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ADVANCED PHYSICAL
CHEMISTRY LABORATORY
LABORATORY MANUAL
These experiments are suites. Each suite consists of two or three experiments.
ID TOPIC
EXPERIMENT
A Activity coefficients
E Enzyme kinetics
K Kinetics
M Micelles
P Permeability
T Transitions in biomolecules
W Acidity of wines
Some of the material required for the experiments will not have been covered in any of your classes. Background research is
an essential part of the experiments.
*May not run this year.
Date/Time
Partners Name:
Phone No.
Experiment/Group rotation.
Week
Group No.Z
1
2
3
4
5
6
Introduction:2
1. Regulations
All accidents and incidents (near misses and spills) must be reported
immediately to a member of the lab staff.
Students are not usually permitted to use the laboratory except during
their scheduled laboratory period.
No student should attempt unauthorized experiments in the
laboratory, or modify any experimental apparatus.
No student can work in a laboratory without a supervisor present
unless they have completed a WHMIS and the Chemistry Department
Safety course, and then only with the supervisors consent.
Any student deemed dangerously incompetent or intoxicated will be
required to leave the laboratory. An incident report will be filed.
Keep walkways clear at all times. Do not leave cupboard doors open.
Do not wave
pipettes
around
(especially
Pasteur
pipettes) around or you
will spray residual any
material around.
2. Personal Safety.
Many of the chemicals in the laboratory are poisonous, whether taken
orally or absorbed through the skin. If any chemical is swallowed, the
supervisor should be summoned immediately. Immediately wash off
any chemical comes in contact with the skin with plenty of water.
Consult the MSDS data sheets for further information. Make sure you
know the location of the eyewash station and emergency shower.
As a minimum students MUST wear safety glasses at all times. You
must provide your own safety glasses. Contact lenses must not be worn.
Other protection such as side shields, goggles or face shields may be
required. Make sure your goggles are sealed against the face.
No food or drink may be bought into the laboratory. Do not chew gum
in the laboratory.
There must be no smoking in the laboratory.
Students should keep their arms, legs and torso covered. Students
should keep their arms, legs and torso covered. Wearing 100% cotton lab
coats is required. Most chemicals will stain or burn your clothing.
You are not permitted to wear open toed shoes in the lab.
Long hair should be tied back at all times.
Unless otherwise informed assume all unknown samples are
dangerous, that is you must wear goggles, gloves and lab coat while
handling them.
Assume all chemicals are corrosive or toxic by ingestion, and take
appropriate precautions.
Never handle chemicals with your bare hands.
While heating a substance in a vessel with a narrow mouth (e.g. a test
tube) ensure that the mouth of the vessel is not pointing at anyone,
including yourself.
When using compressed gas, vacuum equipment, high temperature or
high voltage equipment be especially careful. Ask the laboratory
instructor for help if you are uncertain of any procedure. Strongly
corrosive or toxic materials should only be handled in the fume hood,
with the sash down, and suitable gloves on. Do no kneel to look under
Introduction:3
2. Fire
Students should be aware of the location and use of the fire
extinguishers in the laboratory.
In case of fire, the flames should be extinguished with one of the
extinguishers and the supervisor notified immediately.
If a student's clothing or hair catch fire, use the emergency shower
(make sure you know its location). If this is not possible, smother the
flames immediately with a laboratory coat or a fire blanket (know the
location of the latter)
4. Disposal
Make sure all broken glass or sharps are disposed of in the appropriate
container.
Make sure all materials are disposed of in the appropriate container
(solids, organics, halogenated solvents or inorganics)
Beware of materials that hydrolyze rapidly, e.g. SOCl2 and acid
anhydrides; they cannot be disposed of in containers containing
alcohol or water.
Never add hydrogen peroxide, nitric acid or any other oxidants to
organic materials (unless instructed) or into the organic disposal
container. Acetone and alcohol are a particular problem.
MARKING POLICIES
Allocation of marks will be different from usual labs. and
may vary with the experiment. As above, you start with one
or two marks less than the maximum otherwise an
unrealistic spread of marks will occur. The follow items
will be considered when marking labs.
a) The main body of text should be in a proportionallyspaced (e.g. not Courier) western (e.g. not Cyrillic) serif
font (e.g. Times New Roman, Garamond, School Book),
not a sans serif font (e.g Arial) or any decorative font (e.g.
Brush Script) or anything weird (e.g. Tekton). Use a normal
face, not italicized, hollow or bold. Type size should be 12,
13 or 14pt.
b) Titles should be bold, serif or sans serif, and larger than
the main text; dont bother with anything fancy.
c) There should be a maximum of three fonts in a report
(the main text, titles, symbols). If you want some variation
on the title page you can use bold and italic (sparingly).
d) Keep it simple, no curly borders or color (except maybe
in graphs). Use bold or italic for emphasis, do not
underline.
e) Use tables for your data, dont rely on tabs etc. Do not
box the table, a couple of lines here and there usually does
the job.
f) Note that stuff like 13 CO 2+
3 can only be typed in using
the equation editor learn to use it.
g) The main body should be double or triple spaced (with
a 1 margin all round) so I can insert comments. However, I
will tolerate single spaced reports.
h) Diagrams can be drawn by hand, but keep them neat.
They should be captioned by a line of type. Equations must
be typed in, I suggest you reference the manual (e.g. Exp. G
eq.3) to reduce this burden. All graphs must be done by
computer, but you can manually cut and paste them into
your report if you wish (OLE works nicely, but only on fast
machines with lots of memory).
i) Do not cut and paste lumps of text from your partners
reports. I want to see some originality; in both the content
and the formatting (Also see the section on plagiarism).
Introduction:6
for comments.
Use a fly page.
Insert diagrams, graphs and tables into the text rather
than on separate pages. (Use cut and paste).
Number the pages.
Do not start sentences with numerals (use the word).
Breaks and indenting for paragraphs are your choice.
Number graphs, figures and tables clearly.
You should use the equation editor for equations.
Calculations can be inserted into the text in hand writing
though (they are a real pain to type).
Introduction:7
Introduction.
Typeface.
Introduction:8
Font Size.
As can be seen in the section above that different fonts have
different widths, heights, weights and spread, even though
they have the same point size. Times is large and open
and Caslon small and cramped so Times works well at
10pt, Caslon does not. On the other hand Caslon works
fine at 14pt, and so does Times. I use 12pt for lab.
manuals since they are usually read standing up (and Im a
little short sighted). For reports 10pt is ok if you have a laser
printer, for inkjets 12pt is better.
Spacing.
Spacing between lines is usually set to single (which is
actually variable). For plain text this is ok, but is poor when
youre using super/subscripts or symbols. I find spacing at
exactly 14pt (for 12pt text) works well if the dont center
exact height lines option in Word is off. If youre writing a
draft, double or triple spacing can be useful to allow for
annotations.
Spacing between words is set by using left justification (no
extra spacing) or by using full justification (spacing filled so
line exactly fits the line). Full justification is fine as long as
you dont use narrow columns (a fine example of what can
go wrong is in the opening paragraph).
Misc.
Lines of text should not be more than (on average) eightnine words long. For large format pages that means two or
more columns. Large margins can also help. Setting up
double columns can be a little awkward and slows down the
computer. Some of the lab. manual is not set that way so I
dont expect it in lab. reports.
PLOTTING GRAPHS.
Specific Heat
Example Graph
40.00
Series1
20.00
Series2
0.00
-4.00
1.00
The top graph is Excels default scatter plot. The scales are wrong,
the annotation too large and the plot cluttered. It is almost useless.
You should setup a custom graph in a proper format and use that as
the default..
6.00
Time(s)
Specific Heat
Example Graph
40.00
20.00
0.00
0.00
2.00
4.00
6.00
For the first step we get rid of the grid lines (only leave grid lines if
you need to read data of the graph) and the gray background which
reduces contrast. The legend is also deleted. Again, legends are
useful when working off the graphs, but for reports the legend
should be below the graph along with other information. Note how
the graph rescales when the legend is removed.
Time(s)
Specific Heat
40.00
30.00
20.00
10.00
0.00
0.00
1.00
2.00 3.00
Time(s)
4.00
The final step is to remove both figure and axis boxes, they serve no
purpose. The title has been removed and replaced by text from
Word because you cant do anything fancy in Excel - in this case
simply to put the legend in. Note that it is centered below the graph.
The axes have been tidied up, the span has been corrected, decimal
places reduced and tick marks added. The least square fit lines have
been added. The data points have been changed to black and made
easily distinguishable.
35
Specific Heat
30
25
20
0
2
Time(s)
Introduction:9
These graphs have been cut and paste in. If you do that, make sure
they are a decent size, including the annotation. (see exp.P for some
bad examples. Graphs can be presented on a separate
page if necessary.
PRESENTING TABLES.
Time(s)
4.03
3.25
3.32
3.06
2.6
2.28
1.75
1.31
0.96
0.66
P1 (cmHg)
76.49
76.67
76.57
76.73
76.63
76.52
76.6
76.58
76.6
76.51
Time(s)
4.03
3.25
3.32
3.06
2.60
2.28
1.75
1.31
0.96
0.66
Cv
28.04099
26.34766
26.65888
26.85892
26
25.06601
25.01056
24.84336
24.65
24.25748
P1
(cmHg)
76.49
76.67
76.57
76.73
76.63
76.52
76.60
76.58
76.60
76.51
Cv
28.04
26.35
26.66
26.86
26.00
25.07
25.01
24.84
24.65
24.26
Vent
Time(s)
Pressure, P1
(cmHg)
Cv
J/mol-1
4.03
3.25
3.32
3.06
2.60
2.28
1.75
1.31
0.96
0.66
76.49
76.67
76.57
76.73
76.63
76.52
76.60
76.58
76.60
76.51
28.0
26.4
26.7
26.9
26.0
25.1
25.0
24.8
24.7
24.3
Introduction:10
Center the text and the table. The number of decimals are
made uniform to clean up the appearance and allow a more
meaningful column alignment and spacing. The heavy grid
has also been removed. The grids can serve a purpose in
large multicolumn data look-up table, but generally they just
clutter the table.
USING FIGURES
Figures are rarely prepared in situ they are usually prepared
separately then cut and paste (manually or electronically)
into the document. The only real exceptions are sketches in
your lab. notebook. Regardless of the type of figure they all
need a figure number and caption placed beneath it. The
caption should be no wider than the figure.
There are a number of ways of preparing figures for formal
reports, each with advantages and disadvantages.
a) Hand drawn
b) Drafted
c) Computer drafted.
d) Scanning.
Hand-drawn figures are not usually used in formal reports,
but they are fast and convenient.
Drafting involves special pens, templates, drawing boards
etc. The usual approach is to draw an oversize figure and
Xerox reduce it (that cleans it up a lot). You then manually
cut and paste it into a prepared gap in the report and Xerox
that page. This process is very labor intensive (the chem.
dept at UBC has two graphic artists for this purpose), but it
is the only way to do some types of pictures.
Computer drafting moves all the drafting accessories onto
the computer, however it can be difficult to do chemical
drafting on the computer and the results are often
unsatisfactory. It is most satisfactory if you have prepared
templates of flasks etc. (CorelDraw has a good selection) or
of rings and bonds (ChemDraw). Drawing pictures from
scratch can be a real pain. Be sure to distinguish between
CAD type programs (CorelDraw) used for drafting, and
paint type programs (CorelPaint). The latter is good for
pictures and tends to be poor for equipment figures.
Computer figures can be printed out and manually cut and
paste in or they can be electronically cut and paste in using
OLE. OLE is the best way to go, you dont loose resolution,
but it sucks up computer resources. Also Corel has never
mastered OLE so you cant always use it. You can cut and
paste in bitmap files from just about any program, but be
careful as their resolution is limited (or they become so big
they crash the computer). You need at least 600dpi
resolution to reproduce line drawings properly.
Introduction:11
General
Pre-Laboratory Preparation.
Instruments
a) Record instrument settings (scan time, resolution, scan
width, temp. etc. etc. anything that can be varied).
Anything that doesnt have a value, indicate any
changes. E.g. lamp height was optimized for the
standard sample.
b) State sample cells used (try and get your own). If they
break record that (different cells give different results).
c) If there are any power glitches or the signal seems very
noisy, indicate those along with the time. E.g. at UBC it
used to be impossible to work at 4.30, when everybody
was turning their instruments off. There was also
trouble with big lasers firing; recording times will help
you track these things down.
d) Record the computer file names that any data is held in.
Make them useful, have a convention. If the OS
supports long file names use them. I find it useful to
use a convention like XXYYnnnn.mmm where XX is a
two letter month code, YY is the day (number), nnnn is
a code for the experiment/sample type, mmm is the
extension, usually you wont have a choice about that.
E.g. JL09dx12.spc might mean a spectrum (spc) taken
July 09, of a doxyl-12 spin probe. Most computers are
pretty good about keeping the file date correct so you
may want to just use an 8-letter code instead. However,
its often easier to search for a file name than for a file
date. I like to use an extension nnn where nnn was 000,
001, 002 etc. for a sequence of files. It can be very
difficult to locate three month old data, especially if you
generate 100 files a day.
Preparation
Introduction:12
Computers.
Times
Time is more important than you think. One preparation in
my laboratory, at UBC, didnt work on sunny winter days,
but worked fine on sunny summer days. This was because
the sun would shine into the laboratory at 4pm, in winter,
just when the silver salts were added to the reaction (it was
an 8hr prep.). In the summer, the sun was to high to shine
in. Record reaction times, flushing times, reflux times, start
times, end times, just about any time you can think of.
Results
Miscellany
Record anything else you notice. e.g. 1) samples were yellow
when removed from freezer, but turned a transient red
Introduction:13
PLAGIARISM
You cheat,
Youre dead meat.
For a detailed discussion of plagiarism see the OUC Guide to Plagiarism available from the bookstore. The policy is
basically one of zero tolerance. The penalty for plagiarism is usually a zero for the work involved and a letter of
reprimand. Penalties for theft of papers or repeated plagiarism may be expulsion.
Avoiding plagiarism in chemistry labs. is quite simple; dont copy other peoples work ! That is, do not copy
or read the whole or sections of another person laboratory report, draft or final.
There are cases where it is acceptable to copy other peoples work, in which case you should note the following:
a) If you wish to copy blocks of text from some source, put it in quotes and reference the source. You should not do this a
lot or you will be penalized for poor style. The source of material must not be another students lab. report or from a
tutoring service.
b) If you use any source to assist in writing your labs. make sure you reference those sources in your write up. If you
paraphrase the text from these sources, try to do it as loosely as possible (i.e. keep it close to your personal style). If the
paraphrasing is too similar to the original you may be open yourself to a plagiarism charge. I find it best to read two or
more sources, close those sources and then write out what I understand of what Ive just read. Again, the sources must
not be other students lab. reports, profs. notes etc.
c) You may discuss labs. with other students and you may compare original data and graphs. At a pinch, you can
compare calculations to track down errors. Under no circumstances should you read or copy other students lab. reports
(final or draft), that is considered plagiarism. If you read another students report (but not necessarily copy) it will taint
the style of your write up. It is quite shocking how easy that is to detect, particularly is small classes so dont do it!
d) Do not copy other students graphs or tables either electronically or by Xeroxing. Producing your own graphs and
tables is an important part of this course. (This may be acceptable in other courses, but not this one).
See the supplementary information on others marks penalties.
HOUSE KEEPING
Before starting an experiment, wash, and if necessary dry, any glassware, before use. Remember, the last person to use
the glassware was a student.
At the end of the experiment be sure to clean and wash any glassware. If you found it on the bench, in the cupboard, the
draws, the outside and return it there. If it came from a stock cupboard , put it on the drying rack. Make sure you leave
the balances clean. If any equipment was disassembled when you got it, disassemble it again and return to where you
found it. Leave any equipment or chemicals that the instructor/technician gave you on the bench. Clean up any spills,
paper towels, or any other mess you make. Report any breakages or depleted reagents so we can make replacements.
Failure to do this housekeeping may result in marks being deducted.
INTRODUCTION:14
ERROR ANALYSIS:1
the error is, nor even, exactly, what range a quantity we are
trying to measure lies in. Much error analysis consists of
educated guesses.
Statistical analysis and formal
computational procedures can help, but to use them
properly we must understand that they do not remove
uncertainty, but merely let us express, more clearly, what the
uncertainty is. For instance the statement
[Mn2+] = (1.56 +0.03) x 104 g/ml
does not mean that [Mn2+] lies, for sure, between 1.53 and
1.59 x 10-4 g/ml. It may mean several things, according to
the convention used by the particular writer for the meaning
of +0.03. To avoid ambiguity, state your convention along
with the numerical result, for example,
[Mn2+] = (1.56+0.03) x 10-4 g/ml (95% conf. limits)
ERROR ANALYSIS: 2
Process
Error
+0.03mL
+0.01mL
+0.01mL
+0.02mL
+0.08mL
+0.10mL
+0.10mL
+0.01mL
+0.02mL
+0.0002g
sd x = Vx
and
1 n-1
2
( xi)1 - xi )
ERROR ANALYSIS:3
For our purposes, we will just use first method, i.e. one
standard deviation and the limits not explicitly stated. A
more rigorous approach is recognize that for small data sets
the errors are not normally distributed, they are distributed
according to the Student-t distribution. The confidence
limits should then be written x t dc sd x where t dc is the t
value for a confidence limit of c (0.1 for 90%, 0.05 for 95%
etc.) and d degrees of freedom (number of data points 2,
which you should quote in your results).
OUTLIERS. Sometimes you obtain data thats way outside
the expected range of values. Such data points are called
outliers. There are three reasons for spurious data i) It s real:
While the probability of a data point beyond the five s.d.
limit is small, it is still finite so occasionally you can expect
an odd point. ii) It is real: The data distribution is not
normal. For example, the data may be distributed with a
Poisson or Lorentzian distribution, both of which have long
tails. Under such circumstances, outliers can be quite
common. iii) It is due an error, e.g. contamination, power
bumps. The last case is the most common cause of outliers
and for that reason outliers are often just thrown out. The
problem is that it is not always clear that the data is a glitch
or that it is even an outlier. For highly scattered data, one
should always do an outlier test before rejecting a point.
Also, remember that it may be real data. Although it may be
necessary to throw the point out to keep your statistical
analysis valid, that data point may contain information. For
instance in soil sampling that point may represent an ore
deposit (in fact ore deposit are detected by looking for
outliers). In medicine, the data point may be due to a
diseased specimen, in which case your analysis may provide
a diagnostic for that disease.
My personal preference is to leave outliers in and use robust
estimation methods, which are, by definition, insensitive to
outliers. If your analysis methods are automated in any way
you should use robust methods, because it is very difficult to
automate outlier rejection in noisy data.
= f (x ) f ( y ) f (x ) f '( y )d y
f ( y ) f '( x )d x f '( y ) f '( x )d xd y
Again, for small errors and a smooth function we can drop
the last term.
f '( y )d y f '(x )d x
f (x ) f ( y )1
f ( y)
f (x )
thus
dw
ERROR ANALYSIS: 4
f '( y )d y f '( x )d x
f ( y)
f (x )
y
x
thus
dw
d y dx
y
x
P.S.Phillips October 31, 2011
Z =| A| + |B| + |C|+.
Multiplication or Division Z = A.B.C.
|Z/Z| = |A/A| + |B/B| + |C/C|+.
General Z = f(A,B,C.)
|Z| = |A.df/dA| + |B.df/dB| + |C.df/dC|+.
ERROR PROPAGATION FOR MULTIDIMENSIONAL
FUNCTIONS. We could proceed as above, but it slightly
clearer to use the chain rule. To calculate the effect of a
small error we need to examine the effect of the error on
(i.e. small changes in) the function, which is the same
questions as what is the size of the derivative of the function
with respect to each of its variables. For a function
w=f(x,y,z) we have that
f (x , y , z )
f (x , y , z )
f (x , y , z )
dy )
dw =
dx )
dz
x y ,z
y x ,z
z y ,x
If the errors are small, we can approximate dx to x etc.
This is equivalent to dropping the high order terms in the
Taylor expansion. Its thus straightforward to get the error
for such function. Also, if we recognize that the product, xy,
is a multidimensional function, f(x,y), its easy to see how to
get the error for a product.
hence
sw2
2
w
w
1
(xi - x ) x ) ( yi - y ) y )
N
2
2
1
2 w
2 w
= (xi - x ) ) ( yi - y ) )
y
x
N
w w
2( xi - x )( yi - y ) )
x y
If we now define a covariance between x and y, xy, as
1
(xi - x )( yi - y )
N
so we get for the error propagation function
2
s xy
2
2
2
2 w
2 w
sw s x ++
s y
y
2 w w
++
2s xy
x y
sw2 s x2 ++
s 2y
i.e. we sum the squares of the errors, to get the total error.
ERROR ANALYSIS:5
Z=f(x,y)
2
Error in Z sz
Relative Error in Z
x+a
s x2
sz / z = s x / x
ax+by
2
a2s x2 + b 2s 2y 2abs xy
Multiplication
+axy
2
(sz / z )2 = (s x / x )2 ) (s y / y )2 ) 2s xy
/ xy
Division
+ax/y
2
(sz / z ) = (s x / x )2 ) (s y / y )2 - 2s xy
/ xy
Powers
Exponentials
ax+b
ae+bx
sz / z =s x / x
sz / z =bs x / x
Logarithms
aln(bx)
(as x / x )2
Operation
Simple sums and
differences
Weighted sums
and differences
Table 3. Error propagation formula. It is common to ignore the covariance term if x and y are uncorrelated.
If x and y are correlated, as happens when we do linear
regression, then we must consider the covariance. This is
discussed in the section below.
ERROR PROPAGATION FOR SOME SPECIFIC CASES. As
an exercise, you should derive these formulae. The formula
are for a function Z=f(x,y), which a and b are constants. In
most cases, the covariance term can be ignored.
Other functions can be dealt with by using the error
propagation formula above, but another approach is to
expand the functions directly as a series and work from
there. e.g. ln(x+x)=ln(x)+ln(1+x/x)~ln(x)+x/x as
ln(1+x) = x+.. etc. so for small x the error on ln(x) is
+x/x. Similarly, using the binomial expansion, the error on
1/x is also +x/x. Ill leave it as an exercise for the student to
show that the error on the nth root is +x/n, i.e. it gets less!.
Conversely it rises rapidly with powers of n.
ERROR ANALYSIS: 6
where
2
sab
=-Sx / D
D = SSxx -(Sx )2
and
n 2
x
Sxx = i2
i=1 si
n
1
S= 2
i=1 si
n
x
S x = i2
i=1 si
where
2
sab
=-Sx / D
ERROR ANALYSIS:7
D =
-(Sx )2
nSxx
and
n
= xi2
Sxx
i=1
s2
Sx = xi
i=1
n-2
ERROR ANALYSIS: 8
Measure
Error
Measure
Error
+0.03mL
+0.01mL
+0.01mL
+0.01mL
+0.015mL
+0.02mL
+0.02mL
+0.03mL
+0.05mL
+0.08mL
+0.10mL
+0.12mL
+0.20mL
+0.30mL
+0.50mL
all +1%
all +1%
+0.5%
+0.03mL
+0.05mL
+0.07mL
+0.008mL
+0.01mL
+0.01mL
+0.013mL
+0.015mL
+0.02mL
+0.025mL
+0.03mL
+0.03mL
+0.05mL
+0.08mL
pH meter
Thermometer (0.1C graduation)
Thermometer (1C graduation)
Thermometer (bomb)
Thermometer (digital-absolute)
Thermometer (digital-relative)
Thermometer (thermocouple)
Time interval (manual)
Time interval (computer)
Weighing on a top-loader balance
Weighing on an analytical balance
Viscometer (Canon-Fenske)
UV spectrometer wavelength
UV spectrometer absorbance
+0.1 pH units
+0.02C
+0.1C
+0.01C
+0.1C-1.0C
+0.001C
+0.01C
+0.1s
+0.01s
+1 in last figure
+0.0002g
+0.2%
+1nm
+0.003A or 1%
ERROR ANALYSIS:9
NOTES
ERROR ANALYSIS: 10
2011
Exp. E.
k2
E + S]ES+P + E
k-1
This is a three-parameter mechanism (rate constants k1, k1, and k2). Because we intend to follow the time evolution of
product, the relevant differential equation is
d[P]
(1)
= k2[ES]
dt
We must obtain a relationship between the measurable
quantity, [P], and [ES]. To do this, we first write the material
balance for enzyme, i.e.,
(2)
[E]o =[E] + [ES]
where [E]o is the total (or bulk) enzyme molarity. Equation
(2) states that the enzyme must be present either in its free
form, E, or in its substrate-bound form, ES. Notice that
[E]o is a measurable (and controllable) quantity.
Next, we make the assumption that the dissociation of the
enzyme-substrate complex into free enzyme and
unchanged substrate is faster than the reaction to form
product (and enzyme). This is equivalent to saying that
dt (1 + K/[S]o )
This result predicts that the product concentration initially
rises linearly (when [S]~[S]o) with time and the reaction
velocity is constant and thus is zero order. Catalyzed
reactions often exhibit zero-order characteristics at the
start of the reaction.
The rate of production of P is, of course, the velocity of the
reaction, v. The maximum reaction velocity, vmax is thus
given by vmax=k2[E]0 (i.e. when [S]o is large). Eqn.6 is
often written as
vmax
(7)
v=
(1 + K /[S])
From which it is clear that a plot of 1/v vs. [S] will be linear
with slope K/vmax, intercept (on the y-axis) of 1/vmax and
intercept (on the x-axis) of 1/K. Such linearised plots are
called Lineweaver-Burk plots. (Note that it not only took
four biologists to achieve this simple result, but they also
had the audacity to confuse everything by naming the
equations after themselves).
rate of production of P,
dt [S] + K (1 + [I]/ K I )
or, in more traditional notation
vmax [S]
(11)
v=
[S] + K (1 + [I]/ K I )
In this case the Lineweaver-Burk plot will yield a slope and
x-axis intercept that depends on the inhibitor
concentration, but the y-axis intercept will be 1/vmax,
independent of inhibitor concentration (why?).
The case for non-competitive inhibition is developed as
above. However, we have one further species to consider, IES.
That is the species where the inhibitor is bound to the
enzyme-substrate complex. Once again we modify the
material balance equation and introduce a new equilibrium
constant K I for the dissociation of the complex IES to IE
and S. If we (reasonably) assume that the binding of the
inhibitor to the enzyme is unaffected by the presence of the
substrate on the enzyme so that KI= K I then we get
vmax [S]
(12)
v=
([S] + K )(1 + [I]/ K I )
In this case the Lineweaver-Burk plot will yield a slope and
x-axis intercept that depends on the inhibitor
P.S.Phillips 31/10/2011
(13)
(14)
X
or more explicitly,
X
(16)
[ES(t )] = (1- exp(-Yt ))
Y
The rate equation for product formation is obtained by
combining equations (1) and (15). We get for the reaction
velocity,
d[P] k2 X
(17)
=
(1- exp(-Yt ))
dt Y
(18)
d[P] k2[E]o[S]
=
dt
K M +[S]o
(19)
dt [S] + K (1 + [I]/ K I )
or, in more traditional notation
vmax [S]
(23)
v=
[S] + K (1 + [I]/ K I )
In this case the Lineweaver-Burk plot will yield a slope and
x-axis intercept that depends on the inhibitor
concentration, but the y-axis intercept will be 1/vmax,
independent of inhibitor concentration (why?).
The case for non-competitive inhibition is developed as
above. However, we have one further species to consider, IES.
That is the species where the inhibitor is bound to the
enzyme-substrate complex. Once again, we modify the
material balance equation and introduce a new equilibrium
constant K I for the dissociation of the complex IES to IE
P.S.Phillips 31/10/2011
E + S]ES
k-1
k2
and
d[ES]
= k1[E][S]- k-1[ES]- k2[ES]
dt
The mass balance equation for enzyme gives
[E]o = [E] +[ES] + [AE]
(25)
(26)
(27)
k1
[ES]
[ES]
(28)
d[AE]
k [E]
= 2 o +
+ k3 [AE]
dt
1+K/[S]o 1+K/[S]o
For convenience, we use the definitions
A=
k2[E]o
1+K/[S]o
B=
k2
+ k3
1+K/[S]o
(31)
(32)
The solution of the differential equation d[AE]/dt = AB[AE] with the boundary condition t = 0, [AE] = 0 is
A - B[AE]
ln
=-Bt
or, explicitly,
A
[AE] = (1- exp(-Bt ))
B
(33)
Figure 2. From left to right. Lineweaver-Burk plots a reaction with no inhibitor at various concentrations of a
competitive (middle) and non-competitive inhibitor (right)
After substituting this expression for [AE] in equation (29),
we get, after rearranging,
[ES] =
[E]o + A / B
A/ B
)+
exp( Bt )
1+K/[S]o 1+K/[S]o
(34)
)+
exp( Bt ) (35)
= k2 o
dt
1+K/[S]o 1+K/[S]o
We can integrate this equation to obtain the direct time
dependence of P1. The result appears in the form
P1(t ) = X (t ) )Y (1- exp(-Bt ))
(36)
X = k2 o
(37)
1+K/[S]o
k A
Y= 2 2
B (1+K/[S]o )
(38)
(39)
(41)
2
k2
k + k3
Y = [E]o act 2
K M
1 +
[S]o
(43)
(45)
=
) M 2 3
Y k2 [E]o act
k2 [E]o act
and we can use a plot of 1/ Y vs. 1/[S]o to obtain KM and
k2/(k2 + k3).
Finally, we may recast B, see (32), into a more convenient
form (using the approximation that k3K < (k2 + k3)[S]o;
i.e., KM< [S]o ):
K 1
1
1
=
+
B k2 + k3 k2 + k3 [S]o
(46)
PROCEDURE.
Solutions Needed. Prepare the following solutions.
1. Solution #1. Prepare (or obtain) about 60mL of a TRIS
buffer with pH = 8.5 (0.01 M). You will use this solution as
the reaction medium and also as a solvent blank.
2. Solution #2. Prepare a 3.4 x 10-3 M solution of the
substrate, 4-nitrophenyl trimethylacetate, in acetonitrile.
About 10mL is sufficient for one experiment, although
25mL may be more convenient to prepare. (Use appropriate
caution when using acetonitrile. Wear protective gloves and
work in a fume hood.)
3. Solution #3. Obtain -chymotrypsin solution (about
50mg of -chymotrypsin in 1.0mL of the pH 4.6 acetate
buffer). You will use this directly in the kinetic run.
4. Solution #4. Make up 5 or 10mL of a 2.8 10-5M solution
of 4-nitrophenol solution in the TRIS buffer. You will use
this solution to determine the molar absorptivity of the
product, P1. Note that since the pKa of 4-nitrophenol is
about 7.0, the predominant species in the pH 8.5 TRIS
buffer is the 4-nitrophenoxide ion.
5. Solution #5. Make up 100mL of a 3.0x10-3M solution of
Malthion (MW=330) in acetone from the stock solution of
commercial Malthion (1.5M). Note that Malthion smells
and, more importantly, inhibits the enzyme acetylocholinase; the enzyme that plays a key role in nerve signal
transmission. This is a characteristic of nerve gases and
many modern pesticides; breathing them in or splashing
them on the skin results in central nervous system failure
(a.k.a. death). Be sure to use gloves, make up the solutions
in the fume hood and keep them stoppered when not in use.
Determination of the Molar Absorptivity of 4Nitrophenol. Set the spectrophotometer wavelength to 400
nm, the position at which the hydrolysis product, P1 is
measured. Use two matched, 1cm spectrophotometer cells.
Preferably these should be constructed of fused silica, but
Pyrex cells are acceptable at this wavelength. Add TRIS
buffer to both cells, place them in the spectrophotometer
cavity, and zero the instrument. Remove the sample cell and
rinse and fill it with the 2.8 10-5M 4-nitrophenol
solution in the sample cell (solution #4). Replace the cell in
the spectrophotometer, and record the absorbance.
Determination of the Spontaneous Hydrolysis Rate of
the Substrate. To determine the rate constant of the
uncatalyzed (i.e., spontaneous) hydrolysis of 4-nitrophenyl
trimethyl-acetate, fill a clean sample cell with precisely
3mL of TRIS buffer. Add 100uL of the 3.4 x 10-3M 4nitrophenyl trimethylacetate/acetonitrile solution (solution
#2). Stopper (or cap) the cell, invert it a few times, and place
Substrate
volume (L)
0
20
25
30
50
100
20
25
30
50
100
Inhibitor
volume (L)
0
0
0
0
0
0
40
40
40
40
40
data file in ASCII format. Repeat until all five solutions have
been run. Once you have the data saved, clear it, do not
accumulate the inhibited runs on top of it. You can run out
of memory if you do (the spectrometer can save 10 spectra
only).
Testing an inhibitor. Repeat the runs above, but in addition
add some Malathion inhibitor (solution #6) as indicated in
table 1 (samples 6-10). Again, remember to save the data
file in ASCII format.
Data Analysis. Import the data files to Excel. You will also
need a program that is capable of performing nonlinear
regression analysis on user-defined functions (Origin). We
furthermore assume that each data file (including the
spontaneous substrate hydrolysis blank) has the same time
interval between data points, and the total acquisition times
are nominally equal.
REFERENCES.
QUESTIONS.
1) The mechanism is more complex than the simple
Michaelis-Menten one so we cannot determine if the
Malathion is competitive or non-competitive using a
P.S.Phillips 31/10/2011
NOTES
P.S.Phillips 31/10/2011
Suite G.
(1.1)
where Vao is the initial volume of the analyte, cbo is the initial
concentration of analyte, Vb is the volume of titrant added,
cbo is the concentration of titrant and
co f
a = a ) Ka
1) rf
c o K (1- f )
b= a a
) Kw
1) rf
THERMODYNAMICS OF GLYCINE
contamination between the standard buffers, and swish the
electrode around. Remember to let the reading stabilize.
Glycine has two pKas so we are going to start with a
solution of glycine hydrochloride (you make). You will be
provided with your titrant; accurately made KOH solution
(exact value on the bottle).
We will titrate glycine hydrochloride with KOH over a
pH range of about 2 to 13, and record the pH as a function
of volume of KOH added. You will need to do a rough
titration first to get the two approximate end-points. This
will give you the two pK a values for glycine.
Proceed as follows. Makeup 100 mL of 0.1M glycine HCl
(it need not be exactly 0.1, but you should know what it is
accurately.) Take a 25.0 mL aliquot and adjust the pH < 2.0,
if necessary, with a few drops of the supplied standard 0.1M
HCl (corrosive!). Titrate this solution with the 0.25M KOH
solution provided
Collect data about every 0.5 mL except near the endpoint where the pH changes rapidly. At that point change to
0.1ml increments. The increments can be approximate but
you must know what the increment is to within 0.005mL, or
whatever you can be read the burette to. You may have to let
the pH meter stabilize between readings.
Repeat the titration using 0.1 mL increments either side
of the first end point and about 0.2mL around the second
endpoint. You will need to plot your first data set to establish
what around the endpoint means.
Ask the instructor if they want you to repeat the
experiment with glutamic acid. In that case you make need
to run to pH 8, or do an intermediate titration with 0.2M or
0.5M acid. Discuss your observations in your report.
CALCULATIONS.
Do the following calculations for both the titrations.
1) Determine the end-point and hence the pKas of both
the acids directly from a plot of pH vs. VNaOH.
2) Determine the pKa of both the acids from a plot of
dpH/dV vs. VNaOH. That is, setup Excel to do a simple
derivative using dy / dx ( yi)1 - yi )/(xi)1 - xi ) . You may
have to drop the first few points of the amino acid titration.
Typical results are shown in figure 1.1 below.
3) Repeat the two plots of the titration curve using
CurveFit (if provided by the instructor) with the cubic spline
(with and without tension) and Akima spline. One method
works better than the other.
EXPERIMENT G:1
QUESTIONS.
1) Compare your data to the literature values. Does the pKa
in the 0.3M NaCl differ from water or its literature value?
2) We often talk about sharp and fuzzy to describe slope
changes or breaks. The formal terms to describe lines are
smoothness, monotonicity and continuity. Briefly, describe
the correct descriptions for the terms and support with
some math. Be sure to distinguish between the informal and
formal definitions of smooth.
3) The conductivity data will go through a minimum.
Explain this and the general structure of the titration curve.
(2.1)
(Gly)
DH
HGly(s) ) H+ (aq) dissoln
HGly(aq) ) H+ (aq)
DH rx1
H2Gly + (aq)
DH proton (HGly(aq))
DH
(Gly)
HGly(aq)
HGly(s) dissoln
DH soln
DHdissoc (HGly(aq))
Gly-(aq) ) H+
This is simply the dissolution of glycine and its subsequent
dissociation, normally referred to as the heat of solution. The
dissociation is not complete and has to be accounted for; see
later. The protonation of glycine is not significant under
these circumstances and can be ignored. Note the
distinction between enthalpy of solution (just adding
glycine to water) and enthalpy of dissolution.
Reaction 3.
NH2CH2COOH + NH3CH2COOThat is, dissociation to the zwitterion. It turns out that
P.S.Phillips October 31, 2011
-
and
K1 =
[H+ ][Gly-]
[HGly]
2
(n )0.1
EXPERIMENT G:3
PROCEDURE IN CHRONOLOGICAL ORDER. set for a chart speed of 2cm/min, a sensitivity of 5V and the
This experiment goes faster if you coordinate your efforts so
read all the instructions first. You may want to do a dry run
first (no solutions or glycine); in particular make sure you
can assemble the sample cell.
First, turn on the calorimeter (switch is on rear left) to check
that it's OK. The calorimeter may give a message about rebooting the RAM disk. If it does, just press the <ENTER>
button, then <ENTER> again when it prompts for the date,
then again when it prompts for the time.
PART I. Heat of reaction.
1. One partner should accurately (4dp) weigh 1.00+0.05 of
dried glycine on an analytical balance into a stoppered
weighing vial. You may want to pre-weigh the sample on a
top-loading balance. The glycine has been ground and
dried (and is stored in a desiccator) so minimize exposure
to the air! Don't weigh out any big lumps; they won't fit in
the dish. Also, prepare the standard sample (see below).
2. Meanwhile, your partner should prepare (or otherwise
obtain) a 0.30M solution of hydrochloric acid. Then put
about 120mL of the HCl solution into a flask and warm it to
25.5 to 26C, but no more, by running it under the hot tap.
When done, weigh to 2dp. about 100g of the warm solution
into the Dewar and it back into the calorimeter.
3. Carefully pour the glycine sample into the Teflon sample
dish (don't spill any or get it into the center hole, if you do,
start that sample again. The sample will nearly fill the dish).
Place the sample dish on the bench and hold the sample cell
exactly vertically above it and gently press down on the stem
of the sample cell until the dish is seated in the bell (it
should not take much pressure to do this; do not force it).
Now slip in the glass push rod and press on it firmly so it
sticks in the sample dish hole.
4. Place the assembly carefully into the Dewar and attach
the stirrer belt (at the motor first). Now press
*122<ENTER> (with your finger tip NOT your finger nail),
you will be prompted for the DAC SPAN (DAC = digital to
analog converter), press 1<ENTER> (this means that a
1C change from the offset temperature, vide infra, will
produce an output of 10V). Next press *250<ENTER>
then 5<ENTER>, wait for about 30 seconds then press
*250<ENTER> again followed by 0<ENTER>. (This
recalibrates the thermocouple). Note that this calorimeter is
very sophisticated, but unforgiving, and pressing any other
combination of the buttons will produce all sorts of weird
results. For instance it will recalibrate itself in the middle of
your runs so don't do it. Now, take a few minutes to check
that the (beige) Linear chart recorder is ready. It should be
EXPERIMENT G:4
-m.(245.76 + 1.436(25-T0.63R))
where m is the weight of TRIS and
Tc = Tf - Ti
T0.63R = Ti + 0.63. Tc
Cp = Cp (calorimeter) + Cp (solution)
QUESTIONS.
1) Compare the heat of solution and reaction from your data
with literature values (preferably at the same ionic strength).
2) What is a zwitterion and what significance does this have
for glycine? What if the zwitterions did not dominate, it was
say only 50%, how would that effect the analysis of the data.
4) There are a number of assumptions and approximations
made throughout the experiment. Can you identify them?
EXPERIMENT G:5
EXPERIMENT G:6
PROCEDURE. NOTE! Before performing this experiment, place the wire in the furrow). Note also that there must be
become familiar with the use of the apparatus and the
procedure. Failure to do so could cause an accident resulting
in serious injury. Mount the bomb (see Fig. 3.2) in the bench
clamp and secure it with the Allen key provided.
Make sure there is no internal pressure by unscrewing the
release valve 3 or 4 turns, then remove the large screw cap
(removable bomb head). The inner bomb head is removed
by carefully working it back and forth to free the O-ring
seal. Do not apply force to the release valve as this can be
damaged. When the head is loose, lift it so that the attached
electrodes clear the bomb and place it into the bomb head
support stand (this is the smaller of two stands provided).
Check that the bomb is clean and dry and that the electrodes
are free of any residual fuse wire.
Figure 3.2. The Parr oxygen bomb.
EXPERIMENT G:7
EXPERIMENT G:8
use the lifter to put the bomb into the bench clamp. Use an
aspirator to remove water from the top of the bomb then
carefully unscrew the release valve thus allowing the bomb
to slowly depressurize. Ensure that all pressure is released by
unscrewing the valve at least 5 turns then remove the screw
cap and bomb head, placing the latter in the special stand.
Check that the sample is completely combusted then
carefully remove, straighten and measure the length of the
remaining ends of the fuse wire. Do not weigh any globules
of oxidized iron. Meanwhile your partner should carefully
rinse out the bomb with some deionised water (see the
section on the nitrogen correction to find out how much, but
20mL is probably a good start) and put in a volumetric flask
ready for titration.
Dry the inside of the bomb then repeat the above procedure
with about 1.0g of glycine. Finely grind the sample and
press it into a pellet as described below. Show that pellet
to the instructor.
The pellet of material is prepared with the IR press. If
necessary, the components of the press can be cleaned with
a small amount of water and then methanol.
Pour the roughly weighed ground sample into the die cavity
(beveled edge up) then put the die into the holder and tamp
the sample down with the plunger (beveled side up). Place
the assembly into the IR press and compress the sample to
about 150kbar (2000psi). Release the press then remove and
invert the die holder. and remove the pellet out by gently
tapping the plunger. Immediately clean all the press parts
with water. Then accurately weigh the pellet and combust it
as before.
Repeat the run.
Finally, clean and dry the bomb before putting it away.
26.2
Temperature (C)
26.3
26.1
26.0
23.8
23.7
23.6
23.5
the errors on the fit. Using the time for the vertical line and
the two linear equations, you can get the temperature
difference and the error.
-2
10
12
14
16
18
Time (s)
EXPERIMENT G:9
HA]H+ + A
Kb
MOH]M + + OH
K sp
MA]M + + A
Kw
H2O]H+ + OH
(1.1)
(1.2)
(1.3)
(1.4)
(1.5)
(1.6)
K [HA]
[H+ ] = a
[A ]
(1.7)
then
EXPERIMENT G:10
[HA] = a- [A-]
(1.8)
(1.9)
(1.10)
(1.11)
[H+]3 +(b+Ka)[H+]2
(Kw+Ka(a-b))[H+]-KaKw = 0 (1.13)
which is a cubic, which can be readily solved to give the pH
at any given point in the titration. To rearrange (1.13) into
the form, (1), in the introduction we need to do some house
keeping. If we let Vao be the volume of the weak acid
solution of initial concentration cao and Vb be the volume of
strong base (titrant) added, (concentration cbo ), then
EXPERIMENT G:11
EXPERIMENT G:12
Suite H.
INTRODUCTION. The hydrophobic effect accounts for
the behavior of non-ionic species in water. Polar species
dissolve in water because they can hydrogen bond to water
(enthalpically favorable) and then dispersal (entropically
favorable). This is balanced against the breaking of waters
hydrogen bonds (enthalpically unfavorable), the break up of
the lattice of the polar compound (if solid) and the loss of
entropy caused by the binding of water to solute (solvation).
For small polar molecules G is favorable, but for larger
molecules, where the fraction of polar functional groups is
often small, the molecules are not so soluble. If the fraction
of polar groups is high (e.g. sugars and some proteins) the
molecule will usually be soluble. From these observations,
one may deduce that the hydrocarbon chains are responsible
for the lack of solubility: they do not hydrogen bond and the
entropy of mixing is insufficient to overcome the disruption
of hydrogen bonding. In fact, this is not true. Although
hydrocarbons do not H-bond, the van der Waals forces are
quite large and they should be moderately soluble. This is
where the hydrophobic effect comes in.
Water cannot H-bond to hydrocarbons (note that it just
cannot bond, it is not repelled ), but it can with itself. To
make sure there are no dangling (i.e. unused) H-bonds,
which is energetically unfavorable, the water molecules have
to take on a geometry (on average) to minimize this. Its a
little difficult to envision, but basically the normal and Hbonds (which are interchangeable in water) organize
themselves so that they
form a hollow polygon
(see the figure). The
hydrocarbon is located
in the cavity. This shell
is labile, but under high
pressures, clathrates can
form. The best known is
methane clathrate, a
white crystalline solid
found on the bottom of the deep ocean. It has the amusing
property of being flammable. The net effect of
hydrophobicity is that water becomes highly structured in
the vicinity of non-polar molecules so S becomes
negative, and since H is small in the absence of polar
groups, G becomes negative and non-polar species do not
dissolve.
HYDROPHOBICITY
have hydrophilic (polar) and hydrophobic (non-polar)
regions. They must fold in such a way that the hydrophilic
sections are inside and the polar sections are on the outside.
It is possible to change the nature of hydrogen bonding in
water thereby reducing the hydrophobic effect allowing the
protein to unfold (denature).
We will do two experiments here. One where we study we
study the solubility of n-butanol and n-pentanol in water
as a function of temperature. And the second the where we
study the solubility of toluene (a generic non-polar species)
in water in the presence of species that change H-bonding
in water (co-solvents).
m A moA + RT ln X A
(1)
X HC (sat .)
XW (sat .)
(2)
and
DG o
DG o /T
o
o
S
and
=
D
= DHtransfer (3)
transfer
T
(1/T )
P
EXPERIMENT H:1
EXPERIMENT H:2
CALCULATIONS.
QUESTIONS.
1) There are many reasons to store solutions of biomolecules
in the cold. Is hydrophobicity one of them?
2) We could use Go= Ho-TSo to get, why didnt we ?
(Hint look at the error).
3) At this point, you know what the search direction is. Make
some arguments to show that you could predict it for similar
solvents.
4) Can you think of other measures one could use for the
composition?
X
o
DGtransfer
= mA,W - mA,S =-RT ln A,W
X A,S
Its more convenient to use the concentration scale here.
Since toluene is only slightly soluble and its a constant
volume system, we get
c
o
DGtransfer
=-RT ln A,W
c A,S
We can get the concentration, cS, of toluene in the aqueous
phase from the p p * transition of toluene at 268nm. cS, is
just the concentration or pure water. So we get
A
o
DGtransfer
=-RT ln W
AS
EXPERIMENT H:3
o
for each concentration and plot it vs.
DGtransfer
concentration.
Part 3. PARTITIONING
EXPERIMENT H:4
defined as
K ow =
[X]octanol
[X]water
HA(oct )
which is described by two equations
Ka =
[H + ][A + ]
[HA]
and
P.S.Phillips October 31, 2011
K ow =
[HA]octanol
[HA]water
K ow1 =
C HIo
C HI w
K ow2 =
C HIo
Ctotalw
EXPERIMENT H:5
QUESTIONS.
1) Synthetic membranes can be made with bilayers of
phosphatidyl choline (see fig.1). Would you expect the
following species to be able to penetrate the membrane.;
water, any anion, alcohol, oxygen. Briefly explain why.
If you did the octanol standards you can get at mHIo from
the spectra.
Hence (since C=m/V)
K ow1 =
mHI w Vw
mI 1 V
K ow2 = 1- w 1 + w
mtotal R Vo
DISCUSSION.
1) Tabulate your results. Are the partition coefficients
consistent with the spectra and the Henderson-Hasselbach
equation?
2) Explain in terms of the UV spectra why you see the
colors that you do.
EXPERIMENT H:6
mHIo Vo
Rm
V
= total -(1 ) R) w
mI w
Vo
similarly
Ka =
but
[H+ ][I-]
[HI]
therefore
pH = pK a + log
[I-]
[HI]
APPENDIX.
dG =
\ dG = dH -TdS - SdT
so
\ dH = dU + PdV + VdP
ki
dG =
hence we get
dG = VdP - SdT
(1)
G
=V
T T
so at constant T
P
G(P2 ) = G(P1) ) RT ln 2
(2)
P1
Solids and liquids are incompressible so the volume change
with pressure is tiny, so the change is negligible (1-2 J), but
for gases its quite large.
If we measure changes in G with respect to some
reference state, they become Gs. To simplify things further,
we usually make P1 the reference state, so P1 become Po and
G(P1) becomes Go , so we get for some pressure P
P
(3)
DG(P) = DG o ) RT ln o
P
Chemical Potential.
ni T ,P ,all n
ki
so (4) becomes
U q +w
but
(4)
ki
H U + PV
but
m
G
G
G
dT ))
dP ......
dni
n
T P ,n1 ,n2 ,... P T ,n1 ,n2 ,...
T ,P ,all n
i
i=1
m
G
G
+
+
dT
dP
.......
mi dni
T P ,n1 ,n2 ,... P T ,n1 ,n2 ,...
i=1
(5)
Solutions.
The chemical potential of a substance is the same
throughout a sample at equilibrium, regardless of how many
phases are present. This is very important because it allows
us to say something about complicated systems. In
particular, for a liquid/vapor system, if we know the
chemical potential of the vapor, we know the chemical
potential of the liquid. This is important because we can
calculate (or measure) a lot about gases, even non-ideal
ones, but we know very little about liquids and, currently,
can calculate diddly-squat about them, but if we have the
chemical potential of the vapor, we know everything we
need to know (thermodynamically) about the liquid.
EXPERIMENT H:7
we change X to activity.
Since the liquid is pure, and if we have one mole, its in its
standard state (the pressure is not one bar, its whatever its
vapor pressure is, but we have shown elsewhere that this
effect is negligible for liquids and solids so we can use the
standard state), so
Raoults Law.
(6)
The standard state for the vapor being the vapor at one bar
pressure.
Now lets consider a solution of solvent A and a single nonvolatile solute B (i.e. it has no vapor pressure).
(7)
P
mA (solution) = mAo (liquid) ) RT ln *
P
Some Nomenclature.
There are some evil forces at work that refer to the
hydrophobic effect as hydrophobic forces, or worse bonds.
However, there are some sources of confusion. The first
being intermolecular vs. intramolecular. Intermolecular
refers to between molecules and intramolecular within a
molecule (and is mainly responsible for protein folding).
The next is the distinction between forces, bonds and
effects. A bond involves sharing of electrons and is
directional. Forces are usually electrostatic in nature (ionic
bonds arent really bonds, they are an electrostatic force).
They may or may not be directional. Hydrogen bonds, stacking etc. are electrostatic forces, which we collectively
call intermolecular forces (although they can be
intramolecular forces. -stacking is almost exclusively
intramolecular.) On the other hand the hydrophobic effect is
just that an effect (on entropy). It is caused by H-bonding,
but is not bonding, or even a force. To avoid confusion were
call all the conditions that organize the structure of
molecules, covalent and ionic bonds excepted,
intermolecular interactions.
PA = X A PA*
mA (solution) = mAo (solvent ) ) RT ln( X A )
That is, the solute lowers the vapor pressure of the solvent
(since XA is <1). Note that the identity of B is irrelevant;
vapor pressure is a colligative property. For a real solution,
EXPERIMENT H:8
Suite K.
(which can be done in any order, but one each period). The
first is familiar to you from Chem. 201. Here we will just
repeat the experiment, but explore non-linear fitting and
also the difficulties of distinguishing 1st from 2nd order
reactions.
The second part is new and not so easy. Here will explore the
kinetics of a reversible reaction, which can be analyzed a
number of ways.
The focus is data analysis, dont expect it to come easily.
EXPERIMENT K:1
the H2O2 to the dropping funnel (tap closed). Now lift the
funnel slightly out of the flask and open the tap. When all
the peroxide is drained, drop the funnel back into place,
close the tap immediately, and turn the stirrer on to the
selected speed, and simultaneously start the timer. Record
the times required to produce 2mL, 4mL, 6mL, 8mL
10mL etc. up to 24mL of O2. In each case, adjust the
movable burette to give the same level of H2O in each
burette. Repeat this run to get a duplicate.
In the 201 experiment we did a run to determine the value
of Co, here we will not do that we will calculate it from the
data. We can do that because we have an extra piece of
information, we know that the reaction is first order in
H2O2.
(1)
Vi=CoVoRT/2P
P = Ptotal PHo O
2
o
and PH O is the vapor pressure of water at room
2
temperature.
As the reaction is first order with respect to C,
the
-dC/dt = k'.C
(2)
and
(3)
(4)
CALCULATIONS.
1) Choose one of the two data sets and guess Vi (try 50)
and using (4) do an LSF. Repeat with another (hopefully
better) guess of Vi. Repeat until the calculated data is as
close as you can get it to a straight line. You can generate a
convenient straight line using your guessed Vi and the k
from the LSF. Use the final choice to get the value for k. You
could program the computer to do this, just keep
incrementing Vi until the square of the residuals
(difference between guessed line and the generated line) is a
minimum (the convergence criterion).
2) Use (5) and a non-linear fit to get Vi and k for both
sets of data. (See appendix Non-linear fitting with
Origin). Use the values from question 1 as starting
parameters. Also try the non-linear fit with starting
parameters that are way off to see the effect on the fit. You
should note that while (5) is theoretically correct, its the
incorrect model for this experiment. You need to add an
extra variable, Vo to account for gas loss (or compression) at
the start of the experiment. Vo should be around 1mL.
Non-linear fitting can be done with Origin or by using the
solver in Excel.
3) Do a t-test on your two values for k and Vi and verify
that they are the same within experimental error.
QUESTIONS.
4) Use the data below from 201 Exp. D (some noise has
been added) and fit a straight line for ln([A]) vs. t and a
straight line for 1/[A] vs. t. The data is first order so the
first plot should be linear and the second not, but can you
prove that using the data below?
[A] (M)
69
107
139
173
206
238
271
306
340
375
414
451
t (s)
0.111
0.105
0.109
0.101
0.096
0.095
0.095
0.089
0.090
0.087
0.078
0.080
(5)
EXPERIMENT K:2
1
2
P
R
I
k 1
(1)
EXPERIMENT K:3
[R(0)](2 k1 ) 1t
(e
+ Be 2t )
2 1
[I (t )]
=
and
[R(0)]k1 1t 2t
(e
)
e
2 1
EXPERIMENT K:4
(5)
(6)
where 1
=
X Y
X +Y
k
=
2 =
B 1 1
2 k1
2
2
and
X =k1 + k1 + k2
Y = X 2 4 k1k2
Notice that 1is always less than 2, since both X and Y are
positive. From these equations, we can see that [R] shows a
double-exponential decay (with decay constants 1and
2), and the intermediate I shows an initial build-up (the
negative exponential term), followed by exponential decay.
The values of 1, 2, and A can be estimated from the [R(t)]
curve using "exponential stripping" or, alternatively, by
non-linear regression
The rate constants k1, k-1, and k2 can be obtained from 1,
2, and B using the following equations:
B2 + 1
21
k2
=
A +1
k1
k-=
1 1 + 2 - k1 - k2
k1
=
and
(7)
(8)
[R(0)](2 k1 )
2 1
Because 2 > 1, it follows that the contribution from the
faster component (i.e., the e 2t term) becomes
increasingly less significant, with time, in the decay of R.
Thus, after a sufficient time has elapsed (denoted by t ', see
fig. 2), the decay approximates to a single exponential
function with a decay constant ; 1. i.e. for t>t
where
D=
[R(t)] = D e 1t
thus
ln([R(t)]) = ln D- 1t
(9)
A = Cl
where is called the molar absorptivity coefficient, and l is
the path length of the absorption cell (in centimeters).
The time dependence of the Cr(VI) concentration can be
followed by monitoring its absorbance at 370nm. The
evolution and decay of the thioester intermediate can be
followed, if desired, at 430nm. However, in that case, for a
quantitative analysis, the time dependence of the 430nm
absorbance must be corrected, because Cr(VI) has a small,
but finite, absorption at that wavelength. Note that it is not
necessary to convert the absorbance values at 370nm to
Cr(VI) concentrations because the rate parameters obtained
from the decay curve, i.e., 2 and 1, are pseudo first order
and thus do not explicitly depend on concentration. Also, the
other rate parameter, A, is dimensionless).
The three pseudo-first-order rate constants k1, k-1, and
k2, besides being dependent on the concentration of GSH
and reaction temperature, are also highly sensitive to the pH,
the nature of the buffer and the buffer concentration. Hence,
the reaction conditions have to be chosen carefully in order
for the system to exhibit well resolved, double exponential
kinetics.
EXPERIMENT K:5
assumed that the cell volume is ~3.5 mL). Add the same eqns. 9 and 10). Note: transform only about the first 75% of
solution to the reference cell and place it in the reference this "early-time" portion of the data to avoid negative
compartment. Set the spectrophotometer wavelength at numbers. Now, transform these subtracted values to their
370nm. The sample cell should be kept at constant natural logarithm and plot vs. time. Use linear regression to
temperature (20-25C) during the experiment.
furnish values of 2 and BC (see equation (9)). Next,
3. Place the sample cell in the cell compartment and zero the calculate the amplitude ratio, B.
instrument at 370nm (where the Cr(VI) absorbs). Remove 5. Carry out a non-linear regression analysis of the original
the sample cell and begin data acquisition (ask the data using equation (11) below, a modified version of (8)
instructor if you are unfamiliar with the operation of the UV that incorporates the start time t0 which is the (unknown)
spectrometer). Inject 20L of the Cr(VI) solution into the time delay between starting the spectrometer and mixing
sample cell, stopper it, invert it several times, and place it in the sample; typically 0.5-1.0s. (The data for the first 20the cell compartment. You should do this quickly to ensure 30s may be missing, but that is a different problem). A good
that the data is collected as close to the start as possible. estimate of R(0) can be obtained directly from the data. Use
Continue data acquisition for at least two half-lives (~30- values of 1, 2 and A obtained from linear stripping as
initial guesses. Good starting values are very important
40min), obtaining a total of 500-1000 data points.
4. Ask the instructor to check your run to see if you need to since this is essentially a six dimensional problem. See the
repeat the procedure if necessary. In particular check the notes section at the end.
absorbance is between 0.2 and 1, the 20L may need
[R(0)]( 2 k1 ) 1 (t t 0 ) k1 1 2 (t t 0 )
e
[R(t )]
=
+
e
(11)
changing.
( 2 1 )
2 k1
A370 (t = 0)
3. Copy the rows for t > t' from the time and absorbance
columns to new columns, transform absorbance to the where "370" and "430" denote the absorbance vs. time
natural logarithm, make a plot of ln(absorbance) vs. time (t curves at 370 and 430 nm.
> t'). Perform linear regression to obtain values of C and 1 Once the 430nm data are corrected, you can analyze them
(see (9)). You may wish to combine steps 2 and 3 by doing according to (6). Once again, nonlinear regression analysis is
an interactive LSF to the portion of interest. A broken line fit necessary, and you must supply seed values of the three
parameters 1, 2 the pre-factor and the start time. You
may help also.
can
use 1and 2 values obtained from the previous 370nm
4. Transform the original absorbance data for t < t' into
another column by subtracting D.exp(-1t) from it (see analysis, and you can estimate the pre-factor to (6), D', as
EXPERIMENT K:6
QUESTIONS.
1) Derive equation (4) from equation(2).
2) By substitution, show that equation 5 is a solution of
equation (2)
3) Origins ExpDecay2 function also fits the data as well as
the logarithmic and reciprocal functions. What does this tell
us about the use of fits to determine the mechanism of the
reaction.
4) Find an equation that gives a reasonable fit to the data
below. Theoretically, it should fit to the sum of two
exponentials.
Conc (M) Shift(ppm)
0.000
0.000
0.0002
0.007
0.001
0.061
0.002
0.123
0.004
0.260
0.010
0.517
0.015
0.654
0.020
0.737
0.040
0.980
0.070
1.186
Table 1. Concentration of tetraphenylboron ion in
EPC membranes, and the chemical shift difference.
5) Generate data in Excel using the equation
=
y A1 e xt1 + A2 e xt 2
for x=0-1 in increments of 0.1. Then use Origin to fit the
data to a double exponential. Either write your own function
or use a built in one (ExpDecay2 if I recall correctly). Either
way, use one as the starting value for all four parameters.
Press the 10 iteration button until you get a good fit. Note
the number of iterations taken to converge. Reset the
starting values to one and repeat using the 10 simplex
P.S.Phillips October 31, 2011
NOTES.
Equation 11 is rather formidable to fit, so do a practice run
using Origins built in ExpDecay2 with y0 and x0 fixed and
set to zero. Then, using your starting values, estimated
elsewhere, use equation 11 with t0 set to zero and fixed at
first then let it free later.
For starting parameters I found, by simulation, exponential
stripping, guessing and examination of the data that
R(0)~0.2 (actually its the absorbance at your first data
point), A~1.4, t0~4, 1~0.00034, 2~0.003, k1~0.00005
worked ok if times are in seconds. The fit was very good, but
the parameter errors were large, which implies that the
parameters are not unique. A good fit does not mean good
parameters. See the aphorism on elephants at the start of the
manual for further insight. One partial resolution is to
recognize that the fit is not very sensitive to some of the
parameters and fix those after the preliminary fit. For
instance, you have good initial guess for R(0) and t0 so if the
fit does not shift them too far from the original values you
can fix them after the first iteration. In fact you may have to
do that, fit, then use the fitted parameters as starting values.
You can verify that the fits are not too sensitive to them with
the simulation mode. In fact, you should (if time permits)
check the sensitivity of the fit to all the parameters. Often
with double exponentials, almost any combination of the
two time constants will fit, as long as they are within an
order of magnitude of each other.
This all illustrates a general problem with kinetics. It is
impossible to calculate anything from theory and nearly
impossible to measure anything to better than an order of
magnitude. For simple reactions, kinetics has told us much
about chemistry, but for complex reactions, the returns are
less. Fortunately, enzyme reactions are often simple enough
to be tractable and this keeps the area alive, never-the-less,
it is one area of physical chemistry I find unrewarding.
You need a USB drive for this lab.
EXPERIMENT K:7
USING ORIGIN
Origin can use Excel directly for its data source, by opening
Excel files directly in Origin. However, its best, at first, to use
Origins native mode. You just cut and paste Excel data into
Origins data tables. (Just mark and copy in Excel, then point
to the first cell in Origins table to paste). Like Excel, you can
customize many of the graph items with right or left mouse
clicks. These instructions are for Origin 6. The newer
versions may differ slightly.
EXPERIMENT K:8
EXPERIMENT K:9
EXPERIMENT K:10
Exp. M.
Introduction to Micelles:1
Introduction to Micelles:2
motion sensitive. The oldest of these methods is radiolabeling, where the molecule is radioactive and can be
tracked with a Geiger counter or similar device. More
modern probes use some spectroscopic property such as
paramagnetism or fluorescence.
Labels and probes are essentially identical, the only
difference being that a label is chemically bound to some
part of the system (e.g. 14C is incorporated into an amino
acid). Probes are just distributed in the system by
dissolution or absorption. Here we will restrict the
discussion to fluorometric probes in micellular systems.
Aggregation studies with fluorometric probes.
Consider an aqueous solution of a surfactant that has a
bulk concentration, [S]o which is above the CMC, the
critical micelle concentration. If we make the simple
assumption that the surfactant molecules are present
either as monomeric units or as micelles that contain N
monomers, there will be a concentration of such micelles,
[M], which can be expressed as
[S]o CMC
(1)
N
where CMC is the concentration of free monomers in
solution. In reality, a micellar solution is not a static
system containing only two solutes, monomer and
micelle. Micelles constantly undergo assembly and
dissociation, and at a given instant in time micelles are
characterized by a distribution of aggregates containing
different numbers of monomer units. Thus in equation
(1), N represents the mean aggregation number, and [M]
accordingly represents an average micelle concentration.
Because the numerator in equation (1) can be directly
determined (the CMC can be obtained experimentally),
we could find the value of N if we knew the average
micelle concentration in the system.
[M] =
ne
n!
(2)
=
Po e = e [Q]/[M]
I
(4)
= e [M]
Io
Recapping, Io is the luminescence intensity of the probecontaining surfactant system in the absence of quencher.
It is proportional to the number of micelles containing a
probe. I is the luminescence intensity in the presence of Q
moles per liter of quencher, and it is proportional to the
number of micelles containing a probe, but without a
quencher. Substituting the expression for [M] from
equation (1) into equation (4) and rearranging, we have
I
-[Q]N
ln =
I o [So ] - CMC
(5)
Introduction to Micelles:3
Introduction to Micelles:4
Suite M.
MICELLE PROPERTIES
Part 1. C.M.C. determination by conductivity. Here we will use a UV probe, benzoyl acetone (a.k.a. 1This is a simple titration. We just add standardized
detergent solution to water (or saline) and measure the
conductivity as a function of concentration. Our
detergent will be SDS, sodium dodecyl sulphonate (a.k.a.
sodium lauryl sulphate), NaOSO3C12H25. This is the most
common non-phosphorus clothes detergent. (Most
washing powders consist of SDS, brighteners, perfume,
pH balancers and sometimes bleach). The titration is
rather lengthy and conductivity and micelle formation
are temperature sensitive so it is desirable to do the
titration, and keep all solutions, in a 25C water bath.
Make all solutions in e-Pure water.
1) Make up a 500mL standard solution of SDS, ~0.08M.
Make a concentrate first, and then dissolve that by
stirring. Do not shake! Its a detergent!
2) Pipette 100mL of water into a 250mL measuring
cylinder. Clamp the cylinder and put the electrode in the
cylinder. Make sure the electrodes are completely
P.S.Phillips October 31, 2011
B*
9
8
6
4
2
1
5
0
Ru(bipy) 3 2~
100L
0
100L
Solution
1
2
3
4
5
6
Blank
Standard
I
CMC [So ]
ln =
[Q]N [Q]N
Io
(6)
QUESTIONS.
1) Compare the c.m.c.s from the two methods with each
other and, along with the aggregation number, compare
with the literature values.
2) Why does conductivity change the way it does when
the c.m.c. is reached? Do the two os in calculation 2)
make sense in light of the relative size and charge of the
micelle and monomeric SDS?
3) Suggest other techniques that may be suitable for
measuring c.m.c.
4) If the hydrocarbon core of a micelle is 3nm in
diameter and the core contains one molecule of the
fluorescent probe, calculate the molar concentration of
the probe in the core and comment.
5) Since the data is discontinuous, differentiating the
data may help find the break. How many times would you
need to differentiate to get the break as a peak? If
instructed to do so, use the Cubic Spline option in
CurveFit.
6) We can calculate the aggregation number of a micelle
if we have [surfactant], [micelles] and the c.m.c. Derive
the algebraic relation for this. (Explain your steps)
7) Explain why the NaCl changes the c.m.c. and
aggregation number. What implications do the results
P.S.Phillips October 31, 2011
NOTES
Suite P.
This is a suite of three miscellaneous experiments, but they
are united by a central theme of modeling (see appendix).
All experiments demonstrate a foundational principle with
fairly obvious applications, but are also useful for
demonstrating modeling, as opposed to theory. The
osmosis/permeability demonstrates two very important
processes in biology it is a model for passive transport
through membranes and simple cells and diffusion. This
experiment is short and not terribly exciting. The wine
experiment is straightforward and is about buffering in
complex solutions and how to model them. The
experimental is easy: modeling not so much. The final one is
about glow in the dark stuff. This is a ROB experiment
where you develop the methodology. Its straight forward, we
are pretty sure it works, and you will be given help with the
fluorimeter. It demonstrates phosphorescence, which is not
that well understood. Your data will, in principle, form a
model for the process. We then may be able to develop a
theory.
Potpourri
We are going to exploit this to get a quantitative value for the
permeability of the dialysis tubing to water. We could use
salmon fry in salt water but it would get vetoed. No wait;
salmon are adapted to survive that. Besides they are
inhomogeneous. This is a short experiment. Note Im leaving
a lot of details out deliberately.
A schematic of diffusion across a semi-permeable
membrane (SPM) is shown below
SPM
Water
[A]
Potpourri:1
time so
1 dnS
(4)
JS =
A dt
where A is the area (not to be confused with a species A). By
Fick's first Law of diffusion we have
dc
(5)
J S = DS S
dz
Where DS is the diffusion coefficient and z the distance.
Thus
dnS
d[S]
(6)
= ADS
dt
dz
Now, membranes are pretty thin, say l, so we can
approximate to D[S]/Dz using simple differences
dnSi
D[S] ADS
([So ] [Si ])
=
ADS
=
dt
Dz
l
(7)
AD K
(8)
=
S S ([So ] [Si ])
dt
l
This illustrates another important point; diffusion
coefficients actually dont vary much in solution for small
species so the transport of species across membranes is
mainly dictated by the partition coefficient, thats why these
rather mundane looking constants are so important in real
systems.
The constant DK/l is called the permeance, P, and
represents; well you tell me. (Hint, look at the units. Note
that the permeability is DK, but we dont have l so we settle
for permeance, but some texts confuse the two). It also turns
out to be relatively easy to measure (compared to D and K).
So we get
dnSi
(9)
=
AP ([So ] [Si ])
dt
Replacing nSi with Vi[Si] we finally get
d[Si ]Vi
(10)
=
AP ([So ] [Si ])
dt
which is in terms of readily measurable parameters.
Potpourri:2
n n
dni
=
AP o i
dt
Vo Vi
but n=m/MW (mass over molecular weight) so
(11)
m m
dmi
=
AP o i
(12)
dt
Vo Vi
where (just as a reminder) mi is the mass inside, mo the
mass outside and Vi and Vo are the respective volumes.
Next, we recognize that the m/V terms are just densities
and our observed mass is
mi ,total =mi ,water + mi ,sucrose + mtube +clips
So
dmi dmtotal
=
dt
dt
Now, if we restrict ourselves to short times and make
sure that Vo >>Vi , then both densities will be constant
providing that sucrose does not cross the membrane so
dmtotal
=
AP ( o i ) =
constant, Q
dt
Integrating (13) we get
AP ( o i ) t + R
mtotal (t ) =
(13)
(14)
i.e. a plot of total mass vs. time a straight line (its a zero
order process) that dead ends when the bag is empty. The
slope is AP ( o i ) so you can get P. The R, of course, is
just the mass of the empty bag + clips.
This derivation illustrates, very nicely, the dilemma of
applied thermodynamics. The physical chemistry concepts
needed for real systems (diffusion, permeability etc.) are
quite simple. However, the maths is much worse than in
traditional thermodynamics and is full of pitfalls; this is not
even a complicated system.
QUESTIONS
this case.
3) Find a permeability value for a biological system and
compare it with that of the dialysis tubing.
4) Does our constant density approximation at short time
seem reasonable in light of the experimental results?
5) There is a serious flaw in the design of this experiment
for use with sucrose. What is it? How would you demonstrate
(if possible) that this flaw exists?
6) Following up on the question above. I tried this
experiment with a strong solution of polyvinyl alcohol, MW
about 30000, and also starch of a similar MW. They are
extremely viscous solutions. Would you expect the
experiment to work properly?
7) Plot the initial rate of permeation vs. % concentration of
the sucrose solutions. Would you expect this value to change
or be constant. Explain. Support your answer with numbers.
8) Define diffusion, effusion, permeation (or permeability),
percolation, porosity, osmosis, partitioning, convection and
(ion) conduction. Be sure the definitions distinguish each
process clearly. Incorporate comments on their
relationships, if any. And dont use Wikapedia!
9) How would you test the approximation that sucrose does
not diffuse. Hint use sugar in the tube, and work out how to
analyse for sucrose. Do the experiment if time permits.
10) Plot the permeability vs. sucrose concentration and
comment. Given an explanation of the data if needed.
11) Analyze and present the data. Answer the questions. In
addition write up this experiment for a second year lab, just
the procedure. Ive left a lot of details out.
Potpourri:3
concentrations (remember an ICE box works with moles not derivative off your differential graph. The buffer capacity
concentrations). Compare your results with the calculated (for the purposes of this experiment anyway) is the
results. Plot graphs as needed. Tables are good to.
reciprocal of that value. Get the buffer capacity of the wine
The alcohol should have a small effect. Redo the at the pKa of acetic acid (when wine goes off it generates
calculations assuming that the alcohol doesnt affect the acid acetic acid), pH 3.4 (a typical wine pH) and pH 6.8 (pH of
concentration. Alternatively assume the alcohol dilutes the the mouth).
water, so five mole% water will shift the hydrogen ion
concentration by 5% (youll need to show me how I did that
calculation). If that doesnt work, speculate how alcohol may
influence the activity of the acid. Hydrogen bonding is an
obvious choice, but sucrose does that as well, but you should
find that it does not change the pH.
Non-polar solutes tend not to change the activity of
other species in solution, so we should not see an effect.
However, the concentrations used here will affect the activity
of water re: osmotic pressure so there may be an indirect
effect. We could measure the pKa using a conductiometric
titration, but I suspect for high sucrose concentrations we
would see an effect (not the effect mentioned above). Why
do you think I suspect that?
Buffering capacity is important. It tells us if the wine is
likely to shift in taste as it ages (as the acids change). Also,
Figure 2. Titration of a diprotic acid. The two pKas
the mouth is alkaline, a poorly buffered wine will shift in
are determined from the pH at the equivalence
taste with each mouthful if its poorly buffered. The buffer
points (e.g. V2 and pK2 on the diagram). Note V1
capacity is simply a measure of how well a solution resists
(not marked) is way between 0 and V1. V2 is
pH changes when acid or base is added. Typically for a wine
way between the two equivalence points this is the
its the amount of KOH that needs to be added to raise the
buffer zone.
pH by one point (and is bizarrely expressed in equivalents of
tartaric acid). We will take a physical chemistry approach. MODELING. We can compare the shapes of the titration
Its simply the differential of titration curve. Close to zero curves to see if our model is reasonable. They will not match
means a high buffer capacity in that region. The bigger that because the compositions are different, but the overall shape
region, the better the buffering. The width of the region as should be the same. To test this properly we need to be able
defined by some convenient parameter is the buffer capacity. to match our compositions. However, we are just after
insights, we can skip the chemistry altogether and simulate
See the glycine experiment on how to differentiate data.
If you want a simple reminder of the effects of buffering the titration curves. This is the physical chemistry bit. ICE
take 50mL of water stick a pH electrode in, measure, and will not work so we have to start from the ground up.
then and add 1mLof 0.25M KOH, it should shift the pH Diprotic acids dissociate as follows:
about 3 units. Repeat the experiment with pH 7 buffer. There
K1
K2
H2 A
H + + HA-
H + + A 2should be little effect.
1) Determine the end-point and hence the two pKas of where
a +a a + a 2the acid directly from a plot of pH vs. VHCl. (Use the
H HA
=
K1 =
K2 H A
Henderson-Hasselbach equation look it up). Also refer to
aH2 A
a HA
the figure 2 below.
a
is
the
activity
which
we
will
approximate
to
2) The buffer capacity can be calculated from the inverse
of the differential of the titration curve. Typical results are concentrations and ignore the fact that pH electrodes
shown in figure 1. To get the buffer capacity at a certain pH actually give the activity. We will have another two equations
use your titration data to find the volume corresponding to for the other acid.
K1
K2
the desired pH. Then use that volume to read the value of the
H2 M
H+ + HM-
H + + M 2 P.S.Phillips October 31, 2011
Potpourri:5
K1
a +a a + a 2H HM
=
K2 H M
aH2 M
a HM
[H + ][OH ]=K w = 10 14
Ive never done a simulation past pH 6 so Im not sure if
Maple stays stable in this region; i.e. with this equation
added in.
Since we make up our own solutions we have the total
amount of acid and potassium (we include the potassium
counter ions in with the total acid). We now have seven
equations and seven variables. If you rearrange this you end
up with cubics in [H+]. This is where Maple (or whatever, I
havent had any success with MatLab or MathCad though).
comes in it can solve this kind of thing easily, but there are
two problems. The first is that Maple is quite general so it
will give you all three solutions for the [H+]. You have to
make sure you get the real +ve root only. You then have to get
Maple to generate all the concentrations for given starting
values, in a nice table or an array. To do that you have to put
everything in a loop. Finally, you have to get Maple to plot it,
although it might be easier to cut and paste your table into
Excel. Ask me for my Maple hints page.
Experiment 3. Luminescence.
Potpourri:6
Potpourri:7
Potpourri:8
EXPERIMENT G:1
If possible use two water baths: one you take your sample
from and one heating up to the next temperature while you
are taking the spectrum. (If its an Isotemp bath be sure to
set the safety cutoff knob to maximum.)
EXPERIMENT G:2
REFERENCES
1. F. Meersman, L. Smeller, K. Heremans Biophys. J. 82,
2635-2644 (2002).
2. See help sheet for the PDB programs on the course web
site.
APPENDICES.
INFLECTION POINTS. To determine the inflection
point of a sigmoidal curve dy/dx vs. x. (here y is
absorbance and x temperature). Setup Excel to do a simple
derivative using dy / dx ( yi1 yi )/(xi1 xi ) Typical
results are shown in figure 2. The inflection point is the
maximum of the derivative.
Conformation: The conformation types are listed in PDBWS are Turn, Helix, Coil, Strand. The elements I'm familiar
with are a-helix, Random Coil, Chain and b-pleated
sheet, ribbon. So help me out here; what's what? Whats
with the a and b? Be sure to explain things in terms of
structures and intermolecular interactions (H-bonding,
sulphur bridges, Pi stacking, hydrophobicity etc.)
Also, some of the programs (Ramplot and Swissplot) have
options for Ramachandran plots which seems a neat way of
sorting out the different structural types. So what are
Ramachandran plots (Wikipedia is as good as any place to
start)? Apply them to the species you studied.
At room temperature the more ordered helical structure
exists in preference or the less ordered b-sheet, or more to
the point a random coil. This would appear to violate the
Second Law, but it doesnt; explain. (i.e. explain the
transitions between helices and whatever in terms of
enthalpy and entropy, as well as intermolecular
interactions.
Rationalize the spectral changes you saw in terms of
structural changes (and accompanying changes in
intermolecular interactions) with pH, guanidine, SDS and
temperature.
See if you can find an X-ray structure of myglobin above
its transition temperature (i.e. above 90C). Print it out.
EXPERIMENT G:3
EXPERIMENT G:4
This is what you get lots and lots of stuff (336 structures). You need to narrow it down.
I selected humans and a 1-2 resolution X-ray structure. You still get tons of stuff and need to scroll through
items at the bottom of the page until you get the one you want. Here we have two or three examples with the
same information. Plea ignorance (i.e. say you are a chemist) and select one. In this case exceptional ignorance
as somewhere it decided to insert hemoglobin into the myoglobin list. Better go find that 101 button at the top.
Anyway lets click on 2HHb, or more specifically the the title next to it.
A picture at last. Ive scrolled down so we can see the bottom menus. These give some interesting views,
especially under the surface options. Well get back to that later.
There are other viewing options so lets scroll back up to see them.
Now wait for Java to load. Its called Java because you have to go to coffee while it loads. I think it should be
called C2 (crippled computing) although this applet seems to be well programmed. Anyway after youve done
this a few times you will want to try off-line programs such as Jmol, Pymol, Ramplot, and SwissView are some.
I can put them on disk or they maybe emailable, or you can find them online. To be able to use them we have to
download the data files To do that find the download menu.
Before we go on and discuss the viewing programs (Jmol, Pymol, Ramplot, and SwissView) in detail heres a
few pointers.
The online Jmol is mainly a viewer but it has some dropdown menus at the bottom that make it a very
interesting viewer. I love the way it refers to the standard text book view for proteins as cartoons.
The offline Jmol is just the simple viewer in the options list. If thats what you want go for it.
Kiosk (the other viewer) makes a good screen display for open house.
The Protein Workshop is a supped up viewer. I think it give the best pictures. There are a few bells and whistles
I have checked out yet. When you first run it it installs itself on your computer.
I my humble opinion Pymol is garbage.
RamaPlot (Ramchandran Plot Explorer) is possibly the most interesting. Its old and the visualization is not
fancy but it seems to give stuff other programs dont, or at least the kind of stuff relevant to this course
SwissView (Swiss-PDB Viewer) Lots of options but no fancy pictures.
Qutemol is interesting if you want to see the importance of lighting and shading when rendering 3D molecules.
For other options see
http://en.bio-soft.net/3d.html
http://www.pdb.org/pdb/static.do?p=software/software_links/molecular_graphics.html