Pharmaceutical Organic Chemistry - Lab Report 6 PDF

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UNIVERSITI KUALA LUMPUR ROYAL COLLEGE MEDICINE PERAKFACULTY OF PHARMACY & HEALTH SCIENCESBACHELOR IN PHARMACEUTICAL TECHNOLOGY (HONOURS)PHARMACEUTICAL ORGANIC CHEMISTRY I (RCB 10303)LAB REPORTTITLE: EXPERIMENT 6 – QUALITATIVE ANALYSIS OF AMINO ACIDS AND PROTEINSNO. NAME STUDENT ID.1. ATIYAH SOFI...

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UNIVERSITI KUALA LUMPUR ROYAL COLLEGE MEDICINE PERAK FACULTY OF PHARMACY & HEALTH SCIENCES BACHELOR IN PHARMACEUTICAL TECHNOLOGY (HONOURS) PHARMACEUTICAL ORGANIC CHEMISTRY I (RCB 10303) LAB REPORT TITLE: EXPERIMENT 6 – QUALITATIVE ANALYSIS OF AMINO ACIDS AND PROTEINS

NO. NAME

STUDENT ID.

1.

ATIYAH SOFIA BINTI MOHD HISABURI

59217120454

2.

AZLIN ZUHAYRA BINTI AZHAR

59217120406

3.

NUR ADRIANA BINTI EDDIE

59217120402

4.

NUR AISYA BALQISH BINTI MOHAMMED ALAM SHAH

59217120414

5.

NURFADHLIN HAIFA BINTI FARIZAL

59217120427

6.

NURHAZIQAH BINTI HANAFIAH

59217120428

LECTURER’S NAME: CHM. DR. MAZLIN BINTI MOHIDEEN CHM. DR. SITI HAJAR BINTI MUSA DR. SHARON A/P FATINATHAN

DATE OF SUBMISSION: 18th NOVEMBER 2021

Table of Contents

1.0 Objectives.............................................................................................................................................3 2.0 Materials & Apparatus........................................................................................................................3 3.0 Introduction.........................................................................................................................................4 4.0 Procedures............................................................................................................................................6 4.1 Solubility Tests.....................................................................................................................................6 4.2 Ninhydrin Tests....................................................................................................................................6 4.3 Stability to Alkali.................................................................................................................................6 4.4 Specific Reactions for Individual Amino Acids.................................................................................7 4.4.1 Xanthoproteic Test............................................................................................................................7 4.4.2 Millon’s Test......................................................................................................................................7 5.0 Result/Data...........................................................................................................................................8 5.1 Solubility Tests.....................................................................................................................................8 5.2 Ninhydrin Tests....................................................................................................................................8 5.3 Stability to Alkali.................................................................................................................................8 5.4 Specific Reactions for Individual Amino Acids.................................................................................9 5.4.1 Xanthoproteic Test............................................................................................................................9 5.4.2 Millon’s Test......................................................................................................................................9 6.0 Discussion...........................................................................................................................................10 7.0 Conclusion..........................................................................................................................................11 8.0 Laboratory Questions........................................................................................................................12 9.0 References..........................................................................................................................................13 10.0 Appendices.......................................................................................................................................13

1.0 Objectives 1. To determine the qualitative analysis of amino acids. 2. To determine the solubility of amino acids. 3. To understand the specific tests, involve in detecting amino acids.

2.0 Materials & Apparatus

Materials Distilled water, 0.1N Hydrochloric acid, Sodium hydroxide, Glycine, Tyrosine, Glutamic acid, Cysteine, 0.2% Ninhydrin Solution in Acetone, Nitric acid, 40% Sodium hydroxide, and Millon’s reagent.

Apparatus Test tubes, boiling tubes, litmus paper, beaker, hot plate, and test tube holder.

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3.0 Introduction Amino acid is a compound that contains an amine group (-NH₂), a carboxylic acid group (COOH), and a variable side chain (R-group) where it combined to form a protein. The amine group is connected to the alpha-carbon of the protein which it relatively closes to the carboxyl group.

The primary difference of all the amino acids is their side chain with its own

identification that linked to the alpha-carbon. As amino acid is amphoteric, there are many ways amino acid can be distinguished and widely classified into four types. The four types of amino acids are polar amino acids, non-polar amino acids, acidic amino acids, and basic amino acids. Amino acids are necessary for life to develop. They offer different types of major functions, including serving as protein key components and metabolic intermediates. Besides, amino acid has no net charge at a particular pH where this is called as isoelectric point. It has zero net charge especially when it is at neutral pH. This is because the amino acids need to carry same number of both protonated ammonium groups (positive charge) and deprotonated carboxylate group (negative charge) at the isoelectric point. Thus, it is known as zwitterions. If the amino acids do not carry both positive and negative charge, it will not travel to neither cathode nor anode if it is under the influence of electric field. Furthermore, the isoelectric point also differs in each type of the amino acids, but it is an exception to glycine as other amino acids have an asymmetric carbon atom that connected to four separate groups. There are many types, and several methods can be used to analyze the amino acids, including solubility test, ninhydrin test, stability to alkali, xanthoproteic test, and millon’s test. Each test has different theory and method for example solubility test. Solubility test is a test of the solubility of amino acid that largely determined by the pH of the solution. It is affected by transformation in amino acid that occur at different pH values. Both functional groups are oxidized in acidic solution while in basic solution both are reduced. Generally, amino acids are water soluble to a considerable extent. The solubility of the amino acid also varies due to the structure of side chains especially in water, dilute alkali, and dilute acid. Moreover, ninhydrin is a substance is used to detect ammonia, as well as it can determine the classification of amines (primary and secondary). When it is reacted with ninhydrin, it will cause reduction where the product then will combine with NH₃ and leftover ninhydrin to produce a 4

blue-coloured material. Next, the stability of amino acid to alkali where amino acid does not produce NH3₃ when it reacts with alkali contrary amides and volatile amines. This approach of test can be applied to many types of amino acid, amines, and amides. Adding on information of the test of amino acid, where there is xanthoproteic test perform to detects for aromatic amino acid with an active benzene ring. It is a test where the nitrogen is being removed in the active aromatic amino acids with presence of strong nitric acid, HNO₃. The outcome of the reaction is it will form yellow product. Lastly, the millon’s test where it is specialized for phenolic amino acids for example tyrosine. It works on the basis of the nitration reaction. The reagent of this test is a strong nitric acid, HNO₃ that will undergo nitrification to convert the phenolic amino acids to nitrated amino acid which is in red colour as a positive test. If yellow precipitate of HgO is formed, then it will become a negative reaction which indicate the solution is too alkaline.

5

4.0 Procedures 4.1 Solubility Tests 1. Three test tubes of amino acids sample, glycine was prepared. 2. Each test tube was dropped with water, dilute HCl and dilute NaOH respectively. 3. The changes in each test tube for detecting solubility were recorded. 4. Step 1 to 3 was repeated using different types of amino acids, tyrosine, glutamic acid and cystine. 4.2 Ninhydrin Tests 1. 1 mL of glycine was measured and inserted into a boiling tube. 2. 5 drops of 0.2% ninhydrin solution in acetone was dropped onto the glycine solution. 3. The boiling tube was inserted into a water bath and boiled for over 2 minutes. 4. The boiling tube was allowed to cool, and color change was observed.

4.3 Stability to Alkali 1. 1 mL of glycine was pipetted into a test tube. 2. 1 mL of NaOH was then added to the test tube and allowed to boil. 3. The vapor formed from the boiling process was tested with litmus paper. 4. Step 1 to 3 was repeated by replacing glycine with Tyrosine, Glutamic Acid and Cysteine.

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4.4 Specific Reactions for Individual Amino Acids 4.4.1 Xanthoproteic Test

1. 2 mL of tyrosine was added to a boiling tube. 2. 2 mL of equal volume of concentrated HNO3 was added to the boiling tube containing amino acid. 3. The boiling tube was heat over flame for 2 minutes and the color change was observed. 4. The boiling tube was allowed to cool under the tap and a few drops of 40% NaOH was cautiously added to make the solution strong alkaline. 5. The color change was observed. 6. Steps 1 to 5 were repeated using different types of amino acids such as Tyrosine, Glutamic Acid and Cystine.

4.4.2 Millon’s Test 1. 2 mL of Glycine was added to a test tube. 2. 1-2 drops of Millon’s reagent were added. 3. The tube was warmed in boiling water bath for 10 minutes. 4. A brick red color was recorded for positive reaction. 5. Steps 1 to 4 were repeated using different amino acids such as Tyrosine, Glutamic Acid

and Cystine to replace Glycine.

7

5.0 Result/Data

5.1 Solubility Tests Amino Acid

Distille d water

Sodium 0.1N Hydrochloric Hydroxide acid

Glycine Tyrosine Glutamic acid

Soluble

Cysteine

Insoluble

5.2 Ninhydrin Tests Amino acid

Observation

Glycine

Light Violet

5.3 Stability to Alkali Amino acid

Observation

Glycine

Red litmus paper change to blue

Tyrosine

Red litmus paper change to blue

Glutamic acid

Red litmus paper change to blue

Cysteine

Red litmus paper change to blue

8

5.4 Specific Reactions for Individual Amino Acids

5.4.1 Xanthoproteic Test Amino acid

Observation

Glycine

No change

Tyrosine

Light yellow

Glutamic acid

No change

Cysteine

No change

5.4.2 Millon’s Test Amino acid

Observation

Glycine

Clear and greyish solution

Tyrosine

Red precipitation

Glutamic acid

White precipitation

Cysteine

Brownish solution

Phenylalanine

Yellow precipitation

9

6.0 Discussion The amino acid content of amino acid, peptide, and protein-containing substances is determined via amino acid analysis. Proteins are long linear polymers of amino acids that are linked together by peptide bonds, with a few exceptions. Hydrolyzing these peptide bonds is the initial step in the amino acid analysis process. Separation, detection, and quantification of the released amino acids follows. The first part of this experiment was to determine the solubility of the amino acids using different solvents. Distilled water, Hydrochloric Acid and Sodium Hydroxide were used as the determining solvent. The amino acids that were tested were Glycine, Tyrosine, Cystine and Glutamic Acid for each experiment. From the experiment The second part of the experiment was involving ninhydrin solution for the ninhydrin test

10

7.0 Conclusion The main purposes of this experiment were achieved as the determination of the qualitative analysis of amino acids are obtained. There are few ways to determine the qualitative analysis of the amino acids including solubility test, ninhydrin test, stability to alkali, xanthoproteic test, and millon’s test. By all these tests, the solubility of the amino acids was determined for glycine, tyrosine, glutamic acid, and cysteine in different types of solvent such as distilled water, hydrochloric acid, and sodium hydroxide. All the amino acids given is soluble in sodium hydroxide but ss for the result of solubility of amino acid in distilled water, only glycine is water soluble. Moreover, the result for solubility in the hydrochloric acid, both glycine and glutamic acid is soluble in hydrochloric acid. Furthermore, the result of the specific test of amino acids such as xanthoproteic test and millon’s test was obtained. For xanthoproteic test, only tyrosine gives positive result which the solution change to light yellow colour. Besides, the millon’s test have varied results which clear and greyish solution was formed in glycine, red precipitate formed in tyrosine, white precipitate formed in glutamic acid, brownish solution was formed in cysteine, and yellow precipitate formed in phenylalanine. From this experiment, there are some precautions that need to be taken to avoid error results such as the glassware that are used must be clean thoroughly to avoid any reaction for the next use. Next, all the test tubes and boiling tubes must be labelled with given sample as this can prevent from any confusion that can affect to the result. Lastly, the reagent dropper given and used in the experiment must be far from the test tube’s mouth when dropping the solution to avoid contamination during the experiment.

11

8.0 Laboratory Questions 1. Write the reactions involved in the Ninhydrin Test. The reaction that involved in the Ninhydrin test is a redox reaction where the ninhydrin as an oxidizing agent. Ninhydrin will react with the amino group of the free amino acid. Later, it will oxidize the compound and causes deamination.

2. What type of reaction is responsible for the evolution of alkaline vapor? Write the reaction and explain briefly. It is a reaction between alkali and alkaline earth metal with water which will produce alkaline vapor. For example: Ca + 2H ₂O → Ca(OH)₂ + H₂

3. Write the reactions involved in Xanthoproteic Test. The reactions involved in Xanthoproteic Test is nitration reaction where it will determine the presence of proteins that contain in the solution. The concentrated nitric acid will react with the aromatic amino acids when the solution is hot. The product formed will be in yellow colour and change to deep orange colour (positive result) with addition of strong base.

4. Define “activated benzene ring”. Briefly. The activated benzene ring can be defined as a substituent on the ring that donate electrons.

5. Do all the amino acids with aromatic side chains give a positive result? Why? Yes, because most protein contain one or both tyrosine, tryptophan, phenylalanine, and glutamic acid which it will show positive result.

12

9.0 References 1. Kamineni, S. (2016, October 26). Musculoskeletal ProteinAnalysis Techniques - A Review.

Symbiosis.

Retrieved

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2021,

from

https://symbiosisonlinepublishing.com/rheumatology-arthritic-diseases/rheumatologyarthritic-diseases08.php 2. Vedantu. (2021, January 4). Ninhydrin Test. Retrieved November 16, 2021, from https://www.vedantu.com/chemistry/ninhydrin-test 3. Libretexts. (2020, September 13). Substitution Reactions of Benzene Derivatives. Chemistry

LibreTexts.

Retrieved

November

16,

2021,

from

https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Supplemental_Modules_(Or ganic_Chemistry)/Arenes/Reactivity_of_Arenes/Substitution_Reactions_of_Benzene_D erivatives#:%7E:text=Activated%20rings%3A%20the%20substituents%20on,are %20groups%20that%20withdraw%20electrons. 4.

10.0 Appendices

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