Waylon Fan
BSP2B
EXPERIMENT 4
PROCESS IN BIOCHEMICAL SYSTEM
OBJECTIVES
1. To relate the particle concept of matter to biochemical processes;
2. To describe molecular motion and interaction; and
3. To observe and describe processes that occur in biochemical system:
diffusion, dialysis, osmosis, hydrolysis and lowering of surface tension.
REAGENTS / MATERIALS
2% NaCl
0.1 % NaCl
1% Na2c03
1 M AgNO3
10% NaOH
0.2 % Ninhydrin
KMn04
0.9% NaCl
1% pacreatin
0.5% CuS04
Iodine solution
Macaroni
Defibrinated blood
Microscope
Bile
String
Margarine
Longanisa membrane
Chicken bouillon
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 bond
To 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 acid
To 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.
2.60C; 1 min. 41 seconds
800C; 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 % NaCl
test tube # 3 - 0.9 % NaCl
test 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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Description
Size: 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
sample
Shape: Circle with the
biconcave
Color: 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 40500C 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
#1
#2
#3
Texture after 15 minutes
heating
Slightly soft/slightly viscous
Soft / viscous
Hard / smooth
Color with the Iodine solution
Blue violet
Charcoal / slightly hardens
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% Na 2c03 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 QUESTION
1. 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 5
GENERAL 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 Carbohydrates
Carbohydrates 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 PROPERTIES
OXIDIZED ALKALINE SOLUTIONS OF CUPRIC SULFATE
REDDISH 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 SUBNITRIDES
ATE WHEN ADDED TO REDUCING SUGAR BLACK PPT – FORMATION OF METALLIC BISMUTH
BARFOEDS REAGENT CUPRIC ACETATE IN WEAK ACETIC ACID, REDUCING
MONOSACCHARIDES NOT DISSACHARIDES
MONOSACCHARIDES
DISACCHARIDES
POLYSACCAHR
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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
RESULT
Silver mirror image
EXPERIMENT 5
GENERAL 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

cupric acetate
1% lactose

1% maltose
1% starch

1% Ba(OH)2
6N NaOH

phenolphtalein

NH4OH
1% xylose

picric acid
1% glycogen

dil. HCL

glacial acetic acid
Benedict’s reagent

Fehling’s reagent
Molisch reagent
1% silver nitrate
Nylander’s 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
Glucose
Xylose
Sucrose
Starch
Maltose
OBSERVATION
+ (positive result)upperlayer-lavender;lower-dark purple
+ (positive result) upperlayer-lavender;lower-dark purple
+ (positive result)upperlayer-white;lower-darkest purple
+ (positive result)upper-grey white;lower-black
+ (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?
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.
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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
1% glucose
1% xylose
1% sucrose
1% starch
1% lactose
OBSERVATIONS
OBSERVATION
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3.
Nylander’s test
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.
a.
TEST COMPOUND
1% glucose
1% xylose
1% sucrose
1% starch
1% lactose
OBSERVATION
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
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.
a.
TEST COMPOUNDS
glucose
xylose
sucrose
starch
lactose
glycogen
D.
OBSERVATION
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.
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 minutes
5. 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.
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Was there a change in color? None
Is a positive result obtained? Yes
What does it indicate? A reddish brown precipitate
EXPERIMENT 6
SPECIFIC 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.


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.
Hexoses

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
 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.
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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
RESULT
The 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 6
SPECIFIC 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
two negative tests (left, middle) and a positive test (right)
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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
(+) Positive result
formation of insoluble
crystals
GLUCOSE
(+) Positive result
formation of insoluble
crystals
LACTOSE
(+) Positive result
formation of insoluble
crystals
UNKNOWN
(+) Positive result
formation 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
1. glucose
Group
Monosaccharide
Observed color
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.
a negative test (left) and a positive test (right)
Time
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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 solution
Glycogen : red/ brown solution
Glucose : 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.
A.
B.
C.
gelatinization
liquefaction
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.