Physical and Chemical Methods
of Food Analysis
Assoc. Prof. Dr. PHAN TẠI HUÂN
Faculty of Chemical Engineering and Food Technology
Nong Lam University - HCMC
Lecture 8:
Carbohydrate Analysis
• Reading : Chapter 10 in the Book “Introduction to the
Chemical Analysis of Foods”, Nielsen, S. Suzanne, 1994,
Jones and Bartlett Publishers, London.
1
Carbohydrate Introduction
• A major source of energy
– account for > 70% of the caloric value of the human diet.
– most of the carbohydrate calories should come from starch.
• Imparters of crucial physical properties
– bulk, body, viscosity, stability to emulsions and foams,
water-holding capacity, freeze-thaw stability, browning,
flavors, aromas, and a range of desirable textures (from
crispiness to smooth, soft gels).
• Modifiers of human physiological processes.
• Carbohydrates also provide satiety.
3
2
General Structure
Carbohydrates are hydrates of carbon
• Monosaccharides (simple sugars) cannot be
broken down into simpler sugars under mild
conditions.
• Oligo = "a few" - usually 2 to 10.
• Polysaccharides are polymers of the simple
sugars.
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5
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Important of carbohydrate analysis
• Carbohydrate analysis is important from several
perspectives.
– ingredient labels present accurate compositional
information.
– added components are listed in proper order on the
ingredient label.
– labeled amounts of specific components of consumer
interest are proper.
• Both qualitative and quantitative analysis can be used
to detect adulteration of products.
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Nutrition labeling regulations of USDA
• Total carbohydrate of a food must be calculated by
subtraction of the sums of the weights of crude protein,
total fat, moisture, and ash from the total weight of the
food.
– "Dietary fiber" is the edible plant material not hydrolyzed by
the endogenous enzymes of the human digestive tract (as
determined by the agreed methods e.g. AOAC methods 985.29
and 991.43)
– "Sugars” are defined as glucose, fructose, sucrose and lactose
and the sugar alcohol sorbitol.
– “Other carbohydrate “ formerly called "complex carbohydrate"
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8
Sample preparation
• For most foods, the first step is drying, which also can
be used to determine moisture content.
• For other than beverages, drying is done by placing a
weighed amount of material in a vacuum oven and
drying to constant weight at 55°C and 1 mm Hg
pressure.
• The material is ground to a fine powder, and lipids are
extracted using 95:5 vol/vol chloroform-methanol in a
Soxhlet extractor (Prior extraction of lipids makes
extraction of carbohydrates easier and more complete).
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Sample preparation
18
(A. Mitchell, 2009)
9
Sample preparation
• Foodstuffs and food products are complex,
heterogeneous, biological materials. They may contain
substances that interfere with measurement.
– Compounds absorb light of the same wavelength used for
carbohydrate analysis.
– Insoluble, colloidal material scatters light.
– Other components, especially amino groups of proteins can
react with the aldehyde or ketone group of the sugar (the
nonenzymatic browning or Maillard reaction).
– Substances might ruin the column or other components of the
HPLC system.
19
Extraction of mono- and oligo-saccharides
• For determination of any mono- (glucose, fructose), di-
(sucrose, lactose, maltose), tri- (raffinose), tetra-
(stachyose), or other oligo- (maltodextrins) saccharides
present, the dried, lipid-free sample is extracted with hot
80% ethanol (AOAC Method 922.02, 925.05).
• Extraction is done by a batch process. Refluxing for 1
hour, cooling, and filtering is standard. Extraction
should be done at least twice to check for and ensure
completeness of extraction.
• If the foodstuff or food product is particularly acidic, for
example, a low pH fruit, neutralization before extraction
may be necessary to prevent hydrolysis of sucrose. 20
10
Removal of interfering compounds
• The 80% ethanol extract will contain components
other than carbohydrates
– ash, pigments, organic acids, and perhaps free amino acids
and low molecular- weight peptides.
• These compounds can be removed by treatment with
either acetate or ion-exchange resins.
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Clarification with Lead Acetate
• Evaporate the alcohol in a water bath.
• Add 100ml water and heat to 80°C in 35-40 min to
soften any gummy precipitates and break up insoluble
masses.
• Cool to room temperature.
• Add saturated Pb(C2H3O2)2, solution to produce a
flocculent precipitate (ppt), shake throughly and let
stand for 15min. Wash with water and filter through
dry paper.
• Add Na oxalate to produce a flocculent precipitate of
all Pb. Wash and refilter through dry paper.
• Dilute to a volume with water and mix.
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11
Clarification with Ion Exchange Resins
• Evaporate the alcohol in a water bath.
• Add 15-25ml water and heat to 80°C in 35-40 min to
soften any gummy precipitates and break up insoluble
masses.
• Cool to room temperature.
• Filter the sample through a celite mat, wash and dilute
to appropriate volume in volumetric flask.
• Place a 50-ml aliquot of the ethanol extract in a 250ml
Erlenmeyer flask. Add 2 g of cation-exchange resin
(acid form) and 3 g of anion-exchange resin (hydroxide
form) (AOAC Method 931.02C). Let stand 2 hr with
occasional swirling. 23
Total Carbohydrate Analysis
• Carbohydrates are destroyed
by heat and acid. Continued
heating in the presence of acid
produces various furan
derivatives, which will
condense with various phenolic
compounds, such as phenol,
resorcinol, orcinol, α-naphthol,
and napthoresorcinol, and with
various nitrogen-containing
compounds to produce colored
compounds that are useful for
carbohydrate analysis 24
12
Phenol-Sulfuric Acid Method
• This method is simple, rapid, sensitive, accurate,
specific for carbohydrates (AOAC Method 44.1.30).
• All classes of sugars, including reducing and
nonreducing sugars can be determined. Oligo- and
poly-saccharides react because they undergo
hydrolysis in the presence of the hot, strong acid,
releasing mono saccharides.
• Derivatives will condensed with phenol. A stable
yellow-orange color is produced. Absorbance is
measured at 490 nm (hexose) and 480 nm (pentose).
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Phenol-Sulfuric Acid Method
Outline of Procedure
1. A clear, aqueous solution of carbohydrate(s) is pipetted into a
small tube. A blank of water also is prepared.
2. An aqueous solution of phenol is added, and the contents are
mixed.
3. Concentrated sulfuric acid is added rapidly to the tube so that
the stream produces good mixing. The tube then is agitated.
(Adding the sulfuric acid to the water produces considerable
heat.) A yellow-orange color results.
4. Absorbance is measured at 485 nm.
5. The average absorbance of the blanks is subtracted, and the
amount of sugar is determined by reference to a standard
curve. 26
13
Total Carbohydrate Analysis (cont.)
Anthrone Method
• Derivatives will condensed with 9,10-dihydro-9-
oxoanthracense (anthrone) under acidic condition
(concentrated H2SO4 ). A stable blue-green color is
produced. Absorbance is measured at 620 nm.
Orcinol Method
• Carbohydrates react with orcinol in strongly acidic
condition to give colored complexes. Absorbance is
measured at 670 nm.
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Total Reducing Sugar
• Munson-Walker Method
• Somogyi-Nelson Method
• Alkaline Ferricyanide Method
• Dinitrosalisylic acid (DNS) Method
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14
Total Reducing Sugars
Reducing Sugars
• Sugars that contain a free carbonyl group (aldoses) &
can act as reducing agents
• Reduce other compounds and become oxidized
themselves
• Under alkaline conditions ketoses can act as reducing
sugar as they isomerize to aldoses
• Glucose, fructose, lactose, arabinose and maltose
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Total Reducing Sugars
Why is this important?
• The Maillard Reaction
• Important factor in determining the color and aroma of
many foodstuffs (baked goods)
• Form of non-enzymatic browning
• Condensation reaction between reducing sugars
and amine groups of amino acids and proteins
• Usually requires heat
• The conjugated double bonds result in products that are
brown or yellowish coloration
30
(A. Mitchell, 2009)
15
Munson-Walker Method
• The Munson-Walker method (AOAC Method 906.03)
has various forms.
Reducing sugar + Cu2+ + base => oxidized sugar + Cu2O
• The precipitate of cuprous oxide can be determined
gravimetrically (AOAC Method 31.039), by titration with
sodium thiosulfate (AOAC Method 31.040), by titration
with potassium permanganate (AOAC Method 31.042),
by titration in the presence of methylene blue (the Lane-
Eynon method) and electrolytically (AOAC Method
31.044).
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Somogyi-Nelson Method
• The Somogyi-Nelson method is based on reduction
of Cu2+ ions to Cu+ ions by reducing sugars.
• The Cu+ ions then reduce an arsenomolybdate
complex, which is prepared by reacting ammonium
molybdate [(NH4)6Mo7O24] and sodium arsenate
(Na2HAs07) in sulfuric acid. Reduction of the
arsenomolybdate complex produces an intense,
stable, blue color that is measured at 520nm
• A standard curve of D-glucose can be used.
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16
Somogyi-Nelson Method
• Outline of Procedure
1. A solution of copper(II) sulfate and an alkaline buffer are
added by pipets to a solution of reducing sugars(s) and a
water blank.
2. The resulting solution is heated in a boiling water bath.
3. A reagent prepared by mixing solutions of acidic ammonium
molybdate and sodium arsenate is added.
4. After mixing, dilution, and remixing, absorbance is
measured at 520 nm.
5. After subtraction of the absorbance of the reagent blank, the
A520 is converted into glucose equivalents using a standard
plot of micrograms of glucose versus absorbance 33
Alkaline Ferricyanide Method
• The alkaline ferricyanide method is based on the
principle that reducing sugar in basic solution (pH >
10.5) can reduce ferricyanide (Fe3+) to ferrocyanide
(Fe2+).
• Ferrocyanide can then react with ferric ions to
produce Prussian blue that is measured at 700nm.
• A standard curve of D-glucose can be used.
34
17
Dinitrosalisylic acid (DNS) Method
• Reducing sugars can react with 3,5-dinitrosalisylate
under alkaline condition to produce red-brown colors
that is measured at 540nm.
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Paper and Thin-layer chromatography
Solvent
For Spot A
C
A B
Rf =
S S
A
B
A
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37
High Performance liquid Chromatography
• HPLC gives both qualitative analysis and, with peak
integration, quantitative analysis.
• HPLC analysis is rapid, can tolerate a wide range of
sample concentrations, and provides a high degree of
precision and accuracy.
• HPLC requires no prior derivatization of
carbohydrates, as does gas chromatography, but does
require micron-filter filtration prior to injection.
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19
Stationary Phases for HPLC
Anion-exchange chromatography
• Carbohydrates have pKa values in the pH range
12– 14.
• In a solution of high pH, some carbohydrate
hydroxyl groups are ionized, allowing sugars to be
separated on columns of anion-exchange resins.
• The general elution sequence is sugar alcohols
(alditols), monosaccharides, disaccharides, and
higher oligosaccharides.
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• High-performance liquid chromatogram of some common monosaccharides,
disaccharides, alditols, and the trisaccharide raffinose at equal wt/vol
concentrations separated by anion-exchange chromatography. Peak 1, glycerol; 2,
erythritol; 3, L-rhammose; 4, D-glucitol (sorbitol); 5, mannitol;6, L-arabinose; 7,
D-glucose; 8, Dgalactose; 9, lactose; 10, sucrose; 11, raffinose; 12, maltose.
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Stationary Phases for HPLC
Cation-exchange chromatography.
• Microparticulate spheres of sulfonated resin are used for
cationexchange stationary phases.
• The resin is loaded with one of a variety of metal counter
ions (Ca 2+, Pb2+, or Ag+…), depending on the type of
separation desired.
• The mobile phase used with these columns is water plus
varying amounts (typically <40%) of an organic solvent
such as acetonitrile and/or methanol.
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Stationary Phases for HPLC
Cation-exchange chromatography.
• Operated at elevated temperatures (>80°C) to increase
column efficiency by increasing the mass transfer rate
which effects peak narrowing and improved resolution.
• Carbohydrate elution from cation-exchange resins takes
place in the order of decreasing molecular weight.
Oligosaccharides with a degree of polymerization (DP)
greater than 3 elute first, followed by trisaccharides,
disaccharides, monosaccharides, and alditols.
• There is some resolution of disaccharides, but the real
strength of this stationary phase is in the separation of
individual monosaccharides. 43
Stationary Phases for HPLC
Normal-phase chromatography
• the stationary phase is polar and elution is accomplished
by employing a mobile phase of increasing polarity.
• Silica gel that has been derivatized with one or more of
several reagents to incorporate amino groups is often
used.
• Aminebonded stationary phases that are generally used
with acetonitrile–water (50–85% acetonitrile) as the
eluent are effective in carbohydrate separations.
• The elution order is monosaccharides and sugar
alcohols, disaccharides, and higher oligosaccharides.
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22
Stationary Phases for HPLC
Reversed-phase chromatography.
• The stationary phase is hydrophobic, and the mobile
phase is largely water.
• The hydrophobic stationary phase is made by reacting
silica gel with a reagent that adds alkyl chains, such as
an 18-carbon-atom alkyl chain (a C18 column) or a
phenyl group (a phenyl column).
• Reversed phase chromatography has been used for
separation of mono-, di-, and trisaccharides by groups.
45
• High-performance, reversed-phase liquid chromatogram of
maltodextrins (degree of polymerization (DP)1–9). 46
23
Detectors for HPLC
Refractive index detection
• The refractive index (RI) detector is commonly
employed for carbohydrate analysis.
• RI measurements are linear over a wide range of
carbohydrate concentrations and can be universally
applied to all carbohydrates.
• RI is a bulk physical property that is sensitive to
changes in flow, pressure, and temperature.
• The most significant limiting factor with RI detection is
that gradient elution cannot be used.
• RI detector is not sensitive to low concentrations.
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Detectors for HPLC
Electrochemical detection
• The triplepulsed electrochemical detector, called a pulsed
amperometric detector (PAD), which relies on oxidation
of carbohydrate hydroxyl and aldehydo groups, is
universally used with Anion exchange-HPLC.
• Gradient and graded elutions can be used with the PAD.
The solvents employed are simple and inexpensive
(sodium hydroxide solution, sodium acetate, water).
• The detector is suitable for both reducing and nonreducing
carbohydrates. Limits are approximately 1.5 ng for
monosaccharides and 5 ng for di-, tri-, and
tetrasaccharides. 48
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Detectors for HPLC
Ultraviolet detection
• Carbohydrates do not have chromophores or
flurophores, so they cannot be detected by absoprtion in
UV or by fluorescence.
• Carbohydrates show an maximum absoption in the near-
ultraviolet region of 180-220nm due to carbonyl group.
• Sample extract must be comletely purified since there
are many organic compounds in foodstuff absorb the
same this wavelength
• Only expensive high grade acetonitrile can meet the
transparency requirement in this region of the spectrum.
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Detectors for HPLC
Post-Column or precolumn Derivatization
• involves addition of reagents that will provide colored
compounds whose concentration can be measured using
absorbance (visible) or fluorescence detection.
– Benzylation, benzoylation, arylosazone, O-
methyloximes, p-toluenesulfonylamides are
chromophores: 230 – 280nm.
– Dansylhydrazones, dansylamides and 1-(N-2-pyridyl-
amino)deoxyalditos are fluorophores: 350nm/500nm,
340nm/470nm and 310nm/380nm.
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25
Detectors for HPLC
Mass detection = Evaporative Light-Scattering Detection
(ELSD)
• Mobile phase is removed by neubulization and
evaporation prior to determination of nonvolatile
carbohydrates by light scattering.
• It allows gradient elution and is more sensitive. The
detetion limit can go up to a few tens of nanogram
injected.
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26
Gas Chromatography (GC )
• GC provides both qualitative and quantitative analysis
of carbohydrates.
• Sugars must be converted into volatile derivatives.
• The most commonly used derivatives are the alditol
peracetates and aldonic acid pertrimethylsilyl ethers
from uronic acids.
• Conversion of sugars into peracetylated aldononitrile
(aldoses) and peracetylated ketooxime (ketoses)
derivatives for GC has also been done.
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Acetylation of alditols
• T
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Trimethylsilyl (TMS) derivatives
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Stationary Phases for GC
• Cappilary columns are commercially available for
carbohydrates analysis.
– internal diameter of 0.1, 0.25, 0.32, 0.53mm and lengths of
15, 30 and 60m
– made from fused silica and coated with a polymeric
material.
• Liquid (stationary) phase is coated with a film
thicknesss of 0.2 – 1.0μm covalenthly bonded to the
capilary walls.
• Sample size is from 5nm to 1500 μ m.
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Detectors for GC
• The flame ionization detector (FID) is a common
choice for carbohydrates analysis.
• Electron capture detector and nitrogen detector can be
used in for halogenated derivatives and amino sugars
respectively.
• GC and Mass Spectrometry (MS) are also a good
choice for both qualitative and quantitative analysis
57
Other Modern Carbohydrate Analysis Methods
• Microscale HPLC
– Internal diameter 0.2 – 0.01 mm
• Supercritical-Fluid Chromatography (SFC)
– Mobile phase is supercritical fluid
• High-performance Capillary Electrophoresis (HPCE)
– Generally, this method provides no advantage over HPLC
methods for carbohydrate analysis.
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Enzymatic Methods
• Due to their high specificity and sensitivity, enzyme
assays are ideal for the analysis of food
carbohydrates.
• Kits for several enzymic methods have been
developed and marketed. The kits contain specific
enzymes, other required reagents, buffer salts.
• Limits of detection by methods involving enzyme or
coupled enzyme-catalyzed reactions are generally
low.
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Enzymatic Methods
60
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Enzymic Determination of D-Glucose
61
Total Starch Analysis
• complete conversion of the
starch into D-glucose by
purified enzymes specific for
starch.
• determination of the D-glucose
released by an specific enzyme
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Degree of Gelatinization
of Starch
When starch granules are heated in
water to a temperature specific for the
starch being cooked, they swell, lose
their crystallinity and birefringence,
and become much more susceptible to
enzyme-catalyzed hydrolysis.
Heating starch in water produces
phenomena that result from two
processes: gelatinization and pasting,
often together referred to simply as
gelatinization, which are very
important in determining the texture
and digestibility of foods containing
starch. 63
Degree of Retrogradation of Starch
• Upon storage of a product containing cooked starch, the two
starch polymers, amylose and amylopectin, associate with
themselves and with each other, forming polycrystalline arrays.
This process of reordering is called retrogradation.
• Retrograded starch, like native starch, is acted on very slowly by
the combination of pullulanase plus β-amylase.
• Therefore, the basic method described in study of degree of
gelatinization can be used to determine retrogradation.
• The decrease in reducing power (from maltose released by
action of the enzyme combination) after storage is a measure of
the amount of retrograded starch at the time of analysis and/or
the degree of retrogradation.
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Analysis of Nonstarch Polysaccharides
(Hydrocolloids/Food Gums)
65
E
• T
66
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Physical Methods
• Polarimetry
• Specific Gravity
• Refractometry
• Microscopy
• Visible spectroscopy
• Mass and NIR Transmittance Spectrometry
67
Polarimetry
• Carbohydrates contains an asymmetric carbon atom
and have ability to rotate the plane polarization of
polarized light
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34
Specific gravity
• 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 specified temperature.
• The concentration of a carbohydrate solution can be
determined by measuring the specific gravity of the
solution, then referring to appropriate specific gravity
tables.
• Measurement of specific gravity as a means of
determining sugar concentration is accurate only for
pure substance (AOAC Method 932.14), but it can be,
and is, used for obtaining approximate values for liquid
products . 69
Specific gravity
• The most common is use of a
hydrometer calibrated either in ◦Brix,
which corresponds to sucrose
concentrations by weight, or in
Baumé Modulus (Bé).
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Refractometry
• When electromagnetic radiation passes from one
medium to another, it changes direction (i.e., is bent or
refracted). The ratio of the sine of the angle of
incidence to the sine of the angle of refraction is termed
the refractive index.
• The refractive index varies with the nature of the
compound, the temperature, the wavelength of light,
and the concentration of the compound.
• Use of refractometry to measure concentrations is
accurate only for pure sucrose or other solutions of a
single pure substance
71
Microscopy
• Granule size, shape, and form using a polarizing
microscope, and, in some cases, iodine-staining
characteristics can be used to identify the starch source
• The extent of retrogradation, microstructure , degree of
mechanically damaged during dry milling, the extent of
digestion by enzymes, whether overcooked,
undercooked, or correctly cooked starch-based product.
• Quantitative microscopy has been employed for
analysis of the nonstarch polysaccharides of cereal
grains.
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73
Visible spectroscopy
• 504 nm: wavelength of greatest difference between the
amylose and amylopectin spectra and where the absorbance
due to amylopectin was greater than that due to amylose.
• 548 nm: amylopectin peak.
• 580 nm: peak for 20% amylose and 80% amylopectin.
• 630 nm: amylose peak.
• 700 nm: wavelength of greatest difference between the
amylose and amylopectin spectra and where the absorbance
due to amylose was greater than that due to amylopectin.
• 800 nm: wavelength of greatest absorbance due to amylose
and where the absorbance due to amylopectin approaches
zero.
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Mass and NIR Transmittance Spectrometry
• Mass and NIR transmittance spectrometry have been
used to determine sugar content.
• NIR spectrometry is described in Lecture 4.
• Mass spectrometry is mentioned in Chapter 26 -
Book “Introduction to the Chemical Analysis of
Foods”, Nielsen, S. Suzanne, 1994, Jones and Bartlett
Publishers, London.
75
MALDI-TOF mass spectrum of maltooligosaccharides produced by 76
hydrolysis of starch. Numbers indicate DP. IS, internal standard.
38
