CHAPTER 9: PROTEIN ANALYSIS

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CHAPTER 9 PROTEIN ANALYSIS
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PROTEIN ANALYSIS

• Why are we interested in the overall protein
  content of a food?
• Functionality (examples)
• Disulfide bonds (wheat gluten)

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Functionality..

• Functionality as applied to foods and food
  ingredients is generally ANY property aside from
  nutritional attributes, that influences the
  usefulness in a food.
• The term functional foods has come to have a
  separate meaning in todays pop-culture in
  reference to nutraceuticals.

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Functionality in Proteins

• Hydration properties
• Water-holding capacity
• Viscosity modifiers
• Solubility
• Protein-Protein Interactions
• Gel formations
• Precipitation
• Surface Properties
• Emulsifiers
• Foaming
• Surface tension

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Protein Content of Some Foods
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PROTEIN ANALYSIS

• Proteins are important for nutrition and have
  important functions in our cells
• Proteins weigh from 5000 daltons (5KDa) to gt1
  million
• 1 dalton is 1/12 the weight of 12C (ie. water
  18 Da)
• Proteins are composed of amino acids there are
  20 common ?-amino acids (AA)
• AA are linked via peptide bonds and are composed
  of C, N, H, O and S
• The N in a protein ranges from 13.4 to 19.1 ,
  depending primarily on the number of basic AAs
  present
• Proteins are classified based on
• 1. solubility
• 2. structure
• 3. function
• The N in a food product primarily comes from
  proteins and NPN (covered later)

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• Protein is analyzed for
• 1. determination of biological activity
• 2. investigation of functional properties
• 3. nutritional labeling
• Protein analysis is required for you to know
• 1. total protein
• 2. amino acid composition
• 3. amount of a particular protein in a
  mixture
• 4. protein content during isolation and
  purification
• 5. Nonprotein nitrogen
• 6. Nutritional value of a protein

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Methods for Protein Analysis

• Chemical
• Microbiological
• Enzymatic
• Useful for screening large numbers of samples,
  but gives little information as to functionality,
  efficiency, or applicability to nutritional needs.

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Protein analysis in foods has been mostly done by
determining Nitrogen Content

• Nitrogen is largely unique to protein only
  MAJOR constituent of foods containing N.
• Other nitrogenous compounds (NPN) Chlorophyll,
  nucleic acids, some vitamins, lecithins, urea,
  amino sugars, alkaloids, ammonium ions, etc.
• On the average, food proteins contain 16
  nitrogen. 100 divided by 16 6.25. Therefore
  multiply N content by 6.25 to get protein content.

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Protein analysis in foods has been mostly done
by Determining Nitrogen content

• The most common and well accepted method for
  determining nitrogen in food is the Kjeldahl
  method.(Kel-Dall)
• Labconco is one of the industry leaders in
  Kjeldahl equipment
• http//www.labconco.com/pdf/kjeldahl/index.shtml
• Click on Kjeldahl and download the brochures.
• (A Guide to Kjeldahl Nitrogen Determination
  Methods and Apparatus)

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KJELDAHL NITROGEN

• Based on conversion of protein nitrogen to
  ammonia (NH3). At the same time, carbon and
  hydrogen are oxidized to carbon dioxide and
  water.
• In the presence of sulfuric acid (H2SO4), the
  ammonia picks up a hydrogen forming ammonium
  sulfate (NH4)2SO4.
• Then concentrated sodium hydroxide (NaOH) is
  added to the solution of ammonium sulfate
  sulfuric acid. This raises the pH transforming
  ammonium (NH4) into ammonia.

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KJELDAHL NITROGEN

• When the sample containing ammonia, sodium
  hydroxide and sodium sulfate is heated, the
  ammonia is driven off as a gas.
• We can condense the ammonia gas and introduce it
  into a fluid that contains boric acid and pH
  indicators.
• Ammonia reacts with boric acid to form ammonium
  borate. This is a base.
• Titrate ammonium borate to an endpoint with
  hydrochloric acid (we must know the normality of
  the hydrochloric acid used).

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KJELDAHL NITROGEN

• 3 Stage Process
• 1. Digestion with acid catalysis
• 2. Distillation with steam and alkali
• 3. Titration with acid and indicators.

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KJELDAHL NITROGEN

• Moles of HCl moles NH3 moles N in sample
• Since most proteins contain 16 N, the factor
  6.25 corrects to convert N to protein.
• N X 6.25 Protein or
• N / 0.16 Protein
• Deviations
• Wheat 5.70 Milk 6.38 Gelatin 5.55
• The higher the protein, the lower the factor,
  therefore you need to use the right
  correction/multiplication factor.

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More Conversion Factors

• Whole wheat cereals 5.83
• Flour 5.70
• Pasta 5.70
• Bran 6.31
• Rice 5.95
• Nuts/Seeds 5.46
• Soya 5.71

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KJELDAHL Reactions

• Protein H2SO4 ? (NH4)2SO4 (Digestion)
• NH4 NaOH ?steam and heat? NH3 (g) H2O
  (Distillation)
• NH3 (l) H3BO3 ? NH4 H2BO3- (Trapped)
• H2BO3- HCl ? H3BO3 (Titration)
• How could we determine free nitrogen or NPN?

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KJELDAHL NITROGEN

• 3 Stage Process
• 1. Digestion with acid catalysis
• 2. Distillation with steam and alkali
• 3. Titration with acid and indicators.

N NH3 (NH4)2SO4 NH4 NH3 H2BO-4 HCl
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• KJELDAHL
• Advantages are
• 1. applicable to most food samples
• 2. simple
• 3. inexpensive
• 4. accepted as Official method
• 5. can measure mg levels of proteins
• Disadvantages are
• 1. Measures total N not protein2. Time consuming
  at least 2 hrs3. Poor precision when compared
  to other methods4. Corrosive (dangerous)
  method

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• ALTERNATES TO DISTILLATION AND TITRATION
• 1. NESSLERIZATION reaction of ammonia with
  mercuric iodide to produce ammonium dimercuric
  iodide which can be read in a SPEC (visible _at_
  440nm)
• 2. Reaction with phenol and hypochlorite to form
  indophenol which can be read in a SPEC (visible _at_
  630nm)
• 3. Ion sensitive electrode for ammonia or ion
  chromatography

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Protein analysis in foods has been mostly done by
determining Nitrogen Content

• Other nitrogenous compounds (NPN) Chlorophyll,
  nucleic acids, some vitamins, lecithins, urea,
  amino sugars, alkaloids, ammonium ions, etc.
• How can we determine the background level of
  these compounds using Kjeldahl?
• What about free ammonia or ammonium salts?
• Can we run a standard curve on a Kjeldahl? How?

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LOWRY METHOD

• The Lowry method is a colorimetric method based
  on the formation of a blue color formed
• Tyrosine and/or tryptophan in a protein reduces a
  phosphomolybdic-phosphotungstic reagent
  (Folin-Ciocalteu reagent) in the presence of
  K-Na-tartrate in alkali (Biuret reagent)
• Absorbance values are determined on a
  spectrophotometer at 750 nm. As little as 0.2 mg
  protein in a sample can be determined.

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Lets back up just a bitRemember the Biuret
Method?

• Cupric ions react with peptide bonds under
  alkaline conditions
• (copper sulfate K-Na-tartrate alkali)
• Measure color in SPEC at 540 nm

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• BIURET METHOD
• Advantages
• - Cheaper and faster than Kjeldahl
• - Less problem with color deviations
• - Few substances interfere
• - Does not measure non protein nitrogen (NPN)
• Disadvantages
• - not sensitive to 2-4 mg level
• - bile pigments interfere
• - ammonium salts interfere
• - color depends on protein
• - lipids and carbos can affect clarity of
  solution
• - PROTEIN MUST BE SOLUBLE

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Folin-Ciocalteu?

• Used to determine if a sugar has reducing ability
  or not
• Reduction of phosphomolybdic-phosphotungstic acid
  by reducing groups in a sample.
• Once reduction has occurred, the solution is made
  alkaline and a blue color is formed.
• Can be used directly for proteins, pure sugar
  solutions, or phenolic compounds.

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LOWRY METHOD-Combination of Biuret and
Folin-Ciocalteu assay

• Cu2 is reduced to Cu in the presence of
  proteins at high pH the biuret reagent chelates
  the Cu ion, then the FolinCiocalteu reagent
  enhances the blue color
• Careful addition of reagents and very careful
  timing is very important to this assay.

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LOWRY METHOD- Advantages

• This is the most sensitive spectrophotometric
  method available for determining total protein.
  It is 10 to 20 times more sensitive than UV
  absorption at 280 nm (next topic)
• This method is more specific than most other
  protein methods and is better at handling
  problems from turbidity in protein solutions
• The Lowry method is relatively rapid, requiring
  1-2 hours for analysis
• Widely used in biomedical field

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LOWRY METHOD- Disadvantages

• This method requires careful standardization
  (making a good standard curve) because
• a. The amount of color varies with different
  proteins.
• b. The color is not strictly proportional to the
  concentration
• c. Recent evidence suggests that sucrose, lipids,
  some buffers, monosaccharides and hexosamines
  react to varying degrees with the reagents in the
  Lowry test
• d. High concentrations of ammonium sulfate,
  sulfhydryl compounds, and phosphate can interfere

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UV Absorption (280 nm)

• Most proteins exhibit strong UV light absorption
  at 280 nm because they contain chromophoric
  side chains such as tyrosine, tryptophan, and
  phenylalanine.
• Assuming a reasonably constant level of these
  amino acids in food proteins, the concentration
  of protein in a non-turbid solution is
  proportional to the absorbance
• When we talk of absorbance and concentration
  in the same sentence, what should this
  AUTOMATICALLY make you think of?

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Ill Take Amino Acids for 100, Alex.
PHENYLALANINE
TYROSINE
TRYPTOPHAN
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UV Absorption (280 nm)

• Absorbance according to Beer's Law
• Aabc
• Where A Absorbance
• a absorptivity, a constant that is
  characteristic of a particular chemical species
  at a particular wavelength.b path length in
  cmc concentration of the absorbing chemical
  species

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UV Absorption (280 nm)

• If we assume that the concentration is to be in
  units of Molarity (moles/Liter), then we can use
  the molar absorptivity (e) in place of the
  absorptivity. The equation would then be
• Aebc
• Molar absorptivity can be determined for
  individual proteins if they are pure (no other
  proteins present).
• This can then be used to estimate unknown
  concentrations of that particular protein

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UV ABSORPTION (280 nm)

• Advantages
• - rapid and sensitive
• - nondestructive
• - no ammonium interference
• Disadvantages
• - nucleic and phenolic acids also absorb at 280
  nm
• - amounts of Trp and Tyr vary with protein types
• - turbidity (cloudiness in solution) is a problem
• Applications of method? Not widely accepted for
  general food analysis more useful for research
  purposes by monitoring the extraction or
  separation of proteins

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DYE BINDING METHOD

• Proteins will bind to certain types of dye. When
  this binding occurs, the protein-dye complex will
  precipitate.
• The unbound dye is then easily determined with a
  spectrophotometer using a standard curve with
  varying dye concentrations.
• Using the amount of dye initially added to the
  protein solution, the amount of protein can be
  calculated.

More Protein, Less Color
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Binding selectivity-Example
Cationic groups of the amino acids react with an
anionic sulfonic acid dye (i.e Amido Black) The
more dye that was bound, the more protein present
in the sample.
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• Coomassie Brilliant Blue solution will directly
  bind to specific amino acids and protein tertiary
  structures
• The dye's color changes from reddish-brown to
  blue
• Absorbance at 595 nm read.
• Pros
• Rapid assay
• Useful when accuracy is not crucial
• Cons
• High protein-to-protein variation
• Not compatible with detergents (used to isolate
  proteins)
• Applications Finds use as rapid protein method
  in food and biomedical industry.

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Ninhydrin

• Primary amino groups on the end of proteins,
  peptides, and free amino acids will react with
  ninhydrin.
• This reaction forms a strongly colored purple
  solution referred to as Ruheman's purple. Read at
  570 nm.
• Sample can be tested for the amount of primary
  amino acids currently present, or sample can be
  alkaline hydrolyzed to increase the amount of
  these amino acids.

Common method for cheese
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• Ninhydrin
• Advantages are
• Faster and more convenient that Kjeldahl
• Disadvantages are
• Large dilutions are necessary for spec. reading.
• Proteins differ in the dye binding capacity
• Make standard curve based on predominant primary
  amino acid present in the food.
• NPN, calcium, or phosphorous constituents will
  bind to the dye or to protein, causing
  interference.
• Addition of a metal chelator (i.e.. oxalic acid)
  may help reduce binding.

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Dumas Method

• Measures the Nitrogen released upon combustion of
  the sample (800C). Measured by GC using a
  Thermal Conductivity Detector (TCD). May be used
  for labeling purposes.
• Infrared Spectroscopy
• Presence of peptide bond in protein molecule
  causes absorption of radiation at specific
  wavelength in mid- or near infrared region

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• BICINCHONIC ACID (BCA) METHOD
• Proteins reduce cupric ions to cuprous ions under
  alkaline conditions. Cuprous ions react with BCA
  reagent to give a purple color
• Advantages
• - as sensitive as Lowry but simpler
• - reagent more stable than Lowry
• Disadvantages
• - color not stable with timeprecise timing
• - reducing sugars interfere more than Lowry
• - color variations between proteins occur
• absorbance vs concentration not absolutely linear

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CHAPTER 15 PROTEIN ANALYSIS
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CHAPTERS 15PROTEIN SEPARATION AND
CHARACTERIZATION

• Do you want to separate proteins for commercial
  reasons or research?
• Take advantage of protein solubility, size,
  charge, adsorption characteristics, and logical
  affinities for other molecules for separation.
• Usually will combine methods to significantly
  purify a protein or amino acid
• Industrial applications one-step procedures

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CHAPTERS 15PROTEIN SEPARATION AND
CHARACTERIZATION

• One step can be
• pH precipitation casein from milk
• Size fractionation whey protein
• Salt precipitation-variety of products
• Need to know some specifics for each type of
  protein before it can be effectively separated
• Accurate characterization is then important

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COMMERCIAL SEPARATION OF PROTEINS-example

• Whey/Soy Proteins
• Waste proteins
• Enzyme Recovery
• Other nutritional supplements and food additives

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PROTEIN SEPARATION

• Salting Out Proteins have unique solubility
  profiles in neutral salt solutions.
• Low concentrations of neutral salts may
• Increase the solubility of some proteins
• Precipitate from solution as ionic strength is
  increased.
• Actions are somewhat unique to each protein.
• Ammonium sulfate (NH4)2SO4 is commonly used
  because it is highly soluble and very effective.
• NaCl or KCl may be also be used to salt out
  proteins.

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Salting Out----Ionic Strength

• Ionic Strength ½ S MiZi2
• Mi Molarity of ion
• Zi Charge of ion
• 1M NaCl ½ S (1 X 12) (1 X 12) 1
• 1M CaCl2 ½ S (1 X 22) (2 X 12) 3
• 1M (NH4)2SO4 ½ S (2 X 12) (1 X 22) 3

Na
Cl-
Cl-
Ca2
NH4
SO42-
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PROTEIN SEPARATION

• Two-step procedure Salts or pH precipitation
• Add (NH4)2SO4 at a concentration just below
  precipitation point - more soluble proteins are
  precipitated - protein of interest remains in
  solution.
• Add (NH4)2SO4 at a concentration just above that
  necessary to precipitate the protein of interest.
  Centrifugation recovers protein

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Isoelectric precipitation

• Proteins are electrolytes- solubility
  characteristics are determined by the type and
  charge on amino acids in molecule - selective
  precipitation.
• IEP (Isoelectric precipitation)- pH at which a
  protein has NO net charge in solution it then
  aggregates and precipitates.
• Proteins have different IEPs thus, separate from
  each other by adjusting solution pH.
• Separation of casein from milk- IEP pH 4.6

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Adsorption

• Adsorption to and desorption from the surface of
  a solid support (resin) by an eluting solvent
• Affinity chromatography and Ion-exchange
  chromatography.
• Ion Exchange Either anionic or cationic
  exchange resins can be used.
• Optimize ionic strength and pH for binding of
  protein on interest based on CHARGE.
• - Pass protein sample through the resin- Elute
  with buffer that will remove protein.

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• Key points
• Stationary phase
• Mobile phase
• Charges
• Ions
• Counter-ions
• Binding
• Elution

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Adsorption
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Size Exclusion

• Protein molecular weights range from about 10,000
  to over 1,000,000 Da thus, size is a logical
  parameter to use for separations.
• Dialysis Mainly research applications
• Ultra filtration Membranes with molecular weight
  cutoffs from 500 to 300,000 are available (more
  in a moment)
• Applications Isolating protein concentrates from
  whey. Membrane with 10,000-20,000 cutoff used to
  partially remove lactose, salts, and water which
  concentrates the proteins.
• Alternative Packed column of porous beads (SEC)

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Membrane Separation
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Membrane Separation
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Ultrafiltration
Retentate what remains in the
sampleUltrafiltrate what passes through the
membrane
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Electrophoresis

• Polyacrylamide Gel Electrophoresis
• Electrophoresis defined as the migration of
  charged molecules in a solution, through an
  electrical field.
• Molecular size, shape, and charge affect mobility
  through the gel.
• Zonal electrophoresis - proteins are separated
  into bands by protein migration (dissolved in
  aqueous buffers) through a gel.
• Polyacrylamide gels - most common matrix (can
  also use starch and agarose)

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Electrophoresis

• Polyacrylamide Gel Electrophoresis
• Separation into bands due to friction through the
  gel and charge on protein.
• Magnitude of charge and voltage will also
  determine how far the protein will travel in the
  electrical field.
• Smaller proteins tend to move faster

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Electrophoresis-Isoelectric Focusing

• Separation based solely on charge.
• Modification of electrophoresis.
• Proteins separated by charge in an electric field
  on a gel matrix in which a pH gradient has been
  generated (using ampholytes)
• Proteins are focused or migrate to the location
  in the gradient at which pH equals the pI of the
  protein
• Can determine the pI of a protein and establish
  purity.

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Electrophoresis- SDS Page

• Separation based on size.
• Proteins bind to SDS to become negatively
  charged.
• SDS sodium dodecyl sulfate gt anionic detergent
  (negative charge)
• Proteins move through gel matrix to the anode
  (electrical pole with a positive charge).
• The RATE they move is based on size.
• Good for determining protein composition, purity,
  and estimation of molecular weight.

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• Dialysis- separation based on SIZE. A
  semipermeable membrane permits passage of small,
  but not large molecules.
• Isoelectric point- protein has no charge at pI,
  so it precipitates from solution.
• Ammonium sulfate- as ionic strength increases,
  proteins precipitate.
• Ultrafiltration- separation based on SIZE.
  Pressure applied to a semipermeable membrane
  similar to dialysis.
• Heating- as proteins denature with heat, they
  precipitate from solution.

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• Ethanol addition- certain solvents will decrease
  the solubility of proteins, causing precipitation
  from solution.
• Affinity chromatography- protein is bound to a
  solid support with a specific affinity to the
  protein of interest. Protein is unbound by
  changing the pH, temperature, or ionic (salt)
  strength.
• Size-exclusion chromatography- separation based
  on size using porous beads. Small particles move
  through the bead slowly, large particles move
  quickly.

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Amino Acid Analysis

• Proteins hydrolyze into amino acids (6 N HCl 24
  hr)
• Amino acids separated using chromatographic
  techniques
• Quantified Reactions with ninhydrin (primary
  amines) or by HPLC or GC following
  derivatization (e.g. phenylthiocarbamyl) and UV
  detection.
• Separation
• Ion-exchange chromatography
• Reversed-phase liquid chromatography
• Gas chromatography

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CH. 10 Carbohydrates (CHO)
Can somebody please pass the sugar..NOW !

• Carbohydrates are generated in plants through
  photosynthesis, and are a significant source of
  energy worldwide.
• Carbohydrates (CHOs) represent stored energy for
  plants and animals (starch, glycogen and
  cellulose).
• Cellulose (wood fiber) is similar to starch but
  it differs in the geometric position of the
  bondage between the glucose units.
• Chitin is an important constituent of the
  exoskeleton of lobsters, crabs and many insects.
• CHOs also perform important functions in human
  physiology as part of glycoproteins and
  glycolipids

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Classification of Carbohydrates

• The Saccharides
• Monosaccharide
• Smallest form, non-hydrolysable.
• Oligosaccharide
• Made of several monosaccharides, hydrolysable.
• Polysaccharide
• Very large polymers of monosaccharides

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The MONOsaccharides

• Simple Sugars Monosaccharides are compounds that
  can not be hydrolyzed in to simpler compounds.
• Examples glucose, fructose, galactose and
  glyceraldehyde.
• Monosaccharides are water-soluble crystalline
  compounds
• Generally aliphatic carbonyls (aldehydes
  ketones).
• Polyhydroxy (many OH groups) aldehydes, ketones,
  alcohols, acids, amines simple derivatives.
• Classification based on functional group ketose
  (ketone) or aldose (aldehyde)
• Classification by number of C in molecule
  (triose, tetrose, pentose, hexose etc).

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Physical Nature of Simple Sugars(used in various
analysis techniques)

• Monosaccharides
• Highly water-soluble. Insoluble in most organic
  solvents. Solubility allows a determination
  between density and concentration (hydrometers).
• Many sugars can bend light (refractive index).
• Some can also rotate polarized light.
• Reducing power. Aldehyde or ketone can reduce
  (add an electron) to soluble metals (Fe gt
  Fe).
• Can form colored complexes with other compounds

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Chemical Properties of Reducing Sugars

• Reducing Sugars
• Some monosaccharides can act as Reducing Agents
  (electron donators). (I.e. Glucose and Fructose)
• They reduce Fehlings, Tollens, or Folins
  Reagents
• We will use these properties of sugars for
  understanding their physical properties, for
  characterization, and additional chemistries for
  analysis and characterization.

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Simple Sugars Reducing Abilityp. 149-150

• Some monosaccharides can act as Reducing Agents
  (electron donators). (ie. Glu and Fru)
• These are metal ions dissolved in either acid or
  alkali media (remember from wet ashing
  techniques).
• The extent of metal reduction can be related to
  sugar concentration via a standard curve.
• The Response is either the amount of initial
  metal ion, the amount of reduced metal present,
  or a color formed.

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Examples of Reducing Sugars and Non-Reducing
Sugars

• REDUCING
• D-glucose
• D-fructose (preferably under alkaline conditions)
• Maltose
• Maltotriose

• NON-REDUCING
• Sucrose
• Raffinose
• Cellulose
• Amylopectin

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Oligosaccharides

• Oligosaccharides or compound carbohydrates are
  repeating or mixed units of simple sugars.
• Often made of 2-4 simple sugars, but can be as
  large as 20 units long.
• Even though these sugar chains can be big they
  are still considered relatively low MW compounds,
  and will yield mostly monosaccharides on
  hydrolysis.
• Units are joined via glycosidic linkages.
• Examples sucrose, lactose, maltose, maltotriose,
  stachyose, raffinose

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Polysaccharides

• Polysaccharides or complex carbohydrates are
  generally very large molecular weight molecules
  also composed of monosaccharide chains.
• Important food polysaccharides
• Starch (amylose, amylopectin, dextrin)
• Fiber (cellulose, hemicellulose, lignin)
• Pectin (galacturonic acid polymers)
• Gums (natural and synthetic hydrocolloids)

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Optical Activity of CHOs

• Simple sugars, like most other compounds that
  contain one or more chiral carbons, can rotate
  the plane of polarized light.
• (Note chirality means that a compound cant be
  superimposed on its mirror image 4 different
  function groups attached)
• Rotation left is (-) levorotation
• Rotation right is () dextrorotation
• The extent to which a compound in solution
  rotates the plane of polarized light can be
  measured with a polarimeter

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Polarimetry (p. 169)

• The extent to which a compound will rotate the
  plane of polarized light.
• The concentration of a carbohydrate in solution
  can be determined from the measured optical
  rotation, provided the nature of the compound,
  temperature, and wavelength of light are held
  constant.

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Polarimetry

• LIMITATIONS
• It can be used only on clear liquid samples
• It is accurate only for solutions of pure sugar
  or other compounds whose specific optical
  rotation is known
• Used to quantify a specific CHO
• Can also be used for obtaining approximate values
  of other sugars.

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Refractive Index

• When light passes from one medium to another, it
  changes direction, being bent or refracted.
• The ratio of the sine of the angle of incidence
  to the sine of the angle of refraction is called
• Index of refraction or
• Refractive index

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Refractometer

• By holding the nature of the compound,
  temperature, and wavelength of light constant,
  the concentration of the compound can be
  determined by measuring the refractive index with
  refractometer

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Hydrometers Remember these?

• The hydrometer is an instrument that measures the
  density of a liquid. The scale is adjusted for
  the solid being determined
• Specific Gravity is defined as the ratio of the
  density of a substance to the density of a
  reference substance (usually water), both at a
  specific temperature
• Use in industry to obtain approximate values
• There are different scales such as Baume and
  Balling and are somewhat equivalent to the Brix
  scale for sucrose sucrose. (measured in degrees)
• 1Brix 1 sucrose

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Physical Nature of Sugar PolymersOligo- and
Polysaccharides

• Solubility varies greatly with each compound
• Some soluble in hot, but not cold water (starch)
• Others are not soluble at all in water without
  modification (hemicellulose polymers)
• Most are easily hydrolyzed in acidic conditions
  and are very stable in this media (except for
  some simple sugars)
• These properties make selective chemical analysis
  much easier

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Case 1 Hydrolysis in Orange Juice

• Sucrose hydrolysis occurs quite frequently in OJ.
  
• Sucrose inverts or hydrolyzes to form 1 molecule
  of glucose and 1 of fructose from the heat of
  processing and natural organic acids.
• Results in changes to sweetness
• Fructose and glucose are then succeptable to
  degradation (HMF formation, Fig. 10-3).
• HMF results in brown color formation, a smelly
  aroma, and a bitter/medicinal taste.

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Case 2 Pectin in Jelly

• Pectin is a complex polysaccharide made from
  individual units of galacturonic acid. Has the
  ability to bind water by the formation of
  hydrogen bonds.
• Gelling properties are highly influenced by pH,
  soluble salts (calcium) and presence of sugars.
• 2 primary types of pectin (Low and High methoxyl)

-OCH3
COOCH3
COOCH3
H20
o
O
n
OH
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Low Methoxyl Pectin
Ca

• Carboxyl groups
• use Ca to form ionic
• unions among pectin
• molecules
• Highly pH dependent
• Independent of solids
• content

Ca
Ca
Ca
Methylated pectin
82
High Methoxyl Pectins
High sugar content binds water and allows for
the formation of tri-dimensional structure
83
Nutritional Classification of CHOs

• Digestible Carbohydrates
• Glucose and fructose are absorbed in the small
  intestine.
• Polysaccharides are hydrolyzed before absorption
  and include lactose, maltoligosaccharides, and
  starch.
• Non digestible Carbohydrates Dietary Fiber.
  Fiber is further divided in to soluble and
  insoluble.

84

• NUTRITIONAL LABELING
• Proximate Analyses
• Analyze for Proximates M, F, P, A
• Carbohydrates are calculated by difference!!!
• Nutritional Label
• TOTAL CARBOHYDRATES is a difference calculated
  as
• CHO Food Weight (MFPA)
• In some instances for quality or nutritional
  claim purposes there is a need to obtain
  information about specific components (simple
  sugars, starch, fiber, etc)

85
Sample Extraction

• Extract CHO based on solubility.
• Solvent
• Water
• Hot ethanol (80).curious choice ?
• Most monos and oligos and some polys are highly
  soluble in Water and/or Hot EtOH.
• Most polysaccharides and proteins are not soluble
  in hot EtOH.
• Therefore, Hot EtOH will extract monos and
  oligos, but not polysaccaharides or interfering
  proteins.

86
CHO Analysis Problems

• 1. Dissolved gases, e.g., carbonated or fermented
  products, remove by drawing vacuum prior to
  analysis. 2. Pigment removal - variety of ways
  to remove e.g., charcoal, lead salts, organic
  solvent extraction. 3. Protein removal -
  proteins interfere with reducing and colorimetric
  determinations.
• may add ethanol or acetone (70-80 v/v) - most
  proteins will coagulate and can be filtered or
  centrifuged out.
• precipitation with heavy metals - e.g. alkaline
  Zn hydroxide protein ? ppt.

87
ANALYSIS OF CARBOHYDRATESMost common analysis
Monosaccharides

• SAMPLE PREPARATION
• Purification of samples is common since food
  matrices are complex. Removes interferences.
• MONOSACCHARIDES
• Remove polysaccharides (Hot EtOH), also
  inactivates any hydrolyase enzymes
• Clarification (centrifuge, filter, solvate, etc)
• May need to neutralize organic acids
  (Ca-carbonate, NaOH, etc)
• Remove charged particles (ion exchange)

88
Why Remove Charged Particles?

• Remember that acids result in hydrolysis
  reactions with some sugars
• Dont want any changes to the sugar during
  analysis ie. glucose and fructose suddenly
  appearing in your sample.
• Nice sample clean-up step, gets rid of trash
  and other charged particles that could interfere
  with analysis

89
Ion Exchange Resins

• Treatment 1st with cation exchange resins can
  cause the hydrolysis of oligosaccharides, among
  other reactions. Therefore, the order in which
  you use resins is very important.
• Resin is generally mixed with the sample and than
  filtered out of solution
• Small mini-columns can also be used, and are much
  faster to use
• Anion Exchange resins are therefore used to
  remove organic acids.

90
Orange Juice

• Key points
• Stationary phase
• Mobile phase
• Charges
• Ions
• Counter-ions
• Binding
• Elution

Stationary Phase Contains Anions Anion Exchange
Resin
91
Methods for CHO Analysis

• Chemical Methods (pp. 148-150)
• Enzymatic Methods (pp. 154-155)
• Instrumental Methods (pp. 150-154)

92
Chemical Methods(Spectrophotometric)

• ALKALINE FERRICYANIDE CHO in basic solution (pH gt
  10.5) reduce ferricyanide to ferrocyanide
• Forms Prussian Blue that is measured at 700 nm
• PHENOL SULFURIC ACID reacts with both reducing
  and non-reducing CHO to form various furans
  (furfural, HMF, furaldehyde See Figure 10-3)
  which condenses with phenol into a near pink
  color.
• Read on spec at 490 nm

93
CHEMICAL METHODS

• PHENOL SULFURIC ACID (cont.)
• Applies to most all sugars mono, oligo and
  polysaccharides
• A standard curve should be ran using standard
  sugars in the same proportions as they are
  present in food.
• Example In potatoes, glucose and fructose are
  present in a 11 ratio, therefore prepare a
  standard in the same proportions.
• Concentrations of the standard curve should
  always be higher that the concentration on the
  analysis sample (Dilute if needed)

94
Other Chemical Methods

• ANTHRONE reacts primarily with hexoses
• Read at 620 nm
• Anthrone carbohydrate H2SO4 ? blue-green
  color
• Also measuring furan derivatives
• 3,5-DINITROSALICYLIC ACID reacts with reducing
  sugars in alkali to form brown-red color that can
  be measured on a spec
• RESORCINOL (a phenol) reaction is primarily with
  ketoses to form a colored complex
• ORCINOL (a phenol) reacts with pentoses with 5X
  more color than hexoses

95
Other Chemical Methods

• MUNSON-WALKER
• Carbos are oxidized in presence of copper sulfate
  and alkaline tartarate under controlled
  conditions.
• Alkali required to keep copper in solution.
• Copper oxide is converted to cuprous oxide by
  heating
• Concentration expressed gravimetrically
  (electrolytic deposition) or following a
  titration using sodium thiosulfate and/or
  potassium permanganate.
• Other modifications of this assay exist (p. 150)

96
What are Enzymes?
CHO ANALYSIS ENZYMATIC METHODS

• Enzymes are large proteins produced by living
  cells, plants and other organisms.
• All living organisms require enzymes for growth,
  and for production and utilization of energy.
• Enzymes are biological catalysts.?

97
ENZYME TERMS

• CATALYST substance that increases the reaction
  speed with out participating in it.
• INHIBITORS substance that decreases the
  reaction speed with out participating in it.
• SUBSTRATE the compound which is acted upon by an
  enzyme, usually results in a new or significantly
  altered compound.

98
Enzymatic Degradation of Lactose
LACTOSE
ENZYME- SUBSTRATE COMPLEX
GLUCOSE
GALACTOSE
LACTASE
99
Oxidase Methods

• CHO oxygen ----gt oxidized CHO H2O2
• The enzyme oxidase catalyses the above reaction
• Add glucose oxidase to samples to measure glucose
• Add fructose oxidase for fructose etc.
• We can indirectly measure CHO by the amount of
  hydrogen peroxide given off.
• We can also measure the amount of oxygen consumed
  by using an oxygen electrode.

100
Gas Chromatography(Analysis for individual CHOs)

• Sugars are not volatile, so they require a
  derivatization step to make them burnable.
• Volatile derivatives can be made by a simple
  one-step chemical reaction
• Most common forms acetates, ethyl ethers, and
  trimethsilyl ethers
• Method used depends on sugars you are testing
  for, which depends on the GC temperature needed
  to volatilize the sugar

101
Reduction to Alditol (for reducing sugars)

• STEPS
• Sugars are reduced to alditols using excess
  sodium borohydride, NaBH4 (See Fig 10-7).
• This causes reduction of aldehydes and ketones to
  primary alcohols
• Alditols (the alcohol form) are then acetylated
  with acetic anhydride in order to produce alditol
  peracetates, which can be analyzed by GC (acetic
  acid derivatives are volatile)

102
Other Derivatization Steps

• Acetates
• Treat sugar with acetyl chloride or acetic
  anhydride - Reflux about 4 hours in the presence
  of an organic solvent
• Methyl ethers
• Treat sugar with either methyl iodide/silver
  oxide or dimethyl sulfate/NaOH
• TMS ethers
• Treat sugars with pyridine and a methylsilyl
  (silica based) media.

103
Why HPLC CHO methods are cool?

• HPLC carbohydrate methods have replaced GC
  methods because they dont require a
  derivatization step
• HPLC methods are non-destructive
• GC requires derivatization because carbohydrates
  are not volatile
• GC derivatization steps must be 100 complete to
  obtain good results, which is difficult.

104
HPLC 101 (High Performance Liquid Chromatography)

• Stationary phase (usually a non-ionic resin)
• Mobile phase is usually 100 water
• Compounds elute based on size and affinity to
  stationary phase
• Common sugars
• Sucrose
• Glucose
• Fructose
• Maltose
• Lactose

105
HPLC Detectors for CHO Analysis

• TYPES OF DETECTORS
• Refractive Index Measures the changes in
  refractive index of a solution coming out of and
  HPLC column
• Can be applied to many carbohydrates
• Limitations It is sensitive to changes in flow,
  pressure, temperature, and generally requires
  high CHO concentrations.

106
Refractive Index Detector
Light source
Detector
Flow Cell
Reference Cell
Flow direction
107
How do I choose? GC or HPLC

• HPLC methods are often preferred over GC method
  because they dont require a derivatization step
• GC requires derivatization because carbohydrates
  are not volatile
• GC derivatization steps must be 100 complete to
  obtain good results, which is difficult.
• BUT.some sugars are best analyzed by GC methods
  (ie. sugar alcohols, pentoses)

108
STARCH

• Amylose D-glucopyranose with alpha-1,4 bonds
  between glucose units. Repetitive unit is
  maltose.Generally 200-2500 units
• Amylopectin It is also formed by glucose units,
  but every 12-15 units it has an alpha-1,6 bonds
  which creates branches.

109
STARCH

• Direct Acid Hydrolysis - dilute acid hydrolysis
  of 100-200 mg starch/liter in 0.4N HsSO4 -
  refluxed for 4 hrs. - yields glucose which can be
  quantified by several different methods
  (previously discussed)
• Limited applications may have problem with
  breakdown of other polysaccharides to yield
  reducing sugars including glucose
• Likely the most accurate method Enzymes

110
Here is a quick example
111
Enzymatic Determination of Starch or other simple
sugar

• PRINCIPLE
• Starch is hydrolyzed into glucose units by
  enzymatic conversion
• D-glucose can then be quantified by enzymatic
  methods

112
Enzymatic Determination of Starch

• ADVANTAGES
• Enzymes give a lot of specificity to the assay,
  thus allowing the analysis of very complex
  matrices.
• There are a lot of kits in the market for the
  commercial determination of starch coupled to the
  determination of glucose
• Problems amylase enzymes must be purified so
  that they are not contaminated with other
  polysaccharide degrading enzymes

113
Ch. 8 Crude Fat Analysis
Monoacylglyceride
114

• FAT ANALYSIS

• So, how do we analyze for fat?
• First, we need to know what IS fat?
• Working definition Compounds that are soluble
  in organic solvents (usually ethers). They are
  derived from living organisms and usually contain
  fatty acids.
• Most fats in foods exist as TAGs
  (triacylglycerols), which are non-polar.
• SIMPLE LIPIDS include fatty acid esters with
  glycerol (TAGs, DAG or MAGs), and long chain
  alcohols (waxes).

115
Crude Fat Components

• Fats/Oils- TAGs
• Waxes- long-chain alcohols and fatty acids
• Phospholipids- phosphoric acid esterified to a
  fatty acid chain (phosphatides)
• Glycolipids- simple sugar esterified to a fatty
  acid chain
• Sterols- specialized ring structure, serving in
  biological functioning
• Free Fatty Acids- carbon chain of various
  lengths, serving as a pool from with fats are
  synthesized.

116
Fat Analysis

• Analytical Methods generally rely on extraction
  of the fat from a food and weighing the extracted
  fat
• FDA is interested in a method that is based on
  amount of fatty acids in 100g of food.
• Different foods have to be treated differently.
• Oxidation or other chemical reactions can cause
  deterioration of the lipid and interfere with the
  assay.

117
Sample PreparationPREDRYING

• Sample drying is used to remove water that will
  interfere with sample contact with solvent.
• However, drying can make it difficult for solvent
  to reach fat, therefore long extraction times are
  often called for
• Freeze drying is much better than oven drying
  since it prevents case hardening

118
PARTICLE SIZE REDUCTION

• Samples can be ground after drying to ensure
  efficiency of extraction by increasing surface
  area
• Coffee grinders or blenders with sieves are used
  commonly.
• Some samples are hydrolyzed with acid to achieve
  complete extraction (cocoa, chocolate).
• Dry, cooked products are notoriously problematic
  and give higher lipid values after hydrolysis.
• Hydrolysis breaks covalent bonds and ionically
  bound lipids (bound to proteins and CHOs).

ACID HYDROLYSIS
119
SOLVENT SELECTION

• Solvent selection is important since a solvent
  that is too polar will poorly extract nonpolar
  lipids and will extract non-lipid materials (I.e
  carbohydrates)
• Too nonpolar will be inefficient for more polar
  lipids.
• IDEAL SOLVENT FOR FAT EXTRACTION
• High solvent power for lipids
• Low solvent power for nonlipids
• No residue
• Evaporate easily (low heat of vaporization)
• Low boiling point
• Non flammable / not explosive
• Nontoxic
• Low surface tension with food
• Single compound
• Cheap
• Non-hygroscopic

120

• Solvent Selection

• Ethyl ether is used a lot but is
• Very flammable,
• Explosion hazard
• Forms peroxides
• Expensive.
• Petroleum ether is not an ether (pentane and
  hexane mainly), is not too expensive and is an
  excellent solvent for lipids
• More selective for more hydrophobic lipids
• Non hygroscopic
• Less flammable
• Cheaper
• Mixtures of ethyl ether and petroleum ether are
  common
• Mixtures of chloroform and methanol are also
  common (Bligh-Dyer)

121
SOLVENT SELECTION

• Solvent selection is critical to fat extraction.
• Solvents such as methanol, ethanol, and acetone
  will readily dissolve fats, but would also
  extract large amounts of moisture, CHO, and
  protein.

122
Quick Primer on Solvents

• In very general terms, Polarity refers to how a
  solvent behaves in comparison to water. Measured
  by a Polarity Index (P)

• Solvent P
• Water 10.2
• Chloroform 4.1
• Diethyl ether 2.8
• Hexane 0.1

123

• Continuous Solvent Extraction
• GOLDFISCH Extraction

• Solvent Extraction Solvent from a continuously
  boiling solvent source flows over the sample held
  in a sample thimble. Fat content is measured by
  weight loss of the sample or by weight of fat
  removed.
• Ethyl ether, petroleum ether, hexane, or
  methylene chloride are common solvents
• Extraction times range from 4-16 hrs
• Sample is weighed, mixed with sand to increase
  surface area, and dried in a forced air oven.
• Lipid is extracted by the solvent
• Solvent is removed by evaporation or under
  reduced pressure, then dried at 100C for 30 min.

124
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125

• Semi-continuous Solvent Extraction
• SOXHLET Extraction

• Similar sample prep to Goldfisch method
• Fat is extracted, semi-continuously, with an
  organic solvent
• Sample is in contact with the solvent in the
  extraction chamber for 5-10 min, then siphons
  back to the boiling flask (see diagram)
• Extraction time 5-6 drops per second (4 hr). 2-3
  drops per second (16 hrs).
• Fat content is measured by weigh loss of sample
  or weight of fat removed

126
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127
Specific Fat Extractions

• We will discuss several methods to extract
  lipids.
• Each method is usually designed for a particular
  food matrix
• Primary issues to contend with
• Moisture content
• Type of fats present
• Time
• Efficiency
• Secondary analysis (ie. fatty acid profiling)

128
Fat Extractions In General

• The simplest fat extractions are conducted by
  grinding a sample to the smallest attainable
  particle size, which increases the surface to
  volume ratio.
• Samples are then mixed with an organic solvent to
  extract the fat.
• The solvent in filtered through a hygroscopic
  salt to remove moisture and then evaporated.
• The reside is primarily lipid.
• Percent fat is then calculated as a difference in
  weight from the initial sample.

129
Refractive Index Methodvery rapidnot so accurate

• Used primarily for processed meats
• Fat is extracted with a solvent.
• RI of the solvent is compared to the refractive
  index of the extracted meat
• Values compared to known lipid concentrations
• QC tool only

130
Gravimetric Fat Determination Chloroform-Methanol

• The Bligh, Dyer and Folch Method
• Extract sample with chloroform-methanol-acetic
  acid
• Methanol and acid helps to dissolve non-lipids
• Sample placed into sep-funnel and bi-layer formed
  by adding water
• Chloroform extracts most lipid classes
• No extraction of sugars, amino acids etc.
• Sample is repeatedly washed with water to remove
  all non-lipid components
• Highly accurate method for low fat, high
  phospholipid fish samples.
• Not used extensively for most foods, but is
  considered a rapid method for analysis

131
Mojonnier or Roese Gottlieb Fat Extraction
(MoJo)

• Solid or liquid samples
• Specifically
• Milk
• Cheese
• Bread
• Pasta
• Method
• Ammonium hydroxide - denatures proteins
• Ethanol- breaks gels
• Ethyl ether-mix
• Pet. Ether-mix
• Centrifuge
• Collect ether-repeat 2X

• Method pp. 207-208 in the text
• Beware of Step 9, p. 208. Evaporate the solvent
  in the dish on the electric hot plate at lt 100C
  in a hood.

See Fig. 13-3
Mojo extraction flask
Ether Fat
Sample
132
BABCOCK Milk Fat Extraction

• Used exclusively for milk, ice cream, and cream
  testing.
• Uses sulfuric acid to digest protein, generate
  heat, and release fat.
• Samples are heated and centrifuged to induce a
  bilayer.
• Special flasks are used to determine fat in the
  sample.

133

• GERBER METHOD
• Modified Babcock method. Uses sulfuric acid and
  isoamyl alcohol, which helps prevent charring of
  milk sugars
• DETERGENT METHOD
• Used mostly for milk
• A detergent (dioctyl sodium phosphate) reacts
  with protein to form a protein-detergent complex
  to break up emulsions and release fats.
• The percent fat is measured volumetrically.

134
Supercritical CO2

• A fluid (usually CO2) is brought to a specific
  temperature-pressure combination, to have
  supercritical fluid solvent properties. The
  supercritical fluid dissolves the fat in the
  sample.
• Fat is precipitated from the the supercritical
  fluid by dropping solution pressure, so
  precipitated fat can be dried and weighed
• Advantage- uses no hazardous organic solvents,
  just carbon dioxide at its supercritical state

135
Supercritical State

• Supercritical fluids are, by definition, at a
  temperature and pressure greater than or equal to
  the critical temperature and pressure of the
  fluid.
• CO2 Critical Conditions
• 1,070 psi
• 31C
• Applications using CO2 are typically 32-49C at
  pressures from 1,070-3,500 psi.

136
OTHER METHODSpp. 123-127

• Low resolution NMR (nuclear magnetic resonance)
• X-Ray Absorption
• Dielectric
• Infrared (used a lot)
• Ultrasonic
• Colorimetric
• Density (for oil seeds)

137
CHAPTER 14- FAT CHARACTERIZATION

• PHYSICAL PROPERTIES
• IODINE VALUE
• SAPONIFICATION NUMBER
• ACID VALUE/FREE FATTY ACIDS
• OXIDATION
• HYDROLYSIS
• PEROXIDE VALUE
• OXIDATION TESTS

138

• Why are we interested in analysis of lipids?
• 1. Nutritional importance (omega 3, saturated
  fat, cholesterol, fat sol vitamins).
• 2. Affect oxidative stability of foods
  (stability of many foods is affected by fatty
  acid composition or presence of enzymes that act
  on lipids such as lipoxygenase, lipase etc).
• 3. Affect physical properties of foods (melting
  behavior in chocolate, margarine, etc.)

139

• Why are we interested in analysis of lipids?
• 4. Related to quality in many food products
  (stored fish, vegetable oils etc)
• 5. There are many changes in lipids that occur
  during processing that affect the quality of the
  food product (frying)

140
SAMPLE PREPARATION

• Use previously discussed crude fat extraction
  methods
• Ensure that the samples are visually clear of
  sediment.
• Make sure lipid is free of moisture for
  quantitative measurements
• Avoid heat, light and air during sample storage
  (may cause lipid oxidation)

141
Physical Properties of Lipids

• Each lipid source of fat has at least one
  distinguishing feature that separates it from
  other sources of fat (chemical, sensory, or
  physical).

142
Refractive Index

• Each oil has its own refractive index.
• It is used as a qualitative measure for
  adulteration of pure oils with other oils.
• Quantitation can be done using standard curves of
  pure oils.
• This is an AOAC approved method for determining
  oil in flaxseed, soy, peanuts, palm kernel, meat
  and fish.

143
Melting Point What does it tell us about the
lipid?

• Reflects degree of saturation/stability
• Example
• Wiley Melting Point Good for heavy industrial
  applications such as production scale, deep-fat
  frying

144
Melting Point

• Melting point - temperature at which we get a
  change from solid to liquid. Fats are blends of
  triacylglycerides so there is no sharp melting
  point.
• Dissolution point - more accurate description of
  melting. Solid fat becomes dissolved in liquid
  oil.
• Examples
• Palm oil (fully hydrogenated) 58-60C
• Typical shortening 46C

145
Melting Point- Methods

• Capillary Melting Point Fat is put into
  capillary tube, sealed, and then heated slowly in
  a water bath until completely clear.
• Wiley Melting Point Fat is formed into a
  standard sized disk (1/8 x 3/8) and then
  chilled to solidify. Disk placed in alcohol-water
  bath and slowly heated until the disk becomes a
  sphere.

146
Melting Point- Methods

• Dropping Melting Point Automated (instrumental)
  method where fat is placed in a cup that has a
  0.11 hole in bottom. Cup is heated until oil
  melts and flows out bottom of cup - drops
  interrupt a light path for detection and temp.
  recorded.
• Slip Point (not used in US often) Fat is put in
  capillary tube but not sealed. Placed in temp
  programmed water bath (vertically) and warmed
  until fat plug "slips" up (moves up the
  capillary).

147
Smoke, Flash and Fire Points

• Heat
• Triglycerides ----gt Fatty Acids Glycerol -gt
  Smoke
• Fat is placed in cup and heated.
• Smoke point - temperature when see wisps of smoke
• Flash point temperature where a flash of fire
  is seen on the oil surface
• Fire point - temp where continuous ignition is
  supported (constant burning beyond a flash)
• Free fatty acids (degradation during heating),
  bits of food, emulsifiers etc. will all alter
  (usually lower) these values.

148
Smoke, Flash and Fire PointsWhere there is smoke
Soy Bean Oil
149
Smoke, Flash, and Fire Points What does it tell
us about the lipid?

• Degree of free fatty acid hydrolysis, oxidation,
  oil purity, usage parameters
• Example, smoke specifications for deep-fat frying
  oil
• 225C for light duty
• 235C for heavy duty

150
Cloud Point (Cold Test)

• Some applications require that oil remain liquid
  at refrigeration temps.
• Ie. mayonnaise and salad dressings.
• Official method - hold oil in ice bath and time
  noted until cloudiness is observed. 5 hrs is
  minimum - 20 hrs good
• Rapid method - chill at -60C for 15 min, hold at
  10C - no visible solids after 30 min, product is
  good.

151
Iodine Value

• Measure of the degree of unsaturation in an oil
  or the number of double bonds in relation to the
  amount of lipid present
• Defined as the grams of iodine absorbed per 100-g
  of sample.
• What does it tell us about the oil?
• The higher the amount of unsaturation, the more
  iodine is absorbed.
• Therefore the higher the iodine value, the
  greater the degree of unsaturation.

152
Iodine Value

• A known solution of KI is used to reduce excess
  ICl (or IBr) to free iodine
• R-C-C C-C-R ICl ? R-C-CI - CCl-C-R ICl
  Excess
  (remaining)
• Reaction scheme ICl 2KI ? KCl KI I2
• The liberated iodine is then titrated with a
  standardized solution of sodium thiosulfate using
  a starch indicator
• I2 Starch thiosulfate colorless endpoint
• (Blue colored)

153
Iodine Value

• Used to characterize oils
• Following hydrogenation
• During oil refining (edible oils)
• Degree of oxidation (unsaturation decreases
  during oxidation)
• Comparison of oils
• Quality control

154
Iodine value g absorbed I2/ 100 g fat
Highly saturated
High in 181
High in 181 and 182)
181, 182, 183
181, 182, 183 (longer chains)
What can we conclude about the COMPOSITION or
STRUCTURE of each of these oil types?
155
Automated Iodine Value Determination
Standard Iodine Value A 23 B 44 C 67 D
89 E 111
Consumption over time
Measures IBr or ICl Consumption (neg. peak)
156
Saponification Value