LAB 12 - ISOLATION OF MILK PROTEIN (CASEIN)
- Page ID
- 506289
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \)
\( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)
\( \newcommand{\dsum}{\displaystyle\sum\limits} \)
\( \newcommand{\dint}{\displaystyle\int\limits} \)
\( \newcommand{\dlim}{\displaystyle\lim\limits} \)
\( \newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\)
( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\)
\( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\)
\( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\)
\( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\)
\( \newcommand{\Span}{\mathrm{span}}\)
\( \newcommand{\id}{\mathrm{id}}\)
\( \newcommand{\Span}{\mathrm{span}}\)
\( \newcommand{\kernel}{\mathrm{null}\,}\)
\( \newcommand{\range}{\mathrm{range}\,}\)
\( \newcommand{\RealPart}{\mathrm{Re}}\)
\( \newcommand{\ImaginaryPart}{\mathrm{Im}}\)
\( \newcommand{\Argument}{\mathrm{Arg}}\)
\( \newcommand{\norm}[1]{\| #1 \|}\)
\( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\)
\( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\AA}{\unicode[.8,0]{x212B}}\)
\( \newcommand{\vectorA}[1]{\vec{#1}} % arrow\)
\( \newcommand{\vectorAt}[1]{\vec{\text{#1}}} % arrow\)
\( \newcommand{\vectorB}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \)
\( \newcommand{\vectorC}[1]{\textbf{#1}} \)
\( \newcommand{\vectorD}[1]{\overrightarrow{#1}} \)
\( \newcommand{\vectorDt}[1]{\overrightarrow{\text{#1}}} \)
\( \newcommand{\vectE}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash{\mathbf {#1}}}} \)
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \)
\( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)
\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)The purpose of this experiment is to:
- Casein, the primary protein in milk, is isolated by acid precipitation at its isoelectric point.
- Understand the chemistry behind protein denaturation and separation.
INTRODUCTION
Milk is a complex mixture of water, proteins, fats, carbohydrates, and minerals. The primary protein in milk is casein, which accounts for approximately 80% of the total protein content. Casein is essential for the nutritional value of milk and plays an important role in cheese making.
Casein exists in milk as a colloidal suspension of casein micelles. These micelles are stable at the natural pH of milk (approximately pH 6.6) due to the presence of κ-casein on their surface, which provides both steric and electrostatic stabilization. When the pH is lowered to the casein's isoelectric point (approximately pH 4.6), these micelles become destabilized, leading to the precipitation of casein from the solution.
In this lab, we will use acetic acid to lower the pH of milk, causing the casein to precipitate at its isoelectric point. We will then isolate and analyze the precipitated casein.
Chemical Equations: While there isn't a specific chemical reaction occurring, the process can be represented as:
Casein (soluble) + H+ → Casein (insoluble precipitate)
The acetic acid provides the H+ ions: CH3COOH ⇌ CH3COO- + H+
1) Always wear chemical splash goggles while working on this experiment.
2) Wear gloves while handling chemicals.
3) Use caution when handling hot liquids and equipment.
4) Even in dilute form, Acetic acid can cause skin irritation. Rinse immediately if contact occurs.
5) Ensure proper ventilation when working with acetic acid. All liquids used in this experiment should be handled under a fume hood.
6) Dispose of all waste in the designated containers, as instructed by your instructor.
7) When finished, thoroughly clean your work area and return all equipment and chemicals to their appropriate places.
8) Wash your hands before you leave
CHEMICALS AND EQUIPMENT NEEDED
| EQUIPMENT | EQUIPMENT | EQUIPMENT | CHEMICALS |
|---|---|---|---|
| 1000 mL beaker | pH meter or pH paper | Stirring rod | Skim milk (500 mL) |
| 250 mL beaker | Buchner funnel and filter paper | Graduated cylinder | Acetic acid (5% solution, 50 mL) |
| Hot plate with magnetic stirrer | Vacuum filtration setup | Digital scale | Distilled water |
| Thermometer | Ice bath | 0.1 M NaOH solution (for pH adjustment if needed) |
EXPERIMENTAL PROCEDURE
Part A: Isolation of Casein
1) Measure 100.0 mL of skim milk into a 400.0/600.0 mL beaker. If you are not using skim milk, centrifuge the milk using centrifuge tubes to remove the fat. When centrifuging non-skim milk, the fat collects on top and should be removed before heating the milk in the next step.
2) Heat the milk to 40.0°C while stirring gently on a hot plate with a magnetic stirrer.
3) While the milk warms, obtain a pH electrode, rinse it thoroughly with distilled water, and place it in a 250.0 mL beaker containing approximately 100.0 to 150.0 mL of distilled water.
4) Gradually add 5.0 mL of 5% acetic acid solution to the warm milk while stirring continuously. Keep stirring and monitor the pH using a pH electrode.
5) The milk should start to curdle. If necessary, add more acetic acid drop by drop until the pH reaches 4.6, which is the isoelectric point of casein.
6) Your instructor may use 0.2 M HCl for steps 4 and 5. If using 0.2 M HCl, add it dropwise to the milk until a steady pH of 4.6 is reached.
5) Remove the mixture from the heat and let it stand for 5 minutes.
7) Prepare an ice bath and cool the mixture for 10 minutes, stirring occasionally.
8) Set up the vacuum filtration apparatus with a Buchner funnel and filter paper.
9) Filter the cooled mixture to separate the precipitated casein from the whey.
9) Rinse the precipitate with cold distilled water to remove any remaining acid.
10) Transfer the casein to a pre-weighed filter paper and let it air dry overnight.
11) Weigh the dried casein and calculate the yield.
Part B: Tests
1. Biuret test: Mix a small amount of isolated casein with the Biuret reagent. A positive result (purple color) confirms the presence of protein.
2. Solubility test: Test the solubility of isolated casein in water and dilute NaOH solution.
3. pH test: Check the pH of the whey (filtrate) after casein precipitation.
PRE-LAB QUESTIONS
Name ____________________________________
1. Why do we use skim milk instead of whole milk for this procedure? Can whole milk be used for this experiment? If so, how can we use it?
2. What is the isoelectric point, and why is it important in this experiment?
3. How does acetic acid cause the precipitation of casein?
4. What other methods could isolate proteins from a solution?
5. What is the theoretical yield of casein for skim milk?
DATA AND OBSERVATIONS
Name _________________________Lab Partner(s) ______________________________
Measurement |
Value |
|---|---|
|
Initial volume of milk (mL) |
|
|
Volume of acetic acid used (mL) |
|
|
Final pH of the mixture |
|
|
Mass of empty filter paper (g) |
|
|
Mass of filter paper + dried casein (g) |
|
|
Mass of isolated casein (g) |
1. Calculate the concentration of casein in the original milk sample (g / 100 mL).
Note: Skim milk typically contains about 3.4% protein, of which about 80% is casein.
2. Calculate the percentage yield of casein:
% Yield = (Mass of isolated casein / Expected mass of casein in milk) × 100
% Yield =
POST-LAB QUESTIONS
1. How does the yield of casein you obtained compare to the theoretical yield? Explain any discrepancies.
2. Describe the appearance and texture of the isolated casein. How does it differ from its appearance in milk?
3. What factors might affect the efficiency of casein isolation using this method?
4. How might the pH of precipitation affect the purity and yield of the isolated casein?
5. Discuss potential applications of isolated casein in the food industry or other fields.
6. What other milk components remain in the whey after casein precipitation? How might these be isolated?
7. How would you modify this procedure to isolate other milk proteins, such as whey proteins?
8. Describe how this lab demonstrates the relationship between protein structure and solubility.
Please click here to access the Pre-Lab, Data Tables, and Post-Lab in Word or PDF format. Complete them and upload the lab report according to your instructor's instructions.