GENERAL INSTRUCTIONS

Transcription

1 Read these before doing any work in laboratory Safety: GENERAL INSTRUCTIONS 1) Eye protection must be worn at all times in the laboratory. Minimum eye protection is eye glasses with side shields. Safety goggles provide the best protection and must be worn whenever anyone in the laboratory is performing a hazardous procedure. 2) Never eat, drink, smoke, or chew anything in the laboratory. Avoid contact between objects and your mouth. NO MOUTH PIPETTING! 3) Do not use Bunsen burners unless instructed to do so. 4) Always work in the hood when handling organic solvents. 5) Do not dispose of water immiscible solvents (i.e. ether, hexanes, etc.) in the sink; waste disposal bottles will be provided for these solvents. 6) Contact your instructor concerning the clean up of spills. Clean up spill immediately, following your instructor's directions concerning how to proceed. 7) Work neatly and clean up any messes before leaving the laboratory. 8) Make sure you understand how to use all materials safely before proceeding. Use of Reagents: 1) Always read the label carefully before using any reagent to make sure that you are using the correct material. 2) Do not put pipettes into the general usage reagent bottles. If you wish to obtain some reagent, pour the desired amount into a clean beaker, graduated cylinder, flask, or test tube, and then pipette from this smaller supply. Please be careful not to waste the reagents. Calculate what you will need and then take a SLIGHTLY larger amount. 3) Do not return excess reagents to the reagent bottles. Discard properly at the END of the laboratory period. Distilled Water: When you are instructed to use water in an experimental procedure it is understood that distilled water is to be used. Distilled water is supplied in plastic carboys in the lab and is clearly marked. The amount of distilled water available is limited; please do not waste it. Preparation for the Laboratory Session: To insure efficient use of laboratory time, each student is responsible for reading carefully the appropriate experimental procedures before coming to the laboratory session. This requirement is subject to periodic, UNANNOUNCED ASSESSMENT, in the form of pop quizzes, notebook checks, etc. General Guidelines and Expectations

2 Laboratory Notebook: 1) EACH PERSON MUST KEEP A LABORATORY NOTEBOOK. The notebook should have bound, non-removable pages. (A composition notebook is acceptable.) The purpose of keeping a notebook is to provide experience in the direct recording of experimental data in an organized form. The notebook is not intended as an exercise in busy work. The notebook should be organized such that anyone reading it can clearly understand what procedures were used and what data was obtained. Information in the notebook should be concise and to the point. Each person (regardless of whether or not the lab is done in groups or individually) should bring his/her laboratory notebook to each laboratory session and record data collected during the session directly into the notebook. 2) The instructor may ask to see the notebook during any laboratory period. The NOTEBOOK MUST BE SUBMITTED FOR EVALUATION AT THE LAST SCHEDULED LABORATORY SESSION. 3) The notebook should include the following: a) A Table of Contents. b) The date each experiment was done and who participated in the experiment (i.e.. any lab partners). c) ALL prelab calculations required for the lab IN THEIR ENTIRETY (i.e. if you did a variation of the same calculation 10 times, ALL 10 calculations should be here). b) A description of the procedures used. The instructions should be an integral part of the notebook. It is helpful to summarize the procedures for yourself in the notebook in the form of a flow chart or as brief notes on the procedure prior to the beginning of the laboratory session. By preparing these instructions before coming to the lab session you should be able to address any questions you have with the instructor before beginning the lab. c) All raw data collected in the laboratory should be recorded DIRECTLY in the notebook. Recording data on scraps of paper to be later copied into the notebook is unacceptable laboratory practice. Tables suitable for entering the data should be developed and entered into the lab notebook BEFORE the scheduled lab session. This will help to ensure that all necessary data is collected. d) Notes on any observations, problems, changes in the procedure, etc. should be clearly noted. e) All graphs and calculations required for the laboratory assignment. Note: even if you are handing in graphs and calculations in an assigned report, the originals (or at minimum, a copy) should appear in the lab notebook. f) A brief summary of any conclusions drawn from the experiment. This may be in the form of an outline since it is assumed that you will be writing it up, in most cases as part of your assignment. Formal Reports: For some (not all) of the laboratory experiences, you will be required to submit a formal report. Regardless of whether or not you worked on an experiment alone or with a partner, you will be required to submit your own report. It is not acceptable for two people to hand in identical reports. The laboratory experiences for which formal reports are required are noted on your Lab Schedule. These formal reports should be typed (it is advisable that you learn how to use a word processor on either a PC or Mac) and any tables or graphics should be produced using suitable PC or Macintosh software. The formal report should have a format similar to journal articles published in the ACS journal Biochemistry. You may find it useful to look up 2-3 papers in Biochemistry and look over their general format and style. In general, the report should include all the details a reader would need to repeat the experiment and understand what was learned from the experiment.

3 The report should include the following sections, in the order given below: a) Introduction Your introduction should be as brief as possible while addressing the following questions: i) what questions or concepts were being explored in this experiment? ii) what general techniques were used and what is the theory behind these techniques? (note: avoid describing the details of the methodology here). iii) Any other background your reader needs to know in order to understand the experiment. Note:You should avoid copying sentences from the laboratory manual. b) Materials and Methods Describe the procedure used to do the experiment. Your description should include enough detail to allow a scientist to repeat the procedure from your description. However, avoid tedious and unnecessary detail (e.g. do not routinely tell what size the beaker, flask, etc. was, unless that size or shape is absolutely critical to the success of the experiment.) Make judicious use of tables, as needed, to simplify the description. c) Results and Discussion Note: you may choose to present the results first followed by the discussion, or you may combine the two portions. Make this decision based on how you can best present a clear picture. Present the experimental data you obtained in this section. Unless specifically asked to present a graph or table in a given format, you may choose to present the data in the format (graph, table, figure, etc.) that most clearly shows the points you are addressing with your discussion. Note that each figure or table should be numbered (Figure 1, Figure 2b, Table 1, etc.) and the reader should be referred to the figure using this designation (e.g. as seen in Figure 1...). The figures should also carry a descriptive legend. Avoid showing the same data in every format you can think of, instead, choose the formats you feel best represent the points you are going to raise in the discussion. It is usually useful to tell the reader what the figure/table is telling them in the text of this section (e.g. Figure 1a shows the activity of carboxypeptidase as a function of ph. As can be clearly seen, the activity of carboxypeptidase increases as the ph is raised and is nearly zero at ph's below 3). The discussion portion of this section should include: i) any conclusions you can draw from the data and the rational behind your conclusions. ii) a comparison of your results to what you expected to see and a discussion of why you expected to see a particular result. iii) any explanations you can make about why you didn't get the expected result. iv) A discussion of the most likely sources of error. d) Conclusion: Briefly summarize the conclusions you draw from the experiment. e) Appendices Examples of all calculations should be done here in detail. The instructor will look at this section to check your work, so they should be complete. However, if you are carrying out the same calculation more that once, you need only show one such example. BE SURE TO INCLUDE ALL RELEVANT UNITS. If questions were asked in the write-up which you did not address as part of your discussion, answer those questions here.

4 PREPARATION OF BUFFERS The Henderson Hasselbach Equation: The general definition of an acid is a molecule which can dissociate, releasing a hydrogen ion. Conversely, a base is a molecule which can accept a hydrogen ion. The following equation shows the dissociation of a proton from the weak acid, HA: The dissociation constant K is given as: Rearranging this equation: Taking the logarithm of both sides gives: HA H + +A - K = [H + ][A - ]/[HA] 1/[H + ]={1/K}{[A-]/[HA]} log{1/[h + ]}= log{1/k} + log{[a - ]/[HA]} Now since ph=log{1/[h + ]} and substituting pka=log{1/k} gives: ph=pka+log{[a-]/[ha]} This equation is called the Henderson-Hasselbalch equation. Buffers are mixtures of weak acids and their conjugate bases that resist changes in ph when strong acids or bases are added. They are most effective if substantial amounts of both the protonated and unprotonated forms of the weak acid are present. If this is the case, and a strong acid is added, its protons can be soaked up by the conjugate base of the weak acid. If the base is added, protons from the weak acid can be lost to neutralize the base. This behavior follows from the Henderson-Hasselbach equation since when [A - ] =[HA] in the Henderson-Hasselbalch equation, then log{[a]/[ha]} = log {1} = 0 and we find that ph=pka. In addition, graphing the equation shows that when the ph is near the pk, the ph changes very slowly with changes of [A - ] and [HA]. The mixture thus acts as a buffer near its pka, ie. the mixture buffers the ph from changes in ph as acid or base is added to the buffer for ph's near the pka of the buffer. One frequently finds that the ph expected from a given ratio of weak acid to its conjugate base is not attained in practice. Assuming that various measurement errors are kept minimal, the reason for this is that the "effective concentration" of the two species may not be equal to the calculated concentration due to non-ideal behavior of the solution. The proper name for "effective concentration" is thermodynamic activity. Phosphate ions, which are small and intensely charged, are notorious for having low thermodynamic activities, especially in higher charged forms. At 0.1 M concentration, the effective concentration of HPO4 2- ion may be as low as 40% of the calculated concentration. Since it is the effective concentration that determines the ratio of weak acid to conjugate base, the observed ph for such solutions is quite different from that calculated from the amounts combined. In practice this thermodynamic activity or effective concentration is dependent on the concentration of the buffer, showing a larger deviation from ideality as the buffer concentration is increased. Therefore, in general, the higher the buffer concentration the less ideal the buffer becomes and the farther the measured ph is from what one

5 calculates that the ph should be using the Henderson-Hasselbalch equation. On the other hand the higher the buffer concentration, the more able the buffer is to resist changes in ph upon the addition of acid or base. This laboratory demonstrates these principles. Prelab 1) Using the Henderson-Hasselbach equation, calculate the volumes of 0.1M phosphate solutions to mix to prepare 100 ml each of buffers with ph values 6.0, 6.5, 7.0, 7.5, and 8.0 starting with 0.1M stock solutions of NaH2PO4 and Na2HPO4. The relevant reaction is given below. The pka for this reaction is H2PO4 _ HPO H + Experimental 1) Mix together the amount of 0.1 M stock solutions which you calculated would give 0.1M phosphate solutions at ph values of 6.0, 6.5, 7.0, 7.5 and 8.0. Measure the ph of each buffer you made using a ph meter/electrode and record your results. (Be sure to calibrate the ph meter/electrode using appropriate ph standards before you do this, following the instructor's instructions). 2) Divide each buffer into 2 40 mls portions (Save the 20 mls you had left for the next part). To one portion add 0.5 ml of 1 M HCl; to the other add 0.5 ml of 1.0 M NaOH. Again measure the ph's and record the values you measured in Table II. 3) For each ph, take 10 mls of the remaining 0.1 M phosphate buffers (one 10 ml aliquot for each ph) and dilute it by 10 times with water (a 10 fold dilution) to make a final volume of 100 mls. Record the volume of water you needed to do this dilution and what the final concentration was for the diluted buffers. Measure and record the ph for the diluted solutions on Table III. 4) Finally divide each diluted buffer into 2 40 ml portions. To one portion add 0.5 ml of 1 M HCl; to the other add 0.5 mls of 1M NaOH. Measure and record the ph for the diluted solutions.

6 The Report Before writing the report, you must develop and submit an outline of the report in outline form (i.e. Roman numerals, Arabic numbers, letters, etc.). The outline must include: 1) all data in tabulated form; 2) copies of all graphs you intend to include in the report. 3) a brief synopsis of the major points you intend to make in each required section (i.e. Introduction, Materials and Methods, Results, Discussion, Conclusions, Sample Calculations). ; 4) copies of all sample calculations you intend to include in the report. Note: the required Outline will be graded (for science and content). It will be returned to you before the report is due so that instructor's comments can be addressed in the report. Other considerations: The laboratory report should include the following: 1) A table of the amounts of each the Na2HPO4 and NaH2PO4 that were mixed together to get each ph value. 2) A table and/or graph of the ph's that you measured before and after dilution compared with calculated ph's. 3) An example of the prelab calculation (for instance, show the entire calculation for ph=6). (Sample calculations should appear in the appendix of your report). 4) The following questions should be addressed in the discussion: a) At what ph range does phosphate buffer have it's best buffering capacity? How does your data support this conclusion and why would you expect this to be so? b) Is your 0.1 M phosphate buffer or your diluted buffer better able to buffer against addition of acid and base? How does your data support this? Why would you expect this to be so? c) Which concentration of buffer gives measured ph values closest to those you calculated? Is this what you would expect? Why or why not? d) Discussion of any other trends in data you observed.