Analisis Karbohidrat

Bagian 2 AW-UII

Pendahuluan

Arti penting analisis KH:

Qualitative and quantitative analysis is used to determine compositions of foods, beverages, and their ingredients. Qualitative analysis ensures that ingredient labels present accurate compositional information. Quantitative analysis ensures that added components are listed in the proper order on ingredient abels. Quantitative analysis also ensures that amounts of specific components of consumer interest, for example, -glucan.

Pendahuluan

Perkembangan metode analisis KH:

qualitative color tests, adaptation of the color test for reducing sugars based on reduction of Cu(II) to Cu(I) (Fehling test) to quantitation of reducing sugars, qualitative paper chromatography, quantitative paper chromatography, gas chromatography (GC) of derivatized sugars, qualitative and quantitative thin layer chromatography, enzymic methods, and highperformance liquid chromatography (HPLC). nuclear magnetic resonance, near-infrared (NIR) spectrometry, antibodies (Immunoassays)

Pendahuluan
Pelabelan (FDA): Total carbohydrate calculated by subtraction of the sums of the weights of crude protein, total fat, moisture, and ash in a serving from the total weight of the food in a serving. Other carbohydrate (formerly called complex carbohydrate) is obtained by calculating the difference between the amount of total carbohydrate and the sum of the amounts of dietary fiber and sugars. Sugars are defined as the sum of all free monosaccharides (D-glucose and -fructose) and disaccharides [sucrose, lactose, and maltose (if a maltodextrin or glucose/corn syrup has been added)]

Pendahuluan
Total KH = 100% - (% crude protein + % crude fat + kadar air + kadar abu) KH kompleks = total KH (serat pangan + gula) Serat pangan = jenis KH yang tidak bisa dicerna (selulose, hemiselulose dan polisakaride lain yang berfungsi sebagai bahan pelindung tanaman, serta lignin) Gula = jumlah monosakarida bebas + disakarida/ maltodextrin

Preparasi Sampel (umum)

vacuum oven and drying to constant weight at 55C and 1mm Hg pressure

Soxhlet extractor

Removed contaminants

Mono- dan Oligosakarid a

Metode analisis

Total karbohidrat Total reducing sugar

Phenol-sulfuric method SomogyiNelson method HPLC

GC

Enzimatis Metode khusus

MS

TLC

CE

Ekstraksi sampel mono- & oli

Alasan:
Spektrofotometri Interferen (koloid) dapat menyerap dan/ menghamburkan sinar. Gugus aldehid dan keto pada gula dapat bereaksi dgn gugus amino protein (nonenzymatic browning or Maillard reaction) perubahan warna, merusak gula.

Proses: direfluks 1 jam diingkan saring

Pelarut 80% EtOH (kondisi panas) Pangan yg bersifat asam (buah), dapat ditambahkan kalsium kabonat (menetralkan) sblm ekstraksi, untuk mencegah hidrolisis sukrosa.

Kontaminan dihilangkan dgn teknik pertukaran ion 50-ml aliquot of the ethanol extract in a 250-ml Erlenmeyer lask + 3 g of anion-exchange resin (hydroxide form) + 2 g of cation-exchange resin (acid form) Let it stand for 2 h with occasional swirling. Sisa pelarut alkohol diuapkan dgn rotary evaporator (45-50oC)

Total Carbohydrate: PhenolSulfuric Acid Method Prinsip KH didigesti dgn asam dan
atau suhu panas, tjd reaksi dehidrasi, shg menghasilkan senyawa turunan furan. Lalu dikondensasi dgn senyawa fenolik (fenol, resorsinol, naphtol) dan amin aromatis (anilin, otoluidine) shg menghasilkan warna utk analisis spektrofotometri. Juga dapat utk oligo- dan polisakarida, karena dgn adanya asam dan panas, keduanya menghasilkan monosakarida.

Total Carbohydrate: PhenolSulfuric Acid Method Prosedur umum :

1. 2. 3.

4.

5.

A clear, aqueous solution of carbohydrate(s) is transferred using a pipette into a small tube. A blank of water also is prepared. An aqueous solution of phenol is added, and the contents are mixed. Concentrated sulfuric acid is added rapidly to the tube so that the stream produces good mixing. The tube is agitated. (Adding the sulfuric acid to the water produces considerable heat.) A yellow-orange color results. Absorbance is measured at 490 nm. The average absorbance of the blanks is subtracted, and the amount of sugar is determined by reference to a standard curve. D-glucose is used to prepare the standard curve.

Total Reducing Sugar: SomogyiNelson Method

Prinsip terjadi reaksi reduksi-oksidasi. Gula pereduksi yaitu gula yg mengandung gugus aldehid (donor elektron/ reduktor) mengalami oksidasi, menghasilkan gugus asam karboksilat. Gugus keton merupakan gula pereduksi yg lemah (dlm suasana basa, mengalami isomerisasi mjd aldosa) The SomogyiNelson terjadi reduksi ion Cu(II) mjd Cu(I) oleh gula pereduksi Cu(I) mereduksi kompleks arsenomolybdate menghasilkan warna biru spektro.

Total Reducing Sugar: SomogyiNelson Method

Prosedur umum: 1. A solution of copper(II)

2. 3.

4.

5.

sulfate and an alkaline buffer are added by pipettes to a solution of reducing sugars(s) and a water blank. The resulting solution is heated in a boiling water bath. A reagent prepared by mixing solutions of acidic ammonium molybdate and sodium arsenate is added. After mixing, dilution, and remixing, absorbance is measured at 520 nm. After subtracting the absorbance of the reagent blank, the A520 is converted into glucose equivalents using a standard plot of micrograms of glucose vs. absorbance.

High-performance Liquid Chromatography

Choice for analysis of mono- and oligosaccharides and can be used for analysis of polysaccharides after hydrolysis HPLC gives both qualitative analysis (identification of the carbohydrate) and, with peak integration, quantitative analysis. Terjadi pemisahan analit berdasarkan interaksinya dgn fase diam fase gerak. Fase diam kolom penukar ion, normal phase (amine-bonded silica gel), Reversed-phase chromatography (C18 atau fenil). Detektor Refractive index, Electrochemical detection, ultraviolet (UV) or fluorescence detector, Postcolumn derivatization, Precolumn derivatization.

Gas Chromatography

Sugars must be converted into volatile derivatives.

The most commonly used derivatives are the alditol peracetates (and aldonic acid pertrimethylsilyl ethers from uronic acids). Conversion of sugars into peracetylated aldononitrile (aldoses) and peracetylated ketooxime (ketoses).

Detektor FID Masalah:

reduction of aldehyde groups to primary alcohol groups conversion of the reduced sugar into a volatile peracetate ester or pertrimethylsilyl ether

Neutral Sugars: Outline of Procedure

1.

Reduksi gula mjd alditol

Larutan gula hasil ekstraksi 80% EtOH atau hasil hidrolisis polisakarida direduksi menggunakan sodium/ potasium borohidrida (berlebih) dlm pelarut amm.hidroksida. Suhu 40oC + asam asetat glasial Sisa asam diuapkan

2.

Asetilasi alditol
asetat anhidrida + 1-metilimidazol Suhu ruang + air + diklorometan diuapkan Residu alditol perasetat dilarutkan dalam pelarut organik polar (aseton)

3.

Alditol perasetat siap injek

std internal inositol hexaacetate (ditambahkan sebelum asetilasi)

Hydrolyzates of Polysaccharides Containing Uronic Acids:

Uronic acids are a class of sugar acids with both carbonyl and carboxylic acid functional groups. They are sugars in which the terminal carbon's hydroxyl group has been oxidized to a carboxylic acid. Oxidation of the terminal aldehyde instead yields an aldonic acid, while oxidation of both the terminal hydroxyl group and the aldehyde yields an aldaric acid.

Outline of Procedure:
1.

Reduksi asam uronat mjd asam aldonat (dan aldosa mjd alditol)
Larutan gula hasil ekstraksi 80% EtOH atau hasil hidrolisis polisakarida + Na.karbonat + direduksi menggunakan sodium/ potasium borohidrida (berlebih) dlm pelarut amm.hidroksida. Suhu 40oC + asam asetat glasial Sisa asam diuapkan

2.

Derivatisasi menggunakan trimetilsilil (TMS)

Trimethylsilyation of free aldonic acids gives derivatives of lactones (predominately the 1,4lactone), while trimethylsilyation of the sodium salt produces the ester Siap injek

Enzymic Methods
The kits contain specific enzymes, other required reagents, buffer salts, and detailed instructions that must be followed because enzyme concentration, substrate concentration, concentration of other required reagents, pH, and temperature all affect reaction rates and results. Limit deteksi rendah

Enzymic Methods {GOPOD (glucose oxidaseperoxidase) method} Preparasi sampel:

Dibutuhkan larutan Carrez potassium hexacyanoferrate (K4[Fe(CN)6]) dan zinc sulfate (ZnSO4), lalu penambahan NaOH. Fungsi memecah emulsi, mengendapkan protein, menyerap warna.

Enzymic Determination of D-Glucose:

Enzim glukosa oksidase meng-oksidasi Dglukosa D-glucono-1,5-lactone (gluconodeltalactone). Leuco dye is oxidized to a colored compound which is measured spectrophotometrically.

To measure the amount of D-glucose present, peroxidase is added along with a colorless compound that can be oxidized to a colored compound. In a second enzymecatalyzed reaction, the leuco dye is oxidized to a colored compound which is measured spectrophotometrical ly

Mass Spectrometry

Bukan untuk analisis rutin. Hasil terbaik dibandingkan metode HPLC, CE, dan TLC.

Thin-layer Chromatography
It is particularly useful for rapid screening of several samples simultaneously. Vaccari G, Lodi G, Tamburini E, Bernardi T, Tosi S (2001) Detection of oligosaccharides in sugar products using planar chromatography. Food Chem 74:99

Capillary Electrophoresis

Capillary zone electrophoresis has also been used to separate and measure carbohydrates, but because carbohydrates lack chromophores, precolumn derivatization and detection with a UV or fluorescence detector is required. Generally, this method provides no advantage over HPLC methods for carbohydrate analysis. Klockow A, Paulus A, Figueiredo V, Amado R, Widmer HM(1994) Determination of carbohydrates in fruit juices by capillary electrophoresis and

Tugas individu..
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