LAB 19: AMINES & AMIDES:

ISOLATION OF CAFFEINE FROM TEA, HYDROLYSIS OF UREA PURPOSE: To isolate caffeine from tea.
To utilize green chemistry principles in the recovery of solvents by simple distillation. To perform basic hydrolysis of the amide urea.

SAFETY CONCERNS:
Always wear safety goggles. Amines have pungent odors. Avoid skin contact with aniline, a skin irritant and deadly poison. Wash your hands after use. .

AMINES:
Structure and Classification of Amines:
Amines are derivatives of ammonia, NH3, in which one or more hydrogen atoms of ammonia have been replaced by an organic group. They are classified as primary, secondary, or tertiary, according to the number of groups attached to the nitrogen. NH2 H H CH3
NH3 Ammonia CH3 N H CH3 N CH3 CH3 N CH3 Aniline (an aromatic amine)

Methylamine (primary, 1o)

Dimethylamine (secondary, 2o)

Trimethylamine (tertiary, 3o)

Amines are often found as part of compounds that are physiologically active or used in medications.

OH HO

H N H CH3 N N NH2

H N CH3 Methamphetamine CH3

Neo-Synephrine

Histamine

Amines in Water:
In water, ammonia and soluble amines (with one to four carbons) act as weak bases because the unshared pair of electrons on the nitrogen atom attracts protons. The products are an ammonium ion or alkyl ammonium ion and a hydroxide ion.

H H N H Ammonia + H2O Water

H H N H H Ammonium ion + OH Hydroxide Ion

H CH3 N H + H2O Water CH3

H N H + OH Hydroxide Ion H Methylammonium ion

Methylamine

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Neutralization of Amines with Acids:

Because amines are basic they react readily with acids to form salts. Amine salts are much more soluble in water than the corresponding amines. H H
CH3 N H + HCl Acid CH3 N H H Cl

Amine Salt Amines (free bases) may be regenerated from the salt by treatment with a base such as sodium hydroxide. H H

Amine Base

CH3

N H

Cl

NaOH

CH3

N H

H2O

NaCl

H Amine Salt

Amine Base

ALKALOIDS:
Alkaloids are naturally occurring nitrogen compounds having pronounced physiological activity. Like other simpler amines, alkaloids and bases; the name alkaloid comes from their alkaline properties. These complex molecules come from plants and many have medical uses. They are generally insoluble in water, and are often found in commercial products in the form of their water-soluble acid salts.
Alkaloid + Acid (water insoluble) Alkaloid Acid Salt (water soluble)
Alkaloid Acid Salt + (water soluble) Base Alkaloid (water insoluble)

The pure alkaloid (in its free base form) can be obtained as a precipitate from its acid salt by treatment with a base such as sodium hydroxide. Some of the better known alkaloids include atropine, used to dilate the pupils of the eyes; morphine, a painkiller; heroin, a synthetic modification of morphine that is a painkiller and narcotic; codeine, a painkiller and cough-suppressant; nicotine, a component of cigarette smoke and also a potent insecticide; reserpine, a tranquilizer; and cocaine, a local anesthetic and narcotic.
H H

HO H CH3O N

N H

H2SO4 CH3O

HO H

N HH

SO42-

N 2 Quinine Hydrogen Sulfate: Water soluble salt. Fluorescent under black light.

Quinine: Antimalarial; skeletal muscle relaxant. 0.8-4% in dried Cinchona bark.

You saw base properties of Quinine in a previous lab. Most alkaloids taste bitter, and quinine is frequently used as a standard reference for bitterness in taste studies. The bitter taste of coffee and tea is due to caffeine. The alkaloid you will study in this experiment is caffeine.

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Caffeine:
The alkaloid caffeine is found in coffee, tea, and kola nuts. It is a mild central nervous stimulant causing alertness and the ability to put off sleep. It is sometimes used to treat migraine headaches and can somewhat relieve asthma attacks. Combination pain relievers like Excedrin often contain both aspirin and caffeine.

O H3C O N N CH3 Caffeine N N

CH3 N

NH2 N N N H Adenine (A) H H2N N

O N N N

H Guanine (G)

Coffee beans contain 2-5% caffeine, about the same amount present in tea leaves. Decaffeinated coffee is made when one of several solvents is used to extract the caffeine from the beans prior to roasting. There was once concern about the safety of the widely used solvent trichloroethylene; traces of the solvent remain the decaffeinated coffee. The safer trichloroethane and the much safer liquid carbon dioxide are now used as solvents for the extraction. Cola soft drink beverages are made from extract of the kola (cola) nut; the extract contains caffeine. Cola drink bottlers add phosphoric acid, caramel, sweeteners, and carbonated water. The U.S. Food and Drug Administration (FDA) requires that a "cola" contain some caffeine but limits the amount that can be present. Manufacturers usually remove all the caffeine from the kola extract and then add the correct amount. Caffeine is also added to some non-cola soft drinks. Caffeine has little effect on the blood pressure in minute quantities but large amounts increase blood pressure. Individuals who drink coffee, tea, or cola drinks in large amounts can develop both a tolerance for and a dependence on caffeine. Heavy users can experience withdrawal symptoms of lethargy, headache, and even nausea after 18 hours of abstinence. The structure of caffeine is very similar to that of adenine and guanine--important components of the genetic material DNA. This similarity has raised concern that caffeine might cause cancer or birth defects. So far there is little evidence to support this concern. Some people consider caffeine to be an addictive drug and some religions prohibit use of beverages containing caffeine.

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AMIDES:
Structure and Classification:
Amides are ammonia or amine derivatives of carboxylic acids. They may be classified as simple (nonsubstituted), monosubstituted, or disubstituted.

O R C N H H Nonsubstituted Amide
Some common examples of amides are: O

O R C N R H Monosubstituted Amide
O NH C CH3 O

O R C N R R Disubstituted Amide

CH3 C N H H Acetamide CH3

O C NH2 NH2

O C NH2 Urea

C N CH3 H Benzamide

N-Methylacetamide

Acetanilide

Amide Preparation:
Amides can be synthesized by several methods. When a carboxylic acid reacts with a basic amine an amine salt forms. When amine salts are heated it is possible to drive off water to form amide bonds.

O CH3 C O H +

H H N CH3 Methyl Amine An Amine CH3

O C O

H H N CH3

Acetic Acid A Carboxylic Acid

H Methylammonium Acetate An Amine Salt Heat O CH3 C H N CH3 + H O H

N-Methylacetamide An Amide
The high heat needed to form amides from carboxylic acids and amines causes racemization (loss of handedness) decomposition or unwanted side reactions in compounds with reactive side groups (like alcohols). Thus, milder alternatives for forming amides are required using carboxylic acid derivatives like esters or anhydrides.

O CH3

O +

H H N CH3 Methyl Amine An Amine CH3

O C

H N CH3 +

O H O C CH3 Acetic Acid

C O C CH3

Acetic Anhydride

N-Methylacetamide An Amide

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Amide Hydrolysis:
Amides, like proteins, can easily be hydrolyzed. Amides can be broken down into carboxylic acids and ammonia (or amines). Hydrolysis takes place in either an acid or a base. Acid hydrolysis produces the carboxylic acid and ammonium salt.

O CH3 C

H N H + H O H HCl CH3

O C OH +

H H N H Cl H Ammonium Chloride

Acetamide

Acetic Acid

In a base, the hydrolysis reaction produces the salt of the carboxylic acid and ammonia (or the amine). The odor of ammonia and the reaction of ammonia with litmus paper are used to detect the hydrolysis reaction of unsubstituted amines.

O CH3

H NaOH CH3

O C O Na +

H H N H Ammonia (a gas)

C N H + H O H Acetamide (an unsubstituted amine)

Sodium Acetate

Urea:
Urea is a waste product of protein metabolism; urea is removed from the blood by the kidneys and eliminated in the urine. Urea is used as a fertilizer and to make plastic resins.

Proteins:
The reactions of amines and amides resemble reactions of proteins in body chemistry. In later studies we shall see that proteins are complex amides that are hydrolyzed in the process of digestion to amino acids-compounds containing both an amino (-NH2) group and a carboxyl (-COOH) group. The amino acids are absorbed into the bloodstream and transported to the cells, where they are reformed into proteins needed by the body.
Hydrolysis (Digestion) Amide synthesis (Reactions in cells)

Proteins in Food (Amides)

H2O

Amino Acids (Amines and acids)

Body Proteins (Amides)

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PROCEDURES:
ACTIONS:

NOTES:
1

I. ISOLATION OF CAFFEINE:
1. Pour 200 mL of concentrated black tea solution1 into your largest
Erlenmeyer flask.

Other caffeinated beverages could also be used including coffee, or sodas (such as colas, Jolt or Mountain Dew).
2

2. Mix in sodium carbonate until the solution is basic.2 3. Pour the basic beverage solution into a 250 or 500 mL separatory
funnel and add 25 mL of ethyl acetate, (CH3CO2CH2CH3). Holding the stopper in place, gently mix the contents by inverting the funnel two or three times (burping often).
3

Sodium carbonate is added to remove benzoic and tannic acids that may be present. Monitor the pH with pH paper.
3

The IUPAC name of ethyl acetate (CH3CO2CH2CH3) is ethyl ethanoate.

4

4. Suspend the separatory funnel with an iron ring and allow the ethyl
acetate and water layers to separate. 4

5. Carefully drain the lower aqueous layer into a beaker. Try to not to
leave any of the water layer behind.

You should see two distinct layers form after a few minutes, with the ethyl acetate layer at the top. Sometimes an emulsion may form at the juncture of the two layers. The emulsion often can be broken by gently swirling the contents or by gently stirring the emulsion with a glass rod.
5

6. Pour the top ethyl acetate layer (now containing caffeine) out the
top of the funnel into a clean, dry Erlenmeyer flask.
5

7. Return the previously drained aqueous layer to the separatory funnel

and wash it with another 25 mLs of ethyl acetate.

When using a separatory funnel, always drain the bottom layer out the bottom and pour the top layer out the top.
6

8. Repeat the separation of layers and combine the top ethyl acetate
layer (carrying more caffeine) with the previous layer already saved.

When enough drying agent is added there will be sandy grains of the agent left. If not enough drying agent has been added it will appear clumpy wet at the bottom of the flask.
7

9. To the Erlenmeyer flask of combined ethyl acetate/caffeine layers

add enough drying agent (anhydrous sodium sulfate or anhydrous magnesium sulfate) to dry the layer.

Weigh the watch glass on an analytical balance (to +0.0001g if available). Keep all significant figures.
8

10. Decant the dry ethyl acetate solution from the drying agent into a
100 mL round bottom flask.

11. Weigh7 and record the mass of a watch glass for use later. 12. Clamp the 100 mL rbf of ethyl acetate/caffeine to a ring stand about
6-8 inches above a laboratory burner. Add boiling stones and set up a glass simple distillation apparatus equipped with a thermometer, and water-cooled condenser8. Place a clean dry collection container at the end of the condenser.

13. Heat the flask over the low flame of a Laboratory burner and distill
off the ethyl acetate solvent9 just until a couple mLs remain in the round bottom flask.
9 o

Ethyl acetate boils at about 77 C.

14. Stop the distillation and pour the remaining couple mLs of ethyl
acetate/caffeine onto the weighed watch glass.

15. Allow the remaining ethyl acetate to evaporate from the watch glass
and leave the caffeine behind.

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CH105 Lab 19: Amines & Amides (S12)

16. Weigh the watch glass with caffeine and subtract the mass of
the watch glass to determine the yield of caffeine.
10

10

17. Describe the odor and appearance of the crude caffeine. 11 18. Add your distilled and recovered ethyl acetate solvent to a
designated collection container to be recycled for future use.

The percent (m/v %) of caffeine in your sample can be calculated since you know the grams of caffeine produced and you know the volume of beverage with which you began.
11

II. CHARACTERIZATION OF CAFFEINE

19. Compare the identity and purity of the isolated caffeine to that
of known caffeine by thin layer chromatography on a fluorescent silica plate as follows:7 A. Dissolve a little crude caffeine by adding a couple drops of acetone to your watch glass. Swirl to mix. B. Prepare a 4 cm x 9 cm fluorescent silica TLC plate as shown using a pencil to mark 3 origin locations (A, B, & C). 12 C. On origins A and B with a capillary tube place several spots of your crude caffeine allowing each spot to dry before applying another. 13 D. On origins B and C place several spots of a known sample of caffeine allowing each spot to dry before applying another.14 E. Place the plate into a 400 mL beaker containing 15 mLs of developing solvent (75% ethyl acetate and 25% hexane). 15 F. Cover with a lid or plastic wrap16 and allow the beaker to remain undisturbed as the solvent moves up the plate. G. When the solvent has risen almost to the top of the plate, remove the plate and draw a pencil line along the solvent front quickly before it dries. H. When the solvent has evaporated from the TLC plate, observe it under UV light. Circle each dark spot and draw a picture of the spots on your report sheet. I. Make a conclusion as to the identity and purity of your isolated caffeine.

(Optional) The caffeine may be purified by sublimation--the process of changing from a solid directly to the gaseous state. The vapor produced will solidify on a cold surface. To sublime, place an evaporating dish filled with cold water over a beaker of crude caffeine and heat the beaker on a steam bath. The crystals that form on the underside of the dish are purified caffeine.
12

Handle the plates at the edge only to avoid transferring substances from your fingers and to avoid rubbing the silica from the plate. Any markings or labels must be drawn with pencil rather than pen to avoid separation of any ink pigments. Markings must be made lightly to avoid scraping the silica from the plate.

A
13

B C

~1 cm

When the sample spot dries you can apply again. The spot must be kept small and not allowed to flow into larger spots. Your caffeine will show dark under UV light which can help you judge if enough compound has been applied to the plate.
14

IV. HYDROLYSIS OF AMIDES:

1. Place a few crystals of urea into a test tube and add about 2
mL of 6M sodium hydroxide (NaOH).

2. Place the test tube in a boiling water bath. 3. Wet a piece of pH paper with water and hold it over the mouth
of the test tube.

You are placing spots of your isolated caffeine and spots of known caffeine on the same origin mark B in what is called a cospot. If the two samples you have are different then they will separate by chromatography into two spots. However, if they are the same then only one sample spot will result.
15

Keep the solvent level below the origin so that the solvent does not wash the samples away.
16

4. Heat the tube gently for about 5 minutes. Record any changes
in the color of the pH paper.

5. Cautiously note any odor coming from the tube and

determine the identity of the gas produced.

6. Write the equation for the hydrolysis of urea in base.

CH105 Lab 19: Amines & Amides (S12)

The developing chamber must be covered to avoid evaporation of the solvent from the plate and from the chamber itself. The sides of the TLC plates should not touch the sides of the chamber. The solvent must rise up the plate from the bottom only and not from the sides of the plate.

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CH105 Lab 19: Amines & Amides (S12)

LAB 19: AMINES & AMIDES:

PRE LAB EXERCISES:
Classify each of the following amines as A. primary, B. secondary, C. tertiary,

NAME_____________ DATE______________
D. quaternary.

CH3

___1.

CH3CH2

N CH2CH3

H N
___2.

CH3

CH3

H2N
___3.
NH2

___4.

___5.

If a substituted amide is hydrolyzed in base the products will be ______ A. a carboxylic acid and ammonia B. a carboxylic acid and a 1o or 2 o amine C. a carboxylic acid salt and ammonia D. a carboxylic acid salt and a 3 o amine o o E. a carboxylic acid salt and a 1 or 2 amine F. a carboxylic acid and an amine salt If an amide is hydrolysed in the presence of base and ammonia gas is produced what might we conclude about the structure of the amide? A. The amide was unsubstituted B. The amide was monosubstituted C. The amide was disubstituted D. The amide must have been urea. When preparing a TLC plate what precautions should you use? List all. A. handle plate only by edges to avoid getting fingerprints on the plate. B. handle plate only by edges to avoid rubbing off the silica. C. label the origin spot placement with pen so that it will be permanent. D. keep the spots small by spotting with a tiny capillary tube and then drying.

___6.

___7.

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CH105 Lab 19: Amines & Amides (S12)

LAB 19: AMINES & AMIDES:

REPORT: I. Isolation of Caffeine from Tea:

NAME___________________ PARTNER_________DATE___

m/v % of caffeine in tea (show calculations) mLs sample used = _______________mL gs of Caffeine obtained = _____________g = m/v%

Observations: Describe the caffeine

produced

Chromatography: Recreate the final chromatogram and

label the spots. Make conclusions as to the purity of your isolated caffeine.

B C

Show the equation for the reaction of benzoic acid with sodium carbonate used in your experiment. Use structures (not formulas) for organic compounds. Account for any gases produced.

What is the chemical reasoning for reacting sodium carbonate with acids in a tea? Why?

Explanation/Analysis: Why were the results the way they were?

Explain any anomalies.

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II. Hydrolysis of Urea:

Observations:

Show the equation for hydrolysis of Urea with base. Use structures (not formulas) for organic compounds.

Name the products formed:

___1. According to my results, the pH paper held over the basic hydrolysis of urea ____ A. turned basic B. turned acidic C. stayed the same ___2. The pH paper reacted as it did because of the presence of ________________________ A. Ammonia gas B. methylamine C. sodium hydroxide D. Urea

Explanation/Analysis of results:
Are your experimental results as expected or are there discrepancies? Explain.

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CH105 Lab 19: Amines & Amides (S12)

RELATED EXERCISES:
1.___ What are the products of acid catalyzed hydrolysis of a substituted amide?
A. B. C. D. E. a carboxylic acid and an ammonium salt a carboxylic acid salt and a primary or secondary amine a carboxylic acid and a primary or secondary amine salt a carboxylic acid and a tertiary amine salt a carboxylic acid salt and tertiary amine

2.___ Which of the following amides would produce ammonia upon hydrolysis in base?
A. N-methylacetamide 3. B. benzamide C. N,N-diethylaniline

Draw the equation for the reaction of atropine with sulfuric acid:

CH3 O O C OH H2SO4

Atropine

4.

The artificial sweetener aspartame (NutraSweet) is a dipeptide, a very short protein molecule. The sweetness of aspartame was discovered accidentally in 1965 when a careless chemist licked his dirty fingers. He was fortunate that the compound was not toxic; his company was fortunate that he was curious to discover why his fingers were so sweet. On each arrow in the following aspartame molecule, label the indicated functional group:

A. alcohol E. ester

B. amine F. ether

C. carboxylic acid G. aldehyde

D. amide H. ketone

H H O H H O H N C C N C CH2 C OH O Aspartame (Nutrasweet) CH2

H H NaOH H2O H

C O C

5.

Complete the equation for the basic hydrolysis of aspartame (Nutrasweet)

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CH105 Lab 19: Amines & Amides (S12)