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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
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.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. Table of Contents Introduction………………………………………………1 Background and Theory……………………………………1-4 Equipment…………………………………………………4 Procedure…………………………………………………4-5 2 .
1-3 Propanediol. Arabitol. 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 molecules. Background High Pressure Liquid Chromatography or HPLC is a chemical separation and analysis technique. therefore it is hard to separate the detection of one from the other. In order to determine the progress of the fermentation reaction it is necessary to measure the levels of 1-3 propanediol and glycerol. Typically a mobile phase is selected that the various components are at least partially soluble in. This is problematic because the two molecules have similar atomic structures and physical properties. The fermentation produces a variety of products including Glycerol.Results and Discussion………………………………………5-7 Future Recommendations……………………………………7-8 Conclusion…………………………………………………8 References………………………………………………… 9 Introduction 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. The method utilizes a tightly packed column and high pressures to drive the separation. Acetic Acid. The column is primed with a mobile phase or a solution that does not absorb UV. Fermentation is a two step process with glucose being converted to glycerol and then glycerol converted into 1-3 propanediol. In an 3 .
The second phenomenon is diffusion of compound A and B within the column. compound A and B will diffuse by Fickian diffusion. By changing one or several of these settings. A phenomenon being considered is the binding of different compounds to the column. This will cause compound A and B to diffuse forward and backward at a certain flux. This could allow a user to raise or lower the temperature of the column to create a better separation.isocratic HPLC the composition of the mobile phase is constant. temperature of the column. 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. 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. compounds A and B. The binding coefficient for a compound binding to the column can be defined as: lnK := −H o RT + S + lnφ R φ o Where H and S are retention enthalpy and entropy. R is the gas constant and is the phase ratio. One’s with lower affinity will take less time to elute through the column. a user can increase the separation distance of the peaks and be able to determine the concentration of the sample. The next affect is changing the temperature of the column will also affect the binding affinity of the compound. If enough sample is ejected into the column. a plateau will occur. pH. As the temperature increases the binding coefficient should decrease. where all the binding sites of the column have been bound to a molecule. causing a faster elution time (McCalley). The first affect occurs when the flow 4 . The amount of sample injected into the column will have an affect on the peak’s base length. A change in flow rate can have two different affects on the separation of a solution. one would like to lower the affinity of compound A and raise the affinity of compound B. Each compound has a different affinity to binding to the column. This would allow for a better separation. Absorption is proportional to concentration so after running a few standards the composition of the solution can be determined. Since there is a concentration gradient within the column. A gradient HPLC uses a changing mobile phase composition to drive the separation. To achieve a better separation of compound A and B. Another affect of the sample size is that of overloading the column. A UV detector at the end of the column can then measure absorption due to each component. By running a sample through the column one gets a separation over the length of the column. use a pH gradient. 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). column type and the mobile phase. one’s with high affinity will bind to the column more readily and take longer to elute through the column. There are several ways to affect the binding affinity and diffusion of compound A and B by changing certain settings of the HPLC machine. This phenomenon can be a problem when attempting to get better separation because the peaks will have larger bases. The idea of HPLC is using a column of material that has varying affinity for the components in solution one wants to separate. A user can change the flow rate. sample size. 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. This could cause a better separation if the changes in K differ more in one compound than another.
54 for W taken at the tangents of the peak a=16. Changing the column will change the binding coefficient of each compound. Increasing the flow rate will decrease the base of the peak. This causes the column efficiency to increase. which may increase the separation of the solution. For W taken at ½ the peak height a=5. but not allow as much time for the compound to bind to the column.1 1-3 Propanediol Equipment 5 . The problem with this is that the peak base will increase since there is more time for diffusion to occur. a change of column may be necessary. Glycerol Figure 1. since there is more time for the compounds to separate. If none of these changes provide the necessary separation required. Resolution on an HPLC column is calculated with the following equation.rate is decreased. t 0 is the retention time of a marker chemical that has no column affinity. A decrease in flow rate may cause a better separation. 1 R = ( − α )N 2 [k ( + k )] 1 1 4 1 t N = a R W t −t k= R 0 t0 α= k2 k1 2 The value of a is dependent on the width (W) used.
01 M NH 2SO 4. The mobile phase was . The standard solutions also included the 5 minute spike.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 Procedure This experiment utilized a Waters Wisp 710B auto sampler auto sampler to run sets of HPLC data. The refractive index and UV absorbance were measured with λ 1= 254 nm and λ 2= 310 nm. temperature varying from 40oC to 65 oC. There was a fairly consistent spike or peak around 5 minutes in all of the samples. Data was recorded and analyzed by Empower Pro on an IBM computer. The mobile phase was sparged with helium at 15 mL/min. 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. Results and Discussion The standard solutions of glycerol and PDO all yielded single peaks on their respective HPLC runs. Then equal mixtures of glycerol and propanediol with two different concentrations of 2. Neither glycerol nor PDO begin decomposing until temperatures exceed 50 o C. Standards solutions of glycerol and PDO between 2. All of these samples were loaded into the auto sampler and run through the HPLC machine with flow rate varying from 0.5 and 5 mass percent were made to simulate high and low concentrations produced by the fermentation process.00 mL/min. 6 .5 and 12 mass percent were created. This single peak in each case eluted in the 28 to 35 minute region with an average peak width of 7 minutes.4 to 1.
Instead of focusing on expanding the experiment space for the glycerol/1-3 propanediol separation. Ideally spiked samples should have been run at this temperature and flowrate to determine any change in retention time from the original standards. Furthermore the standard solutions of both glycerol and PDO were never run at the modified flow rates and temperatures. 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. run at lower temperature.6ml/m. Since this may have modified peak areas it was impossible to get accurate concentration readings using the standards. This data was collected at 290nm and at a flow rate of 0. The best separation we managed was at our highest temperature of 65 degrees Celsius: Figure 1. 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.2 These two distinct peak tops are assumed to be 1-3 propanediol and glycerol. Future Recommendations 7 . The modifications of the flow rate and temperature did not cause significant changes in the binding constants for glycerol and PDO on the column. As you can see from figure 1. Anecdotal evidence on the effects of temperature and flow rate support the literature trends of increased separation with higher temperature and lower flow rates. but processing challenges prevented us from obtaining statistically meaningful results. 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.2 a complete or even mostly complete separation was never achieved. Quantitative analysis of the concentrations of the solutions was attempted. although they did not elute at the expected retention times.
it was difficult to find accurate absorbance values for PDO. 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. 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. complete separation was never achieved but an improvement of results of the unit operations lab of 2005 was successful. we have three different suggestions. since literature data is hard to find. exploration of the effects pH changes. Ideally l’s closer to the actual l maxes of the molecules being detected would be used. This would cause a charge difference that could be used to separate the molecules. A high temperature also improved separation. These are. Furthermore. In later experiments. Glycerol has a l max at 260 and 280 nm. and a change of column. we would be able to determine each peak in the different detectors with little to no overlap effects. it can be concluded that a good separation can not be found by using this column. 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. Unfortunately. Conclusion 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. The column that was used was determined by the patent literature concerning PDO and glycerol.For future experimentation concerning this separation. If a more suitable column can be found. 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. We recommend performing Ultra Violet Spectroscopy on standards of PDO and glycerol to determine the best wavelength to use. At this age the column may no longer be performing at peak efficiency.1 ). 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. The separations on a new column should be tested. If neither one of these suggestions lead to a better separation. However. A charged column would be used to perform such a separation. The wavelengths that were used by the detectors were not the correct ones. 8 . In this experiment we did not explore the affects of changing the pH of the mobile phase. changing the wavelength that the detectors used. The ones chosen in this experiment were chosen because they were used by the last lab group. the column used in this experiment is over 3 years old. We would then recommend that the next lab group research to find a more suitable column. If different wavelengths could be used to detect only PDO and glycerol. This would determine the exact concentrations of each component. a better separation may occur.
Volume 902.html 9 .Final Reports “Corn to Polymer” http://rothfus. “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.cheme. Issue ?.edu/uolab/final03/ferm. Issue 2. David V.edu/uolab/final05/ferm.edu/uolab/final04/ferm.cheme. Pg 311 – 321 McCalley.cmu.cmu.edu/asrg/hplc/history.html http://rothfus. “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.pharm.uky.html McCalley.cmu.html http://rothfus.cheme.References “History of HPLC” http://www. David V. Pg 31-46 Unit Operation Lab. Volume 793.