CHM160L/ Experiment #5 Isolation and Characterization of Carbohydrates Arceo, Chrissie Dillena, Katherine Ong, Ardvin Kester I.

Objectives to isolate the starch and glycogen from potato and chicken liver; to use different quantitative tests to observe the reaction of carbohydrates; to understand the principles involved behind each quantitative test Flow Diagram of Procedure 1. Isolation and preparation of sugar samples A. Starch from potato Peel a small potato and grate. Do not use blender. Place the grated potato in a blender and add 100mL of distilled water. Filter using cheesecloth. September 7, 2013

II.

Collect the filtrate and let it stand for starch to settle at the bottom.

Collect starch through decantation. Air-dry starch collected

B. Glycogen from chicken liver

Get weight of liver sample then cut into small pieces. Grind sample with 5-10mL of cold 10% TCA and small amount of sand every 5g of liver. Rinse mortar and pestle wit h 5mL of 5% TCA and pour washings through cheesecloth. Discard the residue.

Get the volume of filtrate.

Slowly add 2 volumes of 95% ethanol per volume TCA extract while stirring.

Allow to stand until precipitate flocculates. If no fluccation occured, add a pinch of NaCl and place in beaker of warm water until it precipitate forms.

Collect precipitate and determine amount of glycogen isolated. Express as grams of glycogen per 10g of fresh sample.

Test for Starch and Glycogen Lugols Iodine reagernt (IKI) Add 2-3 drops of Prepare 1% sample Lugol's iodine solution from each solution to 5mL isolates. solution to be tested.

Observe color changes and record.

Negative test is brown-yellow color of test reagent.

2. Hydrolysis of Starch and Glycogen

A. Acid Hydrolysis Dissolve 0.5g of obtained sample in 50mL water. Add 10mL of 1M HCl to the starch or glycogen solution. Place acidified solution in hot water bath for 25 minutes.

Neutralize mixture with 1M NaOH. B. enzyme hydrolysis Dissolve 0.5 g starch in 50mL water.

Place in a reagent bottle for further analysis. Refrigerate.

Collect about 15mL of saliva in a clean test tube.

Add 45mL of phosphate buffer solution to the saliva and mix well.

Place saliva solution in the starch and glycogen solution and mix.

Place in a reagent bottle for the succeeding experiment.

3. Specific Reactions of Carbohydrates A. Molisch test Place 1 mL of the given sugars into separate test tubes. Add 2 drops of Molish reagent to each tube. Mix well. Gently pour 5mL conc. H2SO4 to the inclined test tube.

A purple color at the junction of sugar and acid indicates a positive result. Disregard a green color if it appears.

B. Benedicts Test Place 1mL of given sugars into separate test tubes. C. Barfoeds Test Place 1mL of given sugars into test tubes. Add 3mL of Barfoed's reagent into all test tubes. Place in boiling water bath for 5 minutes. Cool at room temperature. Add 3mL of Benedict's reagent into all test tubes. Cool at room temperature. Observe color changes.

Place in boiling water bath for 5 minutes.

Observe and record color changes. D. Fehlings Test Place 1mL of sugar solution and add 1mL of Fehling's reagent. E. Seliwanoffs Test Place 1mL of sugar solution and add 5mL of Seliwanoff's reagent into each. Place in hot water bath for 10 minutes. Observe color changes in each tube w/in the 10min heating period.

Place in boiling water bath for 5 minutes.

Observe and record color changes.

Record color changes.

F. Bials Test Place 1mL of sugar solution into separate test tubes. Add 5mL of Orcinol reagent Gently heat test tubes in hot water bath for 15 minutes.

Take note of color changes occuring. G. Osazone Formation

Record observations.

Place 1mL of sugar solution into separate test tubes.

Prepare test reagent in a separate container. Mix 3.0 grams of NaOAc and 2 g of Phenylhydrazine HCl and 10mL distilled water.

Warm with constant stirring until solution is clear.

Add 2 mL of hot test solution to each

Plug test tubes with cotton. Mix well and heat in boiling water bath for 30 minutes.

Cool at room temperature and examine the crystals under the microscope.

Draw and describe your observations.

III.

Table of results Isolation of starch and glycogen Sample Starch from Potato Glycogen from liver Lugols Test Sample Starch from potato Observation/s Amount in grams or % yield

Glycogen from liver

Specific reactions of carbohydrates 1. Molisch Test Sugar sample 0.1M glucose 0.1M fructose 0.1M galactose 0.1M maltose 0.1M sucrose 0.1M lactose 0.1M xylose Hydrolysed starch Hydrolysed glycogen 2. Benedicts Test Sugar sample 0.1M glucose 0.1M fructose 0.1M galactose 0.1M maltose 0.1M sucrose 0.1M lactose 0.1M xylose Hydrolysed starch Hydrolysed glycogen 3. Barfoeds test Sugar sample 0.1M glucose 0.1M fructose 0.1M galactose 0.1M maltose 0.1M sucrose 0.1M lactose 0.1M xylose Hydrolysed starch Hydrolysed glycogen 4. Fehlings Test Sugar sample 0.1M glucose 0.1M fructose 0.1M galactose 0.1M maltose 0.1M sucrose Observation

Observation

Observation

Observation

0.1M lactose 0.1M xylose Hydrolysed starch Hydrolysed glycogen 5. Seliwanoffs Test Sugar sample 0.1M glucose 0.1M fructose 0.1M galactose 0.1M maltose 0.1M sucrose 0.1M lactose 0.1M xylose Hydrolysed starch Hydrolysed glycogen 6. Bials Test Sugar sample 0.1M glucose 0.1M fructose 0.1M galactose 0.1M maltose 0.1M sucrose 0.1M lactose 0.1M xylose Hydrolysed starch Hydrolysed glycogen 7. Osazone formation Sugar sample 0.1M glucose 0.1M fructose 0.1M galactose 0.1M maltose 0.1M sucrose 0.1M lactose 0.1M xylose Hydrolysed starch Hydrolysed glycogen

Observation

Observation

Observation

Guide Questions: 1. Enumerate other ways to hydrolyze carbohydrate samples

2. Name the 2 component of starch.

3. In a tabulated format, identify the reagents used, principle involved and the positive results of the following carbohydrate tests: Qualitative Test Molisch Test Benedicts Test Barfoeds Test Fehlings Test Seliwanoffs Test Bials Test Osazone formation Reagent used Principle involved Positive visible result/s