Biochemistry Lab Techniques

• The manual introduces basic concepts in biochemistry labs and biochemical experiments, aiming to equip undergraduate students with essential techniques.
• It includes eleven experiments focusing on detecting and estimating biomolecules using simple equipment, each set with a theoretical principle, equipment, and chemicals.

Course Objectives

• Designed as a basic biochemistry introduction for clinical chemistry students.
• Employs simple protocols and readily available materials to teach about biochemical substances.
• Aims to foster independent work skills in the lab and knowledge of biochemical analysis principles and theoretical statements.
• Medical lab science students should have manual skills in measuring solutions/biological liquids, centrifugation, and colorimetry.
• It teaches determination of pH, enzyme investigations, and biochemical analysis to develop required skills.

Safety Rules

• Life-threatening injuries can occur in the lab; students must know the correct procedures and actions.

Conduct

• Practical jokes and boisterous behavior are prohibited.
• Running and personal audio/video equipment are not allowed.
• Performing unauthorized experiments is strictly forbidden.
• Sitting on laboratory benches is not allowed.

General Work Procedure

• Know emergency procedures.
• Supervised work is required.
• Experiments/work should be performed following guidance.
• Spills, accidents, and injuries must be immediately reported.
• Experiments must be constantly monitored.
• Exercise caution with hot glassware.
• Never point a test tube containing substances at anyone.
• Pipettes should only be filled using a pipetting device.
• Flammable solvents need to be away from flames.
• Lit Bunsen burners should never be left unattended.
• Heating apparatus, gas valves, and water faucets must be turned off when not in use.
• Equipment or chemicals must not be removed.
• Coats, bags, and personal items must not be left on benches or aisles.
• Sensitivities to chemicals should be reported.
• Keep the floor clear of objects.

Housekeeping

• Work areas should be neat and free of unnecessary objects.
• Laboratory workspaces must be thoroughly cleaned after the session.
• Sink drains should be free of blockages.
• Exits and emergency equipment must not be blocked.
• Equipment should be inspected for damage before use.
• Chemical waste should not go down the sink or into wastebaskets.
• Chemical waste should be placed in labeled containers.
• Broken glassware and sharp objects should be disposed of immediately in designated containers.
• Weigh boats, gloves, filter paper, and paper towels should be properly discarded.

Apparel

• Appropriate eye protection must always be worn.
• Disposable gloves are required when handling hazardous materials; remove gloves before exiting the lab.
• Wear a full-length, long-sleeved lab coat or chemical-resistant apron.
• Shoes should adequately cover the feet; low-heeled, non-slip soles are preferable; avoid sandals, open-toed shoes, or high-heeled shoes.
• Avoid shirts exposing the torso, shorts, or short skirts; long pants are preferable.
• Long hair and loose clothing must be secured.
• Remove jewelry, especially dangling jewelry.
• Synthetic finger nails are discouraged due to their flammability.

Hygiene

• Hands should be kept away from the face, eyes, mouth, and body when using chemicals.
• Food and drink should not be brought into the lab or chemical storage area.
• Lab glassware should not be used for eating or drinking.
• Cosmetics should not be applied in the laboratory or storage area.
• Hands should be washed after removing gloves before leaving.
• All protective equipment should be removed before leaving the lab.

Emergency Procedure

• Know the location of exits, emergency phone, fire extinguishers, alarm systems, fire blankets, eye washes, first-aid kits, and safety showers.
• Follow the established emergency plan and evacuate the building via the nearest exit in case of an emergency or accident.

Chemical Handling

• Verify the correct substance by checking the label before use.
• Chemical-resistant gloves should be worn.
• Label transferred chemicals with contents, concentration, hazard, date, and initials.
• Use a spatula or scoopula for removing solid reagents.
• Avoid directly touching chemicals.
• Never use a metal spatula with peroxides.
• Containers should be held away from the body during transfer.
• Flammable liquids should be heated in a hot water bath, not directly with a flame.
• Add concentrated acid to water slowly.
• Weigh out only the necessary amount of chemical.
• Excess chemicals should be disposed of.
• Never touch, taste, or smell reagents.
• Avoid placing containers directly under the nose or inhaling vapors.
• Don't mix or use uncalled-for chemicals.
• Promptly clean up spills as instructed.
• Dispose of chemicals as instructed.
• When transporting chemicals (especially 250 mL or more), use a secondary container or bucket.
• Don't handle bottles that are wet or too heavy.
• Use equipment correctly, as indicated.

Chemical Waste

• All containers for chemical waste needs to be labeled with "WASTE" or "HAZARDOUS WASTE", chemical name, accumulation start date, and hazard(s).

Storage Don’ts

• Heavy materials, liquid chemicals, and large containers should not be placed on high shelves.
• Storing chemicals on top of cabinets or on the floor is not allowed.
• Items should not be stored on bench tops and in lab chemical hoods, unless actively in use.
• Chemicals should not be stored on shelves above eye level.
• Food and drink with chemicals should not be stored together.
• Chemicals should not be stored in personal refrigerators, or exposed to direct heat/sunlight.

Use of Chemical Storage Containers

• Food containers should never be used for chemical storage.
• Containers should be properly closed.
• Wipe containers down with a paper towel before returning to storage and dispose of the towel after use.

Lab Reports

• Lab reports should adhere to a specified format, including course name, number, experiment title, date, student details, partners, instructor, and lab number on the first page.
• The second page should include an abstract, introduction, materials, methods, results, discussion, conclusion and references.

Lab 1: Carbohydrate Qualitative Tests

• Focuses on identifying carbohydrates through qualitative methods, understanding reducing/non-reducing sugars.

Molisch’s Test

• Serves as a general test for all carbohydrates.
• Monosaccharides rapidly yield positive results; disaccharides/polysaccharides react slower.
• Furfural and its derivatives form due to the dehydrating action of acid on sugars, hydrolyzing polysaccharides/disaccharides into monosaccharides.
• Molisch's reagent dehydrates pentoses to form furfural, and hexoses to form 5-hydroxymethyl furfural to react with α-naphthol to yield a purple.
• Add 1 ml test solution + 2 drops of α-naphthol, then H2SO4 to form a ring.
• Glucose, maltose, arabinose, and starch all display the purple compound at the interface.
• Has limitations, includes not being carbohydrate exclusive.

Benedict’s Test

• All reducing sugars produce positive results.
• Aldehyde or ketone groups undergo conversion to enediol forms under hot alkaline conditions for the cupric ions (Cu+2) convert to a Cuprose ion and ultimately to a red copper(I) oxide as a red precipitate.
• Benedict's reagent includes copper(II) sulfate (CuSO4)·5H2O, which reduces to red copper(I) oxide by aldehydes into carboxylic acids.
• Copper oxide's insolubility causes precipitation leading to solution colors that range from green to brick red.
• 1ml test solution + 1ml Benedict's reagent are heated in boiling water bath for 5 minutes to form a reddish brown ppt.

Barfoed’s Test

• Distinguishes between monosaccharides and reduced disaccharides.
• The test uses copper ions and copper acetate in dilute acetic acid to identify reducing sugars in an acidic solution pH 4.6.
• Reducing monosaccharides are oxidized by copper ions into carboxylic acid and reddish Cu2O (cuprous oxide) precipitates in a weak acidic medium.
• Reducing disaccharides undergo a reaction at a slower rate.
• 1 ml of solution + 2 ml of Barfoed's reagent are placed in a boiling water bath for 2 minutes.

Seliwanoff’s Test

• A test reagent dehydration of ketohexoses to form 5-hydroxymethylfurfural:
• The resulting 5-hydroxymethylfurfural reacts with resorcinol to produce a red compound within two minutes.
• Add 1/2 ml of sample and 2ml of Seliwanoff's reagent, added to a boiling water bath for two minutes results in a red product.
• Although aldohexoses yield identical products, the rxn occurs more slowly.
• With sucrose, avoid vigorous boiling to prevent hydrolyzing sucrose into glucose and fructose to avoid a false positive.

Iodine's Test

• Is a test for polysaccharides where poly-saccharides adsorb I2 and create a color complex, while starch yields a blue color and glycogen a reddish-brown hue.
• On heating, poly saccharide-Iodine complexes lose blue color by complex dissociation.
• 1/2 mL of fresh starch solution, and 1 drop of iodine solution, where dark blue indicates a positive test for starch.
• It also tests for Dextrin using 1/2 mL of the fresh dextrin solution and 1 drop of the iodine solution to indicate a positive test for dextrin.

Bial’s Test

• Distinguishes between pentose sugars using Bial reagent: HCl, orcinol and ferric chloride.
• HCl hydrolyses pentose sugars into a furfural derivative and reacts with orcinol to yield green -yellow
• Hexoses yield red to brown colorations as 1ml of test sugar solutions is added to Bial's reagent heated for 1 minute.

Phenylhydrazene's Test (Osazone Test)

• Uses the formula of 19 involving reactions with the sugars (monosaccharides) react with Phenylhydrazine to create a yield reducing the ones forming aldehyde or ketone.
• Additional phenyl hydrazine used with monosaccharides to oxidize the adjacent OH group, forming a second phenyl hydrazone and bixphenyl hydrazones called osazones.
• Reactions are complete in 3 steps.
• Insoluble osazones are formed, the time required classifies them into the following times as follows:
  • mannose takes 1-5 minutes
  • fructose takes 2 minutes
  • glucose :5 minutes
  • xylose: 7 mins
  • arabinose: 10 mins
  • galactose: 20mins
• Osazone compounds melt at a sharp point with configuration at the remaining carbon that creates a give osazones that yield a carbon structure.
• HPLC or mass spectrometry can assist in identification.
• Reagents requires 1% solutions of sugars, mixture to add of 300 mg in sample and heat. Then the tubes can be examined microscopically.

Hydrolysis of starch using Saliva or mineral acids

• Is a conversion to reducing sugar using Hydrochloric and heat
• Outlines a contrast with enzymes that result in quick rxns due to reagents at various degrees.
• Breaks bonds by:
  • polymers - reverse of condensation attach OH groups
  • break (lyse) bond - releases engery through negative iodine and positivity through (glucose) w/Benedict's et al.
• Performed with Test tube with solid, liquid then adding water, heat tests and cooling then adding quantity pf salt.

Lab 2: Carbohydrate Quantitative Tests

• Focuses on determining the quantity of carbohydrates, understanding reducing/non-reducing sugars.

Estimation of Glucose by Benedict's Method

• Benedicts reagent is copper sulphate, sodium carbonate, sodium or potassium citrate, potassium thiocyanate and potassium ferrocyanide to corresponds to glucose.
• Has procedures using a regent to make to a volume from a combination to a mixture with tests that create different percentages in solids.
• The reagent consists of titrate and its strength based on formula and volume.

Qualitative Test for Cholesterol

• A test test with double bond in one test for color and concentrated acids to form bicholestadien.
• In another test, its a test to form color from pink - green based on Liebermann reaction.

Lab 3: Amino Acids and Protein Qualitative Tests

• Focuses on identification and tests performed.

Elements of protein

• Carbon, hydrogen, oxygen and nitrogen with protein powder heated converting gasses, moisture etc which gives by products.
• Can test color chnges for sulfur by lead, nitrogen by litmus with presence for hydrogen.

Ninhydrin

• Gives blue/purple by with ammonia or yellow with proline.
• Includes steps to add solutions and shake vigorously.

Biuret test uses

• Copper form colored rxns but no single/die.
• Includes copper(II) reagent reacting with lone on N and peptide.
• Peptides measured with a reagent and polypeptide test.

Xanthoproteic test

• Notes a color, procedure for tests on solutions like those with nitro acid to mix, boil/cool to observe.
• Guanido to add drops from to solutions to produce arginine.