Biochem Laboratory Midterm

BIOCHEMISTRY LABORATORY (Midterm)
I. ELEMENTARY COMPOSITION OF PROTEINS

i. CHEMICALS/REAGENTS
• casein
• NaOH · CaO
• diluted HCl
• 0.1N BaCl2
• concentrated HNO3
• [(NH4)2 · MoO4]
ii. TESTS
USING CASEIN:
A. Carbon & Hydrogen (Charring) ✓ when casein was heated, the charring indicated the presence
of Carbon and the formation of moisture indicated the presence of Hydrogen (and Oxygen).
B. Nitrogen (Soda Lime Test) ✓ in a mortar & pestle, casein and soda lime were both mixed then
heated afterwards, the red litmus paper (placed above the mouth of the test tube) turned blue due to the
evolution of Ammonia gas/NH3 and the urine-like odour indicates the presence of Nitrogen.
➡ FUSION PROCESS: heat the fusion solid (2 parts Na2CO3 & 1 part KNO3) and casein slowly then
strongly (in a porcelain crucible) until there is a formation of clear mixture. Cool & dissolve w/ water and
filter. Divide the filter into two parts:
USING FUSION FILTRATE:
C. Sulfur (Reduced Sulfur Test) ✓ add dil. HCl until acidic to the fusion filtrate and heat to boiling then
add 0.1N BaCl2 yielding to the formation of a white
precipitate (Barium sulfate/BaSO4) that indicates the presence of Sulfur.
D. Phosphorus (Ammonium Molybdate Test) ✓ add conc. HNO3 until acidic to the fusion filtrate then
add [(NH4)2 · MoO4] to the mixture and heat yielding to the formation of a yellow precipitate
(Ammonium phospho-molybdate or (NH4)3 PO4 · 12MoO3 indicating the presence of Phosphorus.
iii. DISCUSSION
1. Casein is a white tasteless, odourless protein precipitated from milk. It is used to make plastic,
adhesive and food. It is a predominant phosphoprotein. It is the principal source of the experiment. 2. The
fushion method/process is used to oxidize sulfur to sulfate and phosphorus to phosphate.
Carbon/C — 15% Oxygen/O — 24% Nitrogen/N — 16% Sulfur/S — 1.0 % Phosphorus/P — 0.4%
Hydrogen/H — 7%
iv. SIDENOTES
1 of 9 ||| laboratoryworksbyveronica2016 SO4 + BaCl2 BaSO4 + 2 Cl
3 NH4 + PO4 + 12 MoO4 + 24 H (NH4)3 PO4 · 12MoO3 + 12 H2O
BIOCHEMISTRY LABORATORY (Midterm) II. COLOR REACTIONS OF PROTEINS
• 2% albumin
• 10% NaOH
• 0.5% CuSO4
• 2% peptone solution/pentose (C5H10O5)
• concentrated HNO3
• NH4OH
• Millon’s reagent
• Hopkins-Colé reagent
• 40% NaOH
• Pb(C2H3O2)2
• glacial CH3COOH
• concentrated H2SO4
ii. TESTS
A. Biuret Test/Reaction ✓ mix 2% albumin solution and 10% NaOH then add 0.5% CuSO4 drop by
drop until a dark violet coloration forms. ✓ mix 2% peptone/pentose solution and 10% NaOH then
add 0.5% CuSO4 drop by drop until a light violet coloration forms.
B. Xanthoproteic Reaction ✓ mix 2% albumin solution and conc. HNO3 then heat the solution after
yielding to a yellow solution. ✓ cool the solution then add NH4OH yielding
to an orange solution.
2. Qualitative colour reactions have been devised for the detection of proteins. Due to the complexity of
the protein molecule and the difficulty of obtaining a single, pure protein compound, these tests are used
for specific-chemical groups if the component amino acid/s. Since no single test is absolutely specific for
proteins, it’s necessary to apply several tests. 3. Biuret reaction/test is a general test for proteins (native
and most derived). The chemical structure of the protein molecule is responsible for the positive result
and is due to the varying amount of peptide linkages (-CONH). Any compound containing 2 carbonyl
groups give a positive result for the biuret test. Simple amino acids will not yield a positive result. 4.
Xantoproteic reaction is due to the presence of the phenyl group (-C6H5) in protein molecules. This
involves nitration of the phenyl rings in an aromatic amino acid such as tyrosine, tryptophan &
phenylalanine to form yellow nitrosubstitution products that turn orange w/ an added alkali (NH4OH). 5.
Millon’s Test is a test using the Millon’s reagent where it is prepared by dissolving mercury in HNO3
(concentrated & dilute). Millon’s reagent can either be HgNO3 or Hg2NO3. When added to a protein
solution, the protein is the precipitate known as the Mercury salt. The positive result is due to the phenol
group in Tyrosine. 6. Glyoxylic Acid (CHOCOOH) Reaction or the Hopkins-Colé reaction is prepared
by 10g of powdered Mg + 250mL cold H2O. It is then filtered and acidified with acetic acid to prevent the
partial precipitation of Mg or made by the reduction of oxalic acid with Mg powder. Hopkins- Colé reagent
(magnesium salt of oxalic acid) gives positive results with proteins containing the essential amino acid
“tryptophan” indicating a high nutritive value. When treated with H2SO4, a
2 of 9 ||| laboratoryworksbyveronica2016 C. Millon’s Test ✓ mix 2% albumin solution and
Millon’s reagent then heat the solution yielding from a pink solution to a flesh red flocculent precipitate.
D. Glyoxylic Acid Reaction (Hopkins-Colé) ✓ mix 2% albumin solution with Hopkins-Colé reagent
afterwards incline the test tube then add conc. H2SO4 yielding to a violet ring formation at the junction
of the test tube.
E. Heller’s Ring Test ✓ by inclining the test tube, mix 2% albumin solution and 5 mL of conc. HNO3
yielding to the formation of a white ring.
F. Reduced Sulfur Test ✓ mix 2% albumin solution with 40% NaOH then add 10 drops of
Pb(C2H3O2)2 solution yielding to the formation of a black precipitate (Lead sulfide/PbS).
G. Adamkiewicz Reaction ✓ mix 3 drops of 2% albumin solution with glacial CH3COOH then add
conc. H2SO4 to the solution yielding to the formation of a red/violet ring.

iii. DISCUSSION
1. Amino acids are building blocks of all proteins, and are linked in series by peptide bond (-CONH-) to
form the primary structure of a protein. Amino acids possess an amine group, a carboxylic acid group and
a varying side chain that differs between different amino acids.
S. (Proteins) + 2 NaOH Na2S Pb(CH3COO)2 + Na2S PbS + 2 CH3COONa
violet ring is produced at the point of contact due to the indole nucleus in Tryptophan (condenses w/
aldehyde to form the coloured compound). Hopkins-Colé reaction is a specific test for detecting
tryptophan. 7. Heller’s Ring Test is a test that is used to detect
albumin in urine. 8. Reduced Sulfur Test is a test where its solution of protein contains crystine, cysteine
or methionine when heated with NaOH and splits up to form NaS. 9. Adamkiewicz Reaction is part of a
biochemical test used to detect the presence of the amino acid tryptophan in proteins. When
concentrated sulfuric acid is combined with a solution of protein and glyoxylic acid, a red/purple colour is
produced.
iv. SIDENOTES
has been completely precipitated by alcohol: no more coagulation.
C. Precipitation by Salting-Out ✓ solid (NH4)2SO4 is added to 5mL of 2% albumin solution until
saturated then filter, test the precipitate by Millon’s reaction: a pink precipitate to a red precipitate. ✓
test the filtrate with the biuret test: blue. ✓ SEPARATION OF PEPTONES & PROTEOSES:
saturate 10mL of 10% commercial peptone with solid (NH4)2SO4 stirring all the time, filter off the
proteoses, peptone is found in the filtrate. Make a biuret test on the filtrate: violet coloration.
D. Precipitation by Heavy Metal Ions ✓ in 3 separate test tubes, 2mL of 2% albumin solution is added.
To the first test tube add 2 to 3 drops of Pb(Ac)2: white precipitate. To the second test tube add 3 drops
of 1% AgNO3: white precipitate. To the third test tube add 1% CuSO4: pale blue precipitate. ✓
Repeat the test using fresh samples of albumin and a few drops of 0.1N NaOH before adding the metal
salts: more precipitate.
E. Precipitation by Alkaloidal Reagents ✓ 2mL of 2% albumin is added in 3 separate test tubes and
acidified with a few drops of 0.1N HCl. To the first test tube add 3 drops of K4[Fe(CN)6] · 3 H2O: pale
yellow. To the second test tube add 3 drops of 5% Tannic acid: pale yellow. To the third test tube add 3
drops of Saturated Picric acid: brown.
3 of 9 ||| laboratoryworksbyveronica2016 III. PRECIPITATION REACTIONS OF PROTEINS
• 2% casein
• Pb(C2H3O2)2
• 2% albumin
• 1% AgNO3
• 10% commercial peptone
• 1% CuSO4
• 1N CH3COOH
• 0.1N NaOH
• 5N CH3COOH
• 0.01N HCl
• CH3CH2OH
• K4[Fe(CN)6] · 3 H2O
• (NH4)2SO4
• Tannic acid
• Millon’s reagent
• Saturated Picric acid
• 10% NaOH & 0.5% CuSO4 (Biuret)
(C6H3N3O7)
ii. TESTS
A. Precipitation by Heat Coagulation ✓ place 5mL of 2% casein in a test tube and heat to boiling: no
coagulation ✓ in 3 different test tubes, place 1mL of 2% albumin solution. To the first test tube, add 1
drop of 1N CH3COOH. To the second test tube, add 2 drops of 5N CH3COOH while the third test tube
serves as the control. Boil al 3 test tubes: best coagulation is the test tube with albumin alone.
B. Precipitation by Organic Solvents ✓ add 4mL of CH3CH2OH to 2mL of 2% albumin solution,
remove a small amount of the precipitate and add water: the white precipitate dissolved in water. ✓
set aside the remainder of the precipitate in a stoppered test tube until the next day, test again the
solubility of the precipitate in water: coagulated after settling for a day. ✓ the mixture was filtered
and heated in a water bath to see if coagulation occurs. If no coagulation occurs, it means that the
protein
iii. DISCUSSION
1. Precipitation by Heat Coagulation: occurs best near the isoelectric point of the protein. Albumin
coagulates when heated. Aqueous solutions of proteoses, peptones, gelatin and casein are not
coagulated by heat. This destroys hydrogen ions and hydrophobic interactions. 2. Precipitation by
Organic Solvents: precipitating (denaturing) fixatives act by reducing the solubility of protein molecules
and (often) by disrupting hydrophobic interactions that give many proteins in their protein tertiary
structure. Acetic acid is a denaturant. Alcohols are known to cause shrinkage and hardening of tissue
during fixation while acetic acid alone is associated with tissue swelling, combining the two may result in
better preservation of tissue morphology. The organic solvents precipitate proteins when made to stand
for sometime, they are denature and insoluble in H2O. 3. Precipitation by Salting Out: it is not a
denaturation, it doesn’t destroy structure of proteins; saturated salt solution would cause it to precipitate.
Negative results for peptide bonds and positive results for Tyrosine presence. Ammonium Sulfate
separates peptones & proteoses. 4. Precipitation by Heavy Metal Ions: Egg white is used to precipitate
out the poisonous heavy metal salts and antidote for Hg & Fe poisoning. 5. Precipitation by Alkaloidal
Reagents: basic organic compounds which has N2 rings and have powerful effects on living things. H+
bond & salt bridges are disrupted. 6. Picric acid is capable of destroying proteins (denaturing). This lends
a very strong antiseptic property that when applied to a burn it can help the burn from lesser chances of
infection and therefore heal properly.
7. Tannic acid controls the irritation in the small intestine; produces an anti-stringent effect on the tissues
and prevents absorption of toxins. 8. GENERAL EQUATION IN IONIZATION OF PROTEIN
IN ACID & BASIC MEDIUMS:
iv. SIDENOTES
IV. TESTS FOR CARBOHYDRATES
• arabinose
• fructose
• glucose
• sucrose
• starch
• dextrin
• water
• 10% NaCl
• CH3CH2OH
• 0.1M glucose
• 0.1M sucrose
• 0.7% starch
• Molisch reagent
ii. TESTS
(1) Physical Tests
A. Solubility ✓ the solubility of arabinose, fructose, glucose, sucrose, starch and dextrin is tested in
2mL of water: soluble, 10% NaCl: soluble, alcohol: insoluble (starch & dextrin are soluble).
B. Dialysis ✓ 1mL of 0.1M glucose, 0.1M sucrose and 0.7% starch are added separately in 3 dialysis
tubes, the ends are immersed covered with semi-permeable membrane in 3 100mL beakers filled with
50mL water. Set aside for 15 minutes. Glucose: 1 layer & light Sucrose: 2 layers & lighter w/ pale
violet precipitate/coloration at the bottom; Starch: 2 layers w/ darker precipitate/ coloration at the
bottom.
4 of 9 ||| laboratoryworksbyveronica2016 • 0.02M glucose H | R - C - COOH H+
H R - C |- COOH NH2 +
NH3
|
+
H R - C - COOH OH -
HO
R-C-C-O
|
- NH2 NH2
• 0.02M sucrose
• 0.05% starch acid:
|
• conc. H2SO4
• 3% thymol
• conc. HCl
basic:
||
• Seliwanoff’s reagent
• 0.01M glucose
• 0.01M fructose
• 0.01M arabinose
• 5% glucose
• conc. NaOH
• phloroglucin solution ||||
(2) Furfural Reactions of Carbohydrates
A. Molisch Test ✓ in 4 separate test tubes with 1mL water, add 1mL of 0.02M glucose, 1mL of 0.02M
sucrose and 1mL of 0.05% starch respectively. The first test tube serves as the control. To each of the
test tubes add 2 drops of Molisch reagent and mix thoroughly. Incline the test tube and allow 1mL of
conc. H2SO4 to flow in the side of the tube: violet ring formation (control).
B. Thymol Test ✓ with 0.5mL of the carbohydrate to the test prior, add 3-6 drops of 3% thymol in
alcohol then add an excess 5-10mL of conc. HCl. Boil gently for 2 minutes shaking the mixture at
intervals: pink to dark pink during the boiling period, carmine color.
C. Seliwanoff’s Reaction ✓ in 4 test tubes, add 5mL of Seliwanoff’s reagent to each. To the test tubes
add 1mL water, 1mL of 0.01M glucose, 1mL of 0.01M fructose and 1mL of 0.01M arabinose respectively.
Dip all the four test tubes in boiling water at the same time for 5 minutes. Water: colorless Glucose:
yellow Fructose: orange Arabinose: colorless.
D. Moore’s Test/Action of Conc. Alkali ✓ mix 1mL of 5% glucose and 1mL of conc. NaOH then boil:
yellow to orange to dark brown w/ caramel odor.
E. Tollen’s Phloroglucin Reaction ✓ in 4 test tubes, add 5 mL of phloroglucin solution. Respectively
add 1mL water, 1mL of 0.01M glucose, 1mL of 0.01M fructose and
4. Seliwanoff’s Reaction: called tghe Resorcin-HCl test since it uses HCl as dehydrating acid and
resorcinol as condensation agent, distinguishes
aldoses from ketoses. The product is in red coloration due to the reaction of hydroxyl methyl furfural with
resorcinol. 5. Moore’s Test: it is the action of concentrated alkali. It is the liberation of aldehydes; to
polymerize to form resinous substance: caramel. 6. Tollen’s Phloroglucin Reaction: it is the reaction
used to differentiate pentoses from hexoses (ex. arabinose). Glucose and galactose are aldohexoses &
fructose is a ketohexose.
iv. SIDENOTES
5 of 9 ||| laboratoryworksbyveronica2016 1mL of 0.01M arabinose. All the test tubes are immersed
in boiling water at the same time. Observe the color changes every two minutes during the first 15
minutes then observe at the end of 1 hour: hexoses yield a dark color while pentoses yield a light
color.
iii. DISCUSSION
1. Carbohydrates are generally considered as substances made up of carbon, hydrogen and oxygen in
which the proportion of hydrogen and oxygen is the same as that found in a molecule of water (2:1).
Carbohydrates form a class of organic compounds that include sugars, starch, and cellulose. They are an
important source of metabolic energy and also form part of a number of important molecules and
structures. 2. Furfural is an organic compound derived from a variety of agricultural byproducts including
corncobs, oat, wheat, bran and sawdust. The name furfural comes from the Latin word furfur meaning
bran referring to its usual source. Furfural is an aromatic aldehyde with the chemical formula
OC4H3CHO. It is a colourless oily liquid with the odour of almonds, but upon exposure to air samples
quickly become yellow. 3. Molisch Test: the general test for Carbohydrates. It is a dehydration-
condensation reaction with the formation of furfual violet ring which indicates an Alpha-naphthol reaction.
In other words, it is a color test for sugars, which condenses with alpha naphthol on thymol in presence of
strong sulfuric acid, which converts sugar to furfural derivatives. The reagent: α-naphthol or thymol
dehydrates pentoses to form furfural and dehydrates hexoses to form 5-hydroxymethylfurfural. Furfurals
further react with naphthol present in the test to reagents to produce a purple product.
BIOCHEMISTRY LABORATORY (CuSO4) and Fehling’s solution B (KOH &
Rochelle salt). Shake and immerse all tubes in
(Midterm)
boiling water and heat for 15 minutes: brick red
precipitate (CuO2) except for sucrose and
V. SPECIAL TEST FOR SACCHARIDES starch.
• Fehling’s solution (KOH & Rochelle’s salt)
0.01M lactose blue w/ red ppt
• 0.01M glucose 0.01M lactose blue w/ red ppt
C. Mucic Acid Test ✓ add 1mL of conc.
• Benedict’s solution
HNO3 to 1mL of 5% galactose and heat in a
0.01M sucrose blue w/ less ppt
boiling water bath. Allow to stand until the next 0.01M sucrose blue w/ less ppt
day: white crystals. 0.01M sucrose blue w/ less ppt
SUGAR RESULT
• 0.1M glucose
SUGAR RESULT
• 0.1M glucose
0.01M glucose light blue w/ red ppt
• 0.002M glucose
0.01M glucose light blue w/ red ppt • 0.002M glucose
• Fehling’s solution (CuSO4) 0.03M lactose blue w/ red ppt
• Fehling’s solution (CuSO4) 0.03M lactose blue w/ red ppt
0.01M fructose blue w/ red ppt 0.03M lactose blue w/ red ppt
0.01M fructose blue w/ red ppt
• 0.01M lactose
0.01M fructose blue w/ red ppt
• 0.01M lactose
• conc. HNO3 • 0.03M glucose
• 5% K2CrO4 • 0.03M glucose
• 20% suspension of Baker’s yeast 0.03M glucose blue w/ red ppt
0.03M glucose blue w/ red ppt
• Phosphate buffer 0.03M glucose blue w/ red ppt
• sucrose 0.03M glucose blue w/ red ppt
• glucose 0.03M glucose blue w/ red ppt
• galactose
• 0.03M lactose
• 0.01M fructose
• 0.03M lactose
• 0.01M sucrose 0.03M sucrose blue w/ less ppt
• 0.7% starch 0.03M sucrose blue w/ less ppt
• 0.5g phenylhydrazine mixture 0.03M sucrose blue w/ less ppt
• 5% glucose • 0.03M sucrose
• 0.03M sucrose
• 0.1% dextrin
D. Reduction Test ✓ FEHLING’S TEST: in 5 • 0.1% dextrin
• dil. I2 solution
separate test tubes add 1mL of 0.01M glucose,
• dil. I2 solution
0.01M fructose, 0.01M sucrose, 0.7% starch
• 5% maltose
and water (control) respectively. To all test
• 5% maltose
tubes add 1mL each of Fehling’s solution A
E. Iodine Test for starch and dextrin ✓ add a E. Iodine Test for starch and dextrin ✓ add a
few drops of dil. I2 solution to a 5mL portion few drops of dil. I2 solution to a 5mL portion
each of 0.7% starch and 0.1% dextrin. Warm each of 0.7% starch and 0.1% dextrin. Warm
each tube very gently: starch (upon heating it each tube very gently: starch (upon heating it
was a blue black color then turned to a was a blue black color then turned to a
colorless solution) and dextrin ( upon colorless solution) and dextrin ( upon
heating it was a violet black color then it heating it was a violet black color then it
returns to a greenish black color upon returns to a greenish black color upon
cooling). cooling).
cooling). cooling).
cooling). cooling).
• 5% fructose
E. Iodine Test for starch and dextrin ✓ add a • 5% sucrose
few drops of dil. I2 solution to a 5mL portion ii. TESTS
each of 0.7% starch and 0.1% dextrin. Warm

each tube very gently: starch (upon heating it
was a blue black color then turned to a A. Nitro-Chromic Acid Test ✓ add 3mL of
colorless solution) and dextrin ( upon
heating it was a violet black color then it conc. HNO3 and 5 drops of 5% K2CrO4 in
returns to a greenish black color upon 5mL of 0.01M glucose solution then mix: blue
cooling). solution after one minute.
✓ BENEDICT’S TEST: in 3 separate test
tubes add 5mL of Benedict’s solution to each returns to a greenish black color upon
then 1mL of 0.1M, 0.01M & 0.002M glucose cooling).
respectively. Place in a boiling water bath for 10
minutes: E. Iodine Test for starch and dextrin ✓ add a
• 5% lactose
• 5% lactose
few drops of dil. I2 solution to a 5mL portion
• Barfoed’s solution
• Barfoed’s solution
was a blue black color then turned to a
heating it was a violet black color then it
returns to a greenish black color upon
SUGAR RESULT cooling).
0.01M glucose lightest w/ less ppt

E. Iodine Test for starch and dextrin ✓ add a
E. Iodine Test for starch and dextrin ✓ add a few drops of dil. I2 solution to a 5mL portion
few drops of dil. I2 solution to a 5mL portion each tube very gently: starch (upon heating it
each of 0.7% starch and 0.1% dextrin. Warm was a blue black color then turned to a
each tube very gently: starch (upon heating it colorless solution) and dextrin ( upon
was a blue black color then turned to a heating it was a violet black color then it
colorless solution) and dextrin ( upon returns to a greenish black color upon
heating it was a violet black color then it cooling).
cooling).
cooling).
lactose and 0.03M sucrose respectively. Place
all the test tubes in a boiling water bath and
heat for 1/2 hour:
0.1M glucose sky blue in color
few drops of dil. I2 solution to a 5mL portion
each of 0.7% starch and 0.1% dextrin. Warm 0.002M glucose darkest w/ more ppt
was a blue black color then turned to a
F. Phenylhydrazine Reaction ✓ add 2mL of
heating it was a violet black color then it
sugar solution (5% glucose, fructose, maltose,
cooling). lactose and sucrose) to 0.5g of phenylhydrazine
mixture, shake well and heat in a boiling water
bath for 30 to 45 minutes. Allow the tube to cool
and examine the crystal under the microscope:

lactose and sucrose) to 0.5g of phenylhydrazine

B. Alcoholic Fermentation ✓ add 5mL of 20%
suspension of baker’s yeast in a test tube and

5mL of sugar solution (sucrose, glucose,
galactose) and 5mL of phosphate buffer. Allow
to stand for one hour: evolution of bubbles
which indicated fermentation and is due to
ethyl alcohol and carbon dioxide being
produced.
6 of 9
✓ BARFOED’S TEST: in 7 separate test tubes ||| laboratoryworksbyveronica2016
add 5mL of Barfoed’s solution to each then 5mL
of 0.01M glucose, 0.01M fructose, 0.01M
lactose, 0.01M sucrose, 0.03M glucose, 0.03M
iii. DISCUSSION
1. Nitro-Chromic Acid Test: the test is due to free - CHOH (ketone) groups in the sugar molecule. 2. Alcoholic Fermenta
decomposition of carbohydrates brought about by the action of microorganisms such as yeast, molds, bacteria, etc. This pr
utilized in the manufacture of beverages and other valuable industrial products. The ferments are naturally: D-glucose and
galactose is fermented by specially cultured yeast.
3. Mucic Acid Test: takes advantage of the isomer of
the saccharic acid. 4. Fehling’s Test: the test is for reducing sugars, sucrose is not a reducing sugar because both anome
involved in the glycosidic linkage. 5. Benedict’s Test: a qualitative test for glucose in
urine, positive result in reducing sugars. 6. Barfoed’s Test: the test is used to distinguish monosaccharides from disaccha
boiling may cause it to react with disaccharides. Positive result if the sugar concentration is high enough and the time of he
Fructose reacts faster than glucose; both produce tiny black red precipitate/formation of Cu2O. 7. Iodine Test: the red colo
glycogen or erythrodextrin (dextrin) while the blue color indicates starch. 8. Phenylhydrazine Reaction: useful in identifyin
free aldehyde or ketone group. The shape of the crystals and the time required for osazone formation are important guides
the various sugars.
iv. SIDENOTES
VI. GLYCOGEN
• glycogen solution
• NaCl
• 95% alcohol
5% glucose (4-5 mins) orange ppt
5% fructose (2 mins) orange ppt

• conc. HCl
• NaOH
• CH3COOH
ii. TESTS
USING THE OPALESCENT FILTRATE (GLYCOGEN PREPARED):
5% sucrose (30-35 mins after hydrolysis) orange w/ ppt
A. Iodine Test 5% lactose (osazone soluble in hot H2O) clear orange sample; presence of circles
✓ add 3-5 drops of NaCl and several drops of iodine solution to 5mL of the glycogen solution in a test tube; compa
starch iodine: reddish brown; starch iodine 5% maltose (osazone soluble yields a blue black color. in hot H2O) oran
B. Glycogen + 95% Alcohol ✓ add 6mL of 95% alcohol to 3mL of the glycogen solution in a test tube: white precipitate
C. Benedict’s Test ✓ with the precipitate formed with the 95% alcohol, add Benedict’s solution and boil for a short time:
D. Hydrolysis ✓ hydrolyze the glycogen solution with a few drops of conc. HCl, boiling for 10 minutes. Cool the solution
with NaOH then test with Benedict’s solution: brick red precipitate.
7 of 9
||| laboratoryworksbyveronica2016
iii. DISCUSSION
1. Glycogen is found in the liver and the muscle. It is precipitated by aqueous alcohol; stable in hot alkali. It is animal starch
reducing polysaccharide 2. Iodine Test: starch and glycogen are both polysaccharides of glucose and are non-reducing sug
Benedict’s Test: it gave off a negative result because glycogen is a non-reducing polysaccharide. 4. Hydrolysis: the produc
hydrolysis of glycogen is glucose. Glycogen was hydrolyzed into its individual glucose sugar units. Glucose in an aldose an
sugar becomes a carboxylic acid (gluconic acid) when oxidized.
iv. SIDENOTES
B. Reaction towards Indicators ✓ in two test tubes, add 1mL of fresh coconut oil. To the first, add congo red and to th
and blue litmus paper. Repeat with rancid oil:
• Coconut oil is neutral
• Rancid oil is acidic
C. Formation of Translucent Spot ✓ place 1 drop of coconut oil on a piece of paper and allow to evaporate: tra
disappears.
D. Acrolein Formation ✓ place 0.5g of KHSO4 in a clean dry test tube. Add coconut oil and heat: black/burnt fat odor.
8 of 9 ||| laboratoryworksbyveronica2016 VII. LIPIDS

• coconut oil
• KHSO4
• water
• soap solution
• dil. HCl
• 1% albumin solution
• dil. NaOH
• oleic acid
• cold alcohol
• Hubl’s iodine solution
• hot alcohol
• stearic acid
• CHCl3
• linseed oil
• ether
• glycerol
• CCl4
• 5% aqueous sol. of glycerol
• congo red
• litmus paper
ii. TESTS
A. Solubility ✓ with one drop of coconut oil test its solubility
in the following solvents:

SOLVENT SOLUBILITY
water immiscible
dil. HCl immiscible
dil. NaOH immiscible
cold alcohol slightly miscible
hot alcohol immiscible
CHCl3 miscible
ether miscible
CCl4 miscible
OIL CONGO RED RED LITMUS BLUE LITMUS
coconut oil red color no reaction no reaction
rancid oil more purple no reaction no reaction
(1)
C11H22COOCH2
C11H22COOHCH
| | |
OH OH OH CH2 = CH - CH2
C11H22COOCH2
+ C11H22COOH
(2)
CH2OH
CHOH
+ H2O
+ KHSO4
CH2OH
||
+ KHSO4
CH2 ||CH|
| CHO
E. Emulsification ✓ add coconut oil to the following test tubes:
F. Test for Unsaturation ✓ place 5mL of the acid/oil (oleic, stearic, coconut oil, linseed oil) in chloroform in a test tube a
drop by drop shaking between addition. Make a control by shaking in another tube a mixture of chloroform and iodine with n
• Stearic acid will absorb more iodine.
• Linseed oil contains more unsaturated fatty acids.
• Since linseed oil has more C=C, it can absorb more I2. The more C=C the more unsaturated.
• Stearic acid has no C=C, therefore it can’t abosrb I2, higher than other group structure.
• 1C=C equals to 1 mol of I2; produced colorless product.
• The higher/more I2, the more unsaturated fat.
5. Unsaturation Test: used to detect the presence of double bonds in lipids by adding Hubl’s Iodine solution. 6. The iodine #
absorption, value, or index) is the mass of iodine in grams that is consumed by 100 grams of a chemical substance or is the
the unsaturation of fats; the higher the iodine number, the more unsaturated fatty acid bonds are present in a fat. 7. Glycero
glycerol in nichrome wire with powdered borax yields to a green flame because of the glycerol ester of boric acid. In the nitr
test on 5% aqueous solution of glycerol yields a blue solution because of the presence of the - CHOH groups.
iv. SIDENOTES
9 of 9 ||| laboratoryworksbyveronica2016 G. Glycerol
SUBSTANCE SOLUBILITY TEST TUBE TYPE OF EMULSION
glycerol in water miscible 5mL water + coconut oil temporary
glycerol in alcohol miscible 5mL water + 3mL soap + coconut oil permanent
glycerol in ether immiscible 5mL water + 1mL 1% albumin + coconut oil permanent ✓ perform an acrolein test on glycerol 5mL wate
Na2CO3 + coconut oil temporary
✓ fuse a drop of glycerol in nichrome wire with powdered borax: green flame. ✓ perform the nitro-chromic
aqueous solution of glycerol
iii. DISCUSSION
1. Lipids include both true fats and fat-like substances. True fats contain glycerol and fatty acids in their molecules. Com
TEST TUBE
phospholipids have, besides glycerol and fatty acids, a nitrogenous base and phosphoric acid. CHCl3 + I2
Cholesterol, ergosterol and 7-dehydrocholesterol
oleic acid + CHCl3 + I2 yellow orange 1=/unsaturated
represent the sterols. 2. Formation of translucent spot: it is caused by high
stearic acid + CHCl3 + I3 red high member/saturated
molecular fatty acid. Essential oils will leave a translucent spot. Coconut oil and palmitic acid are coconut oil + C
m
non-volatile. 3. Acrolein Test: a test for fats and oils. When coconut linseed oil + CHCl3 + I2 yello
oil forms glycerol and fatty acid, oil is hydrolyzed into glycerol and fatty acid. Glycerol reacts w/ KHSO4 (reducing agent
and H2O. 4. Emulsification: an emulsion is an intimate mixture of 2 immiscible liquids. In an emulsion, one liquid (disp
dispersed in the other (continuous phase). Emulsion tends to have a cloudy appearance because the many phase interfa
hat passes throug

Biochem Laboratory Midterm

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BIOCHEMISTRY LABORATORY (Midterm)

I. ELEMENTARY COMPOSITION OF PROTEINS

2 of 9 ||| laboratoryworksbyveronica2016 C. Millon’s Test ✓ mix 2% albumin solution and

Pb(C2H3O2)2 solution yielding to the formation of a black precipitate (Lead sulfide/PbS).

conc. H2SO4 to the solution yielding to the formation of a red/violet ring.

C. Precipitation by Salting-Out ✓ solid (NH4)2SO4 is added to 5mL of 2% albumin solution until

B. Precipitation by Organic Solvents ✓ add 4mL of CH3CH2OH to 2mL of 2% albumin solution,

yellow to orange to dark brown w/ caramel odor.

E. Tollen’s Phloroglucin Reaction ✓ in 4 test tubes, add 5 mL of phloroglucin solution. Respectively

few drops of dil. I2 solution to a 5mL portion ii. TESTS

each of 0.7% starch and 0.1% dextrin. Warm

0.01M glucose lightest w/ less ppt

sugar solution (5% glucose, fructose, maltose,

sugar solution (5% glucose, fructose, maltose,

suspension of baker’s yeast in a test tube and

5% fructose (2 mins) orange ppt

8 of 9 ||| laboratoryworksbyveronica2016 VII. LIPIDS

A. Solubility ✓ with one drop of coconut oil test its solubility

in the following solvents:

E. Emulsification ✓ add coconut oil to the following test tubes:

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