152

Underestimate; the desiccant is likely to exchange moisture with the environment towards equilibrium, meaning the desiccant will be affected by exposure to environmental humidity levels; increase weight of dried samples due to moisture absorption from environment

Answer 153

Underestimate; water trapped in the solvent layer will not be measured along with the rest of the water; water will not be released from emulsion and measured.

Answer 154

Underestimate; water trapped on the condenser cannot be measured

Answer 155

Overestimate; humidity from the environment may adhere to apparatus and facilitate the reduction of iodine by sulfur dioxide

Answer 156

Overestimate; water on glassware can extend the titration endpoint

Answer 157

Underestimate; sample will be trapped in the large particles and be unavailable to react

Answer 158

Overestimate; ascorbic acid reduces Karl-Fischer reagent and extends titration endpoint

Answer 159

Overestimate; unsaturated fatty acids react with iodine

Answer 160

* Method → Hydrometer * Principle → Archimedes' principle; yields specific gravity * Justification → Simple, fast, and cheap form of quality control * Cautions → Construction of standard curve needs to be reliable and valid

Answer 161

* Method → chemical titration * Principle → Sample with KFR reacts with sulfur dioxide to reduce iodine; endpoint of titration is detected when no water remains to react with excess iodine * Justification → good for low moisture foods * Cautions → control humidity of the environment; control particle size; prevent water in glassware

Answer 162

* Method → distillation * Principle → water from sample and immiscible liquid are co-distilled. * Moisture distills off, is condensed, collected, and volume measured. * Justification → AOAC method for spices * Cautions → emulsions must break to read volume of water; need clean glassware with no water; use caution with solvents

Answer 163

* Method → refractive index method * Principle → based on the bending of light; the ratio of sines of the angle of incidence and refraction * Justification → good option to measure sugar content in syrup * Cautions → keep temperature and wavelength of light constant; ensure valid and reliable construction of the standard curve

Answer 164

* Methods → NIR or rapid moisture analyzer

Answer 165

Many methods would work (e.g., rapid moisture analyzer) but microwave oven is an example of a good choice here because it is simple, accurate, and can be performed in about 3 minutes (sufficient for given scenario). Accuracy and precision could be ensured by comparing the results to a primary method and run known reference standards (daily). Additionally, the standard deviation and coefficient of variation of the method needs to be determined to ensure it falls within required ranges.

Answer 166

* **Sample not representative** → develop a statistically valid sampling plan; homogenize sample * **Microelement contamination** → clean glassware and porcelain crucibles by soaking in acid; adjust grinder properly to prevent the appearance of metal shavings in samples; avoid exposing samples to metals in the lab; use deionized-distilled water * **Sample residue carryover from previous samples** → pre-ash crucibles to assure complete removal of organic material from previous samples * **Sample loss during pre-ashing drying step** → slowly ramp up the temperatures to prevent splattering of samples with high moisture content

Answer 167

Yes & No. Dry ashing takes more total time to obtain results, but much less technician time and less constant attention than wet ashing. Also, wet ashing is not used to determine %ash, so this is misleading. Tell the boss yes overall wet ashing is faster, but it is not useful for %ash. If speed is important, we need to buy a microwave instrument for dry ashing.

Answer 168

Relatively safe Requires no acids/oxidizers (as wet ashing does) Requires no blank subtraction Requires little attention Can handle large number of samples at once Can use ashed sample for several other analyses

Answer 169

Minerals usually stay in solution Little or no loss from sample volatization Does not require a muffle furnace (which is expensive)

Answer 170

* Testing just one sample → inaccurate result * No drying of sample before ashing → splattering of sample in oven * Not using platinum-tip tongs to handle crucible → iron/chromium contamination * Ashed for excessively long time and at too high a temperature → volatilization of some minerals * Didn't place ashed samples in desiccator to cool before weighting → samples would absorb moisture Instructions 1. Number of replicates to analyze 2. How to treat liquid samples before ashing 3. How to handle crucibles before and after ashing (i.e., type of tongs) 4. time and temperature for ashing (12-18 hours at 500-600 degrees Celsius) 5. How to cool crucibles coming from muffle furnace prior to weighing

Answer 171

Lower the temperature or use a wet-ashing procedure instead

Answer 172

After initial ashing, solubilize ash in water, then filter (with ash-less filter paper) to separate soluble ash from insoluble ash. Re-ash each portion separately (after drying soluble ash). Make sure to separately ash a piece of ash-less filter paper. This will serve as a blank for correction of insoluble ash sample. Combined the weight of soluble and insoluble to get final ash weight.

Answer 173

Use microwave dry-ashing

Answer 174

Use a different material for the crucible (e.g., platinum)

Answer 175

Use a wet-ashing procedure instead

Answer 176

* Ideal characteristics * High solvent power for lipids * Low solvent power for proteins, amino acids, and carbohydrates * Evaporate relatively easily and leave no residue * Have a relatively low boiling point * Be nonflammable and nontoxic in both liquid and vapour states * Penetrate sample particles readily * Be in single component form to avoid fractionation * Be inexpensive * Be nonhygroscopic

Answer 177

**Pre-drying sample** → fats cannot be efficiently extracted from moist foods with ethyl ether; ether saturated with water is inefficient for fat extraction; dried samples are easier to grind; breaks fat-water emulsions to make fat easier to extract **Particle size reduction** → increases the efficiency of extraction **Acid hydrolysis (or alkaline hydrolysis)** → breaks covalently and ionically bound lipids (i.e., bound to proteins and carbs) so lipid is extractable

Answer 178

The proteins and carbohydrates in the shake interact with the fat (causing it to be bound tightly), which makes it difficult to extract. Results may be lower. Modify the procedure by alkaline or acid hydrolysis prior to fat extraction.

Answer 179

**Ammonium hydroxide** → neutralizes the acidic sample and causes proteins to precipitate **Ethanol** → prevents possible gel formation **Ethyl ether** → dissolves lipid **Petroleum ether** → removes moisture from the ethyl extract and dissolves more nonpolar lipids

Answer 180

Nutrition labelling Quantifies each individual fatty acid (sum is total fat content)

Answer 181

Digest protein, generate heat (heat helps maintain fat in a liquid state to help with fat release and extraction), release the fat

Answer 182

Separate fat from the rest of the sample and brings it to the graduated portion of the test bottle so it can be quantified volumetrically

Answer 183

False. Babcock uses volumetric determination of lipid content.

Answer 184

False. Gerber uses volumetric determination of lipid content.

Answer 185

False. Soxhlet uses gravimetric determination of lipid content.

Answer 186

False. Mojonnier uses gravimetric determination of lipid content.

Answer 187

Ideal solvents for fat extraction have: * A high solvent power for lipids * Low or no solvent power for proteins, amino acids, and carbohydrates. * Ability to evaporate readily and leave no residue. * A relatively low boiling point * Nonflammable in both liquid and vapour states. * Nontoxic in both liquid and vapour states. * Single component form to avoid fractionation * Inexpensive. * Nonhygroscopic

Answer 188

* Continuous methods * Fire risk * Channeling * Too dangerous to use ethyl ether → petroleum ether. * Semicontinuous * Soaking effect → no channeling * Too dangerous to use ethyl ether → petroleum ether. _Ideal solvents for fat extraction have:_ *(hard to find solvents to meet all of these characteristics)* * A high solvent power for lipids * Low or no solvent power for proteins, amino acids, and carbohydrates. * Ability to evaporate readily and leave no residue. * A relatively low boiling point * Nonflammable in both liquid and vapour states. * Nontoxic in both liquid and vapour states. * Single component form to avoid fractionation * Inexpensive. * Nonhygroscopic

Answer 189

* **Ethyl ether** * BP 34.6 degrees Celsius * Better solvent for fat than petroleum ether. * Expensive * Danger of explosion and fire hazards. * Hygroscopic * Forms peroxides * **Petroleum ether** * Low boiling point fraction of petroleum * Composed mainly of pentane and hexane * BP 35-38 degrees Celsius * More hydrophobic than ethyl ether * Selective for more hydrophobic lipids * Cheaper * Less hygroscopic than ethyl ether * Less flammable than ethyl ether

Answer 190

I would use petroleum ether as it is less flammable and less toxic than ethyl ether, and I would use Soxhlet because it is the official method, and there is no channeling issues. Further, Goldfish has a significant fire hazard risk associated with its procedure.

Answer 191

The proteins and carbs in the shake interact with the fat (causing it to be bound tightly), which makes it difficult to extract. Results may be lower than expected. Modify the procedure by adding an acid digestion prior to standard fat extraction.

Answer 192

**(a) Ammonium hydroxide** → Neutralizes the acidic sample and dissolves protein **(b) Ethanol** → Prevents possible gel formation **(c) Ethyl ether** → Dissolves the lipid **(d) Petroleum ether** → Removes moisture from the ethyl ether extract and dissolves more nonpolar lipids

Answer 193

* The key application of the GC method is that it is the most common method used for **nutrition labeling**. * Quantifies each individual fatty acid which gives the total fat * Total fat = sum of fatty acids from C4 to C24 calculated as triglycerides, is "total fat" (including weight of glycerol per 3 FA)

Answer 194

(a) Digest protein, generate heat, and release the fat. (b) Separates fat from the rest of the sample and brings it to the graduated portion of the test tube so it can be quantified.

Answer 195

1. principle/basis of the method 2. purpose of analysis 3. sensitivity; detection limit 4. speed, equipment, and technical expertise

Answer 196

1) **DIGESTION** → Digest protein with H₂SO₄ in presence of catalyst → Food + Sulfuric Acid → gives Nitrogen in the form of Ammonium sulfate (NH₄)₂SO₄ 2) **NEUTRALIZATION/DISTILLATION** → Neutralize Ammonium sulfate with Sodium Hydroxide (NaOH) → produce ammonia (NH₃), distill the NH₃ into Boric acid to produce NH₄ and Borate ions 3) **TITRATION** → Titrate Borate ion with dilute HCl to colorimetric endpoint. Volume of HCl needed is indicative of protein content. HCl can be used as indirect measure because that's the titrant used to titrate the borate ion. The quantity of borate ion (generated over the procedure) is indicative of the amount of nitrogen (and thus protein) present. Volume of HCl → moles of borate anions → moles NH₃ → moles (NH₄)₂SO₄ → moles N → protein content

Answer 197

Average MW of N relative to MW of an amino acid is 16%. **100/16 = 6.25** Not all amino acids have the same N content, so foods of different amino acid composition will have different conversion factors. Different conversion factors are needed because the protein composition/amino acid composition differs (some a.a. have more N than others) and thus some proteins have more/less N.

Answer 198

**Bradford** → uses binding of Blue G-250 dye to proteins which results in a dye protein complex (driven by electrostatic & non-polar interactions, particularly to Arginine + aromatic residues); read absorbance at 595nm; relies on amphoteric nature of proteins; the change in the absorbance at 595nm is proportional to the protein concentration of the sample; the dye changes colour from reddish to bluish; measure A₅₉₅, calculate [protein] using a standard curve. **Anionic dye binding** → basic amino acid residues (like His, Arg, Lys) in protein (and the free amino terminal group) react with the anionic sulfonic acid dye and form an insoluble complex (= precipitate out of solution); the unbound soluble dye is measured by absorbance → amount is inversely related to the protein content of the sample → [dye]_total= [dye]_super + [dye]_{protein-bound}

Answer 199

In the anionic dye-binding method, the amount of unbound dye is inversely related to the protein content of the sample. Therefore, a lower absorbance would be associated with a higher protein content, since protein is available to bind the dye. A lower protein content would have a higher absorbance, which is why there is so much unbound dye remaining. In other words, higher protein content sample has lower absorbance reading because more of the excess dye has reacted with the protein, leaving less dye left in solution.

Answer 200

Method: Absorbance at 280nm Chemical basis: Presence of tyrosine and tryptophan; absorbance is related to concentration by Beer's Law.

Answer 201

(a) Dumas method detects nitrogen. Since melamine contains nitrogen Dumas would detect the nitrogen from melamine and protein without differentiating. They weren't measuring melamine, they were measuring nitrogen. Melamine has many nitrogen molecules, which are measured in the Kjeldahl or Dumas methods, and are calculated as protein. These methods cannot distinguish between protein N and non-protein N. (b) Using a combination of methods based on different principles would give different answers if melamine was present, e.g., Dumas method based on nitrogen content, and infrared based on peptide bonds. Or use TCA to precipitate out the proteins (i.e., remove by filtering, centrifuging) then determine non-protein nitrogen using Kjeldahl or Dumas. There should not typically be substantial amounts of non-protein nitrogen in a food sample.

Answer 202

Method: Dumas or Kjeldahl Chemical basis Dumas: Total N, converted to % protein; N is released upon combustion, and measured by gas chromatography using thermal conductivity detector Chemical basis Kjeldahl: Total N, converted to % protein; determine N by method involving digestion, neutralization, distillation, and titration

Answer 203

Method: BCA method (or Lowry or dye binding) Chemical basis BCA: proteins reduce cupric ions to cuprous ions under alkaline conditions; cuprous ions react with BCA reagent to give purple colour

Answer 204

Method: NIR Chemical basis: Presence of peptide bond in protein molecule causes absorption of radiation at specific wavelengths in mid- or near-infrared region

Answer 205

* **Nutrition labelling** → source of energy, sugars & fibre * **Functional properties** → sweetness, texture, viscosity, water-binding capacity, lowering a_w , freezing point suppression, chemical reactivity (colour, flavour, aroma, reducing sugars)

Answer 206

* simple sugars * monosaccharides e.g., D-glucose, D-fructose * disaccharides e.g., sucrose, lactose, maltose * Maltodextrins (oligosaccharides) * Starch (polysaccharides)

Answer 207

* Dietary fiber (indigestible CHO + lignin) * Soluble (pectin, gums, hydrocolloids) * Insoluble (cellulose, hemicellulose, resistant starch, & lignin)

Answer 208

Sorbitol Xylitol Maltitol Sugar alcohols are organic compounds, typically derived from sugars, containing one hydroxyl group attached to each carbon atom. They are white, water-soluble solids that can occur naturally or be produced industrially by hydrogenation of sugars. Since they contain multiple –OH groups, they are classified as polyols. [Wikipedia](https://en.wikipedia.org/wiki/Sugar_alcohol)

Answer 209

**Degree of Polymerization (DP)** Sugars, DP = 1-2 Oligosaccharides, DP = 3-9 Polysaccharides, DP \> 9

Answer 210

Cereals, bread, pasta

Answer 211

Dairy products

Answer 212

Fruits and veg

Answer 213

Meat, poultry, fish, beer.

Answer 214

* **Total CHO** * Calculated by difference * Phenol sulfuric acid method * **Mono & oligosaccharides** * Total reducing sugar analysis e.g., Somogyi-Nelson & Lane-Eynon methods * Analysis of specific mono-, di-, and oligosaccharides by HPLC, GC, or enzymatic methods * **Polysaccharides** * Starch * Fibre (total, insoluble & soluble) * **Physical methods** * Specific gravity * Refractive index

Answer 215

* **Dry the sample** * **Grind to fine powder** → increases surface area * **Extract lipids** (e.g., with chloroform-methanol or hexane etc…, in Soxhlet extractor) → makes carbohydrate extraction easier and more complete * **Extract with hot 80% ethanol** * monosaccharides and oligosaccharides are soluble * polysaccharides and proteins are insoluble * if acidic, include calcium carbonate to neutralize acidity → to prevent sucrose hydrolysis * **80% ethanol extract obtained** * Remove charged contaminants with ion-exchange technique * Use rotary evaporator to remove aqueous alcohol

Answer 216

**%CHO_TOTAL = 100 - %(moisture + ash + fat + protein)** * Calculated by difference from proximate analysis * Always calculated (i.e., not actually measured) * Measure contents of moisture, ash, lipid, and protein, then calculate ‘total carbohydrate’.

Answer 217

* Digest (acid hydrolysis) CHO → monosaccharides * Heating + H₂SO₄ converts monosaccharides into various degradation products (furans) that react with phenol → coloured reaction products (orange, yellow) * Measure absorbance at 490nm * Relate increase in A_490nm with [CHO]_total using a standard curve

Answer 218

* Virtually all CHO react, but to different extents * Accuracy depends on use of appropriate standard (i.e., as similar to sample as possible) * If CHO composition is unknown = use D-glucose * Dose not detect sugar alcohols (e.g., sorbitol, maltitol)

Answer 219

* **Reducing sugar** → any CHO with the hemiacetal group; the free aldehyde (non-cyclic form) of aldoses is available for redox reactions * **Reduction of Cu (II)** → Cu (I) by reducing sugars under alkaline conditions * **Determination of Cu (I)** by VIS absorption or titration * Relate absorbance or volume titrant to [reducing sugar] using a **standard curve**

Answer 220

* **Somogyi-Nelson method** (measure absorbance at 520 nm) * Reaction of Cu (I) with arsenomolybdate → blue pigment * **Lane-Eynon method** * Titration of Cu (II) solution (of known concentration) with sample * Measure volume of sample required to titrate all Cu (II) → Cu (I) * Solution turns from blue to clear

Answer 221

* Each reducing sugar reacts slightly differently * Accuracy depends on use of appropriate standard (e.g., D-glucose or mixture)

Answer 222

* Analysis of specific mono-, di-, and oligosaccharides by HPLC, GC, or enzyme methods * Analysis of total reducing sugars * Reduction of Cu²⁺ → Cu⁺ * Somogyi-Nelson & Lane-Eynon methods

Answer 223

Separate and quantify compounds based on area under the peak.

Answer 224

High Performance Liquid Chromatography * Extract samples & filter * Separate mono- & oligos- based on * Charge (anion exchange) → at high pH (pKa ~12-14) * Polarity (normal phase HPLC)

Answer 225

Samples must be (1) converted to sugar alcohols and (2) esterified to make them volatile = *extensive sample prep!* Note on esterification: Fermentable. Hydrophilic, may cause osmotic stress in the GI tract. There is evidence xylitol can be digested. Reduce sugars like D-Galactose to form the sugar alcohol, then esterify / acetylate to make it more volatile. Note the planar ketone of D-fructose gives rise to a mixture of two alditols depending on which way the attack happens.

Answer 226

Enzyme uses sample as substrate → measure change in Absorbance or change in conductivity

Answer 227

* **Insoluble residue** (proteins, polysaccharides) → from alcohol extraction of fat-free sample * Resuspended in protease buffer * Proteins digested with protease * Add 80% ethanol * **Polysaccharide precipitate** * Suspend in amylase buffer * Remove (or analyse) starch by digestion * Centrifuge * **Residue** = insoluble fiber * **Soluble** = soluble fibre, starch hydrolysis products * Further separation, acid hydrolysis and analysis by HPLC, GC e.g., hydrocolloids, gums

Answer 228

* Principle: * Digest (enzymes) starch into D-glucose * Measure [D-glucose] * **Starch** * Gelatinize in hot DMSO → hot DMSO minimizes formation of resistant starch * **Starch solution** * Digest with alpha-amylase * **Linear and branched fragments of amylose and amylopectin** * Digest with glucoamylase * **D-glucose** * GOPOD reagent * **Colour** * Proportional to amount of D-glucose released

Answer 229

* Dietary fibre ~ sum of the nondigestible components * Plant-derived or not (e.g., synthetic, microbial, algal) * Includes: polysacchardies (all but starch), and oligosaccharides (all but sucrose, lactose & maltose)

Answer 230

* **Insoluble** * Cellulose * Lignin * Insoluble hemicelluloses (and soluble hemicelluloses entrapped in the lignocellulosic matrix) * Resistant starch * **Soluble** * Soluble hemicelluloses (not entrapped) * Pectin (most) * Hydrocolloids (most) * Non-digestible oligosaccharides

Answer 231

* Defat sample if \>10% lipid * (a) Treat mixture with an alkaline protease → digest protein * (b) Treat hot mixture with a thermostable alpha-amylase → digest starch * ( c) Treat mixture with glucoamylase → digest starch * (d) Add 4 volumes 95% ethanol * Collect residue & precipitate by filtration. Wash with 78% ethanol, 95% ethanol, and acetone. Air dry; then oven dry. Weigh. (residue = total dietary fibre) * Subtract weights of protein and ash determined on duplicate samples → perform Kjeldahl & muffle furnace methods on identical/separate samples = correct for trapped protein & ash

Answer 232

* Defat sample if \>10% lipid * (a) treat mixture with an alkaline protease → digest protein * (b) treat hot mixture with a thermostable alpha-amylase → digest starch * ( c) treat mixture with glucoamylase * Filter (obtain residue) * Wash with water to remove anything soluble * Wash with 78% EtOH, 95% EtOH, and acetone. * **Dry and weigh = insoluble fibre** * *Soluble fibre = in the filtrate.*

Answer 233

* After digesting protein & starch from defatted sample * Filter (obtain residue) & wash with water (obtain filtrate) * Precipitate soluble fibre with 78% EtOH * Filter (obtain residue) & wash with 78% EtOH, 95% EtOH, acetone. * Dry and weigh = soluble fibre

Answer 234

* From Archimedes' principle → to float, the standard mass must be displaced by an equal mass of liquid * Standard mass = mass of liquid displaced * Volume displaced = read from scale on hydrometer * Insert into sample of interest, read depth off of scale (e.g., calibrated to specific gravity OR calibrated in Brix (1 Brix = 1 g sucrose/100g sample))

Answer 235

* Based on bending of light (i.e., refraction) as it hits surface of product * By holding temperature and wavelength of light constant, use RI to determine [compound of interest]

Answer 236

* Nutrition labelling → Na, Ca, Fe, K * Food Fortification → Ca, Fe, Zn, I * Food Processing → Na, P, Ca * Water hardness → Ca, Mg * Water Quality → Pb * Toxicity → F, Se, heavy metals

Answer 237

AAS = atomic absorption spectroscopy ICP-OES = inductively coupled plasma-optical emission spectroscopy

Answer 238

* From cutting, grinding (e.g., use aluminum blades) * Use clean glassware (clean with acid rinse) * Use pure water (e.g., distilled, deionized, filtered) and reagents * Run a **reagent blank** - use to subtract out contaminants

Answer 239

_Matching_ → prepare standards in solution with other key components known to be in the sample *

Answer 240

**_Matching_ → prepare standards in solution with other key components known to be in the sample** * Match the background matrix solutions * Contains elements known to exist in the sample at the same level they exist in the sample * Matched solution used to make standard curve for element of interest * Purpose is to overcome problems related to interferences **_Spiking_ → ‘spike’ the sample with a standard (small volume, concentrated)** * Add spikes to the sample at the start of the procedure to account for incomplete extraction and mineral degradation. * Compare the spiked and non-spiked samples (difference should = amount of spike added) * Purpose is to test for matrix effects (i.e., if get less analyte out than put in)

Answer 241

**_Matching_ → prepare standards in solution with other key components known to be in the sample** * Match the background matrix solutions * Contains elements known to exist in the sample at the same level they exist in the sample * Matched solution used to make standard curve for element of interest * Purpose is to overcome problems related to interferences

Answer 242

**_Spiking_ → ‘spike’ the sample with a standard (small volume, concentrated)** * Add spikes to the sample at the start of the procedure to account for incomplete extraction and mineral degradation. * Compare the spiked and non-spiked samples (difference should = amount of spike added) * Purpose is to test for matrix effects (i.e., if get less analyte out than put in)

Answer 243

True. Often wet-ashing. Especially colorimetric methods, instrumental methods. Separation of minerals from food components.

Answer 244

* **Titration** * EDTA complexation e.g., measure Calcium, Magnesium * Precipitation titration e.g., measure Chloride * Forward titration (Mohr) * Backward titration (Volhard) * **Colorimetric** * Measure many different minerals * **Ion selective electrodes** * Measure Na⁺, Ca²⁺, H⁺

Answer 245

* Ions initially bound to **metal ion indicator** * EDTA has greater affinity for the ions * Titrate with EDTA until all ions are freed from indicator * **Indicator turns blue = end point** * Moles EDTA = moles analyte (Ca²⁺, Mg²⁺)

Answer 246

* Titration must occur at pH ~10-11 * EDTA affinity for ions reduces at pH \< 10 * Mg and Ca precipitate near pH 12

Answer 247

* Water hardness (Ca²⁺, Mg²⁺) * Ca²⁺ in fruits and vegetables

Answer 248

* Cl^- is titrated with Ag⁺ (f. silver nitrate, AgNO₃) * Excess Ag⁺ reacts with chromate indicator to form orange precipitate = endpoint * Amount of silver nitrate used relates to amount of NaCl present in sample

Answer 249

Over-titration (→) results in over-estimation of NaCl content

Answer 250

NaCl content in food | (Assumes all Cl^- from NaCl)

Answer 251

* Excess silver nitrate is added to react with all Cl^- * Back titrate excess Ag⁺ with potassium thiocyanate * Ferric ion indicator + excess SCN → red = endpoint * Relate amount of thiocyanate to excess Ag, to calculate amount of Ag⁺ that reacted with Cl^-

Answer 252

Over-titration (→) results in under-estimation of NaCl content

Answer 253

NaCl content of food

Answer 254

False. Over-titration using the Volhard method of mineral analysis results in under-estimation of NaCl content.

Answer 255

False. Over-titration using the Mohr method of mineral analysis results in over-estimation of NaCl content.

Answer 256

Description for sample containing just calcium: EDTA of titrant solution complexes with calcium 1:1; EDTA binds to calcium ions stronger than the indicator binds to calcium. When all the calcium present in the sample has reacted the EDTA titrant, and no calcium is bound to the indicator, the indicator changes from pink to blue. **Moles of calcium in the sample are equivalent to moles of EDTA used in titration.**

Answer 257

Testing water hardness. Calcium in ash of fruits and vegetables.

Answer 258

* Advantages → rapid, especially when done with test strip; inexpensive * Disadvantages → Can be interfering compounds. endpoint is subjectively determined.

Answer 259

A forward titration. Chloride (in NaCl) is titrated with silver nitrate in the presence of potassium chromate. Silver reacts with the chloride; when all chloride is reacted, the excess silver reacts with chromate to form an orange-coloured solid, silver chromate. **Volume and molarity of silver nitrate are used to calculate amount of chloride, which relates to amount of NaCl.**

Answer 260

A backward titration. An excess of a standard solution of silver nitrate is added to a chloride-containing solution. Excess silver nitrates is back-titrated with a standardized solution of potassium or ammonium thiocyanate, with ferric ion as an indicator. **Volume of thiocyanate solution used is proportional to the excess silver. Moles of total silver is equal to the sum of the moles of chloride in the sample and moles of thiocyanate in the titrant.**

Answer 261

Description for just mineral analysis: Chromogen in reagent reacts with mineral of interest to form a soluble coloured compound that can be quantitated by absorption of light at a specific wavelength. ## Footnote **Concentration of mineral of interest is determined from standard curve of absorbance vs. concentration, based on Beer's Law.**

Answer 262

* **Advantages** → does not require expensive equipment, highly trained personnel, or ashing of sample; fewer reagents and less time-consuming than Volhard titration; inexpensive (unless automated equipment) * **Disadvantages** → Subjectivity of determining endpoint of titration (if manual titration)

Answer 263

* **Advantages** → Does not require expensive equipment, highly trained personnel, or ashing of sample; rapid; inexpensive * **Disadvantages** → Subjectivity of determining endpoint of titration. Requires more reagents and time than Mohr titration.

Answer 264

* **Advantages** → applicable to wide variety of minerals; very specific; other minerals usually don't interfere; less expensive than AAS or ICP-OES, but similar accuracy and precision * **Disadvantage** → requires significant technician time.

Answer 265

Principle is the same as for measuring pH (i.e., uses Nernst equation), but by varying the composition of the glass in the sensing electrode, the electrode can be sensitive to a specific mineral. Sensing and reference electrodes (often as combination electrode) are immersed in solution with element of interest; electrical potential that develops at surface of sensing electrode is measured by comparing to reference electrode with fixed potential. Voltage between sensing and reference electrode is related to ion activity, measured in mV. Ion activity is related to ion concentration via the activity coefficient, which is controlled by ionic strength. **Concentration of element is determined using standard curve of mV vs. log concentration**

Answer 266

Salt content of a variety of foods. Test strip version and automated instruments available.

Answer 267

Salt content of a variety of foods. Test strip version and automated instruments available.

Answer 268

Salt content of a variety of foods.

Answer 269

Low-cost method for analyzing single element.

Answer 270

* **Advantages** → can measure anions and cations directly; does not require expensive equipment (only pH meter), highly trained personnel, or ashing of sample; analysis is independent of turbidity, colour, or viscosity. * **Disadvantages** → cannot measure at low concentration; electrode response can be slow; sensing and reference electrode must be specific to element being measured; high rate of premature failure for some electrodes.

Answer 271

* A chromogen reacts with a mineral to form a coloured compound * Measure VIS absorbance * Relate absorbance to [analyte] via: * Molar extinction + Beer-Lambart law (A = elc) * OR, use a standard curve (Abs. vs. Conc.)

Answer 272

* **Limitation** → generally, sample must be pre-ashed to obtain solubilized analyte * **Application** → phosphorus, + a variety of minerals in food

Answer 273

* Electrode selective for a specific ion (e.g., Ca²⁺, Na⁺, Cl^-, K⁺, etc…) * Measure voltage between _sensing_ and _reference_ electrode * Electrical potential (voltage) developed is related to concentration of the ion * Use standard curve to relate potential to [ion]

Answer 274

* Na, Cl (salt content) * Ca in milk * Na, K in wine * CO₂ in sodas * Nitrate in meat and vegetables

Answer 275

* Plot electrode potential (mV) vs. log[ion] * Has positive or negative slope depending on element * At low concentrations → standard curve is not linear; takes longer to give a stable and reliable reading

Answer 276

* ISE is responsive to ionic activity * Ionic strength should be the same in samples and standards * Add ionic strength adjustment (ISA) buffer to all samples and standards. * All measurements should be done at the same temperature → 25 degrees C is often the standard

Answer 277

pH Sample matrix Temperature Analytical conditions Reagents

Answer 278

ISE may be used to detect titration end point (potentiometric titrations)

Answer 279

* Glass membrane → H⁺, Na⁺, Ca²⁺, NO₃^- * Solid state → Cl^-, I^-, Ag⁺, Pb²⁺ * Gas-sensing → NH₃, CO₂, O₂ * Immobilized biosensors → glucose oxidase, urease Note: electrodes are selective, not specific, for the designated ion → possible interferences for each electrode (see examples, ORION ISE specifications)

Answer 280

Colorimetric assays implemented for application to test strips Detect end-point by visual observation of colour Fast, easy, cheap: QC, field analysis

Answer 281

* Components of ‘proximate composition' on nutrition label - protein, fat, carbohydrate * Quantitate also moisture and ash so total carbohydrate can be calculated as follows: * % Total CHO = 100% - (% protein + % ash + % moisture)

Answer 282

* Monosaccharides, sometimes called ‘simple sugars’, are carbohydrates that cannot be broken down into smaller units by hydrolysis. They are soluble in both water and 80% alcohol. * Oligosaccharides are also low MW compounds. They are oligomers of 2-10 monosaccharide units, most often 2-4 units, and yield monosaccharides upon hydrolysis. The monosaccharide units are joined via glycosidic linkages. Most oligosaccharides, like monosaccharides, are **soluble** in hot 80% ethanol. * Polysaccharides are high MW polymers of monosaccharides. Polysaccharides are **insoluble** in hot 80% ethanol. * Both oligo- and poly-saccharides yield monosaccharides upon hydrolysis by hot acid or specific enzymes.

Answer 283

Reducing sugar → a sugar with a free aldehyde group (non-cyclic) or a hemiacetal group * D-glucose → reducing * D-fructose → reducing under alkaline conditions (nonreducing under neutral or acidic conditions) * Sorbitol → non reducing * Sucrose → non reducing * Lactose → reducing * Amylose → non reducing * Cellulose → non reducing *Note: the polysaccharides have a reducing end unit but are nonreducing because of their high MW.*

Answer 284

(a) **Freeze dry sample and extract fat**. Dry sample, without heating, to allow for easy grinding and extracting the fat-soluble substances. The removal of fat-soluble substances with organic solvents (e.g., chloroform-methanol) makes the extraction of polysaccharides more quantitative. (b) **Aqueous ethanol extract**. Sugars, other low MW compounds, and ash, can be removed by dissolving them in hot 80% ethanol, so these compounds don't interfere with the final analysis. (c ) **Protein digestion**. Proteins are solubilized in a buffer, heated to denature them, then hydrolyzed with enzymes (papain or bacterial alkaline proteases) to amino acids and small peptides that are not precipitated with ethanol. (d) **Ethanol precipitation**. Soluble polysaccharides are recovered by precipitation with ethanol. (e) **Starch digestion, then centrifugation.** To remove starch, a polysaccharide that is precipitated with ethanol, it is hydrolyzed with specific enzymes to D-glucose, which will be soluble in 80% ethanol. Centrifugation is used to _isolate insoluble polysaccharides_. (f) **Precipitation.** Polysaccharides that are soluble are precipitated by adding sodium chloride then 4 volumes of ethanol. (g) **Dialysis**. The precipitated polysaccharides are dissolved in water. The solution is dialyzed first against water that contains the sodium aside (to prevent microbial growth), then against water alone to remove the sodium azide. The solution is then freeze dried for further analysis. (h) **Hydrolysis**. The polysaccharides are hydrolyzed with acid to constituent monosaccharides, so individual sugars present can be determined by HPLC or GC. This is a useful tool for indicating what polysaccharides are present.

Answer 285

**Contamination** → use the purest water and reagents possible → also use a reagent blank

Answer 286

Hardness of water would be **underestimated** at pH 11.5 because magnesium and calcium in the water could be precipitated as their hydroxides, so they would not be available to react with the EDTA.

Answer 287

Overshooting the endpoint in a back titration would cause an underestimation of the compound being quantitated. The excess titrate would indicate there was more free Ag⁺, which would indicate that less Ag⁺ was reacted with Cl^- and therefore, that there was less Cl^- (NaCl) present.

Answer 288

1. Potentiometry involves the use of an electrolytic cell composed of two electrodes dipped into a test solution. 2. A voltage develops, which is related to the ionic concentration of the solution. 3. Hydrogen ion concentration (activity) is determined by the voltage that develops between the two electrodes. 4. The Nernst equation relates the electrode response to the activity. 5. Voltage produced by the electrode system is a linear function of the pH. The electrode potential is +59mV for each change of one pH unit.

Answer 289

* Sample stored for 2 weeks → fermentation would occur, so reduced pH (microbes produce acid) * Should have used 2 or 3 standard buffers; using only 1 buffer assumes temperature is related to slope * Should do standards at the same temperature as the sample.

Answer 290

(a) Orange juice - **citric** (b) Yogurt - **lactic** (c) Apple juice - **malic** (d) Grape juice - **tartaric**

Answer 291

Colourimetric methods not appropriate due to the dark colour of tomato juice. * Use potentiometric method & titrate to certain pH → use the mid-point of the abrupt change in pH as the equivalence point

Answer 292

Unboiled carbonated beverage (which would contain carbon dioxide) would have a higher calculated titratable acidity. The NaOH used for titration reacts with carbon dioxide to produce sodium carbonate and sodium bicarbonate, which reduces alkalinity and sets up a buffer system. Also, the bicarbonate breaks down to generate hydrogen ions that will reduce the pH. Unboiled carbonated beverage would have a fading endpoint, because carbon dioxide would continue to react with the NaOH and create more buffer, and hydrogen ion generation continues.

Answer 293

* pH has a strong effect on: * Flavour * Colour * Texture * Stability * Nutritional quality * Food quality * Food safety

Answer 294

pH = -log[H⁺] or pH = -log[H₃O⁺]

Answer 295

False. The further the equilibrium lies to the right, the larger the dissociation constant and the smaller the pK_a.

Answer 296

Acid = a proton donor Base = a proton acceptor Neutralization = ‘the reaction of an acid with a base to form a salt’

Answer 297

Acid dissociation constant The smaller the pK_a value, the stronger the acid. Strong acids have high K_a values.

Answer 298

Strong acids dissociate completely. Weak acids partially dissociate.

Answer 299

* pH meter is a **potentiometer** * Measures voltage (electrical potential) at infinitesimal current * Consists of a reference electrode, indicator electrode, and voltmeter * **[H₃O⁺] is proportional to the change in the electrical potential**

Answer 300

* Electrode potential decreases with increasing pH * Electrode potential increases with increasing [H₃O⁺]

Answer 301

* read the instructions for your particular electrode! * perform 3-point calibration * keep sample solution stirring * most common = combination electrode * most instruments can adjust for temperature

Answer 302

* Whenever control of pH is desired/required * Many assays (e.g., enzyme activity) * Extraction of components (maintain stability, solubility, effect electrostatic interactions) * Foods contain natural buffering agents (e.g., organic acids; proteins)

Answer 303

**pH = pK_a + log[A^-] / [HA]**

Answer 304

* [buffer] * ionic strength (~negligible \<0.1M) * temperature (TRIS buffers are relatively sensitive)

Answer 305

A buffer is effective only around pH = pK_a +/- 1 The strongest buffer capacity is at pH = pK_a

Answer 306

* Flavour * Fruit ripeness (Brix/acid ratio of fruit) * Colour (pH effect) * Microbial stability (pH effect) * Stability (pH effect) * Acids tend to decrease with maturity while sugars increase, so the ratio is a good indicator of fruit maturity.

Answer 307

A variety Most are carboxylic acids (e.g., acetic, citric, malic, lactic) Also → phosphoric acid and carbonic acid (from dissolved carbon dioxide)

Answer 308

* Titrate the food sample with a strong base (e.g., standardized 0.1 N NaOH * As the OH^- neutralizes the acid, the pH rises * Indicator added (phenolphthalein) changes colour ~pH 8-9 = end point * Use moles NaOH to determine amount of acid (reported as % primary acid)

Answer 309

* Difficulty seeing colour change * Dissolved carbon dioxide interferes by reacting with hydroxide to form carbonate * Pre-boil samples to remove