Milk – Experiment 11
Milk
- Most complete food
- Proteins, carbohydrates, vitamins, fats, inorganic
salts
- is a complex dispersion of fat globules and
casein micelles in an aqueous suspension of
whey proteins, soluble caseins, salts, and lactose.
-
Fresh Unboiled Milk
- Enzyme, protease, lipase, lactase, phospotase,
catalase, peroxidase
Casein
- Chief protein of milk
- Can be precipitated by acid, and carrying with it
the milk fat
- is a phosphoprotein, a kind of conjugated
protein. It is the chief protein found in milk.
- Fresh Milk - low specific gravity – contains milk fat
Fat
- Can be extracted by organic solvents
Filtrate from casein and fat
- Contains soluble constituents like lactose and
inorganic salts
Proteins of milk
- Derived from amino acids of the blood,
- The synthesis occurring in the mammary glands
Milk Fat
- Origin is in phospholipid of the blood
Lactose of Milk
- Derived from glucose of the blood
Derived from blood through the process of filtration
- Inorganic salts: calcium, magnesium, sodium,
phosphates, citrates and chlorides
Bone
- Reserve supply of calcium
Human Milk differs from cow’s milk
- Less casein and ash
- More albumin and lactose
Acidity of Milk is due to lactic acid. It produced by the
action of the lactic acid organisms on the milk sugar. It
is now generally believed to be due to the presence of
the carbon dioxide, acid phosphates and casein, all of
which are found in fresh milk and which have an acid.
1. Reaction of Milk
pH - Milk is slightly acidic (fresh milk has a pH of
6.7)
1 ml of milk – red & blue lithmus paper ; congo
red ; phenolphthalein solution
Results:
 red & blue lithmus paper – stays the same
- milk is neither acidic or alkaline
 Congo red – red / pink color solution
- Redox reaction
 Phenolphthalein – colorless
-commonly used indicator for titration
2. Determination of Specific Gravity
- desimeter
Skimmed Milk – higher specific gravity – no milk fat
Milk Fat
- Highest constituent of milk ‘
- More  lighter
- Less  heavier
3.Film Formation
- 5 ml of diluted canned milk in a small beaker and boil
Sour Milk – no film bec. the proteins are already
denatured by additional acetic acid
Fresh Milk – forms film
Factors that affect Film Formation:
 Temperature
 Stirring/ Whisking
 Presence/Absence of milk fat
 Type of milk
Film – milk skim or lactoderm is cause by denaturation
of proteins such as beta-globulin
- Soluble milk proteins denatured and coagulate
with milk fat
- whey proteins denature; at higher temperatures,
casein proteins also denature.
due to a transverse hydration gradient that drives
evaporation and induces stresses in the milk skin, which
are alleviated through wrinkling.
The film, also called milk skin is formed by fat-protein
interaction. When milk is boiled, the water at the surface
evaporates, thereby exposing the fat and protein
molecules, which bind and dry out. At high
temperatures, the proteins tend to clump up. As it is
heated even more, the soft layer dries up. Skin formation
does not form in skim milk because of the absence of
fats; without fat, there is nothing that proteins would
bind to. Only surface fats and proteins contribute to the
film.
In sour milk, lactose from the milk is oxidized by
bacteria into Lactic Acid, which acidifies the solution
leading to the denaturation of the proteins.
4. Coagulation Test
- 1 ml of fresh milk in a tt and acidify with acetic acid.
Heat to boiling
- formation of casein: involves two process:
 Rennin converts the caseinogen into paracasein
or soluble casein
 The calcium salts present in the milk precipitate
the paracasein as casein
Is there coagulation? yes
-coagulation due to the presence of whey protein which
is mainly composed of beta-lactoglobulin
Acidifaction of milk with acetic acid allows the proteins
to reach isoelectric point, lowering the intermolecular
repulsion between proteins, leading to coagulation(or
increased precipitation) when heated.
5. Action of Hot Alkali
- mix: 1 ml of milk with drops of 6 M NaOH
- heat
NaOH: oxidizing agent
Results: presence of brown solution and caramel odor
due to the reducing sugar glucose
- Moore’s test – test with same principles
Moore's Test. Polymerization of aldehyde groups of
sugars (in milk, it is lactose).
6. Preparation of Casein
-10 m of milk
- dilute acetic (1%) – drop by drop – flocculent ppt.
forms ; if excess dissolution may occur
-supernatant fluid (whey)
-add ethyl alcohol : used to remove moisture
- ether – to remove milk fat
Results:
 Water – soluble
 NaOH – soluble ‘
NaOH Solution - Soluble. Isoelectric pH of casein
is slightly acidic. The pH of this solution is far from
the isoelectric pH of casein, leading to increased
molecular repulsion and dissolution of casein.
 NaCl – soluble
Non-Concentrated(10%?) NaCl Solution - Soluble.
Low salt concentration increases the solubility of
substances through the effect called "Salting-in"
 HCl – insoluble
Dilute HCl Solution - Insoluble. Isoelectric pH
of casein is slightly acidic. The pH of this solution is
close to isoelectric pH of casein. Reduced molecular
repulsion
Millon’s test – formation of red ppt.
- Due to the denatured proteins that forms amino
acids tyrosine
dilue acetic acid is added to reach the isoelectric point of
casein. Adding excess acid lowers pH beyond isoelectric
point of casein leading to dissolution.
The precipitated protein is positive with Millon's Test
(red ppt formation)
7. 3-ml portions of whey
a. Coagulation by heat
- Formation of coagulation of the coagulum
(lactalalbumin & lactoglobulin)
Biuret test
- Positive : violet
b.
 Phosphorus– yellow ppt ( ammonium
phosphomolybdate)
Phosphate Ion + Ammonium molybdate =
ammonium phosphomolymdate
 Calcium – white ppt. (calcium oxelate)
Calcium Ion + ammonium oxalate =
calcium oxelate + ammonium ion
 Millon’s test – orange ppt
 Benedict’s Test – Cuprous Oxide
- Red ppt. due to the presence of reducing sugar
Whey from milk contains all other substances found in
milk such as lactalbumin and lactoglobin proteins
(Biuret test, violet sol'n), phosphates (yellow ppt,
Ammonium phosphomolybdate), calcium (white ppt,
Calcium oxalate) and lactose(Benedicts test, orange ppt,
Cuprous oxide) apart from the separated casein.

8. Milk Fat
- transfer filtrate from no. 6 in a evaporating dish
- place in a boiling bath to evaporate the ether
- presence of residue: MILK FAT
- touch residue with a piece of paper: formation of
translucent spot

Fat from milk is non-polar and dissolves in the non-polar

ether. When placed in a evaporating dish at room
temperature, ether evaporated due to its volatility and
left behind a residue - milk fat. Tested with Translucent
Spot Test
 Salivary Digestion - Experiment 12
Saliva Proteins present in saliva:
- Moistens the mucous membrane of the mouth  Amylase
and helps in preventing tooth decay by cleansing  Defensins
the mouth of cariogenic carbohydrates and by  Cystatins
neutralizing lactic acid.  Histatins
- Contains 99% water and less than 1 % solid  Immunoglobulins
- Mucin: chief solid  Statherin
 Lactoperoxidase
- Secreted by 3 pairs of glands:  Lysozyme
 Parotid  Lactoferin
 Submaxillary
 Sublingual 1. Reaction
 Hundreds of buccal glands - Resting saliva: phenolphthalein, litmus and
congo red
- The flow is stimulated by: - Stimulated saliva (chewing paraffin for 5 mins)
 Psychic
 Chemical Resting Saliva
 Mechanical - Saliva found in mouth in the intervals of food
taking and mastication
- It contains a protein, mucin - Basic
- An enzyme, ptyalin Phenolphthalein Litmus Congo Red
- Inorganic salts

Inorganic Salts in saliva has 2 important functions: Violet Red  blue Red w/
 Phosphate acts as buffers that tend to Blue bubbles
maintain the reaction (pH) of the saliva
constant
 Chloride ions are essential as coenzymes for Stimulated Saliva
the salivary amylase. - Saliva secretions during stimulation
- more basic
The flow of saliva (about 1500 cc daily) is the result of - higher pH
stimulation of the salivary glands by the nervous system, Phenolphthalein Litmus Congo Red
as is evidence by the fact that actual contact with food is
unnecessary since sight, odor, or even though will cause
the salivary gland to secrete profusely. Violet Red  blue 2 layers:
Blue red (upper
part)
Salivary Amylase/Ptyalin clear (down)
- Begins the breakdown of starches, responsible
for the digestive function of the saliva
- Principal enzyme of saliva Paraffin – stimulates saliva secretions
- Capable of hydrolyzing cooked starch into The acidic pH is essential in dissolution of enamel to
dextrin and maltose produce dental carries

Starch  soluble starch  dextrin  maltose 2. Test for Mucin

- 3 ml of saliva
The maltose so produced is later hydrolyzed by the - 1-2 drops of dilute acetic acid
intestinal maltase into glucose.
Mucin – is a mucopolysaccharide or glycoprotein that is
chief constituent of mucus
Salivary Digestion - is formed when there is coagulation due to
- Hydrolysis of starch by salivary amylase heating and its function is to lubricate the mouth
(ptyalin) and prevent bacterial build up
- Takes place in buccal cavity
 - produced by epithelial cells and used as food
lubricant Calcium
Result: - clear with moisture (once HCL is dropped)
- cloudy solution with white ppt (Mucic) - Positive
Function: - Name of ppt: Calcium Oxelate (CaC2O4)
Mucin can be a symptom to any abnormalities in the - Dominant metal ion present
body such as cancer
Sulfate
3. Inorganic Matter - Clear
- Acidify 10 ml of saliva with a drop or 2 of acetic - Negative
acid
- Heat to boiling NORMAL PH RANGE OF SALIVA: 5.6 – 7.9
- Filter to remove protein - Several factors influence the pH nature of the
saliva and varies depending to the food and
Test filtrate for CHLORIDES, PHOSPHATES, drinks intake of an individual
SULFATES AND CALCIUM
4. Digestion of Starch Paste
Chloride salts and phosphate salts – most abundant - 10 ml of 1% starch paste in a small beaker
inorganic matter in saliva - add 5 drops of saliva and stri
- iodine (after 5 mins)
Chlorides: AgNO3 + HCl - opalescence of the starch solution disappears due
Phosphates: HCl + (NH4)2MoO4 to the formation of soluble starch
Sulfates: BaCl2 + HCl
Calcium: HCl + K2C2O4 Maltose – responsible to reducing action
10-20 mins – it takes to completely transform starch to
Results: reducing sugar
Chlorides Turbid soln with white Reaction of iodine with Benedict’s Test: Blue (Positive)
ppt.
Phosphate Clear, colorless soln with 1. Boiled Starch + ptyalin = soluble starch
pale yellow ppt 2. Soluble starch + ptyalin = erythrodextrin +
Sulfates Slightly turbid soln maltose
Calcium Clear, colorless soln 3. Erythrodextrin + maltose + ptyalin =
achrodextrin + maltose
4. Achrodextrin + maltose + ptyalin = isomaltose +
Chlorides maltose
- WHITE ppt
- Positive The optimum pH for the action of Ptyalin is 6.7
- Name of ppt: Silver Chloride (AgCl) In a theoretical setup, if starch paste becomes negative in
- Major amylase activator the test with iodine, it is surely positive with the test for
- Chloride ions help in maintaining the osmotic sugar (because starch is completely converted to
balance in the mouth preventing any excess glucose).
inflow or out flow of water from water tissues
5.Influence of Acid
AgNO3 + Cl-  AgCl + NO3 - 2 ml of:
 0.25 % HCl
Phosphates:  0.1 % HCl
- Yellow ppt  0.05% HCl
- Positive  0.025% HCl
- Name of ppt: Ammonium Phosphomolybdate  0.0075% HCl – obtain the greatest digestion
(NH4)3PMo7O40 because it is the least acidic
- pH buffer - place in a water bath for 20 mins at 40 C
- functions: contributes to solubility product of
calcium phosphate, which is crucial in 6.Influence of Alkali
maintaining tooth structure, important as a - repeat test in #5 but use:
buffer and an essential nutrient for oral  2% NaOH
microflora for metabolic pathways  1% NaOH
  0.5% NaOH - Silver nitrate
 0.125% NaOH
 0.065% NaOH Reagent for phosphate test
- neutralize with acetic acid - Ammonium molybdate

Reagent for sulfate test

Acid – has the greatest inhibiting power because it - Barium chloride
disrupts the basicity of the saliva which is necessary for
buffering capacity Reagent for calcium test
- Ammonium oxalate
Gastric juice starts protein digestion while saliva starts
starch digestion What is responsible for reducing reaction in benedict’s test
- Maltose
It is inhibited more by acids, therefore ptyalin is slowly
inactivated by the acidic Gastric Juice in the stomach. Which has greater inhibiting power? Acid or alkali?
Salivary digestion continues in the stomach for 10 to 20 - Acid
minutes due to the slow penetration of acidic Gastric
juice into the bolus (mass of chewed food). After the 10 B.S Ptyalin
to 20 minute period, salivary digestion stops due to the
total inactivation of ptyalin.

Questions:
Saliva is secreted by 3 pairs of glands S.S
- Parotid
- Submaxillary
- Sublingual
Ptyalin
Factors that stimulate salivary flow
- Psychic
- Chemical Erythrodextrin Maltose
- Mechanical

Protein in saliva
- Mucin Ptyalin

Enzyme in saliva
- Ptyalin
archodextrin Maltose
Aka salivary amylase
- Ptyalin

Hydrolyzes starch
- Salivary amylase Ptyalin

Where does salivary digestion occur

- Buccal cavity; fundic end of stomach Isomaltose
Why is saliva basic and not acidic
- If its acidic it will dissolve enamel producing dental
caries Maltose
Used to precipitate mucin out of saliva
- Dilute acetic acid

Function of mucin
- Lubricate mouth & prevent bacterial build up

Abundant inorganic salts found in saliva

- Chlorides & phosphates

Reagent for chloride test

Bile a. Gmelin’s Test
- Very complex and varies according to the Superimpose 1 ml of bile to 1 ml of conc. HNO3.
nutritional state of an individual Production of green, blue, violet, red and reddish-
- Secretion of the liver yellow layers of color at the point of contact. This color
- It is viscid and has alkaline reaction and its color formation is due to the oxidation of bile pigments by
is greenish brown HNO3.
- It’s important constituents:
 Bile acids b. Rosenbach's modification of Gmelin's Test
 Bile pigments bile is instead filtered and a drop of HNO3 is added at
 Inorganic salts (potassium, sodium, the dried cone. Formation of concentric succession of
bicarbonate) colors indicate presence of bile pigments.
 Cholesterol
4. Test for bile acids and bile salts
Organic Constituents: Bile acids - Primary bile acids for humans is Cholic
 Bile salts Acid and Chendeoxycholic acid.
 Bile acids
 Bile pigments There are two main groups of bile acids: taurocholic
acids and glycocholic acids.
Inorganic Constituents
 Chloride a. Pettenkofer's Test or Sucrose-H2SO4 test
 Sulphates Test for bile acids. One drop of sucrose sol'n is added to
 Phosphates 1 ml of dilute bile. H2SO4 is allowed to run down the
side of the tube. Red ring forms at the point of contact.
1. Reaction Shaking makes the whole solution red.
- Bile to litmus paper, congo red and
phenolphthalein b. Foam Test
1:1000 soln of furfural is added to 1 mL of bile solution.
Results Mixture is shaken to form foam. H2SO4 is added to the
Litmus Paper Blue foam. Foam produces a pink coloration. Color formation
Phenolpthalein Yellow indicates the presence of bile salts/acids
Congo Red Red
c. Hay's Test of Surface Tension
Normal pH value of bile: 7 – 8.4 (basic) Surface tension is the tendency of the surface of a liquid
to resist external force.
2. Inorganic Constituents Surface tension is due to the cohesion of water
- Evaporate 10 ml of bile to dryness molecules. Surface tension is inversely proportional to
- Fuse residue with fusion mixture (2 oarts of temperature that is why the water was cooled to 17 °C
sodium carbonate and 1 part potassium nitrate) in the experiment.
- Extract with 10 ml of water Bile salts are emulsifying agents. Emulsifying agents
- Acidify with HNO3 reduce the surface tension of liquids, for example,
Results water, allowing substances that float normally on water,
Chloride White Calcium chloride such as sulfur, to sink.
Sulphate Yellow Barium Chloride
Phosphate Turbid - 5. Cholesterol in Bile
Bile is evaporated to dryness. Ether was placed in the
3. Test for Bile Pigments residue to extract cholesterol since ether is non-polar.
Bile pigments: The ether extract is allowed to evaporate and the
 Bilirubin – gives the red color in the tests residue is tested with Liebermann-Buchard test on
 Biliverdin – gives the green color in the tests chloroform solution. A red ring formed which
 Bilicyanin – gives the blue color in tests progressed to form a green ring.
Normally, bile contains enough chemicals to dissolve a CONSTITUENTS OF BILE
normal amount of cholesterol. Excessive cholesterol in Secretion Components Excretion Components
bile forms crystals since cholesterol can no longer be  Bile Salts  Bile pigments
dissolved which leads to formation of Gallstones.  Sodium Hydrogen  Cholesterol
Gallstones can then lead to blockage of the path of bile. Carbonate
 Water
BILE
- A viscous, yellow to brown, bitter-tasting THE BILE SALTS OR BILE ACIDS
alkaline fluid
- Secreted 500 to 800 cc daily by the liver Bile
- Flows via the bile ducts into the duodenum - Contains no digestive enzymes
- Plays an important role in digestion and
Rate of secretion and of flow of bile absorption of fat and indirectly that of other
- Influenced by the nature of food undergoing foodstuffs
digestion - Alkaline
- The stimulation, probably being influenced by a - Bile, along with the pancreatic juice and
hormone: SECRETIN intestinal juice
o Neutralizes the acid chime from the
Epsom Salts stomach
- Salt that stimulates the flow of bile - Contains a type of compound called the bile
salts or the bile acids
Absence of food:
Most of bile is diverted to the gallbladder BILE SALTS (or bile acids)
(about 30 cc capacity) - The two most important of these substances
- Where it is stored and becomes concentrated to are:
one-tenth its previous volume by: 1. Sodium glycocholate
1. Lymphatic reabsorption of: 2. Sodium tyrocholate
o Water
o Salt - A most important property of the bile salts:
o Cholesterol o They act as wetting agents, lower
o Pigment from it surface tension and facilitate the
2. By the addition of mucin emulsification very little fact digestion
 A secretion from the wall of the by pancreatic lipase (streapsin) occurs
gallbladder in the intestine

Active digestion: - Play an important role in fat absorption by

The contents of the gallbladder are evacuated uniting with the insoluble fatty acids, liberated
into the duodenum and thus provide a from fats by lipase action, to form soluble
concentrated supply of bile compounds known as choleric acids

Bile Choleric acids

- May also be considered as excretion  Upon absorption, separate
o Since it eliminates as waste products of again and the fatty acids unite
cellular action with glycerol in the lymphatic
o Certain substances which it alone can vessels of the villi (lacteals) to
dissolve (e.g. cholesterol) as well as form fats which are transported
other end products by the lacteals to the thoracic
- Contains: duct and eventually to the
o Lipids bloodstream
o Mucin
o Bile pigments
o Certain salts
 Liberated bile salts
 Are then available for further
use by the liver
 Are resecreted into the bile
 Thus undergo a type of
circulation:
BLOOD
↳ LIVER
↳ BILE
↳ INTESTINE
↳ BLOOD
- Possess the property of stimulating the
secretion of bile
o As such, it can be regarded as
cholagogues
 Substance stimulating the flow
of bile
- Also aid peristalsis
- Absence in the adequate bile flow:
o Extensive putrefaction of protein
occurs in the lower portion of the
gastrointestinal tract
 Due in part of the decreased
peristalsis and in part to the
undigested fat
 Forming an oily film
over protein particles
and retarding their
digestion
- Absence of bile
o Causes serious disturbances of
intestinal digestion and absorption
o Feces
 Contain greatly increased
quantity of fat and may be clay
colored and greasy, and leave a
very foul odor
o Constipation
 May result from decreased
peristalsis
THE BILE PIGMENTS
- Consists mainly of:
o Bilirubin
o Biliverdin
- Mostly formed when the liver salvages “organic
iron” from the blood pigments (hemoglobin) of
worn-out red blood cells
In this decomposition of hemoglobin into globin
and hematin, the latter with loss of iron,
changes into bile pigments which impart a
yellow to brown color to bile.
Bilirubin → has a reddish cast
Biliverdin → oxidized product of bilirubin
→ a green pigment
Normal: little or no biliverdin in the bile
Exposure of bile to air: it will turn green due to
oxidation of bilirubin to biliverdin
Oxidation
o Produces a series of colored
compounds, including
bilicyanin
 A blue pigment
Series of colors on bruised skin:
Undoubtedly due to the decomposition of
hemoglobin liberated by injured red cells which
exuded from the capillaries of the injured tissue
Obstruction to the bile ducts
Disease and dysfunction of the liver or an abnormal
destruction of the red blood cells
- Will result in an increased amount of bilirubin in
the blood
o This will diffuse into the skin and will
give them a characteristic yellow color
which known as jaundice or icterus
In the INTESTINES:
- The reducing bacteria change bile pigments to
stercobilin or urobilin
o A brown pigment which accounts for
the characteristic color of feces
Urobilin
- A part of the yellow color of urine is due to this
pigment
- Of secondary importance to another yellow
pigment in urine called urochrome
Diarrhea
- Too frequent elimination
- Does not allow for much reduction of bile
pigments with the consequence that feces then
have a decided yellow instead of brown color,
 the color depending somewhat on the cause of
diarrhea

CHOLESTEROL
- The remaining important excretory compound
of the bile
- Ordinarily soluble in bile

In the presence of foreign substances (such as

injured cells or bacteria):
- The cholesterol tend to crystallize, carrying with
it some bile salts and pigments to form
gallstones

- An alcohol (C27H45OH)

- Like glycerol (C3H5(OH)3), unites with fatty acids

to form esters but it differs in that its esters do
not easily saponify

Lanolin
- The fat of sheep’s wool
- Contains the stearic, palmitic, and oleic esters
of cholesterol
URINE Oxidation
- A filtrate from the blood - when allowed to stand without preservative,
- Serves as a medium for excretion of: urine becomes ammoniacal in odor and alkaline
o Water in pH
o Acids o because of the oxidation of urea to
o Bases ammonium carbonate
o Waste products of metabolism
o Other toxic materials Organic Constituents
- Helps in the maintenance of water balance, acid  Urea
base equilibrium  uric acid
- Serves as an important factor in the  creatinine
detoxification of the body
Inorganic Constituents
COLLECTION AND PRESERVATION OF URINE SAMPLE  Uric acid
24-hour specimen  Sodium urate
- Examined for the study of both the qualitative - precipitate in acidic conditions
and quantitative composition of urine o These precipitates are the primary
- Bladder is emptied at 8:00 AM (urine is components of kidney stones.
discarded)
o All the urine from this time up to Calcium phosphate
8:00 AM the next day is taken as sample - precipitates in alkaline urine

Toluene Specific Gravity Range of normal urine

- A thick layer of this is over-layed on the surface - range of 1.015 - 1.025
to preserve the urine
2. DETECTION OF CREATININE
1. GENERAL CHARACTERISTICS Creatinine
- breakdown product of creatine phosphate
1,000 – 1,500 mL
- Volume of the 24-hour urine
A. NITROPRUSSIDE TEST - Weyl
A. EXAMINE THE SPECIMEN AS TO COLOR, ODOR, 5 ml of urine
TRANSPARENCY AND REACTION + 3 drops SODIUM NITROPRUSSIDE
+ NaOH (to alkalinize)
Responsible for the normal color of urine: = ruby red color which turns yellow
- Urochrome/urobilin pigments (yellow) - indicative of the presence of creatinine
- Uroerythrin pigments (amber)
- Uses sodium nitroprusside
What happens when the urine is allowed to stand for - Red ppt indicates presence of creatinine
some time, exposed to air? - Color fades in normal urine if added with acid
- It smells of ammonia due to certain types of
bacteria present in the water that work on the
urea present in the urine and convert it back
into ammonia, thus the longer it sits the
stronger it gets

Urine pH of 6 when freshly voided due to:

- disodium phosphate - Na2HPO4
- monosodium phosphate - NaH2PO4

Freshly voided urine is clear and transparent.

If transparency is of cloudy flocculate, it might
be because of mucus and epithelial cells
B. PICRIC ACID REACTION - Jaffe 5. DETECTION OF CHLORIDE
5 ml urine 5 ml urine
+ aqueous sol’n of picric acid + acidify with 2 drops of HNO3
+ NaOH (to render the sol’n alkaline) + 2 drops of AgNO3
= red color = White ppt of AgCl formed
- Due to the formation of a red tautomer of
creatinine picrate + excess of NH4OH
- Turns yellow when the sol’n is acidified = Excess NH4OH dissolves AgCl2
- Glucose: gives a similar red color only upon
heating Normal amount of chlorides eliminated in 24 hours:
- 10-15 grams of chlorides are eliminated in 24
- Red ppt due to formation of tautomer hours.
of creatinine picrate o Usually eliminated in the form of NaCl.
- Normally turns yellow when acidified
- If it does not turn yellow, acetone bodies are 6. DETECTION OF PHOSPHATES
present. 10 ml urine
+ AMMONIUM HYDROXIDE (to alkalinize)
Jaffe reaction Warm.
- a colorimetric method used in clinical = Precipitation of earthly phosphates (Ca and Mg salts)
chemistry to determine creatinine levels in occur
blood and urine
- color change that occurred was directly → The earthy phosphates of Ca and Mg separate.
proportional to the concentration of creatinine → Filter off the earthy phosphates.
- however also noted is that several + small amount of MAGNESIA MIXTURE (to the filtrate)
other organic compounds induced similar → Warm the Solution.
reactions = Magnesia mixture precipitates alkali phosphates (Na
and K salts)
3. DETECTION OF PIGMENTS
→ Determine which form of phosphate is present in
AMMONIACAL ZINC CHLORIDE TEST larger amount.
2 ml urine
+ 1 ml of NH4OH = Alkali phosphates are larger in amount compared to
→ Let it stand for a while. Earthly phosphates with a ratio of 2:1
→ Filter.
+ 2 drops ZINC CHLORIDE SOLUTION (to the filtrate) PATHOLOGIC CONSTITUENTS
= greenish fluorescence
- indicates the presence of urobilin 7. ALBUMIN

other pigments normally present in urine: (? Not sure) A. COAGULATION TEST

- urochrome / urobilin 5 ml urine
- uroerythrin → Heat to boiling.
- indican → Filter if urine is not clear.
= if heated portion becomes cloudy, the turbidity may
INORGANIC PHYSIOLOGICAL CONSTITUENTS be due to phosphates

4. SULPHATES + 3-4 drops of VERY DILUTE ACETIC ACID

A. DETECTION IN INORGANIC SULPHURIC ACID → Warm.
5 ml urine = phosphates will dissolve
+ 5 drops of ACETIC ACID = a more flocculent ppt will be produced if only
+ BARIUM CHLORIDE sol’n albumin is present
= ppt of BARIUM SULPHATE
- White ppt of BaSO4 B. HELLER’S RING TEST
5 ml conc. Nitric acid
→ Slant the tube.
→ Very carefully allow an equal amount of urine to
slowly run down the side of the tube.
= urine will float on the nitric acid
= a white ring (precipitated protein) will appear at the
junction of the two liquids
- This confirms the presence of albumin
Is bilirubin normally present in the urine?
Bilirubin is normally ABSENT in urine because
bilirubin is converted to urobilinogen in the intestines
and then absorbed in the bloodstream. The absorbed
pigment is then oxidized to urochrome or urobilin which
makes its way to the kidneys(responsible for urine
color).

Sometimes the white zone does not appear until What does its presence indicate?
allowed to stand for a few minutes. Presence of bilirubin indicates obstruction of
the flow of bile from the gall bladder so that the bile is
8. GLUCOSE absorbed in the blood and bilirubin goes to the kidneys.

BENEDICT’S TEST (semi-qualitative test) 11. ACETONE BODIES

5 ml of Benedict’s reagent
+ 5 drops of urine Ketone bodies
→ Boil vigorously for 2 mins - are brought about by increased lipid
→ Set aside to cool metabolism wherein the utilization of lipids is
incomplete. These bodies are:
Amount of ppt and its color (red, yellow, or green) o Acetoacetic acid (diacetic acid),
- Depend on the quantity of glucose present in o Acetone
the urine o Beta hydroxybutyric acid
- Presence of these substances in urine is called
Benedict's Test - Semi quantitative test. Color depends ketonuria
on the quantity of glucose.
 Red - conc. amt. of glucose There is no satisfactory, simple direct test for b-hydroxyl
 Yellow - glucose substantially present butyric acid in the urine.
 Green - little glucose present
 Blue - no color change; no glucose present Aceto-acetic acid
- Decomposes so rapidly with the formation of
9. BLOOD acetone, that the usual test for acetone are
(demonstration only) also given for aceto-acetic acid

BENZIDINE TEST Test for ketonuria

3 ml of urine - Are tests for either acetone or aceto-acetic acid
→ Heat to boiling. or both
→ Cool. - True for nitroprusside test
→ Treat with an equal volume of a saturated sol’n of
Benzidine in Glacial Acetic Acid NITROPRUSSIDE TEST – Legal’s
+ 1 ml of 3% H2O2 2 ml urine
= development of a blue or green color indicates the + 3 drops of 5% freshly prepared aqueous sol’n of
presence of blood sodium nitroprusside
+ NaOH (to alkalinize)
10. BILE = ruby red color indicates acetone

GMELIN’S TEST + 0.5 ml of acetic acid

1 ml of conc. Nitric acid = if red color persists, ketone bodies are present.
→ Superimpose 1 ml of urine.
→ Do not mix. If the test is made directly on urine, a red color is given
= At the point of contact, various color rings are noted: by creatinine which disappears on the addition of acetic
blue, green, violet, red, and reddish yellow, in the acid.
presence of bile pigments.
 PAPER CHROMATOGRAPHY experiment is the Filter Paper (cellulose in the filter
- Separation of amino acid on the basis of the difference in paper is responsible for the degree of movement)
solubility of amino acid between 2 immiscible solvents 2. Mobile Phase - this is the solvent that flows through
- is an analytical method used to separate colored the stationary phase. This carries the components of
chemicals or substances the mixture. The mobile phase in the experiment is
- two phases: the developing solution
 Stationary Phase (a solid, or a liquid supported on a 3. Rƒ - this is the Retention Factor or the Retardation
solid) Factor. This is the ratio of the distance traveled by
 Mobile Phase (a liquid or gas) the substance to the distance traveled by the
- Type of chromatography: solvent.
 Liquid chromatography
 Gas chromatography A. Preparation of the Developing Chamber
 Ion exchange chromatography Pipette 8 mL of the solvent consisting of 4:1:5: (by volume)
 Affinity Chromatography mixture of butanol, acetic acid, and distilled water
- most effective for the identification of unknown respectively, and introduce into a dry 250 mL beaker.
substances when known samples are run on the same Avoids splashing the liquid on the sides of the beaker.
paper chromatograph with unknowns Cover with a piece of aluminum foil and let stand for 10
minutes for the atmosphere inside to become saturated
with the solvent vapor.
Term Definition
solvent moving through the column  The developing solution consists of 4:1:5 by volume
Mobile phase - the medium that accompanies the mixture of butanol, acetic acid and distilled water.
or carrier analyzed substance as it moves  The Developing Chamber is a 250 mL beaker covered
through the stationary phase with a piece of aluminum foil
substance that stays fixed inside the  The solvent is placed in the chamber and was
Stationary allowed to evaporate for 10 minutes to saturate the
column
phase or chamber with its vapor (since it is volatile). This step
- medium on which the separation
adsorbent is very important since saturating the atmosphere in
occurs
Eluent fluid entering the column the beaker with the solvent vapor stops the solvent
from evaporating as it rises up the paper.
fluid exiting the column (that is
Eluate
collected in flasks)
B. Preparation of the Paper Chromatogram
the process of washing out a compound
Elution through a column using a suitable 1. With minimal handling (fingerprint can obscure the
solvent result) cut a piece of Whatmen filter paper no. 1, 16.50 cm
long and 8.0 cm wide. With a pencil, draw a line 6 mm
mixture whose individual components
Analyte from the lengthwise edge of the paper and 1 cm from each
have to be separated and analyzed
crosswise edge, for handling.
- Phenomenon where the solution
Capillary (sometimes called the eluting
1CM
Action solvent) will begin to rise up the
paper

Principle of separation of different components: Differential

affinities (strength of adhesion) of the various components of
the analyte towards the stationary and mobile phase results
in the differential separation of the components. Affinity, in
turn, is dictated by two properties of the molecule:
6 MM GLY LYS
‘Adsorption’ and ‘Solubility’.
ASP UNKNOWN
Type of Chromatography
2. Mark lightly with pencil equidistant spots along the
 Ascending Paper Chromatography was used in the
lengthwise line of the filter paper.
experiment. Movement of the solvent is due to
capillary action or capillarity
3. Gently and quickly touch the first mark with the point of
a fine capillary tube (0.5 mm diameter) containing 0.5%
Definition of Terms
glycine. Apply approximately 20 micrograms of the sample
1. Stationary Phase - this is the structure that holds the
on the mark, allowing the spot to dry before each
substance that is tested. The stationary phase in the
 application. The wet area should not be more than 2 mm
in diameter.
4. Repeat step no. 3 on the other marks using a different
amino acid for each mark ( 0.5% lysine, 0.5% aspartic acid,
and 0.5% unknown solution)
 Whatmen Filter Paper no.1 was used as the
chromatogram
 Dimensions is 16.50 x 8.0 cm
 With a pencil, draw a line 6 mm from the lengthwise
edge of the paper and 1 cm from each crosswise
edge, for handling
 0.5% of glycine, lysine, aspartic acid and unknown
solution were used in the experiment
Why is the chromatogram developed in an essentially closed
system?
The chromatogram is kept in an essentially closed
system in order to prevent it from acquiring contaminants and
other substances that can hinder in the separation of the
organic pigments. It also prevents the developing solvent used
from evaporating since most solvents used in paper
chromatography are highly volatile, and some can be
flammable and toxic.
C. Development of the Spot
1. With careful handling at the crosswise edges, staple the
paper into a cylindrical form. Do not overlap the edges.
2. Put the cylindrical paper upright into the equilibrated
beaker with the spotted edge at the bottom. The solvent
should wet the lower edge of the paper without reaching
the spots. Put the aluminum foil cover in place and let
stand for 30 - 45 minutes or until the solvent front is 1 cm
away from the upper edge.
3. Remove the paper from the beaker and open-up. Mark
the position of the solvent front before it dries up
completely.
Trace the level of the wet portion in the upper part of the
paper using a pencil.
4. Spray the entire paper lightly with 0.2% ninhydrin
solution and dry it in the oven at 110° C. Heat if necessary
for the color forming reaction. The color should be readily
visible after 20 - 30 minutes.
Expected result: spot will be observed to have moved
upward. It will travel and our purpose is to measure the
separation or travel of the amino acid.
 Solvent moves via capillary action
 Solvent brings along the substances as it moves up
 Ninhydrin (2,2-Dihydroxyindane-1,3-dione) was used
to react with the amino acid to form a colored
compound (when heated at 110 °C).
o Ninhydrin reacts with amino acids to form a
blue-violet compound.
 NINHYDRIN TEST principles:
o Amines (including α-amino acids) react with
ninhydrin to give a coloured product.
o It can be used qualitatively (e.g. for
chromatographic visualisation) or
quantitatively (e.g. for peptide sequencing).
o The α-amino acids typically give a blue-
purple product.
o Proline, a secondary amine, gives a yellow-
orange product.
o The test is sensitive enough that ninhydrin
can be used for the visualisation of
fingerprints.
 The further the spot from the starting line, the
higher the affinity of the amino acid for the mobile
phase and the faster its migration
D. Calculation of the Rƒ value
How to measure the Rf Value:
 Distance traveled by the amino acid: the first dot
made near the 6 mm line until the center of the
colored (violet) spot
 OVER
 Distance traveled by the solvent: from 6 mm line
until the traced portion from the top.
Calculate the Rf value of each amino acid and identify the
unknown amino acid.
1. Distance traveled by substance in mm divided by
the distance traveled by the solvent. This measures
the attraction of the substance to both
the stationary phase and the mobile phase
2. If the substance is strongly attracted to the
stationary phase and not attracted to the mobile
phase, then the substance does not move at all, so,
the Rf is 0.
3. If the substance has no affinity to the stationary
phase, then the substance moves along the solvent
front, so, the Rf is 1.0.
In conclusion, the movement of the substance in
relation to the solvent front (Rf) is dependent upon:
 Solubility of the substance to the mobile phase
(solvent)
o an amino acid that is highly soluble in the
eluting solvent will have a higher affinity for
the mobile phase than an amino acid that is
less soluble in the solvent.
 Affinity of the substance to the stationary phase
(how much it sticks to the cellulose of the filter
paper)
o If an amino acid has a higher affinity for the
mobile phase than the stationary phase, it
will tend to travel with the solvent front and
be relatively unimpeded by the filter paper.
o If the amino acid has a higher affinity for
the paper than the solvent, it will tend to
“stick” to the paper and travel more slowly
than the solvent front.
- these differences in the amino acid affinities
that lead to their separation on the paper.