CHAPTER 9: PROTEIN

ANALYSIS
PROTEIN ANALYSIS
Why are we interested in the overall
protein content of a food?
Functionality (examples)
Disulfide bonds (wheat gluten)
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.
Functionality in Proteins
Hydration properties
Water-holding capacity
Viscosity modifiers
Solubility
Protein-Protein Interactions
Gel formations
Precipitation
Surface Properties
Emulsifiers
Foaming
Surface tension
Protein Content of Some Foods
Milk (dry) 22-25 Rice 7.5-9
Milk (skim) 3.2 Wheat flour 9.8-13.5
Egg (dry) 35 Walnuts 15-21
Beef (dry) 81-90 Potato 10-13
 PROTEIN ANALYSIS
Proteins are important for nutrition and have important
functions in our cells
Proteins weigh from 5000 daltons (5KDa) to >1 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)
 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
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.
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.
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)

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.
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).
KJELDAHL NITROGEN
3 Stage Process:
1. Digestion with acid + catalysis
2. Distillation with steam and alkali
3. Titration with acid and indicators.
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.
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
 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?

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
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 protein
2. Time consuming at least 2 hrs
3. Poor precision when compared to other
methods
4. Corrosive (dangerous) method
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
@ 440nm)
2. Reaction with phenol and
hypochlorite to form indophenol
which can be read in a SPEC (visible
@ 630nm)
3. Ion sensitive electrode for ammonia
or ion chromatography
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?
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.
 Lets back up just a bit
Remember 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

O O O
H N 2+ H N N H
Cu
H R H R 2+ H R
Cu
O OH- O O
H N H N N H

Purple biuret complex

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
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.
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.
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
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
 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?
Ill Take Amino Acids for $100, Alex.
O
PHENYLALANINE

O
TYROSINE

N
HO

TRYPTOPHAN
N
N
UV Absorption (280 nm)
Absorbance according to Beer's Law:
A=abc
Where A = Absorbance
a = absorptivity, a constant that is
characteristic of a particular chemical
species at a particular wavelength.
b = path length in cm
c = concentration of the absorbing
chemical species
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:
A=ebc
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
 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
 DYE BINDING METHOD
Proteins will bind to certain types of dye.
When this binding occurs, the protein-dye
complex will precipitate.
DYE BINDING
Anionic dyes and Bradford Main types

The unbound dye is then easily

Anionic dyes use principle that proteins bind dye
and become insoluble.

determined with a spectrophotometer using

Basic groups bind anionic dye (+ charge at neutral

a standard curve with varying dye

pH)

Unbound dye inversely proportional to protein

concentrations.
Ads: quick, can estimate available lysine, no

Using the amount of dye initially added to

nasty reagents, NPN does not interfere, precise

the protein solution, the amount of protein

Disads: not sensitive, calibration curve
required for each protein, many interferences

can be calculated. more protein

More Protein,
less color

Less Color
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.
 Bradford assay-Coomassie Brilliant Blue

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.
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
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.
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
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
CHAPTER 15: PROTEIN
ANALYSIS
 CHAPTERS 15
PROTEIN 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
CHAPTERS 15
PROTEIN 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
COMMERCIAL SEPARATION OF
PROTEINS-example

Whey/Soy Proteins
Waste proteins
Enzyme Recovery
Other nutritional
supplements and food
additives
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
Salting Out----Ionic Strength

Ionic Strength = S MiZi2

Mi = Molarity of ion
Zi = Charge of ion
Na+ Cl-
1M NaCl = S (1 X 12) + (1 X 12)= 1
Ca2+ Cl-

1M CaCl2 = S (1 X 22)+ + (2 X 122-) = 3

NH4 SO4

1M (NH4)2SO4 = S (2 X 12) + (1 X 22) = 3

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
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
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.
 CATION EXCHANGE
solute molecules
The square molecules

Key points:
have greater affinity
K+
- K+ K+ -K+ K+ for the stationary phase
- - -
- Stationary
because they are + charged
K+ - K+ - - K+
- - K+
phase
K+ -
K+ - K+
- -
-
- - K+- - -K+K+
K+
- -
K+ - - - - - - Mobile phase
-
K+
- K+ --
K+
- - K+ Charges
K+ -
K+
- K+ -
- K+
K+ is the counter ion and Ions
K+ - - -
Counter-ions
- K+ is exchanged for the +
- charged solute
K+ -
- - K+
Binding
molecules
-K+ K+ -
Elution
Adsorption
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.
Membrane Separation
Membrane Separation
Ultrafiltration Retentate what remains in
the sample
Ultrafiltrate what passes
through the membrane
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)
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
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.
Electrophoresis- SDS Page
Separation based on size.
Proteins bind to SDS to become negatively
charged.
SDS = sodium dodecyl sulfate => 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.
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.
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.
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
CH.Can
10: Carbohydrates
somebody (CHO)
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.
Classification of Carbohydrates

The Saccharides
Monosaccharide
Smallest form, non-hydrolysable.
Oligosaccharide
Made of several monosaccharides,
hydrolysable.
Polysaccharide
Very large polymers of monosaccharides
 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).
 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++ => Fe+).
Can form colored complexes with other compounds
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.
Simple Sugars: Reducing Ability
p. 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.
Examples of Reducing Sugars and
Non-Reducing Sugars

REDUCING NON-
D-glucose REDUCING
D-fructose (preferably Sucrose
under alkaline Raffinose
conditions)
Cellulose
Maltose
Amylopectin
Maltotriose
 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
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)
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
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.
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.
 When light passes
Refractive Index 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
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
 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
Physical Nature of Sugar Polymers
Oligo- 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
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.
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.
-OCH3
2 primary types of pectin
COOCH3
(Low and High
COOCH3
methoxyl) H20
O o
n
OH
Low Methoxyl Pectin

COO- Ca++ Carboxyl groups

use Ca++ to form ionic
COO- Ca++ unions among pectin
COOCH3 molecules
Highly pH dependent
COO- Ca++ Independent of solids
content
Ca ++
COO-

Methylated pectin
High Methoxyl Pectins

COOCH3
High sugar content
COOCH3 binds water and
allows for the
COOCH3 formation of tri-
dimensional
COOCH3
structure
COOCH3
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.
 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 (M+F+P+A)

In some instances for quality or nutritional

claim purposes there is a need to obtain
information about specific components (simple
sugars, starch, fiber, etc)
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.
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.
ANALYSIS OF CARBOHYDRATES
Most 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)
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
Ion Exchange Resins
Treatment 1 with cation exchange resins can
st

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.
Orange Juice CATION EXCHANGE
solute molecules
The square molecules

Key points:
have greater affinity
K+
- K+ K+ -K+ K+ for the stationary phase
Stationary - - -
- K+ - - K+
because they are + charged
Phase K+ -
K+ - -
- K+ Stationary
Contains K+K+ -- - K+ - - K+ phase
-
-
- K+ - K+ - -
Anions K+ - -
Mobile phase
- - - -
Anion - K+ - K+
K+ - -
- K+ -
Exchange K+ -
- K+ -
- K+
K+ is the counter ion and
Charges
Resin K+
K+ - - -
-
- - K+
- K+
is exchanged for the +
charged solute Ions
K+ - molecules
-K+ K+ - Counter-ions
Binding
M o st+ c Elution
h
a
rg
ed

l
e
a
s
t
+
c
h
a
r
g
e
d
Methods for CHO Analysis

Chemical Methods (pp. 148-150)

Enzymatic Methods (pp. 154-155)

Instrumental Methods (pp. 150-154)

 Chemical Methods
(Spectrophotometric)

ALKALINE FERRICYANIDE CHO in basic

solution (pH > 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
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 1:1 ratio,
therefore prepare a standard in the
same proportions.
Concentrations of the standard curve
should always be higher that the
 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
HO brown-red color that can be measured on
a spec
OH

RESORCINOL (a phenol) reaction is

primarily with ketoses to form a colored
complex
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.
CHO ANALYSIS:
ENZYMATIC METHODS

What are Enzymes?

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.?
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.
Enzymatic Degradation of
Lactose
LACTOSE ENZYME-
GLUCOSE
SUBSTRATE
COMPLEX
GALACTOSE

LACTASE
 Oxidase Methods
CHO + oxygen ----> 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.
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
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
 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.
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.
 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
 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.
Refractive Index Detector

Light source

Detector

Flow Cell
Reference Cell

Flow direction
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)
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.
 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
 Lactose
H2O
beta-galactosidase

D-galactose + D-glucose
NAD+
galactose dehydrogenase
NADH
galacturonic acid
365nm

Here is a quick example

galactose dehydrogenase also reacts
ENZYMES are useful in analysis because of
their specificity
Reaction affected by pH, time, temperature,
Enzymatic Determination of Starch or
substrate concentration, activators,
inhibitors
other simple sugar
D-GLUCOSE/ D-FRUCTOSE / D-SORBITOL

glucose fructose PRINCIPLE

HEXOKINASE
ADP
HEXOKINASE Starch is
hydrolyzed into
ATP glucose units by
glucose-6-P fructose-6-P
enzymatic
G-6-P de- NADP+ NADP+ conversion
hydrogenase PGI D-glucose can
NADPH NADPH then be
gluconic acid-6-P glucose-6-P quantified by
365nm 365nm enzymatic
PGI = phosphoglucoisomerase methods
 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
Ch. 8 Crude Fat Analysis
H
O

H C O C Fatty Acid Chain

H C OH

H C OH

H
Monoacylglyceride
 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).
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.
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
Sample Preparation

PREDRYING

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
 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.
ACID HYDROLYSIS

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.
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
 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)
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.
 Quick Primer on Solvents

In very general Solvent P

terms, Polarity Water
refers to how a 10.2
solvent behaves in Chloroform
comparison to 4.1
water. Measured
by a Polarity Diethyl ether 2.8
Index (P) Hexane 0.1
 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.
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
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
 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
Refractive Index Method
very 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
 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
Mojonnier or Roese Gottlieb
Fat Extraction (MoJo)

Solid or liquid samples Method pp. 207-208 in the

Specifically text
Milk Beware of Step 9, p. 208.
Cheese Evaporate the solvent in the
Bread dish on the electric hot plate
Pasta
at < 100C in a hood.
Method:
1. Ammonium hydroxide See Fig. 13-3
- denatures proteins Mojo extraction flask
2. Ethanol- breaks gels
3. Ethyl ether-mix
4. Pet. Ether-mix
5. Centrifuge Ether + Fat
6. Collect ether-repeat
Sample
 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.
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.
 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
 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
OTHER METHODS
pp. 123-127
Low resolution NMR (nuclear magnetic
resonance)
X-Ray Absorption
Dielectric
Infrared (used a lot)
Ultrasonic
Colorimetric
Density (for oil seeds)
CHAPTER 14- FAT CHARACTERIZATION

PHYSICAL PROPERTIES
IODINE VALUE
SAPONIFICATION NUMBER
ACID VALUE/FREE FATTY ACIDS
OXIDATION
HYDROLYSIS
PEROXIDE VALUE
OXIDATION TESTS
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.)
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)
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)
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).
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.
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
 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
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.
 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).
 Smoke, Flash and Fire Points
Heat

Triglycerides ----> Fatty Acids + Glycerol -> 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.
Smoke, Flash and Fire Points
Where there is smoke

Soy Bean Oil

800
700
600
Temperature (F)

500
400 Fire
Flash
300
Smoke
200
100
0
0.05 0.1 0.2 0.5 2 5 10
Free Fatty Acid (%)
 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
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.
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.
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)
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
Iodine value: g absorbed I2/ 100 g fat

Iodine Value of some oils (Table 14-2)

Source I2 Value
Beef Tallow <50 Highly saturated
Olive, Palm, < 100 High in 18:1
Peanut
Corn, Cottonseed 100 - 130 High in 18:1 and 18:2)
Linseed, Soybean, > 130
18:1, 18:2, 18:3
safflower, conola
Fish >150 18:1, 18:2, 18:3
(longer chains)

What can we conclude about the COMPOSITION

or STRUCTURE of each of these oil types?
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)
 Saponification Value
Saponification is the process of breaking down
or degrading a neutral fat into glycerol and
fatty acids by treating the sample with
alkali.
Heat
Triacylglyceride ---> Fatty acids + Glycerol
KOH

Definition: mg KOH required to titrate 1g fat

(amount of alkali needed to saponify a given amount
of fat)
Typical values: Peanut = 190, Butterfat = 220
Saponification Value
What does it tell us about the
lipid???

The mg KOH required to saponify

triacylglycerides into glycerol plus fatty
acids is related to:
average fatty acid chain length or
average fatty acid molecular weight
Divide molecular weight by 3 to get
average of the fatty acids present
 LIPID OXIDATION (Fig. 14-2)

35
Lipid System Under
Reactants and Products

30
Oxidizing Conditions
25
Oxygen Uptake
20
Peroxides
15
Secondary Products
10

1 2 3 4 5 6 7 8 9

Time
Free Fatty Acid Titration
Since FFAs are indeed ACIDS, they can
be titrated for similar to a titratable
acidity.
Crude fat is extracted with petroleum
ether, and removed by rotary evap.
Oil sample is weighed and dissolved in
boiling ethanol (~60C). Add indicator
Titrate with standard base.

%FFA = (V) (N) (F) (100)

(S.W.) (1000)
Where, V = mL base, N = [base],
F = acid equiv. wt. of oleic acid
Free Fatty Acids (FFAs)
What does it tell us about the
fat??
Degree of hydrolysis (hydrolytic
rancidity)
Example: good frying oil should have
0.05% max. FFAs (as oleic acid)
High level of FFA means a poorly
refined fat or fat breakdown after
storage or use.
Lipid Oxidation
Addition of oxygen to a lipid will result in
chemical changes that affect flavor, aroma,
viscosity, level of FFAs, and the retention of
fat soluble vitamins.
High iodine values affect the rate of
oxidation.
Can we measure lipid oxidation?
Measure the oxidized lipids (GC, difficult)
Measure the oxidation by-products
(peroxides and maybe FFAs etc.)
Measure effects of oxidation (oxidation of
fat-soluble vitamins)
Measures of Oxidation

Oxidation is a very complex reaction - no

one test will measure all of the reactants or
products.

Some assays measure intermediates while

others measure end products.

Generalized Oxidation Scheme

 Oleic acid

Radical Damage,
Hydrogen
Abstraction

Formation of a
Peroxyl Radical
 Peroxide Value
Measures peroxides and hydroperoxides in an
oil which are the primary oxidation products
(usually the first things formed).

The peroxide value measures the present

status of the oil. Since peroxides are
destroyed by heat and other oxidative
reactions, a seriously degraded oil could have
a low PV.

Plot of PV vs. storage time shows that PV will

peak during oxidation. If you test an oil
without knowing its storage history how can
you tell if the oil is extremely oxidized or
just starting?
 LIPID OXIDATION (Fig. 14-2)

35
Lipid System Under
Reactants and Products

30
Oxidizing Conditions
25
Oxygen Uptake
20
Peroxides
15
Secondary Products
10

1 2 3 4 5 6 7 8 9

Time
 Peroxide Value
The chemistry is simple.
KI + peroxyl radical yields free Iodine (I2)
The iodine released from the reaction is
measured in the same way as an iodine value.
I2 in the presence of amylose is blue.
I2 is reduced to KI and the endpoint determined
by loss of blue color.

Oxygen error occurs when O2 present in the

solution reacts with the KI to produce I2.
Therefore, sample preparation is crucial.
4I + O2 + 4H 2I2 + 2H2O
 Method for Peroxide Value
Obtain a crude fat extract with solvent,
remove solvent. Filter oil.
Dissolve sample in acetic acid and
chloroform (3:2) and add KCl.
Add water to facilitate a bi-layer (water is
on top)
Titrate the liberated iodine with sodium
thiosulfate until clear.
Add starch solution to free up any residual
iodine and titrate again until clear.
PV is expressed as milliequivalents of
peroxide per kg of sample.
PV = (mL thiosulfate titrated) (1000) / S.W.
 Peroxide Value
Degassing solvents should be practiced.

The method sensitive to temperature.

Accepted but variable and results questioned.

What does it tell us about the lipid?

Degree of oxidation-current status
Remember!!! that in an oxidizing sample, the
PV will increase then it will decrease.
Thiobarbituric acid (reactive substances) TBA OR
TBARS
Tests for end products of oxidation aldehydes,
Malonaldehyde (primary compound), alkenals, and 2,4-
dienals

A pink pigment is formed and measured at ~530 nm.

Procedure:
A fatty sample, usually meats, is either extracted or
distilled (heated and vapor condensed) under acidic
conditions.
Distilled sample is reacted with TBA dissolved in acid and
boiled for ~30 min. which will form the pink color.
Read samples spectrophotometrically.
Run against a standard of malonaldehyde.
Thiobarbituric acid (reactive substances)

TBARS is firmly entrenched in meat oxidation research

and in this area is the method of choice.

TBARS measure compounds that are volatile and may

react further with proteins or related compounds.

What does it tell us about the lipid??

Degree of oxidation-current status

High TBA = High Oxidative Rancidity

HEXANAL Determination
Good indictor of the end products of
oxidation (if there are any). Standard method
in many industries.
Aldehyde formation from lipid oxidation.

Done by Gas chromatography

1. Place fat/oil in headspace jar
2. Temper (warm) oil to allow equilibration
3. Inject a sample of the headspace air
4. Quantify hexanal with external standard
Can be automated and very reliable.
Conjugated Fatty Acids

During oxidation, double bond

migration occurs and conjugated fatty
acids are formed.

They absorb light efficiently and can

be monitored in a spectrophotometer.
R C C C C C C C C R
TECHNIQUES OF MEASURING
OXIDATIVE STABILITY

The previous methods determine the status

of oxidation in the sample.

To measure the potential for oxidation,

tests are conducted to measure oxidation
with storage under well controlled well
defined conditions.
TECHNIQUES OF MEASURING
OXIDATIVE STABILITY

Induction Period: is defined as the length of

time before detectable rancidity or time
before rapid acceleration of lipid oxidation
TECHNIQUES OF MEASURING OXIDATIVE
STABILITY
Active Oxygen Method - Air is bubbled through oil or fat
at 97.8C. Time required to reach peroxide value of 100
meq/kg fat determined. (method replaced by OSI)

Oil Stability Index automated Rancimat (instrumental

method). Air bubbled through sample (110C). Oil degrades to
many acidic volatiles (e.g. formic acid) which are carried by the
air into a water trap. Conductivity of the water can then be
assessed.

Oxygen Bomb Sample sealed in bomb, pressurized with O2,

placed in boiling water bath, time until rapid loss of pressure
occurs (indicating oxidation has occurred). Not very common.
LIPID FRACTIONS

For our interests in foods, we can simplify into five main

categories:
1. free fatty acids
2. triacylglycerides (TAGs or triglycerides)
3. partial glycerides (monoglycerides, diglycerides)
4. phospholipids
5. sterols
6. sterol esters

Since these classes differ in their polarity, the most

common means of separation are based on adsorption
chromatography (gas chromatography).
Analysis of Lipid Fractions

Labeling requirements:
Saturated vs. unsaturated fatty acids
polyunsaturated fatty acids
cholesterol

Hot Topics in Labeling

% cis or trans fatty acids
Source of lipids (corn, peanut, canola, )
Fats and fat substitutes e.g. Olestra, Salatrim
Gas Chromatography
Most powerful tool for ID of lipid compounds.
Fatty Acid Composition:
1. First do fat extraction.
2. Make a volatile derivative of the fatty acids
by converting to methyl esters following
saponification.
3. Remove residual moisture with sodium
sulfate (drying salt).
4. Inject sample as compared to a standard
mixture of fatty acids.
5. Compare retention times to standards
Gas Chromatography
Peak ID of FAMEs by GC
ID Component (Acid Methyl ID Component (Acid Methyl Esters)
Esters)
1 C4:0 (Butyric) 20 C18:2n6t(Linolelaidic)
2 C6:0 (Caproic) 21 C18:3n6 ( -Linolenic)
3 C8:0 (Caprylic) 22 C 1 8:3n3 ( -Linolenic)
4 C 10:0 (Capric) 23 C20:0 (Arachidic)
5 C11:0 (Undecanoic) 24 C20:1n9 (cis-11-Eicosenoic)
6 C12:0 (Lauric) 25 C20:2 (cis-11;14-Eicosadienoic)
7 C13:0 (Tridecanoic) 26 C20:3n6 (cis-8;11;14-
Eicosatrienoic)
8 C14:0 (Myristic) 27 C20:3n3 (cis-11;14;17-
Eicosatrienoic)
9 C14:1 (Myristoleic) 28 C20:4n6 (Arachidonic)
10 C 15:0 (Pentadecanoic) 29 C20:5n3 (cis-5;8;11;14;17-
Eicosapentaenoic)
11 C15:1 (cis- 10-Pentadecenoic) 30 C21:0 (Henicosanoic)
12 C 16:0 (Palmitic) 31 C22:0 (Behenic)
For Olestra Potato Chips- How would
you analyze for total fat and saturated
fat?
Not very easily
AOAC Method PVM4:1995:
Uses lipase on the lipid extract to yield
some fatty acids and non-digestible
Olestra
Fatty acids are converted to calcium soaps
and Olestra is discarded
Precipitated calcium soaps are converted
back to fatty acids and analyzed by GC
Cholesterol
Many methods available: TLC, GC, HPLC,
enzymatic, etc.
GC is most common approach:
1. Saponify fat with potassium hydroxide
(cholesterol is in the unsaponifiable
fraction).
2. Extract fraction with benzene or toluene
3. Derivatize to make trimethylsilylethers
4. Injected into a GC