Download MONOsaccharides Simple Sugars

ANALISIS
KARBOHIDRAT
Abdul Rohman
Faculty of Pharmacy, Gadjah Mada University,
Indonesia
http://acadstaff.ugm.ac.id/abdulrohman
Materi Kuliah
Analisis Karbohidrat
Analisis Asam amino dan Protein
Analisis Lipid
Analisis Bahan Tambahan Makanan
Pengawet
Pemanis
Antioksidan
Referensi
Selected papers from:
Journal of Food Analysis and composition,
Food Analytical Methods
INTRODUCTION
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Cx(H2O)y
70-80% human energy needs
Monomers and polymers
Functional properties
 Sweetness
 Chemical reactivity
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).
•
Classification based on functional group :
ketose (ketone) or aldose (aldehyde)
Classification by number of C in molecule
(triose, tetrose, pentose, hexose etc).
•
Reducing Sugars
• Some monosaccharides can act as Reducing
Agents (i.e. Glucose and Fructose)
▫ They reduce Fehling’s, Tollen’s, or Folin’s
Reagents
Examples of Reducing Sugars and Non-Reducing
Sugars
REDUCING
• D-glucose
• D-fructose (preferably
under alkaline conditions)
• Maltose
NON-REDUCING
• Sucrose
• Raffinose
• Cellulose
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.
• Examples: sucrose, lactose, maltose.
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)
Sample Extraction
• Extract CHO based on solubility.
• Solvent:
▫ Water
▫ Hot ethanol (80%)
• 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.
Methods for qualitative Analysis
• Wet chemical techniques (Fehling,
Benedict, Anthron, etc)
• Chromatographic method
Quantitaive Analysis of carbohydrate
• Volumetric
• Enzymatic Methods
• Chromatographic Methods
Volumetri, Luff Schorll
• Metode ini mendasarkan pada sifat mereduksi
gula, misalnya glukosa dan fruktosa.
• Sukrosa tidak bereaksi dengan ion tembaga (II)
komplek, tetapi glukosa dan fruktosa dapat
bereaksi dengan pereaksi ini karena adanya
gugus aldehida pada glukosa dan alfa hidroksi
keton pada fruktosa.
• Untuk dapat dianalisis dengan metode ini,
sukrosa dihidrolisis menjadi glukosa dan
fruktosa.
Reaction during analysis
OH
HO
HO
CH2
CH2
OH
HO
O
O
O
HO
H+/H2O
OH
OH
CH2
HO
HO
HO
O
HO
O
HO
H2
C
OH
C
H2
+
OH
HO
OH
fruktosa
HO
glukosa
sukrosa
Cu2+/CuSO4 + K-Na tartrat + Na2CO3
OH O
HO
OH
+ Cu2O
OH HO
asam glukonat
sisa 2CuSO4 + 4KI
I2 + 2 Na2S2O3
2CuI + I2 + 2 K2SO4
2 NaI + Na2S4O6
OH
endapan kufro oksida
(merah bata)
SPECTROPHOTOMETRICS
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ALKALINE FERRICYANIDE
PHENOL SULFURIC ACID
Anthrone
Dinitrosalycilic
Spectrophotometric technique
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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 which condenses with
phenol into a near pink color.
– Read on spec at 490 nm
Anthrone
ANTHRONE reacts primarily with
hexoses
• Read at 620 nm
• Anthrone + carbohydrate + H2SO4  blue-green
color
• Also measuring furan derivatives
Dinitrosalycilic
• 3,5-DINITROSALICYLIC ACID reacts with reducing
sugars in alkali to form brown-red color that can be
measured on a spec
COO-Na+
HO
OH HO
OH
+
3
+ 3OH-
O
HO
OH
O2N
NO2
D-glukosa
Natrium 2,5-dinitrosalisilat
COO-Na+
OH HO
OH
+
3
-
+ 2H2O
OO
HO
OH
O2N
NH2
D-glukonat
Natrium 3amino-5-nitrosalisilat
Analisis dengan Glukose oksidase
Glukosa oksidase
D-Glukosa + O2
Asam glukonat + H2O2
Peroksidase
H2O2 + O-Diasinidin
2H2O + O-Diasinidin teroksidasi
(Tak berwarna)
H3CO
(coklat)
OCH3
NH2
H2N
O-Diasinidin
H3CO
OCH3
NH
HN
O-Diasinidin teroksidasi
ANALISIS KH DENGAN
KROMATOGRAFI
• KROMATOGRAFI Gas
• Kromatografi Cair kinerja tinggi
Gas Chromatography
(Analysis for individual CHO’s)
• Sugars are not volatile, so they require a
derivatization step to make them “volatile”.
• Volatile derivatives can be made by a simple onestep 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
Step in GC analysis
• Reduction of sugar
• Derivatization
• GC measurement
Reduction to Alditol
(for reducing sugars)
• Sugars are reduced to alditols using excess
sodium borohydride, NaBH4.
• This causes reduction of aldehydes and
ketones to primary alcohols
Derivatization
• Alditols (the alcohol form) are then acetylated with
acetic anhydride in order to produce alditol peracetates,
which can be analyzed by GC (acetic acid derivatives are
volatile)
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.
Analysis CHO using GC
GC Condition
• Column: SE 52
• Injection: the 'on-column system' (cold injector:
Grob);
• the oven temperature was programmed
• The carrier gas was helium with a flow rate of
2.5 ml/min.
High performance liquid chromatography
• HPLC carbohydrate methods have replaced
GC methods because they don’t require a
derivatization step
• HPLC methods are non-destructive
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.
How do I choose? GC or HPLC
• HPLC methods are often preferred over GC
method because they don’t 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.