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! !Kingdom of Saudi Arabia !Ministry of Higher Education ! Prince Salman bin Abdulaziz University ! College of Science and Humanities !Chemistry Department ! ! ! ! ! ! ! !Biochemistry LAB (3010) !practical handout ! ! ! ! ! ! ! ! by Ahoud Alotibi !Lab safety: The work in laboratories requires a full awareness of the importance and seriousness of the materials and equipment used , where much of the materials is characterised as toxic or irritant to the skin or could cause burning. so you must before starting the laboratory work to realize and understand the materials to be used in the each experiment. Also to take cautions and follow all the recommended laboratory safety instructions provided. ! ! ! 1- Everyone who works in a chemistry laboratory should follow these safety precautions: ! • lab coats and gloves must be worn in the laboratory during the experiment. • Long hair must be pinned or tied back (to avoid exposing it to chemicals, cultures, or flames). • Food, drink, and gum are not allowed in the lab. Do not ingest any chemicals and keep hands away from your face when working with them. • After each lab procedure, wash hands diligently with soap and water. • Before each procedure, read, reread, and understand all instructions and procedures before carrying out the experiment . • To avoid contamination, do not return unused chemicals to a reagent bottle. Similarly, never put a pipet, spatula, or dropper into a reagent bottle. Instead, pour some of the reagent into a small clean beaker and use that as your supply. • If spills occur, especially concentrated acid/base spills, immediately inform the teacher. All accidents, no matter how insignificant, should be reported immediately. • • ! ! ! ! Glassware that is chipped/cracked should not be used, notify the teacher immediately. Review chemical safety guidelines before handling chemicals. Read the label on a reagent bottle carefully before using the chemical. ! 2- chemical warning signs of chemicals:! ! ! ! ! ! ! ! ! ! 3- Laboratory tools to be used :! ! ! tonge ماسك ! ! ! ! ! ! ! test tube انبوب اختبار rack حامل انابيب ! ! ! ! ! ! Carbohydrates introduc1on: ! ! ! carbohydrates are the most abundant organic molecules in nature. they have a wide range of func7ons, including providing a significant frac7on of energy in the diet of most organisms, ac7ng as a storage form of energy in the body (in the form glycogen) and serving as cell membrane components. the chemical formula for carbohydrates is (CH2O)n ,that is, consis7ng mainly of carbon, hydrogen and oxygen. ! ! ! classifica1on of carbohydrates: I. according to chemical composi1on: simple and complex. II. according to number of units: mono -‐ Di -‐ Poly saccharide. III. according to posi1on of carbonyl group in mono saccharide: aldoses and ketoses. IV. according to the number of carbon atoms in mono saccharide: triose(3),tetrose(4), pentose(5),hexose(6),heptose(7). ! ! ! ! ! ! ! I. according to chemical composi1on: 1) Simple carbohydrates, oIen called monosaccharides or simple sugars, contain one saccharide unit. 2) Complex carbohydrates are those containing more than one saccharide group. ! ! ! ! II. according to number of units: -‐ Monosaccharides contain one monosaccharide unit. ex: glucose, fructose, galactose. -‐ Disaccharides contain two monosaccharide units. ex: sucrose ,lactose,maltose. -‐ Polysaccharides can contain over 7 or more monosaccharide units. ex: starch, glycogen,cellulose. ! figure 1: example of mono saccharide ! ! ! ! ! ! ! ! ! figure 2: examples of Di saccharides figure3: examples of poly saccharides ! ! III. according to posi1on of carbonyl group: • aldoses: contains terminal aldehyde group in addi7on to R group containing -‐OH. • ketoses: contains a carbonyl group aRached to two R groups having one or more hydroxyl groups. figure 4: example of aldoses and ketoses. ! IV. according to the number of carbon atoms in mono saccharide: They can be classified by the number of carbon atoms they contain; pentoses (5 carbons) such as ribose and hexoses (6 carbons) such as Glucose and Fructose are the most common. ____________________________________________________________________ Reducing sugars: A reducing sugar is a carbohydrate that is oxidised by a weak oxidizing agent which is capable of oxidising aldehydes group in the sugar, such as the Tollen’s reagent and fehling’s reagent in aqueous solu7on. ! ! Any sugar that contains either: 1-‐A free aldehyde group. 2-‐ An α-‐hydroxy ketone group. is considered a reducing sugar. ! The presence of any of these groups allows the carbohydrate to undergo easy oxida7on and If the sugar gets oxidised it causes reduc7on ,Thus the name “reducing sugar”. ! ! Physical Proper1es of Carbohydrates: Solubility: Monosaccharide and disaccharide can be dissolved in water because water is polar and sugars as well. sugars contain OH groups which form hydrogen bonds with water. while polysaccharide cannot be dissolved easily in water, because, it has high molecular weight , which gives colloidal solu7ons in water. ! ! ! ! ! ! chemical proper1es: qualita1ve tests for carbohydrates: ! these tests are done to : -‐ -‐ -‐ -‐ -‐ ! ! ! ! ! differen7ate carbohydrates from lipids and proteins.(molish test) to discriminate reducing sugar from a non reducing sugar.(fehling,tollen test) to differen7ate between mono,Di saccharides.(Barfoid test) to differen7ate between aldoses and ketoses.(seliwanoff test, ammonium molybdate) to iden7fy starch.(iodine test) 1. Molish test: It is the general test for all carbohydrates and it is used to differen7ate between carbohydrates ,proteins and lipids. monosaccharides will give rapid result (purple ring) while disaccharide and poly saccharide react slower. ! method: 1ml test solu7on + 1/2 ml of α-naphthol >> mix well >> add conc. H2SO4 down the side of the tube to form the ring at the interface of the two layers. ! ________________________________________________________________________________ 2. fehling test: This test is used to differen7ate between reducing and non reducing sugars. A reducing sugar reacts with Fehling's reagent to form an orange to red precipitate. Most disaccharides are reducing sugars (e.g. lactose and maltose) while sucrose does not react with Fehling's reagent (Sucrose is a disaccharide of glucose and fructose). Sucrose is non-‐reducing sugar because the anomeric carbon of glucose is involved in the glucose-‐ fructose bond and hence is not free to form the aldehyde in solu7on. ! ! ! method: 2ml Fehling's reagent > heat > + 1ml test solu7on(2min)>>yellow ppt turns red. __________________________________________________________________________ 3. tollen’s test: ! used to differen7ate between reducing and non reducing sugar. reducing sugars form a silver mirror as result of the reac7on. and it can be used to differen7ate between mono and Di saccharide. ! method: 1 ml of the solu7on + 1 ml of tollens reagent and mix well > appearance of silver mirror on cold or simple hea7ng if its a mono saccharide and a long hea7ng in Di saccharide. ! __________________________________________________________________! ! 4. Barfoid test: ! It is a test used to differen7ate between monosaccharides and disaccharides. This reac7on will detect reducing monosaccharides in the presence of disaccharides. Reducing disaccharides (lactose and maltose but not sucrose) undergo the same reac7on but at slower rate. ! method: 1 ml of the solu7on to be tested + 1 ml of freshly prepared Barfoid's reagent > heat on the flame for 2 minutes > red ppt. _________________________________________________________________________ 5. seliwanoff test: ! used to differen7ate ketoses from aldoses. it detect ketohexoses (6 carbon atoms sugar that contains ketone group).The test reagent dehydrates ketohexoses to produce red product within two minutes. aldohexoses undergo the same reac7on but at slower rate. ! method: 1 ml of the solu7on + 3ml Seliwanoff reagent > hea7ng in boiling water for 2 min. >red colour. __________________________________________________________________________! ! 6. ! furfural test: used to differen7ate between mon, Di saccharides. ! ! ! method: ! 1 ml of the solu7on + 1ml of α-naphthol +2ml of conc. HCl > heating > violet colour.! ! ! 7. ammonium molybdate test: ! it is used to differen7ate between aldoses and ketoses. ! method: 2 ml of the solu7on +2ml of ammonium molybdate + 4 drops of ace7c acid > slow heat > apple green colour(glucose) or bluish green colour (fructose). ! __________________________________________________________________________ ! 8. iodine test: ! Test for Polysaccharides (starch).A dark blue colour indicates the presence of starch. If the yellow color of the iodine reagent simply becomes diluted, no starch is present. ! ! method: 2ml of the solu7on +1 drop of the iodine solu7on. > dark blue colour. ! ! ! amino acids and proteins ! ! introduc1on: Amino acids are molecules containing an amine group, a carboxylic acid group and a side chain that varies between different amino acids. Amino acids of the general formula RCH(NH2)COOH are amphoteric, behaving as amines in some reac7ons and as carboxylic acids in others.Amino acids are cri7cal to life. They have par7cularly important func7ons like being the building blocks of proteins and being the intermediates in metabolism. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! figure1: structure of amino acids. There are 20 natural amino acids, All found in proteins having this basic structure, differing only in the structure of the R-‐group or the side chain. The simplest, and smallest, amino acid found in proteins is glycine for which the R-‐group is hydrogen (H). ! ! ! ! Classifica1on of amino acids: 1.Polar amino acids: a-‐Polar charged: i-‐Acidic amino acids (-‐ve) ii-‐Basic amino acids(+ve) b-‐Polar uncharged amino acids ! 2.Non polar/ alipha7c/neutral: 3. Aroma7c amino acid. ! ! ! ! ! ! figure2: classifica7on of amino acids. Some proper1es of Amino Acids: ! -‐Amphoteric Compounds: which mean they can act as acids and bases, due to presence of carboxyl group COOH that able to donate proton(H+), and convert to COO-‐ Also presence of amino group NH2 which is enable to accept this proton(H+) and convert into NH3+. ! -‐solubility: amino acids are soluble in water but Polar amino acids are more soluble in water than non-‐ polar, due to presence of amino and carboxyl group which enables amino acids to accept and donate protons to aqueous solu7on, and therefore, to act as acids and bases. ! ! proteins: Proteins (also known as polypep7des) are organic compounds made of amino acids arranged in a linear chain. The amino acids in a polymer are joined together by the pep7de bonds between the carboxyl and the amino groups of adjacent amino acid residues. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essen7al parts of organisms and par7cipate in virtually every process within cells. Proteins are important in: -‐ catalyzing biochemical reac7ons (enzymes). -‐ structural and mechanical func7ons (ac7n and myosin) -‐ cell signaling. -‐ immune responses. -‐cell adhesion. ! ! qualita1ve tests: these tests are used to differen7ate proteins and amino acids from lipids and to it can be used to differen7ate between amino acids and proteins: ! 1)biuret test. 2)ninhydrin test. ! ! 1)biuret test: proteins react with cuso4 in alkaline medium and gives a posi7ve result (purple colour) while amino acids give dark blue colour. method: 2ml solu7on of protein +1ml of NaoH > Mix well > add 2 drops of cuso4 2) ninhydrin test: in this reac7on both proteins and amino acids will give purple colour but proteins will give it in a slower rate. method: 2ml solu7on + 1/2ml of Ninhydrin solu7on > heat to 1min ____________________________________________________________________ tests used to differen7ate between proteins (albumin,casein): 1) Coagula1on test: if the solu7on coagulate it means the solu7on is albumin and if the result was -‐ve it is casein. method: 2ml of protein solu7on > heat simple ! 2) lead acetate test: ! method: 3ml of protein solu7on +1ml of NaoH 40% > heat > cool down Then add 1ml of pb(CH3COO)2 ! ! ________________________________________________________________________________ the following experiments to differen1ate between Amino Acids: 1)Millon Test: ! This test is specific for tyrosine, the only amino acid containing a phenol group, a hydroxyl group aRached to benzene ring. ! NOTE: all phenols (compound having benzene ring and OH a[ached to it) give posi1ve results in Millon’s test. (Tryptophane & phenylalanine & tyrosine). ! ! method: ! 2ml solu7on+ 1ml Mellon reagent > heat un7l Boiling > Red colour if t is one of the men7oned amino acids. MILLON'S REAGENT IS HIGHLY TOXIC AND HIGHLY CORROSIVE ! 2) Xanthoproteic Test: ! This test is used to differen7ate between aroma7c amino acids (Tryptophane & phenylalanine & tyrosine) which give posi7ve results and other amino acids. ! method: 2ml solu7on + 1/ 2 ml conc HNO3 > heat > yellow colour Then cool down the test tube > add 1/2 conc NH4 + > orange colour. ! HNO3 IS HIGHLY TOXIC AND HIGHLY CORROSIVE ! ! ! ! 3) folin test: ! This test indicates the presence of the amino acid ( arginine). ! ! method: 1 ml of solu7on + 1ml of folin reagent > heat > red colour 4) Sakaguchi Test: ! This test indicates the presence of the amino acid ( arginine). ! method: ! 3ml solu7on + 1ml of NaOH 10% + 3 drops of α-‐naphthol + 2ml of sodium Hypochlorite > Red colour ! !
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