ANALISIS KARBOHIDRAT

1. ANALISIS KARBOHIDRAT Abdul Rohman Faculty of Pharmacy, GadjahMada University, Indonesia http://acadstaff.ugm.ac.id/abdulrohman

2. Materi Kuliah Analisis Karbohidrat Analisis Asam amino dan Protein Analisis Lipid Analisis Bahan Tambahan Makanan Pengawet Pemanis Antioksidan

3. Referensi Selected papers from: Journal of Food Analysis and composition, Food Analytical Methods

4. INTRODUCTION • Cx(H2O)y • 70-80% human energy needs • Monomers and polymers • Functional properties • Sweetness • Chemical reactivity

5. Classification of Carbohydrates The “Saccharides” • Monosaccharide • Smallest form, non-hydrolysable. • Oligosaccharide • Made of several monosaccharides, hydrolysable. • Polysaccharide • Very large polymers of monosaccharides

6. 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).

7. 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

8. 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.

9. 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)

10. 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.

11. Methods for qualitative Analysis • Wet chemical techniques (Fehling, Benedict, Anthron, etc) • Chromatographic method

12. Quantitaive Analysis of carbohydrate • Volumetric • Enzymatic Methods • Chromatographic Methods

13. Volumetri, LuffSchorll • Metodeinimendasarkanpadasifatmereduksigula, misalnyaglukosadanfruktosa. • Sukrosatidakbereaksidengan ion tembaga (II) komplek, tetapiglukosadanfruktosadapatbereaksidenganpereaksiinikarenaadanyagugusaldehidapadaglukosadanalfahidroksiketonpadafruktosa. • Untukdapatdianalisisdenganmetodeini, sukrosadihidrolisismenjadiglukosadanfruktosa.

15. Reaction during analysis

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

17. 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

18. Anthrone ANTHRONE reacts primarily with hexoses • Read at 620 nm • Anthrone + carbohydrate + H2SO4 blue-green color • Also measuring furan derivatives

19. Dinitrosalycilic • 3,5-DINITROSALICYLIC ACID reacts with reducing sugars in alkali to form brown-red color that can be measured on a spec

20. Analisis dengan Glukose oksidase

21. ANALISIS KH DENGAN KROMATOGRAFI • KROMATOGRAFI Gas • KromatografiCairkinerjatinggi

22. 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

23. Step in GC analysis • Reduction of sugar • Derivatization • GC measurement

24. 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)

25. 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.

26. Analysis CHO using GC

29. 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.

31. High performance liquid chromatography • HPLC carbohydrate methods have replaced GC methods because they don’t require a derivatization step • HPLC methods are non-destructive

32. 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.

34. 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.