Your task is to write a lab book for Practical 2 : Lab book – Marking Criteria (10 marks in total): – Perfunctory elements (1 mark) Perfunctory elements to include are: descriptive contents page; page numbering; dates; no blank spaces; experiment titles. These are either present or not and marks will be awarded on completeness. – Completion of specific questions associated with practicals (2 marks) Answer all questions shown in the Practical manual! Marks will be awarded on completeness. – Aims (1 mark) Each part of a practical should have an appropriate aim, not just an overall aim for the whole practical. These should reflect your own belief about the purpose of the experimental procedure undertaken. – Methods (1 mark) This section should accurately reflect the methods used in the practical. You should not just copy the methods from the Practical manual. – Results (2 marks) Results should be recorded in an appropriate way, such as being tabulated or graphed properly. Be mindful of layout and providing units. Note that each table or graph should have an appropriate descriptor, that is each table requires a descriptive title and each graph requires a descriptive figure legend. For example, Table 1: Preparation of standard dilutions of DCIP. – Discussion and Self-reflection (3 marks) Include a detailed discussion of your results for each practical, e.g. include your thoughts on accuracy, precision and/or your confidence in the results presented. Where relevant, discussions should include reference to the broader literature, e.g. “this is similar to many other enzymes, such as enzyme X and enzyme Y, where there is a narrow pH range and where they have maximum catalytic activity. However, it contrasts with enzyme A and enzyme B, which have very broad pH tolerance. The latter come from bacteria, which are subject to a broad range of environmental conditions, therefore, enzyme A and B are likely required for activity over a wider pH range.” Include a Self-reflection section for each practical. In this section you need to first describe what you have learned from your experience engaging with the practical material, and then consider how you would change your behaviour in light of this new learning. You may also wish to consider what you have enjoyed (and why) and what areas you need to improve (and why). You may include a referenced diagram, photograph, or sketch to augment, but these are not to replace your own work/ provided data. It will contain a table of contents. All pages are to be consecutively numbered and blank space is to be at a minimum. The date is to be clearly shown. For laboratory experiments it will be used to note: • experimental background, aim, procedures that are proposed • data recorded and tabulated accurately and succinctly • fully labelled diagrams and drawings • potential sources of error noted/discussed • problems that are encountered, and problem solutions • conclusions and summaries Conclusions should be supported by the scientific literature and referenced in text (i.e. in discussion section, not at the end of the practical). At the end of each practical a brief self-reflections section about your progress in laboratory work will be recorded. Try to include what you have enjoyed, what you have disliked, what you feel confident in, and most importantly what you have had difficulties with and how you plan to resolve the difficulty. Note: No plagiarism Practical 2: Micropipetting Technique and Chemistry of Carbohydrates Introduction This practical will develop your understanding of micropipetting technique and your knowledge of carbohydrates – particularly the structures of monosaccharides and disaccharides. Practical 2 also introduces some analytical techniques that can be used to identify carbohydrates, such as Paper Chromatography, the Benedict’s test, and Osazone crystal formation. Pre-work: Answer all questions in details If I weighed out 1 g of water, what volume of water would I have?Write out in words the name for this symbol (as it relates to the prefix of a unit, not as a Greek letter): µ.If I had 100 µL of water, what percentage of 1 mL of water would this be?Would the answer to Q3 change if the word water were replaced by oil in the question above?What is the boiling point of ethanol?At 20˚C is glucose a solid or a liquid?In your laboratory book draw the structures of α-D-glucopyranose; β-D-galactopyranose; αD-mannopyranose; β-D-fructofuranose; maltose and sucrose (this means draw glucose, galactose, mannose, fructose, maltose and sucrose in their ring forms). You will notice that these structures are all very similar. However, it is possible to conduct a series of chemical tests to distinguish between these structures. This concept will be explored by trying to identify an unknown sugar. Therefore, a series of chemical reactions will be carried out on a number of known sugars and the results compared with the same tests on your unknown sugar. In addition, one of the tests (chromatography) will also be used to explore the nature of the sugars in a ripe banana.What is the stationary phase in paper chromatography?What is the mobile phase in paper chromatography?For the solvent in the chromatography jar, which component(s) is (are) hydrophilic? Which one is more hydrophobic?Why can sugars be stained by silver nitrate?What are the monosaccharides to be tested today?What are the disaccharides to be tested today?Which sample contains polysaccharides? Each group will be allocated an unknown sugar to investigate. Record the letter of the unknown sugar e.g. unknown sugar = C Note: please check the student Data File attached. Part A. Micropipetting Technique : Write Aim/Methods/Results /Discussion and self-reflection(Follow marking criteria and answer all questions ) Background It is typical in modern laboratories to make solutions in small volumes. A micropipettor is a key scientific device that enables this to be done and therefore it is essential that a graduate has adequate micropipetting skills. This practical is an opportunity to learn how to use a micropipettor correctly. A micropipette is used to precisely transfer volumes of liquid to make solutions. Volumes from 1 µL to 1000 µL are commonly transferred this way, but up to 5000 µL can be transferred depending on the pipette used. There are two basic types of micropipettors: (1) air displacement and (2) positive displacement. Air displacement pipettors are most commonly used and so it is essential that you learn to use these correctly and skilfully. The correct technique for using a micropipettor will be demonstrated (please see the demonstration video). Also consider the Check list for pipetting shown below. In your laboratory book, following the demonstration of pipetting, draw a fully labelled diagram of a micropipettor including its disposable tip. Check list for pipetting: Make sure the correct volume is selected by moving the adjusting knob until the required volume is shown on the dial (do not overwind or underwind the pipette).Use a correctly fitting tip and close the lid on the pipette tip box.Hold the pipettor vertically when sampling and expelling solution.Place the pipettor no more than 5 mm below the surface of the liquid being sampled or when expelling the liquid.The push button should be operated smoothly and slowly to avoid introducing air and inaccuracies into the dispensing procedure.Avoid contamination where appropriate by replacing tips Experiment 1 Pipetting Water (Do not forget to include the aim of and write out your methods for this experiment in your laboratory book .Choose either a P1000 or P200 pipettor for this experiment. Choose a volume of water to pipette. Make sure that it is above 50 µL. This could be e.g. 226 µL; 150 µL; 922 µL. The volume is entirely your choice. Make a note of this volume in your laboratory book. Using a balance (please use 4-digit analytical balance for this experiment if available) weigh the volume of water pipetted at your chosen volume. Repeat this so that at least a total of 12 recordings are made. Make sure you record which attempts were your partner’s. Record your observations. Based on the mass that you would expect the volume of water being pipetted to have, evaluate your accuracy, and also evaluate your precision. Determine if there is a difference between each person in the group. Experiment 2 Repeat the above experiment using oil. However, use a P200 pipettor set to an appropriate volume of your choice. Note: 1 mL of water and 1 mL of oil were weighed. Consider any issues mentioned that are associated with pipetting different types of liquids and how pipetting technique may need to be adjusted. Also consider the data provided in the Practical 2 Student Data file attached. Discussion of Procedure Based on your experience, describe how you would change your procedures to obtain the most efficient method for testing your pipetting. Would this be different for pipetting oil compared with water? Discussion Comment on your accuracy and precision in pipetting water compared with oil and justify your answer using evidence. How did your accuracy and precision compare with your partner’s? Consider that a difference was observed, explain the likely reason(s) for this and how could you test it? If you required an accurate volume of oil or other viscous substance, what technique would you avoid and what would you recommend? Explain your reasoning. Note: If taking this prac online please use the data provided in the Practical 2 Student Data file Comments on pipetting – try to keep the following in mind: Choose the correct sized pipettor for the volume to be transferred. (Hint: If the volume desired is less than 20% of the maximum volume for the pipettor, then a different pipettor should be used. e.g. 180 µL requires use of a P200 pipettor rather than a P1000 pipettor)Make sure the correct volume is selected.Use a correctly fitting tip.Understand the function of the “first” and “second” stops when using the pipettor push button.Hold the pipettor vertically when sampling and expelling solution.Place the pipettor no more than 5 mm below the surface of the liquid being sampled or when expelling the liquid.Operate the push button smoothly and slowly to avoid introducing aerosols, air and inaccuracies into the dispensing procedure.To avoid contamination, replace tips – use tip ejector to remove tips. Part B. Paper Chromatography Write Aim/Methods/Results /Discussion and self-reflection(Follow marking criteria and answer all questions ) Background on carbohydrates From the lecture material you will recall that carbohydrates have the general formula (CH2O)n where n is an integer of 3 or greater. If n=3 the carbohydrate is a triose. If n= 4, it is a tetrose, and so on. One important group of monosaccharides (or carbohydrates) consisting of only one unit is the hexoses (n=6). Biologically important hexoses are: glucose, galactose, mannose and fructose. In aqueous solutions, they exist predominantly in a ring form, which is in equilibrium with an open chain aldehyde form in the case of glucose, galactose and mannose, and ketone form in the case of fructose. Glucose and other aldehyde sugars (aldoses) form ring structures, cyclic hemiacetals. When such cyclic hemiacetals form, two different structures are possible depending upon the orientation of the OH group formed by ring closure at C1, the anomeric carbon. These two forms (α and β) are at equilibrium with each other in solution and the interconversion of α and β anomers is called mutarotation, which can be measured by change in rotation of polarised light. Disaccharides consist of two sugar units linked together by ether links. This link is called an Oglycosidic bond. Common linkages are C1 to C4 and C1 to C1. The orientation of the link at C1 often determines the properties of the disaccharide. The link may be either α or β. Disaccharides are hydrolysed to their constituents with mild treatment of acid. Common disaccharides are: maltose (α-D-glucopyranosyl-(1→4)-D-glucopyranose) found in barley grain; sucrose (α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside) found in fruit. Background on Chromatography Chromatography is a technique where different substances are identified or separated by the degree to which they prefer one environment or phase over another. In this case, one environment is a stationary phase (chromatography paper), and the other phase is a liquid which is mobile. A small amount of the sample sugar is spotted onto the paper and allowed to dry. This means that the sample sugar is incorporated in the stationary phase. The mobile phase is then placed at one end of the chromatography paper and allowed to absorb into the paper. It then flows over the sample sugar towards the other end of the paper. If the sample sugar is soluble in the mobile phase, it will move along with the mobile phase. If it is not at all soluble, then it will remain where it was originally on the filter paper. Some sugars are more soluble in the mobile phase than others and therefore move with the mobile phase to different degrees. This can be used to identify an unknown sugar or the sugars in fruit by comparing their degree of movement with that of known sugars. Materials 1x ripe banana per bench Purified water for dilution 10% Standard sugar solutions: glucose, galactose, fructose, sucrose, lactose and maltose 10% Unknown sugar solutions 1, 2 or 3 (choose one of these). 10 mL ethanol Solvent – the mobile phase (isopropanol:acetic acid:water (3:1:1 v/v/v)) Stain (saturated silver nitrate in acetone) Fixative (1g sodium hydroxide in 10 mL of water diluted with 90 mL with ethanol) Procedure: Extraction of sugars from a ripe banana (Steps 1-4) Weigh 5 g of banana.Pulp the banana with a fork, and place into a tube.Add 10 mL of ethanol and mix thoroughly by vortexing and manual shaking. Heat the tube to 80 °C for 20 min. What problem must be overcome with this step? Hint – refer to pre-work. Filter the suspension through a fluted filter paper into a new tube. The filtrate is your ripe banana sugar solution. Do not dilute this. Procedure: Paper Chromatography Dilution, spotting and development (Steps 5-9) Aliquot 1 mL samples of each standard sugar solution and your unknown into separate tubes. Dilute these using purified water to obtain a final concentration of 5%. Record your calculations.Take a sheet of Whatman No. 1 chromatography paper as supplied (15 cm x 28 cm) and mark a line in pencil 2.5 cm from the bottom. Mark nine points on this line (with small crosses) that are 1.5 cm apart.Onto each of the crosses apply 2 µL of one of the diluted standard sugar solutions; the diluted unknown sugar solution and the undiluted filtrate from the banana (each solution will be on a different spot).Record the locations of the sugars. Label in pencil the top part of the filter paper with your initials or name.You will be shown how to prepare and place the chromatography paper into the developing tank. The tank is then sealed as this assists development. Allow the chromatogram to develop for about 1.5 hours. Develop the filter paper with isopropanol:acetic acid:water (3:1:1 v/v/v) solvent mixture.After developing, measure the distance the solvent front has travelled from each original spot (where you applied the sugar solutions). Think about the best way to display these data. Staining and calculation of RF (Steps 11-14) Please note: the steps 11-13 will be carried out by lab Supervisor/demonstrator (in order to prevent any spill of silver nitrate, which could result in indelible stains) Dip the paper in the staining solution (5 mL saturated silver nitrate in 200 mL acetone). Use fresh solution.Dry the paper in the fume hood and dip it into the solution (1g of sodium hydroxide in 10 mL of water and then diluted with 90 mL ethanol). Use fresh solution (~10 mL).Dry the paper at 100°C for 1 min, mark the location of the centre of the sugar spots.Calculate the Retention Factor (RF) for each sugar spot. The RF value is the ratio of distance travelled of the sugar compared to the distance travelled of the solvent. Thus, the formula for RF = Discussion of Procedure Write down your thoughts about the following to demonstrate that you have an understanding of the procedure. Make sure that you do not use any reference sources to compile your answers. We need to know your thinking and why. Do you think that the ripeness of the banana might affect the outcome of the practical? What made you come to this conclusion? Do you think that the amount of banana weighed out was important and why? Is the mobile phase used here hydrophilic or hydrophobic? Why is it relevant to separation of sugars? What would happen if the samples that were applied to the chromatography paper were placed below the level of the developing solution? In some cases, the development front was not parallel to the initial line where the sugar samples were applied. How would the RF values be determined and would they be valid? Results • Determine the most likely sugars to be present in the ripe banana and the most likely sugar to be your unknown based on the RF values. Note: If taking this prac online please use the data provided in the Practical 2 Student Data file to answer these questions. (you should select at least one of the unknowns A-D provided in the Student Data file and interpret the results for Part B, C, and D). Part C. Benedict’s Test for reducing/non-reducing sugars Write Aim/Methods/Results /Discussion and self-reflection(Follow marking criteria and answer all questions ) Materials As for Part B above except for: 1 mL Benedict’s solution (alkaline cupric citrate) Procedure: Benedict’s Test Aliquot 1 mL samples of each standard sugar solution and your unknown into separate tubes.Add 5 drops of Benedict’s solution.Heat tubes in a boiling (100°C) water bath for 2-5 min.Record your observations for each of your sugar solutions. A positive test (red precipitate) indicates the presence of a reducing sugar that reduces cupric ions in the Benedict’s solution to the cuprous state. The Benedict’s test is frequently used clinically to test for sugar in urine. Discussion of Procedure Write down your thoughts about the following to demonstrate that you have an understanding of the procedure. • What is the underlying principle for the colour change when Benedict’s reagent is mixed with a reducing sugar? (Your explanation here only needs to be brief, but mention the reaction that takes place and the functional groups involved) Results Which sugar(s) are non-reducing sugars?What is the main structural feature of the identified non-reducing sugar(s) that differentiates them from reducing sugars? Illustrate this by drawing the structures of specific sugars. Part D. Osazone Formation – Aliquot 1 mL samples of each standard sugar solution and your unknown into separate tubes. Add 0.2 g of phenylhydrazine hydrochloride, 0.3 g of sodium acetate and 1 mL of water to each tube. This must be done in the fume hood.Place the tubes after covering with foil in a boiling water bath for 30 min.Remove the tubes from the water bath and allow them to cool. Observe the colour of any precipitates.Place a drop of the crystalline suspension onto a microscope slide, cover it with a coverslip and examine the crystals under a microscope using the 40x objective.Draw the crystals as they appear under the microscope. Discussion of Procedure What is the underlying principle for osazone formation? Can you see an osazone crystal of sucrose? Based on the crystal structures, what sugar(s) is your unknown likely to be? Overall Discussion By combining your results from the above experiments (Part B, C, and D), identify what your unknown sugar is likely to be and the most likely sugar(s) in ripe banana. Note: If taking this prac online please use the data provided in the Practical 2 Student Data file attached to answer these questions (you should select at least one of the unknowns A-D provided in the Student Data file and interpret the results for Part B, C, and D).
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