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ANALISIS KARBOHIDRAT Abdul Rohman Faculty of Pharmacy, Gadjah Mada University, Indonesia
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 C x (H 2 O) 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 Sugar s 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.
Spectrophotometric technique n n ALKALINE FERRICYANIDE CHO in basic solution (pH > 10.5) reduce ferricyanide to ferrocyanide Forms Prussian Blue that is measured at 700 nm n n 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 + H 2 SO 4 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
Analisis dengan Glukose oksidase
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 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
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, N a BH 4. 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.