Aim: Isolation of Lactose from Milk

In this experiment we will isolate the carbohydrate Lactose [-D-Galactopyranosyl-(1,4)-

D-Glucose] from non-fat powdered Milk. We will then determine if Lactose is a
reducing or non-reducing Sugar. Finally, we will construct models of Lactose and its
constituent monosaccharides to better understand its chemistry.

Lactose

Milk and honey are two of the few substances

with the sole purpose of being a food. Milk is
probably the most nutritionally complete food
that can be found in nature. Whole milk
contains vitamins, minerals, proteins,
carbohydrates and lipids. The only important
elements in which milk is seriously deficient are
Iron and Vitamin C.

Whole milk is an oil-water type of emulsion,

containing about 4% fat dispersed as very small
(5-10 micron 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 absorbed on
the surface 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 Vitamin A and Vitamin 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 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.
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There are three kinds of globular proteins in milk; caseins, lactalbumins, and
lactoglobulins. Casein is a phosphoprotein, meaning that phosphate groups are attached
to some of its amino acid side-chains. These are attached mainly to the hydroxyl groups
of the serine and threonine moieties forming a type of alkyl phosphate. Casein exists in
milk as the calcium salt, calcium caseinate. The salt has a complex structure which forms
soluble micelles. 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;
thus, 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 micelles are neutralized and the
neutral protein precipitates.

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. A third type of protein in milk is the lactoglobulins. These 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 system has developed.

When the fats and proteins have been removed from milk, the carbohydrates remain, as
they are soluble in an aqueous solution. The main carbohydrate in milk is Lactose.
Lactose, also called Milk Sugar, is not found in plants, and is one of the less sweet
sugars. Lactose, being only about 1/6 as sweet as Sucrose, is the reason why milk has a
rather bland taste. This sugar is only formed in the mammary glands of lactating
mammals and is the only carbohydrate that mammals can synthesize.

Sugars, also known as Saccharides (derived from the Greek ), are a simpler
form of carbohydrate that are frequently used as a food, or energy source. Because their
chemical formulas often occur as Cn(H2O)n, they were once thought of as Hydrated
Carbon; (e.g., the sugar Glucose has a chemical formula of C6H12O6 = C6(H2O)6. Lactose
is a disaccharide, meaning it can be hydrolyzed into the two simpler sugars D-Glucose
and D-Galactose. Within Lactose these
monosaccharides are linked via the
number one Carbon of Galactose, in its
 configuration, and the number four
Carbon of Glucose. This linkage is
referred to as a 1,4 glycosidic linkage
and is really nothing more than the
reaction product of a hemiacetal and an
alcohol; an acetal. This acetal linkage is
relatively stable in an aqueous
environment. Nutritionally, the enzyme
Lactase is requires to break-down
Lactose and it is produced in the small
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intestine. Lactase will break Lactose into its Glucose and Galactose components.

Lactose can be removed from whey (milk without fats and proteins) by adding Ethanol.
Lactose is insoluble in Ethanol, and when the Ethanol is mixed with the aqueous solution,
the Lactose is forced to crystallize.

Finally, it should be noted that as represented above, the number one Carbon of both
Glucose and Galactose exist as hemiacetal functionalities. This functionality is labile and
can exist in equilibrium with the aldehyde and alcohol that form it.

Thus, a molecule such as Glucose can exist either as a free aldehyde or a hemiacetal:

(Open Chain Form of Glucose) (Pyranose Form of Glucose)

It should also be noted that when the hemiacetal forms, it can form in two different
stereochemical configurations, the so-called -anomer and the -anomer:
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When in equilibrium, in an aqueous environment, approximately 60% of Glucose

molecules exist as the -anomer, 40% exist as the -anomer and less than 1% exist as the
free aldehyde.

However, it is the free aldehyde form that is important in terms of the redox chemistry of
sugars. When treated with an oxidizing agent, such as Benedict's Reagent, the aldehyde
is oxidized to a carboxylic acid. The copper of Benedict's Reagent is reduced to Cu+1;
causing the color of the Reagent to change from the blue of the Cu2+ form to a
reddish/green of the Cu+1form.

RCHO + 2 Cu2+ + 4 OH- RCOOH + Cu2O + 2 H2O

blue red

In this case, Glucose is said to be Reducing, because it causes the reduction of Benedict's
Reagent. And, because of its color change, Benedict's makes for a nice test for Reducing
sugars. If the hemiacetal had been converted to a glycosidic, or acetal, linkage, it could
not exist in equilibrium with the free aldehyde and it would give a negative Benedict's
test. Such sugars are said to be non-reducing.

In this laboratory we will isolate lactose from non-fat powdered milk. We will then
compare our results for the amount recovered to those listed on the nutrition label of the
milk. In order to better understand the nature of the acetal and hemiacetal linkages which
exist in a molecule of Lactose, we will also build a models of the simple sugars which
comprise Lactose. These models will then be connected via an appropriate Glycosidic
Linkage to form a model of a molecule of Lactose. Finally, we will perform a Benedict's
Test on Lactose to determine if it is reducing or non-reducing. For comparative purposes,
we will also perform the Benedict's Test on several other sugars.
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Procedure

Isolation of Lactose from Milk

1. Take 30 ml of of non-fat milk in a beaker.

2. Heat the milk to 40oC.

3. Add 6mL of 10% Acetic Acid to precipitate out the Casein, and stir the mixture
slowly and briefly. Avoid adding excess Acetic Acid. Work the Casein into a mass
and remove it with a stirring rod or spoon. Place this in a trash can.[Isolation of
Casein]

4. Immediately add 2 g of powdered Calcium Carbonate. Stir the mixture

thoroughly.

5. Heat the mixture almost to boiling for about 10 minutes; stirring continuously. This
should precipitate the remaining proteins.

6. Filter the hot mixture, collecting the filtrate in a 250mL beaker.

7. Stir the filtrate continuously while boiling it down to about 10mL, and then add
10 mL of 95% Ethanol.

8. Carefully heat the solution to 70oC. (Ethanol boils at 78oC.)

9. Filter the warm Ethanol solution, collecting the filtrate in a 125mL flask. And
put aside for one day for lactose.
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