Table 14.2: Reactions of proteins
Experiments Observations Inferences
1. Biuret reaction
To 2 mL of the test solution, add 1 mL of 5% NaOH mix; add two drops of 1% CuSO4 solution
Violet color Indicates the presence of proteins, compounds with two or more peptide bonds, gives a violet color with alkaline copper sulfate solution
2. Xanthoproteic reaction
To 3 mL of the solution, add 1 mL of
concentrated HNO3; boil the contents till it turns yellow; cool and divide it into two parts; to one part add 40% NaOH
Yellow color deepens and changes to orange color in alkaline medium
This indicates presence of aromatic amino acids (Phenylalanine tryptophan tyrosine). On heating with concentrated HNO3, benzene ring undergoes nitration to form yellow derivative; it is increased in strong alkaline medium
3. Aldehyde test (Hopkins-Cole test)
To 1 mL of solution, add a drop of formalin and one drop of mercuric sulfate in H2SO4 mix and add 2 mL concentrated H2SO4 along the sides
Violet ring is formed at the junction
This indicates presence of indole ring containing amino acids (Phe); formaldehyde reacts with oxidized indole ring to give violet-colored complex
4. Sakaguchi reaction
To 2 mL of the solution, add 1 mL of 40% NaOH solution and one drop of Molisch’s reagent; after few min add few drops of alkaline hypobromide solution (bromine water)
Bright red color Indicates the presence of arginine; the guanidine group of arginine reacts with a-naphthol to form a brightred-colored complex
5. Sulfur test
To 2 mL of solution, add 2 mL of 40% NaOH and boil for 1 min; cool and add three drops of 2% lead acetate solution; boil for a min
Black precipitate Indicates the presence of sulfur containing amino acids (cystine cysteine); the sulfur of the proteins in presence of NaOH is changed into Na2S, which forms black lead sulfide when reacted with lead acetate 6. Millon’s test
To 1 mL of solution, add 1 mL of mercuric sulfate in H2SO4 and boil for 1 min; cool and add 1 mL of 1% sodium nitrate
Solution turns red color
Indicates the presence of phenol ring containing amino acid (Tyr); in the presence of H2SO4, phenol
ring reacts with mercuric sulfate to form red mercuric phenolate
7. Pauly`s test (Diazo reaction)
To 0.5 mL sulfanilic acid, add 0.5 mL sodium nitrate, mix well and add 1 mL protein solution, mix and add 1 mL 10% sodium carbonate
A cherry-red color Indicates the presence of tyrosine (Tyr) and histidine; the diazobenzene sulfanilic acid reacts with phenol group of tyrosine and imidazole group of histidine to form red-colored diazotized derivative
Reactions of Albumin (Table 14.3)
Table 14.3: Reactions of albumin
Experiments Observations Inferences
1. Half saturation test
To 2 mL of protein solution is mixed with 2 mL of saturated ammonium sulfate solution and is allowed to stand for 5 min; with the filtrate, perform biuret test
Violet color Egg albumin is not completely precipitated by half saturation; when an inorganic salt (ammonium sulfate) is added, the effective concentration of water or the protein is decreased and the protein gets precipitated 2. Full saturation test
To 2 mL of protein solution, add ammonium sulfate till solution gets saturated; allow to stand for 5 min; perform biuret test with the filtrate
No violet color Egg albumin is completely precipitated by full saturation
Experiments Observations Inferences
3. Heat coagulation test
To 3 mL of solution, add two drops of chlorophenol red; if the solution is pink, add 1% acetic acid till it turns yellow; if it is yellow, add Na2CO3 till it turns pink and make it yellow by adding 1% acetic acid and boil it
A coagulum is formed
Indicates the presence of albumin; coagulation is irreversible when saturated at isoelectric point
4. Precipitation by heat and acetic acid
Fill half of the test tube by albumin solution; boil the upper part of the solution by holding in a slanting position; add few drops of 1% acetic acid
A cloudy white precipitate in the upper part
Proteins are easily denatured when subjected to heat; they are coagulated and precipitated at their isoelectric point
5. Color reactions - -
Reactions of Casein (Table 14.4)
Table 14.4: Reactions of casein
Experiments Observations Inferences
1. Isoelectric precipitation test
To two mL of protein solution, add 2 drops of bromocresol green; add 1% acetic acid drop by drop with mixing, until a green color is obtained
Initially, a deep blue color is formed, which turns to green with the formation of flocculent precipitate
Casein is precipitated at isoelectric pH 4.6; bromocresol green is used as an indicator; its pH ranges from 3.8 to 5.4 and color changes from yellow to blue
2. Acetic acid test
To 3 mL of protein solution, add 1% acetic acid drop by drop
A white precipitate is formed that on further addition of acetic acid gets dissolved
The precipitate is formed at isoelectric pH and dissolves on further addition of acetic acid due to lowering of pH
3. Half saturation test
To 3 mL of protein solution, add equal volume of saturated ammonium sulfate solution and filter; with 2 mL of filtrate, perform biuret test
No violet color Casein gets completely precipitated at half saturation; when an inorganic salt like ammonium sulfate is added to a solution of protein, effective concentration of water or protein is decreased and the protein gets precipitated
4. Modified Neumann’s test
To 3 mL of casein solution, add 0.5 mL of 40% NaOH; boil strongly for few min; cool under tap water and add 0.5 mL concentrated HNO3; add a pinch of ammonium molybdate and warm gently
Canary yellow precipitate is formed
This test detects the presence of organic phosphate; casein is a phosphoprotein and thus answer the test; organic phosphate when boiled with NaOH, is converted to inorganic phosphate; inorganic phosphate then reacts with ammonium molybdate to form ammonium phosphomolybdate
5. Color reactions - -
REACTIONS OF NON-PROTEIN NITROGENOUS SUBSTANCES
Non-protein nitrogenous (NPN) substance includes: 1. Urea.
2. Uric acid. 3. Creatinine.
Contd...
Chapter 14: Qualitative Analysis 141
Reactions of Urea (Table 14.5)
Table 14.5: Reactions of urea
Experiments Observations Inferences
1. Sodium hypobromite test To 2 mL urea solution,
add 4–5 drops of freshly prepared sodium hypobromite NaOBr solution
Brisk effervescence Hypobromite decomposes to nitrogen gas that appear as brisk effervescence
3NaOBr + NH2–CO-NH2 + 2NaOH N2 + Na2CO3 + 3NaBr + 3H2O 2. Specific urease test
Take 3 mL of urea solution, add 2 mL of urease suspension; incubate for 30 min at room temperature; add two drops of phenolphthalein
Pink color Urea is hydrolyzed by urease to form ammonium carbonate, which make the solution alkaline since the solution is alkaline. Phenolphthalein will give pink color
NH2-CO-NH2 + 2H2O 2NH3 + H2CO3 (NH4)2CO3
Reactions of Uric Acid (Table 14.6)
Table 14.6: Reactions of uric acid
Experiments Observations Inferences
1. Phosphotungstic acid reduction test
To 2 mL of solution, add few drops of phosphotungstic acid reagent and 20% Na2CO3
Blue color Uric acid under alkaline condition reduces phosphotungstic acid to tungsten blue
2. Schiff’s test Wet a piece of filter
paper with few drops of ammoniacal silver nitrate solution; dry and add two drops of uric acid solution on the filter paper
Black color Uric acid reduces ammoniacal silver nitrate to black metallic silver
Reactions of Creatinine (Table 14.7)
Table 14.7: Reactions of creatinine
Experiments Observations Inferences
1. Jaffe’s test
Label two test tubes as ‘test’ and ‘control’
Into test, add 1 mL of creatinine solution; mix with 1 mL of saturated picric acid solution, add few drops of 5% NaOH Into control, mix 1 mL of
H2O, 1 mL of picric acid and few drops of 5% NaOH
Orange color
Yellow color
Creatinine reacts with alkaline picrate solution to form orange-colored picrate
Chapter
15
Quantitative Analysis
INTRODUCTION TO
CLINICAL BIOCHEMISTRY
ColorimetryMeasurement of concentration of colored substances in a solution is the basis of colorimetry. Colored solutions can absorb light rays of a particular wavelength called absor- bants. This can be measured by photoelectric equipments.
Beer’s law: The amount of light absorbed by a colored solu-
tion is proportional to the concentration of the solution.
Lambert`s law: The amount of light absorbed by a colored
solution is proportional to the path length through which light passes in the solution.
Beer-lambert law is expressed as: Al = El × C × L Where,
Al = Absorbents of substances at a wavelength of l El = Extension of coefficient at l
C = Concentration of colored substances in that solution L = Length traveled by light in that solution.
Blood Glucose Estimation (Folin-WU Tube Method)
Alkaline copper (cupric ions) is reduced by glucose when boiled with protein-free filtrate to cuprous oxide. The cu- prous oxide in turn reacts with phosphomolybdic acid to form blue-colored oxides of molybdenum. The intensity of color can be determined by measuring in a colorimeter at a wavelength of 680 nm.
Folin-Wu tube with constricted neck can be used to prevent the oxidation of cuprous ion, by atmospheric
oxygen, during the period before adding phosphomolyb- dic acid.
Estimation of Serum Proteins (Biuret Method)
Proteins react with cupric ions in alkaline medium to form a purple-colored complex. The intensity of purple color is the concentration of total proteins present in the serum. Optical density measured at 540 nm. Serum albumin is determined by precipitating the globulin (G) with Na2So4. Albumin in globulin-free solution is estimated by biuret reagent. Differences between total proteins and albumin (A) can found out by A/G ratio.
Estimation of Serum Creatinine (Jaffe’s Method)
Creatinine present in the protein-free filtrate of serum reacts with picric acid in alkaline medium to form an orange-colored complex. The intensity of color is propor- tional to the serum level. Optical density is measured at 540 nm.
Estimation of Urea in Blood
When urea is heated with substance containing two ad- jacent carboxyl groups like diacetyl monoxime, colored complexes are formed. On heating in acidic medium, di- acetyl monoxime decomposes to give hydroxylamine and the diazine, which condenses with urea. Thiosemicarba- zide and ferric ions are used as catalystsm, and pink color is produced. Intensity of the color is proportional to the amount of urea in the sample. Optical density is measured at 540 nm.