Name:‌A‌ lcantara,‌‌Ace‌‌Justine‌‌M.

‌‌ ‌Rating:__________‌  ‌
Section:‌M
‌ E-1313‌‌ D
‌ ate:‌7
‌ /19/21‌  ‌
 ‌
Experiment‌‌#2‌  ‌
Introduction‌  ‌
● Proteins‌‌are‌‌made‌‌up‌‌of‌‌hundreds‌‌of‌‌amino‌‌acids‌‌and‌‌are‌‌a‌‌complicated‌‌
 
substance.‌‌Amino‌‌acids‌‌are‌‌amphoteric‌‌electrolytes‌‌with‌‌carboxyl‌‌and‌‌amino‌‌ 
groups‌‌that‌‌function‌‌as‌‌an‌‌acid‌‌and‌‌a‌‌base,‌‌respectively.‌  ‌
● Color‌‌tests‌‌have‌‌long‌‌been‌‌used‌‌to‌‌determine‌‌the‌‌quality‌‌of‌‌amino‌‌acids.‌‌The‌‌  
reactive‌‌groups‌‌of‌‌amino‌‌acids‌‌are‌‌not‌‌all‌‌the‌‌same.‌‌As‌‌a‌‌result,‌‌the‌‌various‌‌color‌‌
 
tests‌‌produce‌‌responses‌‌that‌‌differ‌‌in‌‌intensity‌‌and‌‌type‌‌of‌‌color‌‌depending‌‌on‌‌  
the‌‌nature‌‌of‌‌the‌‌groups‌‌in‌‌the‌‌[articular‌‌amino‌‌acid‌‌under‌‌investigation.‌  ‌
Objectives‌  ‌
● To‌‌identify‌‌the‌‌amino‌‌acids‌‌that‌‌are‌‌present‌‌to‌‌the‌‌protein‌‌sample.‌  ‌
● To‌‌determine‌‌the‌‌quality‌‌of‌‌amino‌‌acids‌‌that‌‌are‌‌present‌‌to‌‌the‌‌protein‌‌sample.‌  ‌
 ‌
Materials/‌‌Procedures‌  ‌
 ‌
PROCEDURE‌  ‌ REAGENT‌  ‌ GLASSWARES‌  ‌

Biuret‌‌Test‌  ‌ 1ml‌‌of‌‌protein‌‌solution‌  ‌ Test‌‌tube‌  ‌

 ‌ 1ml‌‌of‌‌5%‌‌NaOH‌  ‌  ‌

 ‌ 2-3‌‌drops‌‌of‌‌1%‌‌CuSO4‌  ‌  ‌

Ninhydrin‌‌Test‌  ‌ 1ml‌‌protein‌‌solution‌  ‌ Test‌‌tube‌  ‌

 ‌ 10‌‌drops‌‌of‌‌ninhydrin‌‌
   ‌
solution‌  ‌

Xanthoproteic‌‌Test‌  ‌ 2ml‌‌protein‌‌solution‌  ‌ Test‌‌tube‌  ‌

 ‌ 1ml‌‌of‌‌concentrated‌‌   ‌
nitric‌‌acid‌  ‌

 ‌ 1ml‌‌of‌‌40%‌‌sodium‌‌
   ‌
hydroxide‌‌solution‌  ‌

Millon‌‌Test‌  ‌ 1ml‌‌protein‌‌solution‌  ‌ Test‌‌tube‌  ‌

 ‌ 1ml‌‌mercuric‌‌nitrate‌  ‌  ‌

 ‌ 2‌‌drops‌‌of‌‌sodium‌‌nitrite‌  ‌  ‌
 Sakaguchi‌‌Test‌  ‌ 3ml‌‌protein‌‌solution‌  ‌ Test‌‌tube‌  ‌

 ‌ 2drops‌‌of‌‌40%‌‌NaOH‌  ‌  ‌

 ‌ 4‌‌drops‌‌of‌‌alpha‌‌
   ‌
naphthol‌  ‌

 ‌ 10‌‌drops‌‌of‌‌bromine‌‌
   ‌
water‌  ‌

Sulfur‌‌Test‌  ‌ 1ml‌‌protein‌‌solution‌  ‌ Test‌‌tube‌  ‌

 ‌ 1ml‌‌40%‌‌NaOH‌‌
   ‌  ‌

 ‌ 2-3‌‌drops‌‌lead‌‌acetate‌  ‌  ‌

Pauly’s‌‌Test‌  ‌ 0.5ml‌‌sulphanilic‌‌    ‌
(sulfanilic)‌‌acid‌  ‌

 ‌ 0.5ml‌‌0.5%‌‌sodium‌‌
   ‌
nitrite‌  ‌

 ‌ 1ml‌‌protein‌‌solution‌  ‌  ‌

 ‌ 2ml‌‌10%‌‌sodium‌‌
   ‌
carbonate‌  ‌

Aldehyde/Hopkins‌‌cole‌‌
  1ml‌‌albumin‌  ‌ Test‌‌tube‌  ‌
Test‌  ‌

 ‌ 1‌‌drop‌‌formaldehyde‌  ‌  ‌

 ‌ 1‌‌drop‌‌mercuric‌‌sulfate‌  ‌  ‌

 ‌ 1ml‌‌concentrated‌‌acid‌  ‌  ‌
 ‌
Method/procedures‌‌    ‌
● Biuret‌‌Test‌  ‌
1. Put‌‌1ml‌‌of‌‌protein‌‌solution‌‌in‌‌test‌‌tube‌  ‌
2. Add‌‌1‌‌ml‌‌5%‌‌NaOH‌  ‌
3. Add‌‌2-3‌‌drops‌‌of‌‌1%‌‌CuSO4‌  ‌
4. Mix‌‌well‌ 
● Ninhydrin‌‌Test‌  ‌
1. Take‌‌1ml‌‌protein‌‌solution‌‌in‌‌a‌‌test‌‌tube‌  ‌
2. Now‌‌add‌‌10‌‌drops‌‌of‌‌ninhydrin‌‌solution‌  ‌
3. Heat‌‌the‌‌contents‌‌of‌‌the‌‌test‌‌tube‌‌until‌‌it‌‌boils‌‌strongly‌  ‌
4. Observe‌‌the‌‌formation‌‌of‌‌a‌‌purple‌‌color‌  ‌
● Xanthoproteic‌‌Test‌  ‌
 1. Take‌‌2ml‌‌protein‌‌solution‌‌in‌‌a‌‌test‌‌tube‌  ‌
2. Add‌‌1‌‌ml‌‌of‌‌concentrated‌‌nitric‌‌acid‌‌into‌‌it‌  ‌
3. Heat‌‌the‌‌solution‌‌and‌‌boil‌‌for‌‌1‌‌minute‌  ‌
4. Observe‌‌the‌‌change‌‌in‌‌color‌‌in‌‌the‌‌test‌‌tube‌  ‌
5. After‌‌cooling‌‌it‌‌under‌‌tap‌‌water‌‌divide‌‌contents‌‌to‌‌two‌‌parts‌  ‌
6. To‌‌1‌‌part‌‌add‌‌40%‌‌NaOH‌‌until‌‌solution‌‌becomes‌‌alkaline‌  ‌
● Millon‌‌Test‌  ‌
1. Take‌‌1‌‌ml‌‌of‌‌protein‌‌solution‌  ‌
2. Add‌‌equal‌‌volume‌‌of‌‌mercuric‌‌nitrate‌  ‌
3. Boil‌‌gently‌‌for‌‌20‌‌seconds‌  ‌
4. Add‌‌2‌‌drops‌‌of‌‌sodium‌‌nitrite‌  ‌
5. Observe‌‌the‌‌red‌‌color‌  ‌
● Sakaguchi‌‌Test‌  ‌
1. Take‌‌3‌‌ml‌‌of‌‌protein‌‌solution‌  ‌
2. Add‌‌2‌‌drop‌‌of‌‌40%‌‌NaOH‌  ‌
3. Now‌‌add‌‌4‌‌drops‌‌of‌‌alpha‌‌naphthol‌  ‌
4. Add‌‌10‌‌drops‌‌of‌‌bromine‌‌water‌  ‌
5. Observe‌‌red‌‌color‌  ‌
● Sulfur‌‌Test‌  ‌
1. Take‌‌1ml‌‌of‌‌protein‌‌solution‌  ‌
2. Add‌‌1ml‌‌of‌‌40%‌‌NaOH‌‌into‌‌it‌  ‌
3. Boil‌‌the‌‌contents‌‌for‌‌at‌‌least‌‌3‌‌minutes‌  ‌
4. Allow‌‌it‌‌to‌‌cool‌‌
   ‌
5. Add‌‌2-3‌‌drops‌‌of‌‌Lead‌‌acetate‌  ‌
6. Observe‌‌brown‌‌to‌‌black‌‌precipitate‌  ‌
● Pauly’s‌‌Test‌  ‌
1. Take‌‌0.5ml‌‌sulphanilic‌‌(sulfanilic)‌‌acid‌‌in‌‌a‌‌test‌‌tube‌  ‌
2. Add‌‌equal‌‌volume‌‌of‌‌0.5%‌‌sodium‌‌nitrite‌  ‌
3. Add‌‌1‌‌ml‌‌of‌‌protein‌‌solution‌  ‌
4. Wait‌‌for‌‌1‌‌minute‌  ‌
5. Add‌‌10%‌‌sodium‌‌carbonate‌  ‌
6. Observe‌‌the‌‌cherry‌‌red‌‌color‌  ‌
● Aldehyde/Hopkins‌‌cole‌‌Test‌  ‌
1. Take‌‌1ml‌‌albumin‌‌    ‌
2. Add‌‌1‌‌drop‌‌formaldehyde‌  ‌
3. Add‌‌1‌‌drop‌‌of‌‌mercuric‌‌sulfate‌  ‌
4. Hold‌‌test‌‌tube‌‌slanted‌  ‌
5. Add‌‌1‌‌ml‌‌concentrated‌‌acid‌  ‌
6. Violet‌‌color‌‌ring‌‌at‌‌the‌‌conjunction‌  ‌
Results‌‌and‌‌Discussion‌  ‌
● Biuret‌‌Test‌  ‌
A. Observe‌‌the‌‌violet‌‌color‌  ‌
B. The‌‌biuret‌‌test‌‌is‌‌used‌‌to‌‌detect‌‌the‌‌presence‌‌of‌‌peptide‌‌bonds‌‌in‌‌proteins‌  ‌
 C. This‌‌reaction‌‌is‌‌named‌‌biuret‌‌because‌‌of‌‌the‌‌compound‌‌(NH2CONHCONH2)‌‌  
formed‌‌by‌‌the‌‌condensation‌‌of‌‌two‌‌molecules‌‌of‌‌urea‌‌when‌‌heated‌‌DC‌‌or‌‌NH‌‌is‌‌  
the‌‌peptide‌‌linkage‌‌in‌‌biuret.‌‌At‌‌Least‌‌two‌‌peptide‌‌bonds‌‌in‌‌the‌‌molecule‌‌are‌‌  
required‌‌for‌‌a‌‌positive‌‌test.‌‌Individual‌‌amino‌‌acids‌‌and‌‌dipeptides‌‌will‌‌not‌‌answer‌‌  
this‌‌test.‌‌The‌‌copper‌‌sulfate‌‌is‌‌converted‌‌to‌‌copper‌‌hydroxide‌‌which‌‌collides‌‌with‌‌  
the‌‌peptide‌‌linkages‌‌in‌‌protein‌‌to‌‌give‌‌the‌‌color.‌‌It‌‌should‌‌strictly‌‌avoid‌‌excess‌‌
 
addition‌‌of‌‌copper‌‌sulfate.‌  ‌
 ‌
● Ninhydrin‌‌Test‌  ‌
A. All‌‌alpha‌‌amino‌‌acids‌‌do‌‌purple‌‌color‌‌with‌‌ninhydrin‌‌by‌‌formation‌‌of‌‌a‌‌complex‌‌  
called‌‌ruhemanns‌‌purple.‌  ‌
B. This‌‌reaction‌‌is‌‌often‌‌used‌‌in‌‌the‌‌detection‌‌of‌‌fingerprints‌‌in‌‌forensic‌‌medicine.‌  ‌
● Xanthoproteic‌‌Test‌  ‌
A. The‌‌yellow‌‌color‌‌formed‌‌initially‌‌was‌‌due‌‌to‌‌the‌‌formation‌‌of‌‌nitro‌‌derivatives‌‌of‌‌  
the‌‌benzene‌‌ring‌‌system‌‌containing‌‌amino‌‌acids‌‌that‌‌are‌‌aromatic‌‌amino‌‌acid‌‌  
tyrosine‌‌and‌‌tryptophan.‌  ‌
B. ‌the‌‌color‌‌turned‌‌orange‌‌on‌‌adding‌‌sodium‌‌hydroxide‌‌is‌‌due‌‌to‌‌the‌‌ionisation‌  ‌
C. All‌‌proteins‌‌usually‌‌respond‌‌to‌‌this‌‌test.‌‌This‌‌reaction‌‌is‌‌also‌‌the‌‌basis‌‌of‌‌
 
development‌‌of‌‌yellow‌‌staining‌‌on‌‌skin‌‌by‌‌nitric‌‌acid;‌‌the‌‌nitration‌‌of‌‌
 
phenylalanine‌‌under‌‌these‌‌conditions‌‌normally‌‌does‌‌not‌‌take‌‌place.‌  ‌
● Millon‌‌Test‌  ‌
A. Test‌‌answered‌‌by‌‌compounds‌‌containing‌‌hydroxyl‌‌phenyl‌‌group.‌‌Tyrosine‌‌and‌‌  
proteins‌‌containing‌‌tyrosine‌‌will‌‌give‌‌a‌‌red‌‌color.‌‌The‌‌color‌‌is‌‌due‌‌to‌‌formation‌‌of‌‌  
mercuric‌‌phenolate.‌  ‌
● Sakaguchi‌‌Test‌‌    ‌
A. The‌‌Sakaguchi‌‌test‌‌is‌‌for‌‌the‌‌guanidinium‌‌group‌‌of‌‌arginine.‌‌Arginine‌‌reacts‌‌with‌‌  
alpha‌‌naphthol‌‌and‌‌alkaline‌‌hypobromite‌‌to‌‌give‌‌red‌‌color.‌‌This‌‌reaction‌‌is‌‌  
specific‌‌to‌‌the‌‌guanidinium‌‌group‌‌of‌‌arginine.‌  ‌
B. Sodium‌‌hydroxide‌‌with‌‌bromine‌‌can‌‌also‌‌be‌‌substituted‌‌by‌‌8-10‌‌drops‌‌of‌‌sodium‌‌  
hypobromite.‌  ‌
● Sulfur‌‌Test‌  ‌
A. Alkali‌‌labile‌‌organic‌‌sulfur‌‌in‌‌cysteine‌‌and‌‌cystine‌‌is‌‌converted‌‌to‌‌inorganic‌‌sulfide‌‌  
(sulphide)‌‌ions.‌‌Lead‌‌acetate‌‌reacts‌‌with‌‌it‌‌to‌‌form‌‌black‌‌insoluble‌‌ppt‌‌of‌‌lead‌  ‌
sulfide.‌  ‌
● Pauly’s‌‌Test‌  ‌
A. Pauly’s‌‌test‌‌is‌‌given‌‌by‌‌an‌‌imidazole‌‌and‌‌phenyl‌‌hydroxyl‌‌group‌‌containing‌‌amino‌‌  
acids‌‌tyrosine‌‌or‌‌histidine.‌  ‌
B. Diazobenzene‌‌sulfanilic‌‌acid‌‌formed‌‌from‌‌sulfanilic‌‌acid‌‌reacts‌‌with‌‌these‌‌  
respective‌‌groups‌‌of‌‌histidine‌‌and‌‌tyrosine‌‌under‌‌alkaline‌‌conditions.‌‌It‌‌gives‌‌  
cherry‌‌red‌‌color‌‌with‌‌histidine‌‌and‌‌orange‌‌red‌‌with‌‌tyrosine.‌  ‌
● Aldehyde/Hopkins‌‌cole‌‌Test‌  ‌
A. This‌‌test‌‌is‌‌for‌‌the‌‌indole‌‌nucleus‌‌of‌‌tryptophan.‌‌Aldehyde‌‌reacts‌‌with‌‌oxidized‌‌  
product‌‌of‌‌indole‌‌nucleus‌‌of‌‌tryptophan‌‌to‌‌give‌‌violet‌‌colored‌‌complexes.‌‌Sulfuric‌‌  
acid‌‌with‌‌mercuric‌‌sulfate‌‌is‌‌used‌‌as‌‌oxidizing‌‌agents.‌  ‌
Conclusion‌  ‌
● These‌‌tests,‌‌I‌‌conclude,‌‌display‌‌protein‌‌color‌‌responses,‌‌which‌‌are‌‌used‌‌in‌‌the‌‌  
qualitative‌‌study‌‌of‌‌amino‌‌acids.‌‌As‌‌per‌‌test‌‌there‌‌are‌‌different‌‌colors‌‌that‌‌are‌‌
 
being‌‌produced‌‌by‌‌the‌‌reactions‌‌of‌‌the‌‌solutions.‌  ‌