ANALISIS KARBOHIDRAT Abdul Rohman Faculty of Pharmacy, GadjahMada 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).
Examples of Reducing Sugars and Non-Reducing Sugars Reducing Sugars • Some monosaccharides can act as Reducing Agents (i.e. Glucose and Fructose) • They reduce Fehling’s, Tollen’s, or Folin’s Reagents NON-REDUCING • Sucrose • Raffinose • Cellulose REDUCING • D-glucose • D-fructose (preferably under alkaline conditions) • Maltose
Oligosaccharides • Oligosaccharides or compound carbohydratesare 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, LuffSchorll • Metodeinimendasarkanpadasifatmereduksigula, misalnyaglukosadanfruktosa. • Sukrosatidakbereaksidengan ion tembaga (II) komplek, tetapiglukosadanfruktosadapatbereaksidenganpereaksiinikarenaadanyagugusaldehidapadaglukosadanalfahidroksiketonpadafruktosa. • Untukdapatdianalisisdenganmetodeini, sukrosadihidrolisismenjadiglukosadanfruktosa.
SPECTROPHOTOMETRICS • ALKALINE FERRICYANIDE • PHENOL SULFURIC ACID • Anthrone • Dinitrosalycilic
Spectrophotometric technique • ALKALINE FERRICYANIDE CHO in basic solution (pH > 10.5) reduceferricyanide 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
ANALISIS KH DENGAN KROMATOGRAFI • KROMATOGRAFI Gas • KromatografiCairkinerjatinggi
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, 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 alditolperacetates, 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.
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.