Postlab: -discussion : major steps in procedure + OBSERVATIONS -results of tests, reaction (mech if possible) , explanations -postlab questions : if related

to test, siksik sa results, if not sa huli nlng V. Discussion Biomolecules consist of four classes. (to follow) First are carbohydrates. Carbohydrates are, by weight, the most widespread organic compounds on the Earth. They are typically described as polyhydroxy aldehydes and ketones or substances that hydrolyze to yield polyhydroxy aldehydes and ketones. They exist primarily in their hemiacetal or acetal forms. Those that cannot be hydrolyzed further into simpler carbohydrates are called monosaccharides. Those that yield only 2 molecules of monosaccharide upon hydrolysis are, on the other hand, termed as disaccharides; those that produce 3 molecules of monosaccharide are termed as trisaccharides; those that give a large number of molecules of monosaccharides are called polysaccharides. (Solomons, Frhyle, 2011) Another type of biomolecules is lipids. Lipids are naturally occurring organic molecules that are water-insoluble and can be extracted from organisms by the use of nonpolar organic solvents. These types of biomolecules are defined by their physical property instead of by their structure. (McMurry, 2008) There are many kinds of lipids but in this report, the focus is on triacylglycerols. Triacylglycerols are esters of glycerol (glycerin) in which all three of the hydroxyl groups are esterified. Triacylglycerols can be simple triacylglycerols in which all three acyl groups are the similar. Usually, however, the triacylglycerol is a mixed triacylglycerol in which the acyl groups are dissimilar. (Solomons, Frhyle, 2011) Another type of biomolecules is proteins. All proteins made up of several amino acids connected together in a long chain. Amino acids consist of a basic amino group and an acidic carboxyl group, hence, their name. Amino acids are linked together by forming amide bonds between the NH2 of one amino acid and the CO2H of another. For smaller chains, peptides are usually the term used, and proteins for larger chains. (McMurry, 2008) Last but not the least is nucleic acids. The nucleic acids, deoxyribonucleic acid(DNA) and ribonucleic acid(RNA), are biopolymers composed of nucleotides linked together to form a long chain. Each nucleotide is made up of a nucleoside bonded to a phosphate group, and each nucleoside is made up of an aldopentose sugar connected through its anomeric carbon to the nitrogen atom of a heterocyclic purine or pyrimidine base. (McMurry, 2008). In this experiment, biomolecules are surveyed by conducting several chemical tests on representative compounds from each class.

For carbohydrates, the following tests were conducted: 1. Molisch Test Molisch test can be used for all carbohydrates, and some compounds having carbohydrates in a combinded form. It is rather sensitive and is based on the dehydration of the carbohydrate by sulphuric acid to give an aldehyde (either furfural or a derivative), which then condenses with the phenolic structure giving a red or purple-colored compound on the interface as a positive result. All carbohydrates give a positive reaction, and nucleic acids and glycoproteins also give a positive reaction, as all these compounds are eventually hydrolyzed to monosacharides by strong mineral acids, such as sulphuric acid. Acetic acid will not work since it is not a mineral acid. A negative result only gives information that a carbohydrate is absent. In this experiment, dH2O followed by Molisch reagent( 1 napthol in 95% etOH) was added to 0.05M sugar solution in a test tube. After shaking, concentrated sulphuric acid was added slowly to the sides of the tube. Agitation was avoided. On 0.05 M glucose, lactose and sucrose, formation of layers resulted, pink on top, and orange at the bottom. On the other hand, fructose produced no layers. From these results, only fructose gave a positive result. Supposedly, all carbohydrates given should give a positive result. However, this was not the case. This might be caused by the impurity of the reagents used. The reaction involved is shown below: (reactions)

2. Benedicts Test The Benedict's test allows us to detect the presence of reducing sugars (sugars with a free aldehyde or ketone group). All monosaccharides are reducing sugars; they all have a free reactive carbonyl group. Some disaccharides have exposed carbonyl groups and are also reducing sugars. Other disaccharides such as sucrose are non-reducing sugars and will not react with Benedict's solution. Starches are also non-reducing sugars. The copper sulfate (CuSO4) present in Benedict's solution reacts with electrons from the aldehyde or ketone group of the reducing sugar to form cuprous oxide (Cu2O), a red-brown precipitate, which means a positive result. Benedicts reagent is composed of Na-citrate, sodium carbonate in water, added with a solution composed of copper sulfate and water.

The test involved adding the Benedicts reagent on the test tube containing the sugar solution. The mixture is then boiled for 10 minutes.

For glucose, fructose and lactose, a positive result was observed (formation of brick-red precipitate). Sucrose is negative. Only a blue solution was observed. As expected, glucose, fructose and lactose should give a positive result since their structures contain free aldehyde or ketone group. Since sucrose have both its anomeric carbon involved in the glycosidic bond, it does not have a free aldehyde group which could react with Benedicts reagent, hence giving the negative result.

Reactions (include sucrose para Makita ung structure)

Conversely, sucrose indirectly produces a positive result with Benedict's reagent if heated with dilute hydrochloric acid prior to the test, although after this treatment it is no longer sucrose. The acidic conditions and heat break the glycosidic bond in sucrose through hydrolysis. The products of sucrose decomposition are glucose and fructose, both of which can be detected by Benedict's reagent, as described above. 3. Seliwanovs Test Seliwanovs (Seliwanoffs) test is a chemical test which indicates the presence of keto sugars, hence it helps differentiating aldose from ketose sugars. Ketoses differ from aldoses in terms of their ketone/aldehyde functionality. This test succeeds due to the fact that ketoses dehydrate faster than aldoses when heated. The reagents consist of resorcinol and concentrated hydrochloric acid. HCl serves as a dehydrating reagent converting keto sugars to furfurals. The acid hydrolysis of polysaccharides and oligosaccharides produces simpler sugars. The dehydrated ketose then reacts with the resorcinol forming a complex to produce a deep cherry red color. Aldoses may react slightly to produce a faint pink color but takes a longer time. a yellowish straw or apricot color is not indicative of a positive test. (ANSWER POSTLAB QUESTION) The test involved addition of Seliwanovs reagent to the test tube containing glucose and test tube containing fructose. The test tubes were boiled for 5 minutes. The test tube containing glucose gave a yellow solution after heating, while fructose gave an orange solution. As mentioned above, a positive result should show a deep cherry red color. However, it was not observed on either test tube. Hypothetically, fructose should give a positive result since it is a ketose, while glucose a negative result. The observed result for fructose may be caused by the low E-ketol concentration, since the intensity of the color is proportional to the E-ketol concentration. Therefore, it can be concluded that fructose still gave a partial positive result. (reaction) 4. Barfoeds Test

Barfoed's Test is a chemical test used for detecting the presence of monosaccharides. This reaction will detect reducing monosaccharides in the presence of disaccharides. It is based on the reduction of copper(II) acetate to copper(I) oxide (Cu2O), which forms a brick-red precipitate. RCHO + 2Cu2+ + 2H2O RCOOH + Cu2O + 4H+

The aldehyde group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate.

Barfoeds reagent, Copper (II) acetate in acetic acid, is not as reactive as the Cu2+ Benedicts reagent. Hence, it can only be reduced by monosaccharides but not less powerful reducing sugars. Disaccharides may also react with this reagent, but the reaction is much slower when compared to monosaccharides. Monosaccharides produce the red precipitate in 2 to 3 minutes; disaccharides produce the precipitate in 10 minutes. Thus, one can distinguish monosaccharides from disaccharides based on how fast the redorange precipitate forms.

5. Osazone formation The aldehyde group of an aldose reacts with such carbonyl reagents as phenylhydrazine. Adequate amounts of phenylhydrazine results to the consumption of three molar equivalents of phenylhydrazine, and a second phenylhydrazone group is formed at C2. The resulting produce is termed as a phenylosazone. Phenylosazones crystallize readily (unlike sugars) and are useful derivatives for identifying sugars. The mechanism for osazone formation possibly depends on a series of reactions in which C N behaves very much like C O in giving a nitrogen version of an enol.

Mech

Osazone formation results in a loss of the chirality center at C2 but does not influence other chirality centers; D-glucose and D-mannose, for example, give the identical phenylosazones.

(drawing)

(Solomons and Fryhle, 2011) D- fructose when it tautomerizes to the corresponding more oxidizable aldehyde has the same product as D-glucose and D-mannose.

Illustration

A similar conclusion can be given with the osazones of D-galactose, D-talose, and D________.

Illustration

Different osazones show crystals of different shapes.

The test is done by adding phenylhyrazine-HCl / NaCH3COO in the test tube containing 0.1 g of the sugar. The test tube is placed in a boiling water bath and shaken occasionally. The resulting crystals were not filtered anymore since they were too small and few. After immediately adding the reagent, the test tube containing fructose and test tube containing glucose both gave a yellow solution, while sucrose gave a brighter yellow solution. After some time, fructose gave a brown solution with formation of small black precipitate. Glucose yielded very small brown precipitate at the surface of the yellow solution. Sucrose gave a dark red precipitate. The shapes of the crystals were not observed anymore since the precipitates were too small and few.