lab 9 Chem Isolation of Casein

CHEMISTRY
Isolation of Casein
Investigation
Manual
ISOLATION OF CASEIN
Table of Contents
2
Overview
2
Outcomes
2
Time Requirements
3
Background
4
Materials
5
Safety
5
Preparation
6
Activity 1
7
Disposal and Cleanup
Overview
Students will take a provided sample of milk and through the addition of an acid, determine the pH at the isoelectric point of casein.
The mass of casein will be measured and the percentage of casein
in the milk sample will be calculated. As an extension, students
can test their own milk sample to compare the percentage of
protein.
Outcomes
• Examine the properties of proteins in general and casein
specifically.
• Explain and apply the principles of protein isolation.
Time Requirements
Preparation ………………………………………………………………5 minutes
Activity 1: Isolation of Casein from Dried Milk…………..45 minutes +
Drying Time
Key
Personal protective
equipment
(PPE)
goggles gloves apron
follow
link to
video
photograph stopwatch
results and
required
submit
warning corrosion flammable toxic environment health hazard
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Background
Proteins are complex organic compounds
composed of monomers called amino acids.
Amino acids contain hydrogen, oxygen, carbon,
and nitrogen in three functional groups, with
the general chemical formula NH2–RCH–COOH,
where NH2 is an amine group and the –COOH
is the carboxylic acid functional group. The
–R represents a side chain, which can be an
–H, a carbon chain, or a carbon ring structure.
All amino acids are identical except for the –R
group. All organisms require nitrogen for the
construction of proteins, nucleic acids, and other
compounds.
Twenty amino acids are encoded by the
universal genetic code; these are known as the
natural amino acids. These 20 amino acids
can be linked together by peptide bonds to form
proteins. The linked amino acids can form dipeptides, tripeptides, and polypeptides. The polypeptides may contain any number, sequence,
and/or configuration of amino acids to form a
vast number of proteins. Proteins contained
in foods are a source of prefabricated amino
acid groups. Nine amino acids are considered
essential; these essential amino acids must
be supplied in prefabricated form via the diet.
However, the remaining eleven amino acids can
be synthesized in the human body. The absence
of any of these essential amino acids halts the
synthesis of crucial proteins, resulting in disease,
and in prolonged cases of deprivation, death.
A common source of dietary protein is milk.
Casein is the most prevalent protein in mammalian milks: 80% of the protein in cow’s milk is
casein. Casein is very poorly soluble in water,
which is the base solvent of milk. The casein
in milk is part of a suspension; an undissolved
solid floating in the solvent. The protein is
highly hydrophobic, meaning it is negatively
attracted to water, causing the protein to form
micelles (small bubbles of protein) within the
water. The exact structure of these micelles is
not completely understood, but one hypothesis
is that the κ-casein proteins (one type of casein
protein in the micelles) are arranged such that
their polar (charged) regions are on the surface
of the micelles. Calcium and phosphate ions are
present on the surface of the micelles. These
ions and the polar regions of the κ-casein molecules interact with water molecules, which are
also polar. The interaction of the micelles with
the charged water molecules keeps the micelles
suspended in the liquid phase of the milk. There
are several methods of removing a suspended
solid from solution. One method is to use a
centrifuge. A centrifuge is an apparatus that
can spin a sample at a high speed and essentially force heavy materials to the bottom of a
sample centrifuge tube, similar to how the spin
cycle on a clothes washer helps separate the
clothes from excess water. This method obviously requires expensive laboratory equipment
and would not be suitable for an at-home
investigation.
There is another way to separate these particles,
which involves changing the pH of the milk. In
this investigation, acetic acid will be added to a
milk sample until the isoelectric point, the point
at which the molecule carries no net electric
charge, of the protein is reached. The addition of
acid changes the pH of the solution and disrupts
the intermolecular forces holding the micelles
together. At the isoelectric point, the hydrogen
continued on next page
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ISOLATION OF CASEIN
Background continued
ions (H+) from the acid balance out any negative charges on the protein, breaking up the salt
bridges that give the protein their shape, and
thereby reducing the strength of the intermolecular forces. This process of adding acid denatures the protein, meaning that it is no longer
in its natural molecular conformation (shape).
This disruption pops the protein bubble, and the
insoluble casein coagulates and precipitates out
of solution.
Materials
Included in the materials kit:
2 Filter paper
Dried milk
Glass stirring
rod
Figure 1.
Filtration funnel pH indicator
strips
Casein micelles
suspended in milk;
before the addition
of acid
Network formed
during coagulation;
after the addition
of acid
Casein, while being a source of essential amino
acids, is also the protein primarily responsible
for the formation of cheese; it is the protein that
coagulates to form the solid portion of cheese.
In fact, the method used in this investigation is
similar to ones used in the production of many
cheeses. In this investigation, the percentage
by weight of casein in a milk sample will be
calculated.
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Needed from the chemical kit #1:
Vinegar
continued on next page
Safety
Needed from the equipment kit:
Beaker, 250 mL Medicine cup
Wear your safety
goggles, chemical apron,
and gloves at all times while conducting this
investigation.
Dropping pipet
Read all the instructions for this laboratory
activity before beginning. Follow the instructions
closely, and observe established laboratory
safety practices, including the use of appropriate personal protective equipment (PPE) as
described in the Safety and Procedure sections.
Acetic acid is a corrosive material.
Use this chemical near a source of
running water that can be used as a
safety eye wash or safety shower if any corrosive material comes in contact with skin or eyes.
Plastic cup,
8 oz
Balance
Needed but not supplied:
• Non-fat milk sample (optional)
Reorder Information: Replacement supplies
for the Isolation of Casein investigation can
be ordered from Carolina Biological Supply
Company, kit 580360.
Call 800-334-5551 to order.
Do not eat, drink, or chew gum while performing
this activity. Wash your hands with soap and
water before and after performing the activity.
Clean up the work area with soap and water
after completing the investigation. Keep pets
and children away from lab materials and
equipment.
Preparation
1. With the balance, weigh 10 grams of dry milk
into a 250-mL beaker.
2. Fill beaker to 100-mL line.
3. Stir using glass stir rod until thoroughly
mixed.
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ACTIVITY
Activity 1
A Isolation of Casein from Dried Milk
1. Weigh a small medicine cup, and record the
mass in Data Table 1.
8. Dip a clean stirring rod in the clear, yellow
liquid, and touch it to a pH indicator strip.
Record color and pH in Data Table 1.
9. Weigh a piece of filter paper, and record the
mass in Data Table 1.
2. Add approximately 10 mL of rehydrated milk
to the medicine cup.
10. Assemble the filtration apparatus as shown
in Figure 2 with the weighed filter paper.
3. Weigh the medicine cup with the milk, and
record the mass in Data Table 1.
Figure 2.
4. Calculate the mass of milk in the medicine
cup, and record in Data Table 1.
5. Take the glass stir rod, and dip it into the milk
sample.
6. Touch the milk sample to a pH indicator strip.
Record color and pH in Data Table 1.
7. Add acetic acid drop-wise with dropping
pipet while stirring with the glass stirring rod.
Add drops until a precipitate ceases to form.
continued on next page
Data Table 1.
Dry Milk Sample
Mass of Cup
Mass of Cup + Milk
Mass of Milk
pH of Milk Pre-Acid
pH of Milk Post-Acid
Mass of Filter Paper
Mass of Filter Paper + Casein
Mass of Casein
Percentage of Casein in Milk
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Optional Milk Sample
11. Place the folded filter paper into the funnel.
It may be necessary to wet the funnel with
water to get it to stay.
12. Set the funnel into the 8-oz plastic cup.
13. Pour the milk in the filter, and collect the
solid protein in the filter paper.
14.
Remove the filter paper containing the
protein (casein) from the funnel, and
allow to dry overnight in a cool, dry location
away from pets, children, and food sources.
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Disposal and Cleanup
1. Dispose of solutions down the drain with the
water running. Allow the faucet to run a few
minutes to dilute the solutions.
2. Solid waste may be disposed of in the trash.
3. Rinse and dry the lab equipment, and return
the materials to your equipment kit.
4. Sanitize the workspace.
15. Weigh the dried casein and filter paper, and
record the mass in Data Table 1.
16. Calculate the mass of casein, and record in
Data Table 1.
17. Calculate the percentage of casein in the
milk sample, and record in Data Table 1.
mass of casein
× 100
mass of milk sample
= percentage of casein in milk
18. *Optional* Repeat activity with non-fat or
low-fat fresh milk sample.
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CHEMISTRY
Isolation of Casein
Investigation Manual
www.carolina.com/distancelearning
866.332.4478
Carolina Biological Supply Company
www.carolina.com • 800.334.5551
©2017 Carolina Biological Supply Company
CB780261703
Isolation of Casein
Student Name
Date
1
Data
Activity 1
Data Table 1
Dry Milk Sample
Optional Milk Sample
Mass of Beaker
Mass of Beaker + Milk
Mass of Milk
pH of Milk pre-acid
pH of Milk post-acid
Mass of Filter Paper
Mass of Filter Paper +
Casein
Mass of Casein
Percentage of Casein
in Milk
1. What purpose was served by filtering the milk sample after the addition of
acid?
2. When weighing the protein at the end of the activity, what major assumption
is made about the product? Why might this impact the calculated
percentage of casein in the sample?
3. If a base, such as sodium hydroxide (NaOH) were added to milk, would the
protein precipitate? Why or why not?
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