Download Lab #8 Prelab: Protein, Triglycerides, and Esters Lab

Chemistry 108 Protein, Triglycerides, and Esters Lab Lab #8 Prelab: Protein, Triglycerides, and Esters Lab
Purpose of the lab:
In Part 1, you will make cheese by separating the caseins from milk. In part 2, you will make soap
using the saponification of triglycerides reaction. In part 3, you will synthesize two esters that smell
very good!
Part 1: Proteins in milk: Separating the Caseins from Milk
The name protein is taken from the Greek protelos, which means first. This name is well chosen. Of all
chemical compounds, proteins could certainly be ranked first, for they are the substance of life. Proteins
make up a large part of the animal body, they hold it together, and they run it. They are found in all
living cells. They are the principal material of skin, muscle, tendons, nerves, and blood; of enzymes,
antibodies, and many hormones. (Only the nucleic acids, which control heredity, can challenge the
position of proteins; and the nucleic acids are important because they direct the synthesis of proteins.)
"Chemically, proteins are high polymers. They are polyamides, and the monomers from which they are
derived are the α-amino carboxylic acids. A single protein molecule contains hundreds or even
thousands of amino acids units; these units can be of twenty-odd different kinds. The number of
different protein molecules that are possible, is almost infinite. It is likely that tens of thousands of
different proteins are required to make up and run an animal body; and this set of proteins is not
identical with the set required by an animal of a different kind."
- Morrison & Boyd's Organic Chemistry
In this lab activity we will study proteins - described so eloquently in the above quote from Morrison &
Boyd's Organic Chemistry- from several different viewpoints. In Part 1, we will separate some amino
acids (including an unknown) by using paper chromatography. In Part 2 we will use a pH meter to
measure the acidity of solutions of three amino acids. In Part 3 we will separate
the caseins from milk. Caseins (from Latin caseus "cheese") make up about 80%
of the proteins in milk. It is interesting that proline (see figure), an
unsymmetrical amino acids makes up 20% of the amino acids residues in some
caseins. In proline, the side chain makes a ring structure with the amino group.
The result is a lack of ordered internal protein secondary structure and probably
facilitates attack by digestive enzymes in infants. The rest of the milk protein is a
mixture of soluble proteins that includes the albumins and globulins; these are
commonly called the whey proteins.
Casein consists of four different proteins, usually referred to as α, β. γ, and κ
caseins. The α, β, and κ forms are most abundant, making up roughly 50%, 33%, and 15% of the
casein, respectively. Casein exists in milk as the calcium salt, calcium caseinate. The protein has a
negative overall charge and its charge is balanced by the positive calcium ions. Surprisingly, though, it
is found that calcium ions, at the concentration of Ca2+ normally found in milk, cause α and β casein,
singly or in combination to precipitate. However, in milk, the κ casein is soluble and is thought to
surround the α and β casein forming a soluble micelle. Disruptions, such as denaturation or enzymatic
bond cleavage, of the micelles cause the casein proteins to coagulate; this is what the cheese-making
process does. This can be done with acids, bases, temperature, mechanical agitation, or with certain
enzymes (as in the case of yogurt); we will use acetic acid in the lab activity.
Chemistry 108 Protein, Triglycerides, and Esters Lab Part 2: Saponification of Triglycerides: Soap Making
Soaps are amphipathic molecules used for cleaning and bathing. Soaps molecules are the base form,
the carboxylate ions, of fatty acids. Soaps are made using the saponification reaction that converts a
triglyceride molecule into 3 carboxylate ions (soap molecules) and a glycerol molecule. Since
carboxylate ions have a polar head and a nonpolar tail, they are able to emulsify oil and nonpolar
substances in the cleaning and bathing processes. An example of the saponification reaction is given
below.
Note that the fatty acid residues contained in the reactant triglyceride are arbitrary; any fatty acid
residues could have been used.
This reaction is simply three "hydrolysis of an ester" reactions. We first saw the hydrolysis of an ester
reaction in chapter 6 then once again in chapter 10. In the saponification of triglycerides, the reaction
is catalyzed by a strong base. Since the reaction occurs in a strong base (pH > pKa of fatty acids), it is
the base form of the fatty acids, the carboxylate ions, which are produced. The base used for catalysis
is usually sodium hydroxide. Sodium hydroxide is also called "lye". When one makes soap using
sodium hydroxide, the soap is called "lye soap".
Chemistry 108 Protein, Triglycerides, and Esters Lab History of Soap Making (From Wikipedia):
(Note: I have removed the references from the literature cited, however, they can be found on the
Wikipedia site if you want more information or you wish to use this information in a report for another
class)
Early history. The earliest recorded evidence of the production of soap-like materials dates back to
around 2800 BC in ancient Babylon. A formula for soap consisting of water, alkali, and cassia oil was
written on a Babylonian clay tablet around 2200 BC.
The Ebers papyrus (Egypt, 1550 BC) indicates the ancient Egyptians bathed regularly and combined
animal and vegetable oils with alkaline salts to create a soap-like substance. Egyptian documents
mention a soap-like substance was used in the preparation of wool for weaving.
In the reign of Nabonidus (556–539 BC), a recipe for soap consisted of uhulu [ashes], cypress [oil] and
sesame [seed oil] "for washing the stones for the servant girls".
Ancient Rome. The word sapo, Latin for soap, first appears in Pliny the Elder's Historia Naturalis,
which discusses the manufacture of soap from tallow and ashes, but the only use he mentions for it is
as a pomade for hair; he mentions rather disapprovingly that the men of the Gauls and Germans were
more likely to use it than their female counterparts. Aretaeus of Cappadocia, writing in the first
century AD, observes among "Celts, which are men called Gauls, those alkaline substances that are
made into balls, called soap".
A popular belief claims soap takes its name from a supposed Mount Sapo, where animal sacrifices
were supposed to have taken place; tallow from these sacrifices would then have mixed with ashes
from fires associated with these sacrifices and with water to produce soap, but there is no evidence of a
Mount Sapo in the Roman world and no evidence for the apocryphal story. The Latin word sapo
simply means "soap"; it was likely borrowed from an early Germanic language and is cognate with
Latin sebum, "tallow", which appears in Pliny the Elder's account. Roman animal sacrifices usually
burned only the bones and inedible entrails of the sacrificed animals; edible meat and fat from the
sacrifices were taken by the humans rather than the gods.
Zosimos of Panopolis, circa 300 AD, describes soap and soapmaking. Galen describes soap-making
using lye and prescribes washing to carry away impurities from the body and clothes. According to
Galen, the best soaps were Germanic, and soaps from Gaul were second best. This is a reference to
true soap in antiquity.
Ancient China. Soap, or more accurately a detergent similar to soap, was manufactured in ancient
China from vegetation and herbs. True soap, made of animal fat, did not appear in China until the
modern era. Soap-like detergents were not as popular as ointments and creams.
Middle East. A 12th-century Islamic document describes the process of soap production. It mentions
the key ingredient, alkali, which later becomes crucial to modern chemistry, derived from al-qaly or
"ashes".
By the 13th century, the manufacture of soap in the Islamic world had become virtually industrialized,
with sources in Nablus, Fes, Damascus, and Aleppo.
Medieval Europe. Soapmakers in Naples were members of a guild in the late sixth century, and in
the eighth century, soap-making was well known in Italy and Spain. The Carolingian capitulary De
Chemistry 108 Protein, Triglycerides, and Esters Lab Villis, dating to around 800, representing the royal will of Charlemagne, mentions soap as being one
of the products the stewards of royal estates are to tally. Soapmaking is mentioned both as "women's
work" and as the produce of "good workmen" alongside other necessities such as the produce of
carpenters, blacksmiths, and bakers.
15th–19th centuries. In France, by the second half of the 15th century, the semi-industrialized
professional manufacture of soap was concentrated in a few centers of Provence— Toulon, Hyères,
and Marseille — which supplied the rest of France. In Marseilles, by 1525, production was
concentrated in at least two factories, and soap production at Marseille tended to eclipse the other
Provençal centers. English manufacture tended to concentrate in London.
Finer soaps were later produced in Europe from the 16th century, using vegetable oils (such as olive
oil) as opposed to animal fats. Many of these soaps are still produced, both industrially and by smallscale artisans. Castile soap is a popular example of the vegetable-only soaps derived by the oldest
"white soap" of Italy.
Modern Times. In modern times, the use of soap has become universal in industrialized nations due
to a better understanding of the role of hygiene in reducing the population size of pathogenic
microorganisms. Industrially manufactured bar soaps first became available in the late 18th century, as
advertising campaigns in Europe and the United States promoted popular awareness of the relationship
between cleanliness and health.
Until the Industrial Revolution, soapmaking was conducted on a small scale and the product was
rough. Andrew Pears started making a high-quality, transparent soap in 1789 in London. His son-inlaw, Thomas J. Barratt, opened a factory in Isleworth in 1862. William Gossage produced low-priced,
good-quality soap from the 1850s. Robert Spear Hudson began manufacturing a soap powder in 1837,
initially by grinding the soap with a mortar and pestle. American manufacturer Benjamin T. Babbitt
introduced marketing innovations that included sale of bar soap and distribution of product samples.
William Hesketh Lever and his brother, James, bought a small soap works in Warrington in 1886 and
founded what is still one of the largest soap businesses, formerly called Lever Brothers and now called
Unilever. These soap businesses were among the first to employ large-scale advertising campaigns.
Liquid Soap (Detergent). Liquid soap was not invented until the 1800s. In 1865, William Shepphard
patented liquid soap. In 1898, B.J. Johnson developed a soap formula, and his company (the B.J.
Johnson Soap Company) introduced Palmolive soap the same year. This new soap was made of palm
and olive oils and became popular in a short amount of time; Palmolive became so popular that B.J.
Johnson Soap Company changed its name to Palmolive. At the turn of the century, Palmolive was the
world's best-selling soap.
In the early 1900s, other companies began to develop their own liquid soap. Products such as Pine-Sol
and Tide appeared on the market, making the process of cleaning clothing, counters and bathrooms
easier.
As a detergent, liquid soap tends to be more effective than flake soap, and there is a smaller chance of
residue being left on clothing with liquid soap. Liquid soap also works better for more traditional
washing methods, such as using a washboard.
Chemistry 108 Protein, Triglycerides, and Esters Lab Part 3: Esterification
When an carboxylic acid (R-COOH) and an alcohol (R-OH) are mixed together and heated in the
presence of an acid catalyst (such as H2SO4), the two will react to form an ester (plus H2O). This
process is called esterification. Each ester has its own unique odor, and with a discriminating nose, one
can use this fact to help identify them. In this lab you will be reacting various organic acids (acetic acid
& salicylic acid) with various alcohols (1-pentanol & ethanol). You will make two different esters with
odors that should be familiar to you. You may have noticed that esterification is the reverse reaction
of the hydrolysis of esters seen in the saponification reaction.
Esterification: Addition of an alcohol with a strong acid catalyst creates an ester.
O
||
R–C–O–H
H+
+ H – O – R'
O
||
R – C – O – R' + H2O
PRELAB QUESTIONS:
1. Draw the predominant for the dipeptide Tyr-Phe at pH = 1, 7, and 14.
pH = 1
pH = 7
pH = 14
Chemistry 108 Protein and Esters Lab 2) Draw a triglyceride (do not draw the triglyceride used in the example on page 2 of this prelab, you
can draw any other triglyceride) :
3) Draw the products of the saponification of the triglyceride that you drew in question #2 above:
4. Draw and name the ester that would be formed in the reaction of propanoic acid with ethanol:
(see chapter 10 lecture notes if you need a reminder about naming esters)
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