NT20703

FOOD ANALYSIS &

INSTRUMENTATION
LECTURE 5
 CARBOHYDRATE ANALYSIS
oIntroduction
oImportance of Carbohydrate Analysis
oCarbohydrate Analysis Methods
• MONO- & OLIGOSACCHARIDES
• POLYSACCHARIDES
• DIETARY FIBER
• PHYSICAL METHODS
SUGARS?
STARCHES?
FIBERS?
 INTRODUCTION
 Carbohydrates – compounds containing C,
H2 and O2
 Simple carbohydrates (sugars)

 Complex carbohydrates (starches & fibers)

 Primary source of energy to fuel the body

 Worldwide,carbohydrates account for more

than 70% of the caloric value of the human
diet.
 INTRODUCTION CONT’D
 Basic carbohydrate units –
monosaccharides (glucose, galactose &
fructose)
 Disaccharides – two monosaccharide
units (sucrose, lactose)
 Oligosaccharides – between two and nine
monosaccharide units (Rabinose)
 Polysaccharides – greater than ten
monosaccharide units (starch, glycogen)
 INTRODUCTION CONT’D
EVOLUTION OF ANALYTICAL METHOD FOR
CARBOHYDRATES
Qualitative colour test (Fehling test)
Paper chromatography
GC (Gas chromatography)
TLC (Thin layer chromatography)
Enzymatic methods
HPLC
MS (Mass spectrometry)
 INTRODUCTION CONT’D
 According to nutrition labelling regulations in United
States FDA, “total carbohydrate” content in food
must be calculated by subtraction of sums of weights of
protein, total fat, moisture & ash from total weight of
the food (i.e. carbohydrate is determined by
difference)
 Same applies to nutrition labelling regulations in
Malaysia.

outline
 IMPORTANCE OF CARBOHYDRATE
ANALYSIS
 Carbohydrate analysis is important!
 Qualitative analysis – ensures ingredient labels
present accurate compositional information.

 Quantitative analysis – ensures added components

are listed in proper order on ingredient labels &
proper labeled amounts of specific consumer interest.

 Qualitative & quantitative analysis – to

authenticate (i.e. to detect adulteration) food
ingredients & products.
outline
CARBOHYDRATE ANALYSIS
METHODS

➢ Sample Preparation
➢ MONO- &
OLIGOSACCHARIDES
➢ POLYSACCHARIDES
• Starch
• Nonstarch
➢ DIETARY FIBER
➢ PHYSICAL METHODS
 Extraction !!!

SAMPLE PREPARATION
FLOW DIAGRAM FOR SAMPLE PREPARATION &
EXTRACTION OF MONO- & DISACCHARIDES.
 CARBOHYDRATE ANALYSIS
METHODS
➢ Sample Preparation
• Lipid-free sample is extracted with hot 80% ethanol in the
presence of precipitated calcium carbonate to neutralize any
acidity (AOAC Method 922.02, 925.05)
• Reflux for 1 h, cooling and filtering, done at least twice to
ensure completeness of extraction
• Charged contaminants (ash, pigments, organic acids, free
amino acids and peptides) in 80% ethanol extract are
removed by ion-exchange techniques
• Aqueous alcohol of the ethanol extract is removed under
reduced pressure using a rotary evaporator (45-50C)
• Residue (carbohydrate) is dissolved in a known, measured
amount of water. N
 CARBOHYDRATE ANALYSIS METHODS
1) MONO- & OLIGOSACCHARIDES

I. Total Carbohydrate: Phenol-Sulfuric Acid

Method
II. Total Reducing Sugar: Somogyi-Nelson
Method
III. Specific Analysis
A. HPLC
B. GC
C. Enzymatic Methods
D. Mass Spectrometry
E. Thin-Layer Chromatography
F. Capillary Electrophoresis
 I. TOTAL CARBOHYDRATE:
PHENOL-SULFURIC ACID METHOD
PRINCIPLE & CHARACTERISTICS

 Carbohydrates are destroyed at high acids &/or high

temperatures.
 Continued heating in presence of acid produces various
furan derivatives.
 They can condense with phenol to produce coloured
compounds (yellow-orange) that are useful for
carbohydrate analysis. Absorbance measured at 490
nm.
 Reference standard (calibration curve).
 Method simple, rapid, sensitive, & specific.
 Reagents inexpensive, readily available & stable.
 II. TOTAL REDUCING SUGAR:
SOMOGYI-NELSON METHOD
PRINCIPLE & CHARACTERISTICS
 Most often used method to determine reducing sugars!
 Method based on reduction of Cu2+ ions to Cu+ ions by
reducing sugars.
 Cu+ ions then reduce arsenomolybdate complex,
which is prepared by reacting ammonium molybdate &
sodium arsenate in sulphuric acid.
 Reduction of arsenomolybdate complex produces an
intense, stable, blue colour i.e. measured
spectrophotometrically.
 Standard curve (D-glucose)
 III. SPECIFIC ANALYSIS
A. HPLC (HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY)
 Analysis of mono- & oligosaccharides
 Provide qualitative & quantitative analysis.
 Requires no derivatization of carbohydrates unlike GC.
 Important parameters of HPLC – stationary phase, mobile
phase & detector.
 Stationary Phase – Anion-Exchange Chromatography,
Normal-Phase Chromatography, Cation-Exchange
Chromatography & Reversed-Phase Chromatography.
 Detectors – Refractive Index (RI) detector (commonly
used for carbohydrate analysis), Electrochemical detector,
Post-column derivatization & Pre-column derivatization
(increase detection sensitivity).
HIGH PERFORMANCE, REVERSE-PHASE LIQUID
CHROMATOGRAM OF MALTODEXTRINS (DP1-9)
 III. SPECIFIC ANALYSIS
B. GC (GAS CHROMATOGRAPHY)

 Analysis of mono- & oligosaccharides

 Provide qualitative & quantitative analysis.

 Requires derivatization of carbohydrates (sugars

must be converted into volatile derivatives).
 Derivatives – alditol peracetates (& aldonic acid
pertrimethylsilyl ethers from uronic acids).
 Detector – FID (flame ionization detector)

 Important parameters of GC (stationary phase,

temperature programming & detection).
 III. SPECIFIC ANALYSIS
OTHER METHODS
C. Enzymatic Methods
• enzymatic-catalyzed reactions, specific for starch, used
purified enzyme
• Specific for substance measured (limitation)
D. Mass Spectrometry
• Carbohydrate analysis, not for routine manner.
• MALDI-TOF (matrix-assisted laser desorption time-of-flight)
technique for analysis of homologous series of oligosaccharides.
E. Thin-Layer Chromatography
• identification & quantitation of sugars present in molasses
from sugar beet & cane processing.
• Useful for rapid screening of several samples simultaneously.
F. Capillary Electrophoresis
• Capillary zone electrophoresis used to separate & measure
carbohydrates, but because carbohydrates lack chromophores,
pre-column derivatization & detection with UV or fluorescence
detector is required.
LCMS (LIQUID CHROMATOGRAPHY
MASS SPECTROMETRY)

outline
 CARBOHYDRATE ANALYSIS
METHODS
2) POLYSACCHARIDES - STARCH

 Starch is second only to water as most abundant

component of food.
 Commercial starches available as food additives.
Includes corn (maize), waxy maize, high-amylose corn,
potato, wheat, rice, tapioca, arrowroot, & sago
starches.
 As main component in wheat, rice, barley, oat, corn,
mung bean, & pea flours, sweet potatoes, yams
2) POLYSACCHARIDES - STARCH cont’d
a. Total Starch Analysis Method
 Based on total
conversion of starch into
D-glucose by purified
enzymes specific for
starch
 Determination of D-
glucose released by
specific enzyme
 Does not reveal whether
native or modified food
starch
2) POLYSACCHARIDES - STARCH cont’d
b. Degree of Gelatinization of Starch
 Starch swell when
heated in water to a
specific temperature
(cooked) – result from
two processes:
gelatinization & pasting
 Important – texture &
digestibility of foods
 Degree of gelatinization
is determined by
measuring the amount
of reducing sugar formed
 CARBOHYDRATE ANALYSIS METHODS
2) POLYSACCHARIDES -
Nonstarch Food Gums / Hydrocolloids
 Algins, CMC (carboxymethlycellulose), carrageenans,
guar gum, gum arabic, Inulin, Konjac glucomannan,
locust bean gum, Xanthan gum, pectins etc.
 Use widespread & extensively.

 Use in processed meat products, chocolate products,

etc.
 Analytical method required-
 Determine purity of a gum product
 Ensure label declarations correct
 Monitored gums have not been added to standardized
products in which are not allowed
 2. POLYSACCHARIDES cont’d
Nonstarch Food Gums / Hydrocolloids
 Problematic due to variety of chemical structures,
solubilities, molecular weight.
 Qualitative identification tests, specifications,
analytical methods have been established – none
exclusive
 Not included all approved food gums
 Not all methods to determine total gums can be used if
starch present
 Not all methods can be used to determine all gums
 A general scheme is reported to work successfully
GENERAL SCHEME: FLOW DIAGRAM FOR ISOLATION &
ANALYSIS OF POLYSACCHARIDES.
 CARBOHYDRATE ANALYSIS METHODS
3. DIETARY FIBER
Definition
 Dietary fiber - edible parts of plants or analogous
carbohydrates that are resistant to digestion &
absorption in human small intestine with complete or
partial fermentation in large intestine.
 Includes polysaccharides, oligosaccharides, lignin &
associated plant substances.
 Promotes beneficial physiological effects, such as
laxation, blood cholesterol attenuation, blood glucose
attenuation.
 No polysaccharide other than starch is digested in
human small intestine; so all polysaccharides other
than starch are included in the definition of fiber.
 3. DIETARY FIBER CONT’D
 Major Components
 Cellulose
 Hemicellulose
 Pectins
 Lignins
 Food gums/hydrocolloids
 Resistant Starch
 Analysis Methods / Approaches – essential to remove
all digestible starch for accurate estimates of fiber
1. Gravimetric
2. Chemical
3. DIETARY FIBER
I. Gravimetric Methods
 Crude Fiber
 Only measures cellulose & lignin amount, underestimate
amount of dietary fiber in food
 THEREFORE, inadequate for human food analysis

 Detergent Methods
 Acid detergent fiber and neutral detergent fiber methods –
neither methods includes pectins or food gums
 THEREFORE, not justifiable for human food analysis

 Total, Insoluble & Soluble Fiber (AOAC method) ✓

 Recognition of insoluble fiber and soluble fiber – both
important to human health
 Total dietary fiber = insoluble fiber + soluble fiber
 Remove
starch
and
FLOW protein

DIAGRAM OF
AOAC
METHOD
991.43 FOR
DETERMINING
INSOLUBLE,
SOLUBLE, &
TOTAL
DIETARY
FIBER.

Total Dietary Fiber =

residue wt – (wt of protein + wt of ash)
3. DIETARY FIBER
II. CHEMICAL METHODS
 Englyst-Cummings Procedure
➢ Principle
• Starch gelatinized & digested with enzymes

• Remaining non-starch polysaccharides hydrolyzed

using sulfuric acid as the catalyst to liberate free

monosaccharides
➢ Does not measure lignin (most foods do not contain
lignin)
3. DIETARY FIBER
II. CHEMICAL METHODS CONT’D
Chromatographic Rapid Colorimetric
Procedure Method
➢ 1st approach: ➢ 2nd approach:
Neutral sugars determined All monosaccharides
chromatographically + measured by
uronic acids determined colorimetrically
colorimetrically
➢ Fiber wt = ➢ Fiber wt =
neutral sugar wt + all monosaccharides wt
uronic acids wt ➢ Single-tube assay, special
➢ Provide more detail analytical skills and
chemical composition of equipment not required
fiber (quantitating fiber (other than
content) spectrophotometer)
FLOW DIAGRAM OF ENGLYST-CUMMINGS METHOD FOR
DETERMINING TOTAL DIETARY FIBER
3. DIETARY FIBER
COMPARISON OF METHODS
o AOAC Method
➢ Fiber content can be
overestimated if sugars not
extracted from foods rich in
simple sugars
➢ Include resistant starch
o Englyst-Cummings method
➢ Lowest fiber values (lignin &
resistant starch not included, except
corn flakes, cereal brans)
➢ Less time, technical skills, special
equipment, more reproducible

√ Technical skills available

√ Time constraints
√ Availability of chromatographic equipment (HPLC or GC)
√ Total, soluble and insoluble fiber
→ AOAC & Englyst-Cummings rapid method
√ Major fiber components or Constituent sugar composition
→ Englyst-Cummings chromatographic method outline
 PHYSICAL METHODS
 Microscopy
– starchy foods
 Spectrometry (NIR transmittance spectrometry)
– sugar content
 Specific Gravity, SG

– density of substance, accurate for pure sucrose

or pure substance or approximate values for
liquid products
 Refractive Index, RI

- total solids in solution, same with SG

 Polarimetry

- sucrose concentration, by determination of

special optical rotation outline
END OF LECTURE