BIOCHEMISTRY SBK3013

LAB PROJECT 1: APPLICATION: MAKING pH INDICATOR

MAUREEN SANTIH ANAK AMBANG	D20141067070
SHARIFAH AWANIS BINTI SYED MOHD ASWAD	D20141067053
HEIDI AMELDA ANAK LAGAT	D20141067086

DATE OF EXPERIMENT: 10 MARCH 2017

LECTURER:  DR ROSMILAH BINTI MISNAN

INSTRUCTOR: NUR ATIEKAH BINTI AZAHARI

LAB PROJECT 1: APPLICATION: MAKING PH INDICATOR

Introduction:

A pH indicator is a substance which has one colour when added to an acidic solution and a different colour when added to an alkaline solution. A pH indicator measures whether a substance is acidic or basic, and some indicators can also measure how acidic or basic it is. When the indicator comes in contact with a substance, the indicator's colour corresponds to that substance's approximate pH, which is a scale of zero to fourteen with zero being the most acidic, seven being neutral, and fourteen being the most basic that indicates the acidity or basicity of a substance. In this experiment we make an indicator from red cabbage. The experiment is in two parts. The first part involves some red cabbage. In the second part, we test our own indicator.

Materials:

      i.        0.1 M HCl solution (add 8.3 mL of concentrated HCl solution to enough deionized or distilled water to make 1.0 L of solution)*

     ii.        0.1 M NaOH solution (dissolve 4.0 g NaOH in enough deionized or distilled water to make 1.0 L of solution)*

      iii.        2-propanol (isopropyl alcohol) /acetone /deionized or distilled water

      iv.        Purple cabbage

     v.        Fruits/vegetables/flowers- EACH GROUP HAS TO BRING ANY ONE.

Extracting the indicator

   1. Plant parts that are most pigmented was selected.

For flowers, only a few petals are normally needed. For fruits and vegetables, finely chopped pieces should be used. Use only that portion of the fruit or vegetable that is most pigmented.

   2. 10 mL of solvent and macerate was added.

   3. The plant parts was crushed with mortar and pestle and left for 15 minutes to enhance the extraction.

Suggested solvents: water, 2-propanol, 50-50 water/2-propanol mixture, or acetone. One of these should easily extract the colour. If the extracts are very dilute, the colour can be concentrated by heating the opened bag in a warm water bath or by pouring the extract in a beaker and evaporating on a hot plate.

4. The filtrate from the macerated plant samples is filtered and collected.

Testing the pH range of the indicator

1. 13 test tubes was labelled from 1 to 13.

2. 9.0 mL of distilled or deionized water was placed in all test tubes except #1, #7 and #13.

3. The solutions in the acid range was prepared in the following manner (Refer Table 1)

4. 10.0 mL of 0.1 M HCl was placed in test tube #1. (pH = 1)

b)    1.0 mL of 0.1 M acid was transferred from test tube #1 to test tube #2 and mixed thoroughly. (pH = 2)

c)    1.0 mL of acid solution was transferred from test tube # 2 to test tube #3 and mixed thoroughly. (pH = 3)

d. The making the serial dilutions was continued by transferring 1.0 mL of the most recently diluted acid solution to the next test tube until six acid solutions of pH 1 to 6 have been prepared. Be sure to mix each thoroughly before the transfer.

5. 0.0 mL distilled or deionized water was added to test tube #7. (pH = 7)

6. Solutions of base was prepared in the following manner:

a.    10.0 mL of 0.1 M NaOH was placed in test tube #13. (pH = 13)

b.    1.0 mL of 0.1 M NaOH was transferred from test tube #13 to test tube #12 and mixed thoroughly. (pH = 12)

c.    The making serial dilutions of the base was continued from pH 12 down to pH 8 by transferring 1.0 mL of the most recently diluted basic solution to the next test tube and mixing thoroughly each time.

Test tube	Distilled water	Acid or Base	pH
1	-	10 ml of 0.1 M HCl	1
2	9 ml	1 ml acid from test tube 1	2
3	9 ml	1 ml acid from test tube 2	3
4	9 ml	1 ml acid from test tube 3	4
5	9 ml	1 ml acid from test tube 4	5
6	9 ml	1 ml acid from test tube 5	6
7	10 ml	-	7
8	9 ml	1 ml NaOH from test tube 9	8
9	9 ml	1 ml NaOH from test tube 10	9
10	9 ml	1 ml NaOH from test tube 11	10
11	9 ml	1 ml NaOH from test tube 12	11
12	9 ml	1 ml NaOH from test tube 13	12
13	-	10 ml of 0.1 M NaOH	13

6. The wells of a spot plate was labelled from 1 to 13. Transfer a few drops of each of the solutions prepared in steps 3, 4, and 5 to the corresponding well in the spot plate.

7. A drop or two of the flower/fruit/vegetable extract was added indicator to each well. The pH at which the indicator changes colour was observed and recorded.

Results:

Red gabbage

Red flower

Testing the pH of other liquids

Once the pH ranges of the indicators have been determined, they can be used in acid-base titrations or to test the pH of household chemicals (Each group has to bring several household chemicals to be tested).

Results:

Before

 After

Using red gabbage extract:

Substance	Colour change after 3 drops of extract	pH value
Detergent	Purple	8
Vinegar	Pink	2
Axion (dish wash)	Dark green	13
Hand wash gel	Dark green	13
Shower gel	Orange	14

Using flower extract:

Substance	Colour change after 3 drops of extract	pH value
Detergent	Brown	9
Vinegar	Orange	2
Axion (dish wash)	Dark green	13
Hand wash gel	Red	1
Shower gel	Blue	10

Discussion:

In this experiment, we have made the pH indicator by using the red cabbage given and our own red flower. We made the indicator to indicate the pH value and the colour of our household substances when we drop cabbage extract and flower extract that we have done before.

As a results, when we drop the cabbage extract into detergent, the colour of the detergent change into purple colour. We compare the colour of the detergent with the pH indicator that we have done. The pH indicator indicate that the detergent have the pH value of 8. For the axion, the colour changed into dark green so the pH value showed by the pH indicator is 13 which is basic. As for vinegar, the colour of vinegar changed into pink colour. So, the pH value of vinegar is 2 which means acidic. Hand wash gel gives colour changed into red colour so the pH value showed by the pH indicator is 13 which is it shows acidity. Lastly, shower gel changed into blue colour when we drop the cabbage extract. This show that the pH of shower gel is 14 which means basic substances.

For the second set, when we drop the flower extract into detergent, the colour of the detergent change into brown colour. We compare the colour of the detergent with the pH indicator that we have done. The pH indicator indicate that the detergent have the pH value of 9. For the axion, the colour changed into dark green so the pH value showed by the pH indicator is 13 which is basic. As for vinegar, the colour of vinegar changed into orange colour. So, the pH value of vinegar is 2 which means acidic. Hand wash gel gives colour changed into dark green so the pH value showed by the pH indicator is 1 which is it shows basicity. Lastly, shower gel changed into blue colour when we drop the cabbage extract. This shows that the pH of shower gel is 10 which means very basic.

Conclusion:

The purpose of this experiment is to determine the pH value of the different household substances by using the pH indicator that we done it by using red cabbage extract and flower extract.

Reflection:

Sharifah awanis: from this experiment, I manage to identify the pH value of the household substance that we bought from our house itself. It is an interesting experiment because we manage to identify our surrounding household and we do not use constant substances for each group. So, the result of each group will be different. Moreover, we also learned how to prepare our own pH indicator in this experiment. Although the procedure in making the indicator are quite complicated, but we manage to do it in a group because we cooperate to complete this experiment.

Heidi amelda: From this experiment, I have learnt to be more efficient in conducting the experiment in a group on next time. The instructor also has given a clear instructions for us. I have a great group mates because they gave a great teamwork during conducting the experiment. I am happy to work with them even though we have some problem during the experiment. Overall, everything is fine.

Maureen santih: I like the color changes that happen during testing the pH range of the indicator experiment. But I quite confused during preparation of the solutions because they required transferring of liquid into a lot of test tubes. Fortunately,the instructor guide my team patiently. Overall, I like the experiment.

LABORATORY REPORT 1

SBF 3013 BIOCHEMISTRY

LABORATORY 1

TITLE: ACID BASE (1. Acid Base Titration)

Name	Matric number
Sharifah Awanis bt Syed Mohd Aswad	D20141067053
Maureen Santih ak Ambang	D20141067070
Heidi Amelda ak Lagat	D20141067086

LECTURER: ASSOCIATE PROFESSOR DR ROSMILAH BINTI MISNAN

INSTRUCTOR: NUR ATIEKAH BINTI AZAHARI

Laboratory 1 : Acid base

1.      Acid Base Titration

Introduction:

Titration is the slow addition of one solution of a known concentration (called a titrant) to a known volume of another solution of unknown concentration until the reaction reaches neutralization. In this experiment we used weak acids to react with NaOH.

Weak acid is different from strong acid because it cannot dissociate completely in water. Weak acid such as acetic acid and organic acid can be partially dissociated, while in strong acid, all acid molecules exist as H⁺  and A^-  in a solution. Due to this property, H⁺  concentration in weak acid depends on the coefficient of equilibrium.

The pH can be measured by emerging the tip of pH meter into the solution and read the value in the recorder. The higher the concentration of H⁺ , the lower the pH value.

Objectives:

1.      To observe the property of weak acid with pH changes.

2.      To learn how to use pH meter correctly.

3.      To learn how to prepare buffer system

4.      To experience how to titrate acid-base.

Materials

0.1M acetic acid (pKa=4.76)

0.1M phosphoric acid (pKa= 2.15, 7.20, 12.35)

0.1M NaOH

Calibrated pH meter.

Methods:

Results:

0.1 M acetic acid

Volume of NaOH	pH
1	3.59
2	3.92
3	4.09
4	4.13
5	4.31
6	4.35
7	4.44
8	4.51
9	4.60
10	4.65
11	4.66
12	4.83
13	4.84
14	4.95
15	5.12
16	5.21
17	5.21
18	5.26
19	5.26
20	5.46
21	5.68
22	5.89
23	6.30
24	6.62
25	8.90

0.1 M phosphoric acid

Volume of NaOH	pH
1	2.09
2	2.09
3	2.09
4	2.22
5	2.28
6	2.36
7	2.45
8	2.55
9	2.56
10	2.62
11	2.68
12	3.02
13	3.45
14	4.12
15	6.10
16	6.43
17	6.51
18	6.55
19	6.68
20	6.70
21	6.90
22	7.10
23	7.31
24	7.65
25	7.85
26	8.10
27	8.45
28	9.41
29	11.05
30	11.63
31	11.87
32	12.10
33	12.14
34	12.24
35	12.27
36	12.30
37	12.35
38	12.40
39	12.45
40	12.50

Graph pH vs Volume of NaOH (acetic acid)

Graph pH vs Volume of NaOH (phosphoric acid)

Discussion:

Titration curve for acetic acid and phosphoric acid are shown on the graph papers. When NaOH is added to acetic acid, acetate ion is produced. During titration, the concentration of the acetic will be decreasing. The curve shows a slight linear rising. Now, the solution contains acid and its conjugate base which is acetate ion. This conjugate base acts as a buffer that resists the change in pH upon dilution and addition of both acid and base.

    
From the graph of acetic acid, 1ml of NaOH is the initial point, while at 24.9ml (pH=7.95) is the equivalent point. From 1ml of NaOH to 25ml of NaOH is called as buffer zone. At the equivalent point, all acetic acid is neutralized and only the acetate ion is present in the solution. The equivalent point is the midpoint of the vertical line.

Then, the first point from the titration curve of phosphoric acid is the pure pH of the phosphoric acid. When NaOH is added into the phosphoric acid, H3PO4- is produced. The content in the solution contains acid and its conjugate base which is dihydrogen phosphate. This conjugate base also acts as buffer which resists the change in pH upon dilution and addition of both acid and base.

1ml of NaOH indicates the initial point, at 14.8ml (pH= 5.5) of NaOH indicates the first equivalent point. From point 1ml of NaOH to 15ml of NaOH, it is called as buffer zone.  7.5 ml is the midpoint between 1ml to 15ml which is equal to pKa1 (2.5). At the first equivalent point, all H3PO4-    is being neutralized and only some H2PO4- is present in the solution.

When more NaOH is added, HPO4(2-)  is produced.  is the conjugate base that acts as buffer. At 29.5ml (pH=11.35)  of NaOH is the second equivalent point. From 15ml to 29 ml is called as second buffer zone. The pH of the midpoint between 15ml to 29ml equals to pka2, which is 7.1 at 22 ml. At the equivalent point, only HPO4(2-)     is present in the solution.

When more NaOH is added further, PO4(3-)  will be produced. The solution contains acid and its conjugate base which is phosphate ion. It is acts as buffer. From the graph plotted, we can only see until second equivalent point. But, the graph plotted has reached the third pKa3 (12.35) at 37ml of NaOH.

The graph is different for each acid is because of the difference types of acid that is used. Both of the acid are weak acid. Theoretically, the pKa value for acetic acid is 4.76 which is only one pKa, while phosphoric acid has three pka values that are 2.15, 7.20 and 12.35. The pKa value also indicates the inflection point for the acid. From the graphs plotted, the change in slope for acetic acid graph is small, while for the phosphoric acid graph, the change in slope is larger or steeper. With this comparison, it shows that phosphoric acid is stronger than acetic acid but both acids are weak acids.

Conclusion:

In conclusion, a strength of the acid refers to the ability of the acid to lose a proton. Weak acid is an acid that dissociates incompletely. It also has higher pKa value compared to strong acid. In this experiment, acetic acid is a monoprotic acid because it has only one pKa value which is 4.76. Whereas, phosphoric acid is a polyprotic acid because it has three pKa values which are 2.5, 7.1 and 12.35.

References:

1. Pharmacopoeia, B. (2016). British pharmacopoeia.

2. Berend, K., De Vries, A. P., & Gans, R. O. (2014). Physiological approach to assessment of acid–base disturbances. New England Journal of Medicine, 371(15), 1434-1445.

3. Povar, I., & Spinu, O. (2015). Acid-Base Buffer Properties of Heterogeneous Multicomponent Extraction Systems. Solvent Extraction and Ion Exchange, 33(2), 196-209.

Reflection:

Name: Heidi Amelda

I have learnt how to be patience during conducting this experiment because it requires a lot of time to do the experiment. We faced a little problem during conducting this experiment but we able to handle it. Great teamwork from my group members really help me a lot.

Name: Maureen Santih

In this experiment, we faced some problems such as we do not have enough pH meter that we have to share it with the other group. This make our work become slower as we need to measure the pH of the phosphoric acid and acetic acid every time after adding with 1ml of NaOH Until they reached pH 7(acetic acid) and pH 14(phosphoric acid). We also make some mistakes that we overly titrate more than 1ml of NaOH into the posphoric acid and acetic acid. This may effect the results of the experiment. What I get from this experiment is I get to know that a strength of the acid refers to the ability of the acid to lose a proton. Phosphoric acid is stronger than acetic acid, but both are weak acids. Overall, the experiment is important for me to understand the acid and base.

Name: Sharifah Awanis

This experiment need a lot of patient because i need to measure the pH of the acid repeatedly. The pH value also increased slowly so that it takes a long time to achieve pH 7 for acetic acid and pH 14 for phosphoric acid. with the help of my groupmate, we successfully done this experiment and got the data that we need.