Benedict’s Test : Principle, Reagent Preparation, Procedure and Interpretation
Advanced References and Further Reading 1. Can you think of a situation where it might be useful to be able to measure the concentration of glucose in a solution especially in blood or urine? Once you have a way of quickly and easily measuring the concentration of sugar, then you can determine how much insulin is needed to counteract it. Next question. What would be an easy, visual way to detect the presence of glucose? A test for blood sugar suitable for diabetics should have a similar ease of use.
This brings us via aldehydes to the topic of reducing sugars, since they are the basis of a historically important color-based test for blood glucose. During this process, the aldehyde is oxidized and the oxidizing agent is reduced. Another way of framing this is to say that the aldehyde is the reducing agent in this process.
The list of reagents that can be used to oxidize aldehydes to carboxylic acids is loooong. Of these, a few methods stand out in providing a particularly clear visual indication that the reaction has proceeded to completion. The above tests were also a useful way of distinguishing aldehydes from ketones in the dark days before IR and NMR spectroscopy made this routine.
So what does this have to do with sugars? A simple color change tells you if glucose is present! But what if we want to determine the exact concentration of glucose in a solution of, say, urine or blood? In this case, a slightly different formulation of Benedicts solution is used [ Note 2 ] which results in a colourless precipitate rather than a red color.
A solution of the sample to be analyzed is added, via buret, to a flask containing a known amount of Benedicts solution until the blue color of the Cu II disappears. One researcher recalls that all inductees into the U. So it will also give a positive test for other reducing sugars.
In other words, those sugars are also reducing sugars. Hold on for a second. Et tu, fructose? So why does fructose give a positive test? Great question. Although fructose is a keto sugar, and ketones generally give a negative test with the Benedict, there is an exception.
They are also reducing sugars that give a positive Fehlings, Benedict, or Tollens test picture of lactose positive test is further below. Two main cases: mono and di-saccharides which lack a hemiacetal polysaccharides where the ratio of hemiacetals to acetal linkages is very low e. In contrast, acetals ketals are locked in place and can only be converted back to the aldehyde or ketone with aqueous acid. The poster child for a non-reducing sugar is sucrose , a.
Sucrose is a disaccharide of glucose and fructose. This is obtained by heating glucose in acidic methanol. Starch Sugars are able to form long chains with each other in arrangements known as polysaccharides. Common examples of polysaccharides are starch, cellulose, and glycogen. Hemiacetals are present, but only at the termini of the polymer.
Starch, for example, generally has about individual units of glucose, but only one unit the terminus has a hemiacetal. Therefore these polysaccharides are not considered reducing sugars. For example, starch gives a negative test see below.
Test yourself. Quiz yourself on whether the following sugars are reducing sugars or non-reducing sugars. But if you want to go further down the rabbit hole, I invite you to read further to learn about… 8. One thing about all three tests is that the active reagent is not particularly bench stable and has to be freshly prepared.
The purpose behind using the tartrate is that it coordinates to the copper II and helps prevent it from crashing out of solution. Once prepared, the substance to be analyzed is added, and the mixture is heated for a brief period.
This results in a carboxylic acid and red Cu I which precipitates out as copper I oxide. The ingredients are copper II sulphate, sodium carbonate note: hydroxide is also needed! Note: in the quantitative test, potassium thiocyanate is added, which results in a colourless white precipitate. The test is performed by adding the substance to be analyzed and heating briefly.
The first three lines below describe the procedure. Silver nitrate is converted to silver hydroxide, which forms silver I oxide, Ag2O. Then, addition of aqueous ammonia NH3 results in formation of the silver-ammonia complex which is the active oxidant. The sample to be tested is then added to the freshly prepared active oxidant in a basic solution.
A positive test results in a beautiful mirror of silver metal being precipitated out on the reaction vessel. A variant of this procedure is used for the preparation of mirrors.
How Does It Work? The first thing to note is that all of these procedures occur in basic solution. First, acidic conditions might hydrolyze any acetals present to hemiacetals, giving a false positive test. Secondly, base considerably speeds up the rate of ring-chain tautomerism i. Bottom line here is that adding base has the effect of increasing the concentration of the starting aldehyde.
If I am wrong, please tell me leave a comment. One of the access points for the initiation of a single-electron transfer reaction is a carbon-metal bond, which can be achieved through base-promoted formation of an enolate.
That requires that the aldehyde have a proton on the alpha carbon i. Thus it would appear that the reaction needs to proceed through an enol. So it is likely that a variety of mechanistic pathways can be in operation.
What might a mechanism look like? Maybe, possibly, something like this? Notes Image sources: Benedicts solution. Tollens test. Note 1. The standard way to do it is the Pinnick oxidation. Note 2. The quantitative test apparently employs potassium isocyanate, which results in a colourless precipitate. See the mechanism section. Note 4. The subjection of glutaraldehyde to the Tollens test William D.
The test tubes are then placed in the test tube stand, which is kept in boiling water for minutes. The color in the test tubes are observed and noted down. Image Source: Chemistry Learner. The appearance of a greenish precipitate indicates about 0.
The appearance of the blue color indicates the absence of reducing sugar and represents a negative result. It is very sensitive to even small quantities of reducing sugars 0. A false-positive reaction for urine sample may be obtained due to the presence of reducing substances like uric acid, ascorbic acid or other drugs like levodopa. Iodine test for starch Definition Iodine test, also known as a starch-iodine test, is a chemical test used to distinguish mono- or disaccharides from specific polysaccharides like amylase, dextrin, and glycogen.
Objectives To detect the presence of polysaccharide, primarily starch. Principle Iodine test is based on the fact that polyiodide ions form colored adsorption complex with helical chains of glucose residue of amylase blue-black , dextrin black , or glycogen reddish-brown. Monosaccharides, disaccharides, and branched polysaccharides like cellulose remain colorless. Amylopectin produces an orange-yellow hue.
Iodine on its own is insoluble in water. Addition of potassium iodine results in a reversible reaction of the iodine ion with iodine to form triiodide ion, which further reacts with an iodine molecule to form pentaiodide ion.
Bench iodine solution appears brown, whereas, the iodide, triiodide, and pentaiodide ion are colorless. It is observed that the helix coil or spring structure of the glucose chain is the key to this test. Further, the resulting color depends on the length of the glucose chains.
The triiodide and pentaiodide ions formed are linear and slip inside the helix structure. It is believed that transfer of charge between the helix and the polyiodide ions results in changes in the spacing of the energy levels, which can absorb visible light, giving the complex its color.
The intensity of the color decreases with the increase in temperature and the presence of water-miscible organic compounds like ethanol. On heating, the blue color amylase-iodine complex dissociates but is formed again on cooling because the helical structure is disrupted; thereby amylose loses its iodine binding capacity and the blue color. The blue color reappears on cooling due to the recovery of iodine binding capacity due to the regaining of the helical structure. Test sample.
Benedict’s Test- Objectives, Principle, Procedure, Results
The test tube is placed over the boiling water bath for minutes can be heated directly over flame. Observe for color change in the solution of test tubes or precipitate formation. If it changes color to yellow, then 0. If it changes to orange, then it means that 1 to 1.
Benedict Solution 3%, 500mL.
If the color changes to red, then 1. And if the color changes to brick red, it means that more than 2 percent of sugar is present in the solution.
Reducing sugars present. The red copper I oxide formed is insoluble in water and is precipitated out of solution.
This accounts for the precipitate formed. As the concentration of reducing sugar increases, the nearer the final colour is to brick-red and the greater the precipitate formed. Sometimes a brick red solid, copper oxide, precipitates out of the solution and collects at the bottom of the test tube.
Sodium carbonate provides the alkaline conditions which are required for the redox reaction. Sodium citrate complexes with the copper II ions so that they do not deteriorate to copper I ions during storage. Table sugar disaccharide is a non-reducing sugar and does also not react with the iodine or with the Benedict Reagent. Sugar needs to be decomposed into its components glucose and fructose then the glucose test would be positive but the starch test would still be negative.
2.1.2 The Benedict's Test
If the anomeric carbon consists of aldehyde as a functional group, the sugar is aldose and if ketone is attached, the sugar is ketose. Carbohydrates or sugars can be classified as either reducing or non-reducing on the basis of their reducing property. A sugar is said to be reducing if its anomeric carbon consists of a free aldehyde or ketone group.
In case of disaccharides, some are reducing while others are non-reducing. If anomeric carbon of both the monomers is involved in glycosidic bond, the disaccharide becomes non-reducing.