GENERAL INSTRUCTIONS
Read these before doing any work in laboratory
Safety:
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
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.
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.
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:
HA ⇔ H+ +AThe dissociation constant K is given as:
K = [H+][A-]/[HA]
Rearranging this equation:
1/[H+]={1/K}{[A-]/[HA]}
Taking the logarithm of both sides gives:
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 HPO42- 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
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 7.21.
H2PO4
_
⇔ HPO42- + 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.
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.