Swot Analysis

Course: Principles of Biochemistry I & Biochemistry I Class: BS- Bioinformatics & BS-Biosciences Lab Instructor: Ms. Sadia Jabeen Lab Protocol #

CARBOHYDRATE ANALYSIS
INTRODUCTION:

* Organic compounds are the major components of cells and tissues. They provide energy for life processes, participate in and regulate metabolic reactions, and transmit information. * Organic macromolecules in living organisms can be classified as carbohydrates, proteins, lipids, or nucleic acids etc.
CARBOHYDRATE CHEMISTRY: * A Carbohydrate is an organic compound with the general formula Cn (H2O) n that consists of only carbon, hydrogen and oxygen(CHO), with the carbon, hydrogen and oxygen in the 1:2:1 atom ratio.

* Carbohydrates consist of three main types such as sugars, starches, and celluloses.

* Where sugars and starches serve as energy sources for cells; celluloses are structural components of the walls that surround plant cells.

* The carbohydrates (saccharides/sugars) are divided into four chemical groups: monosaccharide, disaccharides, oligosaccharides and polysaccharides.

* Monosaccharides include Glucose, Fructose, and Galactose etc.

* Disaccharides include Sucrose, Maltose, and Lactose etc.

* Polysaccharides include Starch, Glycogen, Cellulose and Chitin.

* Aldehydes (–CHO) and ketones (= CO) are active groups or functional groups in carbohydrates. Carbohydrates contain many hydroxyl groups as well

* Monosaccharidecontain the free aldehyde or ketone group. Some disaccharides have the free aldehyde group (maltose) and some do not have the free ones (sucrose). The polysaccharides, starch and cellulose, are polymers of monosaccharide linked through theactive groups.

* The chemical properties of Carbohydrates or saccharides vary depending upon the number of hydroxyl groups and the presence or absence of functional groups. These variations are the basis in the development of color reactions to identify the saccharides.

TESTS ON CARBOHYDRATES: * Carbohydrates can be analyzed both qualitatively and quantitatively.

* Regarding QUALITATIVE Carbohydrate testing some simple tests are used to identify the presence/absence of certain carbohydrates or sugars are as under: 1. MOLISCH’S Test 2. BENEDICT’S Test 3. BARFOED’S Test 4. SELIWANOFF’S Test& 5. IODINE Test
GENERAL PROCEDURE: 1. Take five sets of test tubes having 6 test tubes in each of the set. 2. Now take given samples of Glucose, Fructose, Lactose, Maltose and Starch. Each sample solution is of 5% w/v concentration. 3. Also add water in one test tube from each set of test tube which acts as a negative control. 4. Then by using Reagents (Molisch, Benedict’s, Barfoed’s, Seliwanoff’s, and Iodine reagent)one in each set of test tubes serially observe the results.

CARBOHYDRATE ANALYSIS
(QUALITATIVE)
Qualitative carbohydrate analysis involves the detection of various carbohydrates through the following tests. i.e; 1. MOLISCH TEST:
Principle:
Sugars undergo dehydration in the presence of non-oxidizing acids like hydrochloric acid (HCl) and sulphuric acid (H2SO4) to form furfural or hydroxyl methyl furfural. This chemical compound can react with aromatic amines such as ᾳ-naphthol or phenol to give intensely colored compounds.
APPLICATION/USE:
This reaction forms a basis of a general qualitative test for sugar. Or in other words, to detect the presence of carbohydrate in the given solution Molisch’s test is used.
It gives a positive result for all carbohydrates whether free or bound.
REAGENTS:
i. 5 % ᾳ-naphthol in ethanol ii. Conc. Sulphuric acid (H2SO4)
TEST PROCEDURE:
Take 1st set of test tubes and add 2 ml of the given solutions (i.e., Glucose, Fructose, Maltose, Lactose, Sucrose and Starch) in each of the 6 test tubes. Then add 2 drops of an ethanolic solution of naphthol. Carefully run down 1 ml of Conc. Sulphuric acid (H2SO4) along the sides of the tubes and observe the result.
INFERENCE:
Formation of a bluish violet colored ring at the junction of two liquids or development of violet color throughout the solution shows the presence of carbohydrate.
No ring appears in test tube containing distilled water as Blank sample (B).

2. BENEDICT’S TEST:
Principle:
When the reducing sugar is boiled with Benedict’s reagent which is an alkaline solution of cupric sulphate, the blue colored cupric sulphate is reduced gradually to form insoluble cuprous oxide which may be green, yellow, orange or red in color (depending upon the concentration of sugar in the solution).
APPLICATION/USE:
This test is used for the detection of reducing sugars or carbohydrates.
All monosaccharide are reducing sugars but some of the disaccharides are the reducing sugars (e.g., lactose and maltose).
REAGENTS:
i. Sodium citrate ii. Sodium carbonate & iii. Copper sulphate
TEST PROCEDURE: i. Take 2nd set of test tubes and add 1 ml of the Benedict’s reagent solution in each of the six test tubes. ii. Followed by the addition of 1 ml of 5% given solutions (i.e., Glucose, Fructose, Maltose, Lactose and Starch) in each of the 6 test tubes. iii. Then boil for 5 min and allow cooling. iv. And observe the result. INFERENCE: Appearance of Green, Yellow, Orange, or Red ppt indicates the presence of reducing sugar in the given solutions. 3. BARFOED’S TEST: Principle: This test is used to distinguish between monosaccharide from disaccharides as the monosaccharides immediately give cuprous oxide red ppt on heating (for 1-2 min) with Barfoed’s reagent. APPLICATION/USE: The Barfoed’s test is for the determination of reducing sugars Monosaccharide/ Disaccharides. TEST PROCEDURE: i. Take 3rdset of test tubes and add 1 ml of theBarfoed’s reagent solution in each of the six test tubes. ii. Followed by the addition of 1 ml of 5% given solutions (i.e., Glucose, Fructose, Maltose, Lactose and Starch) in each of the 6 test tubes. iii. Then heat for 30 sec and allow standing for 10 min. iv. Then add 5 drops of HCl and heat for 5 min and observe. INFERENCE: i. If Red pptappearsat the bottom of the test tube it indicates that the carbohydrate under test is monosaccharide. ii. If Red pptappears after 15 min of heating, it indicates that the carbohydrate under test is Disaccharide. 4. SELIWANOFF’S TEST: Principle: Ketohexoses like fructose form hydroxyl methyl furfural derivatives with hydrochloric acid (HCl)and form cherry red colored compoundwith resorcinol present in seliwanoff’s reagent. APPLICATION /USE: It is a test for detection of ketonic group (as functional group) of carbohydrates e.g., fructose. REAGENTS: i. Resorcinol ii. Dilute HCl (Hydrochloric Acid)-37% (4M) TEST PROCEDURE: i. Take 4thset of test tubes and add 1 ml of theSeliwanoff’s’s reagent solution in each of the six test tubes. ii. Then add 1 ml of 5% given solutions (i.e., Glucose, Fructose, Maltose, Lactose and Starch) in each of the 6 test tubes. iii. Then boil in a water bath until thedevelopment of required color. iv. Then note down the time required to develop that color in each test tube. INFERENCE: i. If blue green/ peach colored product develops it indicates Aldose sugar. ii. If pink/cherry red colored product is formed then it shows the presence of Ketose sugar. 5. IODINE TEST: Principle: Iodine forms blue colored complex with 1, 4glycosidic linkages present in polysaccharides such as starch and glycogen. APPLICATION/USE: This test is for the detection of polysaccharides such as starch and glycogen. REAGENTS: i. Iodine ii. Potassium iodide TEST PROCEDURE: i. Take 5th set of test tubes and add 1 ml of 5% given solutions (i.e., Glucose, Fructose, Maltose, Lactose and Starch) in each of the 6 test tubes. ii. Then add 2 drops of dilute aqueous solutionof iodine in potassium iodide. iii. Heat to boiling and observe. iv. Then allow to cool at room temperature v. Add few drops of sodium thiosulphateand observe. INFERENCE: i. Appearance of blue-black complex indicates starch. ii. ------------------------------------------------- Appearance of brown color indicates glycogen.

RESULTS: 1. MOLISCH’s TEST: SAMPLE USED ͢→ | Glucose | Fructose | Maltose | Lactose | Starch | Result | Positive | Positive | Positive | Positive | Positive | PRODUCT COLOR → | Bluish violet ring at the junction of two liquids | Bluish violet ring at the junction of two liquids | Bluish violet ring at the junction of two liquids | Bluish violet ring at the junction of two liquids | Bluish violet ring at the junction of two liquids |

2. BENEDICT’S TEST: SAMPLE USED ͢→ | Glucose | Fructose | Maltose | Lactose | Starch | Result | Positive | Positive | Positive | Positive | Negative | PRODUCT COLOR → | Red product (Reducing Sugar) | Red product (Reducing Sugar) | Red product (Reducing Sugar) | Red product (Reducing Sugar) | |

3. BARFOED’S TEST: SAMPLE USED ͢→ | Glucose | Fructose | Maltose | Lactose | Starch | Result | Positive | Positive | | | | PRODUCT COLOR → | | | | | | Red precipitation (within 2-3 min), reducing monosaccharide | // | // | | | | Red precipitation (after 10-15 min), reducing Disaccharide | | | | | |

4. SELIWANOFF’S TEST: SAMPLE USED ͢→ | Glucose | Fructose | Maltose | Lactose | Starch | Result | Positive | Positive | | | | PRODUCT COLOR → | | | | | | Blue-green/ Peach product (Aldose) | // | | | | | Cherry redproduct (Ketose) | | // | | | |

5. IODINE TEST: SAMPLE USED ͢→ | Glucose | Fructose | Maltose | Lactose | Starch | Result | Negative | Negative | Negative | Negative | Positive | PRODUCT COLOR → | | | | | Blue-black complex |