LESSON 1: DIALYSIS  The effect of temperature and pH on the activity of  Record the time required for a positive test
 Record the time required for a positive test for each
Dialysis: common laboratory technique widely used for removing
contaminants from solution, to remove small unwanted molecules
such as salts, reducing agents, preservatives, etc.
֎Works by selective and passive diffusion through a
semipermeable membrane.  colour with iodine till it is completely digested into
֎Used for buffer exchange. The buffer used for dialysis is
called dialysate
Principle of Dialysis
1.Dialysis works by the principle of diffusion.
2.Diffusion - migration of molecules randomly from areas of higher
concentration to lower concentration until equilibrium is reached.  ֎check for the presence of carbohydrates in a given analyte.
3.In Dialysis, the migration of molecules occurs through a
semipermeable membrane, which allow only small molecules to
pass through restricting the movement of large molecules like
proteins.  H2SO4 will dehydrate the sugar allowing it to react with the alcohol
Dialysis Procedures
1.Pre-wet the membrane
2. Load the sample on dialysis tubing
3. Dialyze for two hours
4. Change dialysis buffer and dialyze for 2 hours
5. Change dialysis buffer and dialyze overnight
Water is important to all living systems.
֎Natural solvent for mineral ions and other substances. It is also
the dispersion medium for colloidal cells like protoplasm. ֎Medium
for most biochemical reactions, and is the most abundant
component of the cells. Except for bone tissues and enamel, water
constitutes about 70 percent of the human body.  
LESSON 2: OSMOSIS
Osmosis: diffusion/movement of solvent (water) across a
membrane, the movement solvent molecules from a region of
lower solute concentration to a region of higher solute
concentration.  
 Affected by amount of substance dissolved to it.  it to the test tube with Benedict’s reagent.
 Examples: Red blood cells swelling up when exposed
to fresh water and plant root hairs taking up water.
 Demonstration: soak gummy candies in water. The gel
of the candies acts as a semipermeable membrane.
Diffusion: movement of solute molecules from an area of higher
concentration to an area of lower concentration.  
 Overall effect is to equalize concentration throughout
the medium.
 Examples: Perfume filling a whole room and the
movement of small molecules across a cell membrane.
 Simplest demonstration: Adding a drop of food
coloring to water. Although other transport processes
do occur, diffusion is the key player.
Key Point: Diffusion and osmosis are both passive transport
processes that act to equalize the concentration of a solution
֎Procedure
 Stop the narrow end of a thistle tube with a finger and
fill it with sugar cane juice or a 10% sucrose solution
until the solution reaches up to the base of the tube.
 Cover the mouth of the thistle tube with a cellophane
membrane and keep it in place with a rubber band.
 Glucose, Maltose, Fructose
Suspend the thistle tube in a beaker of water, making
sure that the levels of liquids inside and outside of the
tube are equal.
 in separately labeled test tubes
Observe the difference in solution levels after minutes.
LESSON 3: ENZYMES
THEORY
•Enzymes are complex organic substances produced by an
organism to alter the rate of biochemical reactions that take place
in the cell.
•Show high degree of specificity
•All enzymes have a protein component and are affected by a
number of physical and chemical factors that denature proteins
 Extremes of pH and temperature
 Presence of metal and non-metal ions
 Presence of alkaloidal reagents
 Enzyme and substrate concentrations
֎Temperature: All enzymes are proteinaceous in nature. At a
lower temperature, the enzyme salivary amylase is deactivated and
at the higher temperature, the enzyme is denaturated. Therefore,
more time will be taken by an enzyme to digest the starch at lower
and higher temperatures. Optimum temperature for the enzymatic
activity of salivary amylase ranges from 32 °C to 37 °C. The
optimum temperature means that the temperature at which the
enzyme shows the maximum activity.  At this optimum
temperature, the enzyme is most active and hence, takes less time
to digest the starch.
֎Effect of pH: The optimum pH for the enzymatic activity of
salivary amylase ranges from 6 to 7. Above and below this range,
the reaction rate reduces as enzymes get denaturated. The enzyme
salivary amylase is most active at pH 6.8.  Our stomach has high
level of acidity which causes the salivary amylase to denature and
change its shape. So the salivary amylase does not function once it
enters the stomach.
salivary amylase on starch can be studied by using the
Iodine test.  If we add saliva on starch, the salivary
amylase present in saliva gradually acts on starch and
converts it into maltose.  Starch keeps on giving blue
maltose.  At this point, no blue colour is formed.  This
is the end point or achromic point.
Test Based on the Formation of Furfural and its Derivatives
 I. General Tests for Carbohydrates
1.Molisch Test: general test for carbohydrates
֎The sugars are mixed with a-naphthol. The test tube is inclined,
and concentrated H2SO4 is added along the side of the tube,
causing the formation of a lower layer of acid. The concentrated
forming furfural or hydroxymethul-furfural.
֎Positive: Formation of a purple ring at the interface of the two
liquids will indicated the presence of a carbohydrate.
֎All carbohydrates give positive results to Molisch test. But
monosaccharides give positive test faster
֎ Procedures:
 Mix 4 ml of distilled water and two drops of the
Molisch’s reagent in a test tube. This tube will serve as
the control.
 Place 4 ml of 3% solution of glucose in a second test
tube. Add two drops of the Molisch’s reagent and mix
the contents by gently shaking the test tube.
Incline the test tube and cautiously add about 5 ml of
concentrated sulfuric acid, allowing the acid to run
down the side of the tube. Sulfuric acid is denser than
water and will form a lower layer. Note the color of
the ring formed at the junction if the two liquids.
 In the same manner of adding acid, add sulfuric acid to
the control tube. What do you observe?
Repeat the above test with 3% sample solutions of
fructose, lactose, and sucrose.
 Record all results.
 2.Bial’s Orcinol Test: used to determine the presence of pentoses
and nucleotides and nucleotides that contain pentose sugars.
֎Positive: When pentoses are treated with orcinol, furfurals are
formed and they will yield a blue-green compound in the presence
of ferric ions. The reaction is not specific for pentoses because
other compounds like trioses, uronic acids, and certain heptoses
will also give blue or green products.
 Carbohydrates that yield positive results: Ribulose,
Xylulose, Ribose, Arabinose, Lyxose, Xylose
֎Negative: Hydroxymethyl furfural is formed from hexoses to give
yellow-brown condensation products.
 Carbohydrates that yield negative results: Galactose,
Talose, Mannose, Allose, Altrose, Gulose, Idose,
֎Procedure:
 Place 1 ml of each 3% solution of xylose, and glucose
 Add 3 ml of Bial’s reagent to each test tube
 Carefully heat each tube over a Bunsen flame until the
solution begins to boil. Add one to two drops of 10%
FeCl3 solution.
 Note the color of the product formed
 Record your results in the table
 3.Seliwanoff’s Test: differentiate ketohexoses from aldohexoses.
֎Ketohexoses react faster with the solution containing hydrochloric
acid and resorcinol than aldohexoses.
֎Positive: The dehydrated ketohexoses form a bright cherry red
condensation product
 Carbohydrates that yield positive results: fructose,
psicose, sorbose, sucrose and tagatose
֎Negative: aldohexose yields only a pale pink coloration. In this
test, prolonged heating of samples should be avoided.
 Carbohydrates that yield negative results: allose,
lactose, altrose, glucose, mannose, glucose, idose,
galactose and talose
֎Procedure:
Place 1 ml each of 3% glucose, fructose, lactose and sucrose in
separately labeled test tubes.
 Add 4 ml of Seliwanoff’s reagent to each test tube
 Place the test tubes in a water bath filled with boiling
water and allow them to stay there for exactly 1
minute
 Note the changes and record which test tube gives a
positive result in the shortest time.
 Continue heating and observe the color changes at 1
minute intervals
sample
4. Solubility Test:
 Glucose, lactose and sucrose are soluble in water,
whereas starch is insoluble in water.
LESSON 4: REDUCING PROPERTY OF SUGARS
REDUCING SUGARS: must have a free aldehyde or ketone group
֎Common dietary monosaccharides: glucose, galactose, fructose
1.BENEDICT’S TEST: very sensitive test done under mildly alkaline
conditions
֎Benedict’s Reagent: Composed of CuSo4, Na2CO3, sodium citrate
֎Test for the presence of glucose in urine (green color is the color If
it is a positive result)
֎PRINCIPLE:
 Positive: When Benedict’s solution and simple
carbohydrates are heated, the solution changes to
orange red (moderate amount of reducing sugars) /
brick red/green (traces of reducing sugar). This
reaction is caused by the reducing property of simple
carbohydrates. The copper (II) ions in the Benedict’s
solution are reduced to Copper (I) ions, which causes
the color change.
o Carbohydrates that yield positive results:
Glucose, fructose, and galactose
 Negative Results: Remain blue
o Carbohydrates that yield negative results:
Sucrose, Starch, Cellulose, and other
Polysaccharides
֎Sample: Urine - Aim: To detect the presence of reducing sugar in
urine
֎PROCEDURE:
 Using a pipette, accurately take 5 mL of Benedict’s
reagent and slowly transfer it to the test tube.
Take 5 mL of freshly collected urine by pipette and add
 Place the test tube in boiling water bath for 2-3
minutes
 Observe the color of the solution and note whether a
precipitate was formed
 Avoid prolonged heating
 Record your results
2.BARFOED’S TEST: contains cupric acetate in dilute acetic acid
֎It oxidizes monosaccharides but not oligosaccharides
֎Disaccharides are less oxidized but are oxidized if they undergo
prolonged heating
֎causing hydrolysis of the disaccharides into monosaccharides
֎ produces a positive result
֎used to distinguish between monosaccharide, disaccharides, and
oligosaccharides
֎Positive result: Brick red precipitate
 Carbohydrates that yield positive results: Glucose,
fructose, and galactose
֎Negative result: Remains blue
֎Unlike Benedict’s test, this test is carried out under acidic rather
than basic medium
֎PROCEDURE:
 Place 1 mL each of 3% solutions of glucose, xylose,
fructose, lactose and starch in separately labeled test
tubes
 Add 3 mL of Barfoed’s reagent in each test tube
 Prepare a control tube using distilled water instead of
sugar solution
 Place all the tubes in a boiling water bath for 10
minutes
 Record your observations
3.TOLLEN’S TEST (SILVER MIRROR TEST): Sugars with aldehyde
groups are capable reducing Tollen’s reagent
 Purpose: used to distinguish between an aldehyde and
a ketone.
֎Tollen’s reagent: Ammoniacal solution of silver
֎Positive result: Gray to black precipitate (silver mirror)
 Carbohydrates that yield positive results: allose,
lactose, altrose, glucose, mannose, glucose, idose,
galactose and talose
֎Negative result: Clear Solution
 Carbohydrates that yield negative results: fructose,
psicose, sorbose, sucrose and tagatose
֎PROCEDURE:
 Place 5 drops of 3% solutions of glucose, xylose, and
sucrose in separate test tubes
 Add 2 mL of Tollen’s reagent into each tube
 Preparation: Add 1 drop of NaOH solution to 6 mL of
5% AgNO2. Add dilute NH4O4 (1 mL concentrated
NH4OH + 5 mL water) until the brown precipitate of
silver oxide that forms just dissolves
 This reagent must be prepared fresh and not stored
since it decomposed when left standing and yields an
explosive decomposition product. Discard all leftover
materials.
 Boil for about 5 minutes. Note and record your
observation