BIOCHEMISTRY (LAB)

I. EXPERIMENT 9: ​Color Reactions of Carbohydrates

 A. TABULATION:
GENERAL TEST
TEST  REAGENTS USED  POSITIVE RESULT  PRINCIPLE  CARBOHYDRATES THAT 
WILL GIVE POSITIVE RESULT 

Molisch Test  ● Molisch  Purple complex at the  Sugars undergo dehydration  General tests – all 
Reagent (α-  interface between the two  in the presence of  carbohydrates/sugars should 
naphthol)  solutions  non-oxidizing acids like  have a positive result. 
hydrochloric acid and sulfuric 
    acid to form furfural or  Although this test will detect 
hydroxymethylfurfural. These  compounds other than 
● Concentrated  compounds can react with  carbohydrates (e.g., 
Sulfuric Acid  aromatic amines such as  glycoproteins, a conjugate of 
α-naphthol or phenol to give  sugar and protein), a negative 
  intensely colored compounds.  result indicates the total 
This reaction forms the basis  absence of carbohydrates. 
of a general qualitative test for 
sugar.   

Anthrone Test  Anthrone Reagent
(200g Anthrone in 100mL (Presence of Carbohydrates)
Sulfuric Acid)
Blue-Green Carbohydrates are dehydrated  Kat: All carbohydrates give 
with concentrated H​2​SO​4​ to  positive result 
form “Furfural”, which 
condenses with anthrone to 
form a green color complex 
which can be measured by 
using colorimetrically at 
620nm (or) by using a red 
filter.  
Anthrone reacts with dextrins, 
monosaccharides, 
disaccharides, 
polysaccharides, starch, 
gums, and glycosides. But 
they yields of color where is to 
form carbohydrate to 
carbohydrate. 

From: 

Harisha, S. (2006). An 

Introduction to Practical 
Biotechnology. Retrieved from 
https://books.google.com.ph/b
ooks?id=YqFMQn5UtGQC&pri
ntsec=frontcover#v=onepage&
q&f=false 
 DIFFERENTIATING TESTS FOR ALDOSES AND KETOSES
TEST  REAGENTS USED  POSITIVE RESULT  PRINCIPLE  CARBOHYDRATES THAT 
WILL GIVE POSITIVE 
RESULT 

Seliwanoff’s Test  Freshly Prepared Aldose: ​Faint pink color Kat: Due to concentrated HCl, galactose
Seliwanoff Reagent Ketoses and Aldoses are
(0.05g Orcinol, 30mL Ketose:  dehydrated to give furfural Fructose, and Sucrose
Concentrated Formation of deep red (cherry derivatives. When condensation
Hydrochloric Acid, & red) solution occurs, ketose reacts rapidly to
60mL Water) form a cherry red complex while
aldose reacts slowly and gives
faint pink color.

  Pentose:  “Bial’s reagent uses orcinol,  ribose

Blue-Green Solution HCl, and FeCl3. Orcinol forms 
  (Presence of Pentose) colored condensation products 
Bial’s Reagent with furfural generated by the 
  (0.4g Orcinol, 200mL dehydration of pentoses and 
Concentrated pentosans. It is necessary to 
Bial’s Test  Hydrochloric Acid, use dilute sugar solutions with 
10mL 5% FeCl3) this test (0.02 M).” 

From: 

Harisha, S. (2006). An 

Introduction to Practical 
Biotechnology. Retrieved from 
https://books.google.com.ph/b
ooks?id=YqFMQn5UtGQC&pri
ntsec=frontcover#v=onepage&
q&f=false 

Hexose: ​muddy brown-gray    

condensation product

Tollen’s  ● Concentrated Yellow to Red Solution    

Phloroglucinol  Hydrochloric
Test  Acid

● Tollen’s
Phloroglucino
l Reagent
(0.5%
Phloroglucino
l Reagent)
 TEST FOR REDUCING SUGAR

TEST  REAGENTS USED  POSITIVE RESULT  PRINCIPLE  CARBOHYDRATES THAT 

WILL GIVE POSITIVE 
RESULT 

Barfoed’s Test  Barfoed’s Reagent Monosaccharide:  “ This would detect reducing Glucose 
(6.2g Cupric Acetate, brick red precipitate monosaccharides in the Fructose 
  0.5mL Glacial Acetic Acid, presence of disaccharides.
and 100mL DIstilled Water) Reagent uses copper ions to
detect reducing sugars in an
acidic solution. Barfoed’s
Reagent is copper acetate in
dilute acetic acid.”  

Harisha, S. (2006). An Introduction to 

Practical Biotechnology. Retrieved 
from 
https://books.google.com.ph/books?i
d=YqFMQn5UtGQC&printsec=frontco
ver#v=onepage&q&f=false

A positive test is a dark

red precipitate and is
evidence of a reducing
monosaccharide.

Disaccharide: ​brick    
red precipitate

Benedict’s Test  Benedict’s Reagent
Brick Red Precipitate “Alkaline solutions of copper are  Glucose 
“Cuprous Oxide” reduced by sugars that have a free  Aldoses 
aldehyde or ketone group, with the  Lactose 
formation of colored cuprous oxide. 
Benedict’s solution is composed of 
copper sulfate, sodium carbonate, 
and sodium citrate (pH 10.5). 

The citrate will form soluble complex 

ions with Cu++, preventing the 
precipitation of CuCO​3​ in alkaline 
solutions.” 

Harisha, S. (2006). An Introduction to 

Practical Biotechnology. Retrieved 
from 
 https://books.google.com.ph/books?i
d=YqFMQn5UtGQC&printsec=frontco
ver#v=onepage&q&f=false

in alkaline medium, sodium 

carbonate converts glucose to 
enediol and this enediol reduce 
cupric to cuprous forming 
cuprous hydroxide. This 
solution is kept in sodium 
citrate and on boiling red 
precipitate of cuprous oxide is 
formed. 

https://byjus.com/chemistry/te
sts-of-carbohydrates/ 

Fehling’s Test  Fehling’s A:  Brick Red Precipitate The copper ions present in  Lactose glucose 
copper sulfate  Or fehling’s solution in +3 state is 
solution   Yellow to Orange-Red reduced to +2 oxidation state 
  Precipitate and in alkaline medium it is 
Fehlings’s B:  precipitated as red ​cuprous 
potassium hydroxide  oxide​. 
and potassium  https://byjus.com/chemistry/te
sodium tartrate sts-of-carbohydrates/ 
 

If a reducing agent is 

present, the Cu​2+ ​is reduced 
to Cu​+ a
​ nd forms a red 

precipitate of Cu​2​O. 
Therefore, if Fehling’s 
solution is added to a 
solution containing a 
reducing sugar, a red 
precipitate will form 

 
 

Tollen’s Silver  ● Freshly Prepared Silver Mirror Carbohydrates reacts with  glucose, fructose, lactose
Mirror Test  Silver Nitrate and - deposition of Silver Tollens reagent and form a 
Ammonia TS Metal silver mirror on the inner walls 
of the test tube. This confirms 
the presence of reducing 
sugars. Silver ions are reduced 
 to metallic silver. 
 
https://byjus.com/chemistry/te
sts-of-carbohydrates/ 

Ferricyanide  ● Freshly Prepared Yellow Color    

Test  Saturated (Presence of
Potassium Carbohydrates)
Ferricyanide
Solution

● 10% NaOH

 
Phenylhydrazine  2-,4-dinitrophenylhydrazine Yellow to Orange
Test  Precipitate
(Osazone  “Osazone”
Formation) 
“The ketoses and aldoses react with  glucose
phenylhydrazine  to  produce  a 
phenylhydrazone which further reacts 
with  another  two  molecules  of 
phenylhydrazine  to  yield  osazone. 
Needle-shaped  yellow  osazone 
crystals  are  produced  by  glucose, 
fructose  and  mannose,  whereas 
lactosazone  produces  mushroom 
shaped crystals. Crystals of different 
shapes  will  be  shown  by  different 
osazones. Flower-shaped crystals are 
produced by maltose.” 
https://vlab.amrita.edu/?sub=3&brch=
63&sim=631&cnt=1 

 
 TEST FOR SPECIFIC SUGAR

TEST  REAGENTS USED  POSITIVE RESULT  PRINCIPLE  CARBOHYDRATES 

THAT WILL GIVE 
POSITIVE RESULT 

Test for Fructose  Indole-3-acetic Acid Purple-Violet Color   Fructose 

Reagent (Presence of Fructose)
  (0.5% indole-3-acetic
acid in 95% ethanol)

Iodine Test for  ● Dilute Blue-Violet Color PIC SA BABA  Starch 

Starch  Hydrochloric
Acid From: 
 
● Iodine TS
Harisha, S. (2006). An 
Introduction to Practical 
Biotechnology. Retrieved from 
https://books.google.com.ph/boo
ks?id=YqFMQn5UtGQC&printsec
=frontcover#v=onepage&q&f=fals
e 

  “Oxidation of most   
monosaccharides by nitric acid   
  yields soluble dicarboxylic acids.   
However, oxidation of galactose   
Mucic Acid Test for  Concentrated Nitric Insoluble crystals at the yields an insoluble mucic acid.  Galactose 
Galactose  Acid bottom of the test tubes Lactose will also yield a mucic 
(Presence of Mucic Acid) acid, due to the hydrolysis of the 
  glycosidic linkage between its 
glucose and galactose subunits”. 

From: 

Harisha, S. (2006). An 

Introduction to Practical 
Biotechnology. Retrieved from 
https://books.google.com.ph/boo
ks?id=YqFMQn5UtGQC&printsec
=frontcover#v=onepage&q&f=fals
e 
REFERENCE:

https://books.google.com.ph/books?id=mVuNCwAAQBAJ&pg=PA71&lpg=PA71&dq=Can+Pentose+be+green+in+b
oth+aldose+and+ketose+test?&source=bl&ots=m9pfn8tXNp&sig=ACfU3U1NcwuyQ8PAOykO8a8Eu9yK1zClsQ&hl=
en&sa=X&ved=2ahUKEwjdhonznevoAhWrzIsBHVPeAisQ6AEwCnoECBAQKQ#v=onepage&q=Can%20Pentose%20b
e%20green%20in%20both%20aldose%20and%20ketose%20test%3F&f=false 

Harisha, S. (2006). An Introduction to Practical Biotechnology. Retrieved from 

https://books.google.com.ph/books?id=YqFMQn5UtGQC&printsec=frontcover#v=onepage&q&f=false
 II. EXPERIMENT 10:​ Optical Activity of Sugars

A. PROCESS FLOW
For this learning activity, you will not be required to prepare a schematic diagram
based on the procedure stated above. However, please do watch this video to help you
visualize the actual experiment that we were supposed to perform in the laboratory :
https://www.youtube.com/watch?v=McGd-0kaTxs

After watching the video, please ​write a summary of the steps/process flow
mentioned in the video and include important steps, if applicable.
Below is the outline and some guide questions:

1. Preparation of the Sugar Solution

2. Calibration of the Polarimeter using Deionized Water

o What should be seen in the visual field before you read the dial?
o What should be the reading in the dial for you to say that the polarimeter has been
calibrated?

3. Determination of the Optical Activity of the Sugar Solution

o What should be seen in the visual field before you read the dial?
o How do you read the dial (Which scale represents the whole number and which
scale represents the decimal point)?
o What is the final reading in the video?
o How do you know if you have a positive or negative degree of rotation?

▪ If the sugar has a negative degree of rotation, how will you read the
dial? What is the final reading for the negative degree sample in the video?
 B. THEORETICAL RESULTS
Complete the table. Concentration to be used will be based on the procedure above
(Preparation of Solutions – pages 3 to 4). Show your computations.

CARBOHYDRATE  ANGULAR  SPECIFIC ROTATION  PATHLENGTH  Dextro or Levo? 

ROTATION  [α] D25     
α    

Fructose   Answer: -92.00 m2·rad/kg   20 cm  Answer: L​ evo

 

Glucose  +21.08o  Answer: ​+52.7 m2-rad/kg   2 dm  Answer:

 

Sucrose  Answer: +66.47 m2·rad/kg   10 cm  ​ extro

Answer: D

C. GUIDE QUESTIONS
Answer the following guide questions.

a. Draw and label the parts of a polarimeter.

b. Explain how concentration, pathlength, temperature and wavelength influence specific
rotation.
 Siz wala pa ako makitang reliable source :// -KAYE

c. What are some applications of polarimetry in the pharmaceutical industry?

https://rudolphresearch.com/products/polarimeters/polarimetry-definitions/

Try niyo click link siz pag need niyo more info <33

d. Exercises:

1. Calculate the specific rotation of (2R,3R)-tartaric acid based on the following

observation: A 0.931 g sample of the pure acid was diluted to 10.0 mL with
water and placed in a 10 cm polarimeter tube. The observed rotation using the
589 nm line of a sodium lamp at 25.0 oC was α = +1.12 o

2. The correct answer here is +12.03 m2·rad/kg. Show the correct

solution/computation to arrive at thus answer.

3. 0.050 g of sample is dissolved in 2.0 mL of ethanol, and this solution is placed

in a 2.0-cm polarimeter tube. The observed rotation is clockwise 0.043˚.
Calculate the specific rotation, [α]D.

Aside from the videos in this learning guide, you may also go this page:
https://wiki.anton-paar.com/en/basics-of-polarimetry/
 III. EXPERIMENT 11:​ Isolation of Starch and Glycogen

A. SCHEMATIC DIAGRAM
Create a schematic diagram for the procedures stated above. For the
Isolation of Starch, indicate which between the filtrate and the residue is kept after
filtration. Indicate which between the supernatant and the residue is kept after
decanting.

B. TABULATION​. Complete the table below.

AMYLOSE  AMYLOPECTIN  GLYCOGEN 

Description of Structure    linear structure of glucose chaine
Or
linear polysaccharide
branched structure larger and more frequent branching
Or
Polysaccharide made up of multiple
branches of ​f α-1,4-linked glucose
residues.

Type/s of Glycosidic Bonds  1-> 4 alpha alpha- D 1->4, alpha->1,4

Present  alpha-D 1->6

Monosaccharide Present    d-glucose glucose glucose

Visible Result in the I2 Test    deep blue color reddish brown or purple solution reddish brown color 

Reference for the table:

For Amylose: ​(Pili na lang kayo either sa dalawa para di tayo pare-parehas sa ref)

● D S Jackson, D.S., (2003). Starch Structure, Properties, and Determination. Retrieved from
https://www.sciencedirect.com/sdfe/pdf/download/eid/3-s2.0-B012227055X01141X/first-page-pdf​.

● Luallen, T. (2004). Utilizing starches in product development.Woodhead Publishing Series in Food Science,
Technology and Nutrition, p 393-424. Retrieved from
https://www.sciencedirect.com/science/article/pii/B9781855737310500177​.

For Amylopectin: 

● D S Jackson, D.S., (2003). Starch Structure, Properties, and Determination. Retrieved from
https://www.sciencedirect.com/sdfe/pdf/download/eid/3-s2.0-B012227055X01141X/first-page-pdf​.
 ● Evans, W.C. (2009). Chapter 20 - Carbohydrates.Trease and Evans’ Pharmacognosy, 16th ed. Retrieved from
https://www.sciencedirect.com/science/article/pii/B9780702029332000204​.

For Glycogen:

● Morris, D.L. (1946). COLORIMETRIC DETERMINATION OF GLYCOGEN

DISADVANTAGES OF THE IODINE METHOD. Retrieved from
https://www.jbc.org/content/166/1/199.full.pdf.

C. GUIDE QUESTIONS
Answer the following guide questions:

a. Is 1% NaCl hypertonic or hypotonic relative to physiologic fluids? What is its

possible role in the isolation of starch?
Answer:
https://sciencing.com/osmosis-experiments-potatoes-kids-12066982.html

b. In which solution is cellulose more soluble, in acidic or basic solutions?

Answer:
https://estudogeral.sib.uc.pt/bitstream/10316/29319/1/Cellulose%20solutions.pdf

c. What will be the equation used to compute for percent yield in this
experiment?
Answer:
peris-tortaja 
da2018.pdf 
 

Ganito ba ito? Lol. Di ako sure

https://www.dummies.com/education/science/chemistry/how-to-calculate-percent-yiel
d-in-a-chemical-reaction/

Ganyan din sagot ko sis - moriel

Iba sagot ko sis -Elisha check nyo yung link :(((

 IV. EXPERIMENT 12:​ Hydrolysis of Carbohydrates

A. SCHEMATIC DIAGRAM
Create a schematic diagram for the procedures stated above.

B. TABULATION
Copy and complete the table/s below. Answering the guide questions first may help.

Polysaccharide  Source  Use (Storage or Structural)   Monosaccharide 

(Animal or Plant)    

Starch   Plant -structure: straight or branched glucose

-storage:

Glycogen   Animal - structure: multi-branched glucose

-storage:

Cellulose   Plant -structure: linear chain w/ several b-linked D-glucose units glucose
-storage:

Inulin   Plant -structure: linear fructose polymer fructose

-storage:

Sample   Theoretical Visible Result  Indication 

   

1% Sucrose treated with 3 M 

H2SO4 

1% Sucrose treated with 3 M 

NaOH  

1% Sucrose  

1% Glucose  

Sample  Theoretical Initial  Time for Complete Hydrolysis 

  Iodine Test Visible Result   (indicate which is slower or faster)  
   
Acid-Catalyzed   Enzyme-Catalyzed 

Starch   blue-black color slower faster

Glycogen   brown color

 Sample  Theoretical Visible Result   Indication 
   
Fehling’s Test   Indole-3-Acetic Acid Test    

1% Cellulose treated  blue soln w/ brick red ppt

with 3 M H2SO4 

1% Inulin treated with  blue soln w/ brick red ppt purple soln
3 M H2SO4 

1% Cellulose   blue soln without brick red ppt

1% Inulin   blue soln without brick red ppt

1% Fructose  

Guide Questions. ​Answer the following guide questions.

● Which is expected to catalyze hydrolysis for polysaccharides, an acidic or a

basic medium?

Answer: acidic?

● What is the expected color result of a nonreducing sugar in the Fehling’s test?

Answer:​ ​Red precipitate will form as a positive result for Fehling’s Test. These
changes are evident when aldehyde oxidized to acid and the cuprous oxide that was
formed will be reduced to cuprous ion that changes the color and indicates the
presence of non reducing sugar. -kaye

Check niyo siz waah

Maclean, H. (1906). Observations on Fehling's Test for Dextrose in Urine.

Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1276126/

● What is the enzyme present in saliva that causes carbohydrate digestion? ​(Try
to recall the lecture discussion for this.)

Answer: ​ Carbohydrate digestion starts with the human’s mouth. With the help of the
saliva that releases enzymes it begins to break down carbohydrates. Salivary
amylase is the enzyme present in saliva that is responsible for carbohydrate
 digestion.

● Which is faster, an enzyme-catalyzed reaction or an uncatalyzed reaction?

Answer:

https://www.ncbi.nlm.nih.gov/books/NBK22380/

● During the hydrolysis of starch/glycogen, what will happen if the water bath
used for heating falls below 40​o​C or exceeds 45​o​C?

Answer\
EXPERIMENT 11: from dangco
EXPERIMENT 12: