LAB 9 - TESTS FOR CARBOHYDRATES
- Page ID
- 506286
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)CHARACTERIZATION AND CLASSIFICATION OF CARBOHYDRATES
The purpose of this experiment is to:
- To observe the physical and chemical properties of carbohydrates.
- To recognize and classify carbohydrates as monosaccharides, disaccharides, or polysaccharides.
- To identify the type of carbohydrate present in an unknown sample using various chemical tests.
INTRODUCTION
Carbohydrates are fundamental organic compounds composed of carbon, hydrogen, and oxygen, typically in a ratio where the number of hydrogen atoms is twice that of oxygen atoms. They are polyhydroxy aldehydes and ketones or substances that hydrolyze to yield polyhydroxy aldehydes and ketones. Aldehydes (–CHO) and ketones (–C=O) constitute the major groups in carbohydrates. Their general chemical formula is Cn(H2O)n, reflecting a hydrogen-to-oxygen ratio of approximately 2:1. Carbohydrate molecules are a primary source of energy for most living organisms. Carbohydrates that contain free aldehyde or ketone groups can function as reducing agents in chemical reactions. Due to the presence of chiral carbon atoms, many carbohydrates exhibit optical activity, meaning they can rotate the plane of polarized light. Most simple carbohydrates dissolve readily in water because they can form hydrogen bonds; however, as their molecular size increases, especially in polysaccharides, their solubility tends to decrease due to aggregation and structural complexity.
Carbohydrates are classified into three main categories based on the number of sugar units they contain:
- Monosaccharides are the simplest carbohydrates that consist of a single sugar molecule that cannot be broken down further into smaller carbohydrates. They are usually sweet-tasting and water-soluble. The naturally occurring monosaccharides contain three to seven carbon atoms per molecule. Monosaccharides are reducing sugars and can participate in reactions like Benedict’s test. They serve as a primary energy source and building blocks for larger carbohydrates. Examples include glucose, fructose, and galactose.
- Disaccharides are created when two monosaccharide units undergo condensation, forming a glycosidic bond. They can be broken down back into monosaccharides by hydrolysis of the glycosidic bond with water. Some, like sucrose, are non-reducing sugars. They provide a quick energy source and participate in various metabolic processes. Common examples include sucrose (made of glucose and fructose), lactose (composed of glucose and galactose), and maltose (consisting of two glucose molecules).
- Polysaccharides are long chains of monosaccharide units linked together by glycosidic bonds. They are generally insoluble in water or only partially soluble in water and do not have a sweet taste. They can be branched or unbranched and serve as energy reserves ( storage - starch, glycogen) and structural materials (cellulose) in plants and animals. Polysaccharides are considered complex carbohydrates, known as higher saccharides, and can be hydrolyzed back into their constituent monosaccharides. Examples include starch, glycogen, and cellulose.
This lab uses a series of qualitative tests to determine the presence and types of carbohydrates in both known and unknown samples. Each test focuses on specific structural features of carbohydrates, such as aldehyde or ketone groups, reducing properties, or the formation of polymer chains.
CHEMICAL TESTS FOR IDENTIFICATION OR CLASSIFICATION OF CARBOHYDRATES
For a carbohydrate to be a reducing sugar, the molecule must contain a free anomeric carbon, since it is the open-chain form of the aldehyde that can react (and be oxidized). Reducing sugar can be oxidized.
Molisch’s Test
This is a common test for all carbohydrates larger than tetroses. The test is based on the premise that pentoses and hexoses are dehydrated by concentrated acid. Sulfuric acid to form furfural or hydroxymethylfurfural, respectively. These products condense with α-naphthol to form purple condensation products.
Benedict’s Test
A free aldehyde or keto group in the reducing sugars reduces cupric hydroxide in an alkaline medium to red colored cuprous oxide. Depending on the concentration of sugars, a yellow to green color is developed. All monosaccharides are reducing sugars because they all have a free, reactive carbonyl group. Some disaccharides, like maltose, have exposed carbonyl groups and are also reducing sugars, but less reactive than monosaccharides
Fehling's Test
One test for reducing sugars involves Fehling’s reagent, which contains Cu2+ ions in an aqueous basic solution. If a reducing agent is present, the Cu2+ is reduced to Cu+ and forms a red precipitate of Cu2O. Therefore, if Fehling’s solution is added to a solution containing a reducing sugar, a red precipitate will form. Sometimes, the reaction mixture must be heated to facilitate the formation of the precipitate. The color of the precipitate can vary from red to orange to green (the green color is a mixture of orange and blue precipitate).
Barfoed’s Test
This test is similar to Fehling’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 reagent. Reducing monosaccharides react quickly with Barfoed’s reagent, but reducing disaccharides respond very slowly or not at all. Therefore, it is possible to distinguish between a reducing monosaccharide and a reducing disaccharide using Barfoed’s reagent. A positive test result in a dark red precipitate, indicating the presence of a reducing monosaccharide.
Seliwanoff’s Test
This test involves a dehydration reaction. Seliwanoff’s reagent contains non-oxidizing acid (HCl) and resorcinol. When a ketose (a sugar with a ketone group) is reacted with this reagent, it becomes dehydrated, forming a cherry-red complex (not a precipitate). Aldoses (sugars with an aldehyde group) also react with this reagent, but much more slowly than ketoses. When Seliwanoff’s reagent is reacted with a disaccharide or a polysaccharide, the acid in the solution will first hydrolyze them into monosaccharides, and the resulting monosaccharides can then be dehydrated. Disaccharides and polysaccharides will therefore react slowly with Seliwanoff’s reagent. When conducting this test, it is essential to note the time required for a reaction to occur.
Bial’s Test
Bial’s test is used to distinguish between pentoses and hexoses. They react with Bial’s reagent and are converted to furfural. Orcinol and furfural condense in the presence of ferric ions to form a colored product. Appearance of green color or precipitate indicates the presence of pentoses, and formation of muddy brown precipitate shows the presence of hexoses.
Iodine Test
Iodine forms a blue, black, or gray complex with starch and is used as a test for the presence of starch in experimental samples. The color of the complex formed depends on the structure of the polysaccharide and the strength and age of the iodine solution. Iodine does not form a complex with simpler carbohydrates (monosaccharides and disaccharides). Amylose (starch) is helically coiled in solution, and it is this helical structure that is necessary to form the blue complex with iodine. Monosaccharides and disaccharides are too small to be helically coiled. Amylopectin, cellulose, and glycogen form different colors with iodine – red, brown, or purple.
Osazone Test
Ketoses and aldoses react with phenylhydrazine to produce a phenylhydrazone, which then reacts with two additional molecules of phenylhydrazine to yield an osazone. Different osazones will show crystals of various shapes. Needle-shaped yellow osazone crystals are made by glucose, fructose, and mannose, whereas lactosazone produces mushroom-shaped crystals. Flower-shaped crystals are produced by maltose
Fermentation
Many carbohydrates can undergo fermentation in the presence of yeast. The carbohydrate is the food source for the yeast, and the products of the fermentation reaction are ethanol and carbon dioxide.
C6H12O6 → 2 CH3CH2OH + 2 CO2 (g)
Glucose Ethanol
Fermentation is used in the processes of making beer and wine, where the alcohol produced by the yeast is the desired product. Not all sugars, however, can be used by yeast as a food source. You will test which sugars ferment in the presence of yeast and which ones do not. The evidence of fermentation will be the evolution of carbon dioxide gas. In the test, a quantity of the solution (containing yeast and the sugar to be tested) will be trapped in a small inverted test tube. After a few days, you will check to see if a gas bubble has formed in the test tube. If a gas bubble is present, it indicates that fermentation has occurred.
Hydrolysis of Carbohydrates
Disaccharides and polysaccharides can be hydrolyzed in the presence of acid or specific enzymes. When a disaccharide is hydrolyzed, the products are the individual monosaccharides. When a polysaccharide is hydrolyzed, the products depend on the reaction time, acid or enzyme concentration, and other factors. Polysaccharides are very long and have many glycosidic bonds that can be hydrolyzed. They cannot all be hydrolyzed at the same time, so the product is a mixture of dextrin, maltose, and glucose. If a polysaccharide sample is hydrolyzed completely (which means that it must react for a while), the product is glucose. In this experiment, you will hydrolyze a sample of sucrose and then test it for the presence of reducing sugar. You will also hydrolyze a sample of starch and then test it for the presence of both reducing sugar and starch. A summary of the above test is presented in the table below.
SUMMARY OF CARBOHYDRATE TESTS
Test |
Detects |
Principle |
|---|---|---|
Molisch’s Test |
General test for all carbohydrates |
Carbohydrates are dehydrated by concentrated sulfuric acid to form furfural or hydroxymethylfurfural, which reacts with Molisch Reagent (α-naphthol) to form a violet ring. |
Barfoed’s Test |
Monosaccharides |
Monosaccharides reduce copper(II) acetate in acidic medium to form a red precipitate of copper(I) oxide. |
Benedict’s Test |
Reducing sugars |
Reducing sugars donate electrons to copper(II) ions in an alkaline solution, forming a colored precipitate of copper(I) oxide. |
Fehling’s Test |
Reducing sugars |
Uses a different copper complex to detect reducing sugars by forming a red precipitate. |
Seliwanoff’s Test |
Ketoses |
Ketoses are dehydrated more rapidly than aldoses to form furfural derivatives, which react with resorcinol to produce a cherry-red color. |
Bial’s Test |
Pentoses |
Pentoses form furfural when heated with HCl, which reacts with orcinol and ferric chloride to give a blue-green color. |
Iodine Test |
Starch and polysaccharides |
Iodine forms a blue-black complex with the helical structure of starch. |
Osazone Test |
Reducing sugars |
Reducing sugars react with phenylhydrazine to form characteristic osazone crystals. |
Fermentation Test |
Fermentable sugars |
Yeast ferments sugars to produce ethanol and carbon dioxide. |
Hydrolysis Test |
Disaccharides and polysaccharides |
Hydrolysis breaks glycosidic bonds, converting non-reducing sugars into reducing sugars. |
Food Testing |
Carbohydrates in food |
Combines Molisch’s, Benedict’s, and Iodine tests to identify carbohydrates in food. |
EQUIPMENT AND CHEMICALS REQUIRED
| Equipment | Equipment | Chemicals | Chemicals | Chemicals | Chemicals |
|---|---|---|---|---|---|
| Eight Test tubes (Small and large) | 600 mL Beaker for water bath | 2% Glucose solution | Yeast suspension or Baker’s yeast | Fehling’s A and B solutions | Barfoed’s reagent |
| Test Tube Rack | Hot Plate | 2% Fructose solution | Bial’s reagent | Iodine solution | Seliwanoff’s reagent |
| Bunsen Burner | Microscope | 2% Sucrose solution | Osazone Reagent | Sodium hydroxide | Glacial Acetic Acid |
| Filter Paper | Dropper | 2% Starch solution | Molisch’s reagent | Concentrated sulfuric acid | Three food extract samples |
| Pipettes | Glass rod | An unknown 2% carbohydrate sample | Benedict’s reagent | Hydrochloric acid |
- Always wear safety goggles.
- Handle concentrated acids (H₂SO₄, HCl) with extreme care.
- Use a fume hood when working with volatile or corrosive reagents.
- Dispose of chemical waste as per your institution’s guidelines.
- Do not ingest any chemicals or food samples used in the lab.
- Use test tube holders when heating substances.
- Point test tubes away from yourself and others when heating.
- Know the location of emergency equipment (eyewash, fire extinguisher, etc.).
- Report all accidents or spills to the instructor immediately.
EXPERIMENTAL PROCEDURE
Take eight medium test tubes and label them 1 through 8. For each test, you will use a new sample set. For each sample set, you will add 10 drops of each sample to test tubes: Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and an Unknown Food Carbohydrate. (Only one type in each tube.). Record your results in the data table sheet below.
1. Molisch’s Test (General Test for Carbohydrates)
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Add two drops of Molisch’s reagent to each test tube.
- Carefully add 1 mL of concentrated H₂SO₄ down the side of the test tube.
- Observe the formation of a violet ring at the interface that is the junction of two liquids, which indicates the presence of Carbohydrates
2. Barfoed’s Test (Test for Monosaccharides)
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Add 2 mL of Barfoed’s reagent to each sample, mix well.
- Boil for 1 to 2 minutes in a boiling water bath. Remove the test tubes and allow them to stand for a few minutes.
- Observe for red precipitate (indicates monosaccharides).
3. Benedict’s Test (Test for Reducing Sugars)
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Add 2 mL of Benedict’s reagent to each sample and mix well.
- Heat in a boiling water bath for 5 minutes. Observe color change (green to brick red indicates reducing sugars).
- Cool the solution. Observe the color change from green, yellow, orange, or red, depending on the amount of reducing sugar present in the test sample.
4. Fehling’s Test (Test for Reducing Sugars)
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Mix equal parts of Fehling’s A and B. Add 2 mL of this mixture to each of the samples listed above.
- Heat test tubes in a boiling water bath for a few minutes. When the contents of the test tube come to a boil, mix them well with the stirring rod. (Make sure to clean the stir rod between mixing the test tubes.)
- Observe any change in color or precipitation.
- The production of a yellow or brownish-red precipitate of cuprous oxide indicates the presence of reducing sugars in the given sample.
5. Seliwanoff’s Test (Test for Ketoses)
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Add 2 mL of Seliwanoff’s reagent to each of the test tubes of the samples.
- The mixture should be heated to just boiling in the boiling water bath.
- A cherry red condensation product will be observed in the presence of ketoses in the test sample.
- There will be no significant change in color produced for aldose sugar.
6. Bial’s Test (Test for Pentoses)
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Add 2 mL of Bial’s reagent to each of the test tubes of the samples.
- Heat gently in the hot water bath for 2 minutes.
- The observation of a blue-green colored product indicates the presence of pentoses.
- Hexoses generally react to muddy brown products.
7. Iodine Test (Test for Starch)
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Add two drops of iodine solution to each of the test tubes of the samples.
- A blue-black color is observed in the samples containing polysaccharides.
8. Osazone Test (Crystallization Test)
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Mix 2 mL of the Osazone mixture with two drops of glacial acetic acid in the test tubes containing the samples.
- Heat the mixture in a boiling water bath for 15 -20 minutes.
- Cool the mixture and observe crystal formation under a microscope.
9. Fermentation Test
- Fill the fermentation tubes with the eight sample solutions: Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and an Unknown Carbohydrate Food.
- If fermentation tubes are not available, add the sample solutions to eight large test tubes, ensuring they are at least two centimeters above the height of the test tube.
- Add 0.2 g of baker's yeast or
- Place eight small test tubes upside down inside the large test tubes.
- Cover the mouth of the test tubes completely with filter paper, cardboard, or a glass slide (preferably one that covers the mouth).
- Place your hand over the cover and invert the large test tube.
- When the small test tube inside is filled with the mixture, return the large test tube to an upright position. Set the test tubes aside.
- Add yeast suspension to 5 mL of the sample in a fermentation tube.
- Incubate at 37°C for 1–2 hours.
- Observe gas formation (CO₂) indicating fermentable sugars.
10. Hydrolysis of Disaccharides and Polysaccharides
- Take eight small test tubes and add 10 drops each of Water (blank), Glucose, Fructose, Lactose, Ribose, Sucrose, Starch, and Unknown Carbohydrate Food.
- Boil 2 mL of the sample with 1 mL of dilute hydrochloric acid (HCl) for 10 minutes.
- Neutralize with NaOH.
- Perform Benedict’s test on the hydrolysate to detect reducing sugars.
- Add 0.5 mL of 3 M HCl to 5 mL of a 1 % sucrose solution in a test tube. Mix. Heat and stir the mixture in a boiling water bath for 20 minutes. (You may add deionized water to this solution if the volume starts getting low.) Cool the solution, and add 1 M NaOH until the solution tests neutral on pH paper. Transfer 8-10 drops of this solution to a small test tube. In a separate tube, mix together 1 mL of Fehling’s solution A with 1 mL of Fehling’s solution B. Add this mixture to the small test tube containing your hydrolyzed sucrose, and heat for a few minutes in a boiling water bath. Record your observations. Compare the results of this test with your results for sucrose that has not been hydrolyzed in part 2 of this experiment.
Part B. Testing Food for Carbohydrates
- Prepare extracts of food samples that you got from home (e.g., potato, bread, fruit juice, etc) or your instructor may provide you with one or more.
- Perform the above ten tests on them to identify the type of carbohydrates present.
PRE-LAB QUESTIONS
Name ____________________________________
- What are the structural differences between mono-, di-, and polysaccharides?
- Why is Benedict’s test not suitable for non-reducing sugars?
- What is the principle behind Molisch’s test?
- How does Seliwanoff’s test distinguish between aldoses and ketoses?
- What is the significance of hydrolyzing disaccharides before testing?
DATA AND OBSERVATIONS
Name _________________________Lab Partner(s) ______________________________
| Test | Water(Blank) | Glucose | Fructose | Lactose | Ribose | Sucrose | Starch | Unknown Carbohydrate |
|---|---|---|---|---|---|---|---|---|
Molisch’s TestObservation:Inference: |
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Barfoed’s TestObservation:Inference |
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Benedict’s TestObservation:Inference |
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Fehling’s TestObservation:Inference |
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Seliwanoff’s TestObservation:Inference |
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Bial’s TestObservation:Inference |
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Iodine TestObservation:Inference: |
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Osazone TestObservation:Inference: |
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Fermentation TestObservation:Inference: |
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Hydrolysis TestObservation:Inference: |
| Test |
Food Extract I Name of Food: |
Food Extract II Name of Food: |
Food Extract II Name of Food: |
|---|---|---|---|
Molisch’s TestObservation:Inference: |
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Barfoed’s TestObservation:Inference: |
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Benedict’s TestObservation:Inference: |
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Fehling’s TestObservation:Inference: |
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Seliwanoff’s TestObservation:Inference: |
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Bial’s TestObservation:Inference: |
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Iodine TestObservation:Inference: |
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Osazone TestObservation:Inference: |
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Fermentation TestObservation:Inference: |
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Hydrolysis TestObservation:Inference: |
POST LAB QUESTIONS
- Based on the results, classify the unknown sample as mono-, di-, or polysaccharide.
- Compare the behavior of known and unknown samples in each test.
- Discuss any discrepancies or unexpected results.
- Reflect on the specificity and limitations of each test.
- Suggest improvements or additional tests to enhance the accuracy of identification.
Please click here to access the Pre-Lab, Data Tables, and Post-Lab in Word or PDF format. Complete them and upload the lab report according to your instructor's instructions.