Group X

Memo: Separation of Biological Proteins using High Performance Liquid

Chromatography
10/21/14
The purpose of this experiment was to develop a method to quickly and effectively
separate two proteins, mucor rennet and porcine pepsin, in a High Purity Liquid
Chromatography (HPLC) column by exploiting the difference in isoelectric points of the
proteins. An additional objective was to calculate the concentration of an unknown amount
of porcine pepsin in solution using Beer-Lambert Law (Eq.1).
The Beer-Lambert Law relates concentration of protein in a sample solution, c, to an
absorbance reading, A(t).
A(t)=lc (Beer-Lambert Law)

(Eq. 1)

A schematic of the HPLC process is

described in Figure 1. Two solvents (Acetate
Buffer pH=5.0, Acetate Buffer with 1M NaCl
pH=5.0) were used as the mobile phases to elute
two proteins (Porcine Pepsin, Mucor Rennet)
through the column. The column used in this
experiment employs a diethylaminoethyl (DEAE)
anion exchange resin. The proteins in the column
are anionic when dissolved in the acetate buffer
and will therefore bind to the column. In order to
elute the proteins, the percentage of salt solution is
increased. The HPLC runs at a constant total flow
rate of 3 mL/min. The proteins are injected into the
column (200 L) downstream of the salt solutions.
The eluted proteins are then detected through an
absorbance detector (wavelength = 280 nm).

Figure 1. Schematic of HPLC Purification

Two mobile phases, (Acetate Buffer, Buffer
with 1M NaCl) are mixed. The protein is
injected downstream of the solvents and
eluted through the HPLC column

To understand the specific behavior of the

HPLC used, retention time in the column was
experimentally measured. The residence time
represents the time for the mobile phase to travel
from the injection site to the absorbance detector. HPLC grade acetone was injected into
the column, and since acetone does not interact with the column, it was assumed to flow
through the system at the same rate as the mobile phase. The retention time for this
column was found to be approximately 2 minutes.

To test the validity of the BeerLambert Law (Eq.1), a solution of

known pepsin concentration was
injected into the column. Figure 2
displays the gradient elution trial for a
sample with pepsin concentration of 3.8
mg/mL. As the percentage of salt
solution increased, more and more of
the pepsin eluted from the column until
essentially all pepsin eluted (~20
minutes). The area under the
absorbance curve was quantified and
used to plot the experimental
calibration data point in Figure 3.
Figure 3 shows a linear fitted
relationship between eluted
concentration and absorbance area
using accepted values. Using this curve
the gradient run of known pepsin
solution yielded an elution concentration
3.28 mg/mL (.03 mg/mL). This resulted
an efficiency of 86.3% of pepsin
recovered through HPLC. The
experimental point is within the standard
error of the slope of the calibration line.

for
of
in

By using the calibration data,

Beer-Lambert Law, and knowing the
efficiency of the column, unknown
injection concentration of pepsin can be
determined experimentally through absorbance readings. A protein mixture composed of
15.3 mg/mL of mucor rennet and a sample of unknown pepsin concentration was run
through the column. Gradient and step change elution methods were used to study protein
elution rates and to design a quick and
effective method for separating these two
proteins.
When a gradient elution was used,
the mucor rennet and porcine pepsin were
not perfectly separated. Figure 4 shows
signs of co-elution from 12-15 minutes. The
gradient elution graph is useful because it is
a good indicator of protein charge; it shows
which protein reacts more strongly with the
positively charged DEAE column, and at
what percentage of salt solution certain
proteins tend to elute. Figure 4 shows that
rennet will elute at approximately 20% 1M
NaCl solution, and pepsin at 40%.

For better separations, a step

change method was implemented with
two step changes, one at 30% salt
solution (1M NaCl), which eluted the
rennet first, and an 80% salt solution that
eluted the remaining protein (pepsin).
This method resulted in both a faster and
more effective separation. Figure 5
shows no evidence of co-elution and the
pepsin had finished eluting around 9
minutes compared to about 20 minutes
for the gradient run.
There was some injection error
associated with the step change trial.
During the run, it is suspected that the
computer had begun running the method
approximately two minutes before the sample had actually been injected. The salt
concentration according to the method inputted into the computer and the suspected actual
salt concentration as functions of time are both given in Figure 5.
The injection concentration of pepsin was calculated using calibration data, recovery
efficiency, and the absorbance curves in Figures 4 and 5. The unknown pepsin
concentration was found to be 2.42 and 2.28 mg/mL (.02 mg/mL) for the step change and
gradient method respectively. This results in a pepsin/protein ratio of 13.7%, and 13%
respectively. For identical sample injections, gradient elution yielded a lower elution
concentration of pepsin when compared to the step change method.
In conclusion, the results from the gradient elution method showed that rennet eluted
when the percent salt solution was 20% and the pepsin solution eluted when the salt
solution was 40%. These results were used to develop a faster and more effective
separation. The efficient method had one step change at 30% salt solution to elute rennet
and another at 80% to elute pepsin. The efficient method separated the proteins about
twice as fast. The unknown pepsin concentration was found to be 2.42 and 2.28 mg/mL
(.02 mg/mL) for the step change and gradient method respectively.
References
Pepsin, Worthington Biochemical Corporation. (October 7, 2014). Retrieved from
http://www.worthington-biochem.com/pm/default.html