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EXPERIMENT 4PROCESS IN BIOCHEMICAL SYSTEM

OBJECTIVES

1. To relate the particle concept of matter to biochemical processes;2. To describe molecular motion and interaction; and3. To observe and describe processes that occur in biochemical system: diffusion, dialysis, osmosis, hydrolysis and lowering of surface tension.

REAGENTS / MATERIALS2% NaCl KMn04 Defibrinated blood Microscope0.1 % NaCl 0.9% NaCl Bile String1% Na2c03 1% pacreatin Margarine1 M AgNO3 0.5% CuS04 Longanisa membrane10% NaOH Iodine solution Chicken bouillon0.2 % Ninhydrin Macaroni Thermometer

PROCEDURE

A. Dialysis

1. Obtained one 10-cm long dialysis tubing or gastric lining (longganisa membrane about 2.0-2.5 cm in diameter or colorless cellophane about 25 cm square in size.)

2. If gastric lining to be used, tightly tie a string around its lower portion to close one end. Wet the lining (or dialysis tubing) and open the other end with the stirring rod to check if the pouch or sack is made.

3. Pour about 25 mL of chicken bouillon (previously dissolved in hot water) into the prepared lining pouch and tie a string around the upper potion carefully so that there will be no leak.

4. Wash off with distilled water any spilled solution on the outside of the pouch/sack.5. Place the lining with its contents in a beaker and add distilled water just enough to

immerse about three-fourths of it. (Note: The distilled water to be used must be first checked for negligible amount of Cl. To do this, test 1.0 mL of the distilled water from the large portion and add 1.0 mL of 1.0 M silver nitrate solution. The result must be colorless to neglible turbidity.)

6. Allow to stand for an hour at room temperature, following the set-up given.7. Test the dialysate by doing the following:

a. Test for Chloride ion.To 1.0 mL of the dialysate, add 1.0 mL of AgNO3 solution.

Observation: There is white precipitate, there is clear liquid

b. Test for peptide bondTo 1.0 mL of dialysate, add 2.0 mL of 10% NaOH and 5 drops of 0.5% copper sulfate. Stir the

solution.

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Observation:When add w/ 10% NaOH it was clear, when added with 0.5% copper sulfateformed lavender color w/ blue precipitate.

c. Test for amino acidTo 3.0 mL of the dialysate, add 1 mL 0.2% ninhydrin. Heat in a water bath until a blue color

appears which indicates the presence of amino acid. Observation:Formed 2 layers, immiscible, ninhydrin an upper layer, in lower layer the chicken broth; the ninhydrin turn to blue violet color chicken broth turned to peach color.

Draw the set-up for dialysis and label.

B. Diffusion

1. Place 20 mL tap water in a beaker. Determine its temperature and allow it to stand still after placing the beaker over a piece of graphing paper marked with a dot that coincides with the center of the bottom of the beaker.

2. Using a spatula or glass rod, carefully place a few (2-3) crystals of KMn04 at the bottom of the beaker where the lines of the graphing paper are seen perpendicular to the edge of the container.

3. Note the time required for the colored ions to travel 5 cm along a line toward a center.4. 4. Rinse the beaker and fill it with 20 mL water. Heat the water to the temperature of 700C

to 800C. Again, add crystals of KMn04 to the still water in the same way as step 2 above.. Note the time of migration in the second along the same distance at the higher temperature.

What is the effect of increasing the temperature on the rate of the migration of the solid substance in a liquid medium?

When the temperature is hot / high temp. The rate of migration is faster while if the temperature is cold, the migration is slower.

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Draw the set-up of apparatus and describe the directional movement of the colored particles.

Compare the rate of diffusion at different temperatures. 2.6 0 C; 1 min. 41 seconds 80 0 C; 42 seconds - slow; cold temperature - fast; hot temperature

C. Osmosis

1. To each of four small test tubes, place 5 drops of defibrinated blood. Label the first test tube, # 1 as the control.

2. To the other test tubes, starting with the second, add 20 drops of the following: test tube # 2 - 0.1 % NaCltest tube # 3 - 0.9 % NaCltest tube # 4 - 2.0 % NaCl

3. Shake. Get a small amount of contents from each test tube, place on separate slides and examine under the microscope the appearance of the blood cells.

How do the shape and overall picture of the cell from the three test tubes compare with that of the control?Answer by drawing the appearance of red blood cell as seen under the microscope showing the difference/s in their size and shape. Describe them accordingly. Use the following table.

How the RBC’s compare in size and shape

Drawings

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DescriptionSize: Largest

Shape: circle with biconcave

Color: 1st – Red 2nd – Light green

3rd – white

Size: The smallest among of them

Shape: circle with biconcave

Color: Yellowish white

Size: The second largest among the 4

sampleShape: Circle with the

biconcaveColor: Colorless

Size: Second smallest of the 4 sample

Shape: Circle with the biconcave

Color: colorless

Test tube # 1(control)

Test tube # 2(in 0.1% NaCl)

Test tube #3(in 0.9% Nacl)

Test tube #4(in 2%NaCl)

D. Hydrolysis

1. Place 1 piece of elbow macaroni into each of 3 small test tubes. Add 5 mL of pancreatin solution to the first test tube; 5 mL of 1% Na2c03 to another test tube and 5 mL of saliva to the test tube.

2. Place the three test tubes in a water bath with the temperature controlled at 40-500C for strictly 15 minutes.

3. Pour of the solution and rinse the macaroni pieces with distilled water. Place them in a watch glass equidistant to each other. Observe each.

4. Place a few drops of iodine solution on the surface of each pieces of macaroni and observe the resulting intensity of the color. Make comparisons on the texture and appearance of the threated macaroni pieces after heating and with Iodine solution.

Sample macaroni Texture after 15 minutes heating

Color with the Iodine solution

#1 Slightly soft/slightly viscous Blue violet#2 Soft / viscous Charcoal / slightly hardens#3 Hard / smooth Charcoal / it hardens

E. Lowering of Surface Tension

1. Apply a thin uniform coating of margarine on one 3 slides.2. Cover the margarine on one slide with bile solution, the other with 0.5% Na2c03 and the last one

with soap solution. Let them stand for 30 minutes.3. Pour of the solutions and rinse each slide several times with running water very carefully. Hold

the slide up against the light and observe what happens.4. Make a comparison on degree of disappearance / solubility of the coated margarine soaked with

different solution. Account for observations.

Margarine + bile solution The bile solution is not soluble in margarine, the bile solution faded

Margarine + Na2c03 Margarine and Na2c03 is soluble. The Na2c03 solution faded

Margarine + soap solution Margarine and soap solution is slightly soluble in terms of degree of disappearance, the soap moderately faded.

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SUPPLEMENTARY QUESTION1. Differentiate passive transport and active transport system. Give examples.

Passive transport does not require energy when substance move across in a cell membrane. (example: Hydrated Na+ is larger that K+ and therefore tends to move easily)

2. Is energy always associated with biochemical processes? Justify your answer. No, active transport always occur against an electrical or chemical reagent

3. Discuss the movement of Na+ and K+ ions in the cell. Sodium-potassium exchange pumps moves Na+ out of the cells and K+ into the cells. The result

is the higher concentration of Na+ outside the cell.

EXPERIMENT 5GENERAL REACTIONS OF CARBOHYDRATES

Carbohydrates are the main energy source for the human body. are organic molecules in which carbon, hydrogen, and oxygen bond together Animals (including humans) break down carbohydrates during the process of metabolism to

release energy. Animals obtain carbohydrates by eating foods that contain them.

Simple Sugars All carbohydrates are made up of units of sugar (also called saccharide units). Carbohydrates

that contain only one sugar unit (monosaccharides) or two sugar units Simple sugars are sweet in taste and are broken down quickly in the body to release energy. Two of the most common monosaccharides are glucose and fructose. Glucose is the primary

form of sugar stored in the human body for energy. 

Complex Carbohydrates are polymers of the simple sugars. are long chains of simple sugar units bonded together complex carbohydrates are often referred to as polysaccharides

Starch

is the principal polysaccharide used by plants to store glucose for later use as energy. Plants often store starch in seeds or other specialized organs

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Functions of CarbohydratesCarbohydrates have several functions in cells. They are an excellent source of energy for the many different activities going on in our cells.

Some carbohydrates may have a structural function. The material that makes plants stand tall and gives wood its tough properties is a polymer form

of glucose known as cellulose. Make up the stored forms of energy known as starch and glycogen. Carbohydrates are essential for cells to communicate with each other. They also help cells adhere to each other and the material surrounding the cells in the body. The ability of the body to defend itself against invading microbes and the removal of foreign

material from the body (such as the capture of dust and pollen by the mucus in our nose and throat) is also dependent on the properties of carbohydrates.

Use in wine making

Molisch's Test is a chemical test for the presence of carbohydrates, based on the dehydration of the

carbohydrate by sulfuric acid to produce an furfural derivatives anion with alpha napthol to form colored complexes.

A positive reaction is indicated by a purple ring forming at the interface between the acid and test layers.

RESULT the formation of a purple product at the interface of the two layers.

a negative test (left) and a positive test (right)

ANTHRONE – BLUE OR GREEN COLOR

ALDEHYDE & RETONE HAS REDUCING PROPERTIESOXIDIZED ALKALINE SOLUTIONS OF CUPRIC SULFATEREDDISH BROWN PPT. (CUPRIC TO CUPROUS)

Benedict's reagent

is a solution of copper sulfate, sodium hydroxide, and tartaric acid. Aqueous glucose is mixed with Benedict's reagent and heated.

The reaction reduces the blue copper (II) ion to form a brick red precipitate of copper (I) oxide. Because of this, glucose is classified as a reducing sugar.

 

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RESULT The formation of a reddish precipitate within three minutes.   

 

Results of Benedict's Test with Glucose, Sucrose, and Fructose 

Fehling's solution

is a solution used to differentiate between aldehyde or ketone functional groups. The substance to be tested is heated together with Fehling's solution; a red precipitate indicates

the presence of an aldehyde. Ketones (except alpha hydroxy ketones) do not react. It was developed by German chemist Hermann von Fehling. Fehling's is also used to test for aldoses (a.k.a. reducing sugars).

RESULT

sucrose is added and no change is observed. On the other hand, glucose, a reducing sugar, reacts with Fehling's reagent to form a brick red

precipitate of copper(I) oxide.

NYLANDER – ALKALINE SOLN OF BISMUTH SUBNITRIDESATE WHEN ADDED TO REDUCING SUGAR BLACK PPT – FORMATION OF METALLIC BISMUTH

BARFOEDS REAGENT CUPRIC ACETATE IN WEAK ACETIC ACID, REDUCING MONOSACCHARIDES NOT DISSACHARIDES

MONOSACCHARIDESDISACCHARIDESPOLYSACCAHR

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Tollens' reagent

is usually ammoniacal silver nitrate, but can also be other things, as long as there is an aqueous diamminesilver(I) complex

RESULTSilver mirror image

   

EXPERIMENT 5GENERAL REACTIONS OF CARBOHYDRATES

OBJECTIVES: 

1. The objective of this experiment is to be able to describe the properties and structures of carbohydrates in general.

2.  to differentiate reducing sugars from non-reducing sugars.

MATERIALS: 1% glucose 1% sucrose

conc. Sulfuric acid 1% silver nitrate

cupric acetate 1% lactose

1% maltose 1% starch

1% Ba(OH)2 6N NaOH

phenolphtalein

NH4OH 1% xylose

picric acid 1% glycogen

dil. HCL Nylander’s reagent

glacial acetic acid Benedict’s reagent

Fehling’s reagent Molisch reagent

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PROCEDURE: 

A.       REACTIONS WITH ACIDS 1.      MOLISCH TEST

a. To 1ml of 1% glucose, add 2 drops of Molisch reagent. Mix thoroughly.b. Incline the test tube and allow 1 ml of conc. H2S04 acid to flow down the side of

the test tube. Do not shake. Note the color formed at the junction of the two layers.

c. Repeat the procedure using 1% xylose, 1% sucrose, 1% starch, 1% maltose.  

TEST COMPOUND OBSERVATIONGlucose + (positive result)upperlayer-lavender;lower-dark purpleXylose + (positive result) upperlayer-lavender;lower-dark purple

Sucrose + (positive result)upperlayer-white;lower-darkest purpleStarch + (positive result)upper-grey white;lower-black

Maltose + (positive result)upper-cloudywhite;lower-lavender

a negative test (left) and a positive test (right)

Did all the samples give the same result? All of the test compounds give positive and same result. They are all carbohydrates. Account for the results observed All of them are carbohydrates that is why they all formed violet color at the junction upon adding molisch reagent.

B.        REACTION WITH 1.      MOORE’S TEST

a. to 1 ml of 1% glucose, add 2ml of NaOH, heat in a boiling water bath. What changes in color and odor have you observed?

 It was odorless and clear, turns yellowish in color, after boiling , it turns clear golden brown. It has a slight caramel odor.

b.      To 1 ml of 1% glucose, add 2ml of 1% Ba(OH)2. Place in a boiling water bath. Note the changes in odor and color. Record your observation.

It was odorless and cloudy white and has white precipitate. After boiling, it turns brownish orange and has a caramel odor How do you account for the difference in the results using NaOH and Ba(OH)2? 

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In NaOH, It was odorless and clear, turns yellowish in color, after boiling , it turns clear golden brown. It has a slight caramel odor. In Ba(OH), It was odorless and cloudy white and has white precipitate. After boiling, it turns brownish orange and has a caramel odor.

C.       REDUCING PROPERTY Preparation of Tollen’s reagent: To 5ml of 1% AgNO3 solution, add 1 drop of 6N NaOH, Add ammonia solution dropwise, shaking after each addition until the precipitate initially formed is dissolved. Avoid adding more what is necessary to dissolve the precipitae. 

1.      Tollen’s Test Place 6 drops of 1% glucose into a clean test tube and add 2 ml of tollen’s reagent. Mix well. Allow to stand for 5 minutes. What did you observed? The color of the mixture from dark brown becomes charcoal black with black precipitate. There is a silver mirror result because of aromathic aldehydates.

  2.      Fehling’s Test

a.Prepare Fehling’s reagent by mixing 1 ml of fehling’s A, 1 ml of fehling’s B and 8 ml of distilled water in abig test tube. b. Place 1 ml of Fehling’s reagent in a test tube and add 5 drops of 1 % glucose solution. Heat in a boiling water bath. Note the color produced. c. Reapet the test on 1% xylose, 1% sucrose, 1% starch,a nd 1% lactose solutions. 

TEST COMPOUND OBSERVATION1% glucose1% xylose

1% sucrose1% starch1% lactose

 

OBSERVATIONS 

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3.      Nylander’s test 

a.      mix 15 drops of 1% glucose with an equal amount of nylander’s solution in a test tube. Heat for 5 minutes on a boiling water bath. Note the color produced.

b.      Repeat the procedure on 1% xylose, 1% sucrose, 1% starch and 1% lactose solutions. TEST COMPOUND OBSERVATION1% glucose1% xylose1% sucrose1% starch1% lactose Which compound gave the positive result? Glucose, xylose and lactose gave the positive result.

Which compound did not gave the positive result? Sucrose and starch did not gave positive result.  

4.      Benedict’s test 

a.      to 1 ml of benedict’s reagent in a test tube, add 1ml of 1% of glucose solution. Heat in a boiling water bath in 2 minutes and allow to stand. Note the time required to show a green or red color.

b.      Repeat the test on 1% solutions of xylose, sucrose, lactose, starch and glycogen.  TEST COMPOUNDS OBSERVATIONglucosexylosesucrosestarchlactoseglycogen 

D.       HYDROLYSIS 

1.  Prepare the rice solution by placing a spatula of cooked rice in a mortar and pounding using a pestle. Add 10 ml of distilled water. Stir

2.  Put 2ml of rice solution in a test tube, add 1 ml of 6M HCl and heat in a boiling water bath for 15 minutes to hydrolyzed.

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3.  Neutralize the resulting mixtures with about 10 drops of 6M NaOH. Test by adding a drop of phenolphthalein. A light pink color should be observed.

4.  Add 2ml of fehling’s reagent. Heat in a water bath for 2 minutes5.  In another test tube, mix 2ml of rice solution, 1ml of fehling’s A and 1 ml of fehling’s B.

heat in a water bath for 2 mnutes.  Was there a change in color? None Is a positive result obtained? Yes What does it indicate? A reddish brown precipitate

EXPERIMENT 6SPECIFIC GENERAL REACTIONS OF CARBOHYDRATES

Carbohydrates are classified generally according to their degree of complexity. the free sugars such as glucose and fructose are termed monosaccharides; sucrose and

maltose, disaccharides; and the starches and celluloses, polysaccharides. Carbohydrates of short chain lengths such as raffinose, stachyose and verbascose, which

are three, four and five sugar polymers respectively, are classified as oligosaccharides.

Pentoses are five-carbon sugars seldom found in the free state in nature. In plants they occur in polymeric forms and are collectively known as pentosans.

Hexoses comprise a large group of sugars. Principal among these are: glucose, fructose, galactose and mannose. While glucose and

fructose are found free in nature, galactose and mannose occur only in combined form. The hexoses are divided into aldoses and ketoses according to whether they possess

aldehydic or ketonic groups.

Glucose is widely distributed in small amounts in fruits, plant juices and honey. It is commercially produced by the acid or enzyme hydrolysis of grain and root starches. Glucose is of special interest in nutrition because it is the end-product of carbohydrate

digestion in all non-ruminant animals including fish.

Fructose is the only important ketohexose and is found in the free state alongside glucose in

ripening fruits and honey. Combined with glucose it forms sucrose. Fructose is somewhat sweeter than sucrose and is produced in increasing quantities

commercially as a sweetener. Galactose

occurs in milk in combination with glucose. It is also present in oligo-saccharides of plant origin, in combination with both glucose and

fructose.

Mannose is present in some plant polysaccharides collectively termed mannans.

Disaccharides

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are condensation products of two molecules of monosaccharides.

Sucrose is the predominant disaccharide occurring in the free form and is the principal substance of

sugar cane and sugar beet.

Maltose is a dimmer of glucose, and lactose is a copolymer of galactose and glucose.

Oligosaccharides raffinose, stachyose and verbascose are present in significant quantities in legume seeds. Raffinose, which is the most widespread among the three, consists of one molecule of

glucose.

Polysaccharides represent a large group of complex carbohydrates which are condensation products of

undetermined numbers of sugar molecules.

Iodine test is used to test for the presence of starch. Iodine solution — iodine dissolved in an aqueous solution of potassium iodide — reacts

with starch producing a deep blue-black color

RESULTThe formation of a blue-black complex.

a negative test (left) and a positive test (right)

Bial’s orcinol’s test A test for the presence of a pentose in urine, utilizing oracin, hydrochloric acid, and ferric

chloride; a green color or green precipitate indicates pentose.

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RESULT

The formation of a bluish product. All other colors indicate a negative result for pentoses. Note that hexoses generally react to form green, red, or brown products.

two negative tests (left, middle) and a positive test (right) 

Seliwanoff’s test

A color test helpful in the identification of ketoses, which develop a red color with resorcinol in hydrochloric acid

RESULT

The formation of a red product.

a negative test (left) and a positive test (right)

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EXPERIMENT 6SPECIFIC REACTIONS OF CARBOHYDRATES

OBJECTIVES:

1.   To differentiate the reactions of hexoses from pentoses, aldoses from ketoses.2.   To identify different tests for specific carbohydrates.3.   To identify an unknown carbohydrate sample.

MATERIALS:  

1% FRUCTOSE 1 %GALACTOSE 1 % GLUCOSE 1 % XYLOSE 1 %LACTOSE STARCH SUCROSE SOLUTION GLYCOGEN SOLUTION BIAL'S REAGENT CONC. NITRIC ACID SELIWANOFF'S REAGENT 95% ETHYL ALCOHOL IODINE SOLUTION GLUCOSE POWDER GALACTOSE POWDER LACTOSE POWDER SUCROSE POWDER SODIUM ACETATE

 PROCEDURE:

A. BIAL’S ORCINOL TEST 

1. Pipet 0.5 ml of 1% glucose solution into a test tube and 1.0 ml of Orcinol’s reagent. Place the test tube into a hot water bath. What is the color formed during the first 5 minutes? After 5 minutes, you will observe changes in color, the result is a blue green solution.   2. Repeat the test on 1% fructose, 1% xylose, and an unknown solution. Which of the test compounds gave a positive result? Xylose gave the positive result of having a blue green solution. To which group of carbohydrates does it belong? Xylose belong to the group of pentoses

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 two negative tests (left, middle) and a positive test (right)

B.     MUCIC ACID TEST 

1.   Into 4 separate test tubes, place a pinch of each of galactose, glucose, lactose, and an unknown. Add 1 ml of water and 1ml of conc. Nitric acid.

2. Heat the test tubes with their contents in a boiling water bath for an hour.3. Cool at room temperature. Induce crystal formation by scratching the tube with a clean

stirring rod. Collect some crystal with each tube.4. Examine with a microscope.

 GALACTOSE GLUCOSE LACTOSE UNKNOWN(+) Positive resultformation of insoluble crystals

(+) Positive resultformation of insoluble crystals

(+) Positive resultformation of insoluble crystals

(+) Positive resultformation of insoluble crystals

 Based on the crystals formed, what is the unknown sample? The unknown sample used is sucrose. 

C. SELIWANOFF’S TEST 

1. Place 2 ml of Seliwanoff’s reagent in a test tube and add 2 drops o and its time formation.

2. Repeat the test using 1% fructose, 1% sucrose, 1% starch solution  

To which group of carbohydrates do they belong? Carbohydrates Group Observed color Time1. glucose Monosaccharide Yellow color 2 min

2. fructose Disaccharide Red cherry 3 min

3.sucrose Disaccharides Red cherry 3 min

4. starch Monosaccharides Yellow color 22 min

  Which of the samples reacted most readily to the test? Glucose readily reacted to the test.

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a negative test (left) and a positive test (right)

  D.     IODINE TEST  In a spot plate or watch glass, separately place 5 drops of each of the 1% solutions of starch, glycogen and glucose. To each spot, add a drop of iodine solution. What color was observed with: Starch : bluish black/ blue solutionGlycogen : red/ brown solutionGlucose : light yellow/ colorless solution

a negative test (left) and a positive test (right) 

SUPPLEMENTARY QUESTIONS: 

1. WRITE THE STRUCTURAL FORMULA OF GLUCOSE AND FRUCTOSE.

Glucose Fructose

2. WRITE THE STAGES IN HYDROLYSIS OF STARCH AND THEIR COLOR RWACTIONS WITH IODINE SOLUTION.

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 A. gelatinizationB. liquefactionC. Saccharification

3. EXPLAIN THE FORMATION OF OSAZONE CRYSTALS AND MUCIC ACID. 

The reactions served to identify the sugars by the structure of the crystals and the required time from them upon oxidation with nitric acid hexoses produce crystals that are soluble in dilute acid and H2O.