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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 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 • • • • ALKALINE FERRICYANIDE PHENOL SULFURIC ACID Anthrone Dinitrosalycilic 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 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.