CARBOHYDRATE

ANALYSIS
 INTRODUCTION
Classification of carbohydrates (CHO)
Monosaccarides
Disaccharides
Oligosaccharides
Polysaccharides
Digestible
Non-digestible
Sample Preparation for mono-, di- and
oligosaccharides
Need to isolate the CHO first before the
measurement is made.
Preliminary method commonly used to
many isolation techniques
1. Food are dried under vacuum to prevent
thermal degradation.
2. Ground to a fine powder to enhance
solvent extraction.
3. Defatted by solvent extraction.
 Most commonly used method for isolation
Boil a defatted sample with an 80%

alcohol solution to extract low molecular

weight CHO from foods.
Mono-, di- and oligosaccharides are

soluble in alcoholic solution while the

other components not.
The soluble part are separated from the

insoluble part by filtration method.

 Treating the filtrate with clarifying agents
or by passing it through one or more ion-
exchange resins to remove minor
components such as amino acids;
1. Clarifying agents:
Example: heavy metal salts
Function: to form insoluble complexes
with interfering substances that can
be removed by filtration or
centrifugation.
2. Ion-exchange:
Mono-, Di- and Oligosaccharides are
polar.
Therefore possible to separate by
using the combination of a positively
and negatively charged columns.
 METHODS OF ANALYSIS
CHO content can be determined by
calculating the % remaining after all
the other components have been
measured:
% CHO= 100 - % moisture - % protein -
% of lipid - % mineral
Can lead to erroneous results due to
experimental errors in any of the other
methods.
MONOSACCHARIDES AND
OLIGOSACCHARIDES
metode kromatografi
HPLC
Keuntungan: cepat, dapat mentolerir berbagai
sampel conc, tepat dan akurat dan tidak
memerlukan derivatisasi sebelumnya.
Kekurangan: memerlukan mikron-filter penyaringan
sebelum injeksi
fase stasioner digunakan: Ion kromatografi exhange,
kromatografi fase terbalik dan kromatografi fase
normal
2. GC
sebagian dikonversi ke turunan atsiri
Melibatkan dua langkah persiapan
Pengurangan kelompok aldehida kelompok
alkohol primer
Konversi gula berkurang menjadi peracetate ester
volatile atau pertrimethylsilyl turunan eter
Langkah-langkah ini harus lengkap 100% untuk
memastikan pengukuran yang akurat dan
tepat.
Chemical methods
Based on the fact that many of mono-, di-,
and oligosaccharides are reducing agents
that can react with other components to
yield precipitates or colored complexes.
Non-reducing CHO can be determined
after hydrolysis
3 categories of chemical method: titration,
gravimetric and colorimetric.
A. Titration Methods
Contoh: Metode Lane-Eynon
Digunakan untuk menentukan conc tersebut.
mengurangi gula dalam sampel
kekurangan:
Hasil tergantung pada tepat waktu reaksi, suhu dan
reagen conc. harus dikontrol dengan hati-hati
tidak bisa membedakan jenis taruhan yang berbeda
dari gula pereduksi
tidak bisa langsung menentukan gula non-
mengurangi
Mengalami gangguan dari jenis lain dari molekul
yang bertindak sebagai pereduksi
 Procedure
Di panaskan larutan tembaga
sulfat dan indikator metilen blue

CHO solution

Color changes: blue to white

B. Gravimetric Methods
CHO dipanaskan dengan adanya kelebihan
oksida tembaga dan basa tartrat (untuk
menjaga ion Cu 2+ dalam larutan) dalam
kondisi yang terkendali mengarah pada
pembentukan endapan tembaga oksida:
Mengurangi gula + Cu 2+ + dasar
teroksidasi gula + CuO2 (endapan)
Metode ini memiliki kelemahan yang sama
seperti metode Lane-Eynon
Namun, itu lebih direproduksi dan akurat.
 Konsentrasi ini endapan dapat
ditentukan
Gravimetri - filtrasi, pengeringan
dan berat
Titrimetrically - redissolving
endapan dan titrasi dengan
indikator yang cocok
C. Colorimetric Methods
Dapat digunakan untuk menentukan jumlah
gula (mengurangi dan non-gula pereduksi)
karena adanya zat pengoksidasi (asam
sulfat)
Contoh metode: metode Anthrone dan
Phenol - metode Asam Sulfat
Konsentrasi CHO diukur pada absorbansi
tertentu menggunakan spektrofotometer.
 Anthrone Method
Procedure
Sample + sulfuric acid
+ anthrone reagent

Boiled until a blue-

green color is yielded

Measured the solution absorbance

at 620 nm
Phenol-Sulfuric Acid
Method Procedure
larutan CHO ditambahkan ke
dalam tabung reaksi

fenol + asam sulfat ditambahkan

ke dalam larutan CHO
mengandung tabung uji

Kuning - warna oranye

terbentuk

Diukur pada 420 nm

Somogyi Nelson Method
Determine total reducing sugar
Is based on the reduction of Cu2+ to Cu+
ions by reducing sugars.
Cu+ then reduced an arsenomolybdate
complex which produce blue color that is
measured spectrophotometrically.
D. Enzymatic Methods
Relies on enzyme ability to catalyze specific
reactions
Rapid, highly specific and sensitive to low
concentrations
Little sample preparation needed
Liquid foods directly tested
Solid foods need to be dissolved in water first
Two most common methods
Allowing complete reaction and measure the product
conc.
Measuring the initial rate of enzyme catalyzed
reaction
i. D-glucose/D-Fructose
Glucose is converted to glucose-6-phosphate
(G6P) by enzyme hexakinase and ATP
G6P is oxidized by NADP+ in the presence of
G6P-dehydrogenase (G6P-DH)
G6P + NADP+ gluconate-6-phosphate + NADPH
+ H+
The amount of NADPH formed is proportional
to the G6P conc. and the absorbance can be
measured at 340 nm
Fructose needs to be converted to glucose first
before the analysis.
ii.Maltose/Sucrose
Maltose and sucrose are broken down into
their constituent monosaccharides by -
glucosidase enzyme
Conc. of glucose and fructose are
determined using the previous methods
Problem: oligosaccharides are also
converted to monosaccharides by -
glucosidase enzyme
E. Physical Methods
i. Polarimetry
A device that measures the angle that
plane polarized light is rotated on
passing through a solution
The conc. of CHO in an unknown
sample is determined by measuring its
angle of rotation and comparing it with
the calibration curve.
ii.Refractive Index (RI)
Is velocity of light in a vacuum divided by
the velocity of light in the material
RI of CHO solution increases with
increasing conc.
Temp (20C) and w/length (589.3 nm)
dependent
Used routinely in industry to determine
sugar conc. of syrups, honey, molasses,
tomato products and jams
iii. Density
Density of an aqueous solutions
increases as CHO conc. increases
Routinely used in industry for
determination of CHO conc. of juices and
beverages.
iv. Infra Red
A material absorbs infrared due to
vibration or rotation of molecular groups.
Measurements are normally carried out by
measuring intensity of an infra red wave
reflected from the surface of a sample.
Advantages: non-destructive and rapid.
F. Immunoassays
Low molecular weight CHO are developed
by attaching the CHO of interest to a
protein and then injecting it into an animal
Antibodies specific to CHO molecule is
developed then and can be extracted for
determining the specific CHO
concentration.
Advantages: extremely sensitive, specific,
easy to use and rapid
 POLYSACCHARIDES
Digestible
Important source of energy. E.g. starch
Non-digestible
Cellulose, hemicellulose and pectins
 ANALYSIS OF STARCH
Starch properties
Insoluble in water
High density
It is therefore possible to separate from
other soluble and less dense materials.
Methods of starch separation for
processed foods;
Is similar to isolation of mono- and
oligosaccharides using 80% hot ethanol
solution
Take the sediment as starch components due
to insolubility of starch in ethanol
 For semi-crystalline starch, the sample can
be dispersed in water and heated to a
temp where the starch gelatinizes.
Addition of perchloric acid or calcium
chloride to the water prior to heating
facilitates to solubilization of starch.
 Methods of starch determination
1. Specific enzyme is added to the starch
solution to breakdown the starch to glucose.
The glucose concentration is then analyzed
using the methods described previously.
2. Iodine can be added to the starch to form an
insoluble starch-iodine complex that can be
determined gravimetrically by collecting,
drying and weighing the precipitate formed or
titrimetrically by determining the amount of
iodine required to precipitate the starch
 Analysis of Fibers
Fiber is also known as resistant starch
The basis of many fiber analysis
techniques is therefore to develop a
procedure that mimics the processes that
occur in the human digestive system.
Major components of dietary fiber
Cell wall polysaccharides
Non cell wall polysaccharides
 Sample Preparation and
Analysis
Lipid removal

Fiber
Protein removal analysis

Selective
Starch removal
precipitation of
fibers
 Gravimetric Methods
Crude Fiber Method
Gives an estimate of indigestible fiber in foods
Determine by sequential extraction of a defatted
sample with 1.25% H2SO4 and 1.25% NaOH
The insoluble residue is collected by filtration,
dried, weighed and ashed to correct the mineral
contamination of the fiber residue
Crude fiber measures celllulose and lignin in the
sample but does not determine hemicelluose,
pectins and hydrocolloidsbecause they are
digested by the alkali and acid
Total, insoluble and soluble fiber method
The basic principle: to isolate the fraction of
interest by selective precipitation and then to
determine its mass by weighing
A gelatinized sample of dry, defatted food is
enzymatically digested with -amylase,
amyloglucosidase and protease to break
down the starch and protein components.

Footnote: During gelatinization, starch granules are swell, loss

their crystallinity and birefringence and become much more
susceptible to enzyme-catalyzed hydrolysis.
 Total fiber content of the sample is
determined by adding 95% ethanol to the
solution to precipitate all the fiber.
The solution is then filtered and the fiber is
collected, dried and weighed.
Water - soluble and water - insoluble fibers
can be determined by filtering the
enzymatically digested sample.
 Soluble fiber in the filtrate solution and the
insoluble fiber trapped in the filter
The soluble component is precipitated
from solution by adding 95% alcohol to the
filtrate and is then collected by filtration,
dried and weighed.
 Official method for determine fiber content
and is widely used in food industry
Disadvantages: tends to overestimate the
fiber content of foods containing high
concentrations of simple sugars such as
dried fruit, possibly because they get
trapped in the precipitates formed when
the ethanol is added.
 Chemical Methods
Englyst-Cummings Procedure
A defatted food sample is heated in water
to gelatinize the starch
Enzymes are then added to digest the
starch and proteins
Pure ethanol is then added to the solution
to precipitate the fiber which is separated
from the digest by centrifugation and is
then washed and dried
 The fiber is then hydrolyzed using a
concentrated sulfuric acid solution to
break it down into its constituent
monosaccharides
The concentration is then determined
using the previous methods mentioned.
Total mass of fiber in the original sample is
assumed to be equal to the total mass of
monosaccharides present.
 The concentration of insoluble and soluble
dietary fiber can also be determined by
this method using similar separation steps
as the total, insoluble and soluble
gravimetric method mentioned above.
However, it does not provide information
about the lignin content.