Qualitative Tests Used For Carbohydrates Biology Essay

soft probes Used For doughs Biology EssayCarbohydrates are an essential component of our diet and an important source of energy for us. Most of the things primarily included in our diet get to a large ratio of kales present. Also the patients suffe hem in from Diabetes excrete huge substances of dent in their urine which of necessity to be estimated. The purity of carbohydrates fuel also be checked by methods which can qualitatively estimate particular bootys. Hence the need for carbohydrate estimation arose.We have been estimating carbohydrates qualitatively since school time but it was just confined to observing a food coloring budge or a colou blushing(a)dened pass to detect the aim of a carbohydrate. None of us ever thought that these qualitative campaigns are broad(prenominal)ly sensitive and provide much more tuition than just detecting the presence of a sugar.In this study conducted, two qualitative tests utilize for carbohydrates were studied Benedicts te st and Seliwanoffs test. Benedicts test is a test utilize for detecting the presence of fall down Sugars. The test was per counterfeited using Glucose which is the most common bring down sugar. Sucrose was used as a negative control. Different concentrations of glucose were try ranging from 1.6 M to 1.6 mM and from 4% to 0.25%.Also along with concentration the fall of sugar was varied to find the sensitivity limits and the limitations of the test. The result of Benedicts test is usu whole(prenominal)y a brick trigger-happy precipitate but with some modifications diametrical colours of beginnings and various follows of precipitate could be sight with slight magnetic declination in the concentration and amount of sugar. This suggests that this test being Qualitative is non only an indicator of the presence or absence of a reducing sugar but can also be used to roughly estimate the concentration of sugar present. It can be very useful in estimation of the concentration o f sugar present in the urine of diabetic patients.Seliwanoffs test is a qualitative test used for distinguishing in the midst of Aldoses and ketoses. Ketoses fig a cherry red condensing product whereas Aldoses fight down to form a game-green condensation product, which may further change to a peach product. The test was performed using fructose as the sample sugar. versatile concentrations of Fructose were used ranging from 4% to 0.01% to find the sensitivity limits and the limitations of the test. The test appeared to be sensitive til now at 0.01% turn ining a very faint red colour. on that point was a huge version in the intensity of colour obtained at different concentrations of sugar. But the drawback of the test was that the red colour of the solution was not stable. It intensified with increase in the duration of time.2. INTRODUCTIONCarbohydrates are the most abundant bio molecules on Earth. Each year, photosynthesis converts more than 100 billion metric heaps of CO2 and H2O into cellulose and other plant products. Certain carbohydrates (sugar and starch) are a dietary staple in most parts of the world, and the oxidation of carbohydrates is the central energy- containing r disclosee in most non-photosynthetic cells. Insoluble carbohydrate polymers serve as structural and protective elements in the cell w anys of bacteria and plants and in the connective tissues of animals.Other carbohydrate polymers lubricate skeletal joints and participate in recognition and adhesion between cells. More complex carbohydrate polymers covalently attached to proteins or lipids act as signals that obtain the intracellular location or metabolic fate of these hybrid molecules, called glycoconjugates..Carbohydrates are polyhydroxy aldehydes or ketones, or substances that yield such compounds on hydrolysis. Many, but not all, carbohydrates have the empiricalformula (CH2O)n some also contain nitrogen, phosphorus, or sulphur. There are three major size classes of carb ohydrates infectious mononucleosissaccharides, oligosaccharides, and polysaccharidesClassificationCarbohydrates can be separate on the basis of the various characteristics they possess. They can be classified on the basis of the number of carbon ingredients in the sugar chain, the terminal functional convention in the chain, the number of sugar subunits and the reducing activity of the sugar units. Depending on various basis they are of several typesA. According to the number of carbon atoms in the sugar chain1. Trioses contain 3 carbon atoms (e.g. glyceraldehyde).2. Pentoses contain 5 carbon atoms (e.g. ribose).3. Hexoses contain 6 carbon atoms (e.g. glucose).B. According to the terminal functional assembly in the sugar chain1. Aldoses contain terminal aldehyde group (-CHO) (e.g. glucose).2. Ketoses contain terminal ketone group (C=O) (e.g. fructose).C. According to the number of sugar subunits1. Monosaccharides Monosaccharides, or simple sugars, populate of a single polyhyd roxy aldehyde or ketone unit. The most abundant monosaccharide in nature is the six-carbon sugar D-glucose, sometimes referred to as dextrose.2. Oligosaccharides Consist of short chains of monosaccharide units(2-10) joined by characteristic linkages called glycosidic bonds. The most abundant are disaccharides, with two monosaccharide units. e.g. sucrose (cane sugar), which consists of the six-carbon sugars- D-glucose and D fructose.3. Polysaccharides The polysaccharides are sugar polymers containing more than 20 or so monosaccharide units, and some have hundreds or thousands of units. Some polysaccharides, such as cellulose, are linear chains others such as glycogen, are branched. both glycogen and cellulose consist of recurring units of D-glucose, but they differ in the type of glycosidic linkage and have different properties and biological roles..D. According to the reducing activity of the sugar unitCarbohydrates that can undergo oxidation are called reducing sugars. This depend s on the presence of an exposed carbonylic group.1. Reducing sugars Certain sugars with a free carbonyl carbon can be oxidized by oxidizing agents such as ferric (Fe3) or cupric (Cu2) ion. The carbonyl carbon (anomeric carbon) is oxidized to a carboxyl group. Such sugars capable of reducing ferric or cupric ion are called Reducing sugars. e.g. lactose, maltose.2. Non-reducing sugars Sugars like sucrose contain no free anomeric carbon atom since the anomeric carbons of both monosaccharide units are involved in the formation of glycosidic bond. Therefore it is a Non-Reducing sugar.2.1 Biochemical Tests for CarbohydratesThe biochemical tests for carbohydrates can be divided into two categoriesQualitative Tests- These tests are performed to detect the presence of carbohydrates in a test sample. They are also used to detect the type of carbohydrate present. There are different types of qualitative tests for different types of carbohydrates.e.g. Fehlings and Benedicts test for reducing su gars, Seliwanoffs test for ketoses, Iodines test for starch,Bials test for detecting pentoses etc. They are not very sensitive as compared to valued tests and cannot estimate the exact amount of carbohydrates present but show some difference in the observation with variation in the amount of carbohydrate hence, can be used to make a rough estimate of the amount of sugar present.Quantitative Tests- These tests are the advanced form of qualitative tests and can be used to estimate the amount of carbohydrate present in a given sample. These tests use particular chemicals which form coloured complexes with sugars which can then be read at certain wavelengths using a spectrophotometer. Their absorbance can then be used to estimate the exact amount of carbohydrate present in the sample.e.g. Anthrone test, DNS Method, Phenol- Sulphuric Acid Method etc.2.11 Qualitative TestFlow Chart for classifying an unknown carbohydrateA. Molisch TestMolisch test is used to distinguish between carbohydr ates and non- carbohydrates. It is the preliminary test used to detect the presence of carbohydrates in a sample. tenetIt uses concentrated sulphuric dosageulated as a Dehydrating acid which dehydrates all carbohydrates to form Furfural or 5-hydroxymethylurfural from reply of sulphuric acid with pentoses and/or hexoses. These products condense with -naphthol to yield a purple condensation product.B. Iodine and Potassium Iodide TestThis is a test used particularly to detect starch and glycogen. Starch gives a gloomy-black colur with potassium iodide whereas glycogen gives reddish- chocolate-brown colour.PrincipleStarch contains -amylose, a helical saccharide polymer, and amylopectin. Iodine forms a large complex polysaccharide with the -amylose helix, producing a depressed-black colour. Simple Oligosaccharides and Monosaccharides do not form this complex with Iodine. Thus, the I2/KI test can be used to distinguish starches from other carbohydrates.C. Bials TestBials test is used to distinguish between pentoses and hexoses.PrincipleThis test uses concentrated hydrochloric acid as the dehydrating acid and orcinol with a trace of iron(III) chloride as the condensation reagent.. Pentoses subjected to the test yield a blue or green condensation product, while hexoses yield a muddy brown to grey condensation product.Pentose Dehydration Product Blue or Green condensation product(Furfural)Hexose Dehydration Product pestiferous brown-Grey condensation product(5-hydroxymethylfurfural)D. Seliwanoffs TestSeliwanoffs test is used to distinguish between aldoses and ketoses.PrincipleThis test uses 3N hydrochloric acid as the dehydrating agent as resorcinol as the condensation reagent. When sundry(a) with Seliwanoffs Reagent, Ketopentoses react within 2 minutes to form a cherry red condensation product. Aldopentoses react later 2 minutes to form a blue-green condensation product, which may further change to a peach product.Fructose Hydroxy-methyl Cherry-Red ComplexFurf uralIdentifying Reducing SugarsAll mono and disaccharides with a free aldehyde or keto group act as reducing agents in alkaline solutions. The reducing properties of sugars are dependent upon the presence of actual or potential aldehyde or ketone groups.The enolization of sugars under alkaline conditions is an important consideration in reduction tests. The ability of a sugar to put down alkaline test reagents depends on the availability of an aldehyde or keto group for reduction responses. A number of sugars, in particular disaccharides or polysaccharides have glycosidic linkages which involve bonding between each group, and hence on that point is no reducing group on the sugar such as the case for sucrose, trehalose, inulin, glycogen, starch, and dextrin. In the case of reducing sugars, the presence of alkali causes extensive enolization especially at high pH and temperature. This leads to a higher susceptibility to oxidation reactions than at neutral or acidulent pH. These su gars, therefore, become potential agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and so forth. Reducing sugars can react with many different oxidizing agents. Fehlings test, Benedicts test and Barfoeds test have been used to distinguish between monosaccharides and disaccharidesMonosaccharides fluctuate between a ring open form and a ring closed form. The ketone (-C=O) group, for Fructose and the aldehyde group (-CHO), for Glucose in the ring open forms can be shortend using these tests. Some sugar units in disaccharides also fluctuate between a ring open form and a ring closed form. These disaccharides are also reducing sugars because the ring open form has a ketone or aldehyde to react. Sucrose is one of the few disaccharides that do not have a ring open form so it is a non-reducing sugar. recall Open Fructose and Ring Closed Fructose Ring Open Glucose and Ring Closed GlucoseReducing Sugars are oxidized by copper (II) ions. Benedicts reagent and Fehlings Reagent are mildly basi c solutions whereas Barfoeds Reagent, a mildly acidic solution. The presence of red copper (I) oxide precipitate indicates that the saccharide has reduced the copper (I) ions.E. Fehlings testPrincipleFehlings test uses a mixture of fehlings solution A and B. Fehlings solution A consists of copper(II) sulphate dissolved in dilute sulphuric acid. Fehlings solution B is sodium potassium tartarate dissolved in dilute NaOH. Both the solutions are mixed in equal proportions and used as fehlings reagent. This reagent is used as a general test for detecting reducing sugars. A reducing sugar reduce copper(II) ions to copper(I) oxide, forming a red precipitate.CuSO4Cu++ + SO42 Cu+++ Cu+Glucose(Reducing Sugar)Cu+Cu2O (red precipitate)Cuprous OxideF. Benedicts testPrincipleBenedicts test uses a mixture of copper(II) sulphate, sodium citrate, and sodium carbonate in a mildly basic solution. This reagent is used as a general test for detecting reducing sugars. A reducing sugar reduce copper(II) ions to copper(I) oxide, forming a red precipitate.CuSO4Cu++ + SO4-(Copper Sulphate) (Cupric Ion) (Sulphate Ion)2 Cu+++ Cu+Glucose(Reducing Sugar)Cu+Cu2O (red precipitate)Cuprous OxideG. Barfoeds TestBenedicts test gives positive test results for all reducing sugars. However, not all reducing sugars react at the same rate. With different oxidizing agents, disaccharides are considerably slight reactive compared to monosaccharides. A positive Barfoeds test result is similar to that observed with Benedicts solutions. Monosaccharides give positive Barfoeds test results within 2-3 minutes, while disaccharides do not react under the same conditions.PrincipleBarfoeds test uses copper(II) ions in a fairly acidic medium. If the reaction time is carefully monitored, this test can be used to distinguish reducing monosaccharides from reducing disaccharides. Reducing disaccharides cause the formation of copper(I) oxide later approximately 10 minutes.(CH3COO) 2Cu + 2H2O 2 CH3COOH + Cu(OH)2Cu(O H)2 CuO+ H2O2CuO+Cu2O (red precipitate) (Glucose)Reducing Sugar2.12 Quantitative TestsA. Determination of Total Carbohydrate by Anthrone MethodPrincipleCarbohydrates are commencement ceremony hydrolysed into simple sugars using dilute hydrochloric acid. In hotacidic medium glucose is dehydrated to hydroxymethyl furfural. This compound forms withanthrone a green coloured product with an absorption maximal at 630 nm.HCl anthroneCarbohydrate furfural/hydroxymethyfurfural Green product(630 nm)B. Phenol Sulphuric Acid Method for Total CarbohydratePrincipleIn hot acidic medium carbohydrates are dehydrated to furfural/hydroxymethyl furfural. This forms a green coloured product with phenol and has absorption maximum at 490 nm.H2SO4 phenolCarbohydrate furfural/hydroxymethylfurfural Green product(490 nm)C. Determination of Reducing Sugars by Nelson-Somogyi MethodThe Nelson-Somogyi method is one of the classical and widely used methods for the quantitative determination of reducing sugars.Pr incipleThe reducing sugars when heated with alkaline copper tartarate reduce the copper from thecupric to cuprous state and thus cuprous oxide is formed. When cuprous oxide is treated withArsenomolybdic acid, the reduction of molybdic acid to molybdenum blue takes place. The blue colour developed is compared with a set of standards in a colorimeter at 620 nm.Cu2O+ Arsenomolybdate Molybdenum Blue(Cuprous Oxide) (500 nm)D. Estimation of Reducing Sugar by Dinitrosalicylic Acid MethodThis method is an alternative to Nelson-Somogyi method. It is a simple, sensitive and adoptable method during handling of a large number of samples at a time. However,enzymaticmethods are usually preferred to DNS due to their specificity.Principle3,5-Dinitrosalicylic acid is anaromatic compoundthat reacts with reducing sugars to form3-amino-5-nitrosalicylic acid, which absorbslightstrongly at 540nm.3,5-Dinitrosalicylic acid 3-amino-5-nitrosalicylic acidE. Determination of Glucose by Glucose Oxidase MethodGl ucose is a widely distributed simple sugar with an active aldehyde group. Estimation ofglucose by glucose oxidase gives the true glucose concentration eliminating the birth control deviceby other reducing sugars.PrincipleGlucose oxidase catalyses the oxidation of alpha-D-glucose to D-glucono-1, 5 lactone (gluconic acid) with the formation of hydrogen peroxide. The oxygen liberated from hydrogen peroxide by peroxidase reacts with the O-dianisidine and oxidises it to a red chromophore product.Glucose + O2 H2O2 + Gluconic Acid(glucose oxidase)H 2O2 + O-dianisidine Red-coloured product(peroxidase) (540 nm)3. MATERIALS REQUIREDI) Stock SolutionsGlucose 1.6 MGlucose 4% (w/v)Sucrose 0.1 MFructose 4% (w/v)II) ReagentsBenedicts Reagent metre 1- drop 173 g sodium citrate and 100 g sodium carbonatein about 500 mL water. footfall 2-Heat to dissolve the saltStep 3-Dissolve 17.3 g copper sulphate in about 100 mL water and add it to the above solution with stirring and make up the volume to 1 L w ith water.Seliwanoffs ReagentDissolve 0.05 gm resorcinol in 3N hydrochloric Acid.III) MiscellaneousWater BathTest TubesClampsConical FlasksBeakersContainersTest tube holders4. METHODOLOGY4.1 Benedicts TestStep 1- Pipette out 1 ml of glucose solution in some test tubes so that there is a triplicate for each set.Step 2- Pipette out 1ml of Distilled Water in one test tube which serves as blank and 1 ml of Sucrose solution (0.1M) in another which serves as a negative control.Step 3- Add 2 ml of Benedicts reagent in all the test tubes.Step 4-Transfer all five tubes to the boiling water bath provided and criminal record the time for the mien of precipitate.Step5- Make a subjective analysis of the colours observed. A scale of + to +++++ can be used to record the depth of the colour, from lightest to darkest.4.11 Effect of variation in Carbohydrate Concentration (in M)The concentration of glucose was varied from 1.6 M to 1.6 mM keeping the reaction volume constant to 3 ml. The upper and t he lower sensitivity limits and the optimum molar concentration of glucose was found for the test through subjective analysis.4.12 Effect of Reaction loudnessThe reaction volume was reduced from 3 ml to 1.5 ml for all the concentrations of glucose ranging from 1.6 M to 1.6 mM to find any difference in the sensitivity range and the optimum molar concentration of glucose for the test.4.13 Effect of variation in Carbohydrate Concentration (in %)The concentration of glucose was varied from 4% to 0.25%. Also the reaction volume was changed and the analysis was performed in two sets. The volume of reagent used was 5 ml whereas the volume of samples all concentrations in get A and B were 50l and 25l respectively.4.14 Effect of variation in Sample peopleThe volume of glucose was varied for the same concentrations 4% to 0.25% to see the changes in the colour of solution obtained and at the same time find the upper and lower limits at which the test still remains sensitive. The analysis wa s performed in 6 sets with the following volumes of glucose 200l, 100l, 50l, 25l, 12.5l and 6.25l.4.15 Effect of Reaction Volume on the results obtained.Of the various sets tried 5 ml of Benedicts reagent and 200l of sample gave the best results. To verify the consistency of results obtained the reaction volume was reduced. The analysis was performed in two sets. Set A with 2.5 ml of reagent and 100l of sample and Set B with 1.25 ml of reagent and 50l of sugar sample.4.2 Seliwanoffs TestStep 1- Pipette out 1 ml of fructose solution in test tubes so that there is a triplicate for each set.Step 2-Pipette out 1ml of Distilled Water in one test tube which serves as blank.Step 3- Add 2 ml of Seliwanoffs reagent in all the test tubes.Step 4-Transfer all tubes to the boiling water bath for 2.5 mins.Step 5- Make a subjective analysis of the colours observed. A scale of + to +++++ can be used to record the depth of the colour, from lightest to darkest.4.21 Effect of variation in carbohydrate concentration (in %)The concentration of fructose was varied from 4% to 0.015% keeping the reaction volume constant to 3 ml. The upper and the lower sensitivity limits and the optimum concentration of fructose were found for the test through subjective analysis.5. OBSERVATIONS5.1 Benedicts Test5.11 Effect of variation in Carbohydrate Concentration (in M)A brick red precipitate was observed for all the concentrations of sample ranging from 1.6 M to 3.125 mM. But the last concentration of 1.6 mM showed a negligible amount of precipitate. Also once centrifuged the supernatant was found to be colourless for concentrations ranging from 1.6 M to 0.1 M. The supernatant was observed to be blue in concentrations ranging from 50 mm to 1.6 mM.Glucose(M) glossary potency1.60.80.40.20.10.050.020.0120.0060.0030.001+++++++++++++++++++++++++++++++++++++5.12 Effect of variation in Reaction VolumeA brick red prcipitate was observed for all the concentrations of sample ranging from 1.6 M to 6.25 mM. Concentrations 3.125 mM and 1.5625 mM showed a negligible amount of precipitate. For concentrations 6.25 mM to 0.1 M there was an increase in the amount of precipitate observed with the highest amount formed in 0.1 M of sample. For the following(a) two concentrations i.e. 0.2 M and 0.4 M the amount of precipitate formed was almost equal but again reduced for 0.8 M and 1.6 M glucose sample.Also after centrifugation the supernatant was found to be colourless for concentrations ranging from 1.6 M to 0.1 M. The supernatant was observed to be increasingly blue in concentrations ranging from 50 mm to 1.5625 mM.Glucose(M)Colour Intensity1.60.80.40.20.10.050.020.0120.0060.0030.001+++++++++++++++++++++++++++++++++5.13 Effect of variation in carbohydrate concentrations (in %)There was a brick red precipitate observed in for all the concentrations of sample ranging from 0.25% to 4% in both the sets. The highest amount of precipitate was observed for 1% sample but there was no consistency in results seen.Glucose (%)SetSet B4.02.01.51.00.50.25+++++++++++++++5.14 Effect of variation in Sample VolumeThere was a variation in the colours obtained at different concentrations and volumes of sugar sample used. But sets with 12.5l and 6.25l of sugar did not show any noticeable changes in colour.Glucose(%)Set A Set B Set C Set D Set E Set FSet BSet CSet DSet ESet F4.002.001.000.500.25reddish brownbrowngreengreenish bluedull bluebrowngreengreenish bluedull bluegreengreenish bluedull bluebluebluegreenish bluedull bluebluebluebluedull blueblueblueblueblueblueblueblueblueblue5.15 Effect of reduction in the reaction volumeThere was no change in the observations due to reduction in the reaction volume.Glucose (%)Set A Set B4.02.01.00.50.25reddish brownbrowngreengreenish bluedull bluereddish brownbrowngreengreenish bluedull blue5.2 Seliwanoffs Test5.21 Effect of variation in carbohydrate concentration (in %)A cherry red colour is observed for all the concentrations of fructose used but the intensity of colour obtained decreases with decrease in concentration. The worst concentration used (0.015%) has a faint red colour. Also the colour of the solution intensifies with time if kept after boiling.Fructose(%)Colour Intensity4.002.001.000.500.250.120.060.030.01+++++++++++++++++++++++++++++++++++++++++++++6. RESULTS AND discussion6.1 Benedicts Test6.11 Effect of variation in Carbohydrate Concentration (in M)It can be observed that 0.1 M is the optimum concentration of sugar for Benedicts reaction with the reaction volume of 3 ml since the highest amount of precipitate is formed at 0.1 M.Also the supernatant obtained after centrifugation is colourless which suggests that the reaction is completed and there is no unused reagent left.Concentrations less than 0.1 M show decreasing amounts of precipitate and the colour of the supernatant is also increasingly blue. This suggests that as the concentration of sugar is lowered the amount of unreacted Benedicts reagent increas es which leaves the solution blue even after the reaction completes. 1.6 mM sugar sample shows a negligible amount of precipitate formation which suggests that the reaction is not sensitive for concentrations lower than 1.6 mM.For concentrations higher than 0.1 M the amount of precipitate formed again decreases with increase in concentration which suggests that the concentration is too high as compared to the amount of reagent used and hence no more precipitate is formed after the reaction completes.6.12 Effect of variation in Reaction VolumeThe reaction volume was reduced to half but has no effect on the results of the experiment. 0.1 M is the optimum concentration of sugar for the reaction with the reaction volume of 3 ml since the highest amount of precipitate is formed at 0.1 M.Also the supernatant obtained after centrifugation is colourless which suggests that the reaction is complete and there is no unused reagent left.Concentrations less than 0.1 M show decreasing amounts of precipitate and the colour of the supernatant is also increasingly blue. This suggests that as the concentration of sugar is lowered, the amount of unreacted reagent increases which leaves the solution blue even after the reaction completes. 3mM and 1.6 mM sugar samples show a negligible amount of precipitate formation which suggests that the reaction is not sensitive for concentrations lower than 3 mM with a reaction volume of 1.5 ml.For concentrations higher than 0.1 M, the amount of precipitate formed again decreases with increase in concentration which suggests that the concentration is too high as compared to the amount of reagent used and hence no more precipitate is formed after the reaction completes.6.13 Effect of variation in carbohydrate concentrations (in %)The highest amount of precipitate was formed in 1% sugar sample but there was no consistency in the observations due to incorrect preparation of reagent. During the preparation of reagent instead of mixing Sodium cit rate and Copper Sulphate together in hot water they were dissolved separately and then mixed. Also the solution was not make up to 500 ml with distilled water rather measured amount of water was added to the solution to make it 500 ml which made the solution dilute and hence gave incorrect results.6.14 Effect of variation in Sample VolumeDifferent volumes of sugar were tried for all the different concentrations. There was a variation in the colours obtained. 200l of sugar reacted with 5 ml of reagent showed maximum variation in colours at different concentrations Reddish Brown, Brown, Green, Greenish blue and dull blue obtained at 4%, 2%, 1%, 0.5% and 0.25% respectively.Sets with 12.5l and 6.25l of sugar volume did not show any noticeable changes in colour which suggest that the volume of sugar solution was not enough for the reaction to take place.6.15 Effect of reduction in the reaction volumeReduction in the reaction volume made no difference to the variation in colours obtained at different concentrations of sugar which suggests that Benedicts reagent and glucose can be reacted in this ratio in any volume to estimate the concentration of glucose present in an unknown sample.6.2 Seliwanoffs Test6.21 Effect of variation in carbohydrate concentration (in %)A cherry red colour is observed for all the concentrations of Fructose used but the intensity of colour obtained decreases with decrease in concentration. The highest intensity is observed at 4% sugar concentration. 0.015% which is the lowest concentration of sugar used gives a very faint red colour which cannot be taken as pos