ACM

BIOCHEM LAB MIDTERM REVIEWER - do not give (+) result in Tollen’s, Benedict’s, and
Fehling’s tests

Experiment 4: POLYSACCHARIDES - starch, cellulose, glycogen

I. Introduction (BOOK) Starch

Carbohydrates - plants

- it is a group of biochemical substances that contains - starch — plant — energy

functional groups:
- TYPES:

- Aldehydes
1. AMYLOSE

- Ketones
- straight chain glucose polymer

- Alcohols
- 15% to 20%

- most abundant class of biomolecules on Earth

- alpha (1—>4) linkage

- 3 Main Classes of Carbohydrates

- Monosaccharide
2. AMYLOPECTIN

- Disaccharide
- branched glucose polymer

- Polysaccharide
- 80% to 85%

- Note: all these 3 have differences in terms of - alpha (1—>4) linkage

their physical and chemical properties

- branched point: alpha (1—>6) linkage

Monosaccharides - storage of polysaccharides in plants

- simplest carbohydrate

- physiologically important in metabolism

Iodine Test

- GLUCOSE
- test for presence of starch

- most important form of monosaccharide

- iodine dissolved in KI = deep blue black color

- reaction is the result of the formation of

Polysaccharides polyiodide chains from the reaction of starch and
- Function as CARBOHYDRATE RESERVES
iodine

- Starch and Glycogen

- amylose/straight chain = forms helices

- Function as STRUCTURAL

- Cellulose
Starch Test

- STARCH
- a blue-black color results to starch is present

- serves as a long-term storage medium for food - HYDROLYSIS: Clear

energy in plants
- W/O HYDROLYSIS: Dark Blue

- it accumulates in seeds, tubers, and fruits

- GLYCOGEN
Carbohydrate Test

- serves as a long-term storage medium for food - IODINE TEST - presence of starch

energy in animals
- COOKED STARCH = Dark blue color (presence of
- found in liver and muscle tissues
starch)

- CELLULOSE
- RAW STARCH = Very small amount of change in
- a linear polysaccharide of B-D-glucopyranose color (Light blue)

units found in plants

- it is the principal component of the rigid cell wall Fehling’s Test + Starch

of plants
- no reaction at all

- provides structure rather than energy

- no hydrolysis

- most animals can’t digest cellulose = they lack

suitable enzymes to catalyze its hydrolysis
Starch Hydrolysis + Fehling’s Test

- Termites and Ruminant Animals (cattle, sheep, - starch will be hydrolyzed to glucose = brick red

and goat) are able to use cellulose as food = - positive result

digestive tracts contain enzymes that can break - acid and enzymatic hyrdolysis

down the linkages of cellulose

Cellulose

II. Lecture (PPT) - CHITIN: similar to cellulose in both function and

Polysaccharides
structure

- contains many monosaccharide units bonded to - linkage: beta (1—>4)

each other by GLYCOSIDIC LINKAGE

- function: rigidity to the exoskeleton pf crabs,
- POLYMERS: glucose molecules
lobsters, shrimps, insects, and other arthropods

- not sweet
- linear polymer, no branching, similar to cellulose

 ACM
Iodine Test w/ Cellulose
- solubility in non-polar solvents like chloroform,
- no reaction
carbon tetrachloride

- filter paper
- insolubility in water

- important roles:

Solubility of Cellulose
- major sources of energy in animals and plants

- Cotton: 90% cellulose

- structure of cell membranes

- most indegistible

- Schweitzer’s reagent
Major Roles of Lipids in the Body
- Storage Lipids

Hydrolysis of Cellulose + Fehling’s Test

- Triglycerides, divided into two

- yields beta-D-glucose units

- fats

- positive results
- oils

- they are triesters of glycerol and fatty acids

Glycogen
- significant components of fat storage cells

- storage of carbs in animals

- highly branched
- Membrane Component Lipids

- homopolymer of alpha-D-gluccose
- usually amphipathic and complex

- ethyl alcohol: precipitating agent

- classified into two groups:

- lugol’s iodine test: REDDISH BROWN COLOR/ - phospholipids

YELLOWISH BROWN
- glycolipids

- unhydrolyzed glycogen: (-) from Fehling’s

- cell membranes are made of a lipid bilayer
- hydrolyzed glycogen: (+) from Fehling’s
consisting of phospholipids

III. Results (QUIZ) - Chemical Messengers

- Steroids: characterized by the presence of a

Raw Starch Dark Blue
fused four-ring nucleus

- most common steroid: CHOLESTEROL

Glycogen Brown/Orange-Brown
- Cholesterol: serves as a starting material for the
Heated Starch Solution Colorless formation of HORMONES AND BILE SALTS

Cellulose Amber/Yellow II. Lecture & Results (PPT)

Lipids
Hydrolyzed Starch Brick Red - “adipose” or “taba”

- compounds that consist of mostly of non-polar

groups

- Hydrolysis: the breakdown of starch in glucose

- w/ limited solubility in water, but dissolve freely in
- Cellulose: “Dietary Fiber”
organic solvent

- Unhydrolyzed Starch: No breakdown of molecules

- Polymers: Polysaccharides that contain glucose Structure & Solubility

molecules
- salt and carbohydrates (hydrophilic)

- Filter Paper: Medium to test cellulose

- lipids: big molecule

- Starch: Found in plants for energy storage

- cannot bond w/ hydrogen

- Iodine: Serves as an indicator for starch

- can bond w/ carbohydrates

- Glycogen: Most common polymer in humans and

animals

- Sodium Hydroxide: Reagent used for neutralization Fatty Acids (building blocks of lipids)
of sulphuric acid
- SATURATED

- Smooth: Treated filter paper

- all C-C bonds are single bonds

- UNSATURATED

Experiment 5: LIPIDS - monounsaturated: one C=C bonds

- polyunsaturated: 2 or more C=C bonds present

I. Introduction (BOOK) up to 6 double bonds are present in fatty acids

Lipids
- naturally occuring substances
Simple Triacylglycerol
- grouped together on the basis of their:
- single bond

 ACM
Solubility of Fats & Oils
Mixed Triacylglycerol - sparingly soluble in water but are solube in non-
- double bond
polar organic solvent

Fats & Oils SOLVENT SOLUBILITY SOLUBILITY

- Fats: solid
OF OILS OF BUTTER
- Oils: liquid

Ether miscible soluble

- naturally occuring mixed TRIACYLGLYCEROL

- same general chemical structure

Chloroform miscible soluble
- main difference: PHYSICAL STATE

Alcohol imissicible insoluble

Difference Bet. Fats & Oils
- PHYSICAL STATE
Water imissicible insoluble
- FATS

- predominantly saturated
6N Acid imissicible insoluble
- solids/semisolids @ room temp.

- predominantly obtained from animals

6N Base imissicible insoluble
- tasteless, odorless, colorless

- principle: “LIKE DISSOLVES LIKE”

- OILS

- predominantly unsaturated
Test for Chemical Properties
- liquids @ room temp.
- reaction w/ alcoholic iodine: HALOGENATION
- obtained from plants & fishoils (omega)
REACTION

- odorless, colorless
- determine unsaturated glyceride

- halogen readily reacts with unsaturated glyceride

Physical Determination of Fats & Oils by ADDITION REACTION

- Spotting Effect: practical test for lipids

- Unglazed Paper: opaque (light scattering)

SAMPLE VISIBLE INTERPRETATI
RESULTS ON
SAMPLE SPOTTING EFFECT
Slight change
less
Butter (+) Coconut Oil to a lighter
unsaturated
color
Oil (+)
Color change
Alcohol (—) Linseed/ is lighter than more
Cottonseed Oil that w/ unsaturated
Ether (—) coconut oil
- Coconut Oil: palmitic, lauric, myristic

- Conclusion in Parchment Paper

- Cottonseed Oil: palmitic, oleic, linoleic

- when applied to unglazed paper, it leaves a - these two undergone heat to be converted into oil

parchment translucent spot through w/c

light can pass
- Canola Oil: 6% saturated

- due to the fact that they evaporate much - Coconut Oil: 92% saturated

more slowly than water bcoz the forces that

hold the lipid molecules together are Good Fats vs Bad Fats
stronger, so the spot remains
- studies indicate that TYPE of dietary fat and
- ALCOHOLS
amount of dietary fat are important for balanced
- other substances temporarily coat the diet:

paper and do not leave the paper - current recommended amounts are = total
permanently translucent
fat intake in calories:

- easily evaporate; weak forces that hold - 15% monounsaturated fat

them/ spot will dry up

- 10% polyunsaturated fat

- PAPER FIBERS: shrink

- < 10% saturated fats

- SPOT: disappear
- studies also indicate that:

- Saturated Fats: bad

- Monounsaturated Fats: good

 ACM
- Trans- mono- unsaturated Fats: bad
- organic molecules - building blocks/monomers of
- Polyunsaturated Fats: good/bad
PROTEINS

- omega 3 and 6: good (ESSENTIAL) = tuna/ - STRUCTURE:

salmon
- acidic functional grp: Carboxyl Group (-
COOH)

Saponifications (Soap) - Basic Amino Group (—NH2)

- triglycerides and saponifiable lipids

- Organic R Group or side chain that
- saponifiable lipids are those that can be determines amino acid chemical structure &
hydrolyzed to under basic conditions to form fatty properties

acid salts (SOAP)

- GENERAL STRUCTURE OF AMINO ACID:

- hydrolyzed = naburn fats

- a positive test is indicated by:

- formation of bubbles w/c indicate that a

soap was formed

Effects of MgCl2 on Water

- hard water is WATER that has high MINERAL - there are 20 naturally occuring amino acids

content than SOFT WATER

- at their ISOELECTRIC POINT (pl) or at its
- hard water has high concentrations of Ca2+ and NEUTRAL pH state they exist as ZWITTERIONS
Mg2+ ions
rather than as unionized molecules

- hardness of water is often indicated by the non- - except for GLYCINE; all amino acids are optically
formation of froth when SOAP/TOOTHPASTE is active due to the presence of an ASYMMETRIC
agitated in the water sample
a-Carbon atom.

- all naturally occuring proteins are in an L-amino

Detection of Cholesterol in Food Extracts acid configurations

- SALKOWSKI TEST
- D-amino acids: present in bacterial cell wall &
- when conc. sulfuric acid is added to a antibiotics.

chloroform sol’n of cholesterol, the

chloroform layer shows a RED TO BLUE II. Lecture & Results (PPT)
COLOR
Roles of Amino Acids
- intermediate in metabolism

- LIBERMANN-BURCHARD TEST
- storage & transport of nutrients

- qualitative test for lipids

- test for presence of cholesterol

Amino Acids
- EMERALD GREEN COLOR: (+) result
- Organic Compound/Molecules

- pink, lilac
- serves as the building block or the
- upper layer become RED
monomers of proteins

- results are quite sensitive to the presence of - Amine and Carboxyl

water, keep everything dry

- —NH2 and —COOH

- variable results: coconut oil- green color

Solubility Test
- lard: gives brown color

- color change is gradual, 1st it appears as Tryptophan Miscible Immisicible

PINK coloration, changes to LILAC & finally C

to deep green after.

Tyrosine Miscible Immisicible
H

Arginine Miscible L
Immisicible
O

Experiment 6: QUALITATIVE ANALYSIS OF AMINO Methionine Miscible R

Immisicible
ACIDS O

Proline Miscible F
Immisicible
I. Introduction (BOOK) O

Amino Acids Histidine Miscible R

Immisicible
- our body is made up of amino acids
M

- plays an important role as intermediates in Unknown A Miscible Immisicible

metabolism especially in the STORAGE &
Unknown B Miscible Immisicible
TRANSPORT OF NUTRIENTS.

 ACM
SUMMARY
Qualitative Test
1. Ninhydrin Test TEST RESULT INTERPRETATION
- a-amino acid typically give a BLUE OR PURPLE
Ninhydrin Blue/Yellow Alpha Amino Acid
COLOR

(Blue)

- used for visualization of FINGERPRINTS

Imino Acid (Yellow)

2. Xanthoproteic Acid Xanthoproteic Orange Aromatic Amino
- a method used to determine the amount of Acid
protein soluble in a solution, using a concentrated
NITRIC ACID
Millons Reddish Color (+) Phenol
- (+) result in Amino Acids (ORANGE) carrying cintaining
aromatic groups, especially in the presence of compound
Tyrosine

Histidine Blue/Viloet (+) Histidine

3. Millon’s Test
Sakaguchi Red Mono-substituted
- detect the presence of soluble proteins

guanidine/amino
- a REDDISH-BROWN coloration or precipitation
acid
indicates the presence of TYROSINE residue
which occur in nearly all proteins
Hopkins Cole Purple Ring (+) Tryptophan

4. Histidine Test Folin’s Reddish (+) Methionine

- discovered by KNOOP

- bromination of histidine in acid solution

- heating of alkaline solution develops a BLUE OR

VIOLET COLORATION

5. Sakaguchi Test
- under alkaline, a-naphthol (1-hydroxy
naphthalene) reacts with a mono-substituted
guanidine compound like arginine which upon
treatment with hypobromite or hypochlorite
produces a RED COLOR

- PRESENCE OF MONO-SUBSTITUTED
GUANIDINE CONTAINING AMINO ACID

6. Adamkiewics-Hopkins Test/AcreeRosen Heim

Test
- specific test for TRYPTOPHAN

- the tryptophan reacts with glyoxilic avcid in the

presence of sulphuric acid to give a PURPLE
COLORED product

- colored purple product: HOPKINS COLE TEST

- GLYOXILIC ACID IS PREPARED FROM GLACIAL

ACETIC ACID

7. Folin’s McCarthy Sullivan Test

- reaction also forms the basis for the quantitative
determination of METHIONINE

- PROLINE & HYDROXYPROLINE CONDENSE

WITH ISATIN (ISATIN=ISA)

- REAGENT UNDER ALKALINE CONDITION TO

YIELD BLUE SOLUTION

- SODIUM + METHIONINE + ACID = RED

SOLUTION