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QUANTITATIVE ANALYSIS AND THIN LAYER CHROMATOGRAPHY OF CARBOHYDRATES

Elpa, Jose Fernando; Flores, Joan; Francisco, Kaycee Allen; Gallardo, Mario Emmanuel; Gan, James Viktor Group 4 2-G Pharmacy Pharmaceutical Biochemistry Laboratory

ABSTRACT
Carbohydrates are one of the most important components in many foods. It is important to determine the type and concentration of carbohydrates in foods for a number of reasons. Generally, the experiment was performed in order to determine the components present in a given sample and to correlate those standard sugars presented with that of the acid and enzymatic hydrolyzates using the thin layer chromatography and the Nelsons test. In the Thin layer chromatography performed, the glucose and the acid hydrolyzate had the same Rf value, which means that they both travelled the same length of path from the origin. Even the galactose and fructose had the same Rf value. The ribose travelled the farthest while the galactose and fructose travelled the least. In the Nelsons test, glucose standard curve was plotted using the absorbance readings obtained against the concentrations of the standard solutions.

INTRODUCTION
A carbohydrate is an organic compound with consisting only of carbon, hydrogen and oxygen in which the last two is in the 2:1 atom ratio. It can be viewed as hydrates of carbon. It is divided into four chemical groupings: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. In general, the monosaccharides and disaccharides, which are smaller (lower molecular weight) carbohydrates, are commonly referred to as the sugars. Basically, its primary function is to provide energy for the body, especially the brain and the nervous system. One of the methods used to analyze the sample carbohydrates (TLC). is the thin thin layer layer chromatography Generally,

plant for

contains, the

for

monitoring of

organic or

reactions, for the analysis of ceramides and fatty acids, detection pesticides insecticides in food and water, for the analysis of the dye composition of fibers in forensics, for identifying purity of compounds present in a In given the substance, and for assaying the radiochemical radiopharmaceuticals. interpretation of the results, the components, visible as separated spots in the chromatogram, are being identified by comparing the distances they have traveled with those of the known reference materials. The distance of the start line to the solvent front (=d) is measured, even the distance of center of the spot to the start line (=a). The distance the solvent moved is divided by the distance the individual spot moved. The resulting ratio is called Rf-value.

chromatography is a chromatography technique used to separate mixtures. This technique may be used for the determination of the components

Nelson's test for reducing sugar is a pretty old test and is quite generic in its scope. It basically uses the reduction of some dye compound and then relies on spectrophotometry to determine the level of chemical dye remaining at a specific wavelength. The objectives of the experiment were as follows: 1. To perform thin layer chromatography on the carbohydrate hydrolyzates. 2. To correlate the data obtained from the color tests and thin layer chromatography of the carbohydrate hydrolyzates. 3. To identify the monosaccharide present in the polysaccharide sample. 4. To determine the amount of reducing sugars using Nelsons test and explain the principle involved.

Glucose standard Distilled water B. Procedure 1. Thin-layer chromatography In the developing chamber, 40 mL of the solvent system was placed. The chamber was covered with inverted watch glass and was equilibrated for 10 mins. At the same time, a pencil line was drawn across one end of the TLC plate, about 2 cm from the bottom. Then, equidistant points were marked along the line for the standards, acid, and enzymatic hydrolyzates. In those points, the standards and hydrolyzates were applied five times and ten times, respectively using capillary tubes with drying after every application. Then, the TLC plate was placed in the developing chamber and was allowed to develop until the solvent was about 1 cm from the top of the plate. After the development, the chromatoplate was removed from the chamber and the solvent front was marked with a pencil. The plate was air-dried and was sprayed with p-anisaldehyde visualizing agent. Then, the plate was heated on the hot plate until the sugars became evident by the presence of colored spots. The spots were lightly circled with a pencil. Then, the Rf value was computed. Lastly, the components of acid and enzymatic hydrolyzates were identified. 2. Quantitative analysis In this experiment, Nelsons reagent was prepared by mixing 12.5 mL Nelsons A with 0.5 mL Nelsons B. The 7 test tubes were labeled and were filled with measured amounts of standard glucose solution presented in the table below.

EXPERIMENTAL
A. Compounds used and tested 1. Thin-layer chromatography Acid hydrolyzate Enzymatic hydrolyzate Galactose Glucose Maltose Fructose 9:6:3:1 n-butyl alcohol-acetic acid-ether-water 2. Quantitative analysis Carbohydrate sample (hydrolyzates) Nelsons reagent A Nelsons reagent B Arsenomolybdate reagent 0.5 mL anisaldehyde 9.0 mL 95% CH3CH2OH 0.5 mL H2SO4 0.1 mL CH3COOH Ribose

RESULTS AND DISCUSSION

Table 1: Dilution of samples test tube no. 1 2 3 4 5 6 7 8 Glucose standard (mL) 0 0.1 0.2 0.4 0.6 0.8 1.0 0 Distilled water (mL) 1.0 0.9 0.8 0.6 0.4 0.2 0 0.6 unknown sample (mL) 0 0 0 Figure 1: Thin Layer Chromatography 0 0 0 0 0.4 The figure shows the result of the thin layer chromatography performed. Visible spots appeared on the plate as seen on the figure. Distances travelled by the samples were also illustrated on the sample. Table 2: Thin Layer Chromatography (standards) Then, 1.0 mL Nelsons reagent was added into each prepared tube, and was shaken well. The tubes were heated simultaneously in a boiling water bath for about 20 mins. Afterwards, the tubes were removed simultaneously and were cooled in a beaker of water. Then, 1.0 mL of arsenomolybdate reagent was added into the tubes. The tubes were shaken occasionally for 5 mins. or until the Cu2O precipitate was dissolved. The absorbance of the standards and unknown was taken against a reagent blank at 480 nm. Standard standard unknown determined. curve was constructed Finally, by plotting The table above shows the result of acid and enzymatic hydrolyzates in the thin layer chromatography conducted. The table points out that the enzymatic hydrolyzate travelled farther than that of the acid hydrolyzate. of was absorbance readings against concentrations of solutions. in concentration mg/mL mg/tube and distance travelled by solute Rf value 1.0 cm 1.3 cm 1.2 cm 1.0 cm 1.8 cm distance travelled by solvent 7 cm 7 cm 7 cm 7 cm 7 cm GAL GLU MAL FRU RIB

0.14

0.19

0.17

0.14

0.26

Table 3: Thin Layer Chromatography (acid and enzymatic hydrolyzates)

5 6

0.20 0.27 0.33 0

0.06 0.08 0.1 0

2.200 2.266 2.95 - 0.598

Acid

enzymatic

7 8

distance travelled by solvent (cm)

7 cm

7 cm

The table above shows the different computed concentrations of glucose per tube and per mL, and the absorbance readings of each tube. Spectrophotometer shows direct was used to get the the one absorbance readings of each tube. The table

distance travelled by solute (cm)

proportionality and the

between As

1.3 cm

1.5 cm

concentration

absorbance.

increases, the other also increases. Graph 1: Absorbance vs Concentration

The table above shows the result of acid and enzymatic hydrolyzates in the thin layer The graph shows the direct proportionality of the concentration of glucose to the absorbance. The higher the concentration of the glucose, the higher is the absorbance. chromatography conducted. The table points out that the enzymatic hydrolyzate travelled farther than that of the acid hydrolyzate. Table 4: Concentrations of Glucose and Absorbance Readings

REFERENCES
mg glucose std. per mL (mg/mL) 0 0.01 0.02 0.04 0.049 0.462 1.606 2.170 Absorbance Flitsch, SL & Ulijn, RV (2003). Sugars Tied to the Spot. Nature 421: 219220.

mg glucose test tube no. 1 2 3 4 std. per tube (mg/tube) 0 0.03 0.07 0.13

Harwood,

L.

&

Moody,

C.

Experimental

Organic Chemistry: Principles and Practice (Illustrated edition ed.). pp. 159-173. Vogel, A.I, Tatchell, A.R, Furnis, B.S, etc. Vogel's Textbook of Practical Organic Chemistry (5th Edition).