B. COMPARATIVE REACTIONS OF CARBOHYDRATES B.1 FEHLING’S FEHLING’S AND BENEDICT’S TESTS B.1.1 TEST SUGARS
Objectives: To detect the presence of reducing sugars through a visible po sitive result of
formation of brick red precipitate from each sample.
Presentation of Results:
* Color of reagent: Blue
Samples
Observations
Glucose
Red precipitate
Sucrose
Blue solution
Lactose
Red precipitate
Fructose
Red precipitate
Discussion/Interpretation Discussion/Interpret ation of Results: BENEDICT’S TEST and FEHLING’S TEST a) What is the principle involved in Benedict’s Test and Fehling’s test? test ?
The principle involved in the Benedict’s test is REDOX/Oxidation reaction. REDOX/Oxidation reaction. b) What is the purpose of the tests?
This is a test to detect reducing sugars in a solution
c) What components make up the Fehling’s reagent? The Benedict’s reagent? State the function of each component. Fehling's solution contains copper(II) ions complexed with tartrate ions in sodium hydroxide solution. Complexing the copper(II) ions with tartrate ions prevents precipitation of copper(II) hydroxide. Benedict's solution contains copper(II) ions complexed with citrate ions in sodium carbonate solution. Again, complexing the copper(II) ions prevents the formation of a precipitate - this time of copper(II) carbonate.
d) Provide the type of equation used in the tests
Would be responsible for the brick red precipitate
e) Show the sample equation involved in the reaction between Fehling’s/Benedict’s reagent and your sample/s which gave a positive result.
Samples
Observations
Glucose
Red- Orange precipitate
Sucrose
Blue solution
GLUCOSE
FRUCTOSE
LACTOSE
Lactose
Red – orange precipitate
Fructose
Red – orange precipitate
f) Provide a possible explanation for: (f.1)Positive results obtained with your sample/s
a. Glucose
Benedict solution is light blue because it contains copper sulfate. When it is mixed and heated with a sugar, such as glucose, which has electrons available to donate, the copper will accept the electrons and become reduced, which turns it to brick red. During this process, the blue copper (II) ion is reduced to a red copper (I) ion. While the copper is being reduced, the glucose gives up an electron and is oxidized. Because glucose is able to reduce the copper in Benedict solution, we call it a reducing sugar.
b. Fructose
Fructose is a 6-carbon polyhydroxy ketone. It is an isomer of glucose, therefore the y both have the same molecular formula (C6H12O6) but they differ structurally.
These Disaccharide sugar contains two sugars that contain both aldehyde and a ketone as their first carbon. The ketone here undergoes Tautomerism which means that the ketone functional group hydrolyzes first into an aldehyde and formed aldose. This makes the possibility to react the
sugar in the Benedict’s reagent. And this propert y only happens to an alpha – hydroxy ketone. Lactose
Lactose will yield to a positive result because it is also a reducing sugar even though it comes from the binding of a galactose and glucose, that makes it a disaccharide. And the Benedict’s test is a test for reducing sugars of monosaccharides and some of disaccharides.
(f.2)Negative results obtained with your sample/s Sucrose
Sucrose is a disaccharide derived from glucose and fructose. It has a molecular formula of C12H22O11.
Glycosidic linkage between C1 of glucose and C2 of fructose
Because of glycosidic linkage, there is no presence of free aldehyde, which is a property of a reducing sugar. This makes sucrose a non-reducing sugar, thus having no reaction with the Benedict’s reagent. However, a non-reducing sugar can be hydrolyzed using dilute hydrochloric acid to convert the acetal or ketal into a hemiacetal or hemiketal. After hydrolysis and neutralization of the acid, the product may be a reducing sugar that gives normal reactions with the test solutions.
g) Why is there a need to freshly prepare Fehling’s reagent?
Fehling's solution is always prepared fresh in the laboratory. It is made initially as two separate solutions, known as Fehling's A and Fehling's B. Fehling's A is a blue aqueous solution of copper (II) sulphate pentahydrate crystals, while Fehling's B is a clear solution of aqueous potassium sodium tartrate (also known as Rochelle salt) and a strong alkali (commonly sodium hydroxide).
h) Why is Fehling’s test not used for the analysis of carbohydrates in clinical samples?
Because Fehling’s reagent is, unstable, is only a q ualitative test, auto reduction of cupric hydroxide occur resulting in false positive test. i) State other advantages of Benedict’s reagent over Fehling’s reagent
Results: URINE SAMPLES: j) Differentiate between the 3 kinds of urine samples: fasting, post-prandial and random. Which sample is the MOST suitable for use in a clinical laboratory test? Explain your choice.
Fasting sample – 12hour urine without any food/drink ingestion.
Random – just simple urine. Without any done actions. Post-prandial - The patient is given glucose water in a much diluted quantity and a
blood test is conducted after two to two and a half hours.
The fasting urine is the most suitable because it is the most natural urine that we excrete and it is not yet mix with the chemicals etc. that we ingest.
k) What is Diabetes Mellitus? Differentiate between Type 1 and Type 2 diabetes. Is it possible to have sugar diabetes even if no member of your family has it? Explain your answer
Diabetes mellitus is a kind of metabolic disease that is brought about by either the insufficient production of insulin or the inability of the body to respond to the insulin formed within the system.
What is diabetes mellitus type 1?
Diabetes mellitus type 1 is caused by the loss of beta cells found in the islets of Langerhans in the pancreas.
Beta cells primarily produce insulin and hence, their loss will lead to huge insulin deficiencies in the body.
In most cases, the loss of the beta cells is caused by an autoimmune attack mediated by the body’s own T-cells.
Type 1 diabetes mellitus makes up 10% of all the diabetes mellitus cases in Europe and in North America.
Up until today, there have been no preventive measures found for disease. Most of the patients who acquire this type of diabetes are found in the younger populations. Because of this, Type 1 diabetes mellitus is also no w known as the juvenile diabetes.
What is diabetes mellitus type 2?
Another type of diabetes mellitus is the type 2 diabetes. Type 2 diabetes is generally characterized by the body’s resistance to insulin. This is primarily attributed to the loss of certain insulin receptors in the tissues that are supposed to mediate the entrance of insulin into the body’s cells.
This particular kind of diabetes is the most common kind that afflicts most of the reported cases of the disease. Type 2 diabetes usually leads to hyperglycemia which can be treated by a series of medications that can either improve the body’s insulin sensitivity or improve the liver’s production of insulin in the system.
The last major category of diabetes mellitus is the gestational diabetes. This type of diabetes commonly occurs in pregnant women and may disappear or even progress right after delivery. Gestational diabetes generally manifests the same kind of complication found in type 2 diabetes.
Just like in type 2 diabetes, the body also exhibits a resistance to insulin and can sometimes produce insufficient amounts of the substance as well. This kind of diabetes is fully treatable but may require very close monitoring.
And it is still possible to have a sugar diabetes even is not inherited. DM is also acquired when we’re engaging in a poor lifestyle and abandoned our self in eating too much sweets.
l) Is one considered a diabetic if one obtains a positive result for sugar in a fasting urine sample? Explain your answer.
No.. Because only the amylase activity is the one that is tested on the urine sample. The test is not that accurate. Maybe the test only reacts with the reducing sugar. And not with the other sugar. Sugar can appear in the urine even if blood sugar isn't high. Also, sugar in the urine doesn't reveal how giant blood sugar is. Sugar in the urine can tip a doctor off that you may have diabetes, but it's a really outdated method of detecting and monitoring diabetes.
C. Specific Tests C.1 BARFOED’S TEST: Objective: To detect the presence of reducing sugars through a visible p ositive
result of formation of brick red precipitate from each samples.
Presentation of Results:
Samples
Observations
Glucose
Red precipitate
Sucrose
Blue solution
Lactose
Red precipitate
Fructose
Red precipitate
Discussion/Interpretation of Results: a) What is the principle involved in the Barfoed’s test?
Oxidation b) What is the purpose of the test?
It is used for detecting the presence of monosaccharides. It is based on the reduction of copper(II) acetate to copper(I) oxide (Cu2O), which forms a brick-red precipitate The Barfoeds test will allow us to make a distinction between reducing monosaccharides and reducing disaccharides. It is a test used to differentiate between monosaccharides and disaccha rides. This reaction will detect reducing monosaccharides in the presence of disaccharides. This reagent uses copper ions to detect reducing sugars in an acidic solution. Barfoed's reagent is copper acetate in dilute acetic acid (pH 4.6) c) What components make up the barfoed’s reagent? State the function of each component?
Barfoed’s test uses copper(II) ions in a slightly acidic medium. (an acid media) which delays the reaction of disaccharide. It is based on the reduction of copper (!!) acetate to copper (I) oxide which forms into a brick-red precipitate.
d) Provide the type of equation used in the test?
`
Would be responsible for the brick red ppt.
e) Show your sample equation involved in the reaction between Barfoed’s reagent and your sample/s which gave a positive result
Samples
Observations
Glucose
Brick red precipitate in blue solution
Sucrose
Blue solution
Lactose
Blue solution
Fructose
Brick red precipitate
f) Provide a possible explanation for: (f.1) positive results obtained with your samples
Glucose & Fructose: We all know that glucose and fructose are monosaccharides and in this specific test. Barfoed’s test reacts faster in monosaccharides.
(f.2) negative results obtained with your samples
Sucrose = Sucrose is a not reducing sugar, so that a positive Barfoed test can only be
obtained, when impurities of reducing sugars are present (glucose o re inverted sugar). Lactose is a disaccharides
Disaccharides are less powerful oxidizing agents alkaline medium in Barfoed’s reagent do not favour oxidation reactions so reducing monosaccharide (glucose, fructose) give positive while disaccharides give negative to Barfoed’s test
g) Why is the heating of the mixture limited to 3 minutes only?
If heated long enough (10-15 minutes) disaccharides will give a positive result due to hydrolysis to form monosaccharides. When boiled in water, monosaccharides will react in under five minutes, while disaccharides will take longer.
h) How does this test compare with Fehling’s and Benedict’s tests? Which is more accurate?
Fehling’s and Benedict’s tests are test to detect the presence of reducing sugars. While Barfoed’s test is a specific test, which means that it will only test positive for monosaccharides. Barfoed’s is similar to Fehling’s/ Benedict’s test, except that in Barfoed’s test, different types of sugars react at different rates. Barfoed’s reagent is much milder than Fehling’s / Benedict’s reagent. Reducing monosaccharides react quickl y with Barfoed’s reagent, but reducing disaccharides react very slowly or not at all. Therefore, it is possible to distinguish between a reducing monosaccharide and a reducing disaccharide using Barfoed’s reagent. Therefore, Barfoed’s test is more accurate.
