The document discusses using various plant extracts as natural acid-base indicators in titration experiments. It describes collecting extracts from plants like beetroot, hibiscus, and red cabbage to use as indicators and testing their color change properties in strong acid-strong base, acid-water, and other titrations. The results found the natural indicators performed comparably to synthetic indicators like phenolphthalein, with clear color changes at equivalence points. Therefore, the plant extracts show potential as cost-effective, non-toxic alternatives to traditional chemical indicators for acid-base titrations.

2. ANDHRA UNIVERSITY
SEMINAR -2022
M.TECH BIOTECHNOLOGY
• CONTENTS
AIMS AND OBJECTIVES
INTRODUCTION
MATERIALS &METHODS
RESULTS &DISCUSSION
SUMMARY&CONCLUSION
SUBMITTED TO
BEENA MAM
PROFESSOR
Advanced MicroBiology

4. Aimsand Objectives:
AIM :
To Carry out Acid-base Titration by using different plant extracts as natural indicators.
Objectives:
 To substitute synthetic indicators with natural indicators in performing acid -base titrations.
 To evaluate the indicator property of natural pigments.
 Using different plant extract as indicator is more economical and with the same accuracy of result as that given by syntheticindicator.
 To analyse unknown samples.
 To identify eco friendly , non-toxic natural indicators.

5. INTRODUCTION :
•A compound that exhibits its different colors in solutions
of different acidities. Used to determine the point at
which an acid-base reaction is complete.
(phenolphthalein)
•An indicator is a substance which is used to determine
the end point in a titration.
INDICATOR
•The indicators that are obtained from plants are known
as natural indicators. Natural pigments in plants are
highly colored substances and may show color change .
NATURAL
INDICATORS
•The acid–base equilibrium of a weak acid type
of indicator (HI) in water can be represented as. [I]
The acid, HI, and the conjugate base, I−, have different
colors.
Acid-Base
Indicators

6. Titration :
Titration is a common laboratory method of quantitative chemical analysis to determine the concentration of an identified
analyte. A reagent, termed the titrant or titrator, is prepared as a standard solution of known concentration and volume.
At the equivalence point of a titration, an exactly equivalent amount of titrant has been added to the sample. The
experimental point at which the completion of the reaction is marked by some change is called the end point.
Titrations, classified according to the nature of the chemical reaction occurring between the sample and titrant, include:
Acid-base titrations,
Precipitation titrations
Complex-formation titrations,
Oxidation-Reduction (Redox) titrations.
The basic principle of the titration is the following: A solution - also called titrant or standard solution - is added to sample
to be analyzed. The titrant contains a known concentration of a chemical which reacts with the substance to be determined
In acid-base titration (i.e., the titration of an acid with a base, or vice versa), the indicator is a substance that can exist in
two forms, an acid form and a basic form, which differ in colour. A wide choice of acid-base indicators is available, varying not
only in the colours of the two forms but also in their sensitivity toward acid or base.
Types of titration are on based on acid- base titration are :
1] strong acid-strong base [sulphuric acid – potassium hydroxide]
2] strong acid-weak base [sulphuric acid- water]
3] weak-acid-weak base [Acetic acid – water]
4] strong base-weak acid [potassium hydroxide –Acetic acid]

7.  THE EXTRACTS OF THE FOLLOWING PLANTS ARE USED AS NATURAL INDICATORS :
 Beetroot vulgaris
 Tagetus erecta linn
 Hibiscus rosa sinensus
 Brassica oleraecea
 Gloriosa superba
BEETROOT VULGARIS :
• The Beetroots (Beta vulgaris) were procured from Vegetable Market. The peel of beetroot were discarded and the freshly cut
pieces were used for sample preparations.
• Study Design: Preparation of Natural Indicator.
HIBISCUS ROSA-SINESIS :
• Hibiscus rosasinensis was identified from the flora of Botany Department. Hibiscus rosasinensis was collected for the purpose of
study of natural indicator. It is available throughout the year. Fresh petals were collected in the month of January and February
because it is the blooming season of these plants. Natural indicator was isolated from the Hibiscus rosasinensis . Several plants
pigment like Anthocyanin, Flavonoids occurs in petals of these flowers which acts like natural indicator.
BRASSICA OLARECEA :
• The Red Cabbage were procured from Vegetable Market.
• The extract from boiled red cabbage is used as an acid-base indicator.
TAGETES ERECTA LINN :
• Fresh flowers of Tagetes erecta were collected from the located area and the extract was obtained from the flowers of
tagetes erecta .
GLORIOSA SUPERBA : The flowers were collected through online.

8. Materials& Methods
Materials :
Materials used are chemicals like sulphuric acid , formic acid, potassium hydroxide ,Ethanol and distilled water & the different plant extracts
of Beetroot vulgaris, Brassica oleracea , Hibiscus rosasinensus, tagetes erectalinn, & Gloriosa superba.
0.5 & 1 N Of different Acid-bases solution were prepared :
 Strong acid –strong base (H2SO4–KOH)
 Strong acid – weak base (H2SO4 –H2O)
 Weak acid - strong base (CH3COOH-KOH)
 Weak acid –weak base (CH3COOH - H2O)
NORMALITY :
Normality is define as the number of equivalents per liter of solution.
N = grams solute / Volume solvent in liters * eq.wt
N1V1 =N2 V2
Preparation Of Concentration Of Standard Solutions 0.5 N & 1 N are:
Concentrated Sulphuric acid :
To prepare 1000ml of 1N sulphuricacid from conc sulphuric acid 27.2 ml of conc acid is diluted upto 1000 ml of water. To prepare 0.5N of
sulphuric acid will be use of half of its sulphuric acid conc(13.58) and make up to 1000ml of water. Potassium Hydroxide:
Take 56.11g of KOH for 1N, Dissolve it in around 300 ml water. Transfer to a 1L measuring flask quantitatively ,dilute to 1 litre. To prepare
0.5N of KOH will be use of half of its KOH (28.05) and make up to 1000ml of water.
Potassium Hydroxide:
Take 56.11g of KOH for 1N, Dissolve it in around 300 ml water. Transfer to a 1L measuring flask quantitatively ,dilute to 1 litre. To
prepare 0.5N of KOH will be use of half of its KOH (28.05) and make up to 1000ml of water.
FORMIC ACID :
To prepare 1000ml of 1N formic acid from57.47 ml of is diluted upto 1000 ml of water. To prepare 0.5N of formic acid will be use of
half of its formic- acid (28.73) and make up to 1000ml of water

9. METHODOLOGY :
BEETROOTVULGARIS:
Flow chart:
• Fresh beetroot indicator was prepared
using 100gm of beetroot sample with
distilled water and ethanol and was
tested with Acid-Base titrations and
compared with standard indicator like
phenolphthalein & methyl orange .1N
& 0.5N Sulphuric Acid /formic acid
were the preferred acid while 1N
&0.5N potassium Hydroxide (kOH)
was the preferred base.
Testing fresh
beetroot indicator in
Acid-Base titrations:
• Beetroot powder was prepared by sun
drying and it was used as indicator
after adding hot distilled water and
was tested in Acid-Base titrations. This
powdered sample was preserved in air
tight container .
Testing powdered
beetroot indicator in
Acid-Base titrations:
300 g of chopped beetroot was added to 50 ml of boiling water in a
beaker and was boiled continuously for 45 min
After allowing the beaker to cool for 15 min ,boiled beetroot were
squashed and the liquid was filtered.
The residues were squeezed once again and the liquid was
evapourated to get a highly concentration portion of the indicator

10. Hibiscus rosa sinensus :
HRS
•Flowers were cleaned by distilled water and petals of these flowers were kept in strong sunlight until they get completely
withered.
HRS
•The dried petals were grinded into fine powder with a mechanical blender.
•Dried powder of petals were soaked in 40 ml water for 48 hours and then triturated in mortar and pestle and the resulting.
solution was filtered through muslin cloth.
HRS
•resulted aqueous extract was used as natural indicator in acid-bases.
• The extract was preserved in tight closed container and stored away from direct sunligt .
•Following the similar process ethanolic extract of dried pulvarized petals was prepared, filtered and used as natural ethanolic
indicator.
• The experimental work was carried out by using the same set of glass wares for all types of titration.
•As the same aliquots were used for both titrations the standard indicator, flower extract and the reagents were not calibrated.

11. • Tagetus Erecta linn :
TEL
• The fresh flowers were cleaned and cut into small pieces
TEL
• 100gm of these pieces were macerated with 150 ml of solution containing 9 parts of ethanol and 1 part
of dilute hydrochloric acid for 3 hrs.
• The extract was filtered through muslin cloth & preserved in tightly closed container and stored away
from the direct sun light.
TEL
• The reagents were not calibrated: as same aliquots were used for both titrations i.e. titration by using
standard indicator and fruit extract.
• The ethanolic extract of fresh flowers of Tagetes erectawas screened for its use as indicator for Acid-
Base titration and the results of this screening were compared with the results obtained by using
standard indicators.

12. Brassica olerecea:
BO
• To prepare the red cabbage indicator, blend ¼ head of red cabbage in 300 ml of tap water.
• Strain the resulting mixture.
BO
• Strain the resulting mixture.
BO
• This mixture can be used directly or stored for future use (for long term storage add 1% rubbing alcohol
and refrigerating the solution).

14. Results and discussion :
The following different plant extracts as Natural Acid Base indicators are obtained resultant and discussion as shown
below :
ACID-BASE TITRATION WITH DIFFERENT PLANT EXTRACTS AS INDICATORS :
Synthetic
&
Natural indicators
Acid –Base Titration with different plant extracts as indicators by using 0.5&1 Normality
0.5 N 1N
SA&SB SA & WB WA &
SB
WA &
WB
SA & SB SA & WB WA & SB WA &
WB
phenophthalene 11 12.3 12 8.9 9.3 11.5 10 11.0
Methyl orange 23 1.6 28.4 4.6 13.0 1.4 18.2 2.9
Beet Root vulgaris 29 2.3 26.2 46.2 16.5 1.8 14.6 27.2
Tagetus erecta linn 15 15.3 13.2 38.4 9.8 9.6 10.0 22.0
Hibiscus rosasinesus 20 2.7 14.2 1.2 12.0 1.9 11.0 0.6
Brassica olarecea 11 12.3 12.7 9.8 9.3 11.5 9.8 11.2
Gloriosa superba 8.9 26.4 5.80 26.32 7.18 27.9 5.34 24.0

15. COLOUR CHANGE OF DIFFERENT PLANT EXTRACTS (INDICATOR) IN ACID –BASE TITRATION (START POINT TO END POINT) :
Synthetic indicators
Phenophthalene methyl orange
SA & SB SA&WB WA & SB WA&WB SA & SB SA&WB WA & SB WA&WB

16. NATURAL INDICATORS
Beet root vulgaris Tagetus erecta Hibiscus rosasinensus Brassicaoleraecea Gloriosasuperba
SA &SB SA&SB SA&SB SA&SB SA&SB
SA &WB SA &WB SA &WB SA &WB SA &WB
WA &SB WA &SB WA &SB WA &SB WA &SB
WA & WB WA & WB WA & WB WA & WB WA & WB

17.  Titration results obtained using different plant extracts are shown in four sets of acid –bases like strong acid / strong
base,(sulphuric acid –sodium hydroxide) strong acid / weak base,( sulphuric acid –water /Ammonium hydroxide), weak acid / strong
base,( Acetic acid – sodium hydroxide) weak acid / weak base (Acetic acid –water / Ammonium hydroxide). It was carried out using
two different normality of acids & alkalis viz .0.5 & 1N.
 All types titration shows equivalence point obtained by different plant extracts which are very closely to synthetic indicators.
 The results obtained showed that the routinely used indicator can be replaced successfully by different plant extracts.
 since, the PH of indicator of different plant of aqueous & Ethanolic extracts shows almost neutral .
 All natural components are ecofriendly moreover , colour changes which is observed on addition of plant extracts of aqueous &
ethanolic showed that the colour was maintained for a long time .
 Thus, these indicators are stable. However aqueous extract of plant will be stored at four degree centigrade to avoid microbial
contamination .where as ethanolic extracts can be stored at room temperature.

19. SUMMARY& CONCLUSION:
SUMMARY
• The present work involves the use of Natural Indicators
by using different plant extracts.
• In this work, collection of different plant extracts plays
a major role, i.e.., Beetroot Vulgaris, Tagetes erecta lin,
Hibiscus rosa sinensis , Brassica oleracea , Gloriosa
Superba, Normality.
• Titrimetry analysis studies have also been performed
to study the nature of natural indicators and
comparision was done with synthetic indicators .
• The results obtained by Natural Indicators showed
marked color changes similar to that of standard
indicators phenolphthalein , methyl orange in Acid-Base
titration.
• The different aqueous & Ethanolic plant extract were
collected and used as natural indicators in different
acid base titration like strong acid –strong base,
strong acid-weak base, weak acid – strong base, weak
acid-weak base For detecting the end point. And the
results were compared with the end point of the
synthetic indicators.
• Thus Natural indicator employed in the acid base
titrations was found to be economic, safe, and an
efficient alternative of traditional indicators. In
comparison to this, where as the chemical indicator are
expensive and Hazardous, which proves that different
extracts from natural origin are very advantageous as
Indicators.
CONCLUSION
• The results obtained from current study reveals that the
analytical potential of different plant extracts is very
promoising as seen in acid base titration .It was found
these extracts perfom best in acid base titrations with
sharp and clear color change.The contrast between
there colors in acid and base made the pigments
suitable for acid base indicators.
• The availability and simple extraction procedure with
excellent indicator activity & accurate results would
make this natural indicators suitable subtitutes for
synthetic indicators.