MLS Biochemistry Laboratory - BSMT 2nd Year - Midterm - PDF

MLSBCHML BIOCHEMISTRY LABORATORY BSMT 2ND YEAR | 1ST TERM - MIDTERM | S.Y. 2024-2025 LESSON 1: LABORATORY GLASSWARES INTRODUCTION Other laboratory apparatus is also available to fa...

MLSBCHML BIOCHEMISTRY LABORATORY BSMT 2ND YEAR | 1ST TERM - MIDTERM | S.Y. 2024-2025 LESSON 1: LABORATORY GLASSWARES INTRODUCTION Other laboratory apparatus is also available to facilitate Performing biochemical analysis in the easy handling of laboratory laboratory requires the use of particular glasswares glassware and laboratory equipment. Test tube rack Each apparatus performs a specific purpose which may be similar but most Crucible tong often unique. Test tube holder Students at the beginning of the class Wire gauze must be properly oriented with these Tripod apparatuses and must be able to utilize Specialized automated and them correctly. semi-automated equipment are also utiized in the LABORATORY GLASSWARES biochemistry laboratory to help carry out the experiments There are different types and kinds of glasswares in the laboratory. Spectrophotometer They are commonly utilized to carry Analytical balance Micropipettes out varying functions such as: Measurement of solutions and liquids Graduated cylinder Serological pipettes Carrier or container of reagents Beaker Erlenmeyer flask Reaction vessel Test tube Grounding vessel Mortal and pestle Vessel for heating to extremely high temperatures Crucible with cover Evaporating dish For transferring and mixing chemicals and liquids Stirring rod Glass funnel  MLSBCHML BIOCHEMISTRY LABORATORY BSMT 2ND YEAR | 1ST TERM - MIDTERM | S.Y. 2024-2025 LESSON 1: LABORATORY GLASSWARES INTRODUCTION 𝐩𝐎𝐇 = −𝐥𝐨𝐠 [𝐎𝐇−] Potential hydrogen (pH) is a measure of how acidic or basic a solution is. The range goes from 0 to 14. Acidic – pH < 7 Neutral – pH = 7 Basic – pH > 7 The control of pH is important in organism and their cells because chemical reactions and processes are affected by the hydrogen ion concentration. Acid – a compound that can donate a hydrogen ion Base – a substance that accepts hydrogen ions pH is a measure of the hydrogen ion (H+) concentration in an aqueous solution. pH is also expressed as the negative logarithm of the hydrogen-ion concentration. 𝐩𝐇 = −𝐥𝐨𝐠 [𝐇+] BACKRGOUND Chemists have tried to define acids and bases in relation to their molecular In contrast, pOH stands for potential of structures. hydroxide. SVANTE ARRHENIUS It is a measure of hydroxide ion (OH-) concentration. He defines acids as substances that It is expressed as the negative logarithm produce H+ ions in aqueous solution of the hydroxide-ion concentration.  while bases are substances that It converts the H+ concentrations to pH produce OH- ions in aqueous solution. using the formula: 𝐩𝐇 = − 𝐥𝐨𝐠 [𝐇 +] GILBERT N. (G.N.) LEWIS The precise definition of pH is He mentioned that acids are electron- "the negative common logarithm pair acceptors and bases are electron- of the activity of hydrogen ion in pair donor. solution.” However, the two mentioned definitions have limitations. Thus, the most useful and accepted definition of acids and bases nowadays are those proposed by Johannes Bronsted and Thomas Lowry, and it is known as the Bronsted-Lowry theory. REACTION BETWEEN HCL WITH WATER BUFFERS Buffers prevent changes in pH. HCl (Hyrochloric acid) is an acid Buffers resist changes in the pH because it donates a proton making Cl- even when acids or bases are (Chloride) while water is a base added. because it accepts a proton making Buffers are a mixture of a weak acid or H3O+. alkali and one of its salts. Furthermore, the theory explains that for Example: acetic acid + every acid-base reaction, there is a sodium acetate creation of conjugate acid-base pair. The ability of buffers to resist large In the above example Cl- is the changes in pH is governed by the Le conjugated base of HCl and H3O+ is the Chatellier's principle. conjugated acid of water as shown Le Chatellier's Principle – a principle of below. equilibrium shift due to changes in buffer conditions BUFFERS IN HUMAN BLOOD In our blood, carbonic acid is the most important buffer. This solution maintains our blood pH to facilitate transport of oxygen from the lungs to the cells. pH of blood is normally slightly basic (7.35 – 7.45). SØREN PETER LAURITZ (S.P.L.) SØRENSEN WAYS TO MEASURE Ph He introduced the pH scale that LITMUS TEST measures the strength of an aqueous acidic or basic solution. The Litmus test is a simple test to check if a substance is acidic or basic using litmus paper. There are two types of litmus paper available that can be used to identify acids and bases, red litmus paper and blue litmus paper. Blue litmus paper turns red for acidic pH. Red litmus paper turns blue for basic pH. No color change for neutral pH. INDICATOR PAPER Indicator paper is impregnated with organic compounds that change their color at different pH values. The color shown by the paper is then compared with a color standard usually provided by the manufacturer. pH METER The pH meter should be calibrated first before operating the device. The standard procedure for calibrating a pH meter is to calibrate it at three different pHs (pH 7, pH 4, and pH 10). After calibration, all that needs to be done is to insert the electrodes of the pH meter into the solution to be tested and read the pH flashed on the screen.  MLSBCHML BIOCHEMISTRY LABORATORY BSMT 2ND YEAR | 1ST TERM - MIDTERM | S.Y. 2024-2025 LESSON 1: LABORATORY GLASSWARES INTRODUCTION Hexoses - 6 carbon atoms Aldohexoses – glucose Carbohydrates are polyhydroxy and galactose aldehydes (aldoses) and Ketohexose – fructose polyhydroxy ketones (ketoses). On hydrolysis, disaccharides yield 2 General formula: (CH2O)n monosaccharide units. Classification is based on the number of monosaccharide units they contain: Monosaccharides Disaccharides Oligosaccharides Polysaccharides 2 general classes of carbohydrate test reagents based on the type of reaction involved: 1. Dehydrating acids (2-step analysis) Converts pentoses into furfural and hexoses into 5- hydroxymethylfurfural which then reacts with phenolic compounds Production of highly colored products Molisch’s, Anthrone, Bial’s & Seliwanoff’s tests 2. Copper (II) ions containing solutions Monosaccharide is the simplest sugar CHO reduces copper (II) and cannot be hydrolyzed further. ions into copper (1) oxide. Classification is by number of carbon Reducing sugars include atoms they contain. aldoses containing either a Pentoses – 5 carbon atoms free aldehyde group or a Aldopentoses – ribose cyclic hemiacetal. and xylose  BENEDICT’S TEST INTERPRETATION OF RESULT TEST OBJECTIVES To determine the presence or absence of reducing sugar in the solution All monosaccharides are reducing sugars. A reducing sugar has a free aldehyde group or a free ketone group. BARFOED’S TEST TEST OBJECTIVE Barfoed’s test is a chemical test used to detect the presence of reducing monosaccharides. To distinguish reducing monosaccharides from disaccharides PRINCIPLE OF THE TEST The Barfoed reagent is made up of copper acetate (cupric ions) in a dilute solution of acetic acid (acidic medium). Reducing monosaccharides are strong PRINCIPLE OF THE TEST reducing agent. It reacts within 3 minutes. Benedict’s reagent contains copper (II) Reducing disaccharides have to first get ions in an alkaline solution with sodium hydrolyzed in the acidic solution and citrate to keep the cupric ions in then react with the reagent. It reacts in solution. about greater than 3 minutes. The test is performed by heating the reducing sugar solution with Benedict’s reagent. The alkaline condition of this test causes isomeric transformation of ketoses to aldoses resulting in the reduction of blue cupric ion to cuprous oxide. Positive Result – brick red Monosaccharide – less than 3 minutes Disaccharide – greater than 3 minutes  The difference in the time of appearance Ketoses reacts to produce a cherry red of precipitate thus helps distinguish color. reducing monosaccharides from Aldoses may react slightly to produce a reducing disaccharides. faint pink to cherry red color if the test is prolonged. SELIWANOFF’S TEST The product and reaction time of the TEST OBJECTIVE oxidation reaction helps to distinguish between carbohydrates. To detect the presence of ketohexoses Sucrose and inulin also give a positive in a given sample result for this test as these are To distinguish ketoses from aldoses hydrolyzed by acid to give fructose. BIAL’S ORCINOL TEST TEST OBJECTIVE To detect the presence of carbohydrates To distinguish the pentoses and pentosans from other derivatives of ALDOSE KETOSE carbohydrates like the hexoses An aldose contains A ketose contains Pentose – a monosaccharide with 5 one aldehyde one ketone group carbon atoms. The chemical formula of group per molecule per molecule all pentoses is C5H10O. In Seliwanoff’s test, In Seliwanoff’s test, aldoses react ketoses react with slowly and produce resorcinol to give a a light pink color. deep cherry-red color. PRINCIPLE OF THE TEST PRINCIPLE OF THE TEST The reagent of this test consists of resorcinol in 6M HCl. This test is based on the principle that The acid hydrolysis of polysaccharides under hydrolysis, pentosans are and oligosaccharides yields simpler hydrolyzed into pentoses. sugars. Further, pentoses are dehydrated to Ketoses are more rapidly dehydrated yield furfural, which in turn condenses than aldoses. with orcinol to form a blue-green Ketoses undergo dehydration in the precipitate. In the presence of hexoses, presence of concentrated acid to yield hydroxyfurfural is formed instead of hydroxymethyl furfural that condenses furfural, which upon condensation with with resorcinol. orcinol, forms a muddy brown colored precipitate. The intensity of the precipitation is directly proportional to the concentration in the sample. The intensity of the color developed depends on the concentration of HCl, ferric chloride, orcinol, and the duration INTERPRETATION OF RESULT of boiling.  MLSBCHML BIOCHEMISTRY LABORATORY BSMT 2ND YEAR | 1ST TERM - MIDTERM | S.Y. 2024-2025 LESSON 4: PHENYLHYDRAZONE AND MUCIC ACID TEST PRE-LAB DISCUSSION 1. Prepare the phenylhydrazine reagent by mixing 2 grams of the MUCIC ACID TEST phenylhydrazine hydrochloride, 3 grams of CH3COONa and 10 mL of Materials and Reagents distilled water. Carbohydrate Solution 2. Place the reagent in a warm water Galactose bath. Stir until the solution clears. Lactose 3. In different test tubes, mix 2 drops of Concentrated HNO3 the carbohydrate solution (glucose, Alcohol Lamp fructose, xylose, lactose, sucrose, Microscope and starch) with 4 drops of freshly Procedure prepared phenylhydrazine reagent. 1. Mix 3 drops of the carbohydrate 4. Mix the solutions well. Cover the solution (galactose and lactose) and tubes with cotton. 3 drops of the concentrated HNO3 on 5. Heat them in boiling water bath for a glass slide. 30 minutes. Record the time when 2. Pass the mixture over a small flame yellow crystals first appeared. until it is almost dry. 6. Cool the tubes and observe the Use an alcohol lamp. Do not crystals under the microscope. scorch. 7. Draw the different osazones. 3. Cool the mixture at room temperature. POST-LAB DISCUSSION 4. Examine the crystals under the MUCIC ACID TEST microscope 5. Draw the mucic acid crystals. Mucic acid test is a test that is highly 6. If no crystal appears, let the glass specific and is used for the detection of slide stand until the next period. the presence of galactose and lactose. It is also termed galactaric acid, named PHENYLHYDRAZONE TEST after the product of the reaction. Materials and Reagents Test Purpose: Phenylhydrazine HCl To detect the presence of CH3COONa galactose and lactose in a given Distilled Water sample Test Tubes To distinguish between the Carbohydrate Solutions galactose-containing saccharides Glucose and other sugars Fructose Xylose Lactose Sucrose Starch Microscope Procedure Principles of the Test: Differentiation of reducing sugars is Aldohexoses (monosaccharide) based on the time of appearance of the upon treatment with potent complex and relative solubility in hot oxidizing agents like nitric acid water. yield saccharic acids Examples of reducing sugars: (dicarboxylic acids). Glucose Nitric acid has the capacity to Fructose oxidize both aldehyde and Glyceraldehyde primary alcoholic groups present Galactose at C1 and C6 respectively of Test Objective: galactose to yield an insoluble To detect reducing sugar precipitate (rod-shaped crystals) To differentiate reducing sugars of mucic acid under higher from non-reducing sugars temperature. To distinguish different reducing sugars between each other Principle of the Test The reagent for this test consists of phenylhydrazine in acetate buffer. This test is based on the fact that carbohydrates with free or potentially free carbonyl groups react with phenylhydrazine to form osazone. Reaction: The condensation-oxidation- condensation between three molecules of phenylhydrazine Lactose also yields a mucic acid, due to and carbon 1 and 2 of aldoses the hydrolysis of the glycosidic bond and ketoses yields 1,2- between the glucose and galactose diphenylhydrazone, which is subunits of the carbohydrate. known as osazone. Other monosaccharides like glucose also have a similar structure. However, the resultant precipitate formed in glucose is water-soluble under room temperature. The formation of crystal at the bottom of the tube indicates a positive result, which means that the sample solution has galactose or its derivatives. The absence of such crystals indicates a negative result and represents that the Interpretation of Result: sample does not have galactose or its Osazone appears as yellow- derivatives. The solution might still have colored crystals of characteristic other carbohydrates. shape, solubility, melting point, and time of formation. PHENYLHYDRAZONE TEST Osazone is different for different sugars. Phenylhydrazone or Osazone test is a chemical test used to detect and differentiate reducing sugars.  MLSBCHML BIOCHEMISTRY LABORATORY BSMT 2ND YEAR | 1ST TERM - MIDTERM | S.Y. 2024-2025 LESSON 5: HYDROLYSIS OF DISACCHARIDES AND POLYSACCHARIDES INTRODUCTION Hydrolysis of disaccharides and polysaccharides is essential for various Hydrolysis is a fundamental chemical biological and industrial processes. process that plays a crucial role in the breakdown of complex carbohydrates, such as disaccharides and polysaccharides, into simple sugars. MATERIALS AND EQUIPMENT Test tubes Test tube brush Test tube rack Hot plate 500 mL beaker Disaccharides, which are composed of Test tube holder two monosaccharide units linked Pipette or dropper together, are hydrolyzed into their 10% NaOH constituent sugars HCl solution For example, sucrose, a common Starch solution disaccharide found in table sugar, is Benedict’s reagent hydrolyzed into glucose and fructose. Iodine reagent Similarly, lactose, the sugar found in Spot plate milk, is broken down into glucose and galactose. SAFETY PRECAUTIONS Polysaccharides, on the other hand, are long chains of monosaccharide units The laboratory gown must be worn at all and include starch, glycogen, and times. cellulose. These complex carbohydrates Wear a face mask and disposable nitrile are hydrolyzed into simpler sugars gloves during the experiment. through a series of enzymatic reactions. Read the procedure ahead of the Starch, for instance, is broken down into designated laboratory period. maltose and eventually into glucose, If using acids or bases for hydrolysis, while cellulose is hydrolyzed into handle them with care. Always add glucose units, though this process is acids to water, not the other way more challenging due to the structural around, to prevent exothermic reactions complexity of cellulose. and splashing.  Use enzyme solutions carefully, as Sucrose + HCl – hydrolysis of sucrose some can be irritants. Follow the by HCl should yield glucose and manufacturer’s instructions for handling fructose, but initially, you will have an and disposal. acidic solution. Be familiar with the procedures for Upon adding NaOH, the solution should cleaning up chemical spills. Use spill kits turn neutral, indicated by a color change and follow your institution’s protocols. in litmus paper from red to blue. Follow your institution’s guidelines for the disposal of chemical waste. Do not pour chemicals down the sink unless instructed to do so. PROCEDURES USING LITMUS PAPER Starch + HCl – hydrolysis of starch by HCl produces simpler sugars like 1. Prepare 4 test tubes in a test tube rack. maltose and glucose. The solution Label them where 2 test tubes are for should initially be acidic. sucrose, while the other 2 are for the With NaOH addition, you should starch (with H2O and HCl for both sets observe a color change from red to blue of test tubes). as the solution becomes neutral. The 2. Place 1.5 mL of sucrose to the sucrose- number of drops required to neutralize labeled test tubes, and 1.5 mL of starch the solution gives an indication of the to the starch-labeled test tubes. extent of acid present. 3. Add 10 drops of deionized water and 10 drops of 10% HCl to their respective USING IODINE’S TEST labeled test tubes. 4. Put water in a 500 mL beaker and heat it 1. Using a spot plate, place 5 drops of on hot plate until it boils. each 4 solutions in different spot. 5. Put the test tubes inside the beaker and 2. Add 1 drop of iodine reagent to each be careful not to topple it down. Keep solution in spot plate. Observe what will the 4 test tubes in boiling water bath for happen. about 10 minutes. EXPECTED RESULTS (USING IODINE’S 6. Remove the test tubes from the water TEST) bath and let them cool by placing them in test tube rack. Starch – iodine turns blue-black in the 7. For the test tubes containing HCl, add a presence of starch. If starch was drop of 10% NaOH in it. Then, put a red present before hydrolysis, you should litmus paper. Check if there are any see a blue-black color. sudden changes in the color of the Sucrose – iodine will not change color if litmus paper. sucrose is present, as sucrose does not 8. Add another 5 drops of NaOH to the react with iodine. HCl-labeled test tubes. Check again for Post-Hydrolysis Solutions – after color changes using litmus paper. You hydrolysis, starch should be broken may do for maximum of 20 cumulative down into simpler sugars, which do not drops and check if the solution became react with iodine. Thus, a blue-black neutralized. Take note of the number of color should fade or disappear if starch drops it will take. Check the solution has been hydrolyzed. again using red litmus paper. USING BENEDICT’S TEST EXPECTED RESULTS (USING LITMUS PAPER)  1. Add Benedict’s reagent to the remaining solution in the labeled test tubes. 2. Heat the water placed in a 500 mL beaker using hot plate until it boils. 3. Place the 4 test tubes in the boiling bath water for 3-4 minutes. 4. Remove the test tubes and place them in the test tube rack. Check if there are changes in the color of the solution after boiling. EXPECTED RESULTS (USING BENEDICT’S TEST) Reducing Sugars (e.g., Glucose, Fructose) – Benedict’s reagent changes color based on the concentration of reducing sugars: Blue – no reducing sugars Green/Yellow – low concentration of reducing sugars Orange/Red – moderate to high concentration of reducing sugars Brick Red – high concentration of reducing sugars Sucrose – before hydrolysis, sucrose will not react with Benedict’s reagent (solution remains blue). After hydrolysis, the presence of glucose and fructose should result in a color change, indicating reducing sugars. Starch – starch itself will not change color with Benedict’s reagent. However, after hydrolysis, the resulting glucose and maltose will cause a color change if present.  MLSBCHML BIOCHEMISTRY LABORATORY BSMT 2ND YEAR | 1ST TERM - MIDTERM | S.Y. 2024-2025 LESSON 6: LIPIDS INTRODUCTION Egg yolk Acetone Lipids – they are amphipathic Ethanol molecules that encompass fatty acids Chloroform and derivatives such as diglycerides, Centrifuge triglycerides, phospholipids, and Petroleum ether cholesterol Roles in the human body: PROCEDURE Provide energy for the body Act as chemical messenger 1. Collect egg yolk from 2 eggs in a Involved in the maintenance of breaker and mix well. body temperature 2. Add 50 mL of cold acetone and mix with Involved in membrane layer glass rod and allow to stand for 15 formation minutes. Involved in the formation of 3. Collect the precipitate by centrifugation prostaglandins and mediation of for 5 minutes at 4000 rpm. inflammation 4. Wash the precipitate with sufficient quantity of acetone until the supernatant is clear and colorless. 5. Extract the precipitate with about 100 mL of chloroform-methanol mixture (2:1) for 3-4 hours at room temperature. 6. Filter and collect the filtrate (extract). 7. Evaporate the extract to dryness under a stream of nitrogen or at room temperature. 8. Dissolve the residue in small volume of petroleum ether (10-15 mL) and re- precipitate the phospholipid with addition of cold acetone (about 50 mL) and then allow the precipitate to settle EXTRACTION OF LIPIDS FROM EGG YOLK down at the bottom. After this, collect the precipitate of phospholipid. MATERIALS, REAGENT, AND EQUIPMENT 9. Dry under vacuum and store in dark in a minimum volume of chloroform- Beaker methanol system or as solid. Test tubes Test tube rack PRINCIPLE OF THE TEST Test tube holder Filter paper Many phospholipids are insoluble in Funnel acetone and they precipitate out Petri dish whereas triglycerides, sterol, and Graduated cylinder pigments are soluble in acetone. Stirring rod Egg and egg yolk are very important Tripod sources of Lecithin, which is a generic  term to designate any group of yellow- 1. Get 3 test tubes and label with clarified brownish fatty substance occurring in butter, vegetable oil, and linseed oil. Add animal and plant tissues, which are 2 mL of each to their respective tube. amphiphilic – they attract both water and 2. Add small amount of potassium fatty substances (and so are both bisulphate crystals to each tube. hydrophilic and lipophilic), and are used 3. Heat the mixture directly on a burner. for smoothing food textures, dissolving 4. Observe the formation of a gas and odor powders (emulsifying), homogenizing that will be produced. liquid mixtures, and repelling sticking 5. Record this as your observation. materials. Lecithin can easily be extracted PRINCIPLE OF THE TEST chemically using solvents such as hexane, ethanol, acetone, petroleum Acrolein test is used to detect the ether, benzene, etc., or extraction can presence of glycerol or fat. be done mechanically. When fat is treated strongly in the It is usually available from sources such presence of a dehydrating agent like as soybeans, eggs, milk, marine potassium bisulphate (KHSO4), the sources, rapeseed, cottonseed, and glycerol portion of the molecule is sunflower. dehydrated to form an unsaturated It has low solubility in water, but is an aldehyde, acrolein that has a pungent excellent emulsifier. irritating odor. Positive Result – if glycerol is present MATERIALS, REAGENT, AND EQUIPMENT in the sample, it will give a pungent smell. Test tubes Negative Result – if glycerol is absent Test tube holder in the sample, it will not produce a Test tube rack pungent smell. Bunsen burner Dropper Filter paper Light source Clarified butter Potassium bisulphate Cholesterol Chloroform Acetic anhydride Concentrated sulfuric acid Oleic acid Petroleum ether Copper acetate solution Vegetable oil Linseed oil LIEBERMANN-BURCHARD TEST Olive oil Sunflower oil PROCEDURE Palm oil Corn oil 1. Get a test tube with 2 mL of cholesterol Coconut oil dissolved in chloroform. 2. Add 2 mL of acetic anhydride to the ACROLEIN TEST cholesterol solution and mix completely. 3. Add 5 drops of concentrated sulfuric PROCEDUREs. acid to the previous mixture by tilting the  test tube and dropping on the side of the Appearance of white blue test tube then gently mix the solution. precipitate at the bottom of the 4. Observe for a bluish-green color. upper layer 5. Record this as your observation. PRINCIPLE OF THE TEST To detect the presence of cholesterol A chemical estimation of cholesterol, the cholesterol reacts as a typical alcohol with a strong concentrated acids and the product are colored substances. Acetic anhydride is used as a solvent and dehydrating agent. SPOT OR TRANSLUCENT TEST Positive Result – it indicates cholesterol in a sample by giving bluish- PROCEDURE green color to the solution Negative Result – the color of the 1. Prepare a filter paper and drop the solution will not change clarified butter on it then press another filter paper on top of the other. COPPER ACETATE TEST 2. Hold the filter paper against a light source. PROCEDURE 3. Then, repeat the process with vegetable 1. Get 2 test tubes and label with olive oil oil and linseed oil. and oleic acid. Add 2 mL of each to their 4. Record your observation. respective tube. PRINCIPLE OF THE TEST 2. Add petroleum ether to both test tubes. 3. Shake test tubes to mix the content. A translucent spot test is a preliminary 4. Add copper acetate solution to both test test for the lipids, which is characterized tubes. by a translucent and greasy spot. The 5. Shake both test tubes vigorously. lipid will not wet the filter paper, unlike 6. Observe for blue-green color. water, the lipids will form a greasy or 7. Record this as your observation. translucent spot due to their greasy texture, and penetrate the filter paper. PRINCIPLE OF THE TEST Unlike lipids, the spot of water will This test is used to distinguish between disappear from the paper. oil or neutral fat and fatty acid saturated Positive Result – translucent spot will and unsaturated. appear on the filter paper The copper acetate solution does not Negative Result – translucent spot will react with the oils, while saturated and not appear on the filter unsaturated fatty acids react with copper acetate to form copper salt. Unsaturated fatty acids can only be extracted by petroleum ether. Unsaturated Fatty Acid – 2-layer appearance: Upper layer – green Lower layer – blue Saturated Fatty Acid – 2-layer appearance:  UNSATURATION TEST PROCEDURE 1. Prepare 5 test tubes and label with olive oil, coconut oil, corn oil, palm oil, and sunflower oil. Add 1 mL of each to their respective tube. 2. Add a few drops of bromine water to each tube one by one and make sure to replace the lid of the container. 3. Shake each test tube vigorously. 4. Note if the color of bromine water disappeared. 5. Record your observation. PRINCIPLE OF THE TEST An unsaturation test is used to detect the unsaturated fatty acids or double bonds in a lipid sample. All the neutral fat contains glycerides of fatty acids. Double bonds are found in the structure of unsaturated fatty acids, which become saturated by taking up either bromine or iodine. If the lipid contains more unsatured fatty acids or more double bonds, it will take in more iodine. Positive Result – pink color will disappear by the addition of unsaturated fatty acids Negative Result – pink color will not disappear