BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
LABORATORY REPORT COVER PAGE
GROUP NUMBER: M3
PROJECT TITLE: Freshman Lab Experiment for Protein Molecular Weight Determination by Gel
Exclusion Chromatography(AG)
DATE SUBMITTED: December 21, 2001
ROLE ASSIGNMENTS
ROLE
GROUP MEMBER
FACILITATOR…………………………………..…..Nina Teng
TIME & TASK KEEPER…… …………………….Michael Jue
SCRIBE………………………………………….…Shishir Dube
PRESENTER…………………………………….Marisa Kastner
SUMMARY
The main objective of this experiment was to design a suitable lab experiment that
freshman BE students could perform to determine the Molecular Weight (MW) of a
protein using a given calibration curve. To achieve this objective, the aim was to develop
a calibration curve that could be used by freshmen to determine the MW of a protein
accurate within 2% of the literature value and precise to a confidence interval within 10%
of the calculated value. This was done by running proteins on the Gel Exclusion
Chromotography (GEC) column and constructing a calibration curve that graphed the
individual protein’s elution volume divided by the void volume of the column versus the
log of the molecular weight of the protein. A calibration curve making use of data all
collected in one day as well as one making use of data collected over a number of days
were analyzed. It was found that although it provided are shorter range, the curve using
the data collected in one day was generally more accurate, while the curve using more
data points was more precise. However, neither was within the set objective range. It
was also concluded that while data collected in one day is reproducible, there is a
significant difference in the data collected over a span of days.
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Objectives:
The main objective of this experiment was to design a suitable lab experiment that
freshman BE students could perform to determine the Molecular Weight (MW) of a
protein using a given calibration curve and a Gel Exclusion Chromotography (GEC)
column. The specific aim to achieve this objective was to develop a calibration curve
that could then be used by freshmen to determine the MW of a protein accurate within
2% of the literature value and precise to a confidence interval within 10% of the
calculated value. Specifically, gel exclusion chromatography (GEC) was used to
characterize proteins by their molecular weight based on their individual interaction with
the gel-packed glass column.
Background:
The proteins being employed in this experiment to construct the calibration curve
are Bovine IgG (MW=150 kDa), Bovine Serum Albumin (MW=77 kDa), Hemoglobin
(MW=64 kDa), Ovalbumin (MW=42.7 kDa), Myoglobin (MW=16.9 kDa), Chicken Egg
Lysozyme (MW=14.3 kDa), and Cytochrome C (MW=13.0 kDa). 1 Ovalbumin is also
known as chicken egg albumin and consists of 385 residues and denatures at high
temperatures.2 Myoglobin is a type of α-protein and contains eight helices as well as 153
amino acid residues (121 belonging to the α-helices). 3 Cytochrome C is also a type of αprotein which has four α-helices packed together in a bundle.3
Theory and Methods of Calculation:
All calculations are in the BE Lab Manual.4
Specifically, in his paper on the Determination of Molecular Weights of Proteins by Gel
Filtration on Sephadex, John R. Whitaker provides the linear relationship of V/Vo versus
log (Molecular Weight) where V is the elution volume of the protein and Vo is the void
volume of the column.
Materials, Apparatus, Methods:
 Proteins used:
Protein
MW (Da)
Bovine IgG
150000
Bovine Serum Albumin
77000
Hemoglobin
64000
Ovalbumin
42700
Casein
23600
Myglobin
16900
Chicken Egg Lysozyme
14300
Cytochrome C
13000




Bovine Serum Albumin (2 mg/ml)
Ovalbumin (2 mg/ml)
Myoglobin (2 mg/ml)
Cytochrome C (2 mg/ml)
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.

All other material and apparatus specifications used were from the BE Lab
Manual.4
Results:
Using Gel Exclusion Chromatography (GEC), elution times and corresponding
volumes for Blue Dextran and a set of proteins: Cytochrome C, Myoglobin, Ovalbumin,
Bovine Serum Albumin were collected. The volumes were calculated by multiplying the
observed flow rate by the elution times. Vo, the void volume of the column, was
calculated from Blue Dextran GEC runs and V was calculated from individual GEC
protein elutions. V/Vo was graphed against log(MW) and a calibration curve was
constructed (fig. 1)
Figure 1: Calibration Curve of the data taken during project period
Figure 1 represents the calibration curve of the data taken for the proteins Cytochrome C,
Myoglobin, Ovalbumin, and Bovine Serum Albumin.
Regression analysis was performed on the collected data (table 1). The intercept
was 4.298 +/- 0.231, the slope was –0.489 +/- 0.0515. The R^2 value for the curve was
0.9728. The percent uncertainty is calculated by 100*(CI/calculated value).
Table 1: Regression Analysis Summary for data collected during project period.
Table 1: Regression analysis summary for calibration curve of data collected during project
period.
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Elution data gathered by other lab groups in previous weeks were collected and
then combined with the data taken in the project period to create a calibration curve with
an extended calibration range (fig. 2).
Figure 2: Calibration Curve with an Extended Range
Figure 2: Extended calibration curve incorporating previous GEC elution data taken during
laboratory experiment weeks as well as data taken during the project period.
Regression analysis was performed on the compiled data (table 2). The intercept
was 5.128 +/- 0.286, the slope was –0.653 +/- 0.1276. The R^2 value for the curve was
0.8098.
Table 2: Regression Analysis Summary for Extended Curve.
Table 2: Regression analysis summary for extended calibration curve of both new data collected
during project period and old data from previous weeks.
Ovalbumin and Cytochrome C elution data were collected in this experiment and
analyzed using the calibration curves developed. In addition Hemoglobin elution data
from previous groups was compiled and analyzed in the same way (table 3).
Specifically, all data was analyzed using both the calibration curve developed from the
data collected in the project period (new data) and the curve with both the new and
compiled data from previous experiments (complete data set). For each protein,
molecular weight was calculated with its associated confidence interval and %
uncertainty. In addition, the difference between the calculated molecular weight and the
literature value was also found.
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Table 3: Collected data analyzed using two different calibration curves.
Table 3: Data from the elution of three different proteins analyzed using the calibration curve
developed from data collected in this experiment and analyzed with the calibration curve
developed from data compiled from new and previous experiments.
Elution data for Blue Dextran for each day was analyzed to determine if data
collected on the GEC apparatus were repeatable in the given day (table 4). In Week 1,
the % uncertainty in the three collected Blue Dextran values was 0.293%. In Week 2 the
% uncertainty in the three collected Blue Dextran values was 1.397%. These very small
% uncertainty values indicate that trials are reproducible in the given day.
Table 4: Blue Dextran Reproducibility in a Given Day
Table 4: Blue Dextran values tabulated over two weeks and analyzed for reproducibility on a
given day. The small percent uncertainty values indicate small confidence intervals resulting in
more precise data.
T-tests were run with the data from two different weeks to determine whether
elution volume data was repeatable from day to day (table 5). T-stat is larger than tcritical and as a result, average Blue Dextran elution volumes from two different days can
be deemed significantly different. Thus GEC elution data is not repeatable from week to
week. The ramifications of this will be discussed in the following section.
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Table 5: Blue Dextran Reproducibility over Multiple Weeks
Table 5: T-test run on Blue Dextran elution data collected over 2 days to determine statistical
differences in data between days. Because t-stat is greater than t-critical, the data can be
considered significantly different.
Discussion and Analysis of Results:
The main objective of this experiment was achieved in that a calibration curve
that enables freshman BE students to determine the molecular weights of proteins was
developed. However, the specific aims of developing a calibration curve to determine the
MW of a protein accurate within 2% of the literature value and precise to a confidence
interval within 10% of the calculated value were not attained. Thus, it is concluded that
the calibration curve is not capable of achieving the desired accuracy or precision.
In this experiment, two calibration curves based on the relationship between V/Vo
and the logarithmic function of MW were obtained for analysis and comparison. As
shown in Figure 1, the first calibration curve of three proteins from one trial day
(Cytochrome C, Myoglobin, Ovalbumin, and Bovine Serum Albumin) yielded an
equation of: V/Vo = -0.4892*log (MW) + 4.2976, with the 95% confidence limits for the
slope and y-intercept being 4.298  5.385 % and –0.489  10.519 %, respectively (Table
1). The confidence limits represent factors that pose a significant effect on the precision
of the calculated molecular weight of proteins. Such an effect can be attributed to the
logarithmic (base 10) function of the curve, in which the slight deviations can propagate
exponentially into the final calculation of the molecular weight.
Based on this first curve in Figure 1, the average molecular weights and precision
to a 95% confidence of the ovalbumin, cytochrome C, and hemoglobin were respectively
calculated to be: 45,472.028 Da  16.970%, 12,476.482 Da  28.100%, and 35,234.725
Da  59.378% (Table 3). The respective accuracies of each protein’s molecular weight to
its respective literature values are: 7.724 % (ovalbumin), 9.040 % (cytochrome C), and
45.373 % (hemoglobin). Although the values did not fall within 2% of the desired
accuracy to its literature value, the literature values for ovalbumin and cytochrome C did
fall within the 95% confidence limits of the experimentally obtained molecular weightswhich indicate insignificance between the two values for the two proteins. However, the
confidence intervals are so large that the calibration curve is still not a reliable method of
determining the molecular weight of the proteins accurately and precisely. One factor to
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
note is that the trend of the hemoglobin results is inconsistent with the ovalbumin and
cytochrome C results. This is due to the fact that the hemoglobin results were obtained
from different trial days, as opposed to the other proteins.
The extended calibration curve constructed in Figure 2 consists of data from the
calibration curve in Figure 1, as well as data points from previous laboratory experiments
performed throughout the semester. The curve was determined to be: V/Vo = -0.653*log
(MW) + 5.128, with the 95% confidence limits for the slope and y-intercept being 5.128
 11.479 % and –0.653  19.538 %, respectively (Table 2). These confidence limits are
considerably larger than the values for the first curve by almost twofold, indicating a
higher incidence of error from the exponential function for the calculations of MW from
this extended curve (Figure 2) than from the previous curve (Figure 1).
In comparison to the first calibration curve, this extended curve yields a higher
degree of inaccuracy and precision in determining the molecular weights of the proteins.
The calculated molecular weights and 95% confidence limits of ovalbumin, cytochrome
C, and hemoglobin were respectively found to be: 58,062.498 Da  12.791%, 22,004.159
Da  21.040%, and 47764.699 Da  45.430% (Table 3). The smaller values for the
confidence limits of the molecular weights as compared to the first curve can be
attributed to the effect of plotting more points on the calibration curve, which improves
the uncertainty (precision).
The percent accuracies from the literature values for each protein are as follows:
35.978 % (ovalbumin), 69.263 % (cytochrome C), and 25.946 % (hemogoblin). The
increased inaccuracies of the molecular weights calculated to its respective literature
value for ovalbumin and cytochrome C using this extended curve were most likely
caused by the variations among each molecular weight value obtained from different
experiments performed upon varying conditions. Such variations are evident from the
comparison of the R2 value of 0.973 (Figure 1) attained from the first curve to the R2
value of 0.810 (Figure 2) obtained from this extended curve, which shows less linearity.
Thus, it translates to the higher degree of inaccuracy as compared to the first calibration
curve-which will be discussed in more detail below.
The recommendations to be applied to this experiment would involve factors that
would improve the accuracy and precision of the calibration curve to obtain the best
possible value of molecular weights for proteins. It was concluded from this experiment
that to reduce errors of uncertainty, the number of points for each protein on the
calibration curve must be increased. Thus, a recommendation to the laboratory protocol
would be to repeat as many trials as possible of the same protein within the time allowed
in one day. In addition, the range of the calibration curve must span the molecular
weights of the proteins to be tested.
In terms of accuracy to the literature value, it was concluded from the experiment
that the same experimental conditions must be applied to each protein to reduce the
variability in the molecular weight values obtained for one protein. The low values of
percent uncertainties (Week 1: 0.293 %; Week 2: 1.397%) in Table 1, which displays the
data for the three trials of blue dextran elution volumes obtained from two different trial
days, illustrate that elution trials are reproducible within the same day. However, the ttest results shown in Table 2 for the same data indicate that the elution volumes obtained
from day to day were significantly different, and thereby causing an increased uncertainty
among the repeatability of the experiment over different trial days and conditions. This
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
could be specifically attributed to the change in the conditions of the sephadex gel
column. Thus, the experimental protocol would be modified to ensure that for each trial
performed for one protein, the experimental conditions are kept as constant as possible.
Another possibility in improving the accuracy of the calibration curve would be to
decrease the flow rate, which would allow more data to be obtained for each trial.
However, this is not recommended highly because it would cause a compromise in
uncertainty via reading the peak times. This is due to its effect of producing flatter peaks.
These recommendations would be applied to the freshman laboratory experiments
as well to optimize their experiment as much as possible in hopes that they could attain
their objectives and aims as outlined in the appendix. It would be feasible for the
freshman students to repeat many trials of one protein, since they would only be running
trials of one protein in the two-hour time period allotted. In addition, since the
experiment would be performed in one trial day, the experimental conditions of the
apparatus would have a higher degree of consistency.
Conclusions:
1. Although it provided a shorter range, the curve using the data collected in one day
was more accurate (60.223%, 27.874%, and –19.427%) for two out of the three
proteins.
2. The curve using more data points was more precise (between 4.179% and
13.948% more), as expected because more trials were run.
3. Neither calibration curves were within the set objective range of 2% for accuracy
and 10% for precision.
4. Data collected in one day is reproducible (percent uncertainties of under 1.4%).
5. There is a significant difference in the data collected over a span of days as
evidenced by a t-test.
References:
1. http://www.sigma.com
2. Leach, Sydney J. Physical Principles and Techniques of Protein Chemistry.
1970. New York. pp. 459-470.
3. Hamaguchi, Kozo. The Protein Molecule: Conformation, Stability, and Folding.
1992. Tokyo. pp. 38-55.
4. BE Lab Manual Experiment 2.
Appendices:
Appendix 1: Freshmen Lab Experiment
Appendix 2: Example write-up for freshmen running Ovalbumin
Appendix 3: Example write-up for freshmen running Cytochrome C
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Appendix 1:Freshmen Lab Experiment:
Protein Molecular Weight Determination by Gel Exclusion
Chromatography (GEC)
Challenge: Can you show that the molecular weight of the “unknown”
protein is accurate within 2% of the literature value and precise to a
confidence interval within 10%?
Background:
Gel Exclusion Chromatography:
Chromatography is defined by Merriam-Webster as “a process in which a
chemical mixture carried by a liquid or gas is separated into components as a result of
differential distribution of the solutes as they flow around or over a stationary liquid or
solid phase.”
Specifically, gel permeation chromatography is defined as
“chromatography in which macromolecules in a solution are separated by size on a
column packed with a gel.” In this experiment, gel exclusion chromatography (GEC)
will be used to characterize a protein by its molecular weight based on its interaction with
a gel-packed glass column.
The particular column to be used is a glass cylinder with Sephadex CL-6B as the
gel. The material to be tested (the protein) is added to the top of the column and allowed
to flow through the gel, through a detector, and out to a waste canister. The detector for
this experiment is an ultraviolet flow-cell spectrometer that measures absorbance at the
set wavelength and path length. The readings from the detector will ultimately be used to
determine the molecular weight of the test protein.
Before running the test protein, another material, one whose molecules are too big
to enter the gel (rather they can only reside in the space between gel particles) is run. It
elutes rapidly because of its size. The time it takes for the peak absorbance to arrive
multiplied by the flow rate of the material is the excluded or void volume Vo. Then,
once the test protein is run, the time it takes for the protein to reach peak absorbance
multiplied by the flow rate of the protein is the eluted volume V. The ratio of the eluted
volume to the void volume can be used to find the molecular weight of the protein.
Precision and Accuracy:
Two different measurements for uncertainty are precision and accuracy.
Precision gives the range of values to be expected if the experiment were to be repeated.
Accuracy compares the experimental value to a known quantity (such as a literature
value).
Materials and Apparatus:
Blue Dextran with MW=200,000; 1 mg/ml solution
Protein for which MW will be determined (one of the following)
Protein
MW (Da)
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Bovine IgG
Bovine Serum Albumin
Hemoglobin
Ovalbumin
Casein
Myglobin
Chicken Egg Lysozyme
Cytochrome C
150000
77000
64000
42700
23600
16900
14300
13000
1x50 cm glass column with CL-6B Sephadex in buffer
Buffer (0.1 M NaAc, 0.4 M NaCl, pH=6 with HCl)
UV Monitor, BioRad Model EM-1
Pump, BioRad Model EP-1
BioLogic LP Injection Valve
Reservoir, tubing, stands
LabView program “Chrom.vi”
Procedures (approximately 2 hours to complete):
1. The column should be setup in advance, but check to make sure that the setup is
correct according to the following layout:
a) Buffer tank (flask) to Pump inlet
b) Pump outlet to Injection valve
c) Injection valve to Column head connector (top)
d) Column outlet (bottom) to UV monitor inlet
e) UV monitor outlet to waste container
f) Waste tube from the injection valve to the waste container
2. Connect the electrical signal outlet of the UV monitor to the first channel of the
LabView input board. Use the LabView program called “Chrom.vi” or the
Virtual Bench Data Logger for data collection. Remember to input data
collection rate and the file name.
3. Set the UV Monitor sensitivity (in AUFS) to 0.1. The protein sample solution
should give an output of about 1 volt in this monitor with the current sample loop
size. Adjust the sensitivity as needed to obtain maximum signals without
saturating the monitor.
4. Set the pump to 1.5 ml/min. Run the pump for several minutes to pack the
column.
5. Check that the injection valve is in the “load” position. Inject the Blue Dextran
solution into the Injection valve sample loop until you see dye coming out of the
waste port of the valve. Make sure to avoid air bubbles in the loop.
6. Start the data collection program and turn the valve to the “inject” position to
begin the injection process. After two minutes, return the valve to the “load”
position.
7. Allow the sample to run through the column, the monitor, and into the waste
container. (LabView will plot a curve of voltage vs. time and save the data to a
spreadsheet file.)
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
8. Measure the actual elution volumetric flow rate to convert the data from time to
volume as the independent variable. Time the collection of a fixed volume of
solution into a graduated cylinder and record the flow rate measured in ml/min.
Calculate the void volume, Vo, from the peak of the blue dextran curve.
9. Repeat these procedures for three separate samples of the “unknown” protein
whose molecular weight is to be determined. Calculate the V/Vo for each run of
the protein.
10. Write up a report about the experiment including Summary, Results, Analysis,
and Conclusion (brief statement of findings). Be sure to include a title and a
descriptive caption for any tables or figures included.
Results Section: How do I work up the data?
Record results of Procedures 8 and 9.
Plot the following calibration equation (calculated from known molecular weights
and V/Vo experimental data). Place log (Molecular Weight) on the x-axis and V/Vo on
the y-axis. Then use this equation to determine where on the plot to place each of the
V/Vo points for your protein. Calculate the average Molecular Weight from the three
different trials.
Calibration Equation: V/Vo= -0.6529*logMW + 5.1276
 What is the Molecular Weight of your protein? [can be found using Excel: type
=AVERAGE(range of data)]
 What is the standard deviation? [can be found using Excel: type =STDEV(range
of data)]
Now that a mean value has been obtained, in order to get precision values, we will
make use of the concept of confidence limits. The general convention for confidence
limits is to be 95% confident that the real value is within the calculated limits. These
limits are calculated using a statistic, t, which is a function of degrees of freedom (DF=
number of trials minus 1) and terms of probability (in the case of 95% confidence, 0.05
probability). Using Excel, the value for t can be calculated and the confidence limit can
be found.
To find t in Excel, type
=TINV(p, DF)
where p=0.05 and DF=3-1=2
Then, the confidence interval can be calculated using the formula:
ts/√n where s is the standard deviation and n is the number of trials (3).
You should now have an answer in the form of: average MW +/- (ts/√n).
Analysis: What information can I draw from the data?
What do we write about in the Analysis Section of the write-up?
1. How good was the data?
Accurate? Was the |literature – average| / literature value < 0.02 ?
Precise? Was the CI / averageMW < 0.10 ?
What were potential sources of error?
2. Analysis of the experiment:
How would changing the flow rate have affected your data?
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Do you have any suggestions to improve the experiment?
References:
“Chromatography,” Merriam Webster’s Collegiate Dictionary. www.m-w.com © 2001
by Merriam-Webster, Incorporated.
“Gel Permeation Chromatography,” Merriam Webster’s Collegiate Dictionary. www.mw.com © 2001 by Merriam-Webster, Incorporated.
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Appendix 2: example write-up for freshmen running ovalbumin
Protein Molecular Weight Determination by Gel Exclusion
Chromatography (GEC)
Challenge: Can you show that the molecular weight of the “unknown”
protein is accurate within 2% of the literature value and precise to a
confidence interval within 10%?
By: Jenny Lopez
Mike Jordan
Summary:
The unknown molecular weight of the protein ovalbumin was determined in this
experiment using gel exclusion chromatography and a given calibration curve equation.
The average molecular weight of ovalbumin was experimentally determined to be
47,362.04 Da with a standard deviation of ± 3,490.59 Da. This was accurate to 10.92%
and precise to 17.08%, which differed from the desired values of 2% accuracy and 10%
precision. These average molecular values differed from the given literature molecular
weight value of 42,700 Da by 9.84%. The literature value does not within the confidence
interval of experimentally determined average molecular weight of ovalbumin
Results:
Initially, the blue dextran (MW=200,000 Da) was initially run on the column.
The peak absorption reading of this curve occurred at the time of 7.88 minutes, while the
flow rate was determined to be 1.8 mL/min. The void volume of the column, Vo, was
found by taking the product of the time of the peak absorption reading and the flow rate,
which equaled 14.19 mL. This void volume was used as a basis of determining the V/
Vo value for ovalbumin.
Table 1: Ovalbumin Elution Results
Trial 1
Trial 2
Trial 3
Time (min)
15.821
15.825
16.039
Flow Rate V (mL) Vo (mL) V/Vo
1.8 mL/min 28.53
14.19 2.011
1.8 mL/min 28.48
14.19 2.007
1.8 mL/min 28.87
14.19 2.035
Table1: Shows elution results for ovalbumin with peak absorption time, flow rate, and V/Vo for three
different trials of ovalbumin.
The table above shows the results of peak absorbance time and the volume data
for ovalbumin over three separate trials. The values of V/Vo can then be used to
substitute into the calibration curve to find the experimental molecular weight. Using the
given calibration curve equation of V/Vo= -0.6529*logMW + 5.1276, the following data
was obtained.
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Table 2: Ovalbumin Molecular Weight Values
Trial 1
Trial 2
Trial 3
Average
Accuracy
Precision
Molecular Weight
49,732.81
49,613.45
43,628.14
47,362.04
10.9%
17.08%
Table 2: Ovalbumin molecular weights for three separate trials as well as average value, precision,
and accuracy.
As seen from the table above, the three trials result in an average molecular
weight value of 47,362.04 Da, with a standard deviation of ± 3,490.59 Da. The average
molecular weight is accurate to 10.92% and precise to 17.08%. The actual molecular
weight value is 42,700 Da.
Analysis:
As shown in Table 2, the average molecular weight of ovalbumin did not fall
within 2% accuracy, but instead with an accuracy of 10.9%. It also did not fall within the
intended precision of 10%, but instead has a precision of 17.08%. These average
molecular values differed from the given literature molecular weight value of 42,700 Da
by 9.84%. The literature value does not within the confidence interval of experimentally
determined average molecular weight of ovalbumin.
The main sources of error in the experiment were systematic error, which could
have occurred through machine and human error. The machine error could have caused
inaccurate readings for the flow rate and human error in estimating the peak adsorption
times could have occurred.
The recommendations for future experiments would be to accurately calibrate and
measure the flow rate by using a computerized sensor and timer. Also, to make sure that
the apparatus is set up correctly, especially when adjusting the screw for the flow rate.
Thus, it would be optimal to constantly measure the flow rate for the duration of the
experiment to ensure the values remain constant.
Conclusion:
1) The average molecular weight of ovalbumin was experimentally determined to
be 47,362.04 Da with a standard deviation of ± 3,490.59 Da. This was accurate to
10.92% and precise to 17.08%.
2) These average molecular values differed from the given literature molecular
weight value of 42,700 Da by 9.84%. The literature value does not within the
confidence interval of experimentally determined average molecular weight of
ovalbumin.
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Appendix 3: example write-up for freshmen running Cytochrome C
*Note: this particular data was taken on two separate occasions which accounts for the two different blue
dextran values. The freshmen would running everything on one day resulting in only one value so the
following report is written as such.*
Protein Molecular Weight Determination by Gel Exclusion Chromatography
(GEC)
Challenge: Can you show that the molecular weight of the “unknown”
protein is accurate within 2% of the literature value and precise to a
confidence interval within 10%?
Jessica Freshmon
Tamara Frishman
Summary:
Gel Exclusion Chromatography is a method by which a mixture is separated
based on its particular properties. In this experiment, the time at which peak absorbance
of the protein Cytochrome C occurred was used to determine the molecular weight of
Cytochrome C. The MW and it’s 95% confidence interval was determined to be
12884.08 Da +/- 3649.638 Da. The calculated MW was accurate to 0.892% and precise
to 28.327% meaning the method used gave a value close to the literature value, but it was
not particularly repeatable.
Results:
First the blue dextran, with molecular weight 200000 Daltons was run on the
column. It’s peak absorption reading occurred at time 7.995 seconds. The flow rate was
measured and recorded to be 1.8 mL/min. Therefore, the void volume of the column, Vo,
was 7.995 minutes * 1.8 mL/min = 14.391 = Vo.
The protein Cytochrome C, with molecular weight 13000 Daltons, was then run
three separate times on the column. The following table details the results of the time at
which peak absorbance occurred as well as the resulting volume data.
Table 1: Cytochrome C Elution Data
Time
Flow Rate
(minutes) (mL/min)
V (mL)
Vo (mL)
V/Vo
18.353
1.8
33.035
14.391
2.296
18.132
1.8
32.638
14.391
2.268
18.254
1.8
32.857
14.184
2.316
Table1:Cytochrome C Elution Data recording the time at which peak absorption occurred, flow rate,
and void volume, Vo, from blue dextran used to calculate V/Vo for three trials of Cytochrome C.
BE 309
Lab Report cover
FALL 2001
Download and complete this form. Be sure the report conforms to the Instructions in Report Writer.
Using the calibration curve, V/Vo= -0.6529*logMW + 5.1276, the three trials
result in Molecular Weights (MW) of 12674.22, 14446.91, and 11531.13 Da. The
average MW then is 12884.08 Da with a standard deviation of 1469.176. The 95%
confidence interval from these three trials is 3649.638. Therefore, the results of the
experiment give a MW of 12884.08 Da +/- 3649.638 Da.
Analysis:
Using Microsoft Excel, the calculate value for the MW was found to be accurate
to 0.892% and precise to 28.327%. While the MW was accurate to within 2%, it was not
precise to 10% meaning that the value found was very close to the literature value but it
was not particularly repeatable.
Sources of error include systematic errors such as flow rate and human error such
as determining the time of peak absorbance. Therefore, a recommendation to improve
the experiment would be to monitor the flow rate more often to be sure that it is not
changing and affecting the results of each individual trial. Changing the flow rate would
change the time at which the peaks occur. Also, a faster flow rate would result in sharper
peaks.
Conclusion:
1) The Molecular Weight of Cytochrome C was found to be 12884.08 Da +/3649.638 Da.
2) The calculated MW was accurate to 0.892% and precise to 28.327%.