qualitative estimation

C H A P T E R

2Qualitative Estimation

Section Two

Definition

Carbohydrates are polyhydroxy aldehyde orketone or compounds that yield thesederivatives on hydrolysis.

Classification: See the flow chart 2.1.

TEST FOR MONOSACHHARIDES

Biologically most important monosachharidesare glucose, fructose and galactose.

GLUCOSE

Glucose is the most important and moststudied monosaccharide. It is….• Optically active• Dextrorotatory• Shows mutarotation• Strong reducing agent (due to potential

aldehyde gr.)• Forms osazone with phenylhydrazine

Molisch’s Test

Principle: A strong dehydrating agent likeconc. Sulphuric acid converts sugar tohydroxymethyl furfural.The furfural condenseswith phenolic compounds like α-naphthol togive a colored complex (Fig. 2.1).

I. CARBOHYDRATES

Fig. 2.1: Molisch’s test (for color version see Plate 1)

12 A Complete Workbook on Clinical Biochemistry

Flow chart 2.1: Classification of carbohydrates

Qualitative Estimation 13

Procedure: To 2 ml of glucose solution add 5drops of Molisch’s reagent and mix. Run down2 ml of conc. Sulphuric acid along the sides ofthe test tube. A purple ring appears at theinterface.

Fehling’s Test

Principle: Copper sulphate contained inFehling’s I and Sodium Hydroxide containedin Fehling’s II form copper hydroxide bydouble decomposition which is reduced byglucose to red insoluble cuprous oxide.

Procedure: Add 1 ml of Fehling’s I to 1 ml ofFehling’s II. Add 8 drops of glucose solutionand boil. A red precipitate of cuprous oxideis formed by reduction.

Benedict’s Test

Principle: Under alkaline conditions reducingsugars tautomerize and form enediols whichare powerful reducing agents. They canreduce cupric ions to cuprous form which mayform green, yellow, orange or brick-redprecipitate depending on the amount ofglucose present (Fig. 2.2).

Procedure: To 5 ml of Benedict’s reagent add8 drops of glucose solution and boil for 2minutes. Depending on the amount of sugarpresent a green, yellow, orange or brick-redprecipitate depending on the amount ofglucose present (Fig. 2.3).

Barfoed’s Test

Principle: This is also a copper reduction testin acidic conditions. Aldoses and ketoses canreduce cupric ions even in acidic conditions.Monosachharides react very fast thandisaccharides. This test is used to distinguish

Fig. 2.2: Benedict’s Test (for color version see Plate 1)

Fig. 2.3: Benedict’s test at increasing concentration ofglucose solution (for color version see Plate 1)

reducing monosaccharide from a reducingdisaccharide by controlling pH and time ofheating.

Procedure: To 2 ml of Barfoed’s reagent add 8drops of glucose solution and boil for 2 minand cool under tap. A red scum of cuprousoxide on the top appears. Then add phospho-molybdic acid solution drop by drop mixingwell till the solution is clear. A deep blue colorappears in case of monosachharides (Fig. 2.4).


Osazone Test

Principle: One molecule of phenylhydrazine reactswith glucose to form glucose phenylhydrazonewith which two more phenylhydrazine mole-cules forms glucosazone, aniline and ammonia.

Procedure: In a clear and dry test tube , takeapproximately 0.5 gm of phenylhydrazinemixture (equal parts of phenylhydrazine

chloride and sodium acetate). Add 5 ml ofcarbohydrate solution and 1-2 drops of glacialacetic acid. Mix and place the test tube inboiling water bath for 30 minutes. The crystalswhen examined under the microscope, havethe appearance of bundles of fine needles,arranged in a fan-shaped aggregates (Fig. 2.5).

FRUCTOSE

Fructose occurs with glucose in fruit andhoney. It is….• Levorotatory• Shows reducing property due to functional

keto group• Identical osazone to that of Glucose

– Molisch’s test : Being a carbohydrate givesa positive test

– Fehling’s test: Positive test due toreducing property

– Benedict’s test:ositive test due to reducingproperty

– Barfoed’s test: Positive test due toreducing property

– Osazone test: Identical osazone as Glucose

Rapid Furfural Test

Principle: Conc. Hydrochloric acid convertshexoses to hydroxymethyl furfural. Thisconversion is faster for ketoses. The furfuralderivative condenses with α-naphthol to givethe color.

Prolonged heating will give positive testfor aldose also.

Procedure: To 3 ml of Conc. Hydrochloric acidadd 2 drops of Molisch’s reagent, add 2 dropsof Fructose solution and boil for just 10seconds. A violet or purple color develops.

Fig. 2.5: Glucosazone ( for color version see Plate 2)

Fig. 2.4: Barfored’s ( for color version see Plate 1)


Overheating of the solution is avoided ason boiling aldoses will get converted toketoses by the hydrochloric acid.

Procedure: To 3 ml of Seliwanoff’s reagent add1 ml of fructose. Boil for 30 seconds only.Cool the solution. A cherry red color appears.

TEST FOR GALACTOSE

Galactose is present in milk, lactose, the milksugar. It gets epimerized to glucose in theliver and then utilized as fuel.Galactose.............• Known as brain sugar• Is an epimer of glucose at C4

• Easily obtainable from lactose by theaction of lactase enzyme.

Molisch’s Test

Being a carbohydrate gives a positive result(Flow chart 2.2).

Mucic Acid Test(To be performed by the demonstration)

Principle: The nitric acid oxidizes galactose toan isomer of tetrahydroxyadipic (tetrahy-droxyhexaenedioic) acid that crystallizes outfrom water under the conditions of the test.The particular isomer resulting from galactoseis called mucic acid. Other sugars, glucose,also gives similar acids, called by othercommon names, but they are water-solubleunder conditions of the test.

Procedure: Take 50 mg of galactose in a cleandry test tube. Add 1 ml of distilled waterand 1 ml of concentrated nitric acid to it. Heatit in a boiling water bath for one and halfhours. Allow to stand overnight. Examineunder the microscope. Crystals of mucic acidis found under microscope.

TEST FOR DISACCHARIDES

The important disaccharides are lactose,maltose and sucrose.LACTOSE: Lactose is the milk sugar andoccurs in milk of all mammals. It is…….• Dextrorotatory• Yields glucose and galactose on hydrolysis

(β-1, 4 glycosidic linkage between Galactoseand Glucose)

• Reducing property due to free potentialaldehyde gr.

• Forms osazone– Molisch’s test: Being a carbohydrate

gives a positive test

Seliwanoff’s Test

Principle: Ketohexoses on treatment withhydrochloric acid form 5-hydroxymethylfurfural which on condensation with resorcinolgives a cherry red colored complex (Fig. 2.6).

This test distinguishes between fructoseand glucose.

Fig. 2.6: Seliwanoff’s test (for color version see Plate 2)


Flow chart 2.2: Detection of unknown carbohydrate



– Benedict’s test: Positive test due toreducing property

– Barfoed’s test: No red scum; gives a clearblue solution after adding phospho-molybdic acid.

– Osazone test: Pin cushion with pins/hedgehog/woolen ball appearance ofLactosazone (Fig. 2.7).

– Barfoed’s test: No red scum; gives a clearblue solution after adding phospho-molybdic acid.

– Osazone test:Greenish-yellow “sun-flower” shaped crystals under micro-scope (Fig. 2.8).

SUCROSE: Sucrose occurs in many plantslike sugarcane and beet root. It is…….• Made up of glucose and fructose (α-1,2

glycosidic linkage)• Non-reducing (Reducing group of Glucose

and Fructose are linked together)• Dextrorotatory• Invert sugar (on hydrolysis becomes

levorotatory)– Molisch’s test: Being a carbohydrate

gives a positive test– Fehling’s test: Negative– Benedict’s test: Negative– Barfoed’s test: Negative test being a non

readucing sugar– Osazone test: Negative– Seliwanoff’s test: Faint red color formed

due to presence of keto group ofFructose.

Fig. 2.8: Maltosazone (for color version see Plate 2)

Fig. 2.7: Lactosazone (for color version see Plate 2)

MALTOSE: Maltose is the mainconstituent of malt. It’s easily digested andhence used as infant food. It is…..• Dextrorotatory• Made up of two molecules of glucose

(α-1,4 glycosidic linkage)• Mildly reducing• Forms osazone

– Molisch’s test: Being a carbohydrategives a positive test


– Benedict’s test: Positive test due toreducing property


– Hydrolysis or Inversion test

Principle: Sucrose, which is dextrorotatory, onhydrolysis becomes levorotatory due toFructose.

Procedure: To 5 ml of sample solution add 6drops of Conc. Hydrochloric acid, boil for 1minute, cool and add 6 drops of 40% sodiumhydroxide. With this hydrolysed productperform Benedict’s test.

TEST FOR PENTOSE SUGARS

Bial’s Test

Principle: Hydrochloric acid acts on pentoseforming furfural which binds with orcinol togive a green colored complex.

Procedure: To 1.5 ml of Bial’s reagent add 5drops of pentose ( arabinose) solution. Heatjust to boil and cool. A green color appears.

TEST FOR POLYSACCHARIDES

Polysaccharides are complex carbohydrateswith high molecular weight. They’re tasteless,apparently amorphous and mostly insolublein water.They are…• Non-reducing• On hydrolysis with acids or enzymes they

yield simple hexoses.

Starch: Occurs in plants, particularly in tubers,roots and seeds as reserved food. It is madeup of amylose (1,4 glycosidic linkage) andamylopectin ( 1,6 glycosidic linkage). Onhydrolysis by acid (Conc. HCl) gives thefollowing products.

Starch Soluble starchAmylodextrins ErythrodextrinAchrodextrin Maltose Glucose

On enzyme hydrolysis (Amylase) maltose isthe major end product.

Dextrin: These are intermediate products ofstarch hydrolysis and are composed of lessunits of glucose in their molecule.

Glycogen: (Animal starch) Occurs in the liverand muscles. It is a reserve food in our system.Liver readily converts it into glucose.Glycogen has a highly branched structure likeamylopectin.• Molisch’s test: Being a carbohydrate gives

a positive test• Fehling’s test: Negative• Benedict’s test: Negative

Hydrolysis Test

Principle: After hydrolysis by strong acid orenzyme starch gives rise to simpler hexosesand gives a positive test for Benedict’s test.

Procedure: To 5 ml of starch solution add 2 mlof conc. Hydrochloric acid and boil for 2minutes. Cool under tap, put a red litmus add40% sodium hydroxide till alkaline (red litmuspaper turns blue). Perform Benedict’s test withthe hydrolysed solution.

Iodine Test

Principle: Iodine forms a co-ordinatecomplex between the helically coiledpolysaccharides chain and iodine centrallylocated within the helix due to adsorption.The iodine color obtained with thepolysaccharide depends upon the length ofthe unbranched or linear

→→ →

→→ →


Fig. 2.9: Starch iodine test (for color version see Plate 2)

(α-1,4 linkage ) chain available for complexformation.

Procedure: Take 5 ml of each of the poly-saccharide solutions in different tubes and add2 drops of dil. Iodine to each. Do not shake,compare the colors produced. Starch gives ablue color, Dextrin gives a purplish red colorand Glycogen gives a brownish color (Fig.2.9).


WORKBOOK FOR STUDENTS

TEST FOR MONOSACCHARIDES

TEST FOR GLUCOSE

Experiment Observation Inference

1. Molisch’s test

To 2 ml of glucosesolution 5 drops ofMolisch’s reagentwas added andmixed. Then 2 mlof conc. sulphuricacid was run downslowly along theside of the test tube.

QUESTIONS: (For answers see key at the end of this chapter)1. What are the constituent of Molisch’s reagent?

2. Which compounds give a positive test for this?

3. What is the role of conc. sulphuric acid?

4. If a green colored ring appears, what does that indicate?


2. Fehling’s test

To 1 ml of Fehling’sI, 1 ml of Fehling’sII was added. Toit 8 drops of Glucosesolution was addedand boiled.

QUESTIONS:

(For answers see key at the end of this chapter)

5. What are the constituent of Fehling’s I & II?

6. Which compounds give a positive test for this?



3. Benedict’s test

To 5 ml ofBenedict’s reagent8 drops of Glucosesolution was addedand boiled for2 minutes.

QUESTIONS:(For answers see key at the end of this chapter)

7. What are the constituent of Benedict’s qualitative reagent?

8. What are the roles of each agent in Benedict’s reagent?

9. Why the test is called a semi quantitative test?

10. Interpret the different colors of the precipitates.

11. What is the principle of Benedict’s test?

12. Which non-carbohydrate substances give a positive Benedict’s test?

13. What is the normal quantity of sugar excreted in urine in 24 hrs.?

14. What are the causes of glycosuria?


4. Barfoed’s test

To 2 ml of Barfoed’s reagent 8 drops ofGlucose solutionwas added, boiledfor 2 minutes andcooled under tap.Phosphomolybdicacid was added dropby drop till thesolution was clear.


QUESTIONS: (For answers see key at the end of this chapter)15. What are the constituents of Barfoed’s reagent?

16. What is the purpose of this test?

17. What precaution should be taken during boiling and why?


5. Osazone testApproximately 0.5gm of phenylhy-drazine mixture(equal parts ofphenylhydrazinechloride andanhydrous sodiumacetate)was takenin a dry test tube.To it 5 ml of glucosesolution and 1-2drops of glacial aceticacid was added. Thecontents were mixedwell and the tube wasplaced in a boilingwater bath for 30minutes. The crystalswere then examinedunder microscope.

QUESTIONS: (For answers see key at the end of this chapter)18. Which sugars form osazone?

19. What is the appearance of osazones of different sugars?

20. Why glucose, fructose and mannose give similar osazones?

21. What are the roles of acetic acid and sodium acetate?


TEST FOR FRUCTOSE


1. Molisch’s test(Write as writtenfor Glucose)

2. Fehling’s test


4. Barfoed’s test

5. Osazone test

6. Rapid furfural testTo 3 ml of conc.Hydrochloric acid2 drops of Molisch’sreagent was added.To it 2 drops offructose solution wasadded and was boiledfor 30 seconds only.

QUESTIONS: (For answers see key at the end of this chapter)22. Why strict timing for boiling is followed?

23. For which sugar this test is specific?

24. What is the clinical implication of testing fructose in urine?



8. Seliwanoff’s test

To 3 ml ofSeliwanoff’sreagent 1 ml ofFructose solutionwas added. It wasboiled for 30 secsonly and then cooled.

QUESTIONS:(For answers see key at the end of this chapter)25. What are the constituent of Seliwanoff’s reagent?

26. What is the clinical indication for a Seliwanoff’s test?

TEST FOR DISACCHARIDES

TEST FOR LACTOSE


1. Molisch’s test(Write as written for Glucose andrecord your observation and inference)

2. Fehling’s test



4. Barfoed’s test

5. Osazone test

TEST FOR MALTOSE


1. Molisch’s test(Write aswritten for Glucoseand record yourobservation andinference)

2. Fehling’s test


4. Barfoed’s test


5. Osazone test

TEST FOR SUCROSE


1. Molisch’s test(Write as written for Glucoseand record your observationand inference)

2. Fehling’s test


4. Barfoed’s test

5.Seliwanoff’s test


6. Osazone test

7. Hydrolysis or Inversion testTo 5 ml of sucrose solution6 drops of conc. Hydrochloric acidwas added, was boiled for 1 minuteand 6 drops of 40% sodium hydroxidewas added. To 5 ml of Benedict’sreagent 8 drops of hydrolysed solutionwas added and boiled for 2 minutes.

QUESTIONS:(For answers see key at the end of this chapter)27. Why sodium hydroxide is added in hydrolysis test?

28. Why this is called Inversion test?

TEST FOR PENTOSE


1. Molisch’s test

2. Bial’s testTo 1.5 ml of Bial’s reagent5 drops of pentose solutionwas added. It was heated just .to boil and cooled.


QUESTIONS: (For answers see key at the end of this chapter)29. What is clinical use of this test?

30. What are the causes of pentosuria?

TEST FOR GALACTOSE


1. Molisch’s test

2. Mucic acid test(to be demonstratedonly)

QUESTIONS: (For answers see key at the end of this chapter)31. Which other sugar gives this test positive?

32. What are the causes of galactosuria?

TEST FOR POLYSACCHARIDES

TEST FOR STARCH


1. Molisch’s test

2. Iodine testTo 5 ml starch solution 2 dropsof dilute iodine was added.Preventing shaking colorchange was observed.


3. Starch Hydrolysistest To 5 ml of starch solution 2 ml ofconc. Hydrochloric acid was addedand was boiled for 2 minutes. It wascooled under tap and a red litmus wasput. 40% sodium hydroxide was addedtill alkaline ( i.e. litmus paper turns blue).To 5 ml of Benedict’s reagent 8 drops ofthe hydrolysed solution was added andboiled for 2 minutes.

QUESTIONS: (For answers see key at the end of this chapter)33. What is the practical implication of iodine test?

TEST FOR DEXTRIN


1. Molisch’s test

2. Iodine test

3. Hydrolysis test

ANSWER KEY(CARBOHYDRATES)

1. α-naphthol and ethanol.2. All carbohydrates; at least ‘5C’ should be there; Albumin3. Conc. sulfuric acid dehydrates carbohydrates to form furfural derivatives.4. Excess of α-naphthol may be present.


5. Fehling’s I- copper sulfate; Fehling’s II-potassium hydroxide and Rochelle’s salt

6. Carbohydrates having a potentialaldehyde or keto group.

7. Copper sulfate, sodium carbonate andsodium citrate.

8. Copper sulfate—furnishes cupric ionsSodium carbonate—makes mediumalkalineSodium citrate—Prevents the pre-cipitation of cupric ions as cuprichydroxide by forming a loosely boundcupric-sodium citrate complex which ondissociation gives a continuous supplyof cupric ions. Sodium citrate acts as astabilizing agent.

9. From the color of precipitate approxi-mate amount of sugar present in thesample can be known.

10. Green color—Up to 0.5 g%Green ppt.—0.5- 1.0 g%Yellow ppt.—1.0- 1.5 g%Orange ppt.—1.5 -2 g%Brick red ppt.—> 2 g%

11. Refer text12. Uric acid, creatinine, ascorbic acid,

homogentisic acid.13. Total reducing sugar is 1-1.5 gm/24 hrs.

Glucose is 50-300 mg/24 hrs out of these.14. Presence of reducing sugars in urine is

known as glycosuria and for glucoseonly the term glucosuria is used..Diabetes mellitus, alimentary glycosuria,hyperthyroidism, hyperpituitarism,hyperadrenalism, severe infections,anesthesia, asphyxia, increasedintracranial tension, lactosuria,galactosuria, and pentosuria are fewcauses of glycosuria.

15. Copper acetate, glacial acetic acid andwater.

16. This test distinguishes between reducingmonosaccharide and disaccharides.

17. Over-heating may be avoided as for thesame, disaccharides also give a positivetest.

18. Reducing sugars give a positive test.19. Glucose, Fructose, Mannose—Needle-

shapedLactose—woolen ball/hedge-hog/pincushion with pins appearanceMaltose—Sunflower-petal shaped.

20. 1st and 2nd ‘C’ atoms are involved inthe reaction during osazone formation.

21. Acetic acid and sodium acetate are usedto maintain pH at 5, appropriate forformation of osazone.

22. Prolonged heating will give positive testfor aldoses also.

23. Ketohexoses; Fructose24. It can detect essential fructosuria.25. Resorcinol in dilute hydrochloric acid.26. Specific for fructose. Seminal fluid

fructose can be tested to know the siteof blockade in case of male infertility.

27. To neutralize the solution.28. By hydrolysis due to fructose the optical

rotation changes from dextrorotation tolevorotation.

29. Detects pentosuria.30. Ingestion of large amount of fruits,

essential pentosuria(Enzyme xylitoldehydrogenase deficiency; drugs takebarbiturate and aminopyrine)

31. Due to presence of galactose, lactose alsogives this test positive.

32. Galactosemia, lactosuria33. Check adulteration of milk with starch

and see presence of iodine in iodized salt.

qualitative estimation

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