Republic of the PhilippinesEastern Visayas State University

Tacloban City

THE SOLVENT EXTRACTION OF ROSA ROSA (RED ROSE) FLOWER PETALS AND ITS APPLICATION

AS PH INDICATOR___________________________

A Research Proposal

In Partial Fulfillment

of the Requirements for the Course

(ChE 361)

Chemical Engineering Methods of Research II

___________________________

Submitted by:Sarahster A. Alejandre

Almira O. RipaldaBSChE- 4A

Submitted to:Engr. Nida B. Lacaba, MA

Professor

CHAPTER IINTRODUCTION

BACKGROUND OF THE STUDY

pH is a measure of the acidity or alkalinity of an aqueous

solution. pH measurements are important in various fields such as

medicine, biology, chemistry, food science, environmental

science, oceanography, civil engineering and many other

applications.

In our field, pH indicators are frequently employed in

titrations in analytical chemistry and biology to determine the

extent of a chemical reaction. Because of this, different

indicators are used to measure the pH of solutions. Commonly used

pH indicators are the various commercial indicators that are made

synthetically such as phenolphthalein, methyl red and methyl

orange. These commercial pH indicators are usually of high cost.

Thus, leads to unavailability or limited quantity in the

laboratories. Regarding the, the proponents endeavored ways to

measure the pH of a solution other than using commercial pH

indicators. In a literature they have read, it exemplified

different flowers, fruits and vegetables effectively used as

natural pH indicators because they contain pigments called rose

flower s.

Indicators are dyes or pigments that are isolated from a

variety of sources, including plants, fungi, and algae. Almost

any flower that is red, blue, or purple in color contains a class

2

of organic pigments called rose flower s that change color with

pH. The use of natural dyes as acid–base indicators was first

reported in 1664 by Sir Robert Boyle in his collection of essays

Experimental History of Colours. Indeed, Boyle made an important

contribution to the early theory of acids and bases by using

indicators for the classification of these substances. The idea,

however, may actually have originated much earlier—medieval

painters used natural dyes treated with vinegar and limewater to

make different color watercolor paints.

Rose flower pigments are responsible for the red, purple

and blue colors of many fruits, vegetables, cereal grains and

flowers. They are members of a class of water soluble,

terrestrial plant pigments that are classified as phenolic

compounds collectively named flavonoids. The difference in

chemical structure that occurs in response to changes in pH is

the reason that Rose flower s can be potentially used as pH

indicator, as they change from red in acids to blue in bases. The

unique property of these pigments has been exploited as pH

indicator application of Rose flower s in the study.

Rose is any of the genus Rosa within the family Rosacea.

They form a group of flowering plants that can be erect shrubs,

climbing or trailing with stems that are often armed with sharp

prickles. The Philippines offers different horticulture products3

for both domestic and external trade and Roses are one of these

products usually used for aesthetic purposes. Different species

of Roses are widely cultivated in our country, even in simple

household gardens. The most common variety is the red Rose. Its

vibrant red coloration is caused by the abundance of rose flower

pigments in its petals. Thus, Roses is chosen as the suitable raw

material of the study.

STATEMENT OF THE PROBLEM

The research study aimed to form a natural pH Indicator

sourced from Rosa rosa (red Rose) flower petals extract.

Specifically, the study seeks to answer the following questions:

1. What is the pH of the acidified methanol rose flower extract

and water rose flower extract?

2. What color change does the rose flower extracts exhibit during

pH indication application?

3. Is there any significant difference between the results of the

application of extracted pH indicators with phenolphthalein pH

indicator in determining the equivalence point in different

types of titrimetric analysis?

NULL HYPOTHESIS

1. The efficacy measured in terms of precision in

obtaining sharp color change in titration by using such4

rose flower extract as pH indicators do not give

quantitative results compared with phenolphthalein

synthetic indicator.

2. There is no effective substitute to the frequently used

pH indicators in measuring the precise pH of the solution

other than the phenolphthalein synthetic indicators.

SIGNIFICANCE OF THE STUDY

It is hoped that through a successful result of the

research study, the will give benefit to the following:

1. To the students. They will be able to use the rose flower

extract as an effective and safe substitute to

those expensive commercial indicators in

measuring the pH of a solution.

2. To the institution. They can introduce the rose flower

extract in their science laboratories as an

effective substitute to the frequently used

commercial indicators such as phenolpthalein,

provided that these rose flower extract sourced

from rose petals extract is less expensive and

safe to use.

3. To the community. The study will give great value to the

common flower Rose (rosa). Thus, the study will

give the needed interest to the people of the

community to become motivated in cultivating5

Roses.

4. To the researchers. The success of the study would bring a

sense of fulfillment and accomplishment to the

researchers.

DEFINITION OF TERMS

These are some of the terminologies used that will

help us provide a better understanding of the study:

Acids (often represented by the general formula HA) is

generally a chemical compound that when dissolved in

water will produce a solution with a pH less than 7.

In the modern definition, an acid is a substance that

can give a proton (H + ions) to other substances

(called bases), or may receive a free electron pair of

the bases.

Rose flower s water-soluble vacuolar pigments that may appear

red, purple, or blue depending on the pH. They belong

to a parent class of molecules called flavonoids

synthesized via the phenylpropanoid pathway; they are

odorless and nearly flavorless, contributing to taste

as a moderately astringent sensation.

Bases are chemical compounds that release hydroxide ions

(OH -) when dissolved in water. Base is the opposite

of acid, which is intended to elements / compounds

6

that have a pH more than 7.

Extract a product (as an essence or concentrate prepared by

extracting) especially a solution(as in alcohol) of

essential constituents of a complex material (as an

aromatic plant).

Extraction a separation process consisting in the separation of

a substance from a matrix. .

Flavonoid any of a group of oxygen-containing aromatic

antioxidant compounds that includes many common

pigments (as the rose flower s and flavones).

pH indicator is a halo-chromic chemical compound that is added in

small amounts to a solution so that the pH (acidity or

basicity) of the solution can be determined

visually.It is a chemical detector for hydroniumions (

H3O+) or hydrogen ions (H+) and

changes color depending on whether they donate or

accept proton.

pH a measure of acidity and alkalinity of a solution that

is a pH value of a neutral solution is 7.0. Solutions

with a pH value below 7.0 are considered acidic,

whereas solutions with pH value above 7.0 are basic

(alkaline)..

Purificationis defined as to remove impurities and undesired

7

substances from the extract.

Rose a woody perennial of the genus Rosa, within the

family Rosacea.

Titrimetric analysis refer to quantitative chemical analysis

carried out by determining the volume of solution of

accurately known concentration which is required to

react quantitatively with a measured volume of a

solution of the substance to be determined.

8

SCOPE AND DELIMITATION OF THE STUDY

The study focused on the extraction of rose flower petals

and its application as pH indicator. The extracted rose flower

from Rosa rosa (red rose) petals will be utilized as pH indicator

and its precision will be tested in determining the equivalence

point in different types of titrations(titrimetric analysis)

compared to that of conventional synthetic pH indicators. The

comparison of the extracted pH indicator with the conventional

synthetic indicators will be limited to those available in the

area.

Furthermore, the duration of the research will be limited

to the allotted time of experimentation which is 1st Semester,

School Year 2014-2015. The study will also be limited to the

equipment and facilities available at Eastern Visayas State

University-Main Campus Tacloban City laboratories, and other

authorities and institutions found in the province of Leyte if it

will be necessary.

9

CONCEPTUAL FRAMEWORK

10

Gathering of Rosa rosa (red rose) petalsAcidified Methanolic Extraction of Rose petalsPhenolphthalein Synthetic Indicator TitrationComparison of rose flower extract to phenolphthalein indicator in Titrimetric AnalysisTesting of pH indication applicationDeliberate and Analyze DataForm Conclusion and recommendations

CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

The chapter presents the various literature and studies reviewed

by the researchers for the purpose of gaining more insights relevant

to the present study. Hence, the literature and studies are presented

and discussed showing the similarities and differences in some aspect

of the present study.

RELATED LITERATURE

pH.

According to the New Lexicon Webster’s dictionary of the

English language (Encyclopaedic Edition), pH is a symbol

signifying the negative logarithm (base 10) of the hydrogen ion

concentration of a solution or pure liquid expressed in gram per

liter. The range of value of pH extends from 0 to 14, 7 being the

negative logarithm of the hydrogen ion concentration in pure

water of 25 degree Celsius or neutrality. Smaller numbers signify

increasing acidity while greater one is increasing alkalinity.

To determine the pH (acidity and alkalinity) of the solution

a halochromic chemical com

pound in small amount is being added into a solution. Hence, the

chemical detector is called pH

11

indicator. (Steven S. Zumdahl (2009). Chemical Principles 6th Ed

pages 319–324)

According to acid-base theory Bronsted-Lowry, an acid is a

species that gives (donors)

protons, while bases are acting as the recipient (acceptor)

protons in a proton transfer reaction.

(Analytical Chemistry 8th Ed)

12

pH Indicator.

pH indicators are weak acids that exist as liquid- dyes and

dye infused- paper strips. The liquid form of pH indicators is

usually added directly to a solution, while the paper form is

dipped into a solution and then removed for comparison against a

colour or pH key. (General Chemistry: principles of modern

applications. 9th Ed. New Jersey: Pearson Education, Inc, 2007)

According to Steven S. Zumdahl (2009), pH indicator has an

effective pH range, where the change in colour is apparent and

encompasses the pH of the equivalence point of the solution being

titrated. (Chemical Principles (6th ed.). pp. 319–324)

Meanwhile, according to Geiger et.al many naturally

occurring pH indicators can be obtained from plants or plants

part that contain chemicals from the naturally-coloured rose

flower family of compounds. (Department of Agriculture, June

2005.)

Rose flowers.

Rose flower s (aare water-soluble vacuolar pigments that may

appear red, purple, or blue depending on the Ph. They belong to a

parent class of molecules called flavonoids synthesized via

the phenyl propanoid pathway; they are odorless and nearly

flavorless, contributing to taste as a moderately

astringent sensation. Rose flower s occur in all  tissues of13

higher plants, including leaves, stems, roots, flowers,

and fruits. (Andersen et.al 2001)

Rose flower can be obtained from the rose petals through

the process of extraction. According to Xuejun Pan et.al,

extraction is a separation process consisting in the separation

of a substance from a matrix. It may refer to Liquid-liquid

extraction, and Solid phase extraction. (Chemical Engineering and

Processing, Volume 42, Issue 2, February 2003, pp 129-133)

On the other hand, most commonly used indicators are the

various commercial indicators that are made synthetically. The

indicator such as phenolphthalein, methyl red and methyl orange

are usually in high cost. Thus, lead to its unavailability or

limited quantity in the laboratories.

Therefore, it is indeed the researchers endeavor to develop an

alternative pH indicator from extract rose flower that is not

expensive, yet capable in measuring the pH of the solution.

Roses. Rose (member of Rosa) a family of rosaceae is

believed to have an rose flower compound. They form a group of

plants that can be erect shrubs, climbing or trailing with stems

that are often armed with sharp prickles. Flowers vary in size

and shape and are usually large and showy, in colours ranging

from white through yellows and reds. (Britannica.com. 2007)

14

RELATED STUDIES

Comparison of Natural and Synthetic Indicator.

The works of Sarita Kadam et al, deals about a comparative

study of natural and synthetic indicators. The study highlights

the exploit of the acidified methanolic extract of the flowers of

few medicinal plants as an acid-base indicator in acid-base

titrations. The natural indicator is easy to extract as well as

easily available. Promising results were obtained when it was

compared against standard synthetic indicators. Titration shows

sharp colour change at the equivalence point. The equivalence

points obtained by the flowers extract coincide with the

equivalence points obtained by standard indicators. These natural

indicators are found to be a very useful, economical, simple and

accurate for the said titration. (Der Pharma Chemica, 2013, 5

(1):296-299).

Another study related to the extraction of naturally

occurring pH indicator is conducted by Josh Hurwitz at Riverview

High School Sarasota Fl.USA which deals about extraction of

rose flower s in the two Hydrangea Macrophylla (grown in two

closed soil conditions).

Hydrangea macrophylla commonly known as ‘big leaf’ hydrangea

is known for its two distinct colours. The same plant can exhibit

brilliant pink leaves and deep blue sepals. The plant can switch

15

colours, depending on soil conditions. The mechanism for the

change, and also the original pigmentation is credited to rose

flower . The Extracts were obtained by heating the sepals in

ethanol, and measurements were taken ultimately, it was found

that the red colour in hydrangea macrophylla is the result of

protonated rose flower . The concentration of the acidic pigments

was nearly twice the concentration of the blue extract.

(Extended-Essay%20%281%29.pdf)

Continued study is conducted by Anne Marie Helmenstine (n.d)

which pointed out that red cabbage contains a pigment molecule

called flavin (an rose flower ). The water-soluble pigment is

also found in apple skin, plums, poppies, cornflowers, and

grapes. Very acidic solutions will turn rose flower a red

colour. Neutral solutions result in a purplish colour. Basic

solutions appear in greenish-yellow. Therefore, she stated that

it is possible to determine the pH of a solution based on the

colour it turns the rose flower pigments in red cabbage juice

(http://web.a.ebscohost.com/)

Rose flower Extraction.

Another study conducted by Sushma Bondre et.al (2012) which

deals about the extraction and purification of Rose flower

pigments from Black Raspberries (Rubus occidentalis) as natural

pH indicator. Homogeneous and fine powder was obtained from the

liquid nitrogen blending process. After extraction and

purification of the plant material, a coloured solution was 16

obtained. Depending on the nature of the predominant rose flower

and its concentration, the coloration of the solution is pink/red

or purple. The solution obtained after extraction was dark

brownish red coloured. Two different methods were used for

extraction viz. extraction with

acetone/chloroform partitioning and extraction with methanol. In

the acetone/chloroform procedure, the upper phase shows

colouration due to extraction of pigment in acetone while

chloroform containing layer is only slightly coloured. The

extraction was followed by partial purification procedures using

Column chromatography and TLC. The column chromatography process

was optimized for methanol extracts. The fractions collected from

column analysis were subjected to thin layer chromatography. TLC

analysis revealed the presence of Rose flower s in all fractions

in varying concentrations but only fraction-4 was shown to have

the R f value close to that of pure Rose flower s. (International

Journal of Advanced Biotechnology and Research.

ISSN 0976-2612, Online ISSN 2278-599X, Vol.3, Issue3, 2012, pp

698-702. (http://www.bipublication.com)

In the study conducted by Vankar et.al made used rose

flower extracted from rose flowers as natural indicators for

acid-base titration. They used a convenient method of extraction

of rose flower from Rose flowers (Rosa rosa) using methanolic

solution of 3% citric acid which gave better yield of rose flower17

s than the methanolic solution of 0.1% hydrochloric acid. It has

been shown that by changing the pH of the extract from 2-9, the

hue colour changes from dark pink to mehdi green. The extract

showed wavelength maxima change from 517 nm (dark pink) to 592 nm

(green) between the Ph ranges 2 to 9. The intrinsic pH of the

extract was 2.88. Significant changes were observed when stannous

salt was added to the rose rose flower extract. The

spectroscopic data of the extracted rose flower pigments

indicate a shift in wavelength maxima

of the colours by addition of stannous salt and the complex with

a change in pH of the aqueous medium.

(chemistry.about.com/od/acidsbase1/a/red-cabbage-ph-

indicator.htm)

The studies mentioned above are somewhat similar to the

present study for it also dealt with the extraction of rose

flower compound from a natural source to form a pH

indicator. However, they differ in some aspect of the present

study like the method of extraction proce raw materials and the

locale of the study.

18

CHAPTER III

METHODOLOGY

The chapter focuses on the methods applied in forming pH

indicator from rose flower extracts isolated from Rose flower

petals.

Research Design

The study will be conducted with the experimental research

method. Experimental studies are done in carefully controlled and

structured environments and enable the causal relationships of

phenomena to be identified and analyzed. The variables can be

manipulated or controlled to observe the effects on the subjects

studied. One method of rose flower extraction will be employed

viz. methanol extraction. The rose flower extracts obtained will

be further purified using column chromatography. The rose flower

extracts’ color development in varying pH will be observed using

standardized solutions. In testing the pH indication application

of the extracts, the researchers will conduct, set-up 1

(negative control), where the rose flower extracts will be

utilized as pH indicators in different types of titrations and

the mean equivalence point obtained will be compared to that of

set-up 2 (positive control) where the titrations will employ the

use of specific synthetic indicators. The set-up 1 and set-up 2

will be carried out three times.

19

Materials, tools and equipments

The raw material used in the study is the fresh Rose

petals. While the chemicals used are the Methanol, Liquid

Nitrogen, Chloroform, Sodium Hydroxide, Acetic Acid, De-ionized

water, Hydrochloric Acid, Ammonium Hydroxide, Hexane, Alumina,

and Silica Gel. The equipments and apparatus used are the mortar

and pestle, test tubes, beakers, Pasteur pipette, microcentrifuge

tubes, dropper thermometer clamps, Spectrophotometer, pH meter

and Centrifuge machine.

Gathering of Rose flower petals

Fresh rose flowers will be collected from the local gardens

of the province of Leyte, and will be authenticated by the

College of Arts and Sciences, EVSU (Tacloban City Campus).

Preparation of raw material for Rose flower Extraction

The plant material will be frozen with Liquid Nitrogen, powdered

using a mortar and pestle suitable for use under extremely low

temperatures. The use of liquid nitrogen minimizes Rose flower

degradation by lowering the temperature and providing a nitrogen

environment. The fine powder maximizes pigment recoveries due to

its high surface area and favors disruption of cellular

compartments. Later on from the crushed material rose flower

will be extracted by following Methanol extraction method (Neff,

Chory 1998) 20

Rose flower Extraction Procedure

Using scissors remove individual flowers from the rose

plant and remove the petals from the flowers using tweezers.

Measure the weight and place in a 1.5 mL microfuge tube. Place

the tube directly into liquid N2 to freeze plant tissue.

Remove tubes from the liquid N2 and quickly grind the plant

tissue with a pestle. Add 300 micro-L of Methanol 1%HCl and

continue to grind the plant tissue. Allow extraction to occur

overnight in a dark refrigerator.

Add 200 micro-L De-ionized H20 to each tube. Next, go to

the fume hood and add 500 micro-L of chloroform to each tube.

Spin the tubes in a centrifuge set at the highest rpm for 2-5

minutes.

Return to the fume hood and extract the supernatant/aqueous

(top) solution from the tube using a pipette. To do the start by

setting the pipette to 400 micro-L. Slightly tilt the tube to

one side, place the tip of the pipette just below the surface and

remove the supernatant while moving the tip toward the bottom of

the tube. Place the supernatant in a new 1.5 mL microfuge tube.

Bring the volume up to 800 micro-L by adding 400micro-L of a 60%

Methanol 1% HCl : 40% De-ionized H20 solution to each tube.

21

Rose flower Extract Purification

Preparation of Column Chromatography System

Construct a column chromatography system by making a straw

stand and clamp in 96-well tray. Use a plastic column tip (with a

small wad of polyester fiber in the tip) on the end of a straw as

the chromatography column. Rinse with water. Allow to drain to a

small cup or beaker. Transfer neutral aqueous slurry of fine-mesh

resin to the column an try to make the column about 5 cm long.

Add a little water to the column and then push wetted ¼”

circle of filter paper (already punched out for you) onto the top

of the resin. Use slim straw to guide the circle down and to

gently tamp it on to the top of the resin.

Add a little more water and allow water to drain under

gravity to make sure there is good flow through the column and

that the water level stops at the paper circle. Note: Capillary

action will stop the column from draining completely. It is

important that the liquid level not go below the circle or

channeling will occur. Cut above 4cm off the column straw.

Rose flower Extract Purification

Obtain 3 drops of rose flower rose flower extract. Use a

well from the 96-well tray. Use a thin-stem pipet to transfer 1

drop of the extract to the top of the column. Allow it to flow22

into the packing.

Wash out the thin-stem pipet an fill it about ½ full with

0.1 M HCl/methanol solution. Add the solution to the column

straw. Allow the rose flower s to elute under gravity and collect

the colored fraction in a well of a 24-well tray.

Checking of Wavelength Maxima Change and Maximum Absorption

Later on, the extracts obtained will be subjected to column

chromatography and the absorbance and maxima wavelength change

will be checked using a spectrophotometer for fraction exhibiting

pinkish color to check the rose flower content of the obtained

extracts. The absorbance of each tube will be read at 530nm (rose

flower s peak) and 657nm(nonspecific absorption of photosynthetic

pigments).The blank should be 480micro-L Methanol 1%HCl and

320micro-L De-ionized H20 for a total of 800micro-L.

pH indication application

Color development in pH 1-12

The color development of the extracts in varying pH will be

observed. The purified and unpurified rose flower extracts will

be accurately measured as 1 ml and taken in different, clean test

tubes. The pH of solution in each test tube will be adjusted by

1N HCL and 1N NaOH sequentially from 1 to 12 using the acid and

base and pH meter for accurate pH Adjustments. The contents in

each tube will be properly mixed and the color development will

be observed and 23

recorded.

Application of extracted Rose flower as pH indicator in

different types of Titration

Obtaining equivalence point in different types of titration

of the rose flower s in comparison with synthetic indicators will

be tested. The extracts viz. purified and unpurified will be

preserved in tight closed container and stored away from direct

sun light. The experiment will be carried by using the same set

of glassware’s for all types of titrations. As the same aliquots

will be used for both titrations i.e. titrations by using

standard indicators and rose flower extract. The equimolar

titrations will be performed using 10 ml of titrant with three

drops of indicator. A set of three experiments each for all the

types of acid base titrations will be carried out. The mean and

standard deviation for each type of acid base titrations will be

calculated from results obtained. Strong acid-strong base (HCl -

NaOH), strong acid- weak base (HCl - NH4OH), weak acid-strong

base (CH3COOH- NaOH) and weak acid-weak base (CH3COOH- NH4OH)

titrations will be employed. Each titration will be carried out

three times by using 1N strength of acid and alkali and results

were recorded as mean ± SEM.

Statistical Treatment

Analysis of Variance (ANOVA) will be used to determine if

there is a significant difference in acquired equivalence point24

between purified and unpurified methanolic rose flower extracts

(negative control) compared to synthetic indicator (positive

control). The basic principle of ANOVA is to test for differences

among the means of the two set-ups by examining the amount of

variation within each of these samples, relative to the amount of

variation between the samples.

While, the t-test of independence will be also used to

determine if there is a significant difference between the

purified and unpurified extracts’ rose flower content, the main

component of the natural pH indicator.

SUMMARY OF TABLES

Table 1. Intrinsic pH of extracts pH of sampleUnpurified Methanolic

Rose flowerExtract(Sample A)

Sample A1

Sample A2

Sample A3

Purified Methanolic Roseflower Extract (Sample

B)

Sample B1

Sample B2

Sample B3

Table 2. Comparison of Roseflower Content

Wavelength MaximaChange

Maximum Absorption

UnpurifiedMethanolicRose flowerExtract(Sam

ple A)

Sample A1

Sample A2

Sample A3

PurifiedMethanolicRose flower

Sample B1

Sample B2

25

Extract(Sample B)

Sample B3

HCl:

Hydrochloric Acid, CH3COOH: Acetic Acid, NaOH: Sodium Hydroxide,NH4OH: Ammonium Hydroxide, Sample A: Unpurified Rose flowerExtract, Sample B : Purified Rose flower Extract

26

Table 3. Color Development in varying pH Application

UnpurifiedMethanolic Roseflower Extract

PurifiedMethanolicRose flower

ExtractSample A1 Sample B1

Color of ExtractColor Change as a Function of pH

pH value of StandardSolution

Color Development of Solution

pH 1pH 2pH 3pH 4pH 5pH 6pH 7pH 8pH 9pH 10pH 11pH 12

Table4.Equivalencepointin

different

typesof

Titration

Acid(Titrant)Vs. Base(Titrate)

Synthetic

Indicator

used (Positive

control)

Mean

±S.D.*of

EquivalencePoint

Color

Change

Negative

Control

Group

Mean±S.D.*of

EquivalencePoint

ColorChange

pH

StrongAcid-StrongBase

HClvs.NaOH

SampleA1

SampleB1

StrongAcid-WeakBase

HClvs.NH4OH

SampleA1

SampleB1

WeakAcid-StrongBase

CH3COOH vs.NaOH

SampleA1

SampleB1

WeakAcid-WeakBase

CH3COOHvs.NH4OH

SampleA1

SampleB1