Chemlab Practicals (Experiment 5-8) (FINALS)

Preparation of Formaldehyde

1. heat a copper in oxidizing (blue) flame

2. dip it into 1 ml methanol (CH₃OH) + water

3. stopper the test tube after dipping the wire

4. repeat process 10 times

2CH₃OH + O₂ ——> 2CH₂O + 2H₂O

Schiff’s Test

-test for presence of aldehyde
-differentiate aldehydes and ketones

-positive result= MAGENTA SOLUTION
-negative result= CLEAR SOLUTION

SCHIFF’S REAGENT= fuchsin decolorized by sulfur dioxide SO2)

• 1 ml Schiff’s solution + ½ prepared formaldehyde (HCHO)
  —> MAGENTA SOLUTION

• 1ml 1 ml Schiff’s solution + 5 drops benzaldehyde (C₆H₅CHO)
  —> MAGENTA SOLUTION (very slow reaction)

• 1 ml Schiff’s solution + 5 drops acetone (CH₃COCH₃)—> NEGATIVE

Resorcinol Test

-formaldehyde only (HCHO)

formaldehyde (HCHO) + .5% resorcinol solution + conc. H₂SO₄ (inclined test tube) ——> (TWO LAYERS) RED FLOCCULENT PRECIPITATE AT THE JUNCTION OF TWO LATERS

Tollen’s Test (Silver Mirror Test)

-gives positive result for all aldehydes and benzaldehyde (C₆H₅CHO)
-warmed in water bath
-positive result= SILVER MIRROR ON WALLS OF THE TEST TUBE
-negative result= CLEAR SOLUTION

TYPE OF REACTION:
Oxidation

TOLLEN’S REAGENT= Ammoniacal Solution of Silver Nitrate [(NH₃)₂Ag]

• 1 ml Ammoniacal Solution of Silver Nitrate (NH₃)₂Ag solution (Tollen’s Reagent) + 5 drops of formalin (37-40% of formaldehyde in water)
  —> SILVER MIRROR ON WALLS OF THE TEST TUBE

• 1 ml (NH₃)₂Ag solution (Tollen’s Reagent) + 5 drops benzaldehyde (C₆H₅CHO)
  —> SILVER MIRROR ON WALLS OF THE TEST TUBE

• 1 ml (NH₃)₂Ag solution (Tollen’s Reagent) + 5 drops acetone (CH₃COCH₃)—> NEGATIVE

EQUATIONS:

HCHO + 2(NH₃)₂Ag + 3 OH —→ HCOOH + 2Ag + 4NH₃ + 2H₂O

C₆H₅CHO+ 2(NH₃)₂Ag + 3 OH —→ C₆H₅COOH+ 2Ag + 4NH₃ + 2H₂O

CH₃COCH₃ + 2(NH₃)₂Ag + 3 OH —→ NEGATIVE

Formaldehyde on Protein Substances

-formaldehyde only
-When protein is immersed in formalin, it forms chemical cross-links between proteins

• ("fixing" them in place and preventing degradation)

• that’s why formaldehyde is used for preservation

-positive result= proteins become insoluble in formalin due to the formation of cross-links between amino acids

Gelatin sheets 2 SET UPS
1ML FORMALIN + 1 ML WATER + GELATIN
=gelatin sheet hardened
=less soluble because of the formalin

2 ML WATER + GELATIN
=gelatin sheet softened
= more soluble in water

Fehling’s Test (exp 5)

-warmed in water bath
-positive result= BRICK RED PRECIPITATE
-negative result= REMAINED BLUE

TYPE OF REACTION:
Oxidation

FEHLING’S SOLUTION= Copper Sulfate (CuSO₄) + Sodium Hydroxide (NaOH) + Rochelle’s Salt

• 1 ml Fehling’s Solution + 5 drops of formalin (37-40% of formaldehyde in water)
  —> BRICK RED PRECIPITATE [CUPROUS OXIDE or COPPER (I) OXIDE] Cu₂O

• 1 ml Fehling’s Solution + 5 drops benzaldehyde (C₆H₅CHO)
  —> NEGATIVE

• 1 ml Fehling’s Solution + 5 drops acetone (CH₃COCH₃)—> NEGATIVE

EQUATIONS:

HCHO + Cu(OH)₂ —→ HCOOH + Cu₂O (Cuprous Oxide or Copper (I) Oxide + H₂O

C₆H₅CHO+ Cu(OH)₂ —→ NEGATIVE

CH₃COCH₃ + Cu(OH)₂ —→ NEGATIVE

Auto Oxidation of Benzaldehyde

Benzaldehyde (C₆H₅CHO) is exposed to the atmosphere in a watch glass
-positive result= TURNED TO BENZOIC ACID (C₆H₅COOH)

EQUATION:

Addition of Sodium Bisulfite

TYPE OF REACTION:
Addition

-positive result= TURBIDITY/ CLOUDINESS (WHITE PRECIPITATE)

• .5 ml of acetone (CH₃COCH₃) + (Sodium Bisulfite) NaHSO₃——> ACETONE SODIUM BISULFITE

Formation of Phenylhydrazone (

TYPE OF REACTION:
Addition

-positive result= YELLOW ORANGE PRECIPITATE (YELLOW SOLIDS)

• 3 drops of acetone (CH₃COCH₃) + 2,4-dinitrophenylhydrazine (C₆H₅NHNH₂)—-> ACETONE PHENYL HYDRAZONE

Iodoform Test

-test for compounds with methyl carbonyl group
(—CH₃CO—)

TYPE OF REACTION:
Haloform Reaction

-positive result= YELLOW PRECIPITATE (YELLOW SOLIDS)

• 3 drops of acetone (CH₃COCH₃) +8 drops of 10% Sodium Hydroxide (NaOH) + Iodine Solution (I₂₎
  —-> IODOFORM (CHI₃) yellow precipitate
  

  EQUATIONS:
  CH₃COCH₃ + 4NaOH + 3I₂ ——> CHI₃ + CH₃COONa + 3NaI + 3H₂O
  

• CHI₃= Iodoform

• CH₃COONa = Sodium Acetate

• NaI = Sodium Iodide

Biuret’s Test

- general test for proteins
- test for peptide bonds (—CONH)
- rgts: CuSO₄ + NaOH

-positive result= ROSEPINK TO VIOLET COLORATION SOLUTION

• 1% Solution of Egg Albumin + Copper (II) Sulfate CuSO₄ + 10% Sodium Hydroxide (NaOH)
  —-> ROSEPINK TO VIOLET COLORATION SOLUTION

Xanthoproteic Test

- general test for amino acids
- solution turns yellow due to the nitration of benzene ring in amino acids like tyrosine, tryptophan, phenylalanine
-rgts: HNO₃ + NH₄OH

-positive result= DARK YELLOW TO ORANGE PRECIPITATE(when neutralized with NH₄OH)

• 1% Solution of Egg Albumin + Conc. Nitric Acid HNO₃ + Conc. Ammonium Hydroxide NH₄OH
  —-> DARK YELLOW TO ORANGE PRECIPITATE

Millon’s Test

- general test for the presence of phenolic/phenol group
- proteins containing tyrosine
-rgts: HgNO₃/[Hg (NO₃)₂

MILLON’S REAGENT= Dissolving Mercury (Hg) in Nitric Acid (HNO₃)
=mercuric (HgNO₃)and mercurous nitrates [Hg (NO₃)₂]

-positive result= FLESH TO RED COLOR PRECIPITATE

• 1% Solution of Egg Albumin + Millon’s Reagent (HgNO₃/[Hg (NO₃)₂])
  —-> FLESH TO RED COLOR PRECIPITATE

Molisch’s Test (exp 6)

-general test for carbohydrates
-glyco-protein is present
- 2 ml egg albumin + Molisch Rgt + 2 drops conc. sulfuric acid (INCLINED/ AT THE SIDE OF TEST TUBE)
-rgts: Molisch Rgt + H₂SO₄

MOLISCH REAGENT= Alpha napthol in alcohol

-positive result= VIOLET RING/PRECIPITATE AT THE JUNCTION OF TWO LAYERS

• 1% Solution of Egg Albumin + Molisch’s Reagent
  —-> VIOLET RING/PRECIPITATE AT THE JUNCTION OF TWO LAYERS

Sulfur Test

-general test for cysteine, cystine, or methionine
-when heated with sodium hydroxide (NaOH), splits up the sulfur to form Sodium Sulfide (Na₂S)
-2 ml egg albumin + 6 ml NaOH (BOIL)+ CH₃COOH (COVER WITH FILTER PAPER SOAKED IN LEAD ACETATE Pb(CH₃COO)₂)

-positive result= BLACK PRECIPITATE (PbS [LEAD (II) SULFIDE]

• 2 ml egg albumin + 6 ml Sodium Hydroxide NaOH (BOIL)+ Acetic Acid CH₃COOH (COVER WITH FILTER PAPER SOAKED IN LEAD (II) ACETATE [Pb(CH₃COO)₂]
  —-> BLACK PRECIPITATE (PbS [LEAD (II) SULFIDE]

Effects of heat in proteins

-albumin is a simple protein
-coagulated by heat, soluble in water, and dilute in salt solution

• 2 drops of Egg Albumin + heated —→ Coagulates (turbid/cloudy)

Heller’s Ring Test

-commonly used to detect albumin in urine
-Nitric Acid (HNO₃) at lower portion of the test tube, with Protein (albumin) above
- egg albumin + conc. HNO₃
-rgts: concentrated HNO₃

-positive result=WHITE PRECIPITATE OF COAGULATED PROTEIN/RING AT JUNCTION OF TWO LAYERS

• 1 drop egg albumin + 1 drop conc. HNO₃ (DROPPED AT THE SIDE OF THE TEST TUBE)
  —-> WHITE PRECIPITATE / RING AT JUNCTION OF TWO LAYERS

Heavy-Metal Salts

• 1 drop egg albumin + 3 drops Copper (II) Sulfate (CuSO₄)
  —> BLUE PRECIPITATE

• 1 drop egg albumin + 3 drops Lead (II) Acetate [Pb(CH₃COO)₂]
  —> WHITE PRECIPITATE (LEAD ALBUMINATE)

• 1 drop egg albumin + 3 drops Silver Nitrate (AgNO₃₎
  —> WHITE PRECIPITATE (SILVER ALBUMINATE)

Alkaloidal Reagents

• 3 drops egg albumin + 2 drops 5% Tannic Acid
  —> LIGHT BROWN PRECIPITATE

• 3 drops egg albumin + 2 drops 5% Potassium Ferrocyanide
  —> PRUSSIAN BLUE PRECIPITATE

• 3 drops egg albumin + 2 drops Concentrate Picric Acid
  —> YELLOW PRECIPITATE

Molisch’s Test (exp 7)

-general test for carbohydrates
-sulfuric acid H₂SO₄ acts as a dehydrating agent forming furfural derivatives
- 1 ml substance + Molisch Rgt (a-naphthol in alcohol) + 3 mL conc. sulfuric acid (INCLINED/ AT THE SIDE OF TEST TUBE)
-rgts: Molisch Rgt + H₂SO₄

MOLISCH REAGENT= Alpha napthol in alcohol

-positive result= VIOLET RING/PRECIPITATE AT THE JUNCTION OF TWO LAYERS

• 1 mL of H₂O + Molisch’s Reagent
  —-> NEGATIVE

• 1 mL of Glucose (Monosaccharide)+ Molisch’s Reagent
  —-> VIOLET RING/PRECIPITATE AT THE JUNCTION OF TWO LAYERS

• 1 mL of Sucrose (Disaccharide) + Molisch’s Reagent
  —-> VIOLET RING/PRECIPITATE AT THE JUNCTION OF TWO LAYERS

• 1 mL of Starch (Polysaccharide) + Molisch’s Reagent
  —-> VIOLET RING/PRECIPITATE AT THE JUNCTION OF TWO LAYERS (slow reaction)

Phenylhydrazine Test (exp 7)

-general test for carbonyl group
-Osazone formation
-reducing sugars form osazone crystals
-presence of aldehyde and ketone group
-glucose, fructose, and mannose yield the same osazone crystals
-1 ml glucose + Phenyl hydrazine (C₆H₅NHNH₂)—>
GLUCOSAZONE/ GLUCOSE PHENYLHYDRAZONE

rgts: Phenyl hydrazine (C₆H₅NHNH2)

-positive result= YELLOW- ORANGE PRECIPITATE/CRYSTALS

• 1 mL of Glucose (Monosaccharide)+ Phenyl hydrazine (C₆H₅NHNH2)
  —-> YELLOW- ORANGE PRECIPITATE/CRYSTALS (GLUCOSAZONE/ GLUCOSE PHENYLHYDRAZONE)

Action of Alkali (Moore’s Test)

-test for reducing sugars
-when heated with an alkali NaOH, it turns yellow to orange to dark brown
-gives caramel odor
-liberation of aldehydes to form a resinous/viscous substance; caramel
- 5 mL substance + 25% NaOH
-rgts: 25% NaOH

-positive result= CARAMEL ODOR

• 5 mL 10% glucose solution + 25% NaOH
  —-> CARAMEL ODOR

• 5 mL sucrose (non reducing sugar)+ 25% NaOH
  —-> NEGATIVE

Benedict’s Test

-test for reducing sugars
-monosaccharides and disaccharides (except sucrose) have free aldehyde and ketone group
-reduce alkaline materials and transformed into organic acid
-metals —> acid

TYPE OF REACTION:
Oxidation

BENEDICT’S REAGENT= Sodium Carbonate Na₂CO₃, Sodium Citrate, Copper (II) Sulfate CuSO₄

-positive result= RED PRECIPITATE (CU₂O)
CUPROUS OXIDE/COPPER (I) OXIDE

• 2 drops of Glucose (C₆H₁₂O₆) + Benedict’s solution —→ RED PRECIPITATE (CUPROUS OXIDE/ COPPER (I) OXIDE

• 2 drops of Lactose (C₁₂H₂₂O₁₁) + Benedict’s solution —→ RED PRECIPITATE (CUPROUS OXIDE/ COPPER (I) OXIDE

• 2 drops of Sucrose [non-reducing sugar] (C₁₂H₂₂O₁₁) + Benedict’s solution —→ NEGATIVE (REMAINED BLUE)

• 2 drops of Starch [non-reducing sugar] (C₆H₁₀O₅)_n + Benedict’s solution —→ NEGATIVE(REMAINED BLUE)

EQUATIONS:

C₆H₁₂O₆ + Cu(OH)₂ —→ gluconic acid (C₆H₁₂O₇) + Cu₂O (Cuprous Oxide or Copper (I) Oxide + H₂O

C₁₂H₂₂O₁₁+ Cu(OH)₂ —→ lactonic acid (C₁₂H₂₂O₁₂) + Cu₂O (Cuprous Oxide or Copper (I) Oxide + H₂O

C₁₂H₂₂O₁₁+ Cu(OH)₂ —→ NEGATIVE

C₆H₁₀O₅+ Cu(OH)₂ —→ NEGATIVE

Hydrolysis of Sucrose

-in the presence of HCl Hydrochloric Acid, disaccharides and polysaccharides react with water to yield monosaccharides
-breaking down of Disaccharide into monosaccharide: for sucrose: FRUCTOSE AND GLUCOSE

• 5 mL Dilute sucrose C₁₂H₂₂O₁₁ + HCl (BOIL) then (COOLED)

-positive result= REACTS WITH BENEDICT’S TEST AND SELIWANOFF’S TEST

Benedict’s test (hyrolyzed sucrose)

-test if the hydrolyzed sucrose has broken down into monosaccharides: glucose and fructose
-its glycosidic bond has now broken down
-neutralized with NaOH

• Hydrolyzed sucrose + NaOH (TO NEUTRALIZE) —>

• Hydrolyzed sucrose + Benedict’s Solution—→ BRICK RED PRECIPITATE

Seliwanoff’s Test (hyrolyzed sucrose)

-test if the hydrolyzed sucrose has broken down into monosaccharides: glucose and fructose
-test for presence of FRUCTOSE
-neutralized with NaOH

SELIWANOFF’S REAGENT= Resorcinol + HCl

• Hydrolyzed sucrose + NaOH (TO NEUTRALIZE) —>

• Hydrolyzed sucrose + Seliwanoff’s Reagent—→ MAHOGANY RED PRECIPITATE

Hydrolysis of Starch

-in the presence of HCl Hydrochloric Acid, disaccharides and polysaccharides react with water to yield monosaccharides
-breaking down of polysaccharide into monosaccharide: GLUCOSE

• 20 mL starch solution + 1 mL conc. HCl

  • IODINE TEST=

    • continuously drop hydroyzed starch solution on I₂ (Iodine solution) until it gives no color

    • soluble starch—> blue

    • amylodextrin —> purple

    • erythrodextrin—> red

    • achrodextrin —> no color

      • ACHROMATIC POINT=gives no color

    • maltose—> no color

    • glucose —>no color

      • FULLY HYDROLYZED

Benedict’s test (hyrolyzed starch)

-test if the hydrolyzed sucrose has broken down into monosaccharides: glucose and fructose
-its glycosidic bond has now broken down
-neutralized with NaOH

TYPE OF REACTION:
Oxidation

BENEDICT’S REAGENT= Sodium Carbonate Na₂CO₃, Sodium Citrate, Copper (II) Sulfate CuSO₄

• Hydrolyzed starch + NaOH (TO NEUTRALIZE) —>

• Hydrolyzed starch + Benedict’s Solution—→ BRICK RED PRECIPITATE

Miscibility

Acrolein Test

- when heated with Potassium Hydrogen Sulfate (KHSO₄) dehydrating agent— undergo hydrolysis'
- dehydration then gives an irritating odor
- the compounds formed are fatty acids and acrolein (unsaturated Aldehyde 2-propenal)
- dehydrating the glycerol part
rgts: KHSO₄/ dehydrating agent

TYPE OF REACTION:
Elimination (??)

- positive result: IRRITATING/PUNGENT ODOR

• 3 drops coconut oil + .2 g of KHSO₄ —→ MILD ODOR, COOKING OIL

• 3 drops CH₂OHCHOHCH₂OH+ .2 g of KHSO₄ —→ IRRITATING ODOR

  EQUATIONS:

  CH₂OHCHOHCH₂OH+ KHSO₄ —→ CH₂CHCHO + K₂SO₄ + H₂O

- CH₂CHCHO= Acrolein (2-Propenal)
- K₂SO₄ = Potassium Sulfate

Unsaturation (huble’s test)

-the more iodine is being absorbed by the substance, the more unsaturated it is
-the more iodine is being decolorized, the lighter the oil, the more unsaturation
-unsaturated compounds absorb iodine because of halogenation
-unsaturated compounds are either double or triple bonded

TYPE OF REACTION:
Halogenation

LEVEL OF UNSATURATION

1. Linseed Oil + I₂ solution —→ light brown oil

2. Cottonseed Oil + I₂ solution —→ light brown oil

3. Olive Oil + I₂ solution —→ dark brown oil

4. Coconut Oil + I₂ solution —→ dark brown oil

Saponification

-basic hydrolysis
-Fat is heated with NaOH— glycerol and metallic salt form

TYPE OF REACTION:

Basic Hydrolysis

Coconut Oil is heated with NaOH— glycerol and metallic salt form

• 15 mL Coconut Oil +25 mL 10% NaOH solution—-. thick solution
  - heat the remaining mixture and stir
  - reaction is complete if result has thickened