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BENG 162: Biotechnology Lab

Fall 2012

GROUP #7
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF ALBUMIN AND MYOGLOBIN
Nicolas, A., Etcheverry
UCSD Bioengineering
La Jolla, California, USA

ABSTRACT
High performance liquid chromatography (HPLC) is a protein
separation method that takes advantage of the interaction of proteins in
with solid stationary beads to separate the proteins based off a variety
of factors. The proteins can be separated by size, hydrophobicity,
positive or negative charge, as well as specific binding interactions.
This experiment produced protein peaks for albumin and myoglobin at
their respective time points as well as a peak to valley ratios of 3.156
and 11.58, respectively.

INTRODUCTION
The main objective of this experiment is to separate the proteins
albumin and myoglobin using high performance size exclusion liquid
chromatography and analyze the results displayed by the
chromatogram. The interaction of the proteomic components in the
mobile phase with the stationary phase, made up of an agarose bead
containing column, promotes the separation of proteins, and the
ultraviolet/visible (UV/VIS) detector measures and records the
absorbance of the effluent in order to produce a chromatogram.

METHODS
Water from the Milli-Q is run through a vacuum filter (.45 µm),
and the eluent used in the procedure is .15M NaCl in H2O. The HPLC
lamp is turned on by adjusting a few setting on the HPCL Method
screen, and the indicator light confirms that the lamp is on.
Before running the standards and sample, priming the pump is
required in order to remove bubbles in the tubing of the system. A 10
ml syringe is placed on the Pump A draw off valve, the purge drain
valve is opened, the prime selector valve is turned to PRIME LINES,
and 10 ml of NaCl eluent are drawn into the syringe. Once all the air
bubbles are removed, the sample injection port is placed in the LOAD
position, the syringe is placed back on the valve, and 10 ml are drawn
into the syringe. The priming selector valve is turned to PRIME
PUMP, and 9 ml of the eluent are pushed through the syringe and into
the waste compartment. The priming selector valve is changed to
OPERATE and the syringe is removed. The purge drain valve is
turned back to its original position.
The method file is opened and set to run for about 45 minutes at
.5ml/min, and the Gel Filtration Standard by Biorad is diluted to the
proper concentration. Keeping in mind that the Shodex SB-804
Column (Model HQSB84) works more efficiently at protein
concentrations lower than 5 mg/ml, the standard stock concentration is
diluted from 36 mg/ml to 2 mg/ml. The standard solution contains the
following proteins: thyroglobulin (670 kDa), gamma globulin (158

kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B-12
(1.35 kDa). The sample to be tested, containing the proteins albumin
and myoglobin, has a stock solution of 5 mg/ml and it is also diluted to
a concentration of 2 mg/ml where the albumin to myoglobin ratio is
1:1.
Once the samples and standards are prepared, 40 µl of the protein
standard is loaded into the machine using a Hamilton 50 µl syringe in
order to perform size exclusion chromatography. After the standard is
run, the sample containing albumin and myoglobin is loaded in the
same manner and run for about 45 minutes to an hour. Due to the
machine experiencing some complications with respect to maintaining
the expected flow rate of .5 ml/min, the flow rate is adjusted to .75
ml/min on the computer screen at approximately 13 minutes in for
both the standards and the sample. The flow rate is also determined by
having a graduated cylinder capture the flow through in order to record
the volume per time. Once the runs are over, and the data is exported,
the column is stored in a NaCl solution.

RESULTS
After running the 2 mg/ml protein standard concentration for
approximately 53 minutes, the chromatogram shown in Figure 1 was
produced. The computer interface indicates that the solution was
flowing at .5 ml/min, but the flaws in the flow rate were made known
prior to beginning the experiment. The flow rate was adjusted as
described above for both the standards and the samples, and the
average flow rates were calculated and recorded. The average flow rate
for both the standard and sample were both individually determined to
equal .22 ml/min. According to the data displayed by the
chromatogram in Figure 1, thyroglobulin, the first protein expected to
elute due to its high molecular weight, did not appear as a peak
indicating the possibility of some error with respect to the standard.
The four other proteins appeared as peaks, and the peak separations in
minutes were calculated by determining the time differences between
the peaks of the respective proteins. Also, the chromatogram for the
standards was adjusted by adding .006 to all of the absorbance values
in order to standardize the graph at zero.
The peak to valley ratios were calculated by dividing the peak
absorbance values by the lower valley absorbance values to the right
or the left of the peak. Vitamin B-12 showed the best separation with a
peak to valley ratio of 36.04 shown in Table 1. In order to determine
the area of the peaks for both the standards and samples, Figures 1 and
2 were both opened with ImageJ, and the drawing tool was used to
create triangle shaped areas underneath the peaks. The measure tool
was used to determine the pixels in the triangular areas, and a

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converting factor (76.0was determined by using the same function to
determine the pixels in a straight line from 0 to 10 minutes. Table 1
showed that Vitamin B-12 had the largest area followed by Gamma
Globulin indicating that these proteins were present in the highest
concentration in the standards.
The sample chromatogram in Figure 2 showed that the protein
albumin (67 kDa) eluted at 38.87 minutes, and myoglobin (17.2 kDa)
followed shortly 3.01 minutes later. Table 2 illustrates that myoglobin
has a greater protein concentration than albumin as shown due to the
area differences. The peak areas were calculated by multiplying the
areas by the flow rate of .22 ml/min, and the ratios were calculated
after and placed in the graph. According to this, the concentration of
myoglobin is greater than albumin by a factor of 3.870. The peak to
valley ratio for myoglobin was also much larger indicating that it had
better separation.

Absorbance (A.U.)

0.02

Gamma
Globulin

0.015

Myoglobin

Vitamin B-12

Ovalbumin

0.01
0.005
0

0

20

Time (min)

40

60

Fig. 1: Standards Chromatogram with calculated average flow rate of
.22 mL/min. Composition of Standard: thyroglobulin, gamma globulin,
ovalbumin, myoglobin, and vitamin B-12.

Absorbance (A.U.)

0.04

Myoglobin

0.03
0.02
Albumin

0.01
0
-0.01

0

10

20

30

40

50

Time (min)

Figure 2: Sample Chromatogram with calculated average flow rate of
.22 ml/min. Composition of sample: Albumin and Myoglobin.

Thyroglobulin
Gamma Globulin
Ovalbumin
Myoglobin
Vitamin B-12

Peak
(min)
N/A
36.47
38.75
41.60
50.60

Peak
Sep.
{Right} (min)
N/A
2.283
2.850
9.000
N/A

Peak: Valley
{Left}
N/A
N/A
1.652
5.644
36.04

Peak: Valley
{Right}
N/A
2.664
3.453
35.62
N/A

Area
(A.U.*min)
N/A
392.8
263.4
343.8
421.6

Table 1: Characteristics of Standards Chromatogram

Albumin
Myoglobin

Peak
(min)
38.87
41.88

Peak
Sep.
{Right} (min)
3.01
N/A

Peak:
Valley
3.156
11.58

Area
(A.U.*min)
82.11
317.8

Peak Area
Ratio
.2584
3.870

Table 2: Characteristics of Sample Chromatogram

DISCUSSION
The most intriguing result in the experiment was the lack of a
peak for the protein Thyroglobulin in the standards chromatogram.

Due to the large size of the protein, it is possible that the protein
became stuck in the filter and did not run through the column. One
must also consider the possibility of said protein degrading in the
standards solution mixture before being run through the HPLC system.
The noise in the first 10 minutes of the standards run can lead to this
conclusion.
Another very important factor to consider is the lack of
consistency with the flow rate. A high flow rate is necessary to
separate the proteins efficiently, and the calculated flow rate using the
graduated cylinder method was much lower than the expected .5
ml/min flow rate indicated on the computer screen. Even the increase
to .75 ml/min failed to provide an increase in flow rate for proper
separation. Defects in the tubing could be affecting the pressure of the
machine, which therefore affects the flow rate based off of fluid
mechanic properties. A loose hinge or spring or even the lining of the
tubing could be affecting the pressure leading to a decrease in flow
rate.
The most important data in the tables is the peak to valley ratio
which directly shows how well separated the proteins are from one
another. Vitamin B-12 was the protein with the highest separation as
well as the lowest valley to its left, so its peak to valley ratio of 36.04
is very close to the optimal results with respect to protein separation.
Albumin and myoglobin did not have peak to valley ratios this high
indicating that the separation could be improved. The peak to valley
ratios could be improved by increasing the flow rate and addressing
some of the issues mentioned above. Other proteins in the standard
solution failed to exhibit ideal separation as well.
Albumin reached its highest peak at 38.87 minutes, and
myoglobin had its highest peak at 41.88 minutes. Due to albumin’s
molecular weight of 67 kDa, its max peak height should fall in
between the standards gamma globulin (158 kDa) and ovalbumin (44
kDa). Albumin elutes at a later time and does not fall within the time
range of 36.47 and 38.75 minutes, but this may be due to the
inconsistencies with respect to the flow rate and pressure of the
machine. Myoglobin (17.2 kDa) eluted at a similar time in both the
standard and the sample run indicating that there are slight time
differences with respect to protein eluting per run.
The peaks in the standards did not exceed absorbance values of
.02, and albumin in the sample run reached a peak of ~.01. Due to the
relationship of peak height and concentration, the standard proteins
had higher concentration in the standard solution compared to albumin
in the sample solution. Myoglobin from the sample had an absorbance
peak value of ~.037 as well as the largest area, indicating that its
concentration was very high in the sample solution. Area underneath
the peaks relates directly to protein concentration, so Myglobin in the
sample solution had the highest concentration.
The experiment’s strengths can be attributed toward its providing
a unique method to separate proteins in solution, but its weaknesses lie
in the mechanical flaws of the HPLC system. Other methods to
improve separation could involve taking advantage of other possible
biochemical properties of myoglobin and albumin in order to attempt
other chromatography methods besides size exclusion.

REFERENCES
Micou, Melissa. A Laboratory Course in Tissue Engineering.
Boca Raton: Taylor and Francis Group, 2013. Web. 5 Nov. 2012.
<https://ted.ucsd.edu/bbcswebdav/pid-207124-dt-content-rid8403729_1/courses/BENG162_FA12_Micou/Chapter%2019%20Tech
nical%20Communication%282%29.pdf>.
Micou, Melissa. High Performance Liquid Chromatography
(HPLC) (2008):1-13. Web. 5 Nov. 2012.<https://ted.ucsd.edu/bbcsw
ebdav/pid-207124-dt-content-rid-8403728_1/courses/BENG162
FA12_Micou/BENG%20162%20HPLC%20Lab%20Guide%202012.p
df

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