Laboratory Experiment 1 - Protocol PDF

MD100 – Medical Biochemistry I Lab Exercise #1: Introduction to Techniques – Use of Pipetman, Sample Preparation, and Dilutions Introduction Pipettes are basic tools of a fully equipped laboratory. They are used for handling and distributing small volumes of liquids. Typically, sample...

MD100 – Medical Biochemistry I Lab Exercise #1: Introduction to Techniques – Use of Pipetman, Sample Preparation, and Dilutions Introduction Pipettes are basic tools of a fully equipped laboratory. They are used for handling and distributing small volumes of liquids. Typically, sample volumes are measured in microliters, or 1/1000 of milliliter. e.g.: 1L = 1000ml, 1ml = 1000μl => This means that: 1L = 1000000μl OR 106μl Such small volumes require precise pipetting. Therefore, in order to perform your experiments accurately, it is essential that you have excellent knowledge of pipette use. The pipettes operate on the principle of air displacement and use detachable and disposable tips. There are different pipette models depending on the minimum and maximum volume they can carry. The maximum volume they carry is usually mentioned on the push button. EXPERIMENTAL PROCEDURE PROTOCOL Material/Equipment: 1. Pipetman (P1000, P200, P10) 2. Pipette tips 3. Distilled Water 4. 1.0 M CuSO4 solution 5. Cuvettes 6. Balance 7. Weighing boats 8. Spectrophotometer 9. Waste containers PART A: You need to weigh accurately the following volumes of distilled water and complete the tables. For the first table, use P1000. TABLE 1. Volume 1000 μl Volume 650 μl Volume 358 μl Reading 1 Reading 2 Reading 3 Average How precise are your measurements?.................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................... Remember that pipettes have a precision of 1%. Anything more than this is due to human error! For the second table, use P200. What do you see? TABLE.2 Volume 200 μl Volume 65 μl Volume 47.5 μl Reading 1 Reading 2 Reading 3 Average How precise are your measurements?............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ PART B: Sample Preparation Outline: Students will be organized into small groups where they will calculate and prepare solutions from solutes and solvents or by diluting denser solutions. Introduction: Solutions may be liquid or gaseous mixtures. In a liquid solution the liquid that forms the bulk of the solution volume is called the solvent. If the solvent is water, then the solution is called an aqueous solution. The substance dissolved in the solvent is called solute. Since living organisms consist mainly of water, the study of biochemistry involves to a large extent the study of aqueous solutions in various biochemical processes. Therefore, knowledge of the preparation of aqueous solutions is essential. The concentration of a solution refers to the amount of a solute per unit of solution and can be expressed as: Solution concentration = amount of solute / amount of solution The amount of both the solute and the solution can be expressed in units of mass or weight or volume. The main units of concentration Name Unit % by mass = mass/mass (%w/w) g of solute / 100 g of solution % By volume = volume/volume (%v/v) ml of solute / 100 ml of solution % Weight by volume = mass/volume (%w/v) g of solute / 100 ml of solution Molarity (Μ) mol of solute in 1L solution Examples % By mass (%w/w) = g of solute / g of solution * 100 It is often used for commercially available reagents. It is also used if the solution to be prepared is to be used at widely differing temperatures (because its concentration will be independent of changes in ambient temperature). 1. What is the % by mass concentration of 100g solution when 5 g of NaOH are dissolved in water? % By mass (w/w) = 5g NaΟΗ/100g * 100 = NaΟΗ 5% (w/w) % by volume (%v/v) = ml of solute/ml of solution * 100 Used when preparing liquid solutions 1. What is the % by volume concentration when 50ml of pure alcohol is dissolved in water to form a 100ml solution? % by volume (%v/v) = 50ml pure alcohol/100ml * 100 = 50% pure alcohol (v/v) % weight by volume (%w/v) = g of solute/ml of solution * 100 Used widely when preparing solutions with solids 1. What is the % weight by volume concentration of 100ml solution when 2 g of NaCl is dissolved in water? % weight by volume (% w / v) = 2g NaCl / 100ml * 100 = 2% NaCl (w / v)  M = mol of solute/1L 1. What is the Molarity of 100ml solution when 3mol CaCl2 is dissolved in water? First, we need to convert ml to L 1000ml = 1L thus 100ml = 0.1L M = 3mol CaCl2 / 0.1L = 30M CaCl2 Exercises: 1. What is the % by mass concentration of 25g solution when 0.5 g of NaOH is dissolved in water? _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ 2. What is the % by volume concentration when 50ml of pure alcohol is diluted with 100ml of water? _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ 3. What is the % weight by volume concentration of 50ml solution when 5g of NaCl is dissolved water? _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ 4. What is the Molarity of 100ml solution when 11g of CaCl2 are dissolved in water? ***First, we have to convert g to mol and ml to L ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ 5 PART C: Dilutions When a solvent is added to a solution, the amount of solute remains constant while the volume of the solution is increased. Thus, the final solution has a lower concentration than the original one. To create lower concentration solutions, we use the following dilution formula: C1*V1 = C2*V2 C1 = Initial Concentration V1 = Initial Volume C2 = Final Concentration V2 =Final Volume BEWARE: the units in both sites of the equation must be the same Exercise 1: Prepare 100ml of 0.1M NaOH from 1M NaOH solution. How many ml of 1M NaOH should you use? …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… Exercise 2: We want to prepare 50ml of 1X NaOH from 10X NaOH solution. How many ml of NaOH 10X do we need for this dilution? ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………………………………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………......................................................................................................................................... Exercise 3: You are given CuSO4 5Μ. If the MW of CuSO4 is 159.61, how many grams of this compound have been used for the preparation of 10ml CuSO4 5M? ………………………………………………………………………………………………… ………………………………………………………………………………………………… 6 …………………………………………………………………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………......................................................................................................................................... Exercise 4: Using CuSO4 5M make 10ml CuSO4 2Μ. How much volume of CuSO4 5M do you need? …………………………………………………………………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………......................................................................................................................................... Exercise 5: Using the solution CuSO4 2Μ that you have already prepared do the following parallel dilutions: 1/2, 1/5, 1/10, 1/20 and 1/50. The volume of each new solution should be 10ml. What is the concentration of each new CuSO4 solution? …………………………………………………………………………………………………...…………………………………………………………………………………………………..…………………………………………………………………………………………………..…………………………………………………………………………………………………..……………………………………………………………………………………………….… ………………………………………………………………………………………………….. ………………………………………………………………………………………………….. ………………………………………………………………………………………………….. 7