Research

8
QUARTER 3
Module 2: Laboratory Techniques and
Methods (Chemical)
Research 8
Self-Learning Module (SLM)
Quarter 3-Module 2:
Laboratory Techniques and Methods (Chemical)
First Edition, 2021

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 Development Team of the Module

Writer: Anna Mae T. Valdez

Evaluator: Aurelia S. Garcia

Reviewer: Amerfina D. Nelmida

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8
Research 8
Quarter 3-Module 2:
Laboratory Techniques and
Methods (Chemical)
(Week 3-4/Day 11-20)
Introductory Message
Welcome to the Grade 8 Research Self-Learning Module on
Laboratory Techniques and Methods (Chemical) which deals with
spectrophotometry, titration, and extraction!

The hand is one of the most symbolized part of the human body. It
is often used to depict skill, action, and purpose. Through our hands we
may learn, create, and accomplish. Hence, the hand in this learning
resource signifies that you as a learner is capable and empowered to
successfully achieve the relevant competencies and skills in Research 2.
Your academic success lies in your own hands!

This module has the following parts and corresponding icons:

What I Have
Learned
What I Need To Know

This will give you an idea of the

skills or competencies you are
expected to learn in the module.
This part includes an activity that
What I Know aims to check what you already
know about the lesson to take. This
What’s In is a short review which helps you
link the current lesson with the
previous one.
In this portion, the new lesson will
be introduced to you in various
What’s New ways such as a story, a song, a
poem, a problem opener, an
activity, or a situation.

This section provides a brief

What is It discussion of the lesson. This aims
to help you discover and
understand new concepts and
What’s More skills.
This comprises activities for
independent practice to assess
your understanding and skills of filled in to process what you
topic. learned from the lesson.
the This includes questions or
blank sentence/paragraph to be i

This section provides an activity

What I Can Do which will help you transfer your

new knowledge or skill into real life

situations or concerns.

Assessment This is a task which aims to

evaluate your level of mastery in
achieving the learning competency.
This contains all the answers of the
Key Answer
activities in the module.

At the end of this module, you will also find: references which contains
the list of sources used in developing this module.
 ii
What is so special about using this module?

You will work on your own way and pace. You have read it right! This
module is like an ordinary classroom lesson, but without the actual
classroom and without the teacher. You should also remember that this
module was designed to provide you with fun and meaningful learnings
despite of what we are facing today in this time of pandemic. Do not
worry because as you will independently learn from this module, you will
be guided properly along the way.

Before you proceed, please carefully read the following reminders

which will serve as your guide in this module:

1. Use the module with care. Do not put unnecessary mark/s on any
part of the module. Use a separate sheet of paper in answering the
exercises/ activities.

2. You have one week to answer and use this module. Make sure to
accomplish the given tasks step-by-step.

3. Read the instructions carefully before doing each assigned task.

4. Do not skip the What I Know part and answer it with the best that
you can.

5. Be honest in answering the activities given in this module and in

checking your answer using the Answer Key.

6. Return this module to your teacher once you are through with it.

If you encounter difficulties in using the module, do not hesitate to

consult your teacher. I hope that you will gain meaningful learning
experience and deep understanding of the relevant competencies through
this module.
Good luck and have fun!

iii

What I Need to Know

Before starting any experiment, you should understand the entire

procedure that you will be following. You need to make sure that you have
the proper equipment, and that you know how to use it. When you are
trying an unfamiliar procedure for the first time, it is a good idea to
practice at least one "dry run" without chemicals. That way, you can
make sure you have all the materials you will need at hand, and that your
workflow will proceed smoothly. You will greatly reduce the risk of an
accident by carefully planning. Thus, using proper laboratory techniques
will increase your level of safety in the lab.

This module focuses on three important topics under chemical laboratory

techniques and methods which are as follows:
Lesson 1: Spectrophotometry
Lesson 2: Titration
Lesson 3: Extraction
After going through this module, you are expected to:

 Identify standard methods and techniques used in

performing experiments.

More specifically, you are expected to:

 Define spectrophotometry, titration, and extraction

method.
  Demonstrate proper procedure and technique in
performing spectrophotometry, titration, and
extraction for experimental research.

Before going on, check how much you know about this topic.
Answer the pretest on the next page in a separate sheet of paper.

1
 What I Know

Direction: Read each question carefully and write the letter of your
answer on a separate 1 whole sheet of paper.

1. Leny does not know the concentration of the solution she will use
in the experiment. Her teacher advised her to measure the
absorbance of the solution to determine its concentration. What
device can she use to do it?
A. Autoclave C. Hot air oven
B. Centrifuge machine D. Spectrophotometer

2. Aldwin learned from his grandmother that paragis can cure

stomachache. Based on his research, medicinally active portions of
plants can be separated using selective solvents through standard
procedures. What method is this?
A. Plant Extraction C. Plant Homogenization
B. Plant Testing D. Phytochemical Analysis

3. During an experiment, the teacher asked the learners to find out

the concentration of the liquid in the beaker. What method is used
in determining the concentration of an unknown solution?
A. Centrifugation C. Filtration
B. Distillation D. Titration

4. Which of the following principles BEST describes

spectrophotometry?
A. Transmission properties of a material is qualitatively
measured.
B. As concentration increases, absorption decreases
exponentially.
C. Each compound absorbs or transmits light over a certain
range of wavelength.
D. Light absorbed by the sample is equal to the concentration of
sample in the solution.

5. Which serves as indicator in a titration experiment?

A. change in color B. smell of the solution

C. volume of the known solution

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 D. volume of the unknown solution

6. Liza performs a titration experiment with her classmates. What

should they do so that the known solution in the burette will not be
added directly to the unknown solution in the conical flask? A. Put
an indicator to the unknown solution.
B. Slowly pour the known solution to the graduated glass tube.
C. Adjust the precision tap of the burette to slowly add the
solution.
D. Just put the solution in to the burette without adjusting the
precision tap.

7. How is the pure extract of plant samples obtained to make sure

that the bioactive components are retained? A. Use decoction
process.
B. Pound the sample using a mortar and pestle.
C. Grind the plant sample and boil it for 30 minutes.
D. Soak the plant sample for 10 minutes and squeeze it.

8. Why is there a need to add an indicator to the unknown solution in

a titration experiment?
A. To easily determine the endpoint.
B. To achieve faster chemical reaction.
C. To stop chemical reaction between the two solutions.
D. To measure accurately the concentration of the unknown
solution.

9. Why is it important to determine the absorbance of a sample

solution? A. To calculate the exact wavelength of light. B. To know
the amount of the blank sample.
C. To identify the transmission of the solution.
D. To determine the amount and concentration of the solution.

10. Why is there a need to use a blank solution in measuring the

transmission and absorbance rate of the sample with food
coloring? A. It acts as the source of transmission and absorbance.
B. It improves the reading process of the unknown concentration.

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 C. It serves as the reference in measuring the transmission and
absorbance rate.
D. It increases the concentration of the solution measured at
different wavelengths.

What’s In

In the previous module, you have learned about laboratory

techniques and methods which focuses on aseptic technique, typical
transfer of bacteria and microbiological techniques. These methods are
very useful in conducting your experimental research soon. With this, I
hope you could identify the best method for your study as we continue
discussing the different chemical laboratory techniques and methods. Yes,
you are right! This is how it works in research! So, have fun and enjoy
learning about them!

What’s New

Activity 1: How much do you know me?

Direction: Define the following laboratory methods based on the given
pictures below.

1. SPECTROPHOTOMETRY-

4
2. TITRATION-

3. EXTRACTION -

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Lesson
Spectrophotometry
1

What is It

Spectrophotometry is an experimental technique that is used to

measure the concentration of solutes in a specific solution by calculating
the amount of light absorbed by those solutes. This technique is powerful
because certain compounds will absorb different wavelengths of light at
different intensities.
By analyzing the light that passes through the solution, you can
identify dissolved substances in solution and how concentrated those
substances are.

Materials Required:

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 2. Cuvette

3. Blank solution

Reagents:

Cobalt (II) Crystal chloride

Violet

Hexaaquacobalt Rose
(II) ion bengal

Ferrocene Coumarin

GENERAL PROCEDURE:

Part 1: Preparing the Samples

1. Turn on the spectrophotometer. Most

spectrophotometers need to warm up
before they can give an accurate reading.

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 Turn on the machine and let it sit for at least 15 minutes before
running any samples.
• Use the warm-up time to prepare your samples.

2. Clean the cuvettes or test tubes. If

you are doing a lab for school, you may
be using disposable test tubes that do not
need to be cleaned. If you are using
cuvettes or reusable test tubes, make
sure they are properly cleaned before
use. Rinse each cuvette thoroughly with
deionized water.
• Take care with cuvettes as they can be quite expensive,
particularly if they are made from glass or quartz. Quartz
cuvettes are designed for use in UV-visible spectrophotometry.
• When handling the cuvette, avoid touching the sides the light
will pass through (generally, the clear sides of the container). If
you accidently touch these sides, wipe the cuvette down with a
kimwipe (which are formulated to prevent scratching the
glass).

3. Load the proper volume of the

sample into the cuvette. Some
cuvettes have a maximum volume of 1
milliliter (mL) while test tubes may have
a maximum volume of 5 mL. If the laser
producing the light is passing through
the liquid and not an empty part of the
container, you will get an accurate
reading.
• If you are using a pipette to load your samples, use a new tip
for each sample to prevent cross-contamination.

4. Prepare a control solution. Known as

a blank, the control solution has only the
chemical solvent in which the solute to
be analyzed is dissolved in. For example,
if you had salt dissolved in water, your
blank would be just water. If you dye the
water red, the blank must also contain

8
 red water. The blank is the same volume as the solution to be
analyzed and kept in the same kind of container.

5. Wipe the outside of the cuvette.

Before placing the cuvette into the
spectrophotometer, you want to make
sure it is as clean as possible to avoid
interference from dirt or dust particles.
Using a lint free cloth, remove any water
droplets or dust that may be on the
outside of the cuvette.
Part 2: Running the Experiment

1. Choose and set the wavelength of

light to analyze the sample with. Use
a single wavelength of light
(monochromatic color) to make the
testing more effective. The color of the
light chosen should be one known to be
absorbed by one of the chemicals
thought to be in the test solute. Set the
desired wavelength according to the specifications of your
spectrophotometer.

2. Calibrate the machine with the

blank. Place the blank into the cuvette
holder and shut the lid. On an analog
spectrophotometer, there will be a screen
with a needle that moves based on the
intensity of light detection. When the
blank is in, you should see the needle
move to the right. Record this value in
case you need it for later. With the blank still in the machine, move
the needle to zero using the adjustment knob.

3. Remove the blank and test the

calibration. With the blank removed the
needle should stay at 0 (zero) or the
digital readout should continue to read 0.
Place the blank back into the machine
and ensure the needle or readout does
not change. If the machine is properly
calibrated with your blank, everything
should stay at 0.

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 4. Measure the absorbance of
your experimental sample.
Remove the blank and place the
experimental sample into the machine.
Slide the cuvette into the designated
groove and ensure it stands upright. Wait
about 10 seconds until the needle is
steady or until the digital numbers stop
changing. Record the values of % transmittance and/or absorbance.

5. Repeat the test with successive

wavelengths of light. Your sample may
have multiple unknown compounds that
will vary in their absorbance depending on
wavelength. To eliminate
uncertainty, repeat your readings at
25 nm intervals across the spectrum. This
will allow you to detect other chemicals suspected to be in the
solute.

Part 3: Analyzing the Absorbance Data

1. Calculate the transmittance and

absorbance of the sample.
Transmittance is how much of the light that
passed through the sample reached the
spectrophotometer. Absorbance is how
much of the light has been absorbed by
one of the chemicals in the solute.

2. Plot the absorbance values versus the

wavelengths on a graph. The absorbance
value is plotted on the vertical y-axis
against the wavelength of light used for a
given test plotted on the horizontal x-axis.

3. Compare your absorbance spectrum

plot to known plots of specific
compounds. Compounds have unique
absorbance spectrum and will always
produce a peak at the same wavelength

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 every time they are measured. By comparing your plots of unknown
compounds to those of known compounds, you can identify the
solutes that compose your solution.

Note: You may visit this link https://www.youtube.com/watch?

v=xHQM4BbR040 to watch an actual video on how to perform
spectrophotometry method.

Determination of Molar Absorption Coefficient:

1. Select a blank cuvette and place it in the spectrophotometer. Close

the lid.
2. Click on 0 ABS 100%T button, the instrument now reads 0.00000 A.
3. Choose a solution with known concentration and measure the
absorbance between the wavelengths 350 nm to 700 nm.
4. Record the wavelength at the maximum absorbance value.
5. Calculate the value of molar absorption coefficient, using the
equation

Determination of Unknown Concentration:

1. Set the wavelength to the value corresponding to maximum

absorbance (recorded above).
2. Place the cuvette with same solution but at an unknown
concentration.
3. Read the absorbance for this wavelength.
4. Calculate the concentration with the help of the equation,

molarity
5. Enter the calculated concentration value in the given box.
(Note: Should enter the value correct to four decimal places)
6. Repeat the same procedure for a second solution

Observations and Calculations:

Concentration of Solution =.............................M

Absorbance of known conc. solution =...............................A
Cuvette length =.............................cm
Wavelength at maximum absorbance value =.........................nm
Molar Absorption Coefficient, =...................... M −1 cm−1
Absorbance of given solution =..............................A
Therefore, concentration (c) =.............................M

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Result:

Concentration of the given solution =...............................M

Points to Remember while Performing the Experiment in a Real

Laboratory:

1. Always wear lab coat and gloves when you are in the lab. When you
enter the lab, switch on the exhaust fan, and make sure that all the
chemicals and reagents required for the experiment are available. If
they are not available, prepare the reagents using the components
for reagent preparation.
2. Make sure to clean all your working apparatus with chromic acid and
distilled water and ensure that all the apparatus are free from water
droplets while performing the experiment.
3. Make sure to calibrate the electronic weigh balance before taking
the measurements.
4. Ensure that the spectrophotometer is working properly.
5. Ensure that you are handling the cuvette with tissue paper. Never
touch it with your hand.
6. Wipe the cuvette with tissue paper before
placing the spectrophotometer.
7. Clean all glassware with soap and distilled water. Once the
experiment is completed recap the reagent bottles. Switch off the
light and exhaust fan before leaving the lab.
8. Discard the used gloves in a waste bin.

What’s More

Activity 2: Performing Spectrophotometry

Direction: Identify the step-by-step procedure in
performing spectrophotometry method by arranging
it from A-J. Write your answer in a separate sheet of pad
paper.

________1. Calculate the value of molar absorption coefficient, using the

equation

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________2. Click on 0 ABS 100%T button, the instrument now reads
0.00000
A.
________3. Select a blank cuvette and place it in the spectrophotometer.
Close the lid.
________4. Record the wavelength at the maximum absorbance value.
________5. Enter the calculated concentration value in the given box.
________6. Calculate the concentration with the help of the equation,

molarity
________7. Read the absorbance for this wavelength.
________8. Place the cuvette with same solution but at an unknown
concentration.
________9. Set the wavelength to the value corresponding to maximum
absorbance (recorded above).
______10. Choose a solution with known concentration and measure the
absorbance between the wavelengths 350 nm to 700 nm.

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Lesson
Titration
2

What is It

Titration is a technique for determining the concentration of a solution

by measuring the volume of one solution needed to completely react with
another solution. It involves the addition of solution of known
concentration from burette to the measured volume of analyte.

PRINCIPLE OF TITRATION

Titration is based on the complete chemical reaction between the

analyte and the reagent (titrant) of known concentration.

Analyte + Titrant = Product

TERMS USED IN TITRATION

• Analyte – The solution of unknown concentration but known

volume.
• Titrant- The solution of known concentration.
• Standard Solution- A solution of known concentration.

SET-UP FOR TITRATION

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PROCEDURE

1. Pour 20 mL of acid solution into the 50

mL beaker and rinse the 15.00 mL
volumetric pipet three times with a
small amount of it.

2. Make sure to place your index finger on

the end of the pipet. Fill the pipet with
the acid solution above the calibration
mark. Dry the outside of the pipet with
a wipe. To obtain an accurate reading,
you should have the calibration mark
(meniscus) at eye level, i.e., your line of
sight should be parallel with the mark.

3. Transfer the 15.00 mL of the acid

solution into the clean (rinsed with
distilled water) 250 mL Erlenmeyer
flask. Repeat the above process (once
or twice).

4. Add 2-3 drops of phenolphthalein

indicator to each Erlenmeyer flask.

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5. Clamp the burette to the burette
stand.

6. Close the stopcock.

7. Rinse the 50.00 mL burette using a small

quantity of the base solution (use the
glass funnel to avoid spillage).

8. Turn and rotate the burette so all inside

surfaces have come into contact with the
base solution.

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9. Drain and discard the base solution into
the waste beaker. Rinse the burette
three times with a small quantity of this
solution. Make sure that the tip of the
burette is rinsed.

10. Remove the funnel and put the waste

beaker under the tip of the burette.
Open the stopcock and allow some
solution to run through (ensure that the
burette tip is filled with the base
solution and contains no air bubbles).

11. Close the stopcock and wait a few

seconds for drainage to be complete,
then note the reading on the burette to
two decimal places (Vinitial).

12. Place the Erlenmeyer flask under the tip

of burette and put the Kimwipe under
the Erlenmeyer flask (white
background).

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13. Titrate to the end point.

14. When you are close to the end point use

the drop-by-drop approach and rinse the
last drop from the tip of the burette at
the side of the Erlenmeyer flask with the
distilled water.

15. Over titrated end point – dark pink.

16. Close the stopcock and wait a few

seconds for drainage to be complete,
then note the reading on the burette to
two decimal places (Vfinal).

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19
 What’s More

Activity 3: Performing Titration

Direction: Identify the step-by-step procedure in performing titration
method by arranging it from A-J. Write your answer in a
separate sheet of pad paper.

________1. Add 2-3 drops of phenolphthalein indicator to each Erlenmeyer

flask.

________2. Remove the funnel and put the waste beaker under the tip of
the burette. Open the stopcock and allow some solution to run
through (ensure that the burette tip is filled with the base
solution and contains no air bubbles).

________3. Drain and discard the base solution into the waste beaker. Rinse
the burette three times with a small quantity of this solution.
Make sure that the tip of the burette is rinsed.

________4. Turn and rotate the burette so all inside surfaces have come
into contact with the base solution.

________5. Rinse the 50.00 mL burette using a small quantity of the base
solution (use the glass funnel to avoid spillage).

________6. Clamp the burette to the burette stand.

_______7. Make sure to place your index finger on the end of the pipet. Fill
the pipet with the acid solution above the calibration mark. Dry
the outside of the pipet with a wipe.

_______8. Pour 20 mL of acid solution into the 50 mL beaker and rinse the
15.00 mL volumetric pipet three times with a small amount of it.

________9. Transfer the 15.00 mL of the acid solution into the clean (rinsed
with distilled water) 250 mL Erlenmeyer flask. Repeat the above
process (once or twice).

_______10. Close the stopcock.

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Lesson
Extraction
3

What is It

Preparation of medicinal plants for experimental purposes is an

initial step and key in achieving quality research outcome. It involves
extraction and determination of quality and quantity of bioactive
constituents before proceeding with the intended biological testing. The
primary objective of this is to evaluate various methods used in the
preparation and screening of medicinal plants in our daily research.
Although the extracts, bioactive fractions, or compounds obtained from
medicinal plants are used for different purposes, the techniques involved
in producing them are generally the same irrespective of the intended
biological testing.

Medicinal plants are extracted and processed for direct consumption

as herbal or traditional medicine or prepared for experimental purposes.
The concept of preparation of medicinal plant for experimental purposes
involves the proper and timely collection of the plant, authentication by
an expert, adequate drying, and grinding. This is followed by extraction,
fractionation, and isolation of the bioactive compound where applicable. In
addition, it comprises determination of quantity and quality of bioactive
compounds.

However, the choice of solvent depends on the type of plant, part of

plant to be extracted, nature of the bioactive compounds, and the
availability of solvent. In general, polar solvents such as water, methanol,
and ethanol are used in extraction of polar compound, whereas nonpolar
solvents such as hexane and dichloromethane are used in extraction of
nonpolar compounds.

PROPERTIES OF SOLVENT OF EXTRACTION

1. Water. It is the most polar solvent and is used in the extraction of a

wide range of polar compounds.

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 2. Alcohol. It is also polar in nature, miscible with water, and could
extract polar secondary metabolites.

3. Chloroform. It is a nonpolar solvent and is useful in the extraction

of compounds such as terpenoids, flavonoids, fats, and oils.

4. Ether. It is a nonpolar solvent and is useful in the extraction of

compounds such as alkaloids, terpenoids, coumarins, and fatty
acids.

5. Ionic liquid (green solvent). This is a unique solvent of extraction

and is highly polar and extremely heat stable. It can remain in a
liquid state even at 3,000oC and usable where high temperature is
applicable. It has extreme miscibility with water and other solvent
and is very suitable in the extraction of polar compounds.

Commonly used methods in the extraction of medicinal plants

1. Maceration. This is an extraction

procedure in which coarsely powdered drug
material, either leaves or stem bark or root
bark, is placed inside a container; the
menstruum is poured on top until
completely covered the drug material. The
container is then closed and kept for at least
three days. The content is stirred
periodically, and if placed inside bottle it
should be shaken time to time to ensure
complete extraction. At the end of
extraction, the micelle is separated from
marc by filtration or decantation. Subsequently, the micelle is then
separated from the menstruum by evaporation in an oven or on top
of water bath. This method is convenient and very suitable for
thermolabile plant material.

2. Infusion. This is an extraction process such

as maceration. The drug material is grinded
into fine powder, and then placed inside a
clean container. The extraction solvent hot or
cold is then poured on top of the drug
material, soaked, and kept for a short period
of time. This method is suitable for extraction
bioactive constituents that are readily soluble.
In addition, it is an appropriate method for
22
 preparation of fresh extract before use. The solvent to sample ratio
is usually 4:1 or 16:1 depending on the intended use.
3. Digestion. This is an extraction
method that involves the use of
moderate heat during
extraction process. The
solvent of extraction
is poured into a clean container
followed by powdered drug
material. The mixture is placed
over water bath or in an oven at
a temperature about 50o C.
Heat was applied
throughout the extraction process to decrease the viscosity of
extraction solvent and enhance the removal of secondary
metabolites. This method is suitable for plant materials that are
readily soluble.

4. Decoction. This is a
process that involves
continuous hot extraction
using specified volume of
water as a solvent. A dried,
grinded, and powdered
plant material is placed
into a clean container.
Water is then poured and stirred. Heat is then applied throughout
the process to hasten the extraction. The process is lasted for a
short duration usually about 15min. The ratio of solvent to crude
drug is usually 4:1 or 16:1. It is used for extraction of water soluble
and heat stable plant material.

5. Percolation. The apparatus used in this

process is called percolator. It is a
narrowcone-shaped glass vessel with
opening at both ends. A dried, grinded,
and finely powdered plant material is
moistened with the solvent of extraction in
a clean container. More quantity of solvent
is added, and the mixture is kept for a
period of 4h. Subsequently, the content is
then transferred into percolator with the
lower end closed and allow to stand for a
period of

23
 24h. The solvent of extraction is then poured from the top until the
drug material is completely saturated. The lower part of the
percolator is then opened, and the liquid allowed to drip slowly.
Some quantity of solvent was added continuously, and the
extraction taken place by gravitational force, pushing the solvent
through the drug material downward. The addition of solvent
stopped when the volume of solvent added reached 75% of the
intended quantity of the entire preparations. The extract is
separated by filtration followed by decantation. The marc is then
expressed, and final amount of solvent added to get required
volume.

6. Soxhlet extraction. This process is

otherwise known as continuous hot
extraction. The apparatus is called
Soxhlet extractor made up of glass. It
consists of a round bottom flask,
extraction chamber, siphon tube, and
condenser at the top. A dried, grinded,
and finely powdered plant material is
placed inside porous bag (thimble) made
up of a clean cloth or strong filter paper
and tightly closed. The extraction solvent
is poured into the bottom flask, followed
by the thimble into the extraction
chamber. The solvent is then heated from
the bottom flask, evaporates, and passes
through the condenser where it
condenses and flow down to the extraction chamber and extracts
the drug by coming in contact. Consequently, when the level of
solvent in the extraction chamber reaches the top of the siphon, the
solvent, and the extracted plant material flow back to the flask. The
entire process continues repeatedly until the drug is completely
extracted, a point when a solvent flowing from extraction chamber
does not leave any residue behind. This method is suitable for plant
material that is partially soluble in the chosen solvent and for plant
materials with insoluble impurities. However, it is not a suitable
method for thermolabile plant materials.

Advantages: Large amount of drug can be extracted with smaller

amount of solvent. It is also applicable to plant
materials that are heat stable. No filtration is required,
and high amount of heat could be applied.

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 Disadvantages: Regular shaking is not possible, and the method is
not suitable for thermolabile materials.

7. Microwave-assisted extraction. This is

one of the advanced extraction
procedures in preparation of medicinal
plants. The technique uses mechanism of
dipole rotation and ionic transfer by
displacement of charged ions present in the
solvent and drug material. This method is
suitable for extraction of flavonoids. It
involves the application of electromagnetic
radiation in frequencies between 300 MHz
and 300 GHz and wavelength between 1cm
and 1 m. The microwaves applied at
frequency of 2450 Hz yielded energy between
600 and 700 W. The technique uses
microwave radiation to bombard an object, which can absorb
electromagnetic energy and convert it into heat. Subsequently, the
heat produced facilitates movement of solvent into the drug matrix.
When polar solvent is used, dipole rotation and migration of ions
occur, increase solvent penetration, and assist extraction process.
However, when nonpolar solvent is used, the microwave radiation
released will produce only small heat; hence, this method does not
favor use of nonpolar solvents.

Advantages: Microwave-assisted extraction has special

advantages such as minimizing solvent and time of
extraction as well as increase in the outcome.

Disadvantages: This method is suitable only for phenolic

compounds and flavonoids. Compounds such as
tannins and anthocyanins may be degraded because
of high temperature involved.

8. Ultrasound-assisted extraction.
This process involves application of
sound energy at a very high frequency
greater than 20 KHz to disrupt plant cell all
and increase the drug surface area for
solvent penetration. Consequently,
secondary metabolites will be released. In
this method, plant material should dry first,
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grinded into fine power, and sieved properly. The prepared sample is
then mixed with and appropriate solvent of extraction and packed
into the ultrasonic extractor. The high sound energy applies

26
 hasten the extraction process by reducing the heat requirements.

Advantages: Ultrasound-assisted extraction is applicable to small

sample; it reduces the time of extraction and amount
of solvent used and maximizes the yield.

Disadvantages: This method is difficult to be reproduced; also, high

amount of energy applied may degrade the
phytochemical by producing free radical.

What’s More

Activity 4: Performing Extraction Method (2nd Performance

Task)
Direction: Demonstrate the proper way of extracting a plant sample.
Record this through video and send your output via email
(annamaejtavas@gmail.com). Do not forget to put the steps
and use improvised equipment or materials if necessary.
 24

What I Have Learned

Activity 5: My Insights
Direction: Answer the questions below briefly but substantially.

1. What is the use of spectrophotometry and titration method in

laboratory experiments?

2. Why is there a need to choose the right method and solvent in

extracting plant samples?

What I Can Do

Activity 6: My Method
Direction: Identify the standard methods and techniques to be used in
performing experiments for your research topic. Support your
chosen methods with related literatures (which explains why
these methods are the most appropriate methods to be used
for your study).

EXAMPLE:

Research Topic:

Methods: Solvent Extraction (Maceration) & Phytochemical Screening

Related Literature:

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 Assessment

Direction: Read each question carefully and write the letter of your
answer on a separate 1 whole sheet of paper.

1. Why is there a need to use a blank solution in measuring the

transmission and absorbance rate of the sample with food coloring?
A. It acts as the source of transmission and absorbance.
B. It improves the reading process of the unknown concentration.
C. It serves as the reference in measuring the transmission and
absorbance rate.
D. It increases the concentration of the solution measured at
different wavelengths.

2. Why is it important to determine the absorbance of a sample

solution? A. To calculate the exact wavelength of light. B. To know
the amount of the blank sample.
C. To identify the transmission of the solution.
D. To determine the amount and concentration of the solution.

3. Why is there a need to add an indicator to the unknown solution in

a titration experiment?
A. To easily determine the endpoint.
B. To achieve faster chemical reaction.
C. To stop chemical reaction between the two solutions.
D. To measure accurately the concentration of the unknown
solution.

4. Leny does not know the concentration of the solution she will use in
the experiment. Her teacher advised her to measure the
absorbance of the solution to determine its concentration. What
device can she use to do it?
A. Autoclave C. Hot air oven
B. Centrifuge machine D. Spectrophotometer

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5. During an experiment, the teacher asked the learners to find out
the concentration of the liquid in the beaker. What method is used
in determining the concentration of an unknown solution?
A. Centrifugation C. Filtration
B. Distillation D. Titration

6. Liza performs a titration experiment with her classmates. What

should they do so that the known solution in the burette will not be
added directly to the unknown solution in the conical flask? A. Put
an indicator to the unknown solution.
B. Slowly pour the known solution to the graduated glass tube.
C. Adjust the precision tap of the burette to slowly add the
solution.
D. Just put the solution in to the burette without adjusting the
precision tap.

7. Aldwin learned from his grandmother that paragis can cure

stomachache. Based on his research, medicinally active portions of
plants can be separated using selective solvents through standard
procedures. What method is this?
A. Plant Extraction C. Plant Homogenization
B. Plant Testing D. Phytochemical Analysis

8. Which of the following principles BEST describes

spectrophotometry?
A. Transmission properties of a material is qualitatively
measured.
B. As concentration increases, absorption decreases
exponentially.
C. Each compound absorbs or transmits light over a certain
range of wavelength.
D. Light absorbed by the sample is equal to the concentration of
sample in the solution.

9. Which serves as indicator in a titration experiment?

A. change in color B. smell of the solution

C. volume of the known solution

D. volume of the unknown solution

10. How is the pure extract of plant samples obtained to make sure
that the bioactive components are retained? A. Use decoction
process.
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 B. Pound the sample using a mortar and pestle.
C. Grind the plant sample and boil it for 30 minutes.
D. Soak the plant sample for 10 minutes and squeeze it.

Key Answer

References
Printed Materials:

D. A., et al. (2014). Introduction to Research Education (9th ed.). (L.

Ganster, Ed.) United States of America.

Caintic, H. E., Cruz, J. M., & Baguio, S. S. (2008). Scientific Research

Manual. Quezon City: C & E Publishing Inc.

Websites:

Juncker, M. (2019, September 6). How to Do Spectrophotometric Analysis.

WikiHow. https://www.wikihow.com/Do-Spectrophotometric-
Analysis#/Image:Do-Spectrophotometric-Analysis-Step-4-Version-
3.jpg

Ravanfar, R. (2018, June 8). Extraction and fractionation of anthocyanins

from red cabbage: ultrasonic-assisted extraction and conventional
percolation method. Journal of Food Measurement and
Characterization. https://link.springer.com/article/10.1007/s11694-
018-9844-y?error=cookies_not_supported&code=514c1e52-75f8-
4568-ad60-323d0cf4b402

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Hussain, M. K. (2019). Techniques for Extraction, Isolation, and
Standardization of Bio-activ. SpringerLink.
https://link.springer.com/chapter/10.1007/978-981-13-72056_8?
error=cookies_not_supported&code=76259c2e-fbea-43a2-
8cf9c5045498df0d

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