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TABLE OF CONTENTS

TABLE OF CONTENTS .............................................................................................................................. 1


CHAPTER ONE ........................................................................................................................................... 3
SYNTHESIS OF ASPIRIN AND ASSAY OF ASPIRIN TABLETS.......................................................... 3
INTRODUCTION .................................................................................................................................... 3
CHEMICALS AND EQUIPMENT FOR THE SYNTHESIS OF ASPIRIN ........................................... 4
OBJECTIVES ........................................................................................................................................... 5
PROCEDURE FOR THE SYNTHESIS OF ASPIRIN: ........................................................................... 5
RESULTS AND CALCULATIONS ........................................................................................................ 7
DETERMINATION OF PERCENTAGE PURITY OF SYNTHESIZED ASPIRIN ............................ 11
PERCENTAGE CONTENT OF ASPIRIN TABLETS .......................................................................... 13
CALCULATION OF PERCENTAGE CONTENT OF TABLETS ....................................................... 15
DISCUSSION OF RESULTS................................................................................................................. 17
CONCLUSION ....................................................................................................................................... 19
CHAPTER TWO ........................................................................................................................................ 20
DETERMINATION OF THE PERCENTAGE CONTENT OF PARACETAMOL TABLETS USING
UV/VISIBLE SPECTROPHOTOMETRY................................................................................................. 20
INTRODUCTION .................................................................................................................................. 20
Discovery of Paracetamol ................................................................................................................. 20
Preparation of Paracetamol ............................................................................................................. 21
Paracetamol as Poison ...................................................................................................................... 22
PROCEDURE ......................................................................................................................................... 24
PREPARATION OF SOLUTIONS TO DETERMINE THE CALIBRATION CURVE ...................... 25
DISCUSSION ......................................................................................................................................... 31
CONCLUSION ....................................................................................................................................... 32
CHAPTER THREE .................................................................................................................................... 33
LIMIT TEST ON SYNTHESISED ASPIRIN USING THIN LAYER CHROMATOGRAPHY .............. 33
INTRODUCTION .................................................................................................................................. 33
PROCEDURE ......................................................................................................................................... 38

1
RESULTS AND CALCULATIONS ...................................................................................................... 39
CALCULATION OF RF VALUES ........................................................................................................ 39
DISCUSSION ......................................................................................................................................... 40
CONCLUSION ....................................................................................................................................... 42
REFERENCES ....................................................................................................................................... 43
APPENDIX ............................................................................................................................................. 44
APPENDIX B ......................................................................................................................................... 48

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CHAPTER ONE

SYNTHESIS OF ASPIRIN AND ASSAY OF ASPIRIN TABLETS

INTRODUCTION

Aspirin (acetylsalicylic acid) is a synthetic organic compound derived from salicylic acid.

Salicylic acid is a natural product found in the bark of the willow tree and was used by the ancient

Greeks and Native Americans, among others, to counter fever and pain. However, salicylic acid is

bitter and irritates the stomach.

A German chemist named Felix Hoffman is credited with being the first to synthesize

aspirin in 1897. Hoffman's father had severe arthritis but could not tolerate salicylic acid he was

taking for pain relief. The name given for Hoffman's new compound was A-spirin. Apparently

this comes from acetylation (A-), together with Spirin, part of the name for Meadow-sweet

(Spiraea ulmaria), a plant rich in salicylates.

Friedrich Bayer, the employer of Hoffman, patented the name and began marketing the

product in 1899. It was a huge success and sales grew rapidly. Bayer's company set up by himself,

is generally reckoned to have been the first pharmaceutical company, and the production of aspirin

is generally accepted to have laid the foundation of the modern pharmaceutical industry.

In this experiment, aspirin (acetylsalicylic acid, C9H8O4 ) was synthesized, purified, and

the percentage yield determined. The purity of the product was confirmed by measuring its melting

point range.

3
The reaction that is used for the synthesis is shown below. In this reaction, an excess of

acetic anhydride (C4H6O3) was added to a measured mass of salicylic acid (C7H6O3) in the

presence of a catalyst, sulfuric acid (H2SO4). The mixture was heated to form the acetylsalicylic

acid (C9H8O4) and acetic acid (C2H4O2). After the reaction took place, water was added to destroy

the excess acetic anhydride and cause the product to crystallize. The aspirin was then collected,

purified by recrystallization, and its melting temperature measured.

CHEMICALS AND EQUIPMENT FOR THE SYNTHESIS OF ASPIRIN

Chemicals used Aspirin Tablets

Salicylic Acid Equipment Used

Acetic Anhydride Retort Stand

Sodium Hydroxide Burette

Ethanol 20ml and 10ml Pipettes

Hydrochloric Acid White Tile

Sodium Carbonate 500ml Volumetric Flask

Sulphuric Acid 200ml volumetric Flask

Methyl Orange and Methyl Red Indicators Water Bath

Distilled Water Mortar and Pestle

4
Washing Bottle Melting Point Apparatus

20ml Beaker Capillary Tube

Weighing Balance Petri Dish

250ml Conical Flask Spatula

Measuring Cylinder Glass rod

OBJECTIVES:

1. To synthesize aspirin (acetyl salicylic acid) from salicylic acid

2. To determine the percentage purity of the synthesized aspirin

3. To determine the percentage content of aspirin tablets

PROCEDURE FOR THE SYNTHESIS OF ASPIRIN:

1. 5g of dry salicylic acid and 7mL of acetic anhydride were placed in a 200mL Erlenmeyer

flask. Three (3) drops of concentrated sulphuric acid were added. The flask was swirled

gently to ensure thorough mixing.

2. The mixture was warmed in a water bath to about 50-60˚C with stirring for about 15

minutes. The mixture was allowed to cool, ensuring occasional stirring.

3. 75mL of distilled water was added to the mixture, stirred and filtered at the pump.

4. The product was recrystallized by dissolving the solid in about 15mL of hot alcohol and

the resulting solution poured into 40mL of warm water.

5. The clear solution obtained was allowed to cool slowly, enabling the crystals to separate

out as beautiful needles.

6. The crystals were filtered at the vacuum pump, allowed to drain well and dried in an oven

at 50˚C.

5
7. The yield was determined as follows:

Actual mass of salicylic acid weighed = 5.0154g

Mass of crude aspirin synthesized = 4.5081g

Mass of pure aspirin recovered after recrystallization = 2.6303g

𝐦𝐚𝐬𝐬 𝐨𝐟 𝐩𝐮𝐫𝐞 𝐚𝐬𝐩𝐢𝐫𝐢𝐧


% Yield = 𝐦𝐚𝐬𝐬 𝐨𝐟 𝐜𝐫𝐮𝐝𝐞 𝐚𝐬𝐩𝐢𝐫𝐢𝐧 × 100

𝟐.𝟔𝟑𝟎𝟑𝐠
% Yield =𝟒.𝟓𝟎𝟖𝟏𝐠 × 100

= 58.3461%

8. The melting point was determined to be in the range 135℃ - 136℃.

PORCEDURE FOR THE ASSAY OF ASPIRIN TABLETS

1. 0.5M HCl was standardised using analar sodium carbonate (Na2CO3) and the standardized

HCl was also used to standardise 0.5M NaOH.

2. Twenty (20) tablets of aspirin were weighed individually and collectively to determine the

mass of each tablet and the total mass of the twenty tablets in order to determine the weight

distribution. The tablets were then powdered using mortar and pestle.

3. An equivalent of 0.5g aspirin was accurately weighed into a conical flask.

4. 50mL of 0.5M NaOH was added to the flask, which was immersed in boiling water-bath

for about 10 minutes.

5. The solution was cooled and the excess back-titrated with 0.5M HCl phenol red as

indicator.

6. A blank determination was carried out by heating, cooling and titrating a further 30mL of

0.5M NaOH. The difference between the titrations represented the amount of alkali

required by the aspirin. Methyl orange was used as an indicator for the standardization of

the NaOH.

6
RESULTS AND CALCULATIONS

Standardisation of HCl

Reaction equation:

Na2CO3(aq) + 2HCl(aq) → 2NaCl(aq) + H2O(l) + CO2(g)

Pipette volume used = 20mL

Table 1: Results from the Standardisation of HCl with Analar Na2CO3

Burette Reading/mL 1 2 3

Final Reading 24.50 30.60 26.90

Initial Reading 3.80 10.60 7.10

Titre 20.70 20.00 19.80

(𝟐𝟎.𝟎𝟎+𝟏𝟗.𝟖𝟎)𝐦𝐋
Average titre = = 19.90mL
𝟐

Milliequivalence Calculation

106g Na2CO3 in 1000mL ≡ 2M HCl

26.5g Na2CO3 in 1000mL ≡ 0.5M HCl

2.65g Na2CO3 in 100mL ≡ 0.5M HCl

0.0265g Na2CO3 in 1mL ≡ 0.5M HCl

Percentage purity of Na2CO3 = 99%

But mass of Na2CO3 = 2.65g

𝟏𝟎𝟎
This implies the mass of 100% Na2CO3 = × 2.65g
99

= 2.6768g Na2CO3 (nominal weight)

Actual weight of Na2CO3 measured = 2.6800g

7
Determining the factor of HCl

Let F = factor, V = volume

𝐀𝐜𝐭𝐮𝐚𝐥 𝐰𝐞𝐢𝐠𝐡𝐭
F(Na2CO3) = 𝐍𝐨𝐦𝐢𝐧𝐚𝐥 𝐰𝐞𝐢𝐠𝐡𝐭

𝟐.𝟔𝟖𝟎𝟎𝐠
= 𝟐.𝟔𝟕𝟔𝟖𝐠

= 1.0012

F(HCl) × V(HCl) = F(Na2CO3) × V(Na2CO3)

𝐅(𝐍𝐚𝟐𝐂𝐎𝟑)× 𝐕(𝐍𝐚𝟐𝐂𝐎𝟑)
F(HCl) = 𝐕(𝐇𝐂𝐥)

𝟏.𝟎𝟎𝟏𝟐×𝟐𝟎𝐦𝐋
= = 1.0062
𝟏𝟗.𝟗𝟎𝐦𝐋

Standardisation of NaOH with the Standardised HCl

Reaction equation: NaOH(aq)+ HCl(aq) → NaCl(aq) + H2O(l)

Volume of pipette used = 20mL

Table 2: Results from the Standardisation of NaOH

Burette Reading/mL 1 2 3

Final Reading 21.70 21.70 22.00

Initial Reading 5.50 0.30 0.40

Titre 21.60 21.40 21.60

(𝟐𝟏.𝟔𝟎+𝟐𝟏.𝟔𝟎)𝐦𝐋
Average titre = = 21.60mL
𝟐

Milliequivalence Calculation

36.5g HCl in 1000mL ≡ 1 M NaOH

18.25g HCl in 1000mL ≡ 0.5M NaOH

8
9.125g HCl in 500mL ≡ 0.5M NaOH

Percentage purity of HCl = 36%

Mass of HCl = 9.125g

𝟏𝟎𝟎
This implies mass of 100% HCl = × 9.125g
𝟑𝟔

= 25.3472g HCl

𝐦𝐚𝐬𝐬
Density (𝛒) =𝐯𝐨𝐥𝐮𝐦𝐞

𝐦𝐚𝐬𝐬
Volume =𝐝𝐞𝐧𝐬𝐢𝐭𝐲(𝛒)

Mass of HCl = 25.3472g

Density = 1.18g/mL

𝟐𝟓.𝟑𝟒𝟕𝟐𝐠
Volume =𝟏.𝟏𝟖𝐠/𝐦𝐋 = 21.4806mL

Actual volume of HCl measured = 21.50mL

The 21.50mL HCl was diluted with distilled water to the 500mL mark of the 500mL volumetric

flask.

Preparation of NaOH

40g in 1000mL ≡ 1M NaOH

20g in 500mL ≡ 1M NaOH

9
10g in 500mL ≡ 0.5M NaOH

0.02g in 1mL ≡ 0.5M NaOH

% Purity of NaOH = 96%

𝟏𝟎𝟎
Mass of NaOH to be weighed = × 10g = 10.4167g
𝟗𝟔

Actual mass of NaOH weighed = 10.4551g

The 10.4551g NaOH was dissolved in distilled water and topped up to the 500mL mark of the

volumetric flask.

Determining the factor of NaOH

F(HCl) = 1.0062

V(HCl) = 21.60mL

F(NaOH) = ?

V(NaOH) = 20mL

F(NaOH) × V(NaOH) = F(HCl) × V(HCl)

𝐅(𝐇𝐂𝐥)× 𝐕(𝐇𝐂𝐥)
F(NaOH) = 𝐕(𝐍𝐚𝐎𝐇)

(𝟏.𝟎𝟎𝟔𝟐)× (𝟐𝟎𝐦𝐋)
= (𝟐𝟏.𝟔𝟎𝐦𝐋)
= 0.9317

10
Table 3: Results from the Determination of Blank

Burette Reading/mL 1 2 3

Final Reading 33.40 36.60 32.20

Initial Reading 0.40 4.50 0.00

Titre 33.00 32.10 32.20

𝟑𝟐.𝟏𝟎+𝟑𝟐.𝟐𝟎
Average titre = = 32.15mL
𝟐

DETERMINATION OF PERCENTAGE PURITY OF SYNTHESIZED ASPIRIN

Volume of HCl that neutralised 30mL of blank (NaOH without aspirin) = 32.15mL

Nominal weight of synthesised aspirin = 0.50g

Actual weights of synthesised aspirin weighed:

i. 0.5052g

ii. 0.5047g

For 0.5052g of the synthesised aspirin weighed;

Volume of HCl that reacted with the synthesised aspirin = (32.15 – 20.20) mL

= 11.95mL

Equivalent volume of NaOH that reacted with the synthesised aspirin = 11.95mL × 1.0062

= 12.0241mL

180g of acetyl salicylic acid (ASA) in 1000mL ≡ 2M NaOH

45g ASA in 1000mL ≡ 0.5M NaOH

0.045g ASA in 1mL ≡ 0.5M NaOH

If 1 mL NaOH ≡ 0.045g NaOH

11
𝟏𝟐.𝟎𝟐𝟒𝟏𝐦𝐋
Then 12.0074mL NaOH ≡ × 0.045g = 0.5411g
𝟏𝐦𝐋

𝐦𝐚𝐬𝐬 𝐨𝐟 𝐩𝐮𝐫𝐞
% Purity of synthesised aspirin =𝐦𝐚𝐬𝐬 𝐨𝐟 𝐢𝐦𝐩𝐮𝐫𝐞 × 100

𝟎.𝟓𝟒𝟏𝟏
= 𝟎.𝟓𝟎𝟓𝟐 × 100 = 107.11%

For 0.5047g of the synthesised aspirin weighed;

Volume of NaOH that reacted with the ASA = (32.15 – 20.10) mL×1.0062

= 12.05 mL× 1.0062

=12.1247 mL = 12.12 mL

0.045g ASA ≡ 1 mL NaOH

𝟏𝟐. 𝟏𝟐𝟒𝟕𝐦𝐋
∴ 12.1247 mL ≡ × 0.045g
𝟏 𝐦𝐋

= 0.5456g

𝟎.𝟓𝟒𝟓𝟔𝐠
% Purity of the synthesised aspirin = 𝟎.𝟓𝟎𝟓𝟐𝐠 × 100

= 107.99%

𝟏𝟎𝟕.𝟏𝟎+𝟏𝟎𝟕.𝟗𝟗
Average % purity of synthesised aspirin = = 107.55%
𝟐

12
PERCENTAGE CONTENT OF ASPIRIN TABLETS

Table 4: Uniformity of Weight Test

𝐱−𝐭
Weight/g Deviation (x-t) Percent Deviation (| | × 𝟏𝟎𝟎)/%
𝐭

1 0.3879 0.0002 0.00516

2 0.3867 -0.0010 0.2579

3 0.3876 -0.0001 0.0258

4 0.3838 -0.0039 1.0059

5 0.3865 -0.0012 0.3095

6 0.3830 -0.0047 1.2123

7 0.3811 -0.0066 1.7023

8 0.3864 -0.0013 0.3353

9 0.3964 0.0087 2.2440

10 0.3926 0.0049 0.0126

11 0.3889 0.0012 0.3095

12 0.3852 -0.0025 0.6448

13 0.3860 -0.0017 0.4385

14 0.3880 0.0003 0.0774

15 0.3891 0.0014 0.3611

16 0.3786 -0.0091 2.3472

17 0.3856 -0.0021 0.5417

18 0.3882 -0.0005 0.1289

19 0.3855 -0.0022 0.5674

13
20 0.3861 -0.0016 0.4127

Weight of 20 tablets = 7.7544g

7.7544g⁄
Average weight per tablet = 20 = 0.3877g

Weight of Active Pharmaceutical Ingredient (API) per tablet = 300mg = 0.30g

If 0.3877g ≡ 0.3g

𝟎.𝟓𝐠
Then 𝟎.𝟑𝐠 × 0.3877g = 0.6462g

Expected weight of aspirin powder to be weighed = 0.6462g

Actual weights of powdered aspirin tablets measured =

i. 0.6467g

ii. 0.6470g

iii. 0.6464g

Table 5: Results from Titration involving Aspirin Tablets

Burette Reading/mL Determinations

1 2 3

Final Reading 20.60 40.40 21.50

Initial Reading 0.50 20.60 1.20

Titre 20.10 19.80 20.30

14
CALCULATION OF PERCENTAGE CONTENT OF TABLETS

Volume of HCl needed to neutralize 30mL of blank (NaOH without aspirin) = 32.15 mL

Nominal weight of aspirin powder = 0.6462g

For 0.6467g Aspirin Powder Weighed;

0.6462g aspirin powder contains 0.50g ASA

𝟎.𝟔𝟒𝟔𝟕𝐠
Therefore 0.6467g aspirin powder contains 𝟎.𝟔𝟒𝟔𝟐𝐠 × 0.50g = 0.5004g ASA

Volume of HCl that reacted with NaOH = (32.15 – 20.10) mL = 12.05 mL

Equivalent volume of NaOH that reacted with aspirin = 12.05 mL× 1.0062 = 12.1247 mL

1 mL NaOH ≡ 0.045g ASA

𝟏𝟐.𝟏𝟐𝟒𝟕𝐦𝐋
12.1247 NaOH ≡ × 0.045g = 0.5456g
𝟏𝐦𝐋

𝟎.𝟓𝟒𝟓𝟔𝐠
% Content of Aspirin Tablet = 𝟎.𝟓𝟎𝟎𝟒𝐠 × 100g = 109.033%

For 0.6470g Aspirin Powder Weighed;

0.6462g ≡ 0.5g ASA

𝟎.𝟔𝟒𝟕𝟎𝐠
0.6470g ≡ × 0.5g = 0.5006g
𝟎.𝟔𝟒𝟔𝟐

Volume of HCl that reacted with NaOH = (32.15 – 20.30) =11.85 mL

Equivalent volume of NaOH that reacted with ASA = 11.85 mL × 1.0062

15
= 11.9235mL

1 mL NaOH ≡ 0.045g ASA

𝟏𝟏.𝟗𝟐𝟑𝟓𝐦𝐋
11.9235mL ≡ × 0.045g = 0.5366g
𝟏𝐦𝐋

𝟎.𝟓𝟑𝟔𝟔𝐠
% Content of Aspirin tablet = 𝟎.𝟓𝟎𝟎𝟔𝐠 × 100 = 107.19%

For 0.6464g Aspirin Powder Weighed

If 0.6462g ≡ 0.50g

𝟎.𝟔𝟒𝟔𝟒𝐠
Then 0.6464g ≡ 𝟎.𝟔𝟒𝟔𝟐𝐠 × 0.50g = 0.5002g

Volume of HCl that reacted with NaOH = (32.15 – 19.80) mL = 12.35 mL

Equivalent weight of NaOH that reacted with aspirin = 12.35 mL × 1.0062 = 12.4266 mL

1 mL NaOH ≡ 0.045g ASA

𝟏𝟐.𝟒𝟐𝟔𝟔𝐦𝐋
12.4266mL ≡ 𝟏𝐦𝐋
× 0.045g = 0.5592g

𝟎.𝟓𝟓𝟗𝟐𝐠
% Content =𝟎.𝟓𝟎𝟎𝟐𝐠 × 100 = 111.795%

𝟏𝟎𝟗.𝟎𝟑𝟑+𝟏𝟎𝟕.𝟏𝟗𝟎+𝟏𝟏𝟏.𝟕𝟗𝟓
Average percentage content = = 109.34%
𝟑

16
DISCUSSION OF RESULTS

Aspirin, a drug widely known for its analgesic, antipyretic, and anti-inflammatory

properties, is a compound derived from two acids namely acetic acid and salicylic acid.

The experiment consisted of three (3) parts: (1) the preparation of the pertinent solutions,

(2) the standardization of 0.50M NaOH and 0.50M HCl solution, and (3) the analysis of the aspirin

sample and the percentage content of aspirin tablets. The first part of the experiment was the

preparation of Acetylsalicylic Acid (Aspirin). A white, milky mixture was obtained when salicylic

acid, acetic anhydride and sulphuric acid (a catalyst) were mixed. The mechanism of the reaction

is:

This shows that the oxygen in salicylic acid attacks one of the carbons in acetic anhydride. Also,

the mechanism shows how acetic acid was separated from the acetylsalicylic acid.

17
In the first part of the experiment, heating of the mixture was done and a clear yellow liquid

was obtained. Heating was employed so that salicylic acid would melt and react with acetic

anhydride. On the other hand, water was added after heating (not at the start of the experiment).

This was to prevent the reaction of acetic anhydride with water at the start of the experiment, if

this had happened, no aspirin could have formed. In this manner, acetic anhydride was decomposed

after the formation of aspirin.

After cooling to room temperature and adding distilled water, the liquid became whitish

(cloudy) with white precipitates. This addition of water is very important in purification and

isolation of the crystals from the liquid. Since aspirin is insoluble in cold water, only the acetic

anhydride was decomposed by the water. Purification was needed to eliminate any salicylic acid

and acetic anhydride that did not react, as well as the acetic acid product and sulphuric acid. In this

part, purification was not yet complete (it was continued on the recrystallization part). Isolation

was done through vacuum filtration and white, needle-like crystals were obtained.

The crude/impure product was then weighed and it weighed 4.5081g.There was a

percentage recovery (yield) of 58.3461%.

The crude aspirin synthesised was recrystallized. The recrystallization constituted a

continuation of the purification process. The recrystallization was done by dissolving the product

in 15mL of hot alcohol and poured into 40mL of water, which was allowed to cool slowly. The

cooling process enabled the aspirin to crystallise out as white needle-like crystals, leaving behind

the impurities.

To be able to analyse the composition of an aspirin sample or the amount of acetylsalicylic

acid, the hydrolysis of the sample by alkali into neutrality was required. The hydrolysis of the

aspirin often uses large amounts or excess NaOH because such a reaction is slow and sufficient

18
amount of NaOH reacting with acetylsalicylic acid would just yield water as the product. The

whole process is called back-titration. It is a process in which the excess of standard solution used

to consume an analyte is determined by titration with a second standard solution. Back-titrations

are often required when the rate of reaction between the analyte and reagent is slow or when the

standard solution lacks stability.

The melting point range (135˚C - 136˚C) obtained indicated that the synthesised aspirin

was pure since it matched the specification in the British Pharmacopoeia. The percentage purity

calculated was 106.95%, meanwhile the BP range is 99.5% - 101.0%.This shows there could be

impurities in the aspirin synthesised. The deviation as compared to BP value could be due to faulty

titration and some amount of moisture that could not be eliminated after drying the product.

From the calculations, the average percentage content of the aspirin tablets was 109.34%.

This was quite a deviation from the specified range (95% – 105%) indicated by the British

Pharmacopoeia. This deviation could be attributed to errors during preparation of solutions.

These errors may include inaccurate weighings, probable faulty titration. The balance was not

really giving stable values. These constitute the sources of error from which the deviation could

have stemmed.

CONCLUSION

The synthesised aspirin was impure because its percentage purity of 106.9% exceeded the

BP specification. The aspirin tablet also failed. The percentage content of the tablets exceeded

the BP range of 95%–105%.

19
CHAPTER TWO

DETERMINATION OF THE PERCENTAGE CONTENT OF

PARACETAMOL TABLETS USING UV/VISIBLE

SPECTROPHOTOMETRY

INTRODUCTION

Discovery of Paracetamol

The painkilling properties of paracetamol were discovered by accident when a similar

molecule (acetanilide) was added to a patient's prescription about 100 years ago. But since

acetanilide is toxic in moderate doses, chemists modified its structure to try and find a compound

that was less harmful but which still retained the analgesic properties. One of these compounds is

N-acetyl-para-aminophenol, which is also known as acetaminophen in the US and paracetamol

(from para-acetyl-amino-phenol) in the UK. When mixed with codeine it goes by the trade name

Tylenol.

20
Acetanilide Paracetamol Aniline

In fact, in the body, the original compound, acetanilide is partially converted into a mixture

of paracetamol and aniline. The paracetamol provides the painkilling properties, but the aniline is

toxic. Paracetamol has a very similar structure to aspirin, and because of this they are recognised

by the same enzyme. This enzyme is responsible for the biosynthesis of prostaglandins, which are

involved in the dilation of blood vessels that causes the pain experienced in a headache. Reduction

of the amount of prostaglandin, therefore, helps prevent headaches and other pain.

Preparation of Paracetamol

Paracetamol is one of the most common drugs used in the world, and is manufactured in

huge quantities. The starting material for the commercial manufacture of paracetamol is phenol,

which is nitrated to give a mixture of the ortho and para-nitrotoluene. The o-isomer is removed

by steam distillation, and the p-nitro group reduced to a p-amino group. This is then acetylated to

give paracetamol.

21
Paracetamol as Poison

Because paracetamol is a potent drug that is available without prescription, it is often used

in suicide attempts, and in this respect it is potentially more dangerous than other over-the-counter

drugs such as aspirin. This is because paracetamol overdoses often cause liver failure, and there

have been many cases where attempted suicides have awakened from an overdose and changed

their minds, yet still died a few days later from liver damage.

The reasons for this poisoning are to do with the process by which paracetamol is

eliminated from the body. It is first metabolised to a quinone imine:

22
This compound is extremely toxic, and like other such compounds is eliminated in the liver

by reaction with a tripeptide, glutathione. If insufficient glutathione is available, the toxic quinone

will not be eliminated and begins to react with cellular proteins and nucleic acids in the liver,

eventually causing irreparable damage.

However, two compounds, methionine and N-acetylcysteine can boost levels of the vital

glutathione in the liver, and so can be used as antidotes for paracetamol poisoning if the overdose

23
is discovered in time. A new formulation of paracetamol is now being marketed in the UK which

incorporates methianine, such that the drug carries its own antidote with it!

OBJECTIVES:

1. To find the uniformity of weight of twenty (20) paracetamol tablets

2. To prepare dry sample for UV reading

3. To calculate the percent content of paracetamol in tablets

PROCEDURE

A. VERIFICATION OF BEER’S LAW

1. 0.1006g of paracetamol powder was weighed and used to prepare the following

concentrations:

0.00025%

0.0050%

0.00075%

0.001%

0.0015%

2. Absorbances of the respective solutions were obtained at 257 nm.

3. A curve of absorbance against concentration using Microsoft Excel was plotted.

4. The slope of the curve was found and coefficient of correlation determined.

B. UV-VISIBLE SPECTROPHOTOMETRIC ANALYSIS OF PARACETAMOL

TABLETS

1. Twenty (20) paracetamol tablets were powdered (ground) and weighed

24
2. 0.15g of the paracetamol powder was added to 50mL of 0.1M NaOH, diluted with

100mL of water, and shaken for about five minutes. Sufficient water was then

added to produce 200mL.

3. The resulting solution was mixed properly, filtered and diluted with water to 100mL

4. 10mL of the resulting solution was added to 10mL of 0.1M NaOH and diluted to

100mL with water.

5. The absorbance of the resulting solution was measured at a maximum wavelength

of 257nm using the appropriate NaOH solution (0.1MNaOH) as the reference

PREPARATION OF SOLUTIONS TO DETERMINE THE CALIBRATION

CURVE

a) Preparation of 200mL 0.1M NaOH

40g in1000mL ≡ 1M NaOH

4.0g in 1000mL ≡ 0.1M NaOH

0.8g in 200mL ≡ 0.1M NaOH

Percent purity of stock NaOH = 96%


100
Therefore the nominal weight to be weighed = × 0.8g = 0.8333g
96

b) Preparation of paracetamol solutions

0.1g of paracetamol was dissolved in 100mL 0.1M NaOH to give 0.1% paracetamol

solution

For 0.01% from 0.1%,

0.01
× 100mL = 10mL
0.1

25
10mL of the 0.1% solution of paracetamol was diluted with0.1M NaOH to100mL to produce

0.01% paracetamol solution.

i. To prepare the required 0.001%,

0.001
× 100mL = 10mL
0.01

10mL of the 0.01% paracetamol was diluted to 100mL with 0.1M NaOH to give the

required 0.001% as stated in procedure, a above.

ii. To prepare the required 0.0015%,

0.0015
× 100mL = 15mL
0.01

15mL of 0.01% paracetamol solution was diluted with 0.1M NaOH to yield the required

0.0015% as stated in the procedure A above.

iii. To prepare 0.00075%;

0.00075
× 100mL = 50mL
0.0015

50mL of the 0.0015% (prepared in ii above) was diluted with 0.1M NaOH to 100mL to

prepare 0.00075% paracetamol.

iv. To prepare 0.00025% ,

0.00025
× 100mL = 25mL
0.001

25mL of the 0.001% (prepared in i above) was diluted with 0.1M NaOH to 100mL to

produce the required 0.00025%

v. To prepare 0.00050%,

0.0005
× 100mL = 5mL
0.01

26
5mL of already prepared 0.01% paracetamol solution was diluted with 0.1M NaOH to 100mL to

yield the required 0.00050% paracetamol solution.

WEIGHT UNIFORMITY TESTS FOR PARACETAMOL TABLETS

WEIGHT/g DEVIATION PERCENT DEVIATION/%

1 0.6756 0.0192 2.9505

2 0.6538 -0.0620 0.3961

3 0.6444 -0.0120 1.8282

4 0.6678 0.0014 02133

5 0.6509 -0.0055 0.8379

6 0.6770 0.0206 3.1383

7 0.6503 -0.0061 0.9293

8 0.6829 0.0265 4.0372

9 0.6598 0.0034 0.1579

10 0.6577 0.0013 0.1980

11 0.6487 -0.0077 1.1731

12 0.6392 -0.0172 2.6204

13 0.6627 0.0063 0.9598

14 0.6451 -0.0113 1.7215

15 0.6451 -0.0151 2.3000

16 0.6617 0.0053 0.8074

17 0.6469 -0.0095 1.4473

18 0.6536 -0.0028 0.4266

27
19 0.6629 0.0065 0.9902

20 0.6305 0.0259 3.9458

Weight of 20 tablets = 13.1284g

𝟏𝟑.𝟏𝟐𝟖𝟒
Average weight of 20 tablets = = 𝟎. 𝟔𝟓𝟔𝟒𝐠
𝟐𝟎

RESULTS

CONCENTRATION/% ABSORBANCE

0.00025 0.176

0.00050 0.352

0.00075 0.469

0.0010 0.698

0.0015 0.967

28
CALIBRATION CURVE
1.2

1 y = 638.54x + 0.0216
R² = 0.9928

0.8
Absorbancee

0.6

0.4

0.2

0
0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 0.0014 0.0016
Concentration/%

29
The equation from the graph is

y =638.54x + 0.0216

The Correlation Coefficient, R2 = 0.9928

Slope Determination

A = ƐbC………………….. 1

y = mx + c……………….. 2

Comparing equations 1 and 2,

c = 0.0216 (intercept)

C = concentration = x, Ɛ = absorptivity

Ɛb = m (slope) but b (path length) = 1 cm, implying that Ɛ = 638.54

The slope therefore represents the absorptivity of the paracetamol.

Absorbance for 0.00075% paracetamol prepared from the tablet = 0.498

This implies,

0.498 = 638.54x +0.0216

𝟎.𝟒𝟗𝟖−𝟎.𝟎𝟐𝟏𝟔
x= 𝟔𝟑𝟖.𝟓𝟒

x = 7.4608 × 10-4

30
This implies that the concentration of paracetamol in the tablet is 7.4608 × 10-4%.

𝟕.𝟒𝟔𝟎𝟖 × 𝟏𝟎−𝟒
Therefore the % content of the tablet = × 𝟏𝟎𝟎
𝟕.𝟓 × 𝟏𝟎−𝟒

= 99.4773%

DISCUSSION

According to Beer–Lambert’s law, a calibration curve of absorbance versus the

concentration of analyte in a series of standard solutions should be a straight line with an intercept

of zero and a slope of ɛb.

According to this law, A = ƐbC.

Where A = absorbance, b = path length and C = concentration.

Sufficiently low concentrations of the analyte and the standard were prepared for the following

reasons:

i. The Beer–Lambert law is valid only for low concentrations of the analyte. As such very

low concentrations were used in the analysis. This is because at higher concentrations,

the individual particles of each analyte no longer behave independently of each other.

The resulting interaction between the particles of the analyte may change the analyte’s

absorptivity.

ii. A second contribution is that the analyte’s absorptivity depends on the sample’s

refractive index. Because the refractive index varies with the analyte’s concentration,

the values of the absorptivity and the molar absorptivity may change. For sufficiently

31
low concentrations of the analyte, the refractive index is essentially constant and the

calibration curve is linear.

A blank was also used in measuring the absorbance to correct the transmittance for

the sample in order to account for the loss of radiation due to scattering, reflection or absorption

by the sample’s matrix.

The percentage content obtained for the paracetamol tablets is 99.5%. The

paracetamol passed successfully according to the BP. The BP range is 95.0% to 105.0%.

CONCLUSION

The percentage content of the paracetamol tablets was approximately 99.5%, which fell in

the BP range. The UV Spectrophotometric method is therefore quite an efficient method for the

assay of paracetamol tablets. The solvent NaOH used was also very useful for the determination.

32
CHAPTER THREE

LIMIT TEST ON SYNTHESISED ASPIRIN USING THIN LAYER

CHROMATOGRAPHY

INTRODUCTION

Chromatography is defined as an analytical technique which involves the separation of

components in a sample by distribution of the components between two immiscible phases namely

a stationery phase and a mobile phase. The stationery phase consists of a fixed bed of small

particles with a large surface area while the mobile phase is a fluid that moves constantly through,

or over the surface of the stationery phase. The components are separated because of their

differential affinities for the two immiscible phases.

Chromatographic systems achieve their ability to separate mixtures of chemicals by

selectively retarding the passage of some compounds through the stationery phase while allowing

others to move more freely. Therefore by determining the retardation of each separated compound

an evaluation of the separation can be achieved. A factor namely the Retardation Factor (Rf) for

each of the separated or eluted substances has then been introduced to enable the qualitative

evaluation of the separation or chromatogram. The retardation factor of an eluted substance is

defined as a measure of that fraction of the total elution time that it spends in the mobile phase. In

terms of parameters easily obtainable from the chromatogram, the Rf is defined as the ratio of the

distance traveled by the mobile phase in a particular time.

𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐓𝐫𝐚𝐯𝐞𝐥𝐥𝐞𝐝 𝐛𝐲 𝐒𝐮𝐛𝐬𝐭𝐚𝐧𝐜𝐞


Rf = 𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐓𝐫𝐚𝐯𝐞𝐥𝐥𝐞𝐝 𝐛𝐲 𝐌𝐨𝐛𝐢𝐥𝐞 𝐏𝐡𝐚𝐬𝐞

33
Thin-layer chromatography, one of the chromatographic techniques, involves the same

principles as column chromatography; it also is a form of solid-liquid adsorption chromatography.

In this case, however, the solid adsorbent is spread as a thin layer (approximately 250 um) on a

plate of glass or rigid plastic. A drop of the solution to be separated is placed near one edge of the

plate, and the plate is placed in a container, called a developing chamber, with enough of the eluting

solvent to come to a level just below the "spot." The solvent migrates up the plate, carrying with

it the components of the mixture at different rates. The result may then be a series of spots on the

plate, falling on a line perpendicular to the solvent level in the container (see, for example, the

following Figure). The retention factor (Rf) of a component can then be measured as indicated in

the figure below.

34
This chromatographic technique is very easy and rapid to perform. It lends itself well to

the analysis of mixture composition and may also be used to advantage in determining the best

eluting solvent for subsequent column chromatography. However, volatile compounds (bp <

100EC) cannot be analyzed by TLC.

The same solid adsorbents used for column chromatography may be employed for TLC;

silica gel and alumina are the most widely used. The adsorbent is usually mixed with a small

amount of "binder"--for example, plaster of Paris, calcium sulfate, or starch--to ensure adherence

of the adsorbent to the plate. The plates may be prepared before use, or commercially available

pre-layered plastic sheets may be used. In either case, the plates or sheets are dried for an hour or

more in an oven at 110EC before use. This procedure removes moisture that is adsorbed on the

adsorbent and produces a surface that is more effective in adsorbing and separating the components

of the mixture being analyzed. The relative eluting abilities of the various solvents that may be

used are the same. The eluting power required of the solvent is directly related to the strength of

adsorption of the components of the mixture on adsorbent.

35
A distinct advantage of TLC is the very small quantity of sample required. A lower limit

of detection of 10-9 g is possible in some cases. The spot of sample is applied to the plate with care,

which is normally not large, for the same reason that the amount of sample placed at the top of a

column should not be too large. However, larger samples such as 0.5 mg may be used on larger

TLC plates, which have thicker coats of adsorbent. In these cases isolation of the components

may be achieved by scraping the separated spots from the plate and eluting (extracting) them with

an appropriate solvent. It is usually necessary to repeat this process a number of times in order to

obtain even several milligrams of material. Such a procedure would offer a method of

identification of the various components, however, because enough material could be collected to

allow spectra to be obtained.

Often it is useful for the purpose of identification to run TLC chromatograms of knowns

and unknowns side by side. Other useful applications involve running multiple aliquots of samples

collected from a chromatographic column or samples taken from a reaction mixture in order to

follow reaction progress as a function of time.

Detection of spots on the chromatogram is easy for colored materials, and a number of

procedures are available for locating spots of colorless materials. For example, irradiation of the

plate with ultraviolet light will permit location of the spots of compounds that fluoresce.

Alternatively, the solid adsorbent may be impregnated with an otherwise inert, fluorescent

substance. Spots of materials that absorb ultraviolet light but do not fluoresce will show up as

black spots against the fluorescing background when the plate is irradiated with ultraviolet light.

Other detecting agents are more often used. These agents may be sprayed onto the chromatograms,

causing the spots to become readily apparent. Examples of detecting agents used in this way are

36
sulphuric acid, which causes many organic compounds to char, and potassium permanganate

solution. Iodine is another popular detecting agent. In this case the plate is placed in a vessel whose

atmosphere is saturated with iodine vapor. Iodine is adsorbed by many organic compounds, and

their spots on the chromatogram become colored (usually brown). Since these spots usually fade,

it is a good idea to circle the spots with a pencil while they are still visible in order to have a

permanent record of the chromatogram.

Under a given set of conditions (adsorbent, solvent, layer thickness, and homogeneity) the

rate of movement of a compound with respect to the rate of movement of the solvent front, Rf, is

a property of that compound. The value is determined by measuring the distance traveled by a

substance from a starting line in the before-mentioned figure. The property has the same

significance as retention time in a GC experiment.

EQUIPMENT USED  Pair of Scissors, Pencil and Ruler

 Chromatank
 50ml Measuring Cylinder
 UV Lamp
 Three 10cm × 20cm Aluminum TLC

Plates with silica gel coating CHEMICALS USED

 Filter Paper
 Pure Aspirin Powder
 Watch Glass
 Crude Aspirin Powder
 Capillary Tube
 Conc. H2SO4
 Stirring Rod
 Ethanol (96%)
 Micro Pipette
 Ethyl Acetate
 200ml beaker
 Distilled Water
 500ml Volumetric Flask

37
PROCEDURE

1. Three 10cm x 20cm Aluminum TLC plates were obtained. A thin line was drawn 1cm from

the bottom of each plate.

2. Three chroma tanks were each filled with 10mL of a mixture of ethanol and ethyl acetate

in the ratios of

i. 7:3,

ii. 1:1, and

iii. 3:7 respectively. Each tank was then covered to allow the solvent enough time to

saturate the environment of the TLC plates.

3. Very small amounts of the synthesised aspirin, the pure aspirin and the salicylic acid were

each dissolved in separate test–tubes using 1mL each of the 7:3 ethanol and ethyl acetate

mixture as prepared in step 2 (i) above. Each of the resulting solutions was then spotted

separately on the faint line on one of the plates using capillary tubes. The plate was allowed

to dry and then placed in the chromatank containing the 7:3 ethanol and ethyl acetate

mixture.

4. The chroma tank was covered with a glass tile and enough time allowed for the solvent to

elute.

5. The TLC plate was removed and dried at room temperature.

6. The procedure was repeated for the 1:1 and 7:3 mixtures of the ethanol and ethyl acetate

prepared in steps 2(ii) and (iii) above using the other two TLC plates. The diameter of each

spot was ensured around 2mm and at equal intervals. Three spots were made on each TLC

plate, each spot representing the pure aspirin, the synthesised aspirin, and the salicylic acid.

7. A UV Lamp was used to view the spots which were carefully circled with pencil.

38
8. Their Rf values were determined.

RESULTS AND CALCULATIONS

SOLVENT DISTANCE MOVED/cm

(ethanol : ethyl Solvent Salicylic Pure aspirin Supposed synthesised Supposed

acetate ratio) acid aspirin (x) impurity (y)

7:3 9.5 9.1 7.8 7.6 9.2

1:1 9.2 8.8 7.1 7.0 8.8

3:7 8.5 - - - -

CALCULATION OF RF VALUES

𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐓𝐫𝐚𝐯𝐞𝐥𝐥𝐞𝐝 𝐛𝐲 𝐒𝐮𝐛𝐬𝐭𝐚𝐧𝐜𝐞


Rf = 𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐓𝐫𝐚𝐯𝐞𝐥𝐥𝐞𝐝 𝐛𝐲 𝐌𝐨𝐛𝐢𝐥𝐞 𝐏𝐡𝐚𝐬𝐞

1. 7:3 ethanol + ethyl acetate mixture

i. Pure aspirin

𝟕.𝟖𝐜𝐦
Rf = 𝟗.𝟓𝐜𝐦 = 0.82

ii. Supposed synthesised aspirin (x)

𝟕. 𝟔𝐜𝐦
Rf = = 0.80
𝟗. 𝟓𝐜𝐦

iii. Supposed impurity (y)

𝟗.𝟐𝐜𝐦
Rf = 𝟗.𝟓𝐜𝐦 = 0.97

iv. Salicylic acid

39
𝟗. 𝟏𝐜𝐦
Rf = = 0.96
𝟗. 𝟓𝐜𝐦

2. 1:1 ethanol + ethyl acetate mixture

i. Pure aspirin

𝟕.𝟏𝐜𝐦
Rf = 𝟗.𝟐𝐜𝐦 = 0.77

ii. Supposed synthesised aspirin

𝟕.𝟎𝐜𝐦
Rf = = 0.76
𝟗.𝟐𝐜𝐦

iii. Supposed impurity

𝟖.𝟖𝐜𝐦
Rf = 𝟗.𝟐𝐜𝐦 = 0.96

iv. Salicylic acid

𝟖.𝟖𝐜𝐦
Rf = 𝟗.𝟐𝐜𝐦 = 0.96

3. 3:7 ethanol + ethyl acetate mixture

Rf =??

There is no Rf value because there was no separation.

DISCUSSION

Three different mixtures of ethanol and ethyl acetate were used as solvents for the analysis in

the following ratios:

i. 7:3

ii. 1:1 and

iii. 3:7

40
This was to help determine which of these solvent systems would be more efficient in the

separation of the compounds in the samples spotted.

For the 1:1 solvent system, the spot for the pure aspirin yielded an Rf value of 0.77 while the

spot for the synthesised aspirin yielded two separations with Rf values of 0.76 and 0.96. The 0.76

Rf value was tagged as x and the 0.96 Rf value was also tagged as y as can be seen from the table

above. Meanwhile the spotted salicylic acid also produced an Rf value of 0.96.

Comparing these Rf values shows that the separation tagged as y has the same Rf value as the

salicylic acid spot. This implies that separation y was highly likely to be salicylic acid which may

have been left in the synthesised aspirin probably due to inefficient washing and recrystallization.

This could also be due to decomposition of some of the synthesised aspirin, since it was kept for

some weeks before carrying out this analysis. Separation x could also be concluded to be the actual

aspirin synthesised judging from the proximity of its Rf values to that of the pure aspirin spot.

The same conclusions were arrived at using the Rf values for the 7:3 solvent system. The 3:7

solvent system produced no separations. No spots were identified after viewing under the UV lamp

using the 3:7 solvent system. Comparing the Rf values and separations obtained using the three

solvent systems implies that the 1:1 solvent system was the most efficient. The 1:1 solvent system

produced Rf values that were much closer to those of their respective standards (pure aspirin and

the pure salicylic acid) used. For example the Rf value for separation x was exactly the same as

that for the pure salicylic acid spot (0.96) using the 1:1 solvent system. The 7:3 solvent system

gave Rf values of 0.97 for separation x and 0.96 for the salicylic acid spot. The 3:7 solvent system

was the worst since it gave no identifiable separations.

41
Ethanol is more polar than the ethyl acetate because of the presence of the OH group. In 3:7

mixture, the percentage of the ethanol in the mixture was quite small and so its polarity was

overcome by the relatively non-polar ethyl acetate hence the incidence of no separation.

CONCLUSION

The synthesised aspirin contained some amount impurity because of the presence of

salicylic acid in it.

42
REFERENCES

1. D.A. Skoog, D.M. West, F.J. Holler and S.R. Crouch, Fundamentals of Analytical

Chemistry, 8th ed. California: Brooks/Cole – Thomson Learning, 2004.

2. D.C. Harris, Quantitative Chemical Analysis, 7th ed. New York: W.H. Freeman and

Company, 2010.

3. R.A. Day, Jr. and A.L. Underwood, Quantitative Analysis, 6th ed. New Jersey:Prentice-

Hall International, Inc., 1991.

4. Analytical Chemistry Laboratory Manual (2007 Edition). Institute of Chemistry,

University of the Philippines Diliman.

5. British Pharmacopoeia (2002) volume I and II CD–ROM, Her Majesty’s Stationery

Office, London, UK.

43
APPENDIX

SPECTRA OBTAINED FOR THE VARIOUS CONCENTRATIONS OBTAINED FOR

THE STANDARD AND THE ANALYTE

1. 0.0010%

44
2. 0.00075%

3. 0.0015%

45
4. 0.00050%

46
5. SPECTRA FOR ALL CONCENTRATIONS OF STNADARD

6. SPECTRA FOR 0.00075% OF PARACETAMOL TABLET

47
APPENDIX B

LIMIT TEST ON SYNTHESISED ASPIRIN USING TLC

1:1 Ethanol + Ethyl Acetate Mixture Solvent System

48