MBC 223 Level 3 Laboratory Manual 2024-2025 PDF

MBC 223 Level 3 LABORATORY MANUAL 2024-2025G Prepared by Osama Fahmi Al-Kadomi and Mohamed Babiker  Dear Student These are instructions you should follow for your own safety There are important points that should be followed...

MBC 223 Level 3 LABORATORY MANUAL 2024-2025G Prepared by Osama Fahmi Al-Kadomi and Mohamed Babiker  Dear Student These are instructions you should follow for your own safety There are important points that should be followed by all students when they come to the Biochemistry Laboratories:  Come to the laboratory ON TIME without any delay.  Always bring the laboratory coat and lab uniform being assigned by your deanship and wear it before interring the lab to protect your self against any chemical substances. No body is allowed to enter the lab without these lab uniforms.  No food or drinks are allowed inside the laboratory. All pagers and mobile telephones should be turned off while in the laboratory.  On the list for presence, sign only your name. Do not attempt to sign for other students. If you do so, you will be considered absent.  Most of the experiments require calculations. Therefore, bring your calculator with you to complete the results, which should be checked by the laboratory supervisor before your leave.  After finishing the experiment, you are required to clean your working area by pouring the used solutions in the sink and moving dirty glassware to their place.  Reports for the experiment should be given at the lab period.  If you were absent at any laboratory session without an excuse you cannot submit a report for the experiment. 2  ‫ْٕاك ذعييَاخ يجة ٍزاعاذٖا ىذ‪ ٙ‬دض٘رك ٍخرثزاخ قسٌ اىنيَياء اىذي٘يح اىطثيح‪:‬‬ ‫‪.1‬الحضور في وقت المختبر حسب الجدول الدراسي المعلن‪.‬‬ ‫‪.2‬إحضار معطف (‪ )Lab Coat‬المختبر وكذلك الزي الموحد المتفق ليةق معقك مقب بداةق كق‬ ‫مختبققر وإرتدا ق قب ق الققدخو ليمختبققر وذلققك لحماة ق م بنققك م ق المققواد المنققتخدم ققا‬ ‫التجربق ‪.‬حةق لق ةنقم ألحقد بالقدخو مقا لقل ةيتقزل باليبقاس الرنقم وذلقك حنقت تعيةمققا‬ ‫لمادة الكية ‪ ,‬رجو م الجمةب االلتزال بيبس الكمامه م عا لإلحراج المتباد ‪.‬‬ ‫‪.3‬عدم اآلكل والشرب داخل المختبر وكذلك إطفاء الجوال‪.‬‬ ‫‪.4‬عندما يطلب منك توقيع حضورك للمختبر‪ ،‬وقع عن نفسك فقط‪ ،‬وإال فقدت حقك في الحضور‪.‬‬ ‫‪.5‬إحضار الحاسبة معك إثناء حضورك للمختبر‪ ،‬وذلكك حتكي يتسكني لكك عمكل الحسكابات المتعلقكة‬ ‫بالتجربككة ليككتم مشككامدتقا مككن قبككل مشككر مككاد المختبككر‪ ،‬حيككل لككن يسككمب لحككد بككالخرو قبككل‬ ‫مشامد النتائج‪.‬‬ ‫‪.6‬عند االنتقاء مكن التجربكة ومشكامد النتكائج مكن فيكل مشكر مكاد المختبكر‪ ،‬وقبكل خروجكك مكن‬ ‫المختبكر‪ ،‬عليككك برمكي المككواد المسككتقلكة فكي سككلة المقمكك‪،‬ت‪ ،‬وككذلك رمككي مككا تبقكي مككن المككواد‬ ‫السائلة في الحوض المائي‪ ،‬ووضع الزجاجيات في السكلة المخصصكة لكذلك ن وذلكك حتكي يبقكي‬ ‫المكان نظيفا ليتم إستعمالة من قبل زم‪،‬ئك في المجموعات الخرى‪.‬‬ ‫‪.7‬‬ ‫‪.8‬ةتل تقدةل التقرةر العمي المتعي بالتجرب ف هاة ك مختبر او حنت ما ةقتل االتفقا ليةق ‪,‬‬ ‫وال ةتل التوقةب إال بعد تنيةل التقرةر لمدرس المادة‪.‬‬ ‫‪.9‬في حالة غيابك عن المختبر‪ ،‬وبدون عذر رسمي يتم قبوله مكن قبكل مكدرل المكاد ‪ ،‬ال يحك لكك‬ ‫تقديم تقرير التجربة‪.‬‬ ‫مع تمنياتنا للجميع بالتوفي والنجاح‬ ‫‪3‬‬  ‫ذعييَاخ ٕاٍح ىالٍرذاّاخ اىعَييح‬ ‫ىذ‪ ٙ‬دض٘رك االٍرذاّاخ اىعَييح األسث٘عيح ٗاىْٖائيح يزج‪ٍ ٚ‬زاعاج ٍا ييي‪:‬‬ ‫‪ -1‬االىرزاً تاىيثاس اىزسَي اىَقزر ىل ٍِ قثو مييرل ٍع ٍعطف اىَخرثز(ىِ يسَخ‬ ‫ألدذ تذخ٘ه االٍرذاُ تذَّٖٗا)‪.‬‬ ‫‪ -2‬اىذض٘ر في اى٘قد اىَعيِ ٗاالىرزاً تاىَناُ اىَعيِ دسة اىق٘ائٌ اىَثثرح عي‪ٚ‬‬ ‫أت٘اب اىَخرثزاخ ‪ /‬ى٘دح اإلعالّاخ‪.‬‬ ‫‪ -3‬إدضار انمواد األساسيح انتانيح نالمتذان‪:‬‬ ‫آنح داسثح‪.‬‬ ‫أ‪-‬‬ ‫قهم دثر جاف ازرق ‪ /‬أسود‪.‬‬ ‫ب‪-‬‬ ‫قهم رصاص‪.‬‬ ‫خ‪-‬‬ ‫مسطرج‪.‬‬ ‫ث‪-‬‬ ‫مذايح‬ ‫ج‪-‬‬ ‫ساعح يدويح او ساعح إيقاف‪.‬‬ ‫ح‪-‬‬ ‫نه يسمخ تاستعمال انجوال أحناء االمتذان‪ ,‬ديج سوف يتم سذة‬ ‫ر‪-‬‬ ‫انجوال في تدايح االختثار مه كم طانة وإيداعه في مكان خاص‪.‬‬ ‫ديث ىِ يرٌ ذذاٗه ٕذٓ اىَ٘اد تيِ اىجَيع‪ ,‬ىذا يزج‪ ٍِ ٚ‬اىجَيع االىرزاً تَا‬ ‫ٗرد أعالٓ ىَا فئ ٍصيذح اىجَيع‪.‬‬ ‫ٍع ذَْياذْا ىيجَيع تاىر٘فيق ٗاىْجاح‬ ‫ٍذرسي اىَادج اىعَييح‬ ‫‪4‬‬ Week No.* Exp. No. Topic 1 0 Orientation 2 1 Laboratory safety 3 2 Most commonly used instrument in the laboratory/ Part-1: 1- Pipettes 4 3 Most commonly used instrument in the laboratory/ Part-2: 2- pH meter 5-6 4-5 Most common used instrument in the laboratory/ Part-3-A: 3- Spectrophotometer: Maximum absorbance 7 6 Most common used instrument in the laboratory/ Part-3-B: 3- Spectrophotometer: Demonstration of Beer-Lambert's law 8 7 Determination of total protein concentration Determination of total protein by Biuret method 9 Quiz1 10 8 Determination of serum albumin 11 Determination of LDH activity 12 9 Determination of serum glucose 12-13 10-11 Determination of total lipids 1) Determination of serum cholesterol 2) Determination of serum triglycerides 14 Quiz2 15 11 Final Practical Exam * Subjected to change, you will be informed later about the new time. 5  Lab No.1 Introduction: LABORATORY SAFETY Introduction: The function of the clinical chemistry laboratory is to perform qualitative and quantitative analysis on the body fluids such as blood, urine and spinal fluid as well as feces, tissues, calculi and other materials. If the results are to be useful to the physician in the diagnosis and treatment of disease, the test must be performed as accurately as possible. This requires good instrumentation. Essential factors include purity of reagents, solvents and suitability of containers, reliability and quality of measuring devices and methods. There should also be an understanding of chemical reactions and hazards present in each test due to materials, equipment and the reaction of the materials. There is a simple rule. This is WHAT YOU DON’T KNOW CAN HURT YOU. By following basic safety precautions you can minimize the probability and consequences of an accident. Various types of hazards are encountered in the Clinical Laboratory. They must be Identified, Labeled and Dealt with. The types of hazards include: - 1- Biological IF 2. Chemical we 6 3. Electrical 4. Fire 7  Identification and Classification: There are 9 classes of hazardous material, which have been identified and classified by the United Nations (UN). These are: - Class Type 1 Explosives 5.11.61 2 Compressed Gases 8 3 Flammable Liquids 4 Flammable Solids 9 5 Oxidizer Materials 6 Toxic Materials Wish 7 Radioactive Materials 10 8 Corrosive Materials 9 Miscellaneous Materials disswimi 11  Labeling of Hazards: Chemicals must carry labels based on the UN classification. These are described as below. These are diamond – shaped, with a digit imprinted on the bottom corners that identify the UN hazard class (1 – 9). Get The hazard is identified more specifically in printed words placed along the horizontal axis of the diamond. at These supplement the identification of the hazardous material on the label. The artwork appears in the top half of the diamond. 12 To operate a Clinical Laboratory safely, prevention of laboratory employee exposure to infectious agent is essential. Exposure to infectious agent results from the following. 1. Accidental puncture with needles. 2. Spraying of infectious materials. 3. Centrifugal accidents. mainfunction o t f 13 4. Cuts or scratches from contaminated vessels. Universal Precautions: This specifies how clinical laboratories should handle infectious agents. In general all human blood and other potentially infectious materials must be treated as if they are known to contain infectious agent. Some of the more serious agent is:- 1. HBV (Hepatitis B viruses). T.WS 46J 2. HIV (Human Immunodeficiency Virus). jul 26 WI.INT 3. Blood-borne pathogens. 4. All specimens; Blood, serum, plasma, blood products, vaginal secretions, semen, Cerebroespinal fluid (CSF), sinovial fluid, etc. 14 Personal Protection Equipment: 15 Laboratory personnel must use Barrier Protection to prevent skin and mucous membrane contamination. These barriers are known as personal protection equipment. They include among others the following. 1. Hand gloves 2. Gowns 16  3. Laboratory coats 4. Face shields or masks 17 5. Eye protection 6. Mouthpieces to 18 7. Resuscitation bags 8. Pocket masks 19 9. Other ventilation devices 4 1 69 20 Safety recommendations: 1. Never perform mouth pipetting. 2. Do not mix infectious materials by bubbling air through them. 3. Use barrier protection whenever necessary. 21 4. Wash hands frequently and whenever gloves are removed. 5. Avoid using syringes whenever possible. i 6. Dispose of needles in rigid containers. 22 7. Do not eat, drink or smoke in the laboratory. 8. Clean and decontaminate all surfaces with appropriate disinfectants. 9-Use biohazard disposable techniques. 23 Proper storage and use of chemicals is essential to avoid dangers resulting in burns, explosions, fires and toxic fumes. The followings are guidelines in handling laboratory chemicals. 1. Always handle chemicals producing toxic vapors or fumes in a fume hood. 2. Bottles of chemicals and solutions should be transported with a trolley. 3. Glass containers with chemicals should be transported in rubber or plastic containers that protect them from breakage and contain the spill if breakage occurs. 24  4. Never holds a bottle by its neck. 5. Acids must be diluted through slow addition to water. Never add water to concentrated acid. water to acid Hinia 6. All bottles containing reagents must be labeled properly with the name and concentration of the reagent, the initials of the person who prepared it and the date of preparation. If necessary the expiration date should be included. 25 7. Handle mercury with special care. 8. Cylinders of compressed gases require special care in handling. 26 Fire hazards: The best solution to fire problems is prevention. Every laboratory should have the necessary equipment to extinguish or confine a fire in the laboratory. The following are the necessary equipment to fight fire. 1. Safety showers 27 2. Fire blankets 3. Fire Buckets Sand 3. Fire extinguishers 28 Classification of fires and fire extinguishers requirements. Type of hazard Class of fire bded Extinguisher 1. Ordinary combustible A Water, Dry chemical foam, loaded steam. 2. Flammable Liquids/Gases B Dry chemical, CO2, loaded steam, Halon1211 or 1301 foam. 3. Electrical Equipment C Dry chemical, CO2, Halon 1211 or 1301 foam. 4. Combination of Hazards A & B Dry chemical, Steam foam. A & C Dry chemical B & C Dry chemical, Co2, Halon A,B & C Triplex dry chemical 29 30 Chemical Safety Symbols A guide to meanings of common chemical safety/hazard symbols. The ones shown are the European/international standard. Poisonous Stay away from foodstuffs Harmful material to be kept away from edible material. Environmental hazard Dangerous when wet Relatively rare with laboratory This generally means that it will react chemicals (most of which pose some fairly violently with water... environmental hazard if not got rid of correctly), these require particular care to be taken on disposal. Corrosive Flammable Gas Avoid contact with the skin. Bear in Safety symbol used for the transport or mind that these can (under some storage of a flammable gas. circumstances) rust chemical cupboards. Explosive Non flammable gas Again, fairly self-explanatory, though Safety symbol used in the transport of fairly seldom seen in the average lab. non flammable (and hence often non Bear in mind that noise and movement hazardous, at least out in the open) can also trigger explosion (not just gases. sparks/flames!). Flammable or extremely Organic Peroxide flammable Chemical safety symbol used in the Chemicals to be stored in flame- transport and storage of organic resistant cupboards. Volatile solvents peroxides. can be a particular problem as they are prone to spread around from unsealed containers. Irritant or Harmful Corrosive This symbol covers a wide range of Transport of corrosive materials - again, (sometimes relatively minor) hazards - avoid contact with the skin. with precautions such as avoid contact with the skin, do not breathe. Oxidizing chemical Inhalation Hazard Oxidizing chemicals are materials that Inhalation hazard transport/storage spontaneously evolve oxygen at room symbol. temperature or with slight heating, or that promote combustion. To be kept away from flammable chemicals at all costs! 31 Poisonous Gas Marine Pollutant Used for transport of a poisonous gas - Marine pollutant - do not dispose of in on gas cylinders, or sometimes as an sewer system. indicator on vehicles. Miscellaneous danger Explosive Catch-all symbol for all other dangers Used in the transport of explosive (usually specified in the space). materials. Poison Spontaneously Combustible More general symbol for the transport Spontaneously combustible material of poisonous materials (not necessarily (treat with great caution!...). a gas). Flammable Solid Flammable Liquid Flammable solid. Used in the transport of flammable liquids. 32  LAB NO. 2 INTRODUCTION TO THE MOST COMMONLY USED INSTRUMENTS IN THE LABORATORY(1) OBJECTIVES For students to become familiar with the use of the following: 1. Pipettes and pipetting techniques. 2. pH -meter. 3. Spectrophotometer I- Pipettes These are designed to deliver the exact amount of solution. Has only one point of graduation, i.e. it is used to deliver only one volume such as 0.5 ml, 1 ml, 2 ml…etc. Volumetric pipettes contain a bulb in the middle of the stem. They are considered very accurate type of pipettes and are usually used in preparing standard solutions or in measuring blood or serum. When delivering the solution of the volumetric pipette, the remaining drop at the tip should not to be blown out (Fig. 1- 1.a) These are two types: i). Mohr pipettes, which are graduated along the stem, but not at the tip. They are less accurate than the volumetric pipettes, but can deliver small fractions of total capacity of the pipette. (Fig. 2-1.b). ii). Serological pipettes, which are calibrated along the stem including the tip. This type of pipette is usually used in preparing dilutions. (Fig. 1-1.c) A B C Fig.2-1: Glass pipettes; volumetric pipette (a) Mohr pipette (b), and Serological pipette (c) 33 B- Automatic Pipettes Are Two Types: 1- Adjustable pipettes: which can be calibrated to deliver any amount of solution which is written on the side of the pipette ;( e.g. 20 μl pipette can be used to deliver solutions from l μl to 20 μl, 100 μl can also be used to deliver solutions from 10 μl to 100 μl, Fig. 2-2.a) 2- Fixed volume pipette: which deliver only the exact volume which is written on the top of the pipette (e.g. 20 μl, 100 μl, 1000 μl, Fig. 1-2.b B A Fig. 2-2: Automatic Pipettes; adjustable (a) and fixed volume (b) Different sizes of pipette tips 34 Use of pipettes The instructor will demonstrate to the students the various types of pipettes and proper way of using them in the laboratory. In general, the following are some of the conditions, which should be followed when using a pipette: A –Use of glass pipettes: 1. Do not use mouth. Use pipette bulbs in aspirating solutions. 2. Pipettes must be clean from any dirt or grease that might be in or outside the pipettes. 3. After aspirating the required volume, wipe off the surface of the pipette with tissue paper. 4. Always read the bottom of the meniscus for clear solutions, and top for colored or viscous ones. 5. The graduation point and your eyes should be in a horizontal position. 6. When delivering solutions, let the tip of the pipette touch the inner surface of the container and let the solution flow by capillary action. 7. Always choose the proper pipette in measuring the required volume; i.e., use smaller pipette for small volumes 35 B- Use of automatic pipettes 1- Select the proper pipette for the desired volumes for the sample or reagents mentioned in the procedure (yellow labeled pipette for the small volumes from 10 µl to 100 µl, while the blue labeled pipette for the volumes from 100 µl to 1000 µl). 2- Adjust the pipette according to the volume needed in the experiment. 3- Fix the proper tip to the pipette and tight very well. 4- All the pipettes have the same procedure for use, the have two steps for aspirating and delivering the solutions. 5- Press the pipette plunger with your finger to the first step outside the solution, then put the pipette into the sample / reagent in vertical positions. 6- Release your finger solely for aspirating the sample / solution. 7- Transfer the pipette in a vertical to the test tube and put the tip inside the edge of the tube, press the pipette plunger to the first step to deliver the solution, and press the plunger to the second step to assure that all the sample / reagent is delivered into the test tube. 8- Press on the pipette ejector, to eject the used tip into the trash. 36 Setting the volume: The counter displays three figures to be read from top to bottom. Additional to the figures on the lower wheel are printed graduations to enable a volume setting in the range increment of ach pipette model. Yellow Pipette Measurement Range = 10 – 100 µl (0.01 - 0.1 ml) 1 0 0 0 5 1 0 0 0 100 µl (0.1 ml) 50 µl (0.05 ml) 10 µl (0.01 ml) Blue Pipette Measurement Range = 100 – 1000 µl (0.1 – 1 ml) 1 0 0 0 5 1 0 0 0 1000 µl (1 ml) 500 µl (0.5 ml) 100 µl (0.1 ml) White Pipette Measurement Range = 1000 – 5000 µl (1 – 5 ml) 5 2 1 0 5 5 0 0 0 5000 µl (5 ml) 2500 µl (2.5 ml) 1500 µl (1.5 ml) 37  Conversion unite 1g = 1000 mg c aka we.de 1L = 1000 ml IIeW dm 1L = 10 dl 86 1 dl = 100 ml According to above information convert the following values: 10 g/dl to g/l =100g/l 750 mg/l to mg/dl = 75 mg/dl 855mg/dl to g/l moog obs =8.55 g/l 112 Wg 180 mg/dl of glucose to SIU mmol/l re weightin mol gram = mg/dl *10/ mw = 10 mmol/l0 molecular wight 180 mg It mW 18g dl L 18g 10 180 mmol 6 38  LAB NO. 3 INTRODUCTION TO THE MOST COMMONLY USED INSTRUMENTS IN THE LABORATORY(2) II-- pH-meter pH- meter is an instrument that is used to measure the pH (acidity or alkalinity ) of any solution.. It is composed of the following: 1. Combination electrode: a) Glass-bulb electrode b) Reference electrode. c) Temperature electrode. 2. Sensitive meter or measuring device. PH log H me Fig.3-1: pH meter Alternative methods for determining the pH of solutions are: 1- Indicators: indicators are materials that are specifically designed to change in color when exposed to different pH values. The color of wetted sample paper is matched to a color on a color chart to infer a pH value. pH paper is available for narrow pH ranges (for example, 3.0 to 5.5 pH, 4.5 t0 7.5 pH and 6.0 to 8.0) , and fairly wide pH ranges of 1.0 to 11.0 pH. However, the pH paper is typically used for preliminary and small volume measuring. It cannot be used for continuous monitoring of a process. Though pH paper is fairly inexpensive, it can be attacked by process solutions, which may interfere with the color change? 39 2- Colorimeter: this device uses a vial filled with an appropriate volume of sample, to which a reagent is added. As the reagent is added, a color change takes place. The color of this solution is then compared to a color wheel or spectral standard to interpolate the pH value. The colorimeter can be used for grab sample measuring, but not continuous on-line measuring. It is typically used to determine the pH value of water in swimming pools, spas, cooling towers, and boilers, as well as lake and river waters. 40  Principle of pH meter; if Indian The glass-bulb electrode (Fig.3-2) contains a solution of a certain fixed pH or hydrogen ion concentration, usually 0.1 N HCl. When the electrodes are placed in a solution of unknown pH, an electric potential is produced between them, dependant on the hydrogen ion concentration of the solution in the glass bulb, and the hydrogen ion concentration of the test solution). This is measured with the aid of the reference electrode. The potential of the reference electrode is compared to the potential of the pH electrode (the glass-bulb electrode), and is measured by means of a meter. The meter is an electronic voltmeter (potentiometer) that measures millivolts (mV). Results are read from an arbitrary pH scale of 0-14 pH units, or from a millivolt scale. A reading of 0 mV is equivalent to a pH of 7.0. The most important step before using a pH is its calibration using a known pH solution. The instructor will demonstrate to you how to calibrate and how to use the pH meter properly. glass bulb Fig.3-2: The Components Of pH Electrode 41 Procedure for the preparation / maintenance and calibration of pH meter Step 1- Remove the electrode from its short term storage solution. Step 2- Rinse the electrode with distilled water (A). Tap off the residual rinse water and blot the electrode dry with a Kim wipe or lint free cloth (B). 5 I I 55937 HIM 42 Step 3- Expose the fill hole by removing the fill the hole plug. Step 4- Verify that the fill solution is not more than one inch (2.5 cm) from the fill hole. If additional fill solution is needed go to Step 5. If the fill solution is within one inch of the fill hole go to Step 6. 43 Step 5- Using a transfer pipette; add fill solution until the level is within one inch of the fill hole. Not all pH electrodes require the same fill solution. The correct fill solution must be used for the electrode to function properly. Check the owner’s manual to verify what the correct fill solution for your electrode is. 44 Step 6- Place the electrode in a pH 7.00 buffer solution (A and B). Press the calibration button on the pH meter (C). Wait for the buffer value to appear on the meter display (D). Step 7- Remove the electrode from the buffer (A). Rinse the electrode thoroughly with distilled water (B). Blot the electrode dry with a kimwipe or a lint free cloth (C). 45 Step 8- Place the electrode in a pH 4.00 buffer solution (A and B). Press the calibration button on the pH meter (C). Wait for the buffer value to appear on the meter display (D). Step 9- Remove the electrode from the buffer (A). Rinse the electrode thoroughly with distilled water (B). Blot the electrode dry with a kimwipe or a lint free cloth (C). 46 Step 10- Place the electrode in a room temperature, representative portion of the sample being measured (A). Press the read button on the meter (B). Wait for the pH value to lock onto the meter screen (C). Note that the actual sample temperature is also displayed. Record the sample pH. Step 11- Remove the electrode from the sample. Rinse the electrode completely off using distilled water. For viscous samples it may be necessary to rinse the bulk of the sample off using tap water, and then rinse with distilled water. This step is critical to ensure other samples and buffer solutions are not contaminated with residual product clinging to the electrode. Blot the electrode dry with a kimwipe or lint free cloth. 47 Step 12- Repeat Step 8 through Step 11 for each additional measurement made. Continue to Step 13 when all samples have been measured. Step 13- Close the fill hole by replacing the fill hole plug. Step 14- Replace the electrode into the short term storage solution. 48 Note: these steps will be demonstrated to you during lab period Note:  pH standards are based upon room temperature samples. The pH of a sample may vary significantly depending upon temperature. An ATC (automatic temperature compensator) will not correct for the specific product temperature variation. An ATC is built to compensate for the electrode performance variations due to temperature, and is not able to compensate for the sample variations. For this reason it is necessary for samples to be as close to room temperature (250C) when measurements are made. Experiment: You will be provided with different solution and unknowns in order to measure the pH of each, record the results. 49  LAB NO. 4 INTRODUCTION TO THE MOST COMMONLY USED INSTRUMENTS IN THE LABORATORY(3) III- Spectrophotometry Spectrophotometer is one of the most commonly used instruments in the biochemistry laboratory; its main function is to estimate the concentration of many substances (carbohydrates, proteins, etc) in a solution. In order to understand the use and function of the spectrophotometer, some basic knowledge of light source should be known. Almost all biochemical experiments eventually use spectrophotometry to measure the amount of a substance in solution. Spectrophotometry is the study of the interaction of electromagnetic radiation with molecules, atoms, or ions. EG Light or electromagnetic radiation has a wave and particles nature. The wavelength.λ, of light is the distance between adjacent peaks in the wave. The frequency, ν, is the number of waves passing a fixed point per unit of time (Fig. 4-1) e WE Fig.4-1: Wave nature of light These parameters can be further defined by the equation: λ = c/ν where c is the speed of light Photons of different wavelength have different energies. These energies can be calculated by the equation: E = hc / λ =hν Where h is Plank’s constant. Therefore, the longer the wavelength, the less energy the light has and vice versa. 50 Table 4-1 shows the relationship between the wavelength of light and the common types of electromagnetic radiation. As you can see, those regions where the wavelength is very short correspond to the types of radiation that you know are powerful and often harmful, such as X-rays, γ-rays, and ultraviolet radiation. Most compounds have a certain characteristic wavelength or wavelengths of light that they absorb. This process can be diagrammed as in Figure 3-2. Thus the solution looks green to us because green light (blue and yellow) is transmitted while the red light is absorbed. A solution may contain many compounds that absorb at many different wavelengths, but if a compound we are interested in absorbs at a unique wavelength, we can determine its concentration even in a solution of other compounds. Table 4-1: Classification of Electromagnetic Radiation Radiations Fig.4-2: Absorption of light by a solution. 51 Beer –Lambert Law t.sk Consider a ray of light of initial intensity (I0) passing through a solution in a transparent vessel. Some of the light is absorbed, so that the intensity of the transmitted lights (I) is less than I0. wakens s't'd www os4ibsssb ww8iwsosb 65WIbW4 SHALINI WITH ibd The ratio of I to 10 is known as transmittance (T) and depends upon the path length of the light through solution. So transmittance is a measure of the amount of light that is allowed to pass through a solution. The Lambert's Law states that when a ray of monochromatic light (single wavelength) passes through an absorbing medium, its intensity decreases as the length of the medium increases. A similar Law of Beer states that when a ray of monochromatic light passes through absorbing medium, the intensity decreases as the concentration of the absorbing medium increases. These two laws are combined in the form of Beer - Lambert law and is expressed as: A = abc Q 180 ww Ed sina.com iosnei S EW III 64 I box IIII WITH 52  6 aug Log I0 / I = abc transmitted light incidentlight I I b 350 700 nm. Fig.4-3: Relationship between I, I0, And C for A Solution Absorbing Monochromatic Light Where: I0 : intensity of incident light. I : intensity of transmitted light. a : extinction coefficient(I M in 1 cm path). b : path length through solution(1 cm path). c : concentration of absorbing solution log I0 / I is usually called the absorbance, and is abbreviated A This Law: A = abc Is called the Beer-Lambert Law 0 As the concentration of the colored solution in the cuvette is increased, I and consequently %T, is decreased. The relationship between concentration and %T is not linear, as shown in Figure 4-4, but if the logarithm of %T is plotted against the concentration, a straight line is obtained. lambent law Beerlaw 53  4 Is Fig.4-4: Percent transmittance (%T) as a function of concentration III Fig.4-5: Absorbance (A) As a Function of Concentration Fig.4-6: Note that the Law is not obeyed at high concentrations 54 Reagent blank: not 0 WWI If I A reagent blank is a control in which everything is included except the substance for which we are testing. One problem often encountered in spectrophotometry is that there is an absorbance at a given wavelength not due to the substance of interest. We handle that by mixing up all of the solutions in a tube except that substance and then reading the absorbance. The absorbance of the reagent blank is then subtracted from the other readings, or directly we zero the spectrophotometer directly with the reagent blank, and we take the readings of the substance(s) which will be the final reading already subtracted from the reagent blank. III. Components of the Spectrophotometer As mentioned earlier, spectrophotometer is an instrument that is capable of measuring absorbance or transmittance of light (Fig.2-6). Spectrophotometer is generally composed of the following major parts: 1- Light Source: This part of the instruments emits visible or UV light depending on the source itself. For example a tungsten lamp emits light from 400-750 nm, while a deuterium lamp emits in the UV region. 2- Monochromator: This part is similar to a prism or a filter which function in isolating only one single light, i.e. one wavelength. 3-Entrance Slit: This part functions in passing a very fine beam of isolated wavelength. 4-Exit Slit: This part functions in passing a very fine beam of isolated wavelength 5- Cuvette holder: This is where the sample of the colored solution is inserted. The cuvette can be either in the round shape like a test tube or square form.When measuring absorbance in the visible region, the cuvette is usually made of glass, while in measuring absorbance at the low UV region cuvettes are made of quartz. 6- Photo Cell: This part converts light energy to electric energy so it can be measured by the measuring device. 7- Galvanometer This is where the electric pulses are received and converted on scale either to absorbance or to transmittance units. Note: all the above mentioned are the basic components of the spectrophotometer are summarized in (Fig.4-8) 55 Fig. 4-7-a: Spectrophotometer (model Ultrospec-II) man min's rostral.MU 0 a is IF Cotton a.mn i ii o ik jfgfn f C was.ws IÑdwi i T d I T Fig. 4-7-b: Spectrophotometer (model Novaspec-III) 56 Entrance slit Exit slit 1 2 3 4 5 6 7 Fig.4-8 : Components of the Spectrophotometer 1= Light Source 2= Entrance Slit 3= Monochromator 4= Exit Slit 5= Sample Cuvette holder 6= Photo cell or Detector 7= Galvanometer 57  EXPERIMENT-1 To determine the wavelength of maximum absorption of two dyes. Objectives: By the end of this practical the student should be able to: 1. Determine the absorbance pattern of two different dyes; Bromophenol Blue Farid and Methyl orange over the wavelengths 350nm to 700nm. 2. Record these absorbencies and the wavelengths. Reagents and Materials: 1. Bromophenol Blue (10mg /L). 0 66 2. Methyl Orange (10mg/L). 3. 4. Cuvette. Distilled Water- Blank 09.810414min merit 5. Spectrophotometer. Distal tibia Procedure: water www.I 1. Switch on the spectrophotometer and leave to stabilize for 10 minutes. 2. Set the wavelength at 350 nm. 5025 3. Using distilled water as blank set absorbance (A) at zero. 4. Remove the blank and put the test Bromophenol Blue solution into the holder.jb.J.mg 5. Record the A over the range of wavelengths 350 to 700 at 50 nm intervals, zero after each reading. 6. Repeat steps 3 to 5 using Methyl Orange solution. Results: # Wavelength (nm) Absorbance Bromophenol Blue Methyl Orange 1 350 2 400 3 450 4 500 5 550 6 600 7 650 8 700 58  Experiment-2 (STANDARD CALIBRATION CURVE) Objectives: By the end of the practical students should be able to: 1. Prepare serial dilutions of 10 mg/L Bromophenol Blue. 2. Calculate the concentration of the dye in each dilution. 3. Measure the absorbance of each dilution. 4. Plot the standard calibration curve (absorbance against concentration). Reagents: 1. 10 mg/L Bromophenol Blue. 2. Distilled water. Procedure: Tube 1 2 3 4 5 6 Bromophenol Blue (ml) UI - 1.0 2.0 3.0 4.0 5.0 Distilled water (ml) V2 5.0 4.0 3.0 2.0 1.0 -  Add the reagents as indicated, and mix well.  Adjust the spectrophotometer to zero at 591 nm against tube # 1 as blank.  Plot the absorbance against the concentrations.  Draw the calibration curve Absorbance of Bromophenol Blue === Conc. of Bromophenol Blue( mg/L) === 59  LAB NO. 5 DETERMINATION OF TOTAL PROTEIN CONCENTRATION by Biuret method Introduction: Serum total protein, also called plasma total protein or total protein, is a biochemical test for measuring the total amount of protein in blood plasma or serum. Protein in the plasma is made up of albumin and globulin. The globulin in turn is made up of α1, α2, β, and γ globulins. These fractions can be quantitated using protein electrophoresis, but the total protein test is a faster and cheaper test that estimates the total of all fractions together. The traditional method for measuring total protein uses the biuret reagent, but other chemical methods such as Kjeldahl method, dye-binding and refractometry are now available. The measurement is usually performed on automated analysers along with other laboratory tests Beer- Lambert's Law As indicated from the previous experiment, Beer-Lambert Law states that: A = abc From this formula, one should know the value of (a) in order to be able to calculate the concentration of the substance being assayed. Usually in the lab the value of (a) is not given. So to be able to determine the unknown concentration, a known standard should be assayed along with the unknown sample. The formula that is used in the laboratory to calculate the concentration of the unknown sample is derived from the law is: A unknown C unknown = X C standard A standard Another way of finding the concentration of any substance is to draw a standard curve. This method requires measuring the absorbance of several known concentrations of standard; the absorbance is plotted against concentration on a graph paper. The concentration of the unknown sample can be measured from this graph. 60  The Beer - Lambert's law states that absorbance is directly proportional to the concentration of substance if the light path is kept constant. To achieve this relationship, the following conditions must be followed: ` 1- Monochromatic light must be used through all the experiment. 1- Clean glass tubes or cuvettes must be used. 2- Homogenous tubes must be used, i.e. same diameter and same thickness. 3- Colored solutions of substances must also be homogenous, i.e. samples must be mixed well 4- Concentration of the substance should not be very high that gives a very dark color, which adversely affects absorbance value. In such cases the sample should be diluted to reduce the absorbance, and the result is multiplied by dilution factor. So, if any of the above conditions is not achieved, linearity of Bee-Lambert's law will not be maintained. 61  Determination of Total Protein by Biuret Method Principle: Alkaline media Protein in sample + Biuret reagent blue-violet complex (2 or more peptide bonds) (Copper sulfate, blue color) (Biuret reaction) The intensity of the color produces is directly proportional to the total protein concentration in the patient’s sample The biuret reagent contains sodium potassium tartarate to form a complex with cupric ions and maintain their solubility in alkaline solution. Iodide is included as an antioxidant. Fig.5-2: Biuret Reaction (!) blue-violet colored Complex (2) 62 Clinical significance to A- Total protein may be elevated due to: Ye 1- Chronic infection (including tuberculosis) 2- Adrenal cortical hypofunction 3- Liver dysfunction 4- Sarcoidosis 5- Dehydration (diabetic acidosis, chronic diarrhea, etc.) 6- Respiratory distress 7- Hemolysis 8- Alcoholism 9- Leukemia 8.5 B- Total protein may be decreased due to: 1- Malnutrition and malabsorption 2- Liver disease 3- Diarrhea 4- Severe burns 5- Loss through the urine in severe kidney disease 6- Low albumin 7- Low globulins 8- Pregnancy Reagents (Human method): Reagent Color reagent: Sodium hydroxide 200 mmol/l Potassium sodium tartrate 32 mmol/l Copper sulfate 12 mmol/l Potassium iodide 30 mmol/l Irritant R 36/38 Standard Protein standard concentration 8 g/dl (80g/l) Sodium Azide 0.095% Reagent preparation and stability: Reagent and standard are ready for use. They are stable even after opening up to the expiration date when stored at 2 – 25 0C. Contamination after opening must be avoided. Specimen: Serum, heparinized or EDTA plasma Stability in serum: Up to 1 month at 2-8 0C, 1 week at 15 – 25 0C. 63  LAB S Procedure 5 WI 020 Reagents Blank Standard Sample # Sample # ima Distilled Water ( μl ) Protein Standard ( μl) O 20 - - 20 - - - - Sample # ( μl ) - - 20 - Sample # ( μl ) - - - 20 Biuret Reagent ( ml ) 1.0 1.0 1.0 1.0 Mix well and let stand for 10 min at room temperature (RT). Adjust the spectrophotometer to zero at 546 nm (520-580) against blank. o Measure absorbance of unknown and standard. A final color is stable for 30 minutes. Record your results for further calculations Blank Standard Sample # Sample # Absorbance ==== Concentration (g/dl) ==== DJI Calculation: Absorbance of the Sample Total Protein (g/ dl) = x Standard Concentration Absorbance of the Standard Jw Wb Reference Value (Normal Value): Normal born babies 84.6 – 7.0 g/dl 8 00 Children from 3 years and adults 86.6 – 8.7 g/dl I Too Jma Blank Distalwater BinetReagent ProteinStD Reagent GWestic six _sa p 11 Reagent 4319481 salteB Reagent 64 normal Ji J 2 stjv.ci Jim rang I_Ws IIIa hw 1dm at