Experiment 1

Carbohydrates
(2023 ver.)
Objectives
To illustrate the chemical properties of carbohydrates.
To distinguish the reactivity and properties of monosaccharides and polysaccharides.

Reagents
These reagents will be listed again in their respective subheadings

glucose, sucrose, fructose, lactose, galactose, starch, phenylhydrazine, Molisch reagent,

concentrated sulfuric acid, Fehling’s reagents A and B, Barfoed’s reagent, Seliwanoff’s
reagent, iodine in potassium iodide solution, 10% trichloroacetic acid, dilute
hydrochloric acid, 10% sodium hydroxide, phenolphthalein

Materials
medicine droppers, test tubes, test tube rack, test tube holder, filter paper, beakers,
graduated cylinder, crucible tongs, cork stopper, hot plate, hot water bath

● 📷
To the students
If you take photos, it is a good idea to photograph before and after each test with a
white background.
● Wear gloves especially during cleanup or washing of glassware.
● Bring a sample of fresh chicken liver. Freshness will affect the results! Keep it
refrigerated or cool. About 500 grams will be enough for the whole class.
● ⚠Be responsible to know the contents and hazards of your reaction mixtures! Label
your samples properly and make sure to clean them before returning them or giving
them to someone else.
● ⚠Be sure to discard the wastes in their proper waste containers when indicated.
-Procedures-
Part 1. Qualitative tests and analysis of the unknown
(one test compound per test tube)
Obtain an unknown sample from your instructor. This is to be subjected to the following
qualitative tests for its identification. For standard comparison, the same tests and reactions
would also be carried out on 1% solutions of glucose, sucrose, fructose, lactose, galactose, and
starch (unless otherwise noted in each procedure). Distilled water will be used as the negative
control. Use small test tubes. Use 250 ml beaker half-filled with water for your hot water bath.

1. Osazone Test (Class work – for each group: one assigned test sample
and your unknown)
Test compounds: unknown sample, glucose, sucrose, fructose, lactose, galactose
Reagents: distilled water, phenylhydrazine reagent ⚠
⚠: Phenylhydrazine is toxic and can be absorbed through the skin.
Materials: test tubes, droppers, hot water bath

Procedure: To separate test tubes, add 10 drops of the assigned test solution or unknown
sample. To these, add 30 drops of freshly prepared phenylhydrazine reagent. Place the test
tubes in a boiling water bath for about 30 minutes. Record the time when crystals first appear. If
no crystals appear after 30 minutes, place in an ice bath. Note the color of the crystals. Place
some crystals on a glass slide and observe under the microscope. Sketch their appearance. You
can also take a photo through the eyepiece.

Cleanup: Discard the waste as non-halogenated organic waste.

2. Molisch Test (perform on all the test compounds below)

Test compounds: unknown sample, glucose, sucrose, fructose, lactose, galactose, starch,
cotton, distilled water
Reagents: Molisch reagent, concentrated H2SO4 ⚠
⚠: highly corrosive
Materials: test tubes, droppers, test tube rack

Procedure: To separate test tubes, add 1 drop of Molisch reagent to 30 drops of test solution (or
the piece of cotton). Incline the container and slowly pour 1 mL of concentrated H2SO4 down
the side so that the acid forms a layer at the bottom. Do not shake or mix. Note the color at the
junction of the two liquids.

Cleanup: Discard the waste as non-halogenated organic waste.

3. Fehling’s Test (perform on all the test compounds below)
Test compounds: unknown sample, glucose, sucrose, fructose, lactose, galactose, starch,
distilled water
Reagents: Fehling’s reagent A, Fehling’s reagent B ⚠
⚠: alkaline, caustic
Materials: test tubes, droppers, hot water bath, test tube holder

Procedure: To 10 drops of each test solution or unknown sample in separate test tubes, add 30
drops of Fehling's reagent (5 mL Fehling's solution A + 5 mL of Fehling's solution B + 5 ml
distilled H2O). Shake the mixture and immerse in boiling water for about 2-3 minutes. Record
the results.

Cleanup: Dispose in heavy metal or inorganic waste. Clean up the test tube immediately after
this test, since it becomes considerably harder to clean if left to stand for a while.

4. Barfoed’s Test (perform on all the test compounds below)

Test compounds: unknown sample, glucose, sucrose, fructose, lactose, galactose, distilled water
Reagents: Barfoed’s reagent
Materials: test tubes, droppers, hot water bath, test tube holder

Procedure: Mix 30 drops of Barfoed's reagent and 20 drops of each test solution in individual
test tubes. Place in boiling water bath. Remove the test tube once it becomes cloudy or changes
color. Note the length of time for the reaction to take place. If there are no visible changes
within 10 minutes, continue heating for another 5 minutes. If change in color is difficult to
detect, compare the color of solution to that of the blank sample. (Blank sample is 30 drops of
Barfoed’s reagent with 20 drops of distilled water)

Cleanup: Dispose in heavy metal or inorganic waste. Clean up the test tube immediately after
this test, since it becomes considerably harder to clean if left to stand for a while.

5. Seliwanoff’s Test (perform on all the test compounds below)

Test compounds: unknown sample, glucose, sucrose, fructose, lactose, galactose, distilled water
Reagents: Seliwanoff’s reagent
Materials: test tubes, droppers, test tube rack, hot water bath

Procedure: To 5 drops of each of the test compound in individual test tubes, add 20 drops of
Seliwanoff's reagent and place in a boiling water bath for approximately 1 minute. Record the
results.

Cleanup: Discard the waste as non-halogenated organic waste.

6. Iodine Test (perform on all the test compounds below)

Test compounds: unknown sample, glucose, sucrose, fructose, lactose, galactose, starch,
cotton, distilled water
Reagents: iodine in potassium iodide solution
Materials: spot plate, droppers

Procedure: Add 5 drops each of test compound or solution and the unknown sample on
separate wells on a spot plate. Add 2 drops of I2 in KI solution. Observe

Cleanup: Carefully flush the waste down the sink with large amounts of running water.

Part 2. Isolation of glycogen from chicken liver

Take a piece of cold chicken liver weighing about 20 grams (it does not have to be exactly 20
grams). Mince the liver, transfer to a mortar, and add about 10 mL of trichloroacetic acid ⚠
(about 1 mL / gram of liver). Do not allow the sample to get too warm, use an ice bath if
necessary. Using the mortar and pestle, grind the liver into a paste. With a small spatula,
transfer this to a centrifuge tube and centrifuge at 3000 RPM for about 2 minutes. Your
glycogen sample will be the supernatant.

⚠: Trichloroacetic acid is corrosive and may be absorbed through skin. Beware of splashes
when grinding the liver.

Perform the following tests on the supernatant (no need to use other test samples):
A. Molisch Test
● Discard the waste as halogenated organic waste
B. Fehling's Test
● Use Fehling's reagent: 2 mL Fehling's A + 2 mL of Fehling's B + 2 ml distilled H2O.
● Discard the waste as heavy metal waste
C. Iodine Test
● Continuously add iodine to cause a permanent color to remain in your sample.
Interpret the result in comparison with distilled water as a negative control.
● Discard the waste as halogenated organic waste

Part 3. Hydrolysis of polysaccharides

Place 2 ml of 1% starch solution in a test tube. Add 1 ml of dilute HCl. Heat in a water bath and
place 2 drops of the reaction mixture on a spot plate at 0, 5, 10, 15, 20 and 25 minutes of
heating.
● Add 1 drop I2 in KI solution into each. Record changes in color. (The waste can be
flushed down the sink with copious amounts of water)
Cool the reaction mixture and add 1 drop of phenolphthalein. Add 10% NaOH until the solution
turns faint pink.
● Perform Fehling’s Test on the neutralized, hydrolyzed sample. Discard the waste as
heavy metal waste.

References

(1) Former DLSU Department of Chemistry Laboratory Manual (LBYKMBI)

(2) Roe, J. H., Bailey, J. M., Gray, R. R., & Robinson, J. N. (1961). Complete removal of
glycogen from tissues by extraction with cold trichloroacetic acid solution. J. Biol. Chem.
236(5), 1244–1246.

Document history
11 May 2023: Prepared by Laurenzo D.V. Alba
16 May 2023: Revised by Laurenzo D.V. Alba for clarity, changed disposal for Molisch test
24 May 2023: Revised the procedure for Part 2 (Isolation of Glycogen) to include modifications
6 Jan 2024: Revised the procedure for Part 2 (Isolation of Glycogen) to include modifications
Experiment 2
Proteins and Amino Acids
(2023 ver.)
Objectives
To illustrate the chemical properties of proteins and amino acids.
To distinguish the reactivity and properties of proteins and amino acids.

Reagents
These reagents will be listed again in their respective subheadings

Valine, phenylalanine, tyrosine, tryptophan, arginine, cysteine, 10%, 20%, and 1M

sodium hydroxide, 0.1% copper (II) sulfate, 0.1% ninhydrin, nitric acid, Millon-Nasse
reagent (mercuric nitrate in conc. Nitric acid), 0.1% NaNO2, glyoxylic acid, conc. Sulfuric
acid, a-napthol in alcohol, NaOBr, lead acetate, 5M acetic acid, ethanol, picric acid,
trichloroacetic acid, 2% FeCl3, (NH4)2SO4, NaCl, HCl, n-butanol:HOAc:H2O solution

Materials
medicine droppers, test tubes, test tube rack, test tube holder, filter paper, beakers,
graduated cylinder, crucible tongs, cork stopper, hot plate, hot water bath, centrifuge,
Whatman No. 42 filter paper, watch glass, 500-mL beaker, blow dryer

● 📷
To the students
If you take photos, it is a good idea to photograph before and after each test with a
white background.
● Wear gloves especially during cleanup or washing of glassware.
● Bring a sample of fresh egg and skimmed milk. 2 eggs and 250 mL of skimmed milk will
be enough for the whole class.
● ⚠Be responsible to know the contents and hazards of your reaction mixtures! Label
your samples properly and make sure to clean them before returning them or giving
them to someone else.
● ⚠Be sure to discard the wastes in their proper waste containers when indicated.

-Procedures-
Part 1. Qualitative tests and analysis of the unknown
Obtain an unknown sample from your instructor. This is to be subjected to the following
qualitative tests for its identification. For standard comparison, the same tests and reactions
would also be carried out on 3% solutions of valine, phenylalanine, tyrosine, tryptophan,
arginine and cysteine (unless otherwise noted in each procedure). Distilled water will be used as
the negative control. Use small test tubes. Use 250 ml beaker half-filled with water for your hot
water bath.

1. Biuret Test (perform on all test compounds below)

Test compounds: unknown sample, 1% albumin, 3% valine, distilled water
Reagents: 10% NaOH⚠, 0.1% CuSO4
⚠: corrosive
Materials: test tubes, droppers, hot water bath

Procedure: To each test tube, add 10 drops of sample and 10 drops of 10% NaOH solution.
Then, add one drop of 0.1% CuSO4 solution. Mix well and observe.

Cleanup: Discard the waste as heavy metal or inorganic waste.

2. Ninhydrin Test (perform on all the test compounds below)

Test compounds: unknown sample, 1% albumin, 3% valine, 3% phenylalanine, 3% tyrosine, 3%
tryptophan, 3% arginine, 3% cysteine, distilled water
Reagents: 0.1% ninhydrin solution ⚠
⚠: irritant, harmful if absorbed through the skin
Materials: test tubes, droppers, test tube rack, hot water bath, test tube holder

Procedure: In separate test tubes, add 10 drops of 0.1% ninhydrin solution and 20 drops of
sample. Heat the mixture in a water bath until color change occurs. Note the length of time it
takes for the solution to change color.

Cleanup: Discard the waste as non-halogenated organic waste.

3. Xanthoproteic Test (perform on all the test compounds below)

Test compounds: unknown sample, 1% albumin, 3% valine, 3% phenylalanine, 3% tyrosine, 3%
tryptophan, distilled water
Reagents: concentrated HNO3 ⚠, 20% NaOH
⚠: highly corrosive
Materials: test tubes, droppers, hot water bath, test tube holder

Procedure: In separate test tubes, add 10 drops of sample and 5 drops of concentrated HNO3.
Note observations. Place the test tubes in boiling water bath for 5 minutes. Note the color of
the solution. Cool the solution then add 20% NaOH dropwise until the solution changes color.
Record observations.

Cleanup: Carefully flush the waste down the sink with large amounts of running water.

4. Millon-Nasse Test (perform on all the test compounds below)

Test compounds: unknown sample, 1% albumin, 3% tyrosine, 3% valine, distilled water
Reagents: Millon-Nasse reagent ⚠, 0.1% NaNO2
⚠: corrosive, toxic if swallowed or inhaled, skin and eye irritant
Materials: test tubes, droppers, hot water bath, test tube holder

Procedure: In separate test tubes, add 5 drops of Millon-Nasse reagent and 10 drops of sample.
Heat the mixture in boiling water bath for 5 minutes. Cool and add 2 drops of 0.1% NaNO2. Note
observations and any color change.

Cleanup: Dispose in heavy metal waste.

5. Hopkins-Cole Reaction (perform on all the test compounds below)

Test compounds: unknown sample, 1% albumin, 3% valine, 3% tryptophan, distilled water
Reagents: glyoxylic acid, concentrated H2SO4 ⚠
⚠: corrosive, skin and eye irritant

Materials: test tubes, droppers, test tube rack

Procedure: In separate test tubes, mix 10 drops of glyoxylic acid with 10 drops of sample. To
each mixture, carefully add 2 mL of concentrated H2SO4 by pouring it carefully down the side of
the inclined tube so that the layers do not mix. Allow the tube to stand for approximately 10
minutes at room temperature. Record observations

Cleanup: Dilute sample before carefully flushing the waste down the sink with large amounts of
water.

6. Sakaguchi Test (perform on all the test compounds below)

Test compounds: unknown sample, 1% albumin, 3% valine, 3% arginine, distilled water
Reagents: 10% NaOH, a-naphthol in alcohol solution ⚠, NaOBr solution
⚠: corrosive, irritant
Materials: test tubes, droppers, test tube rack
Procedure: In each test tube, add 10 drops of sample and 5 drops of 10% NaOH to ensure an
alkaline medium. Add 2 drops of dilute a-naphthol in alcohol solution and mix thoroughly. Add 3
drops of NaOBr solution to the homogenous mixture. Note the color and the time it appears.
Record observations.

Cleanup: Discard the waste as non-halogenated organic waste

7. Lead Acetate Reaction (perform on all the test compounds below)

Test compounds: unknown sample, 1% albumin, 3% valine, 3% cysteine, 3 strands of hair,
distilled water
Reagents: 20% NaOH, 10% Pb(OAc)2
Materials: test tubes, droppers, test tube rack, hot water bath, test tube holder

Procedure: In separate test tubes, add 10 drops of sample (for hair sample, place 3 strands in a
test tube). Then add 10 drops of 20% NaOH and 1 drop of Pb(OAc)2. Heat solution in a water
bath for 5 minutes. Note the color of the solution.

Cleanup: Dispose in heavy metals or inorganic waste.

Part 2. Precipitation Reactions

1. Heat and Acid
Test compound: 10% Albumin
Reagents: 5M HOAc
Materials: test tubes, droppers, hot water bath, test tube holder

Procedure: In two separate test tubes, place 10 drops of 10% albumin in each. In one of the test
tubes, add 1 drop of 5M HOAc. Place all solutions in hot water bath for 5 minutes. Record
observations and compare results.

Cleanup: Dilute sample before carefully flushing the waste down the sink with large amounts of
water.

2. Alcohol
Test compound: 10% albumin
Reagents: ethanol
Materials: test tubes, droppers, test tube rack

Procedure: In two test tubes, add 10 drops of 10% albumin solution each. In one of the test
tubes, add 10 drops of ethanol. Record observations.

Cleanup: Dilute sample before carefully flushing the waste down the sink with large amounts of
water.

3. Alkaloidal Reagents
Test compound: 10% albumin
Reagents: picric acid⚠, trichloroacetic acid ⚠
⚠: corrosive, irritant
Materials: test tubes, droppers, test tube rack

Procedure: Prepare three test tubes. In all three test tubes, add 10 drops of 10% albumin in
each. In one of the test tubes, add 2 drops of picric acid solution, label. In the other test tube,
add 2 drops of trichloroacetic acid, label. Record observations, compare with the control
solution (test tube containing 10% albumin only)

Cleanup: Discard the waste as non-halogenated organic waste (mixture with picric acid) and
halogenated organic waste (mixture with trichloroacetic acid).

4. Heavy Metal Salts

Test compound: 10% albumin
Reagents: 2% CuSO4, 2% FeCl3
Materials: test tubes, droppers, test tube rack

Procedure: In three separate test tubes, add 5 drops of 10% albumin. In one of the test tubes,
add 2% CuSO4 dropwise until a precipitate forms. In the other test tube, add 2% FeCl3 dropwise
until a precipitate forms. Record observations, compare with the control solution (test tube
containing 10% albumin only)

Cleanup: Dispose in heavy metal or inorganic waste.

5. Salting Out
Test compound: 10% albumin
Reagents: (NH4)2SO4, NaCl
Materials: test tubes, droppers, test tube rack

Procedure: In three test tubes, add 10 drops of 10% albumin each. In one of the test tubes add
solid (NH4)2SO4 until no more of the salt dissolves, mix intermittently. To the other test tube, add
solid NaCl until no more of the salt dissolves, mix intermittently. Record observations, compare
with the control solution (test tube containing 10% albumin only).

Cleanup: Dilute sample before carefully flushing the waste down the sink with large amounts of
water.

6. Precipitation at the Isoelectric Point

Test compound: skimmed milk
Reagents: 0.05M HCl ⚠, 1M HCl ⚠, 1M NaOH ⚠
⚠: corrosive, irritant
Materials: test tubes, droppers, test tube rack, centrifuge, pH meter

Procedure: Measure 20 mL of skimmed milk and place in a 50-mL beaker. Using a pH meter,
measure the initial pH of the skimmed milk. Handle the electrode carefully, rinsing it between
measuring solutions. Add 0.05M HCl dropwise until pH reaches 4.5, mix intermittently. Transfer
the mixture into 2 centrifuge tubes, divide solution equally. Centrifuge solution at 3000 RPM for
3 minutes. Discard the supernate. Add 10 drops of 1M HCl to one test tube, in the other test
tube, add 10 drops of 1M NaOH. Record observations.

Cleanup: Dilute sample before carefully flushing the waste down the sink with large amounts of
water.

Part 3. Amino Acid Analysis

Reagents: n-butanol:HOAc:H2O solvent solution (100:22:50), Ninhydrin solution ⚠
Materials: Whatman No. 42 filter paper, 500-mL beaker, watch glass, pencil, capillary tubes
⚠: irritant, harmful if absorbed through the skin

Procedure: Prepare a piece of filter paper (Whatman No. 42) about 5 by 4 square inches. Using
a pencil, draw a line on one side of the paper 1 cm from the edge. Mark 6 points along this line,
where the standard solutions and unknown will be placed.

Using capillary tubes, place a drop of the amino acid solution on a marked point. As
much as possible, maintain the same volume of solution spotted on the filter paper. One
solution for each mark along the line. Allow samples to dry. Form a cylinder by connecting one
end of the paper with the other, do not overlap the paper, secure with masking tape.

Prepare a 500-mL beaker and a watch glass as its cover. Place enough amount of solvent
(n-butanol:HOAc:H2O, 100:22:50) in the beaker to reach 0.5 cm height.

Slowly place the cylinder of filter paper inside the beaker with solvent. Make sure that
the line is not submerged in the solvent. Cover beaker with a watch glass. Do not disturb the
system while the run is ongoing. When the solvent is about 2 cm from the upper edge of the
filter paper, remove the paper from the bath and mark the solvent front using a pencil. Dry the
paper by allowing the solvent to evaporate. When the filter paper is thoroughly dry, spray the
front with ninhydrin solution. Gently dry the paper using a blow dryer. Carefully encircle the
spots that appear. Calculate the Rf value of each amino acid. Determine the identity of the
unknown amino acid.

References

(3) Former DLSU Department of Chemistry Laboratory Manual (LBYKMBI)

Document history
23 May 2023: Prepared by Bernadette Cecilia Morillo
Experiment 3
Buffers
(2023 ver.)
Objectives
To prepare a buffer with the specified concentration and pH.

Reagents
Distilled water, anhydrous sodium hydrogen phosphate, sodium dihydrogen phosphate
dihydrate, 1M hydrochloric acid, 1M sodium hydroxide

Materials
Analytical balance, spatulas, medicine droppers, beakers, graduated cylinder, volumetric
flask, stirring rod

To the students
● Do not assume that volumes are additive for liquids with different compositions.
● Dissolving solids can increase or decrease the volume of the solution.
● Make sure to adjust the pH of your solution before topping off the volume to the desired
amount.
● Remember to use algebra for your calculations.

What are buffers?

A buffer is a solution which consists of a definite proportion of conjugate base [A-] to weak acid
[HA] with a pH near the pKa of the weak acid. Since the ratio [A-]/[HA] ranges from 10-1 to 101,
the two species are always present in considerable amounts. Together they resist large change
in pH by partially absorbing the H+ and OH- ions added to the system, as shown by the following
equations:

H+ + A- 🡪 HA
(added)
OH- + HA 🡪 A- + H2O
(added)

Buffered solutions do change in pH but the change is much less than that which would occur if
no buffer was present.
Choice of Buffer System
The following criteria are used in choosing a buffer system for a particular reaction:

1. A high buffering capacity at the desired pH (i.e. choose the weak acid whose pKa is
closest to the desired pH).
2. The buffer system must not affect the participants in the reaction under consideration.
3. Although a high buffering capacity is required, it is not always possible to use a relatively
concentrated buffer. Consider the individual concentration of the buffer salt and acid
needed to obtain a suitable buffer capacity. Concentration ranges from 0.010 to 1.0 M.
4. Use a high a concentration as is compatible with the features of the system.
5. Too high concentration of salt frequently inhibits activity of enzymes and other
physiological systems.
6. The solubility of the buffer component may also limit the concentration which can be
employed.

Calculation of the necessary amounts of buffer components (This part

does not need to be included in the pre-lab journal)
Consider the Henderson-Hasselbalch equation below:
−
[𝐴 ]
𝑝𝐻 = 𝑝𝐾𝑎 + [𝐻𝐴]

Where:
[A-] is the concentration of your conjugate base
[HA] is the concentration of your acid
pH is your target pH
pKa is the pKa of your acid-conjugate base system as shown below:

HA ⇌ H+ + A-

For this experiment, you will make a buffer solution with the following parameters.
pH = 7.0
Concentration = 0.10 M
Volume = 100.00 mL

Different buffer systems (or conjugate acid-base pairs) have different pKa values. After we
specify a pH value for our buffer, we will choose a suitable conjugate acid-base pair.
● The best conjugate acid-base pair for your chosen pH is the one with the closest pKa to
your pH.
Experiment 3 Worksheet: Buffer Calculations (This is a worksheet,
submit this page ahead of our experiment and copy it to your journal)

Name: _________________________________ Section: _____________

_________________________________ Group Number: _______
_________________________________

Remember to indicate the units!

Acid-Base system you will use in this buffer
For the chemicals below, write the formulas that do not include the metal ion.
Chemical formula of conjugate acid (1)
Chemical formula of conjugate base (2)
pKa of the conjugate acid-base system (3)
Target pH of the buffer system 7.0

Calculations with the Henderson-Hasselbalch Equation

log10([A-]/[HA]) (4)
Concentration ratio, [A-]/[HA] (5)
Buffer concentration, [A-] + [HA] 0.10 M
[A-] (6)
[HA] (7)
Total volume of the solution 100.00 mL
Moles of A- (8)
Moles of HA (9)

Selection of reagents
For the chemicals below, write the full chemical formulas.
Na salt of conjugate acid (formula) (10)
Na salt of conjugate acid (molar mass) (11)

Na salt of conjugate base (formula) (12)

Na salt of conjugate base (molar mass) (13)

Mass of conjugate acid to be used: (14)

Mass of conjugate base to be used: (15)
Procedure (This part will go to your pre-lab journal)
Weigh the necessary amount of the sodium salts of your conjugate acid and base in a small
beaker (50-100 mL). Dissolve them together in about 50 mL of distilled water. Measure the pH
with the probe of the pH meter (be sure to rinse out the probe with distilled water before and
after use). While measuring the pH, adjust the pH to the desired level by adding HCl or NaOH
dropwise while stirring the solution.

Once the desired pH has been achieved, transfer the contents of the beaker to a 100-mL
volumetric flask. Rinse out the beaker with a small amount of distilled water and transfer this
too to the volumetric flask. Carefully fill the flask to the mark with distilled water. (Note: If you
overfill this, you might have to start over from the very beginning!)

Transfer the contents of this volumetric flask into an Erlenmeyer flask, then cover it tightly with
foil and Parafilm (if available). Label this with your section and group number, or keep it in your
group’s locker until next meeting.

Cleanup: Carefully flush any waste down the sink.

References

(4) Former DLSU Department of Chemistry Laboratory Manual (LBYKMBI)

Document history
30 May 2023: Prepared by Laurenzo D.V. Alba
Experiment 4
Enzyme Digestion
(2023 ver.)
Objectives
To demonstrate the catalytic activities of digestive enzymes

Reagents
These reagents will be listed again in their respective subheadings

Distilled water, Fehling’s Reagent A, Fehling’s Reagent B, iodine solution, 2% starch,

0.10M HCl, 0.2% HCl, 0.5% Na2CO3, bile salt solution, 2% pepsin, pancreatin solution,
methyl red solution

Materials
medicine droppers, test tubes, test tube rack, test tube holder, beakers, graduated
cylinder, crucible tongs, stopper, hot plate, hot water bath, watch glass, 500-mL beaker,
pH meter, plastic Pasteur pipettes

● 📷
To the students
If you take photos, it is a good idea to photograph before and after each test with a
white background.
● Wear gloves especially during cleanup or washing of glassware.
● Bring a sample of fresh egg and all-purpose cream. 1 egg and a 250 mL pack of
all-purpose cream will be enough for the whole class.
● ⚠Be sure to wash the glassware thoroughly at the end of the period with detergent
and water. Our experiments contain biological materials which may pose infection
hazards to other people.
● ⚠Be sure to discard the wastes in their proper waste containers when indicated.
-Procedures-
1. Digestion of Proteins
Test compounds: albumin from fresh egg
Reagents: 2% pepsin, 0.10 M HCl
Materials: test tubes, plastic Pasteur pipette, droppers, graduated cylinder, boiling water bath,
water bath at 37° C

Procedure1: Carefully draw the raw albumin into a plastic Pasteur pipette. With the albumin in
the stem of the pipette, immerse the pipette (holding by the bulb) in boiling water until the
albumin solidifies and turns white. Cut the pipette stem into 3 equal parts as shown below, and
discard the bulb:

Then, prepare three test tubes as follows:

Test Tube label Distilled water (mL) 2% pepsin (mL) 0.10 M HCl (mL)
K 5.0 0 1.0
L 0 5.0 1.0
M 1.0 5.0 0

Carefully squeeze the cooked albumin out of the stems. Immerse each cut separately into test
tubes K, L, and M. Incubate at 37° C for 90 minutes. Observe for any change in color and
appearance of the albumin. If there is a change in appearance of the solidified albumin,
measure its distance from the surface.

When possible, leave the samples overnight at room temperature and observe again the next
day. Be sure to discard and clean up right away after to avoid bacterial fouling.

Cleanup: Discard the liquid waste in the sink. Dispose the pipette stems as regular solid waste.
2. Digestion of Lipids
Test compounds: all-purpose cream
Reagents: 5% pancreatin, bile salt solution
Materials: test tubes, droppers, graduated cylinder, water bath at 37° C

Procedure: Label four large test tubes W, X, Y, and Z, and prepare their contents as follows:

Test Tube label All-purpose Distilled water Pancreatin Bile salt solution
cream (mL) (mL) solution (mL) (mL)
W 2.0 7.0 0
X 2.0 5.0 2.0
Y 2.0 0 7.0 0
Z 2.0 0 5.0 2.0
● It is easier to pour all-purpose cream when it is warmed up.
● If it is too difficult to measure the volume of the cream, just estimate it.

Stopper and gently shake each test tube to mix the contents. Note the initial appearance or take
photos of each mixture. Measure the initial pH by inserting the probe of the pH meter into the
test tube. Incubate at 37° C for 45 minutes. Measure the pH again for each test tube after
incubation. If pH measurement is unavailable, place 3 drops of methyl red solution on each test
tube and note the color of the drops on contact with the mixture (do not mix the methyl red
into the reaction).

Cleanup: Discard the liquid waste in the sink with large amounts of water.

3A. Digestion of Carbohydrates: Hydrolysis of Starch by Salivary

Amylase
Test compounds: 2% starch solution
Reagents: human saliva ☣, Fehling’s solutions A and B, iodine in KI
Materials: test tubes, droppers, graduated cylinder, beaker, water bath at 37° C, boiling water
bath

Procedure: In a small beaker, collect about 3 mL of saliva from a healthy volunteer. (A larger
sample is necessary if the collected saliva is frothy.) To this, add 6 mL of distilled water and mix
well. This will be the source of salivary amylase. Label four test tubes A, B, C, and D, and prepare
their contents as follows:

Test Tube label Distilled water (mL) 2% Starch (mL) Saliva solution (mL)
A 4.0 2.0 -
B 4.0 2.0 -
C - 2.0 4.0
 D - 2.0 4.0

Stopper and gently shake each test tube to mix the contents. Note the initial appearance or take
photos of each mixture. Incubate at 37° C for 30 minutes.

For the contents of Test Tubes A and C, perform the Fehling’s Test (refer to the procedures in
Experiment 1). Be sure to use only the recommended amount of test sample in the proper
ratios. Record your observations.

To the contents of Test Tubes B and D, add two drops of iodine in KI. Record your observations.

Cleanup: Discard the Fehling’s Test mixtures as inorganic or heavy metal waste. Discard the
other liquid waste into the sink with large amounts of water. Be sure to wash the glassware in
contact with human saliva with detergent and water.

3B. Digestion of Carbohydrates: Hydrolysis of Starch by Pancreatic

Amylase
Test compounds: 2% starch solution
Reagents: 5% pancreatin solution, 0.50% Na2CO3 solution, 0.2% HCl, Fehling’s solutions A and B,
iodine in KI
Materials: test tubes, droppers, graduated cylinder, beaker, water bath at 37° C, boiling water
bath

Procedure: Label four test tubes A, B, C, and D, and prepare their contents as follows.

Test Tube Distilled 5% 0.50% 0.2% HCl 2% starch

label water (mL) pancreatin Na2CO3 (mL) (mL) solution (mL)
(mL)
E 4.0 - - - 2.0
F 2.0 2.0 - - 2.0
G - 2.0 2.0 - 2.0
H - 2.0 - 2.0 2.0

Stopper and gently shake each test tube to mix the contents. Note the initial appearance or take
photos of each mixture. Incubate at 37° C for 30 minutes.

Take a portion of the contents of test tubes E, F, G, and H, and, perform the Fehling’s Test (refer
to the procedures in Experiment 1). Be sure to use only the recommended amount of test
sample in the proper ratios. Record your observations.

To the remaining samples in test tubes E, F, G, and H, add two drops of iodine in KI. Record your
observations.
Cleanup: Discard the Fehling’s Test mixtures as inorganic or heavy metal waste. Discard the
other liquid waste into the sink with large amounts of water.

References

(5) Protein Digestion (n.d.). Flinn Scientific. Retrieved from

https://www.flinnsci.com/protein-digestion/dc10123/
(6) Former DLSU Department of Chemistry Laboratory Manual (LBYKMBI)

Document history
08 September 2023: Prepared by Laurenzo Alba
06 January 2024: Revised Part 1 (Digestion of proteins) – albumin can now be squeezed out of
the tube after heating.
Experiment 5
Effect of Substrate Concentration on Enzyme
Activity (2023 ver.)
Objectives
To observe the effect of substrate concentration on enzyme activity
To generate a standard curve and determine the sample concentration

Reagents
Glucose, 3,5-dinitrosalicylic (DNS) acid, 5% Pancreatin Solution, 1 mg/mL starch solution,
phosphate buffer (from Experiment 4), 0.9% NaCl solution

Materials
medicine droppers, test tubes, test tube rack, test tube holder, beakers, thermometer,
hot plate, graduated cylinder, Vis spectrophotometer, 25-mL volumetric flasks

To the students
• If you take photos, it is a good idea to photograph before and after each test with
a white background.
• Wear gloves especially during cleanup or washing of glassware.
• Use the appropriate glassware for measuring. This experiment is quantitative and
ensuring accuracy of volumes is important.
• ⚠Be responsible to know the contents and hazards of your reaction mixtures!
Label your samples properly and make sure to clean them before returning them or
giving them to someone else.
• ⚠Be sure to discard the wastes in their proper waste containers when
indicated.
• ⚠Do not use funnels to transfer your samples into the volumetric flasks. Doing
so might cause the sample to escape up the gap between the flask and the funnel
stem.
-Procedures-

Part 1. Preparation of the Standard Curve

Test compounds: glucose solution (1 mg/mL)
Reagents: dinitrosalicylic acid (DNS)
Materials: test tubes, 25 mL volumetric flasks, droppers, graduated cylinder, beaker, boiling
water bath

Procedure:
1. Using large test tubes, prepare the following samples:
Test Tube Volume (mL)
Glucose Solution (1 Distilled Water DNS
mg/mL)
Blank 0.00 5.00 1.00
1 0.10 4.90 1.00
2 0.50 4.50 1.00
3 1.00 4.00 1.00
4 2.00 3.00 1.00
5 3.00 2.00 1.00
6 4.00 1.00 1.00
7 5.00 0.00 1.00

2. Mix well. Cover the test tubes with aluminum foil and place in a boiling water bath for 5
minutes.
3. Cool the samples to room temperature then cool by immersing in tap water.
4. Transfer each solution to a 25 mL volumetric flask. Dilute to mark with distilled water.
Mix well.
5. Determine the absorbance of the solutions against a blank at 540 nm using a Vis
Spectrophotometer (Spectronic 20).

Data Processing:
1. Plot the absorbance versus concentration of glucose (mg/mL)
a. A linear graph will be obtained based on the equation A = mx + b; where A =
absorbance, x = concentration of glucose (mg/mL), m = slope, b = y-intercept
2. Obtain the values of m and b. These will be used in Part 2: Enzyme Assay.

Cleanup: Solutions with DNS are to be thrown in the non-halogenated organic waste bottles.
Part 2. Enzyme Assay
Test compounds: starch solution (1 mg/mL) (substrate)
Reagents: 5% pancreatin solution (enzyme), 0.10 M pH 7.0 phosphate buffer (from Experiment
3), 0.9% NaCl solution, DNS reagent
Materials: test tubes, droppers, graduated cylinder, beaker, water bath at 37° C, boiling water
bath

Procedure:
1. Using large test tubes, prepare the following samples. The 5% pancreatin solution should
be added last.
Test Tube Volume (mL)
Label 1 mg/mL Starch 5% Pancreatin Phosphate NaCl solution
Solution Solution Buffer
Blank 0.00 0.50 6.00 0.50
1 0.50 0.50 5.50 0.50
2 1.00 0.50 5.00 0.50
3 2.00 0.50 4.00 0.50
4 3.00 0.50 3.00 0.50
5 4.00 0.50 2.00 0.50
6 5.00 0.50 1.00 0.50

2. Place in a 37ºC water bath and allow hydrolysis to proceed for 15 minutes.
3. To stop the hydrolysis, quickly add 1.00 mL of DNS reagent to each test tube.
4. Cover each test tube with aluminum foil and place in boiling water bath for 5 minutes.
5. Cool the samples to room temperature then cool by immersing in tap water.
6. Transfer each solution to a 25 mL volumetric flask and dilute up to the mark with
distilled water. Mix well.
7. Determine the absorbance of the solutions against a blank at 540 nm using a Vis
Spectrophotometer (Spectronic 20).
Data Processing:
1. Calculate the concentration of glucose using the equation from the standard curve
2. Calculate the velocity (mg/mL•min) using the following formula:
𝑚𝑔
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 (𝑖𝑛 𝑚𝐿 )
a. 𝑉 = 15 𝑚𝑖𝑛𝑢𝑡𝑒𝑠
3. Plot velocity vs substrate (Michaelis-Menten plot). Determine Km and Vmax.
4. Plot 1/V vs 1/S (Lineweaver-Burke plot). Determine Km and Vmax.

Cleanup: Solutions with DNS are to be thrown in the non-halogenated organic waste bottles.
References

(1) Former DLSU Department of Chemistry Laboratory Manual (LBYKMBI)

Document history
04 September 2023: Prepared by Bernadette Cecilia Morillo
Experiment 6 (2023 Ver.)
Simple DNA Extraction and Properties of Nucleic
Acids
Objectives
To understand the basic concepts involved in DNA extraction
To successfully extract DNA

Reagents
Table salt, distilled water, liquid detergent, papain or commercial meat tenderizer, 95%
ethanol (about 1 liter), 10% NaOH, 0.1% CuSO4, 0.1% Ninhydrin

Materials
Blender/mortar and pestle, strainer or cheesecloth, filter paper, funnel, 250/125-mL
Erlenmeyer flask, beakers, graduated cylinders, stirring rod, centrifuge (if necessary),
test tubes, droppers, cold water bath

• 📷
To the students
If you take photos, it is a good idea to photograph before and after each test
with a white background.
• Wear gloves especially during cleanup or washing of glassware.
• Use the appropriate glassware for measuring.
• Bring frozen chicken liver/peas/broccoli/banana/strawberries. This will be your
DNA source. Dried or canned products cannot be used!
• Use a 250-mL beaker half filled with tap water for the hot water bath.
• ⚠Be responsible to know the contents and hazards of your reaction mixtures!
Label your samples properly and make sure to clean them before returning them or
giving them to someone else.
• ⚠Be sure to discard the wastes in their proper waste containers when
indicated.
-Procedures-
Test compounds: frozen chicken liver OR frozen peas OR frozen broccoli OR frozen banana
Reagents: ice-cold distilled water, liquid detergent, NaCl, papain, 95% ethanol, 10% NaOH, 0.1%
CuSO4, 0.1% ninhydrin

Lysis of Sample Tissue (once for the whole class)

Obtain a clean blender to create a lysate of the sample. If using tough-skinned fruits, remove
the skin of the fruit before blending.

If using a blender, 500 g of sample will be enough for the class. For every 500 g of sample, add 5
g of table salt, and 1 L of cold distilled water in the blender. Blend on high for 20 seconds. Pass
the sample through a cheesecloth to remove large debris.

Disruption of Cell Membrane

Add 20 mL of liquid detergent into the solution and swirl to mix. Allow the mixture to stand for
10 minutes. Each group should take the solution into 2 large test tubes labeled A and B (the
solution must fill at least 2 large test tubes half-full). One test tube (A) will be used for DNA
extraction, the other (B) will be for qualitative tests.

Elimination of Proteins and Test for Proteins and Amino Acids (Biuret
and Ninhydrin Test)
To test tube A, add a pinch of enzyme (papain) and stir gently. Use the contents of test tube B as
the test sample for the Biuret Test and Ninhydrin Test (the procedures for these can be found in
Experiment 2). Record observations.

Precipitation of DNA
Tilt test tube A and slowly pour 95% ethanol down the side of a centrifuge tube. A layer on top
of the mixture should form. Continue pouring until there is an equal amount of ethanol and
mixture in the test tube. Observe the formation of white precipitate. Record these
observations.
● You need to collect the white precipitate.
● If the precipitate is stringy, you can use the sharp end of a barbecue stick to spool it and
take it to a separate container.
● If the precipitate is diffuse, centrifuge the mixture at 2000 RPM for about 30 seconds.
The precipitate will collect on the side of the tube and between the water and ethanol
layers. Use a pipette or decant the mixture to collect only the DNA precipitate. (Note:
the precipitate at the bottom of the tube should be discarded).

Cleanup: Solutions may be thrown into the sink with running water except for solutions treated
for Biuret Test (heavy metal waste) and Ninhydrin Test (organic waste).
II. Qualitative Tests
Sample preparation. For your DNA test solution, disperse the white DNA precipitate into 5 mL
of distilled water. If your original DNA sample contains liquid (such as ethanol), add enough
distilled water to make 5 mL of sample.

1. Test for Nucleoproteins

Test compounds: DNA test solution, distilled water
Reagents: 10% NaOH⚠, 0.5% CuSO4
⚠: corrosive
Materials: test tubes, plastic Pasteur pipette, droppers,

Procedure: To 20 drops of DNA solution, add 10 drops of 10% NaOH and 5 drops of 0.5% CuSO4
solution. Observe.

Cleanup: Discard the waste as heavy metal or inorganic waste.

2. Test for Nucleic Acid Hydrolysis Products

Test compounds: DNA test solution
Reagents: 10% H2SO4⚠
⚠: corrosive
Materials: test tubes, plastic Pasteur pipette, droppers, graduated cylinder

Preparation of hydrolysate: To 3 mL of DNA solution, add 5 drops of 10% H2SO4. Boil gently for 3
minutes. Perform the following tests on this solution.

Cleanup: After doing the experiments below, dispose the unused solution in the sink with
plenty of water.

2-1. Test for purine bases (only do this in an open-air lab or under the fume hood)
Test compounds: DNA test solution, distilled water
Reagents: concentrated HNO3⚠, 10% KOH⚠
⚠: corrosive
Materials: test tubes, plastic Pasteur pipette, droppers, boiling water bath

Procedure: Place 10 drops of hydrolysate in a test tube and add 3 drops of concentrated nitric
acid. Place in boiling water bath and evaporate until almost dry. Cool to room temperature and
add 5 drops of 10% KOH. Observe any color change.

Cleanup: Discard the waste in the sink with plenty of water.

2-2. Test for pyrimidine bases (do not perform for now)
Test compounds: DNA test solution, distilled water
Reagents: Bromine water (Br2•H2O)⚠, saturated Ba(OH)2⚠⚠
⚠: corrosive, ⚠⚠: toxic
Materials: test tubes, plastic Pasteur pipette, droppers

Procedure: To 10 drops of hydrolysate, add 20 drops of Br2•H2O and 1 drop of saturated

barium hydroxide solution. Observe results.

Cleanup: Discard the waste as halogenated organic waste.

2-3. Test for ribose

Test compounds: DNA test solution, 1% ribose, 1% glucose,
Reagents: Bial’s orcinol reagent⚠
⚠: corrosive
Materials: test tubes, plastic Pasteur pipette, droppers, boiling water bath

Procedure: Mix 20 drops of Bial’s Orcinol reagent to 20 drops of test sample. Cover with marble.
Place the tubes in boiling water bath until a color develops in any of the test solutions. Note this
color change.

Cleanup: Discard the waste in the sink with plenty of water.

2-4. Test for deoxyribose

Test compounds: DNA test solution, 1% ribose, 1% glucose,
Reagents: diphenylamine reagent
Materials: test tubes, plastic Pasteur pipette, droppers, boiling water bath

Procedure: Mix 2 mL of diphenylamine reagent to 10 drops of test solution. Cover with marble.
Place the tubes in boiling water bath until a color develops in any of the test solutions. Note this
color change.

Cleanup: Discard the waste in the sink with plenty of water.

2-5. Test for phosphate

Test compounds: DNA test solution, 1% ribose, 1% glucose,
Reagents: 10% HNO3⚠, 5% (NH4)2MoO4
⚠: corrosive
Materials: test tubes, plastic Pasteur pipette, droppers, boiling water bath

Procedure: To 20 drops of test sample, add 20 drops of 10% HNO3 and 20 drops of 5%
(NH4)2MoO4 solution. Heat to boiling. Let stand for a few minutes and note the results.

Cleanup: Discard the waste as inorganic or heavy metal waste.

References

(1) Former DLSU Department of Chemistry Laboratory Manual LBYKMBI)

(2) DNA purification. Promega.com. Retrieved from
https://worldwide.promega.com/resources/guides/nucleic-acid-analy
sis/dna-purification
(3) How to extract DNA from anything living. Utah.edu. Retrieved from
https://learn.genetics.utah.edu/content/labs/extraction/howto/

Document history

11 July 2023: Prepared by Bernadette Cecilia Morillo

17 July 2023: Edited by Laurenzo Alba
3 Sept 2023: Edited by Laurenzo Alba (notes on DNA precipitation)

Experiment 7
Lipids (2023 extended ver.)
Objectives
To illustrate the chemical properties of lipids
To distinguish the reactivity and property of fatty acids and phosphates

Reagents
Conc. HNO3, conc. H2SO4, chloroform, DCM, 1% lecithin, 1% bile salt solution, Br2•H2O,
n-butyl alcohol, 0.05% a-tocopherol in DCM, alcoholic KOH, 10% NaOH, KHSO4, acetic
anhydride, 5% (NH4)2MoO4, Glycerol, coconut oil, stearic acid, olive oil, lecithin,
cholesterol

Materials
medicine droppers, test tubes, test tube rack, test tube holder, filter paper, beakers,
graduated cylinder, crucible tongs, cork stopper, hot plate, hot water bath, funnel,
crucible, Bunsen burner, iron ring, iron stand

To the students
• If you take photos, it is a good idea to photograph before and after each test with
a white background.
• Wear gloves especially during cleanup or washing of glassware.
• Use the appropriate glassware for measuring.
• Make sure that the test tubes are as dry as possible.
• Use dichloromethane (DCM/CH2Cl2) as the negative control.
• Use a 250-mL beaker half filled with tap water for the hot water bath.
• ⚠Be responsible to know the contents and hazards of your reaction mixtures!
Label your samples properly and make sure to clean them before returning them or
giving them to someone else.
• ⚠Be sure to discard the wastes in their proper waste containers when
indicated.

-Procedures-
Qualitative Tests
Unknown samples will not be used for this experiment. Use small, dry test tubes. Use
DCM/CH2Cl2/Dichloromethane as negative control when indicated.

1. Solubility Test
Test compounds: glycerol, coconut oil, stearic acid, DCM
Reagents: distilled water, chloroform

Procedure:
Prepare and label the test tubes as follows:
Test Tube Test Compound Reagent
A-1 3 mL glycerol 3 mL distilled water
B-1 3 mL coconut oil 3 mL distilled water
C-1 3 mL stearic acid 3 mL distilled water
D-1 3 mL DCM 3 mL distilled water
E-1 3 mL unknown sample 3 mL distilled water
- - -
A-2 3 mL glycerol 3 mL chloroform
B-2 3 mL coconut oil 3 mL chloroform
C-2 3 mL stearic acid 3 mL chloroform
D-2 3 mL DCM 3 mL chloroform

Shake the test tubes and allow them to stand for 1 minute. Observe the solution for layers.
Record observations.

Cleanup: Solutions with DCM, chloroform, and/or unknown are to be thrown in the
halogenated organic waste bottles. The rest can be disposed as non-halogenated organic waste.

2. Emulsification Test
Test compound: coconut oil
Reagents: 1% lecithin, distilled water, 1% bile salt solution

Procedure:
Prepare and label the test tubes as follows:
 Test Tube Contents
A 20 drops of test compound + 20 drops of distilled water
B 20 drops of test compound + 20 drops of 1% bile salt solution
C 20 drops of test compound + 20 drops of 1% lecithin solution

Shake each mixture and let stand for 1 minute. Record observations

Cleanup: Dispose of the waste into the sink.

3. Unsaturation Test
Test compounds: olive oil, oleic acid, coconut oil, DCM
Reagents: Br2•H2O (bromine water)

Procedure:
In separate test tubes, add 5 drops of the test compounds (add a pinch if the compound is
solid). Add Br2•H2O dropwise until a faint permanent brownish color is visible in the aqueous
part of the mixture. Record number of drops needed to reach this point. Use the same dropper
for each test compound. Record observations.

Cleanup: Dispose of the waste as halogenated organic waste.

4. Saponification Test
Test compounds: olive oil, coconut oil, DCM
Reagents: alcoholic KOH, distilled water

Procedure:
In separate test tubes, add around 1 mL of each test compound. In each test tube, add 1 mL of
alcoholic KOH. Mix solutions thoroughly and allow to stand for 10 minutes with periodic mixing.
Add 3 mL of distilled water, stopper, and shake vigorously. Observe for soap formation or
lathering.

Cleanup: Solutions with DCM and/or unknown are to be thrown into the halogenated organic
waste bottles. The rest can be disposed in the sink.

5. Test for Free Fatty Acids

Test compounds: olive oil, coconut oil, DCM
Reagents: 10% NaOH, distilled water
Procedure:
In a test tube, add 2-3 drops of phenolphthalein solution then add 10% NaOH dropwise until
solution turns pink. Then add 5 drops of the test compound and shake mixture thoroughly.
Perform the same procedure to the different test compounds. Record observations.

Cleanup: Solutions with DCM and/or unknown are to be discarded into the halogenated organic
waste bottles. The rest can be disposed in the sink.

6. Acrolein Test
Test compounds: glycerol, coconut oil, stearic acid
Reagents: KHSO4

Procedure:
In separate test tubes, add 2 drops of test compound (or a pinch if it is solid). To each test tube,
add a pinch of KHSO4. Ensure that relatively the same amount of KHSO4 is added to each test
tube. Heat the test tubes gently (~40C) for 3 minutes then heat in boiling water. Describe the
odor produced.

Cleanup: Dispose of the waste into the sink.

7. Lieberman-Burchard Test (Acetic Anhydride Reaction)

Test compounds: cholesterol, DCM
Reagents: DCM, acetic anhydride, conc. H2SO4

Procedure:
In a test tube, place a pinch of cholesterol and add 10 drops of DCM. Mix well. Add 3 drops of
acetic anhydride and 1 drop of conc. H2SO4. Mix well. Note changes in color. In separate test
tubes, add 10 drops of the unknown sample in one and 10 drops of DCM in the other. To both
test tubes, add 3 drops of acetic anhydride and 1 drop of conc. H2SO4. Mix well. Note changes in
color. Record observations

Cleanup: Dispose of the waste as halogenated organic waste.

8. Salkowski Test
Test compounds: cholesterol, DCM
Reagents: DCM, conc. H2SO4
Procedure:
In a test tube, place a pinch of cholesterol and add 10 drops of DCM. Mix well. Add 10 drops of
conc. H2SO4. Mix well. Note changes in color. In separate test tubes, add 10 drops of the
unknown sample in one and 10 drops of DCM in the other. To both test tubes, add 10 drops of
conc. H2SO4. Mix well. Note changes in color. Record observations.

Cleanup: Dispose of the waste as halogenated organic waste.

9. Test for Phosphate

Test compounds: lecithin, DCM**
Caution: **Skip this sample if using an airconditioned laboratory without fume hood.
Reagents: 5% (NH4)2MoO4, conc. HNO3

Procedure:
Incinerate a small amount of lecithin (pea-sized) in a porcelain crucible by using the intense heat
of a Bunsen burner or directly over a hot plate. Allow it to cool down to room temperature.
Extract the cooled residue by adding 3 mL distilled water into the crucible, swirling the liquid
around, then filtering it. To 1 mL of filtrate, add 10 drops of freshly prepared 5% (NH4)2MoO4 and
2 drops conc. HNO3. Heat to boiling in a boiling water bath and allow to stand for a few minutes.
Observe the formation of a yellow precipitate.

In separate test tubes, add 1 mL of unknown sample and DCM respectively. To each test tube,
add 10 drops of freshly prepared 5% (NH4)2MoO4 and 2 drops of conc. HNO3. Heat to boiling and
allow to stand for a few minutes. Record observations.

Cleanup: Solutions with DCM and/or chloroform are to be thrown in the halogenated organic
waste bottles. The rest can be disposed as inorganic or heavy metal waste.

10. Test for Vitamin E (Modified Furter-Meyer Test)

Test compounds: 0.05% a-tocopherol in DCM, DCM
Reagents: n-butyl alcohol, conc. HNO3
Procedure:
In separate test tubes, add 10 drops of 0.05% a-tocopherol in DCM, 10 drops of unknown
sample, and 10 drops of DCM respectively. In each test tube, add 2 mL of n-butyl alcohol and 5
drops conc. HNO3. Mix well and place test tubes in 80°C water bath for 10 minutes. Note
changes in color. Record observations.

Cleanup: Dispose of the waste as halogenated organic waste.

References

(1) Former DLSU Department of Chemistry Laboratory Manual (LBYKMBI)

(2) P. Kumar. Qualitative and Quantitative Tests for Lipids.
https://www.biologydiscussion.com/lipids/tests/qualitative-and-quantita
tive-tests-for-lipids/13050

Document history
26 June 2023: Prepared by Bernadette Cecilia Morillo
11 July 2023: Edited by Laurenzo Alba
17 July 2023: Edited by Laurenzo Alba (v3)
06 Jan 2024: Edited the procedure for Test for Unsaturation