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EXPERIMENTAL A.

Samples used Acid Hydrolysate, Enzymatic Hydrolysate,n-butyl alcohol:acetic acid:ether:water (9:6:3:1), Standards(1% concentration): dextrin, maltose and glucose, p-anisaldehyde visualizing agent: 0.5 mL aniasldehyde, 9.0 mL 95% ethanol, 0.5 mL H2SO4, and O.1 mL acetic acid. B. Procedure 1. Thin Layer Chromatography 40 mL of the solvent was placed into the developing chamber. A watch glass was then used as a cover on the chamber. It was then equilibrated for 10 minutes. A light pencil line was then drawn across one end of the TLC plate, at a distance of about 2 cm from the bottom. Equidistant points were then marked across the plate for the origin of the standards and the hydrolysates. The standards were applied for five times while both hydrolysates were applied for ten times with the use of capillary tubes. Both the standards and hydrolysates were dried before another application was made. The TLC plate was then placed inside the chamber. The setup was then left to develop until the solvent front was about 1 cm from the top of the TLC plate. The solvent front was marked with a pencil after removing the chromatoplate from the developing cahmber. The chromatoplate was dried and sprayed with p-anisaldehyde, a visualizing agent. It was then placed inside an oven and heated at temperatures within the range of 100150C for about 10 minutes. The spots which appeared were encircled lightly with a pencil. The Rf values were then computed. The values of the products were compared to the standards. The hydrolysates were then identified.

2. Qualitative Analysis Nelsons reagent was prepared by mixing 12.5 mL of Nelsons A was added to 0.5 mL Nelsons B. 7 test tubes were labeled. Ample amounts of the corresponding volume of the glucose solution were then distributed to the test tubes. 1.0 mL of Nelsons reagent was added to each test tube. It was then wobbled to mix well. The mixtures were then simultaneously heated in a boiling water bath for 20 minutes. The test tubes were simultaneously removed from the water bath. 1.0 mL of arsenomolybdate was added to each tube. The tubes were occasionally shaked until the Cu2O precipitate was dissolved. The absorbance of the standards and unknown were read against the blank at 480 nm. A glucose standard curve was then constructed by plotting absorbance readings against concentrations of standard solutions. The concentration of the unknown was then determined in mg/mL. RESULTS AND DISCUSSION A. Thin-layer Chromatography This type of chromatography is based on the phenomenon of adsorption. It results to faster development in separating and identifying liquids as compared to paper chromatography. Thinlayer chromatography also gives of a better resolution against paper chromatography. There are two basic components that comprise a chromatography set-up, a mobile phase and a stationary phase. The stationary phase in a thin-layer chromatography is in a powdered adsorbent form which is fixed to an aluminum, glass, or plastic plate. In Column and Thin Layer chromatographies, the stationary phase is polar. The polarities of both the component of a mixture and the solvent used as the mobile phase are the determining factors in how fast the compound travels. It is also used in the identification and characterization of an unknown. This can be carried out by comparing Rf values to a standard with the use of a particular solvent system. An increase in the polarity of the solvent system will cause the components of the mixture to move faster. An ideal solvent

Figure 1: Thin-layer chromatography setup

system is simply a system that separates the components.N-butyl alcohol:acetic acid:ether:water (9:6:3:1) is a very polar solvent system thus making the components move faster. The standards used for this experiment were maltose and glucose. Sugars are known to be polar, thus both standards moved at a fast rate due to the polarity of the solvent system.

B. Quantitative Analysis In doing the quantitative analysis of carbohydrates for this experiment Nelsons method was used. This method is based on the capacity of the free reducing groups of sugars in a carbohydrate sample to reduce Cu+2 in an alkaline solution. The data obtained with the use of this method is sensitive and reproducible. The amount of free reducing sugars in the sample is directly related to the molybdenum blue formed. This coloration is formed due to a series of oxidation reduction reactions. This method is measured colorimetrically.

Glucose Standard Curve


Figure 2: Structure of Maltose 0.7 0.6 0.5 Absorban ce 0.4 0.3 0.2 Figure 3: Structure of Glucose The data shown on table 1 shows the result of the thin layer chromatography. In order to identify the unknown the Rf values of the hydrolysates were compared to that of the standards. The Rf value nearest to that of the standards is the identity of the unknown. Table1: Results of the Thin-layer Chromatography STANDARDS HYDROLYSATES MalGluAcid Enzytose cose matic Distance 64 64 64 64 traveled by the solvent Distance 20 31 24 16 traveled by the solute Rf Value 0.31 0.48 0.375 0.25 Identity of MalGluGluGlucomponents tose cose cose cose Due to some errors of the members of the group the enzymatic hydrolysates Rf value is closer to that of maltose. The unknown is supposed to be identified as glucose. y = 3.5093x 0.1 R = 0.5138 0 0 0.05 0.1 0.15 Concentration (mg/mL)

Figure 4: Line graph of the absorbance of the standard and the hydrolysates