An investigation of change in pH and antimicrobial effect by changing mass of Soapwort

(Saponaria officinalis) extract during the production of natural homemade soap by cold process

1. Research Topic
An investigation of change in pH and antimicrobial effect by changing mass of Soapwort (Saponaria
officinalis) Extract during the production of natural homemade soap by cold process.

2. Introduction
I've been facing allergic skin and respiratory problems since I was little. So, the use of cleaning materials
such as soap and shampoo has always been difficult for me. After learning the saponification reaction in
the 10th organic chemistry unit in IB chemistry lesson, our teacher taught us how to make our soap. I
researched to make a suitable soap for my skin during this experiment. According to Wiselkomolmat,
some plants contain saponin active ingredient which has cleaning properties. One of these plants,
Soapwort (Saponaria officinalis), attracted my attention because it is a local plant that grows abundantly
in my country. I also learned in the Wisetkomolmat’s article that the Soapwort plant was used for the
treatment of skin problems. In this assessment, I decided to investigate the change in the pH value of the
homemade soap that I will prepare based on the change in the amount of Soapwort. Besides, homemade
soap prepared using Soapwort extract needed to have disinfectant and antimicrobial properties. Although
its antibacterial effect appears to be a part of biology, I have only included biochemistry in the experience
because in generally, biochemistry is a sub-branch of chemistry.

3. Research Questions
• To what extent the change in mass of Soapwort (Saponaria officinalis) extract is effective to change
the pH of natural homemade soap produced by cold process?
• How does the change in mass of Soapwort (Saponaria officinalis) extract affect the antimicrobial
activity of natural homemade soap produced by cold process?

4. Hypothesis
In some parts of the plants, saponin can be found as an active ingredient. In research by Dorota, the roots
of Soapwort (Saponaria officinalis) contain high amounts of saponin which can be determined as a foam
producer natural detergent. Consequently, due to the increase in the amount of saponin in Soapwort to be
used in soap making, the cleaning property, and pH value will increase too. However, this increase in pH
value will show itself as exponential. According to Veda, the plants that contain saponin have high
antimicrobial activity. Based on this article, the antimicrobial activity of natural homemade soap will
increase with the increase in the amount of saponin in Soapwort (Saponaria officinalis).

5. Connection between Research Question and Scientific Problem

The base used in the structure of soaps causes’ skin irritation over time. The additives used in soaps to
increase the antimicrobial effect are a threat to aquatic life and human health. My goal in this experiment
was to prepare an ecological product that is environmentally sensitive and will not harm human health. In
my research, I tested the effectiveness of soapwort in soap making. If I can observe that the Soapwort
plant enhances the sudsing efficacy of the soap and imparts antimicrobial properties to the soap, I will
have gained and made an environmental product.

6. Background Information
• Physical and Chemical Properties of Soapwort (Saponaria officinalis)
Saponaria officinalis is a long-lasting plant belongs to Caryophyllaceae family, generally called as
soapwort (Priya et al. 1). It is a 50-60 cm high, very branched, pile rooted, small flowering herbaceous
perennial plant and blooms in June and July. Outer surface of plant roots has white or yellowish color and

1
 it has fragile structure. Roots are removed from the soil in May-August, washed and dried to use it (Battal
et al. 76). The main elements in the composition of the Soapwort are sugars, resins and saponins of the
triterpene class, called "albosaponin".
• Economic Usage of Soapwort (Saponaria officinalis)
Some species are fairly important in economy. Saponin-rich and economically important usage of these
plants is named as “Çöven” by public. These plants extract is used to make detergent as fabrication, fire
extinguisher, liquor, soap and to obtain commercial Saponin (Özçelik et al. 57). The different parts of S.
officinalis plant have been used in pharmaceutical industry for various diseases like roots as blood
purifier, diuretic; roots and leaves for skin diseases; and plant for respiratory disorders since ancient times
(Khare et al. 582) Especially roots are rinsed with water to form permanent foam (Sezik et al. 43).

• Saponification Reaction
Saponification is the chemical reaction in which
triglycerides are combined with a strong base to form fatty
acid metal salts during the soap-making process. Vegetable
oils and animal fats are fatty esters of triglycerides. The
alkali breaks the ester bond and releases the fatty acid salt
and glycerol (Sirajo et al. 41479). Sodium hydroxide is
used in formation of hard soap while potassium hydroxide
is used in formation of soft soap. Figure 1. Chemical Structure of Saponin
(Oscar Bunez Nunoz, 2017)

Figure 2. Saponification Reaction (Deb, 2017)

7. Materials

Table 1. Apparatus and Chemicals

Soapwort extract (Saponaria officinalis)
Burette (50.0 ± 0.1 𝑚𝐿) Ring stand
(±𝟎. 𝟎𝟏𝐠)
NaOH (Sodium hydroxide; 0.1 M) Volumetric Flask (500.0 ± 0.5 𝑚𝐿) Funnel
HCl (Hydrochloric acid; 11.36 M stock solution) Beaker (100.0 ± 0.5 𝑚𝐿) Ruler
H2O (Distilled water; 600.0±0.1 mL) Beaker (500.0 ± 0.5 𝑚𝐿) Bunsen burner
Phenolphthalein (5 drops) Electronic Balance (±0.01 𝑔) Glass Rod
Measuring Cylinder
Vegetable oil (100.00±0.01 g) Water Bath
(100.0 ± 0.5 𝑚𝐿)
TI-84 Plus CE Calculator Erlenmayer Flask (250.0 ± 0.5 𝑚𝐿 ) Labels
Logger Pro Software Separatory Funnel (250 mL) Wire brush
Hand Blender (Philips) Thermometer Gloves
Filter paper: MN 640 mm/the diameter 125 mm Incubator Lab coat
Glass container to boil Soapwort roots Blood agar 5% Safety goggles
1.000 mL Pipette
Silicon cup as a molder for soap Tripod stand
(+0.001 mL)
Cotton bud
2
 Figure 3. Action plan for experiment

8. Variables
❖ 1:0 ratio consists of 33.00 g water and 0.00 g soapwort extract. 1:1 ratio 16.50/16.50, 1:2 ratio
11.00/22.00, 1:3 ratio 8.25/24.75, 1:4 ratio 6.60/26.40 and 0:1 ratio 0.00/33.00 water (g) and
soapwort (g) extracts it is formed.
Table 2. Variables
How the variable will be
Variables Impact to the study
controlled and changed
The water/Soapwort extract ratios are
Independent The water/Soapwort
(1:0, 1:1, 1:2, 1:3, 1:4, 0:1) differentiated to observe the pH
variable extract mass ratio
change.
As the water/Soapwort extract mass
Dependent
Change in pH Measured with titration ratio of soaps changes, the pH will
variable
change too.
25.0 0C at measured by In order to avoid any problems while
thermometer preparing the soaps.
Temperature The optimum temperature for the
40.0 0C measured by
saponification reaction in cold
thermometer
process.
100.0 mL water measured In order to observe the pH change
Amount of water
with a graduated cylinder with different amount of extracts.
Mstock = 11.36 moldm-3 In order to observe the amounts of
Concentration of HCl
(0.440 ml stock solution volume to be compare with other
(0.01 M)
will be diluted to 500 ml) soaps.
Controlled
To ensure that the soaps were of
variable Size of the silicon cup standard silicon cup
the same size.
12.90 ± 0.01 g measured
In order to obtain soaps with the
Mass of NaOH (12.90 g) with digital weighing scale
same pH value
and added to soaps.
In order to make the observations
Amount of 5 drops with dropper, works more robust in terms of color,
Phenolphthalein between 7.2-10.5 pH range titration was performed with
phenolphthalein.
Kept constant to observe the effect
Pressure Kept at 1 atm
of the independent variable.
3
 9. Risk Assessments
Table 3. Risk Assessments
• HCl and NaOH is a strong acid and strong base, they both have corrosive property. To
prevent the harmful effects of NaOH and HCl; glove, eye goggles and lab coat were used.
• NaOH and HCl have high concentration and hazardous vapor for respiratory system.
Therefore, the experiment should be done in a fume hood.
Safety Issues
• Measures were taken to wash the acid or base with plenty of water in case of skin or eye
contact. The remaining works were completed near the eye wash stations.
• While preparing solution of HCl from stock solution, acid is added on water to prevent
the splashing and for minimizing the unwanted reaction, the burette is washed
• The software used in the experiment was licensed to be an ethical study.
Ethical Issues
• During the experiment, the sources were shown correctly in the bibliography.
Environmental • The HCl and NaOH solutions were once again diluted to avoid damaging the aquatic life
Issues when spilled after the experiment.

10. Method
Preparation of 0.1 mol dm-3 500 mL of NaOH Solution:
𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑖𝑛𝑔 𝑀𝑊 𝑜𝑓 NaOH = 23.01 + 16.00 + 1.01 = 40.01 𝑔𝑚𝑜𝑙 −1
𝑴 = 𝒎𝒐𝒍𝒂𝒓𝒊𝒕𝒚 (𝒎𝒐𝒍 𝒅𝒎−𝟑 ) 𝒏 = 𝒎𝒐𝒍𝒆 (𝒎𝒐𝒍) 𝑽 = 𝒗𝒐𝒍𝒖𝒎𝒆 (𝒅𝒎𝟑 )
𝒎 = 𝒎𝒂𝒔𝒔 (𝒈) 𝑴𝑾 = 𝒎𝒐𝒍𝒂𝒓 𝒎𝒂𝒔𝒔 (𝒈𝒎𝒐𝒍−𝟏 )
𝒏 𝑛
𝑴=𝒗 0.1 = 0.5 𝑛 = 0.05 𝑚𝑜𝑙
𝒎 𝑚
𝒏 = 𝑴𝑾 0.05 = 40.01 𝑚 = 2.00 𝑔 𝑁𝑎𝑂𝐻
1. Calculate the mass of NaOH needed to prepare 0.1 M 500 mL NaOH solution.
2. Use electronic balance to measure out 2.00 g of NaOH and put 2.00 g NaOH into a 100 mL
beaker.
3. Measure 10 mL distilled water by using 50 mL measuring cylinder and pour it into the beaker.
4. Mix with glass rod until all of the NaOH dissolves completely.
5. Pour the solution into the 500 mL volumetric flask by using funnel.
6. Fill the rest of the volumetric flask with distilled water up to 500 ml by paying attention to
meniscus level.

Preparation of HCl solution from stock solution (0.01 mol dm-3500 mL)
𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑖𝑛𝑔 𝑀𝑊 𝑜𝑓 𝐻𝐶𝑙 = 1.01 + 35.05 = 36.06 𝑔𝑚𝑜𝑙 −1
%𝟑𝟓 stock HCl 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝐻𝐶𝑙 = 1.17 𝑔𝑐𝑚−3

𝑴 = 𝒎𝒐𝒍𝒂𝒓𝒊𝒕𝒚 (𝒎𝒐𝒍 𝒅𝒎−𝟑 ) 𝒅 = 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 (𝒈 𝒄𝒎−𝟑 ) 𝒎𝒂𝒔𝒔%: 𝒑𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 𝒃𝒚 𝒎𝒂𝒔𝒔

𝟏𝟎 ∗ 𝒅 ∗ 𝒎𝒂𝒔𝒔 % 1O ∗ 1.17 ∗ 35
𝑴𝒔𝒕𝒐𝒄𝒌 = 𝑴𝒔𝒕𝒐𝒄𝒌 = = 𝟏𝟏. 𝟑𝟔 𝑴
𝑴𝑾 36.06
𝑴𝒔𝒕𝒐𝒄𝒌 × 𝑽𝒔𝒕𝒐𝒄𝒌 = 𝑴𝒇𝒊𝒏𝒂𝒍 × 𝑽𝒇𝒊𝒏𝒂𝒍
11.36 × (𝑉𝑠𝑡𝑜𝑐𝑘 ± 0.001) = 0.01 × (500.0 ± 0.1) 𝑉𝑠𝑡𝑜𝑐𝑘 = 0.440 ± 0.001 𝑚𝐿 𝑠𝑡𝑜𝑐𝑘 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

1. Measure 100 mL distilled water with 100 mL measuring cylinder and pour into the 250 mL
beaker.
2. Measure 0.440 mL from stock HCl solution with the 0.001 mL pipette.
3. Pour into a 250 mL beaker which contains 100 mL of water and stir with glass rod.
4. Pour the solution into 500.0 mL Volumetric Flask. Fill the rest of the flask (until 500 mL) with
water to dilute the HCl solution.
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Preparation of Soapwort extract
➢ The extract obtained from this method will be used in soap.
1. Purchase 100.00 g of soapwort roots from a local herbalist by weighing with electronic balance.
2. Brush the roots with wire brush for removing its dust and wash with tap water.
3. Place the roots and add 500 mL distilled water in a glass container.
4. Place the container on tripod stand and heat it with the help of a Bunsen burner until half of the
water evaporates. Filter the extract solution and pour in a 500 mL beaker to store.

Figure 4. The photograph of Soapwort roots and foam of saponin during the boiling

Preparation of a Natural Homemade Soap in Cold-Process

➢ In this method, production of soap with only 1:0 water/Soapwort extract ratio will be described.
➢ Step 2 will be varied for soaps with 1:1 (16.50 g water + 16.50 g Soapwort extract), 1:2 (11.00 g
water + 22.00 g Soapwort extract), 1:3 (8.25 g water +24.75 g Soapwort extract), 1:4 (6.60 g
water + 26.40 g Soapwort extract) and 0:1 (0.00 g water + 33.00 g Soapwort extract).
Volumetric Flask Beaker
1:0 (33.00 g water + 0.00 g Soapwort extract) Soapwort extract /
Water mass ratio
1. Weigh 12.90 g NaOH with the electronic balance into a 250
mL beaker. Silicon
2. Measure 33.00 mL distilled water with 50 mL measuring Cup
cylinder.
3. Add water into the beaker and dissolve the NaOH by stirring
with glass rod.
4. Measure the temperature of the solution with the thermometer
until it decreases the 40 0C.
5. Measure 100.00 g of vegetable oil with 100 mL measuring Figure 5.The photograph
cylinder. taken during the experiment
6. Add oil onto the NaOH-water solution. Measuring Cylinder
7. Mix the suspension with hand blender for 2 minutes until it becomes homogeneous.
8. Pour the mixture into the silicon molds.
9. Leave the soap for 1 week until it becomes solid.
10. Repeat the method from step 1 to 9 for 2 more trials.

Titration Method
1. Control the stop cock of a 50.0 mL burette and make sure that it’s close.
2. Fill the burette with 0.01 M HCl solution with the help of the funnel.
3. Weigh 5.0 grams of soaps from 0:1, 1:1, 1:2, 1:3, 1:4, 1:0 soap samples with electronic balance.
4. Dissolve each soap in 100.0 mL distilled water in different six 250 mL Erlenmeyer.
5. Add 5 drops of phenolphthalein into the 100 mL of each soap solutions in the Erlenmeyer.
6. Make a titration with the soap solutions until the color change from pink to colorless.
7. Record the volume of HCl solution that used to neutralize the soap solutions.
8. Repeat the method step 1 to 7 for 2 more trials.
5
 Preparation of Soapwort soaps for antimicrobial analysis
➢ In this method, preparation of soap with only 1:0 for
antimicrobial analysis will be described.
➢ Step 1 will be varied for soaps with 1:1, 1:2, 1:3, 1:4 and
0:1.
1. Weigh 5.0 grams of soap from 1:0 (33.00 g water + 0.00 g
Soapwort extract) with electronic balance.
2. Dissolve the soap in 100.0 mL distilled water in 250 mL
Erlenmeyer.
3. Divide a tile (40x40cm) on floor in a classroom into 18
equal pieces with using a ruler and a pen.
4. Take a sweep sample from the tile with the help of a cotton
buds. Figure 6. The photograph of
5. Inoculate the sample on 5% blood agars. blood agars in the incubator
6. Measure 1.0 mL with the micropipette and dribble the soap
solution into the agar.
7. Place the blood agar into an incubator at 37 0C for 24 hours. Repeat the steps from 1 to 8 for 2 more
trials.

11. Analysis

11.1 Raw Data

Table 4. Burette Reading of Volume of HCl Solution Against Duration of Titration

Mass ratio of water/Soapwort ( ±0.01g) Trials Vinitial of HCl (±0.1 ml) Vfinal of HCl (±0.1 ml)
1 0.0 1.4
1:0
2 2.0 3.2
(33.00 g water + 0.00 g Soapwort extract)
3 4.0 5.3
1 0.0 1.8
1:1
2 2.0 3.7
(16.50 g water + 16.50 g Soapwort extract)
3 4.0 5.8
1 0.0 3.2
1:2
2 4.0 7.3
(11.00 g water + 22.00 g Soapwort extract)
3 8.0 11.5
1 0.0 6.3
1:3
2 7.0 13.0
(8.25 g water + 24.75 g Soapwort extract)
3 13.0 19.5
1 0.0 9.5
1:4
2 10.0 20.0
(6.60 g water + 26.40 g Soapwort extract)
3 20.0 31.0
1 0.0 12.6
0:1
2 13.0 26.0
(0.00 g water + 33.00 g Soapwort extract)
3 26.0 38.7

Raw Data show that the amount of HCl consumed increases as the amount of soapwort in the soap
increases.

6
Table 5. Number of Bacteria Colonies in Blood Agars versus Mass of Water/Soapwort Extract Ratio
Mass of water/Soapwort extract ratio ( ±0.01g) Trials Number of the bacteria colonies
1 319
1:0
2 327
(33.00 g water + 0.00 g Soapwort extract)
3 329
1 250
1:1
2 247
(16.50 g water + 16.50 g Soapwort extract)
3 253
1 147
1:2
2 154
(11.00 g water + 22.00 g Soapwort extract)
3 149
1 76
1:3
2 68
(8.25 g water + 24.75 g Soapwort extract)
3 81
1 59
1:4
2 51
(6.60 g water + 26.40 g Soapwort extract)
3 64
1 30
0:1
2 34
(0.00 g water + 33.00 g Soapwort extract)
3 41

11.3 Processed Data

❖ Example Calculations for 1:0 ratio: (33.00 g water +0.00 mL Soapwort extract)
𝑽 𝒖𝒔𝒆𝒅 𝑯𝑪𝒍(𝒎𝑳) = 𝑽 𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝑯𝑪𝒍(𝒎𝑳) − 𝑽 𝒇𝒊𝒏𝒂𝒍 𝑯𝑪𝒍(𝒎𝑳)
𝑉 𝑢𝑠𝑒𝑑 𝐻𝐶𝑙(𝑚𝑙) = 1.4 − 0.0 = 1.4 𝑚𝐿 𝑓𝑜𝑟 𝑓𝑖𝑟𝑠𝑡 𝑡𝑟𝑖𝑎𝑙
This calculation is repeated for each trial and for each sample.
𝐕 𝐭𝐫𝐢𝐚𝐥 𝟏+𝐕 𝐭𝐫𝐢𝐚𝐥𝟐+𝐕𝐭𝐫𝐢𝐚𝐥 𝟑
𝑪𝒂𝒍𝒄𝒖𝒍𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑯𝑪𝒍 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 𝒖𝒔𝒆𝒅: 𝟑
1.4 + 1.2 + 1.3
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝐻𝐶𝑙 𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑢𝑠𝑒𝑑: = 1.3 mL
3
𝑨𝒃𝒔𝒐𝒍𝒖𝒕𝒆 𝑼𝒏𝒄𝒆𝒓𝒕𝒂𝒊𝒏𝒕𝒚: 𝑽𝒎𝒂𝒙 𝒖𝒔𝒆𝒅 𝑯𝑪𝒍 − 𝑽𝒎𝒊𝒏 𝒖𝒔𝒆𝒅 𝑯𝑪𝒍
±1.4 − ±1.2
𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 = = ±0.1
2
𝑨𝒃𝒔𝒐𝒍𝒖𝒕𝒆 𝑼𝒏𝒄𝒆𝒓𝒕𝒂𝒊𝒏𝒊𝒕𝒚
𝑷𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 𝑼𝒏𝒄𝒆𝒓𝒕𝒂𝒊𝒏𝒕𝒚: ( ) × 𝟏𝟎𝟎
𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑯𝑪𝒍 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 𝒖𝒔𝒆𝒅
0.1
𝑥100 = ±7.7%
1.3
Same calculations are done for other samples.

7
 Table 6. Volume of HCl Solution Used against Duration of Titration
Absolute Percentage
Mass of water/Soapwort extract ratio VHCl used
Trials Vaverage HCl Uncertainty of Uncertainty of
( ±0.01g) (± 0.2 ml)
Volume of HCl Volume of HCl
1 1.4
1:0
2 1.2 1.3 ±0.1 ±7.7%
(33.00 g water + 0.00 g Soapwort)
3 1.3
1 1.8
1:1
2 1.7 1.8 ±0.1 ±5.6%
(16.50 g water + 16.50 g Soapwort)
3 1.8
1 3.2
1:2
2 3.3 3.3 ±0.2 ±6.1%
(11.00 g water + 22.00 g Soapwort)
3 3.5
1 6.3
1:3
2 6.0 6.3 ±0.3 ±4.8%
(8.25 g water + 24.75 g Soapwort)
3 6.5
1 9.5
1:4
2 10.0 10.2 ±0.8 ±7.8%
(6.60 g water + 26.40 g Soapwort)
3 11.0
1 12.6
0:1
2 13.0 12.8 ±0.2 ±1.6%
(0.00 g water + 33.00 g Soapwort)
3 12.7
❖ Example Calculations for 1:0 ratio: (33.00 g water +0.00 mL Soapwort extract)
❖ 100.0± 0.5 mL soap solution is neutralized with 1.3± 0.1 mL 1.0x10-2 mol dm-3 HCl solution.
❖ Hydrochloric acid is a monoprotic strong acid and 100% dissociates in water. NaOH is a strong
base and 100% dissociates in water. Therefore, during the calculations 𝒌 𝒂𝒄𝒊𝒅 𝒂𝒏𝒅 𝒌 𝒃𝒂𝒔𝒆 will
be accepted as constant by 1.
HCl (aq) Cl- (aq) + H+(aq)
𝟎.𝟓
❖ 𝑽𝒔𝒐𝒂𝒑 = 𝟏𝟎𝟎. 𝟎 ± 𝟎. 𝟓 𝒎𝑳 △ 𝑽𝒔𝒐𝒂𝒑 = 𝟏𝟎𝟎.𝟎
𝒙𝟏𝟎𝟎 = 𝟎. 𝟓%
𝟎.𝟏
❖ 𝑽𝒂𝒄𝒊𝒅 = 𝟏. 𝟑 ± 𝟎. 𝟏 𝒎𝑳 △ 𝑽𝒂𝒄𝒊𝒅 = 𝟏.𝟑
𝒙𝟏𝟎𝟎 = 𝟕. 𝟕%

𝑵𝒆𝒖𝒕𝒓𝒂𝒍𝒊𝒛𝒂𝒕𝒊𝒐𝒏; 𝒌 𝒂𝒄𝒊𝒅 × 𝑴 𝒂𝒄𝒊𝒅 × 𝑽𝒂𝒄𝒊𝒅 = 𝒌 𝒃𝒂𝒔𝒆 × 𝑴𝒃𝒂𝒔𝒆 × 𝑽 𝒃𝒂𝒔𝒆

1 𝑥 0.01 𝑚𝑜𝑙 𝑑𝑚−3 𝑥 (1.3𝑥10−3 ±7.7%) 𝑑𝑚3 = 1 𝑥 𝑀𝑏𝑎𝑠𝑒 𝑚𝑜𝑙 𝑑𝑚−3 𝑥 (100.0 𝑥10−3 ± 0.5%) 𝑑𝑚3
𝑀𝑏𝑎𝑠𝑒 = 1.3 𝑥 10−4 𝑚𝑜𝑙 𝑑𝑚−3 % 𝑢𝑛𝑐 = 0.5% + 7.7% = 8.2%
1.3 𝑥 10−4
𝑴𝒃𝒂𝒔𝒆 = 𝑥8.2 = 1.1 𝑥 10−5 upper limit 𝑀𝑏𝑎𝑠𝑒 = 1.3 𝑥 10−4 + 1.1 𝑥 10−5 = 1.4 𝑥 10−4 𝑚𝑜𝑙 𝑑𝑚−3
100

lower limit 𝑀𝑏𝑎𝑠𝑒 = 1.3 𝑥 10−4 − 1.1 𝑥 10−5 = 1.2 𝑥 10−4 𝑚𝑜𝑙 𝑑𝑚−3
𝒑𝑶𝑯 = − 𝐥𝐨𝐠[𝑶𝑯− ] = −𝑙𝑜𝑔 1.4 𝑥 10−4 = 3.85 min 𝑣𝑎𝑙𝑢𝑒 𝑓𝑜𝑟 𝑝𝑂𝐻
𝒑𝑶𝑯 = − 𝐥𝐨𝐠[𝑶𝑯− ] = −𝑙𝑜𝑔 1.2 𝑥 10−4 = 3.92 max 𝑣𝑎𝑙𝑢𝑒 𝑓𝑜𝑟 𝑝𝑂𝐻
max 𝑝𝑂𝐻 + min 𝑝𝑂𝐻 3.85 + 3.92
𝑝𝑂𝐻 = = = 3.9
2 2
𝒑𝑯 = 𝟏𝟒. 𝟎 − 𝒑𝑶𝑯 𝑝𝐻 = 14.0 − 3.9 = 10.1
8.2
❖ 𝑎𝑏𝑠. 𝑢𝑛𝑐. 𝑜𝑓 𝑝𝐻 = 100 𝑥 10.1 = 0.8 Final pH is 10.1 ± 0.8
❖ Calculations for other data were made accordingly.

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 Table 7. pH level against mass of water/soapwort extract ratio
M NaOH Percent Absolute
Mass of water/Soapwort extract ratio −4
VHCl 𝑥 10 pOH pH (%) Unc. Unc. of
( ±0.01g) -3
(mol dm ) of pH pH
1:0
1.3 1.3 3.9 10.1 8.2 0.8
(33.00 g water + 0.00 g Soapwort extract)
1:1
1.8 1.8 3.7 10.3 5.6 0.6
(16.50 g water + 16.50 g Soapwort extract)
1:2
3.3 3.3 3.5 10.5 6.6 0.7
(11.00 g water + 22.00 g Soapwort extract)
1:3
6.3 6.3 3.2 10.8 5.3 0.6
(8.25 g water + 24.75 g Soapwort extract)
1:4
10.2 10.2 3.0 11.0 8.3 0.9
(6.60 g water + 26.40 g Soapwort extract)
0:1
12.8 12.8 2.9 11.1 2.1 0.2
(0.00 g water + 33.00 g Soapwort extract)

In Table 7, it is observed that the pH value increases due to the increasing mass of soapwort
extract.

Figure 7. pH of Soapwort Soaps versus mass of soapwort extract used

In Figure 7, it is observed that pH value increases due to increasing soapwort extract mass.

𝐍 𝐭𝐫𝐢𝐚𝐥 𝟏+𝐍 𝐭𝐫𝐢𝐚𝐥𝟐+𝐍 𝐭𝐫𝐢𝐚𝐥 𝟑

𝑪𝒂𝒍𝒄𝒖𝒍𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝑵𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒃𝒂𝒄𝒕𝒆𝒓𝒊𝒂 𝒄𝒐𝒍𝒐𝒏𝒊𝒆𝒔: 𝟑
319 + 327 + 329
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝐻𝐶𝑙 𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑢𝑠𝑒𝑑:
3
= 325 number of the bacteria reproduced in blood agar for 1: 0 soap sample

• This calculation is repeated for each sample.

9
 Table 8. pH of Soapwort Soaps versus Average Number of Colonies
pH of Soapwort
Mass of water/Soapwort extract ratio (±0.01g) Average number of the colonies
Soap
1:0 (33.00 g water + 0.00 g Soapwort extract) 10.1 325
1:1 (16.50 g water + 16.50 g Soapwort extract) 10.3 250
1:2 (11.00 g water + 22.00 g Soapwort extract) 10.5 150
1:3 (8.25 g water + 24.75 g Soapwort extract) 10.8 75
1:4 (6.60 g water + 26.40 g Soapwort extract) 11.0 58
0:1 (0.00 g water + 33.00 g Soapwort extract) 11.1 35

Figure 8. The Number of the Bacteria Colonies versus pH of the Soapwort Soaps

11.4 Uncertainty Calculations

In this study, uncertainty values from the instruments used were calculated and random error value was
reached. Three trials made in this experiment to reduce the random errors.
• 2.00 g NaOH (± 0.01 g) • 500.0 mL NaOH in Volumetric Flask (± 0.5 mL)
• 100.00 g Vegetable oil (± 0.01 g) • 0.440 mL HCl in Pipette (± 0.001 mL)
0.01
Electronic Balance (NaOH): 2.00 ± 0.01g Percentage Uncertainties: 2.00 𝑥 100 = 0.5%
0.01
Electronic Balance (Vegetable oil): 100.00 ± 0.01g Percentage Uncertainties: 100.00 𝑥 100 = 0.01%
0.5
Volumetric Flask: 500 ± 0.5 mL Percentage Uncertainties: 500 𝑥 100 = 0.1%
0.001
Pipette: 0.440 ± 0.001 mL Percentage Uncertainties: 0.440 𝑥 100 = 0.2%
Total Uncertainty=0.5% + 0.01% + 0.1% + 0.2% = 0.81% = 𝟎. 𝟖 % from the equipment.

12. Conclusion
According to Table 7. and Figure 7., there is a correlation between the mass of soapwort extract with the
pH of the soaps. In other words, the higher the pH values, the higher the volume of used volume to
dissolve the solution. The pH was measured as 10.1 when 1.3 mL HCl was used at a 1:0 water/soapwort
10
extract ratio. Secondly, the pH was measured as 10.3 when 1.8 mL HCl was used in a 1:1 ratio. 3.3 mL
HCl with a ratio of 1:2 ratios was used and pH 10.5 was measured. 6.3 mL of soap was used in 1:3 ratios
and the pH was measured as 10.8. The HCl used in the 1:4 water/soapwort extract ratio was 10.2 mL,
while the pH was 11.0. Finally, 12.8 mL HCl was used in soap with a ratio of 0:1, while the pH was
measured as 11.1. Although the pH values increased 0.3 regularly until the soap having a ratio of 1:4, it
increased by 0.1 when it came to soap having a ratio of 0:1 ratio. So, as the amount of soapwort extract
used in the produced soaps increased, the volume of HCl used in the titration also increased. In this
context, the pH of the soaps increased. The decrease in the slope of the increase in pH values after 1:3
ratio reflects that the amount of soapwort used within the soap is approaching the optimum value. I have
realized that increasing amount of soapwort extract at the ratio values after 26.40 g soapwort extract will
not have much effect on pH. When Figure 7 and Table 7 are examined, it is seen that there is no
significant difference between 1:4 soap and 0:1 soap pH value. When the amount of soapwort extract
used in soap in a ratio of 1:4 was increased to 33.00 g, the pH change was not significant after this point
even though the pH value increased from 11.0 to 11.1. As a result, it was observed that the effect of
soapwort extract on pH reached the maximum effect value. Therefore, the pH value of 0:1 in Figure 7 is
not shown on the graph. From this point it was seen that the optimum value was reached.
According to Table 8 and Figure 8, bacterial colonies decreased as pH values increased. Soaps with a pH
of 10.1 (1:0 ratio) has average of 325 bacterial colonies. Secondly, an average of 250 bacterial colonies
were observed in soap with a pH of 10.3 (1:1 ratio) and an average of 150 bacterial colonies in soap with
a pH of 10.5 (1:2). However, 58 bacteria were found in soap with a pH of 11.0 (1:4 ratios) and 35 bacteria
were found in soap with a pH of 11.1 (0:1 ratio). There is no significant difference between the
antibacterial effects of soaps prepared at a ratio of 1: 4 (33.00 g extract) and 0: 1 as in the same pH
change (26.40 g extract). On average, 58 bacterial colonies were found on agar added in 1:4 soap
solutions and on average agar added in 0:1 soap solution. As a result, it can be stated that 1:4 soap
prepared by using 26.40 g soapwort extract achieves optimum antibacterial effect.
13. Evaluation
Majority of natural homemade soaps have a pH range of 9.01 to 10.00 (Tarun et al. 442). However, due to
the alkaline property contained in the soapwort itself, the pH is very likely to fall out of this range.
Therefore, in this research the literature value of pH is accepted as 10.0. Also, the theoretical value is
compared with the pH of the soap having a ratio of 0:1 which is 11.1, because the soap with this ratio is
made only from soapwort;
|experimental value−theoretical value| |11.1−10.0|
%𝑒𝑟𝑟𝑜𝑟 = 𝑥 100 %𝑒𝑟𝑟𝑜𝑟 = 𝑥 100 = 11.0%
𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑣𝑎𝑙𝑢𝑒 10.0
The systematic error value will be found by subtracting the error value created by the test instruments
(random error) from the total error value;
𝑠𝑦𝑠𝑡𝑒𝑚𝑎𝑡𝑖𝑐 𝑒𝑟𝑟𝑜𝑟 = 11.0 − 0.8 = 10.2 % = ~ 10 %
Although the systematic error obtained as a result of the experiment was high, the value in the percentage
error showed that the research question was a reliable question. Referring to Figures 7 and 8, it can be
said that the alkalinity property increases when the soapwort/water ratio of soaps increases and thus the
pH value increases. Furthermore, the high correlation value indicated in Figure 7 (0.9980) demonstrates
the accuracy of the processed data because this value indicates a strong correlation between mass of
soapwort extract used in soaps and pH of the soaps, which is the ideal description of logarithmic graph.
My processed data is thus consistent with established scientific theories. Hence the mass of soapwort
extract used in soaps, as an independent variable, was effectively controlled.
While the antibacterial effect analysis was carried out in the experiment, the bacterial clusters forming a
white zone with a diameter of 1 mm were accepted as 1 unit of bacteria. In this assay, unlike pH analysis,
3 trials were performed. Reducing the number of trials also increased the random error in the experiment.
In addition, the proximity of some white bacterial colonies on the agar made counting difficult. In each
blood agar the number of the colonies counted and Figure 8 were drawn with the average of bacterial
colony formed on agar due to the pH change of soap. In addition, the 1:1, 1:2 and 1:4 water/soapwort
ratio data values in the graph are not on the line. Difficulties in colony counting and working with dilute
11
 soap solution caused deviations from the results. As a result, the number of bacterial colonies grown on
agar decreased significantly due to the decrease in pH. In addition, the correlation value of the graph in
Figure 8 is 0.9944, which shows that there is a strong correlation between the increasing amount of
soapwort extract in the prepared soaps and the antibacterial effect of these soaps. The reliability of the
second research question and accuracy of the hypothesis were also proved.
14. Weaknesses and Improvements
➢ The studying with very low concentration of HCl solution during titration increased the systemic error
margin. If a drop counter was used, the result would be more accurate and precise.
➢ Bacterial counting process was very difficult due to the fact that bacterial colonies of 1 mm in diameter
were very close to each other. Therefore, when colony counting on agar, the solution can be diluted 1:10
or 1: 100 to reduce the number of bacteria growing. Then the actual number of bacteria can be reached by
calculating the ratio. In cases where the number of bacteria is 30-300, the area to be counted on the agar is
divided into squares and counting can be done with a backlit colony counter using a magnifying glass.
With the help of this colony counter instrument measurement can be more accurate.
➢ According to Michelle Hack, natural soaps must be kept for 4-8 weeks. Although the drying time of the
soaps was between 4 and 8 weeks, the complete drying of the soaps could not be observed due to the time
limit of the experiment. As a result, the pH value of the soaps was higher than the actual values. It is
thought that the pH value of the soap will decrease and the effect of damaging the skin will decrease if the
waiting time is extended.
15. Modifications and Extensions for Further Experiments
➢ In further experiments, soaps can be produced with hot process and pH of the soaps can be compared.
This may be less irritating for the skin.
➢ Other types of plants with high saponin content (like soapnut) can be investigated and the pH values of
the soaps obtained from these plants and their effects on the skin can be examined.
➢ The sunflower oil I used in this soap had a certain super fatting rate. This super fatting rate determines the
lubricity of soap. By using different types of oil also may change the pH of the soaps.
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