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The solubility of amino acids and proteins is largely dependent on the solution pH.

The structural changes in

an amino acid or protein that take place at different pH values alter the relative solubility of the molecule. In
acidic solutions, both amino and carboxylic groups are protonated. In basic solutions, both groups are
deprotonated. Amino acids are essentially soluble in water. Their solubilities in water, dilute alkali and
dilute acid vary from one compound to the other depending on the structure of their side chains.
Ninhydrin (triketohydrindene hydrate) is a chemical used to detect ammonia or primary and secondary
amines. Amino acids also react with ninhydrin at pH=4. The reduction product obtained from ninhydrin
then reacts with NH3 and excess ninhydrin to yield a blue colored substance. This reaction provides an
extremely sensitive test for amino acids.
Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can
undergo reactions that are characteristics of benzene and benzene derivatives. One such reaction is the
nitration of a benzene ring with nitric acid. The amino acids that have activated benzene ring can readily
undergo nitration. This nitration reaction, in the presence of activated benzene ring, forms yellow product.
This test is mostly applied to tyrosine, tryptophan, phenylalanine and glutamic acid.
The Biuret Test positively identifies the presence of proteins (not less than two peptides). The reaction in
this test involves the complex formation of the proteins with Cu2+ ions in a strongly alkaline solution. This
test is mostly applied to gelatin, casein and albumin.
The precipitation of a protein occurs in a stepwise process. The addition of a precipitating agent and steady
mixing destabilizes the protein solution. Mixing causes the precipitant and the target product to collide.
Enough mixing time is required for molecules to diffuse accross the fluid.
The precipitation of a protein by neutral salt is commonly known as salting-out method. Addition of a
neutral salt, such as ammonium sulfate, compresses the solvation layer and increases the protein-protein
interaction. As the salt concentration of a solution is increased, more of the bulk water becomes associated
with the ions. As a result, less water is available to take part in the solvation layer around the protein, which
exposes hydrophobic parts on the protein surface. Therefore, proteins can aggregate and form precipitates
from the solution. The amount of neutral salt required to cause protein precipitation varies with the nature of
the protein and the pH of the solution. Apply this test to all the proteins provided.
Milk is a food of exceptional interest. Not only is milk an excellent food for the very young, but

humans have also adapted milk, specifically cows milk, as a food subtance for persons of all ages.
Many specialized milk products like cheese, yogurt, butter, and ice cream are staples of our diet.
Milk is probably the most nutritionally-complete food that can be found in nature. This property is
important for milk, since it is the only food young mammals consume in the nutritionally significant
weeks following birth. Whole milk contains vitamins (principally thiamine, riboflavin, pantothenic acid,
and vitamins A, D, and K), minerals (calcium, potassium, sodium, phosphorus, and trace metals),
proteins (which include all the essential amino acids), carbohydrates (chiefly lactose), and lipids (fats).
The only important elements in which milk is seriously deficient are iron and Vitamin C. Infants are
usually born with a storage supply of iron large enough to meet their needs for several weeks. Vitamin
C is easily secured through an orange juice supplement.
Whole milk is an oil-water type of emulsion, containing about 4% fat dispersed as very small (5-10
microns in diameter) globules. The globules are so small that a drop of milk contains about a million
of them. Because the fat in milk is so finely dispersed, it is digested more easily than fat from any other
source. The fat emulsion is stabilized to some extent by complex phospholipids and proteins that are
adsorbed on the surfaces of the globules. The fat globules, which are lighter than water, coalesce on
standing and eventually rise to the surface of the milk, forming a layer of cream. Since vitamins A and
D are fat-soluble vitamins, they are carried to the surface with the cream. Commercially, the cream is
often removed by centrifugation and skimming and is either diluted to form coffee cream (half and
half), sold as whipping cream, converted to butter, or converted to ice cream. The milk that remains
is called skimmed milk. Skimmed milk, except for lacking the fats and vitamins A and D, has
approximately the same composition as whole milk. If milk is homogenized, its fatty content will not
separate. Milk is homogenized by forcing it through a small hole. This breaks up the fat globules and
reduces their size to about 1 to 2 microns in diameter.
Thus, about two thirds of all the fatty acids in milk are saturated, and about one third are unsaturated.
Milk is unusual in that about 12% of the fatty acids are short-chain fatty acids (C2-C10) like butyric,
caproic, and caprylic acids.
Additional lipids (fats and oils) in milk include small amounts of cholesterol, phospholipids, and
lecithins (phospholipids conjugated with choline). The structures of phospholipids and lecithins are
shown. The phospholipids help to stabilize the whole milk emulsion; the phosphate groups help to
achieve partial water solubility for the fat globules. All the fat can be removed from milk by extraction
with petroleum ether or a similar organic solvent.

Proteins may be classified broadly in two general categories: fibrous and globular. Globular
proteins are those that tend to fold back on themselves into compact units that approach nearly
spheroidal shapes. These types of proteins do not form intermolecular interactions between protein
units (H bonds, and so on) as fibrous proteins do, and they are more easily solubilized as colloidal
suspensions. There are three kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins. All
are globular.
Casein is a phosphoprotein, which has phosphate groups are attached to some of the amino acid
side-chains. These are attached mainly to the hydroxyl groups of the serine and threonine moieties.
Actually, casein is a mixture of at least three similar proteins, principally caseins. These three proteins
differ primarily in molecular weight and amount of phosphorus they contain (number of phosphate
Casein exists in milk as the calcium salt, calcium caseinate. This salt has a complex structure. It is
composed of , , and caseins which form a micelle, or a solubilized unit. Neither the nor the
casein is soluble in milk, singly or in combination. If casein is added to either one, or to a combination
of the two, however, the result is a casein complex that is soluble owing to the formation of the micelle.
A structure proposed for the casein micelle is shown on the following page. The casein is thought
to stabilize the micelle. Since both and casein are phosphoproteins, they are precipitated by
calcium ions. Recall that Ca3(PO4)2 is fairly insoluble.
The casein protein, however, has fewer phosphate groups and a high content of carbohydrate bound
to it. It is also thought to have all its serine and threonine residues (which have hydroxyl groups), as
well as its bound carbohydrates, on only one side of its outer surfaces. This portion of its outer surface
is easily solubilized in water since these polar groups are present. The other portion of its surface binds
well to the water-insoluble and caseins and solubilizes them by forming a protective colloid or
micelle around them. Since the entire outer surface of the micelle can be solubilized in water, the unit
is solubilized as a whole, thus bringing the and caseins, as well as casein, into solution.
Calcium caseinate has its isoelectric (neutrality) point at pH 4.6. Therefore, it is insoluble in
solutions of pH less than 4.6. The pH of milk is about 6.6; therefore casein has a negative charge at
this pH and is solubilized as a salt. If acid is added to milk, the negative charges on the outer surface
of the micelle are neutralized (the phosphate groups are protonated) and the neutral protein
Ca2+Caseinate + 2HCl Casein + CaCl2
The calcium ions remain in solution. When milk sours, lactic acid is produced by bacterial action
(see below), and the consequent lowering of the pH causes the same clotting reaction. The isolation
of casein from milk will be carried out in this experiment.

The casein in milk can also be clotted by the action of an enzyme called rennin. Rennin is found in
the fourth stomach of young calves. However, both the nature of the clot and the mechanism of clotting
differ when rennin is used. The clot formed using rennin, calcium paracaseinate, contains calcium.
Ca2+Caseinate + rennin Ca2+Paracaseinate + a small peptide
Rennin is a hydrolytic enzyme (peptidase) and acts specifically to cleave peptide bonds between
phenylalanine and methionine residues. It attacks the casein, breaking the peptide chain so as to
release a small segment of it. This destroys the water-solubilizing surface of the casein, which
protects the inner and caseins and causes the entire micelle to precipitate as calcium
paracaseinate. Milk can be decalcified by treatment with oxalate ion, which forms an insoluble calcium
salt. If the calcium ions are removed from milk, a clot will not be formed when the milk is treated with
The clot, or curd, formed by the action of rennin is sold commercially as cottage cheese. The liquid
remaining is called the whey. The curd can also be used in producing various types of cheese. It is
washed, pressed to remove any excess whey, and chopped. After this treatment, it is melted, hardened
and ground. The ground curd is then salted, pressed into molds, and set aside to age.
Albumins are globular proteins that are soluble in water and in dilute salt solutions. They are,
however, denatured and coagulated by heat. The second most abundant protein types in milk are the
lactalbumins. Once the caseins have been removed, and the solution has been made acidic, the
lactalbumins can be isolated by heating the mixture to precipitate them. The typical albumin has a
molecular weight of about 41,000.
A third type of protein in milk is the lactoglobulins. They are present in smaller amounts than the
albumins and generally denature and precipitate under the same conditions as the albumins. The
lactoglobulins carry the immunological properties of milk. They protect the young mammal until its own
immune systems have developed.
When the fats and the proteins have been removed from milk, the carbohydrates remain, as they are
soluble in aqueous solution. The main carbohydrate in milk is lactose.
Lactose, a disaccharide, is the only carbohydrate that mammals synthesize. It is synthesized in the
mammary glands. Hydrolyzed, it yields one molecule of D-glucose and one of D-galactose.
In this process, one molecule of glucose is converted to galactose and joined to another of
glucose. The galactose is apparently needed by the developing infant to build brain and nervous
tissue. Brain cells contain glycolipids as a part of their structure. A glycolipid is a triglyceride in which
one of the fatty acid groups has been replaced by a sugar, in this case galactose. Galactose is more
stable (to metabolic oxidation) than glucose and affords a better material for forming structural units in

Although almost all human infants can digest lactose, some adults lose this ability on reaching
maturity, since milk is no longer an important part of their diet. An enzyme called lactase is necessary
to digest lactose. Lactase is secreted by the cells of the small intestine, and it cleaves lactose into its
two component sugars, which are easily digested. Persons lacking the enzyme lactase do not digest
lactose properly. As it is poorly absorbed by the small intestine, it remains in the digestive tract, where
its osmotic potential causes an influx of water. This results in cramps and diarrhea for the affected
individual. Persons with a lactase deficiency cannot tolerate more than one glass of milk a day. The
deficiency is most common among blacks and older whites.
Lactose can be removed from whey by adding ethanol. Lactose is insoluble in ethanol, and when
the ethanol is mixed with the aqueous solution, the lactose is forced to crystallize. This is the process
used in this experiment to isolate lactose from milk.
When milk is allowed to stand at room temperature, it sours. Many bacteria are present in milk,
particularly lactobacilli. These bacteria act on the lactose in milk to produce the sour lactic acid. These
microorganisms actually hydrolyze lactose and produce lactic acid only from the galactose portion of
the lactose. Since the production of the lactic acid also lowers the pH of the milk, the milk clots when it
Agitate the solution gently after each addition to dissolve the ammonium sulfate.
II. By salts of Heavy Metals:
Heavy metal salts usually contain Hg2+, Pb2+, Ag1+, Tl1+, Cd2+ and other metals with high atomic
weights. Since salts are ionic, they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a
protein usually leads to an insoluble metal protein salt. Apply this test to all the proteins provided.
III. By Acid Reagents:
The precipitation of a protein in the presence of acid reagents is probably due to the formation of insoluble
salts between the acid anions and the positively charged protein particles. These precipitants are only
effective in acid solutions. Apply this test to all the proteins provided.
6) Unknown Part:
- Take an unknown solid from your assistants and please DO NOT forget to write your unknown number
in your lab reports.

- Carry out the amino acid and protein tests in a reasonable sequence to determine your unknown solid
(Please DO NOT trust on your solubility observations and physical appearances of your uknown
Lactic Acid
Many "cultured" milk products are manufactured by allowing milk to sour before it is processed. For
instance, milk or cream is usually allowed to sour somewhat by lactic acid bacteria before it is churned
to make butter. The fluid left after the milk is churned is sour and is called buttermilk. Other cultured
milk products include sour cream, yogurt, and certain types of cheese.
Isolation of a Protein, Casein, and a Sugar, Lactose, from Milk
In this experiment, you will separate several of the chemical substances found in milk. First, you
will isolate a phosphorus-containing protein, casein. The remaining milk mixture will then be used as a
source of a sugar, -lactose. After you isolate the milk sugar, you will make several chemical tests on
this material. Fats, which are present in whole milk, are not isolated in this experiment because
powdered nonfat milk is used.
First, the casein is precipitated by warming the powdered milk and adding dilute acetic acid. It is
important that the heating not be excessive or the acid too strong, because these conditions also
hydrolyze lactose into its components, glucose and galactose. After the casein has been removed, the
excess acetic acid is neutralized with calcium carbonate, and the solution is heated to its boiling point to
precipitate the initially soluble protein, albumin. The liquid containing the lactose is poured away from the
albumin. Alcohol is added to the solution, and any remaining protein is removed by centrifugation. Lactose crystallizes on cooling.
A Bchner funnel is a piece of laboratory equipment used in filtration.[1] It is traditionally made of
porcelain, but glass and plastic funnels are also available. On top of the funnel-shaped part there is a
cylinder with a fritted glass disc/perforated plate separating it from the funnel. The Hirsch funnel has a
similar design; it is used similarly, but for smaller quantities of material. The main difference is that the
plate of a Hirsch funnel is much smaller, and the walls of the funnel angle outward instead of being vertical.
A funnel with a fritted glass disc can be used immediately. For a funnel with a perforated plate, filtration
material in the form of filter paper is placed on the plate, and the filter paper is moistened with a liquid to
prevent initial leakage. The liquid to be filtered is poured into the cylinder and drawn through the perforated
plate/fritted glass disc by vacuum suction.
The main advantage in using this type of filtration is that it proceeds much more quickly (several orders of
magnitude) than simply allowing the liquid to drain through the filter medium via the force of gravity. It is
essential that the amount of liquid being used to be limited to less than what would overflow the flask,
otherwise the liquid will be drawn into the vacuum equipment. If the vacuum is provided by a water flow
device, an overflow of the liquid could result in the spilling of a hazardous liquid into the wastewater

stream, a potential violation of the law, depending on the liquid. The potential for overflow and the potential
for water to be drawn back into the flask can be reduced by using a trap between the flask and the vacuum
It is used in organic chemistry labs to assist in collecting recrystallized compounds. The suction allows the
wet recrystallized compound to dry out such that the pure dried crystal compound is left remaining.
However, it is often the case that further drying is required, by an oven or other means, in order to remove
as much residual liquid as possible.
It is often used in combination with a Bchner flask, Bchner ring and sinter seals. A vacuum tight seal and
stability of the Bchner flask and filter are essential during the filtration process. A Bchner ring can be
used with Bchner funnels, flasks, glass crucibles and gooch crucibles. The wide flange and large surface
contact ensures an excellent vacuum tight seal whilst the rings are easy to remove and offer excellent
support to even the largest funnels.
It is commonly thought to be named after the Nobel Laureate, Eduard Buchner (without umlaut), but it is
actually named after the industrial chemist Ernst Bchner.

There are six tests for the detection of functional groups in amino acids and proteins. The six tests
are: (1) Ninhydrin Test (2) Biuret Test (3) Xanthoproteic Test (4) Millons Test (5) Hopkins-Cole Test
and (6) Nitroprusside Test.
We divide the food we consume into three main classes: carbohydrates, the bodys most readily available
energy source; lipids, the bodys principal energy reserve; and proteins, the bodys source of energy for
growth and cellular maintenance.
Proteins also make up the second largest portion of cells, after water. They are large polymeric compounds
that cells synthesize from various building blocks called amino acids.
The general structure for an amino acid is shown in the following figure:

Note that all amino acids contain carboxylic acid groups (COOH), amino groups (NH2), and
substituent or replaceable side chains (R). Twenty different amino acids, which differ only in the
structures of their side chains, are used by human cells to build proteins. The side chain structure determines
the class of the amino acid: nonpolar, neutral, basic, or acidic.
Human cells can synthesize most of the amino acids they need to build proteins. However, about 8 amino
acids called essential amino acids cannot be synthesized by human cells and must be obtained from food.
Amino acids incorporated into proteins are covalently linked by peptide bonds. Peptide bonds are amide
bonds formed between the carboxylic acid group of one amino acid and the amino group of a second amino

Equation 1 below shows a peptide linkage formation between two amino acids:

The peptide bond is indicated. Note that, because every amino acid contains at least one amino group and
one carboxylic acid group, it is possible for a peptide bond to form in two different ways. For example, with
glycine and valine, it is also possible for the peptide bond to form between the carboxylic acid group of
valine and the amino group of glycine, producing valylglycine.
Proteins are composed of hundreds of amino acids linked by peptide bonds, forming a peptide chain. We
define the direction in which the amino acids link by referring to the two ends of the chain as the Nterminus and the C-terminus. The N-terminus is the terminal amino acid in the chain that contains the only
amino group not part of a peptide bond.
The C-terminus is the other terminal amino acid in the chain, containing the only carboxylic acid group not
part of a peptide bond. Note that the N-terminus and the C-terminus are not determined by the side chains.
The number of constituent amino acids and the order, in which they are linked starting from the N-terminus,
are referred to as the proteins primary structure.

I. Amino Acid and Protein Solubility:

The physical properties of amino acids and proteins are mainly a result of their structure, both in the solid
state and in various solutions. In this part of the experiment you will investigate the solubility of selected
amino acids and proteins in various solutions. Using your data you will compare amino acid and protein
structural characteristics.
Solubility as a Function of Solution pH:
The presence of amino and carboxylic acid groups enables amino acids to accept protons from and donate
protons to aqueous solution, and, therefore, to act as acids and bases. Because proteins contain both acidic
and basic side-chains, they too can donate or accept protons. A molecule that functions simultaneously as an
acid and a base is known as an amphoteric molecule.
In neutral aqueous solutions, amino acids act as amphoteric molecules. For example, an amino acid with a
neutral side chain contains two charges: one positive, due to the protonation of the amino group, and one
negative, due to the dissociation of the carboxylic acid proton. This double ionic form of an amino acid is
the zwitterionic form. Following figure shows an amino acid in the zwitterionic form.

Amino acids in zwitterionic form have many physical properties that are also associated with ionic salts. For
example, zwitterionic amino acids are colourless, nonvolatile, crystalline solids with melting points above
200C, usually melting with decomposition. They are soluble in water but not in nonpolar organic solvents
such as cyclohexane.

Compared to organic amines and carboxylic acids of similar molecular weight, amino acids have much
lower melting points and are highly soluble in polar organic solvents, but only slightly soluble in water. The
amino and carboxylic acid groups of constituent amino acids, as well as the nature of various side-chains,
allow proteins to possess some of these same properties. However, there are many other factors that must be
considered when discussing protein solubility.
The solubility of amino acids and proteins is largely dependent on the solution pH. The structural changes in
an amino acid or protein that take place at different pH values alter the relative solubility of the molecule. In
acidic solutions, both amino and carboxylic groups are protonated. In basic solutions, both groups are unprotonated. Following figure shows an amino acid in acidic, neutral, and basic solutions.

The pH value at which the concentrations of anionic and cationic groups are equal is the isoelectric point for
that amino acid or protein. Amino acids and proteins are least soluble at their isoelectric points. Most of the
proteins found in human tissues and fluids have isoelectric points below pH 7.0 (below human body pH)
and, therefore, exist mostly in their anionic forms.

II. Chemical Reactions of Amino Acid and Protein Functional Groups:

Certain functional groups in amino acids and proteins can react to produce characteristically coloured
products. The colour intensity of the product formed by a particular group varies among proteins in
proportion to the number of reacting functional or free groups present and their accessibility to the reagent.
Now we will discuss various colour-producing reagents (dyes) to qualitatively detect the presence of certain
functional groups in amino acids and proteins.
Ninhydrin Test:
Amino acids contain a free amino group and a free carboxylic acid group that react together with ninhydrin
to produce a coloured product. When an amino group is attached to the first, or alpha, carbon on the amino
acids carbon chain, the amino groups nitrogen atom is part of a blue-purple product, as shown in Equation
2. Proteins also contain free amino groups on the alpha carbon and can react with ninhydrin to produce a
blue-purple product.

Amino acids that have secondary amino group attachments also react with ninhydrin. However, when the
amino group is secondary, the condensation product is yellow. For example, the amino acid proline, which
contains a secondary amino group, reacts with ninhydrin, as shown in Equation 3. Blue-purple and yellow
reaction products positively identify free amino groups on amino acids and proteins.

Biuret Test:
The biuret test for proteins positively identifies the presence of proteins in solution with a deep violet
colour. Biuret, H2NCONHCONH2, reacts with copper (II) ions in a basic solution to form a deep violet
complex. The peptide linkages in proteins resemble those in biuret and also form deep violet complexes
with basic copper (II) ions in solution. The general or biuret complex formed between the protein linkages
and the copper (II) ion of the biuret test is shown in following figure.

Xanthoproteic Test:
Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can
undergo reactions that are characteristic of benzene and benzene derivatives. One such reaction is the
nitration of a benzene ring with nitric acid. The amino acids tyrosine and tryptophan contain activated
benzene rings and readily undergo nitration.

The amino acid phenylalanine also contains a benzene ring, but the ring is not activated and, therefore, does
not readily undergo nitration. This nitration reaction, when used to identify the presence of an activated
benzene ring, is commonly known as the xanthoproteic test, because the product is yellow.
Xanthoproteic comes from the Greek word xanthos, which means yellow. The intensity of the yellow colour
deepens when the reaction occurs in basic solution. This reaction is one of the reactions that occur if you
spill a concentrated solution of nitric acid onto your skin. The proteins in skin contain tyrosine and
tryptophan, which become nitrated and turn yellow.
Millons Test:
Millons test is a test specific for tyrosine, the only amino acid containing a phenol group, a hydroxyl group
attached to a benzene ring. In Millons test, the phenol group of tyrosine is first nitrated by nitric acid in the
test solution. Then the nitrated tyrosine complexes mercury (I) and mercury (II) ions in the solution to form
a red precipitate or a red solution, both positive results. Proteins that contain tyrosine will, therefore, yield a
positive result.
However, some proteins containing tyrosine initially forms a white precipitate that turns red when heated,
while others form a red solution immediately. Both results are considered positive. Note that any compound
with a phenol group will yield a positive test, so one should be certain that the sample that is to be tested
does not contain any phenols other than those present in tyrosine.
Hopkins-Cole Test:
The Hopkins-Cole test is specific for tryptophan, the only amino acid containing an indole group. The
indole ring reacts with glyoxylic acid in the presence of a strong acid to form a violet cyclic product. The
Hopkins-Cole reagent only reacts with proteins containing tryptophan. The protein solution is hydrolyzed
by the concentrated sulphuric acid at the solution interface. Once the tryptophan is free, it reacts with the
glyoxylic acid to form the violet product.
Nitroprusside Test:
The nitroprusside test is specific for cysteine, the only amino acid containing a sulfhydryl group (SH).
The group reacts with nitroprusside in alkaline solution to yield a red complex.