🧪 Experiment 1
The cell is the fundamental unit of life.
The cell contains protoplasm which is subdivided to:
* Nucleus
* Cytoplasm.
All cells have a plasma membrane or cell membrane.
* Plasma membrane gives mechanical strength, shape, and some protection to the cell.
* Preventing substances from entering the cell or leaking out of the cell.
* The plasma membrane regulates the transfer of nutrients inside the cell through the following processes:
Diffusion - It is the net movement of a particular substance from a region of higher concentration to a region of lower concentration
Osmosis - It is the movement of water from the solution with the free osmotically active particles to the solution with fewer particles
* Osmotic Concentration - the number of osmotically active particles contained per unit volume.
* Osmotic Pressure - the pressure required to stop the movement of water through a semipermeable membrane due to osmosis.
Hypertonic and Hypotonic are used to describe the relative concentration of solutes.
* Hypertonic - High concentration outside the cell which causes the cell to shrink due to the solute outside is absorbing the solute inside
* Hypotonic - Lower concentration outside the cell, which causes the cell to swell or burst due to overabsorption of the solute.
Dialysis - It is the diffusion of solute molecules across a differentially permeable membrane
Surface Tension - It is the elastic-like force on the surface of a liquid caused by the attraction (cohesion) between the liquid's molecules, especially at the surface.
1. The Cell
Acquire glass slides of animal cells and plant cells and examine them under a microscope. Then, sketch and label the important parts of the cell.
2. Dialysis
Soak a piece of cellophane (10x10”) in water to soften it.
Fold the soaked cellophane similar to filter paper. Pour in 50mL of 1% NaCl then pour 50mL of 1% starch solution.
Suspend the cellophane containing the mixture from a buret clamp into the 500mL beaker with distilled water. The distilled water must be at the same level as the surface of the starch.
After one hour, pipette a 2mL of the distilled water into the beaker (dialysate) and test it by adding a few drops of silver nitrate solution. Pipette another 2mL but this time test it with iodine solution.
Lastly, observe and record the visible result.
3. Osmosis
Skin a potato, then cut it into small pieces approximately 1cm. Then divide them into 4 groups of 10. Each group will be immersed for 30 minutes in a different solution:
Group 1 - distilled water (Con. H20 100%)
Group 2 - 2% saline solution (Con. H20 98%)
Group 3 - 4% saline solution (Con. H20 96%)
Group 4 - 8% saline solution (Con. H20 92%)
Then dry it with a paper towel and weigh it to the nearest 0.01g, that’s the initial weight. Put them back in the solution again for 30 minutes, dry them, and weigh them up, that’s the final weight
Calculate the weight percent change with the formula
4. Diffusion
Put 5mL of water in a test tube, incline the test tube carefully and add 2mL of 2% potassium permanganate solution. Note the time it takes for the color to change entirely.
5. Surface Tension
Prepare 2 test tubes, each with 1mL of distilled water and 1mL of linseed oil. Drop 1mL of soap solution in test tube 1 then shake both test tubes and let them stand for a few minutes. Do the same, but this time use chloroform instead of linseed oil. Note how long it takes for the drops to coalesce (mix) in each test tube
1. The Cell
Plant
Animal
Cell Wall
Centrioles
Large Vacoule
Smaller Vacoule
Chloroplast
Lysosomes
Rectangular
Round/Irregu;ar Shaped
2. Dialysis
SAMPLE
RESULTS
EXPLANATION
Dialyzate + AgNO3 Solution (test for presence of chloride Ions)
Colorless to milky white precipitate (positive results)
Chloride ions have diffused through the dialysis membrane, meaning they are small enough to pass through.
Dialyzaate + Iodine solution
(test for presence of starch)
No color change
No starch is present in the dialyzate, indicating that starch did not pass through the membrane due to its large molecular size.
3. Osmosis
The level of solution liquid inside the thistle tube rose
4. Diffusion
Trial 1: 5 minutes 15 seconds
Trial 2: 5 minutes and 16 seconds
5. Surface Tension
SAMPLE
TIME
EXPLANATION
1mL distilled water + linseed oil
80 seconds (Oil remains separate or quickly separates)
Soap lowers surface tension, allowing the oil to disperse and form an emulsion. Without soap, the oil remains in droplets due to high tension.
1mL distilled water + linseed oil + soap
Did not separate (Forms stable emulsion, drops take longer to coalesce)
1mL distilled water + chloroform
119 seconds (Already mixes quickly)
Chloroform has low surface tension and partial miscibility with water, so soap has minimal effect. Drops coalesce quickly in both cases.
1mL water + chloroform +soap
210 seconds (Mixes more readily)
1. In terms of cellular structure, what is the difference between plant and animal cells?
* Animal cells don’t have a cell wall, chloroplasts, and vacuoles. Plant cells don’t have lysosomes and centrioles.
2. Explain why the dialysate gave a positive reaction with silver nitrate and a negative reaction with Iodine solution.
* Because the Chlorine anion in dialyzate reacts and forms a white precipitate of silver nitrate. This means the chlorine anion is small enough to pass through the dialysis membrane unlike with iodine which either has big or no starch
3. Explain how soaps lower the surface tension of fats and oils
* Due to soap’s amphiphilic nature it breaks the strong hydrogen bond with water allowing water to mix with oil thus reducing oils surface tension.
* Hydrophilic part (H20): Water-loving
* Hydrophobic part (Oil): Water-hating
* SOAP = SURFACTANT
4. How does the kidney maintain the body’s internal environment
* Homeostasis and Filtering
* Osmosis
5. What is the effect of suspending the cells in hypotonic, and isotonic environment
* Hypotonic = Swells
* Isotonic = As is
VIDEO LINKS
Osmosis
https://youtu.be/9U3CONhEbaA
Diffusion
https://youtu.be/BHZZyDMeu1M
Hot vs Cold water: https://youtu.be/IgbR-K1ff-w
Surface Tension
https://coolscienceexperimentshq.com/mixing-oil-water/
🧪 Experiment 2
* Water = ~70% of the human body.
* It's a universal solvent crucial for life.
* Acts as the medium for biochemical reactions (metabolism, respiration, etc.).
* The pH of water-based solutions affects enzyme activity, protein structure, and cellular processes.
* pH = “potential of hydrogen” → measures [H⁺] concentration in a solution.
* Scale: 0 (acidic) → 7 (neutral) → 14 (basic)
* A change of 1 pH unit = 10x change in H⁺ concentration.
* Determines how acidic or basic a solution is.
* Even slight changes in H⁺ levels can disrupt biological function.
* Cells must regulate pH tightly to maintain homeostasis.
* A buffer maintains a stable pH when small amounts of acid/base are added.
Made of:
* A weak acid + its conjugate base, or
* A weak base + its conjugate acid (usually as a salt).
How Buffers Work:
* When acid is added, the conjugate base neutralizes H⁺.
* When base is added, the weak acid donates H⁺ to neutralize OH⁻.
Molar Concentration of Buffer Components
Higher concentration = higher buffer capacity
More "stuff" to neutralize incoming H⁺ or OH⁻
Ratio of Conjugate Base to Acid
Ideal ratio = 1:1
Buffers can still work within a range (usually pKa ± 1)
LAB RESULTS
SAMPLES
pH using pH paper
pH using pH meter
fresh milk
6.5
6.6
urine
6.0
6.2
pineapple juice
3.5
3.6
softdrink
3.0
2.9
vinegar
2.5
2.6
toothpaste
8.0
8.2
Distilled water
7.0 (neutral)
6.8
saliva
6.5
6.8
toner
5.5
5.6
* pH paper gives an approximate reading (rounded to 0.5), while a pH meter gives a more precise value.
* Soft drinks, vinegar, and pineapple juice are acidic.
* Toothpaste is slightly basic — helps neutralize acids in the mouth.
* Distilled water should be ~7 but may read lower due to CO₂ absorption forming carbonic acid.
* Toner (depending on ingredients) is usually slightly acidic for skin compatibility.
* Urine and saliva vary slightly depending on diet and hydration.
Solution
Actual pH
PH with NaOH
^ in PH
(a) distilled water
7.0
11.5
+4.5
(b) 0.19 M NaAc
8.9
12.0
+3.1
(c) 0.10 M Hac
2.9
8.5
+5.6
(d) acetate buffer
4.74
5.00
+0.26
1. Distilled water has no buffering agents, so the addition of NaOH causes a large jump in pH. It cannot resist changes in pH.
2. NaAc (sodium acetate) is a salt of a weak acid, slightly basic, but no acid present to buffer the added OH⁻ → large pH increase.
3. Acetic acid alone is acidic, but has no conjugate base to neutralize OH⁻ → strong pH rise with NaOH addition.
4. Acetate buffer (CH₃COOH + CH₃COONa) has both acid and base components, so it can neutralize added OH⁻, resisting pH change. Shows the least Δ in pH.
* Buffer solutions show very little change in pH.
* Acetate buffer is effective because it contains both the acid (HAc) and its conjugate base (NaAc) → it neutralizes added base (OH⁻).
* The larger the Δ in pH, the weaker or absent the buffering system.
Solution
Actual pH
PH with HCl
^ in PH
Buffer Capacity
Acetate Buffer
4.74
4.60
0.14
Strong
The acetate buffer is a mixture of:
* Acetic acid (CH₃COOH) – weak acid
* Sodium acetate (CH₃COONa) – conjugate base
When HCl is added, it increases the H⁺ ion concentration in the solution. However, the acetate ion (CH₃COO⁻) in the buffer reacts with the added H⁺, forming more acetic acid:
* This neutralizes most of the added acid, so the pH drops only slightly (small Δ in pH).
* Because of this small change, the buffer capacity is strong — meaning it can resist acidification effectively
LAB REPORT QUESTIONS:
1. Why should distilled water be boiled and cooled before determining its pH?
* Boiling distilled water removes dissolved gases such as carbon dioxide (CO2). CO2 can dissolve in water to form carbonic acid (H2CO3), which lowers the pH of the water. By boiling and then cooling the water, you ensure the pH measurement reflects only the water itself, not the influence of dissolved gases.
2. What are the factors that affect buffer capacity?
* Concentration: Higher buffer component concentration increases capacity.
* Acid-to-base ratio: Optimal capacity when the ratio is close to 1 (pH=pKa).
* pH range: Effective within ± 1 pH
3. At what pH does buffer solution exhibit maximum buffer capacity?
* Occurs when pH=pKa, where acid and conjugate base concentration are equal.
4. At what pH does buffer solution exhibit maximum buffer capacity?
🧪 Experiment 3
PROTEINS — one of the major constituent of living cells
* Macromolecules that contain amino acids (as a building blocks joined together by peptide bonds)
* Has variety of functions:
* Catalyze biochemical reactions
* Regulate the activity of various organs in the body
* Counteract the adverse effects of antigens
* Transport molecular oxygen and serve as structural materials of the muscle, skin and hair
Reactions which are:
Example
Collectively called / refers to
Due to colloidal nature of proteins
E.g. Salting out and heat coagulation
Denaturation
Due to the presence of specific chemical groups in the protein molecule
Different color tests to identify the amino acid or proteins
Due to the presence of specific chemical reaction between the protein molecules
E.g. Precipitation by strong mineral acid, salt of heavy metal or alkaloidal reagents
Denaturation
The a-helix structural pattern of polypeptides at the secondary level
* Is maintained by weak hydrogen bonds between amino acids on one part of the chain and those another part forming a stable molecule
* The linkages responsible for the tertiary structure of protein are a function of the nature of amino acid side chains within the molecule. Color Reactions of Proteins and Amino Acids
*
Bonds that stabilize the tertiary structure proteins are:
1. Salt linkages
2. Hydrogen Bonds
3. Disulfide linakages
4. Hydrophobic interactions
Various physical and chemical methods break these bonds and disrupt the secondary and tertiary structure of proteins. Such proteins are said to be denatured.
DENATURED – any change that alters the unique three-dimensional configuration of protein molecules without causing a concomitant cleavage of peptide bonds.
* Denatured proteins aggregate and become visible as a precipitate
Many proteins are coagulated by heat, strong acids, and alcohol.
(Masyado madami procedures, basahin nalang natin HAHAHHAHAHA)
Color Reactions of Proteins and Amino Acids
TEST NAME
HOW IT WORKS
POSITIVE FOR
POSITIVE EXCEPT FOR
COLOR GRADIENT
BIURET TEST
(protein detection)
Due to the coordination of complex formed by cupric ions (in the reagent) and the amino groups
The test is taken from organic compound called biuret which is represented with the formula:
Positive for all compounds that contain two or more peptide bonds
Threonine and serine
Urea and amino acids (except threonine and serine) do not give a positive reaction.
characteristic purple color
NINHYDRIN TEST
(to detect and identify the presence of amino acids and amine))
When ninhydrin is heated in the presence of amino acids, a blue violet colored complex is formed (yellow for proline and hydroxyproline).
A-amino group is responsible for this test.
All proteins or protein derivatives, as well as ammonia and amines, give positive results, thus indicating that a-amino group is responsible for this test.
a blue violet colored complex is formed (yellow for proline and hydroxyproline).
XANTHROPROTEIC TEST
(aromatic amino acid detection)
This test involves nitration of the amino benzene ring with concentrated nitric acid to produce yellow orange derivatives of nitrobenzene.
Positive for those amino acids that contain benzene ring or aromatic ring.
yellow orange derivatives of nitrobenzene
HOPKIN’S COLE TEST
(amino acid tryptophan detection)
This test is due to the presence of the indole ring of the tryptophan.
The reagent used contains glyoxylic acid.
HOOCCHO, which condenses with indole derivatives, in the presence of strong acids like sulfuric acid, to form a violet colored complex
Tryptophan amino acid
Violet colored complex
MILLON’S TEST
(tyrosine amino acid detection)
This test depends on the presence of phenolic groups in the molecules. These groups are easily nitrated by a solution of mercuric and mercurous nitrates and nitrites in concentrated nitric acid.
The color produced was due to a mercury complex of nitrophenyl derivatives.
the detection of tyrosine-containing proteins in a given sample
Inorganic ions, such as ammonium ions and chloride ions interfere with this test and therefore it is not useful in urine analysis.
A white precipitate is produced which becomes brick red upon prolonged heating.
SAKAGUCHI TEST
(arginine detection)
This test depends on the presence of guanidine or guanido group.
In alkaline solutions, compunds that contain this group combine with a-naphthol and sodium hypobromite or hypochlorite (an oxidizing agent) to give a red to an orange colored complex.
Arginine-containing proteins in a given sample
red to an orange colored complex.
LEAD ACETATE TEST
(test for sulfur / Cysteine and cystine)
In strongly alkaline solutions, the sulfuhydryl or disulfide groups are converted to inorganic sulphide, Na2S.
This further reacts with lead acetate to form a brownish-black precipitate of lead sulfide, Pbs.
detect the presence of sulfur-containing amino acids, particularly cysteine and cystine, in proteins
brownish-black precipitate of lead sulfide, Pbs.
TEST
SAMPLE
RESULTS
EXPLANATION
BIURET TEST
Albumin
✔
Contains proteins
Gelatin
✔
Contains proteins
Tyrosine
✖
Does not contain more than one protein
Phenylalanine
✖
Does not contain more than one protein
NINHYDRIN TEST
Albumin
✔
Albumin is a protein composed of amino acids that have free amino groups.
Ammonium Water
✔
Has ammonia/amines (result is blue rather than purple)
Urea
✖
Urea has amino groups, but they are involved in a stable structure and do not react strongly with ninhydrin.
XANTHROPROTEIC TEST
Albumin
✔
Contains aromatic amino acids like tyrosine and tryptophan.
Gelatin
✖
Lacks or has very low levels of aromatic amino acids.
Tyrosine
✔
Has a phenol ring that is easily nitrated.
Phenylalanine
-
Benzene ring is less reactive than tyrosine; weak or no reaction.
HOPKIN’S COLE TEST
Albumin
✔
Albumin contains tryptophan residues.
Gelatin
✖
Gelatin lacks or has very little tryptophan.
Tyrosine
✖
Tyrosine is not detected by this test; it lacks the indole ring of tryptophan.
Trypthopan
✔
Direct source of tryptophan; strong positive reaction.
MILLON’S TEST
Albumin
✔
Albumin contains tyrosine residues.
Tyrosine
✔
Tyrosine has a phenol group that reacts strongly
Phenylalanine
✖
Lacks a phenol group; no reaction
Phenol
✔
Phenol directly reacts with Millon’s reagent.
LEAD ACETATE TEST
Albumin
✔
Albumin contains sulfur-containing amino acids (e.g., cysteine).
Gelatin
✖
Gelatin lacks significant amounts of sulfur amino acids.
COAGULATION/ DENATURATION
SAMPLE
RESULTS
EXPLANATION
1, By heat
Albumin + distilled water heated in a water bath + Biuret reagent
Violet/purple color (slower reaction)
Protein still present; heating denatures (coagulates) the protein but peptide bonds remain intact.
Albumin (heated) + distilled water + Biuret reagent
Violet/purple color
denatured proteins still yield a positive Biuret test because peptide bonds are not broken.
2. By Acid
Albumin + HCl
White precipitate forms
HCl denatures albumin by disrupting ionic and hydrogen bonds, causing it to coagulate.
Albumin + H2SO4
White precipitate forms
HCl denatures albumin by disrupting ionic and hydrogen bonds, causing it to coagulate.
3. By Alcohol
Albumin + ethanol
White precipitate forms
Ethanol causes albumin to denature and coagulate by disrupting hydrophobic interactions and hydrogen bonds.
Gelatin + Ethanol
Little or no visible change
Gelatin is less structured and more heat-stable; it may not precipitate as easily in ethanol at room temperature.
PRECIPITATION
SAMPLE
RESULTS
EXPLANATION
1, By heavy metal
Albumin + lead acetate
White precipitate
Lead acetate reacts with sulfhydryl groups (–SH) in albumin, forming lead sulfide (PbS), which is insoluble.
Albumin + mercuric chloride
White precipitate
White precipitate Mercuric chloride reacts with the thiol groups (–SH) of albumin, forming mercury salts that precipitate out of solution.
Albumin + silver nitrate
White precipitate
White precipitate Silver nitrate reacts with sulfide groups in albumin, forming silver sulfide (Ag₂S), which is a black precipitate.
2. By Alkaloidal Reagent
Albumin + picric acid
Yellow precipitate
Picric acid (trinitrophenol) precipitates albumin by forming complexes with the protein.
Albumin + tannic acid
Brown precipitate
Tannic acid, a polyphenol, reacts with albumin to form an insoluble complex, resulting in a brown precipitate.
1. Surgical instruments are sterilized by heating them and alcohol is used as a disinfectant in cleansing the skin prior to an injection. Why are these methods effective against microorganisms?
* Sterilizaition by heat and disinfection by alcohol denatures the proteins and disrupts the hydrogen bonds.
2. Egg white and milk are used as an antidote for heavy metal poisoning. Offer an explanation.
* Heavy Metals binds to the proteins of the egg (Chelation)
3. Both picric acid and tannic acid are used in the treatment of burns. Offer an explanation.
* Acts as Astringent meaning it shrinks tissues and acts as a protective barrier.
4. Suppose you were given two jars, one containing amino acid and the other containing a polypeptide. Tell how you would distinguish between them by the use of ninhydrin test and biuret test.
* Ninhydrin can detect amino acids and produces a blue-violet color while using the Biuret reagent it can detect if the jar contains a polypeptide/proteins and produces a purple color.
5. Why does nitric acid stain the skin yellow?
* Xanthroproteic reaction includes the nitration of aromatic amino acids like tyrosine.
6. What is the formula of the colored precipitate obtained in the sulfur test or lead acetate test?
* Pbs (lead)
YOUTUBE VIDEO REFERENCES
BIURET TEST
Egg Albumin and Gelatin
https://youtu.be/RSAo9qPV5R4?t=37 and Simulation: https://amrita.olabs.edu.in/?sub=73&brch=8&sim=140&cnt=4
NINHYDRIN TEST
Egg Albumin and Gelatin
Simulation: https://amrita.olabs.edu.in/?sub=73&brch=8&sim=140&cnt=4
XANTHROPROTEIC TEST
Egg Albumin and Gelatin
Simulation: https://amrita.olabs.edu.in/?sub=73&brch=8&sim=140&cnt=4
HOPKIN’S COLE TEST
Tryptophan
https://youtu.be/7w3xLnFsB7s
MILLON’S TEST
Egg Albumin and Gelatin
Simulation: https://amrita.olabs.edu.in/?sub=73&brch=8&sim=140&cnt=4
SAKAGUCHI TEST
Arginine
https://youtu.be/LbuyTl7xSqs
LEAD ACETATE TEST
Gelatin, Casein and Albumin
https://youtu.be/j5lx_vGGjOQ
🧪 Experiment 4
MILK
* is a mixture of many types of proteins, most of them present in very small amounts
MILK PROTEINS
* classified into three main groups of proteins based on their widely different behaviors and forms
* Caseins (80% of Milk Proteins)
* Whey Proteins
* Minor Proteins
CASEIN
* a heterogeneous mixture of phosphorus-containing proteins in milk
* present in milk as calcium salt and calcium caseinate
* a mixture of alpha, beta, and kappa caseins to form a cluster called micelle
💌Alpha, Beta, and Kappa Caseins
* different types of casein proteins that are present in milk
* classified based on their molecular structure and properties
Alpha-casein (α-casein):
* forms the solid structure of cheese curds by interacting with other casein proteins
* plays a major role in the texture and firmness of cheese
Beta-casein (β-casein)
* maintains the smoothness and uniform distribution of casein proteins within the milk
Kappa-casein (κ-casein):
* prevents premature coagulation of milk proteins
*yung mga naka-box, NOT INCLUDED talaga sa book (ni-research ko lang for better understanding)
MICELLE
* responsible for the white opaque appearance of milk
💌Expounded Definition and Explanation of Micelle:
* A small clump or cluster of casein proteins found in milk
* These micelles stay suspended and evenly spread out in the liquid because their surfaces carry negative charges, which make them repel each other
* This repulsion keeps the milk stable and prevents the proteins from clumping together
Like proteins, casein is made up of hundreds of individual amino acids, which each may have a positive or a negative charge, depending on the pH of the milk.
ISOELECTRIC POINT (IEP)
* pH value wherein all the positive charges and all the negative charges on the casein protein will be in balance, so that the net charge on the protein will be zero
* pH at which the protein is least soluble
IEP IN CASEIN = 4.6
* pH value at which acid casein is precipitated
* In milk (pH = 6.6), the casein micelles have a negative charge and are quite stable
* During the addition of acid to milk, the negative charges on the outer surface of the micelle are neutralized (the phosphate group are protonated), and the neutral protein precipitates. (explained further below)
💌Definition of Protonated:
* When a molecule or group (like a phosphate group) gains a hydrogen ion (H⁺)
💌Explanation of the Reaction of the Milk to the Acid:
* When acid is added to milk, the extra hydrogen ions (H⁺) from the acid attach to the phosphate groups on the surface of the micelles, making them neutral — a process called protonation.
* As the negative charges are neutralized, the micelles stop repelling each other and start to clump together.
* This clumping is called precipitation, which is what happens when milk curdles.
Lactic acid bacteria (like Lactobacillus)
* Feed on the milk sugar called lactose
* Digest the lactose and produce a byproduct
Lactic Acid
* Byproduct of the fermentation process
* gradually lowers the pH of the milk.
* When the pH reaches about 4.6, which is the IEP of casein, the casein proteins lose their charges and can no longer stay dissolved. As a result, the casein precipitates forming curd.
Normal range of Percentage of Casein: 3% to 5%
BIURET TEST
* A chemical test used to detect the presence of protein in a solution, specifically the peptide bonds that link amino acids together in proteins
* The test involves adding a solution of copper(II) sulfate (CuSO₄) to the sample being tested.
* When proteins are present, the copper ions (Cu²⁺) from the copper sulfate react with the peptide bonds in the protein.
The presence of peptide bonds causes a color change in the solution from blue to purple, due to the formation of a complex between copper ions and the protein's peptide bonds.
*See the results and expected color reactions for the Biuret test on the last page.
XANTHOPROTEIC TEST
* used to detect the presence of aromatic amino acids in proteins — specifically tyrosine, tryptophan, and to a lesser extent, phenylalanine
* casein is a protein and contains aromatic amino acids like tyrosine
* concentrated nitric acid (HNO₃) is added to the casein solution
* nitric acid reacts with the aromatic rings of amino acids like tyrosine and tryptophan, resulting in the formation of yellow-colored nitro derivatives.
* the mixture is heated to accelerate the reaction.
* heat helps break the aromatic amino acid's bonds, allowing for a stronger reaction with nitric acid, which intensifies the color change
* after heating, alkali (e.g., NaOH or ammonia) is added to the solution
* The addition of alkali intensifies the yellow color, turning it into a bright orange
*See the results and expected color reactions for the Xanthoproteic test on the last page.
Biuret Test on Casein
SOLUTION
EXPECTED COLOR
EXPLANATION
ROLE OF EACH CHEMICAL
0.1 % copper sulfate solution
Light Blue
This is the natural color of copper(II) sulfate in water. No protein is present, so no Biuret reaction takes place.
CuSO₄: Source of Cu²⁺ ions; alone, it does not react without protein and an alkaline environment.
Casein + Sodium hydroxide
Cloudy white
Casein may not fully dissolve and can appear cloudy. No color change occurs yet because copper ions have not been added.
Casein: Protein with peptide bonds.
NaOH: Makes the solution alkaline and starts denaturing the protein.
Casein + NaOH + CuSO₄
(Biuret test)
Purple
(stirred)
A purple complex forms when Cu²⁺ ions react with peptide bonds in an alkaline environment, indicating the presence of protein.
Casein: Provides peptide bonds.
NaOH: Creates high pH and unfolds protein.
CuSO₄: Supplies Cu²⁺ ions that bind to peptide bonds.
After adding more CuSO₄ to the same solution
Deeper Purple
(no picture)
Initially, the purple color may intensify slightly. If too much CuSO₄ is added, the solution may turn bluish due to excess unreacted Cu²⁺ ions.
Extra CuSO₄: Adds more Cu²⁺ ions. Once all peptide bonds are used, excess ions remain free, shifting the color toward blue.
Xanthoproteic Test on Casein
SOLUTION
EXPECTED COLOR
EXPLANATION
ROLE OF EACH CHEMICAL
Casein + Concentrated Nitric Acid
Yellow (theoretical)
Nitric acid reacts with the aromatic rings in the tyrosine (and possibly tryptophan) in the casein, forming a yellow nitro derivative.
Casein: Contains aromatic amino acids like tyrosine, which reacts with nitric acid.
Nitric acid (HNO₃): Reacts with the aromatic amino acids, causing the yellow color.
Casein + Nitric Acid (After Heating)
Deeper Yellow (theoretical)
Heating intensifies the reaction, helping to break down the protein structure, ensuring more aromatic rings react with the nitric acid, creating a stronger yellow.
Heating: Speeds up the reaction and allows for more complete interaction between nitric acid and aromatic amino acids.
Casein + Nitric Acid + Sodium Hydroxide (3 M NaOH)
Orange (theoretical)
Sodium hydroxide (NaOH) turns the yellow solution orange, indicating the formation of xanthoproteic salts, which intensify the yellow color.
Sodium hydroxide (NaOH): Neutralizes the acidic solution, forming an orange color as the xanthoproteic compound reacts with the alkali.
PROCEDURE
YOUTUBE VIDEO REFERENCE
Extraction of Casein
https://youtu.be/Agc4S5hTb6g?feature=shared&t=31
Biuret Test on Casein
https://youtu.be/3R0I8KpgDGI?feature=shared&t=155
Xanthoproteic Test on Casein (real results showed faint color change, while another video showed no color change)
Positive Faint Yellow Color Change:
https://youtu.be/tlzIx5a5OAo?feature=shared&t=264
No color change:
https://youtu.be/7kpWYsKsNEM?feature=shared&t=10
LAB REPORT QUESTIONS:
1. "Little Miss Muffet sat on a tuffet, eating her curds and whey." What was she eating and where did It come from?
* Curds
* solid parts of milk that clump together
* Whey
* leftover liquid
2. In order for the ninhydrin test to work, what functional group must be present?
* Amino/Amine Group must present for me ninhydrin test to work
* The ninhydrin test detects the presence of primary and secondary amino groups (amines), wherein it forms a blue-purple compound called Runemann's purple.
3. According to the equation for the ninhydrin reaction with an amino acid, an aldehyde and CO2 are products. What parts of the amino acid give rise to these products?
* Carboxyl Group (COOH) – forms CO2
* R- Group (sidechain) – forms the aldehyde
4. Using the percentage you obtained for casein in milk, how many grams of casein are in a glass of milk (175 g)?
5. Below is the structure of phenylalanine. Why should this amino acid give a yellow color with concentrated nitric acid? What part of the molecule becomes nitrated (le., adds a —NO2
* Benzene Ring
* undergoes Nitration when reacted with
HNO3 (nitric acid )
* Amino acids containing aromatic rings like tyrosine and tryptophan, turn yellow when treated with concentrated nitric acid due to a nitration reaction where the aromatic ring is attacked and forms yellow - colored nitro denvarves.
6. Does albumin and casein contain tyrosine? How do you know? Yes
* Both turn yellow in the xanthoproteic test which defects amino acids like tyrosine
* Millon's Test can also be used to detect the presence of phenolic compounds. Particularly tyrosine, which is an amino acid containing a phenol group.
🧪 Experiment 5
* A method of separating and identifying amino acids.
* It involves the distribution of the solute between the polar liquid phase, which is strongly absorbed on the cellulose fibers of the paper and the eluting solvent.
* The paper is sprayed with ninhydrin solution to determine the position of the amino acid.
MOBILE PHASE
* Moves up the paper and the amino acid is carried to a particular position on the paper.
FACTORS AFFECTING THE LOCATION OF THE AMINO ACIDS
* The pH
* The temperature
* The concentration of the solvent
* The time of the chromatography
AMINO ACIDS
* Amino acids are organic molecules that serve as the building blocks of proteins. Each amino acid has the same basic structure:
* A central carbon atom (α-carbon),
* An amino group (-NH₂),
* A carboxyl group (-COOH),
* A hydrogen atom, and
* A variable side chain or R group that determines the identity and properties of the amino acid.
Rf VALUE
* The Rf value (retention factor) is used in chromatography (e.g., paper or thin-layer chromatography) to help identify compounds like amino acids based on their movement through a medium.
Rf Value =
POLARITY OF AMINO ACIDS
* More polar substances tend to interact more strongly with the polar stationary phase (e.g., paper), and move more slowly — resulting in a lower Rf value.
* Less polar substances interact less with the stationary phase and move farther with the non-polar solvent — leading to a higher Rf value.
MORE POLAR VS. LESS POLAR AMINO ACIDS
The polarity of amino acid depends on the nature of its R group:
TYPE
Characteristics
Examples
More polar
* Hydrophilic
* R groups can form hydrogen bonds or are charged (acidic or basic).
Serine, Glutamic acid, Lysine
Less polar
* Hydrophobic
* R groups are nonpolar hydrocarbons or rings.
Valine, Leucine, Phenylalanine
Key Differences:
* Polarity affects solubility
* More polar amino acids dissolve better in water
* Polarity affects behavior in chromatography
* More polar amino acids stick to the stationary phase and move slower (lower Rf values).
* Less polar amino acids move faster with the solvent (higher Rf values)
* Polarity influences structure and function in proteins
* Polar amino acids tend to be on the outside of proteins (interacting with water), while nonpolar ones are often buried inside.
KEY CONCEPTS
* The stationary phase (paper) is polar.
* The mobile phase (solvent) is non-polar or less polar.
* Molecules with higher polarity stick more to the paper and move less → low Rf value.
* Molecules that are less polar move further with the solvent → higher Rf value.
REASONING BEHIND THE Rf VALUES
1. Polarity is the main driver of movement in paper chromatography.
2. Ionic compounds or amino acids with charged side chains (like lysine and glutamic acid) bind strongly to the paper.
3. Natural substances like pusit, which contain a mixture of amino acids, show Rf values corresponding to the dominant or most abundant amino acid in the mixture.
4. MSG, being a salt of glutamic acid, behaves slightly differently — its ionic nature makes it stick even more, explaining why it had the lowest Rf value (0.20).
LAB REPORT QUESTIONS
1. For what purpose is the developing solvent used?
* It carries the amino acids which serve as its mobile phase.
PAPER CHROMATOGRAPHY RESULTS
SPOT
Distance by Solute (cm)
Distance by Solvent (cm)
Rf Value
POLARITY & EXPLANATION
Lysine
1.9
9.2
0.21
Very polar (basic) — The amino group in the side chain makes lysine highly polar, so it binds strongly to the polar paper and moves less with the solvent.
Glutamic Acid
2.4
9.2
0.26
Very polar (acidic) — Although also polar, the slightly longer side chain and acidic nature let it move a bit further than lysine.
Aspartic Acid
2.1
9.2
0.23
Very polar (acidic) — Slightly less movement than glutamic acid due to a shorter side chain; still highly polar.
Vetsin (MSG)
1.8
9.2
0.20
Highly polar (ionic) — MSG is the sodium salt of glutamic acid, making it ionic and strongly attracted to the polar paper, which limits its movement more than regular glutamic acid.
Pusit
2.0
9.2
0.22
Mixture of polar amino acids — Contains proteins rich in polar amino acids (e.g., lysine, glutamic acid). The observed Rf is close to lysine, indicating similar average polarity.
2. What factors affect the Rf value of the different solute used?
* Stationary phase
* Temperature
* Nature of chromatography plate
3. Explain the Rf value obtained on the basis of the structure of amino acids used.
* More polar amino acids → less soluble in solvent = lower Rf
* Less polar amino acids → more soluble in solvent = higher Rf