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Toaz - Toaz

Toaz
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organic- bio chemistry (CHEM153L)

145 Documents
Students shared 145 documents in this course
Academic year: 2019/2020
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Xavier University - Ateneo de Cagayan

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EXPT 6. ANALYSIS

1.) Why must the solution to be tested with ninhydrin be neutral?

  • In the Ninhydrin test, the samples were neutralized with NaOAc and was added with Ninhydrin solution. The samples must be neutral because Ninhydrin is a chemical used to detect ammoniaor primary and secondary amines. Amino acids also react with the Ninhydrin solution at pH=4. That is why the samples should be neutralized, so that all α- amino acids that react with ninhydrin (triketohydrindene hydrate) can be detected.

2.) What is the use of the marble in the ninhydrin reaction?

  • The test tube was stoppered with a marble to prevent evaporation during the heating period of the solution since it was heat to a boiling water bath for 1-2 minutes.

3.) A very dilute solution of CuSo 4 is used in the biuret test. Why?

  • The Biuret Test is done to show the presence of peptide bonds, which are the basis for the formation of proteins. These bonds will make the blue Biuret reagent turn purple. CuSO4 reacts with compounds containing two or more peptide bonds to give a violet colored product which is due to formation of co-ordination complex of cupric ions with unshared electron pairs of peptide nitrogen and O2 of water.

4.) Are the Xanthoproteic and Millon-Nasse tests satisfactory for use in the urinary examination for protein? Why?

5.) Why does the bromine water be avoided in the test for free tryptophan?

  • The Bromine water tests free tryptophan in solutions. Free tryptophan interacts with bromine water and n-amyl alcohol to form a pinkish lavender complex. However, the pink color may be masked by the color of the reagent if excess reagent is added. The colored complex is soluble at the alcohol layer.

6.) Which test can be used to show up to what stage the hydrolysis of a protein proceeds?

7.) Discuss each test to detect groups in proteins and amino acids and write equations to support results.

Ninhydrin Test:

Ninhydrin is a strong oxidizing agent used in amino acid analysis for the precise determination of protein quantities. It is mainly used as a detector in liquid chromatography methods coupled with ninhydrin post-column derivatization systems. The reagent which is initially yellow reacts with free alpha amino groups present in all amino acids, proteins, or peptides and forms a deep blue or purple colored complex known as Ruhemann’s purple.

The samples that were used for the test were neutralized with solid NaOAc with 2-3 drops of ninhydrin solution. It was then covered with marbles and was heated in a boiling water bath for 2-3 minutes. Colors deep blue and purple were observed in the solution.

Biuret Test:

The Biuret Test positively identifies the presence of proteins (not less than two peptides). The reaction in this test involves the complex formation of the proteins with Cu2+ ions in a strongly alkaline solution. The samples were mixed with 0 mL of 10% NaOH with 1-2 drops of 0% CuSO4. The reason why the samples were mixed with NaOH because the biuret test for proteins solution reacts in a basic solution to form a deep violet complex.

Hopkins-Cole Reaction:

This chemical test is used to identify the presence of tryptophan, the only amino acid containing in dole group. When the protein solution is mixed with Hopkins- Cole reagent the in-dole ring reacts with the glyoxylic acid in the presence of concentrated sulfuric acid and forms a violet or purple colored product, signifying a positive result. The protein solution is hydrolyzed by the concentrated H 2 SO 4 at the solution interface. Once the tryp-tophan is free, it reacts with the glyoxylic acid to form the violet product.

Bromine Water Test

It is used to detect the presence of unsaturated compounds of alkene and alkyne. Such a test for alkenes works via the mechanism of making alkenes or hydrocarbons, having a minimum of one double bond that undergoes addition reactions. The alkenes and hydrocarbons combine with bromine to impart a colorless appearance to this element. As for the change in bromine's color, alkenes are colorless and therefore, their combination with bromine causes the latter to lose color, as well as it gets consumed in the reaction process. The color of the alcohol layer that was observed was colorless. The equation for this reaction is:

H2 = CH2 ---> H2BrC - CbrH

Pauly Reaction:

In five test tubes, casein, albumin, histidine, tyrosine and a blank were treated with sulfanilic acid with NaNO2and was cooled in an ice bath for 3 minutes and made to alkaline with Na2CO3. The principle behind the Pauly’s reaction is diazotized. Sulfanilic acid will bediazotized with the addition of NaNO2and Na2CO3and formed diazotized component component reacts with the imidazole ring of histidine and a phenol group of tyrosine to form dark red compound. However, in the experiment, instead of forming a red compound, yellow product was obtained. Thus, a negative result was observed.

Lead Acetate Reaction:

Four test tubes were obtained with casein, albumin, a blank, and an untreated piece of hair were added with NaOH and Pb (OAc)2. Covered with marble, the samples were heated to boiling under a water bath for a few minutes. The albumin and the test tube which contains the hair gave a positive result. Sulphur containing amino acids upon boiling with NaOH, yields Na 2 S. This reaction is due to the partial conversion of the organic Sulphur into inorganic.

Sakaguchi Reaction:

The Sakaguchi reagent is used to test for a certain amino acid and proteins. The amino acid that is detected in this test is arginine. Since arginine has a guanidine group in its side chain, it gives a red color with α-naphthol in the presence of an oxidizing agent like bromine solution Based on our observation, the casein solution turned color red after it was mix thoroughly with 0 bromine water.

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Toaz - Toaz

Course: organic- bio chemistry (CHEM153L)

145 Documents
Students shared 145 documents in this course

University: Xavier University - Ateneo de Cagayan

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EXPT 6. ANALYSIS
1.) Why must the solution to be tested with ninhydrin be neutral?
- In the Ninhydrin test, the samples were neutralized with NaOAc and was
added with Ninhydrin solution. The samples must be neutral because
Ninhydrin is a chemical used to detect ammoniaor primary and
secondary amines. Amino acids also react with the Ninhydrin solution at
pH=4. That is why the samples should be neutralized, so that all α- amino
acids that react with ninhydrin (triketohydrindene hydrate) can be
detected.
2.) What is the use of the marble in the ninhydrin reaction?
- The test tube was stoppered with a marble to prevent evaporation during
the heating period of the solution since it was heat to a boiling water bath
for 1-2 minutes.
3.) A very dilute solution of CuSo4 is used in the biuret test. Why?
- The Biuret Test is done to show the presence of peptide bonds, which
are the basis for the formation of proteins. These bonds will make the
blue Biuret reagent turn purple. CuSO4 reacts with compounds
containing two or more peptide bonds to give a violet colored product
which is due to formation of co-ordination complex of cupric ions with
unshared electron pairs of peptide nitrogen and O2 of water.
4.) Are the Xanthoproteic and Millon-Nasse tests satisfactory for use in the
urinary examination for protein? Why?
5.) Why does the bromine water be avoided in the test for free tryptophan?
- The Bromine water tests free tryptophan in solutions. Free tryptophan
interacts with bromine water and n-amyl alcohol to form a pinkish
lavender complex. However, the pink color may be masked by the color
of the reagent if excess reagent is added. The colored complex is soluble
at the alcohol layer.

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