Lab Manual
Lab Manual
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
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
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 each test tube, 10 drops of the assigned test solution and the unknown sample
to 30 drops of freshly prepared phenylhydrazine reagent. Place the test tube 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.
Procedure: To individual test tubes, add 1 drop of Molisch reagent to 30 drops of each 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.
Procedure: To 10 drops of each test solution and the unknown sample, 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.
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.
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.
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.
⚠: 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
• Discard the waste as halogenated organic waste
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
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
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.
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.
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.
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.
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.
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.
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.
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 compounds: 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 compounds: 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).
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)
5. Salting Out
Test compounds: 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.
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.
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
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.
H+ + A- à HA
(added)
OH- + HA à A- + H2 O
(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.
[𝐴$ ]
𝑝𝐻 = 𝑝𝐾! + log"#
[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)
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)
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.
References
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
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:
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
Immerse each cut stem 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 at the cut ends of the pipette stem. If there
is a change in appearance of the solidified albumin, measure its distance from the cut ends.
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.
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.
Procedure: Label four test tubes A, B, C, and D, and prepare their contents as follows.
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
Document history
08 September 2023: Prepared by Laurenzo Alba
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.
-Procedures-
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 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:
!"
%&'%(')*!)+&' &- ./0%&1( (+' )
a. 𝑉 = "4 5+'0)(1
!#
Cleanup: Solutions with DNS are to be thrown in the non-halogenated organic waste bottles.
References
Document history
04 September 2023: Prepared by Bernadette Cecilia Morillo
Experiment 6
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-
1. Solubility Test
Test compounds: unknown sample, 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
E-2 3 mL unknown sample 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
3. Unsaturation Test
Test compounds: unknown sample, 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 the solution is decolorized. Record number of drops needed
to decolorize solution. Use the same dropper for each test compound. Record observations.
4. Saponification Test
Test compounds: unknown sample, 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: unknown sample, 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.
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: unknown sample, glycerol, coconut oil, stearic acid
Reagents: KHSO4
Procedure:
In separate test tubes, add 2 drops of each 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.
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
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.
Procedure:
Incinerate a small amount of lecithin (pea-sized) in a porcelain crucible by using the intense
heat of a Bunsen burner. 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: unknown sample, 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.
References
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)
Experiment 7 (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
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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. From experience, frozen peas work best. 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
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.
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.
Procedure: To 20 drops of DNA solution, add 10 drops of 10% NaOH and 5 drops of 0.5% CuSO4
solution. Observe.
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.
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.
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.
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-
analysis/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)