Laboratory Report Cover Page s2

PROJECT FINAL REPORT COVER PAGE

GROUP NUMBER: T4

PROJECT TITLE: Column Chromatography Optimization

DATE SUBMITTED: 12/18/00

ROLE ASSIGNMENTS

ROLE GROUP MEMBER

FACILITATOR………………………..Alan Lee

TIME & TASK KEEPER………………Alan Lee

SCRIBE………………………………..Eugenia Koo

PRESENTER………………………….Eugenia Koo

SUMMARY OF PROJECT CONCLUSIONS

The major constraint of this lab is being pressed for time to complete the trials. Each protein requires 1-2 hours to finish eluting. The first week was devoted to packing G-100 and running it over night. However G-100 falters due to its extremely slow flow rate. The next two weeks were devoted to G-75 and CL-6B, however we only had 1 and half lab period to do it due to the class arrangement. Nevertheless it was still not enough. Thus the two 0.2ml/min trials were actually run on Wednesdays as was blue dextran and lysozyme for G-75.

We had spent time on collecting data for the control for both columns. This took up to more than half of the lab period. Also the ovalbumine is too impure to give an accurate elution volume. Too much time was spent on the control and on a protein that does not work.

To optimize this experiment the main focus is to collect as much data at the least possible time. Future researchers should do a meta research on the existing elution volumes from the class data. It will guide the experimenters to choose which proteins to use and serve as the control. Experimenter will only do experiments on the mixed solution. Further focus might be on gels that allow fast flow rate therefore maximizing the amount of data collected.

The CL-6B column is the column of choice to carryout experiment for this lab. CL-6B is consistently providing a more accurate elution volumes than G-75 when run at 0.2ml/min. With more accurate and consistent elution volumes, it will accurately estimate the molecular weight of the protein.

OBJECTIVES

The objective of this experiment is to optimize molecular weight determinations and protein separations using gel exclusion chromatography. In essence, this is an optimization of Experiment #2: Protein Separation-Column Chromatography performed by lab groups during the semester. The various parameters that affect the experiment were examined and observed under several conditions.

Our objective was achieved through a series of specific aims:

· Comparison of columns packed with different chromatography gels (Sepharose CL-6B or Sephadex G-75-50) to determine which results in the best separation of proteins of mixes solutions

· Examination of the effect of flow rate upon the elution of proteins and their separation when injected in the form of a mixed solution

The achievement of our specific aims will ultimately result in the completion of overall objective: to find the optimal setup that would result in the complete separation of mixed proteins that occurs with the least amount of dilution of proteins and time needed for elution. Our overall objective is to determine which gel and flow rate will provide the best results for Experiment 2 and greatest possible learning experience for students, so that they may observe Gel Exclusion Chromatography used to determine molecular weight of substances, separate a mixture, and carry out a quantitative mass balance determination for a specific protein in the column.

BACKGROUND

Chromatography is a useful method for protein fractionation. A specific matrix composed of porous beads separates proteins based on their size. This method retards molecules and is known as gel-filtration chromatography. Gel filtration columns are packed with many tiny porous beads. Molecules small enough to enter the pores linger inside successive beads. Larger molecules remain in the solution and flow around the beads, there motion unretarded. They are able to pass through the column more rapidly, thus eluting sooner.1 Chromatographic techniques are ideally suited for the separation of proteins, the overall factor that governs the efficacy of the gels being examined. Proteins of different sizes can be separated by their differential interaction with a gel.2

In Whitaker’s article “Determination of Molecular Weights of Proteins by Gel Filtration on Sephadex” he arrived at many conclusions that were used in the planning and execution of this experiment. Most importantly, he found that there is a linear correlation between the log value of a protein’s molecular weight and the ratio of the eluted volume to void volume (V/V0). He also found that V/V0 is independent of protein concentration, column size, and ion exchange adsorption, but that V/V0 is dependent upon temperature of the system for some proteins.3

Whitaker’s examination of the parameters of his experiment yielded results that facilitated further conclusions.

Determination of the Void Volume:

· V/V0 was found to be a constant for each protein regardless of column size

· one sharp peak permits V0 to be readily determined with a reasonable degree of accuracy

Effect of Sample Size and Concentration

· V/V0 is independent of sample size and concentration

· only microgram quantities of protein are needed for molecular weight determinations if sensitive detection methods are used

Effect of Ionic Strength on Elution Volume

· many substances give elution volumes different from those expected on the basis of molecular size only due to the adsorption to the gel matrix and to the small amount of ionizable groups on the gel

· ionizable groups effect can be eliminated by proper pH and/or ionic strength

Effect of Temperature on Elution Volume

· some proteins are affected by varying temperatures while other are not

· no significant difference was found in elution volumes were temperature variation was 2-3 °C

Effect of Solute Size on Elution Volume

· there is a good correlation between log of molecular weight and V/V0 on Sephadex G-75

· for G-75, log MW = 0.660 ± 0.054 (V/V0-1) + 4.785 ± 0.040

· the linear relationship between V/V0 and log MW does not hold for solutes which can only penetrate the gel only slightly

The information provided by Whitaker’s article on G-75 provides a basis for evaluating the results run through the Sephadex G-75-50 column and their accuracy. Reliability in the data obtained from the Sepharose CL-6B gel is found by comparison to known values for the molecular weight of the proteins used in the experiment.

THEORY AND METHODS OF CALCULATION

As proteins are eluted through the gel chromatography columns, solution is passed through the BioRad UV Monitor. The output is shown in the form of voltage and these values are converted to absorbance (AU) based on the pre-set monitor sensitivity.

For example:

If the sensitivity is 0.1:

1V = 0.1 AU à Absorbance = Voltage Output ¸ 10

If the sensitivity is 0.2

1V = 0.2 AU à Absorbance = Voltage Output ¸ 5

The time needed for a protein to elute is inversely related to its size (molecular weight). As a result, larger proteins’ movements through the column are not retarded by the beads of the gel matrix and have lower elution volumes. Proteins with lower molecular weights are caught in the bead pores, impeding their elution and thus causing larger elution volumes.

Elution Volume (ml) = Time to maximum absorbance (min) * flow rate (ml/min)

The conclusion for what is the optimal setup for the experiment will be based on the comparison of the Sephadex G-75 and Sepharose CL-6B chromatography gels to one another. Primarily, the comparison will lie in their ability to separate proteins injected into the column if the form of a mixed solution composed to several proteins. The ability of the gels to separate proteins can be observed through the elution volumes of the proteins in comparison to their elution volumes when run through columns separately by themselves.

MATERIALS, APPARATUS, METHODS

The apparatus and materials used mirror those required for Experiment 2 of the BE 309 Lab Manual2. The equipment used to perform the elution of the proteins include:

· 2 - 50 cm glass columns, filled with Sepharose CL-6B or Sephadex G-75-50 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, assorted tubing, stands, etc.

· LabView program “Chrom.vi” or Virtual Bench Data Logger

The proteins used were changed in order to observe the elution for a range of molecular weights:

· Blue Dextran (MW=2,000,000; 1mg/mL solution)

· Chicken Egg Lysozyme (MW=14,300; 3mg/mL solution)

· Ovalbumine (MW=45,000; 20mg/mL solution)

· Bovine Serum Albumin (MW=77,000; 30mg/mL solution)

The observations made of the elution of proteins were done in the same manner as for the Lab Manual experiment. The same injection routine was followed in order to duplicate the procedure normally used. For each column, a procedural protocol was performed to obtain the data needed for the experiment.

With each column:

· Determine maximum flow rate

· Determine V0 using Blue Dextran Dye

· Run each protein separately and together as a mixed solution

· Elution of individual proteins will occur at 1/3 the original concentration

· Repeat trials as time allows

From calculations, the lowest maximum flow rate of the two column gels was 0.5 ml/min (for G-75-50). Thus, each column was run at 0.5 ml/min as well as 0.2 ml/min for the determination of flow rate effect upon elution of proteins.

RESULTS

Figure 1

Sephadex G-75-50, Serial Dilution

Figure 1 is a composite of the individual elution curves from a serial elution in the Sephadex G-75 column. Ovalbumine is not used in the data analysis because there were two peaks from the curve. The first ovalbumine peak is before the albumin elution peak. This indicates that parts of ovalbumine have a greater molecular weight than albumin, while from literature, it is known that ovalbumine is 3,200 g/mol smaller. This phenomenon is due to the impurities in the stock ovalbumine protein. The albumin and lysozyme elution curves from this graph are used as the experimental control to compare the mixed solution elution volumes.

Figure 2

Sephadex G-75, Mixed Solution

Figure 2 is the mixed protein solution curve run at 0.5ml/min in the G-75 column. There are only two peaks, the first peak is identified as albumin and the second peak is lysozyme. Figure 1 suggests that ovalbumine is directly under albumin, thus it will not have a dramatic effect on the elution peak of the albumin. Thus, elution peaks for albumin and lysozyme can be estimated from the curve.

Figure 3

Sephadex G-75, Mixed Solution

Figure 3 is the mixed protein solution curve run at 0.2ml/min in theG-75 column. The shape of the curve is similar to the 0.5ml/min, where there are only two elution peaks. Likewise only the elution volumes of albumin and lysozyme are estimated.

Figure 4

Sepharose CL-6B, Serial Elution

Figure 4 is the composite of individual elution curves from a serial elution in the Sepharose CL-6B column at .05ml/min. The shape of the ovalbumine curve is different from the G-75 column. The first ovalbumine peak appears even before the albumin begins to elute. The second peak is not directly below the elution peak of albumin, thus it is possible that the elution volume of albumin will be incorrectly estimated. The elution peaks of albumin and lysozyme will serve as controls to compare the mixed solution elution volume

Figure 5

Sepharose CL-6B, Mixed Solution

Figure 5 is the mixed protein solution curve run at 0.5ml/min in the CL-6B column. As expected the general shape is similar to Figures 2 and 3. The flat peak on the albumin is due to problem of the setup where the absorbance of the protein has gone beyond the sensitivity range of the Lab View equipment.

Figure 6

Sepharose CL-6B, Mixed Solution

Figure 6 is the mixed protein solution curve run at 0.2ml/min in the CL-6B column. Also like Figure 5, there is a shoulder on the left side of the albumin curve. The same shoulder can be observed in Figure 3. Thus that shoulder is due to impurities rather than the contribution of the ovalbumine.

Table 1

Table 1 shows the numerical values of the elution peaks, flow rates and elution volumes with their respective uncertainties. Only the albumin and the lysozyme are compared to the experimental controls. The uncertainty of the elution peaks is determined from the range time where the maximum absorbance occurred. If there were no repeats, uncertainty is set at 20 seconds or 30 seconds for 0.5ml/min trial or 0.2ml/min, respectively. The uncertainty of the flow rate was generated from the confidence interval of the flow rates from the eight-lab groups.

The elution volumes for the trials run at 0.2ml/min in solution for the G-75 column are significantly lower than the experimental control. The elution volume for mixed proteins run at 0.5ml/min elution volume is not significantly different from the control. The resulting elution volumes of lysozyme and albumin for the rate of 0.2ml/min are off by as much as the 11 ml and 4 ml, respectively.

The elution volume for albumin run at 0.5ml/min and 0.2ml/min is not significantly different from the control. Although the albumin run at 0.5ml/min has triple the percentage of uncertainty compared to the rest of the data, the uncertainty is caused by the flat top on the albumin curve, which contributes to the large error in estimating the peak time.

ANALYSIS

An advantage of CL-6B is that it can be run at almost 4 times the flow rate of Sephadex G-75. Since we did not find any difference in the elution volumes between the different flow rates, the elution peak should not be significantly different if it was run at a CL-6B maximum flow rate. Thus it is possible to obtain the same accurate data 4 times faster. Since the G-75 column’s maximum flow rate is at 0.5 ml/min it will take approximately 1-2 hours for a sample to completely elute through a 40 ml elution column. Thus it is not suitable for a 6 hr laboratory, since only 2-3 trials can be conducted in that period of time. In that same time frame CL-6B can run 8-12 trials. The ability to run more trials dramatically improves statistical analysis of the confidence interval for estimation of molecular weight.