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University of the Immaculate Conception

Davao City Revision Date: October 2019
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Document: LABORATORY MANUAL

Title: PCH 400 PHARMACEUTICAL & MEDICINAL ORGANIC CHEMISTRY

List of Experiments

Experiment no Topics Scores:

1 ENZYMES ACTIVITY IN SALIVARY DIGESTION
2 ENZYMES ACTIVITY IN GASTRIC DIGESTION
3 ENZYMES ACTIVITY IN INTESTINAL DIGESTION
4 PREPARATION AND IDENTIFICATION OF ETHYL
ALCOHOL FROM DEXTROSE
5 COLOR REACTIONS OF VARIOUS STEROIDS
6 IDENTIFICATION OF ANALGESIC USING TLC
7 PREPARATION AND IDENTIFICATION OF METHYL SALICYLATE
8 SYNTHESIS OF ACETYLSALICYLIC ACID
9 THE PREPARATION OF ACETAMINOPHEN
10 PREPARATION OF BENZOCAINE
11 CATEGORIES OF GENERAL ANESTHESIA
12 PREPARATION OF BENZYL ALCOHOL AND BENZOIC ACID
13 PREPARATION OF SULFANILAMIDE
14 CHROMATOGRAPHIC PLATE IDENTIFICATIOON OF SULFANILAMIDE
15 COLOR REACTIONS OF ANTIBIOTICS
16 ANTIBACTERIAL ACTIVITY OF PEN G
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SAFETY RULES IN THE CHEMISTRY LABORATORY

1. Be sure the science teacher is present in the laboratory.
2. Know the location of all emergency equipment.
3. Wear goggles and other safety garments always when you are in the
laboratory. Do not wear contact lenses.
4. Tie back long hair and remove coats. Do not wear open-toed shoes or
sandals in the laboratory.
5. Read each activity carefully before starting work to
become familiar with the experimental procedure and safety
precautions.
6. Perform only those procedures directed by your teacher.
Use only the recommended amounts of chemicals and no more.
7. Always keep your work area neat and uncluttered. Keep
materials away from the edge of the laboratory bench or
worktable
8. Do not eat, drink, chew gums, or apply cosmetics in the
science laboratory. Do not taste anything.
9. When instructed to test for the odor of a substance, hold
the container away from you and fan a small amount of the vapor toward with your
hand.
10. When heating in a test tube, hold it at an angle and move it
through the flame: point the mouth of the test tube away from others
and from yourself.
11. Handle hot glassware with tongs, test tube holders, or
potholders as directed in the procedure. Remember that hot glass
looks like cold glass.
12. Extinguish all burner flames before using flammable materials.
13. Turn off burners that are not in use.
14. Note carefully the method of disposal of chemicals in each
activity and follow directions given by your teacher.
15. Wash your hands thoroughly after you work in a science laboratory.
“SAFETY FIRST.”
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University of the Immaculate Conception
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Document: LABORATORY MANUAL

Title: PCH 400 PHARMACEUTICAL & MEDICINAL ORGANIC CHEMISTRY

Learning Activity No. 1

ENZYMES ACTIVITY IN SALIVARY DIGESTION
Specific Learning Objectives:
1.
2.
3.

Time Allotment: 3 hours

Theoretical Background:
Enzymes are catalysts produced as a result of cellular activity that occurs in abundance in plant
and animal tissue. By general agreement most enzymes end in “ase,”. Chemically they are
proteins and are classified in six major categories.
1. Oxidoreductases, also known as dehydrogenases or oxidases, include alcohol
dehydrogenases and glutamic dehydrogenase in the liver.
2. Transferases, which catalyze the transfer of one-carbon groups, include aldehydes, ketones,
phosphorus and sulfur groups.
3. Hydrolases catalyze hydrolysis of ester, ether, peptide, and other bonds, including such
examples as pepsin and rennin.
4. Lyases catalyze removal of groups from substrates by means other than hydrolysis, leaving
double bonds. They include ketone, aldehyde, and carbon-oxygen lyases.
5. Isomerases, including enzymes, catalyze interconversion of optical and geometric isomers.
6. Ligases catalyze the linking together of two compounds coupled to the breaking of a
pyrophosphate bond. They include Succinate Co-A and Acetyl Co-A.

Saliva
Three pints of saliva: that’s how much the average healthy person makes daily. It’s 99% water
and 1% proteins, enzymes, and electrolytes. It contains the enzyme amylase which breaks down
select starches into maltose and dextrin, initiates fat breakdown, and starts digestion. It also
contains role-specific proteins (eg, antibacterial histatins, protective statherins, lubricating
mucins).
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Drugs and Dry Mouth

Several hundred medications can cause or exacerbate xerostomia,including antihypertensives,
antidepressants, analgesics, tranquilizers, diuretics, and antihistamines. These drugs affect the
saliva’s role in digestion. Dry mouth, especially when it is chronic, drastically alters patients’
lives. They will find that the sore mucous membranes and gums, cracked lips and split corners
of the mouth, and a rough, painful tongue make eating impossible. When teeth feel like razors,
spicy foods set off alarms, and sleep eludes them because they wake to sip water, they need
help. Pharmacists can recommend appropriate and soothing interventions.

Learning Resources:
Starch Fresh Saliva 10% Potassium iodide
Water 2% starch solution 2% Ferric chloride
Toluene Ice Diluted HCl
Iodine solution 10% Sulfuric acid 1% Mercuric chloride

Procedure:
The series of exercises in this experiment will serve to identify enzyme activity of body fluids.
I. Classic Wolgemuth method to determine the erythrodextrin reaction with iodine.
1. Suspend 2 g of soluble starch in 25 ml of water.
2. Gradually heat to boiling while stirring and continue until solution is nearly clear.
3. Cool to room temperature and dilute to 100 ml.
4. Preserve with a few ml of toluene.
5. Next dilute 1 ml of 0.1 N I2 solution to 120 ml, which gives you a N/1200 I2 solution.
6. Label seven test tubes 1 through 7 (16 x 150 mm) and measure off 2.5, 2.0, 1.6, 1.2, 0.8,
0.4, and 0.2 ml of fresh saliva respectively.
7. To these tubes add, in the same order, 0.0, 0.5, 0.9, 1.3, 1.7, 2.1, 2.3 ml of distilled water
to make equal volumes of 2.5 ml.
8. Shake well to mix and then rapidly add 2.5 ml of a 2% soluble starch solution. Shake
each tube well and place in a water bath at 40°C.
9. Keep tubes in the bath for 30 minutes, then at once immerse in ice water and add 2 ml
of N/1200 I2 solution to each.
10. Shake each tube well and note the minimum concentration of saliva at which the
erythrodextrin reaction persists.
From your data calculate the number of ml of 2% starch that 1 ml of your saliva will hydrolyze
to the achromic point in 30 minutes at 40°C. Show the calculations.
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II. The second activity of this experiment will show the presence of nitrites (N02) in saliva.
1. To 1 ml of saliva add 2 drops of 10% H2S04.
2. Mix well and add 2 drops of a 10% KI solution and a drop of starch solution.
3. The nitrous acid (HN02) formed liberates the iodine that is detected by the starch.
4. Record the color reaction. Perform 3 trials.

III. The third activity shows the presence of sulfocyanates in saliva.

1. To 1 ml of saliva add 1 drop of 2% FeCl3 solution and acidify slightly with HCl.
2. If a pink to red color develops, it is due to the sulfocyanates forming a complex ion with
the ferric ions or to a precipitate of ferric phosphate.
3. If due to sulfocyanates, 2 to 5 drops of 1% HgCl2 solution will convert the colored
compound to colorless mercuric sulfocyanate.
4. Record results. Perform 3 trials
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Learning Activity No. 2

ENZYMES ACTIVITY IN GASTRIC DIGESTION
Specific Learning Objectives:
1.
2.
3.

Time Allotment: 3 hours

Theoretical Background:
Digestive enzymes are classified based on their target substrates:
• Lipases split fatty acids off fats and oils.
• Proteases and peptidases split proteins into small peptides and amino acids.
• Amylases split carbohydrates such as starch and sugars into simple sugars such
as glucose.
• Nucleases split nucleic acids into nucleotides.
In the human digestive system, the main sites of digestion are the oral cavity, the stomach, and
the small intestine. Digestive enzymes are secreted by different exocrine glands including:
• Salivary glands
• Gastric glands in the stomach
• Secretory cells(islets) in the pancreas
• Secretory glands in the small intestine

Whilst for many clinicians, acid secretion is the most important aspect of gastric function that is
studied in clinical practice, there are other facets that should be considered in drug
development and measurements of gastric function.

Pepsin is one of the major enzymes involved in gastric digestion and has a precursor called
pepsinogen. Evidence for the existence of the two forms is based upon the difference in
behavior of the two toward acids. Alkali destroy pepsin, whereas acids protect and aid it. Alkali
protect pepsinogen, whereas acids destroy it by converting it into pepsin.

Physiologic functions of gastric exocrine secretions.

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Physiologic function Secreted product

Killing or suppression of growth of ingested micro-organisms H+
Facilitation of duodenal inorganic iron absorption H+
Stimulation of secretin release H+
Suppression of antral gastrin release H+
Initiation of peptic hydrolysis of dietary proteins H+, pepsin
Liberation of vitamin B12 from dietary protein H+, pepsin
Binding of vitamin B12 for subsequent ileal uptake Intrinsic factor
Initiation of hydrolysis of dietary triglycerides Gastric lipase
Protection against noxious agents Mucin, NaHCO3−,
water, components of
the mucus gel
The function of the stomach includes initiation of digestion by exocrine secretions such as acid
and pepsin, which are under the control of the endocrine secretion of hormones that also
coordinate intestinal motility. The stomach also stores and mechanically disrupts ingested food.
Various techniques have been developed to assess gastric physiology, the most important of
which is assessment of acid secretion, as well as gastric motility and gastric emptying. The
influence of drugs on gastric function and the effect of gastric secretion and mechanical actions
on the bioavailability of novel compounds are of critical importance in drug development and
hence to clinical pharmacologists. The control of acid secretion is essential in the treatment of
peptic ulcer disease as well as gastroesophageal reflux disease (GERD); pH-metry can be used to
determine the necessary dose of an acid suppressant to heal mucosal damage. Disturbed
gastric myoelectric activity leading to gastroparesis can cause delayed gastric emptying, often
found in patients with diabetes mellitus. Electrogastrography (EGG) may be used to evaluate
the influence of prokinetics and other drugs on this condition and aid in determining effective
therapy.

Learning Resources:
Gastric mucous membrane 1 N Sodium chloride Evaporated milk
Sea sand 0.1 N HCl 1N Potassium Oxalate
10% Sodium carbonate 1N Sodium carbonate 1N Calcium chloride
Cheesecloth 0.1 NSodiumcarbonate Renin
Water Egg White
Procedure:
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I.
1. In this experiment take 10 g of well-washed gastric mucous membrane, add about 15 g
of sea sand and grind in a mortar.
2. Gradually add to this 100 ml of water containing two drops of a 10% Na2C03 solution.
3. Mix well and strain through cheesecloth.
4. Next measure off four 5 ml portions of this extract into 25 x 200 mm test tubes and label
them 1 through 4.
5. To tube 1
a. add 13.4 ml of water and 1.6 ml of a 1 N NaCl solution and mix well.
b. You now have 20 ml of a solution containing 1.6 ml of NaCl, but which is neutral
and has not been acted upon by acids or bases.
c. Since there is no acid medium, no pepsin activity is evident.
6. To tube 2
a. add 11.4 ml of water, 2 ml of 1 N HC1, and 1.6 ml of 1 N NaCl and mix well.
b. Here we have 20 ml of solution that is 0.1 N HCl and also contains 1.6 ml of 1 N
NaCl.
c. This solution should digest protein readily, but it does not tell us whether the
original extract contains pepsin or pepsinogen or both.
7. To tube 3
a. add 9.4 ml of water and 1.6 ml of a 1N Na2C03solution and mix well.
b. This gives you 16 ml of a 0.1 N Na2C03 solution. If pepsin is present in the
original extract, it should be destroyed, but the pepsinogen is not destroyed.
c. Next add 3.6 ml of 1 N HC1 and 0.4 ml of water.
d. You now have 20 ml of a 0.1 N HC1 solution again containing 1.6 ml of 1 N NaCl.
e. In case pepsin is present you have favorable conditions for its action on proteins.
8. To tube 4
a. add 0.6 ml of 1 N HC1 and 0.4 ml of water and mix well.
b. If pepsinogen is present, it should be converted to pepsin. Next add 1.6 ml of a
1N Na2C03 solution and 2.4ml of water and mix well.
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c. You now have 10 ml of a 0.1 N Na2C03 solution. If pepsinogen is still present, it

will not be destroyed by the Na2C03, but if the pepsinogen has previously been
converted to pepsin, it will now be destroyed by the base.
d. Next add 3 ml of 1N HC1 and 7 ml of water and shake.
e. The final solution is of a 0.1 N HC1 solution containing 1.6 ml of 1 N NaCl.
f. This last addition of acid is made in order to render the solution acid so that
pepsin can act if present.
9. Introduce 0.5 g of dried egg white into each of the four tubes. Mix well and incubate in a
water bath at 45°C for 60 minutes.
10. Agitate the tubes frequently and observe the relative rates of solution in the four tubes.
The differences in rates of digestion should be quite distinct. (Hint: two of the four tubes
should show some real enzyme activity.)
11. Record all observations

II. The second portion of the gastric enzymes experiment involves renin, another enzyme of
great importance in gastric digestion. Its efficacy depends on the presence of calcium salts for
precipitation of end products.
1. To illustrate this action, prepare four 25 x 200 mm test tubes as shown by the following
table:
Tube Milk Additions
1. 10 ml 2 ml of water
2. 10 ml 1 ml 1 N K2C204 and 1 ml water
3. 10ml 1.25ml 1N CaCl2 and 1 ml K2C204
4. 0 1 ml 1 N K2C204 and 1 ml water

2. Set the four tubes in a water bath at 40 deg c and add 1 ml of a renin solution prepared
by grinding 5 grams C of renin with sand and adding 25 ml of water.
3. Record the results of curdling action after 15 minutes have elapsed.
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Learning Activity No. 3

ENZYMES ACTIVITY IN INTESTINAL DIGESTION

Specific Learning Objectives:

1.
2.
3.

Time Allotment: 3 hours

Theoretical Background:
The small intestine is where most chemical digestion takes place. Most of the digestive
enzymes in the small intestine are secreted by the pancreas and enter the small intestine via
the pancreatic duct.
The three pancreatic enzymes
• Pancreatic proteases (such as trypsin and chymotrypsin) - digest proteins.
• Pancreatic amylase - which helps to digest sugars (carbohydrates).
• Pancreatic lipase - which helps to digest fat.
Chemical Digestion in the Small Intestine
These enzymes enter the small intestine in response to the hormone cholecystokinin, which is
produced in response to the presence of nutrients. The hormone secretin also causes
bicarbonate to be released into the small intestine from the pancreas to neutralize the
potentially harmful acid coming from the stomach.
The three major classes of nutrients that undergo digestion are proteins, lipids (fats), and
carbohydrates.
Proteins
Proteins are degraded into small peptides and amino acids before absorption. Their chemical
breakdown begins in the stomach and continues through the large intestine. Proteolytic
enzymes, including trypsin and chymotrypsin, are secreted by the pancreas and cleave proteins
into smaller peptides. Carboxypeptidase, a pancreatic brush border enzyme, splits one amino
acid at a time. Aminopeptidase and dipeptidase free the end amino acid products.
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Lipids
Lipids (fats) are degraded into fatty acids and glycerol. Pancreatic lipase breaks down
triglycerides into free fatty acids and monoglycerides. Pancreatic lipase works with the help of
the salts from bile secreted by the liver and the gallbladder. Bile salts attach to triglycerides and
help to emulsify them; this aids access by pancreatic lipase because the lipase is water-soluble,
but the fatty triglycerides are hydrophobic and tend to orient toward each other and away from
the watery intestinal surroundings. The bile salts act to hold the triglycerides in their watery
surroundings until the lipase can break them into the smaller components that are able to enter
the villi for absorption.
Carbohydrates
Some carbohydrates are degraded into simple sugars, or monosaccharides (e.g., glucose,
galactose) and are absorbed by the small intestine. Pancreatic amylase breaks down some
carbohydrates (notably starch) into oligosaccharides. Brush border enzymes take over from
there. The most important brush border enzymes are dextrinase and glucoamylase, which
further break down oligosaccharides. Other brush border enzymes are maltase, sucrase, and
lactase. Lactase is absent in most adult humans and for them lactose, like most poly-
saccharides, is not digested in the small intestine. Some carbohydrates, such as cellulose, are
not digested at all, despite being made of multiple glucose units. This is because the cellulose is
made from beta-glucose that makes the intermonosaccharidal bindings different from the ones
present in starch, which consists of alpha-glucose. Humans lack the enzyme for splitting the
beta-glucose-bonds—that is reserved for herbivores and bacteria in the large intestine.

Learning Resources:
10% Pancreatin or Toluene Bile
Pancreatic lipase Olive oil
Cheesecloth Distilled water

Procedure:
Pancreatin or pancreatic lipase acts in the body to metabolize fatty substances.
1. To illustrate this action, prepare 100 ml of a 10% solution of pancreatin.
2. Allow it to stand for 24 hours and strain any residue through cheesecloth.
3. Then add 1 drop of toluene and allow it to digest overnight at 40°C.
4. Now prepare nine test tubes according to the table below, using olive oil or another
suitable fatty acid substance.
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Tube Olive Oil(ml) Water(ml) Others Enzyme solution in mL

l 0.5 4.5 0 5.0
2 0.5 4.5 0 5.0 (boil 5 min)
3 0 4.5 0 5.0
4 0 9.5 0 0
5 0.5 9.5 0 0
6 0.5 8.5 1ml bile 0
7 0.5 3.5 1ml bile 5.0
8 0 8.5 1ml bile 0
9 0 3.5 1ml bile 5.0