Isolation and Characterization of Carbohydrates (Isolation, General Tests, and Hydrolysis of Strach) Jenny Lynn A.

Karunungan, Daniel Adam Levy, Josell Mary Engelee Lim, Alphonse Leandro Lomotan, Ma. Eloisa Mateos Group 6 2B Medical Technology Biochemistry Laboratory
In the experiment,Qualitative Color Reactions was performed in order to analyze the chemical goups responsibe for the color reaction of the protein Gluten. Different tests such as Biuret, Ninhydrin, Xanthhoproteicm Millons, HopkinsCole, Sakaguchi, Nitroprusside, Fohls, Test for Amides, and Pauly yielded different colored solutions as results. The different results was due to the difference in the side chains present in Gluten. Paper Chromatography was also conducted to be able to separate and determine the amino acid constituents of the acidic, basic, and enzymatic hydrolysate of Gluten, a protein found in wheat flour, based on the polarities of Proline, Alanine, Cysteine, Tyrosine, and Glycine. A 1.5 cm margin had been drawn across the longer bottom edge of the chromatograph paper. Eight equidistant points were plotted along the line where the given amino acids and protein hydrolysate samples had been applied. The TLC plate was then introduced in the solvent system consisting of 1-Butanol,acetic acid and water with a ratio of 4: 1: 5. Then, Rf values for the standards were computed and it was determined that there were 3 amino acids present in the acid hydrolysate of gluten and they are : Alanine (Rf value = 0.19), Tyrosine (Rf value = 0.33) and an amino acid with a Rf value of 0.43 (unknown since it did not match with any of the used standards of amino acids). For the Alkaline Hydrolysate of gluten, Cystein (Rf value = 0.11) and Tyrosine (Rf value = 0.34) was identified. And lastly, for enzymatic hydrolysate of gluten, Tyrosine (Rf value = 0.31) and Glycine (Rf value = 0.13) was identified.

ABSTRACT

INTRODUCTION

coloration disappears. The mixture was cooled and the resulting changes were observed.

4. Acid Hydrolysis

EXPERIMENTAL A. Compounds tested (or Samples used)

The samples used in this experiment are 3 medim sized potatoes, acid hydrolysate, and enzymatic hydrolysate. The isolated starch appeared as a white amorphous precipitate. The acid and enzymatic hydrolysates appeared both as non-viscous solutions.

5 drops of conc. HCl was added to 5 mL of the glycogen solution contained in a test tube. The tube was covered with a cotton boil and was boiled in a water bath for 30 minutes.The viscosity of the solution before and after heating was noted. The solution was then neutralized using dilute NaOH and the hydrolysate was then kept in a refrigerator until it was subjected for Benedicts Test.

B.Procedure 1. Isolation of Starch from Potatoes

5. Enzymatic Hydrolysis

Three medium sized potatoes were comminuted and were placed in a 600 mL beaker. 100 mL of water was poured to the beaker and was mixed. The mixture was strained using cheesecloth and the filtrate was collected in a jar. The starch was then allowed to settle in the jar. The collected starch was then subjected to Molischs Test, Iodine Test, Acid Hydrolysis, and Enzymatic Hydrolysis.

2. Molischs Test

10 drops of Molischs reagent was added into 1 mL of glycogen solution. 2 mL conc. H2SO4 was then carefully poured to the side of the tube to form a layer. A violet ring formed at the junction of the two liquids was then observed and noted. 5 drops of 0.01 M I2 was added into 1 mL of the glycogen solution while observing the appearance of a red coloration. The mixture was then warmed in a water bath while noting if the

2.3 mL of saliva was added to 10 mL of isolated glycogen solution in a beaker and was allowed to stand for 30 minutes. The viscosity of the solution before and after 30 minutes was noted. The solution was introduced in a dialyzing bag and was suspended until the next meeting in a small beaker containing 50 mL of distilled water. After dialyzing the solution, the viscosity of the residue inside the bag was noted and the bag was discarded.1 mL of the dialysate was concentrated using an open flame. The concentrated enzymatic hydrolysate was then subjected to Benedicts Test.

RESULTS AND DISCUSSION

Isolation of starch from potatoes was done by mixing grinded potatoes with 100 ml of distilled water and filtrating out the starch with the use of a cheesecloth. The filtrate collected in a jar was allowed to settle and white amorphous precipitates appeared. The precipitate was known

3. I2 Test

to be the starch, an insoluble polysaccharide found in most plants. It consists of many glucose units connected by a glycosidic bond. It consists of two types of molecules: the linear and helical amylose and the branched amylopectin. Amylose is a spiral polymer made up of D-glucose units. It makes up 20% - 30% of the structure of starch and is insoluble in water. Amylopectin, on the other hand is soluble in water and is a highly branched polymer of glucose. It makes up 70% 80% of the structure in starch and since it is soluble, it is not suitable for the storage of energy in plants.

Figure 3. Molischs Test Reaction Ddddddd

The enzyme used to hydrolyze the isolated starch was the enzyme salivary amylase found in human saliva. Salivary amylase also called Ptyalin catalyze only the hydrolysis of -1,4 glycosidic linkages in the amylose and amylopectin components of starch. The hydrolysis does not proceed directly from polysaccharides to monomer units; rather, partial hydrolysis products of intermediate size are obtained. These products are maltose and dextrin. Maltose consists of two glucose units in -1,4 linkage; dextrin is made up of several glucose units joined by -1,6 linkage in addition to -1,4 linkages. When the hydrolysate was subjected to Benedicts test, a test for the presence of reducing sugars (e.g dextrin and maltose), no change in color was observed, the solution appeared as a blue coloration indicating a negative result. This means maltose and dextrin was not present and the enzyme was not able to catalyze the starch.

Figure 1. Structure of Amylose Molecule

Figure 2. Structure of Amylopectin Molecule

The isolated starch was subjected to Molischs Test, a general test for the presence of carbohydrates. Upon addition of Molischs Reagent, a solution of alpha-naphthol in 95% ethanol, and concentrated H2SO4, the starch is hydrolyzed to yield their monomer unit, glucose. Glucose is then dehydrated to from hydroxymethylfurfural since glucose is a form of hexose. The hydroxymethylfurfural will then react with alpha-naphtol forming a violet colored condensation or ring.

Figure 4. Rection of Starch with Ptyalin

REFERENCES
Enzymatic Hydrolysis Reaction http://www.sigmaaldrich.com/technicaldocument s/articles/analytix/enzymatic-food-analysis.html Retrieved 02/25/2014 The Basics of Amylase http://en.wikipedia.org/wiki/Amylase Retrieved 02/25/2014 Starch http://en.wikipedia.org/wiki/Starch Retrieved 02/25/2014 Molischs Test and Reaction Principle http://en.wikipedia.org/wiki/Molisch's_test Retrieved 02/25/2014 Carbohydrates http://www.pjmcelligottcom.com/carbohydrates. doc Retrieved 02/25/2014