High Pressure Liquid Chromatography or HPLC is chemical separation

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					  HPLC separation
   of fermentation
products glycerol and
   1-3 propanediol

    Transport Lab
     Spring 2006
      Team 10
       Vincent Chou
        Corey Mays
       Robert Wong
       Carmen Jones

Fermentation of corn starch is a method of producing 1-3 propanediol (PDO) from
glycerol. To monitor the 1-3 propanediol levels the fermentation products are put
through high pressure liquid chromatography (HPLC). However, these two molecules
have very similar in structure and physical properties. So to generate better separation of
the two major products of the fermentation process the flow rate and temperature were
changed and implemented on known mixtures of these two major products. These
experiments agreed with the findings about how these factors would effect the separation
and helped the group get more acquainted with the HPLC machine.
Table of Contents


Background and Theory……………………………………1-4



Results and Discussion………………………………………5-7

Future Recommendations……………………………………7-8


References………………………………………………… 9

Fermentation of corn starch with Pichia farinosa and Klebsiella pneumoniae is a method
of producing 1-3 propanediol. This substance is useful in the creation of polymers which
can be woven into polyester fibers. Fermentation is a two step process with glucose
being converted to glycerol and then glycerol converted into 1-3 propanediol. The
fermentation produces a variety of products including Glycerol, Arabitol, Acetic Acid, 1-
3 Propanediol. In order to determine the progress of the fermentation reaction it is
necessary to measure the levels of 1-3 propanediol and glycerol. This is problematic
because the two molecules have similar atomic structures and physical properties;
therefore it is hard to separate the detection of one from the other. The method utilized in
this experiment is high pressure liquid chromatography (HPLC). This experiment
analyzes HPLC separations in order to determine the best methods for detecting these


       High Pressure Liquid Chromatography or HPLC is a chemical separation and
analysis technique. The method utilizes a tightly packed column and high pressures to
drive the separation. The column is primed with a mobile phase or a solution that does
not absorb UV. Typically a mobile phase is selected that the various components are at
least partially soluble in. In an isocratic HPLC the composition of the mobile phase is
constant. A gradient HPLC uses a changing mobile phase composition to drive the
separation. When using a mobile phase that is more polar then the column it is called
reverse phase HPLC if the column is more polar then the mobile phase it is normal phase
HPLC. The idea of HPLC is using a column of material that has varying affinity for the
components in solution one wants to separate. By running a sample through the column
one gets a separation over the length of the column. A UV detector at the end of the
column can then measure absorption due to each component. Absorption is proportional
to concentration so after running a few standards the composition of the solution can be

         A phenomenon being considered is the binding of different compounds to the
column, compounds A and B. Each compound has a different affinity to binding to the
column, one’s with high affinity will bind to the column more readily and take longer to
elute through the column. One’s with lower affinity will take less time to elute through
the column. To achieve a better separation of compound A and B, one would like to
lower the affinity of compound A and raise the affinity of compound B. This would allow
for a better separation.
         The second phenomenon is diffusion of compound A and B within the column.
Since there is a concentration gradient within the column, compound A and B will diffuse
by Fickian diffusion. This will cause compound A and B to diffuse forward and
backward at a certain flux. This phenomenon can be a problem when attempting to get
better separation because the peaks will have larger bases.
         There are several ways to affect the binding affinity and diffusion of compound A
and B by changing certain settings of the HPLC machine. A user can change the flow
rate, temperature of the column, pH, use a pH gradient, sample size, column type and the
mobile phase. By changing one or several of these settings, a user can increase the
separation distance of the peaks and be able to determine the concentration of the sample.
         The amount of sample injected into the column will have an affect on the peak’s
base length. As more of a sample is injected into the solution will cause the base of the
peak to increase due to more sample being in the stationary phase (McCalley). This
effect should not affect the separation of a solution if the sample size is kept relatively
small and close to the optimum sample size. Another affect of the sample size is that of
overloading the column. If enough sample is ejected into the column, where all the
binding sites of the column have been bound to a molecule, a plateau will occur. This will
cause that run to provide no useful data and the column will have to be run till all the
sample has left the column.
         The next affect is changing the temperature of the column will also affect the
binding affinity of the compound. The binding coefficient for a compound binding to the
column can be defined as:
            o           o
         H         S
lnK                       ln
         RT         R

          Where H and S are retention enthalpy and entropy, R is the gas constant and      is
the phase ratio. As the temperature increases the binding coefficient should decrease,
causing a faster elution time (McCalley). This could cause a better separation if the
changes in K differ more in one compound than another. This could allow a user to raise
or lower the temperature of the column to create a better separation.
          A change in flow rate can have two different affects on the separation of a
solution. The first affect occurs when the flow rate is decreased. This causes the column
efficiency to increase, since there is more time for the compounds to separate. The
problem with this is that the peak base will increase since there is more time for diffusion
to occur. Increasing the flow rate will decrease the base of the peak, but not allow as
much time for the compound to bind to the column. A decrease in flow rate may cause a
better separation.
          If none of these changes provide the necessary separation required, a change of
column may be necessary. Changing the column will change the binding coefficient of
each compound, which may increase the separation of the solution.

Resolution on an HPLC column is calculated with the following equation.

R   1   N 2 k 1  k 
     t 
N  a R 
     W 
   t t
k R 0
The value of a is dependent on the width (W) used. For W taken at ½ the peak height
a=5.54 for W taken at the tangents of the peak a=16. t0 is the retention time of a marker
chemical that has no column affinity.

Glycerol                     1-3 Propanediol
Figure 1.1


Waters 600 pump
Waters 600 controller
Waters 2410 refractive index detector
Waters Wisp 710B auto sampler
Waters 2487 dual λ absorbance detector
Shodex SH-1011 column
Empower Pro
IBM computer


This experiment utilized a Waters Wisp 710B auto sampler auto sampler to run sets of
HPLC data. Standards solutions of glycerol and PDO between 2.5 and 12 mass percent
were created. Then equal mixtures of glycerol and propanediol with two different
concentrations of 2.5 and 5 mass percent were made to simulate high and low

concentrations produced by the fermentation process. The mobile phase was .01 M
NH2SO4. The mobile phase was sparged with helium at 15 mL/min. All of these
samples were loaded into the auto sampler and run through the HPLC machine with flow
rate varying from 0.4 to 1.00 mL/min, temperature varying from 40oC to 65oC. The
refractive index and UV absorbance were measured with λ1= 254 nm and λ2= 310 nm.
Data was recorded and analyzed by Empower Pro on an IBM computer.

Results and Discussion

       The standard solutions of glycerol and PDO all yielded single peaks on their
respective HPLC runs. This single peak in each case eluted in the 28 to 35 minute region
with an average peak width of 7 minutes. There was a fairly consistent spike or peak
around 5 minutes in all of the samples. The standard solutions also included the 5 minute
spike. Since those solutions were relatively pure except for the component inserted PDO
or glycerol it seems unlikely unless the compounds were degrading that this spike was
caused by another chemical in the injection solution. Neither glycerol nor PDO begin
decomposing until temperatures exceed 50o C.
       Instead of focusing on expanding the experiment space for the glycerol/1-3
propanediol separation, this lab allowed us to become familiar with running and
calibrating the Waters machine to further continue the analysis of the fermentation
products in unit operations lab. Anecdotal evidence on the effects of temperature and
flow rate support the literature trends of increased separation with higher temperature and
lower flow rates. The best separation we managed was at our highest temperature of 65
degrees Celsius:

Figure 1.2

        These two distinct peak tops are assumed to be 1-3 propanediol and glycerol,
although they did not elute at the expected retention times. This retention time shift may
have been caused by the increased temperature which should have decreased molecule
binding affinity for the column under equivalent flow rates. Ideally spiked samples
should have been run at this temperature and flowrate to determine any change in
retention time from the original standards, run at lower temperature. This data was
collected at 290nm and at a flow rate of 0.6ml/m. Quantitative analysis of the
concentrations of the solutions was attempted, but processing challenges prevented us
from obtaining statistically meaningful results. The peaks overlapped to a large degree
making it impossible to get any more then a general idea of the relative concentrations of
glycerol and PDO. As you can see from figure 1.2 a complete or even mostly complete
separation was never achieved. The modifications of the flow rate and temperature did
not cause significant changes in the binding constants for glycerol and PDO on the
column. Furthermore the standard solutions of both glycerol and PDO were never run at

the modified flow rates and temperatures. Since this may have modified peak areas it
was impossible to get accurate concentration readings using the standards.

Future Recommendations

           For future experimentation concerning this separation, we have three different
suggestions. These are, changing the wavelength that the detectors used, exploration of
the effects pH changes, and a change of column.
           The wavelengths that were used by the detectors were not the correct ones.
Glycerol has a  max at 260 and 280 nm. The ones chosen in this experiment were
chosen because they were used by the last lab group. Ideally ’s closer to the actual 
maxes of the molecules being detected would be used. Unfortunately, it was difficult to
find accurate absorbance values for PDO. If different wavelengths could be used to
detect only PDO and glycerol, we would be able to determine each peak in the different
detectors with little to no overlap effects. This would determine the exact concentrations
of each component. We recommend performing Ultra Violet Spectroscopy on standards
of PDO and glycerol to determine the best wavelength to use, since literature data is hard
to find.
           In this experiment we did not explore the affects of changing the pH of the mobile
phase. In later experiments, the pH should be changed to see if it is able to see if the
change of charge will have an effect on the separation. Running at high pH should cause
a charge difference in the two molecules by removing the hydrogens from all of the
alcohol groups (see figure 1.1). This would cause a charge difference that could be used
to separate the molecules. A charged column would be used to perform such a
separation. The exact effects on charge with respect to pH were never determined due to
a lack of information on the pH of alcohols. A more in depth study of the pKa’s could be
manually determined for the two compounds using titration.
           If neither one of these suggestions lead to a better separation, it can be concluded
that a good separation can not be found by using this column. We would then recommend
that the next lab group research to find a more suitable column. The column that was
used was determined by the patent literature concerning PDO and glycerol. If a more

suitable column can be found, a better separation may occur. Furthermore, the column
used in this experiment is over 3 years old. At this age the column may no longer be
performing at peak efficiency. Since the two compounds are so similar in nature it would
only take a slight degrading of the column interior to decrease the separation to unusable
levels. The separations on a new column should be tested.


The separation of glycerol and PDO is a difficult separation that can be improved by
lowering the flow rate to generate higher pressure which generates better binding affinity
to the column for the different molecules. A high temperature also improved separation.
However, complete separation was never achieved but an improvement of results of the
unit operations lab of 2005 was successful. We were also able to get better acquainted
with the HPLC machine and all its abilities which will continue to improve the success of
separations in the future.


“History of HPLC” http://www.pharm.uky.edu/asrg/hplc/history.html

McCalley, David V. “Effect of temperature and flow-rate on analysis of basic compounds
in high-performance liquid chromatography using a reversed-phased column” Journal of
Chromatography A. Volume 902, Issue 2. Pg 311 – 321

McCalley, David V. “Influence of sample mass on the performance of reversed-phase
columns in the analysis of strongly basic compounds by high-performance liquid
chromatography” Journal of Chromatography A. Volume 793, Issue ?. Pg 31-46

Unit Operation Lab- Final Reports “Corn to Polymer”