analisis karbohidrat abdul rohman faculty of pharmacy, gadjah mada university, indonesia
TRANSCRIPT
ANALISIS KARBOHIDRAT
Abdul RohmanFaculty of Pharmacy, Gadjah Mada University, Indonesiahttp://acadstaff.ugm.ac.id/abdulrohman
Materi KuliahAnalisis KarbohidratAnalisis Asam amino dan ProteinAnalisis LipidAnalisis Bahan Tambahan Makanan
PengawetPemanisAntioksidan
INTRODUCTION
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
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
HO
CH2
OH
OH
O
OH
O
OH
HO
HO
CH2
HO
O H+/H2O
OH
HO
HO
HO
CH2
HO
O +
HOCH2
HO OH
O H2C
OH
HO
sukrosaglukosa
fruktosa
Cu2+/CuSO4 + K-Na tartrat + Na2CO3
OHHO
OHHO
OH
O
OH
asam glukonat
+ Cu2O
endapan kufro oksida (merah bata)
sisa 2CuSO4 + 4KI 2CuI + I2 + 2 K2SO4 I2 + 2 Na2S2O3 2 NaI + Na2S4O6
Spectrophotometric technique
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 reacts primarily with hexoses • Read at 620 nm• Anthrone + carbohydrate + H2SO4 blue-
green color • Also measuring furan derivatives
Anthrone
• 3,5-DINITROSALICYLIC ACID reacts with reducing sugars in alkali to form brown-red color that can be measured on a spec
Dinitrosalycilic
O
HO OH
HO OH
HO
D-glukosa
COO-Na+
OH
NO2O2N
+ 3OH-+
Natrium 2,5-dinitrosalisilat
-OO
OH
HO OH
HO
D-glukonat
COO-Na+
OH
NH2O2N
+ 2H2O+
Natrium 3amino-5-nitrosalisilat
3
3
Analisis dengan Glukose oksidase
D-Glukosa + O2 Asam glukonat + H2O2
H2O2 + O-Diasinidin 2H2O + O-Diasinidin teroksidasi (Tak berwarna) (coklat)
Glukosa oksidase
H2N
H3CO OCH3
NH2
Peroksidase
HN
H3CO OCH3
NH
O-DiasinidinO-Diasinidin teroksidasi
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 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)
•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 StepsAcetates •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.
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.