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

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

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

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 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

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, 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.

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.