BE 309 M4 Fall 2000

Mass Balance Optimization and Hemoglobin Analysis on GEC

PROJECT FINAL REPORT COVER PAGE

GROUP NUMBER: M4

PROJECT TITLE: Mass Balance Optimization and Hemoglobin Analysis on GEC

DATE SUBMITTED: 12/18/00

ROLE ASSIGNMENTS

ROLE GROUP MEMBER

FACILITATOR……………………………...Bindu George

TIME & TASK KEEPER……………………Jeffrey Wu

SCRIBE………………………………………Laura Kaplan

PRESENTER…………………………………Tony Yeung

SUMMARY OF PROJECT CONCLUSIONS

The primary objective of this project is to optimize the mass balance procedure used to analyze chromatography elution curves. Secondly the molecular nature of the first peak in the original hemoglobin is studied. The results indicate that the Simpson’s rule is the most feasible and accurate method of performing a mass balance. However, even using this rule we did not obtain our goal of + 2% uncertainty for both hemoglobin and albumin. The biggest uncertainties in the mass balance are largely due to the extinction coefficient uncertainties. For example, the total uncertainty for albumin is + 5.81% and the extinction coefficient uncertainty is + 4.5%. The average percent recovery for albumin is 100.54 + 1.04%S.D. This means that our goal of + 2% precision is achieved and there is high repeatability in the albumin trials. However, the percent recovery for the more refined hemoglobin is 98.51 + 5.71 %S.D. and does not achieve our goal of + 2% precision. This indicates a low repeatability in the more refined hemoglobin trials.

Finally, the conclusions made on the analysis of the hemoglobin small peak seen in the elution curve include the following. First, the small peak exists in the elution curves of both the original and the more refined hemoglobin, though about 10x more concentrated in the original sample. Second, the molecular weight of the peak is determined to be 920,000 and the extinction coefficient is approximately 1900 AU*mL/g. Lastly, the fractionated hemoglobin solutions determine that this peak does not dissociate, thus placing it more likely due to an impurity.


Objective

Since Gel Exclusion Chromatography (GEC) is widely used in biochemistry and other related fields, it is beneficial to develop a protocol to obtain an accurate and precise mass balance on a chromatography elution curve. Developing such a procedure would also assist determining impurities that might be in a solution. The primary objective of this project is to optimize the mass balance procedure using bovine albumin and hemoglobin. Using this optimized procedure, the elution curve of the original hemoglobin, which elutes an unexpected two peaks, is studied. The following goals are set at the onset of testing:

•  To obtain a percent recovery of 100 + 2% uncertainty on GEC for all mass balance trials.

•  To achieve a precision of ± 2% on multiple trials of bovine albumin and more refined hemoglobin.

•  To examine the nature of the first peak shown in the original bovine hemoglobin elution curve.

- We hypothesize that the small peak of the hemoglobin elution curve is due to a sample impurity.

Background

The two proteins studied in this project are bovine serum albumin and bovine hemoglobin. Bovine Serum Albumin has a molecular weight of 77,000 and functions in maintaining osmotic pressure between the blood vessels and tissues. Hemoglobin is a tetramer composed of globins and four heme groups. Its molecular weight is 64,000 and is important in oxygen transport within the body. In this project, two forms of hemoglobin are examined in order to study the nature of the first peak. The H-2500, “original”, hemoglobin came in the form of Lyophilized powder. The H-2625, “more refined”, sample is in substrate powder form. Furthermore, there are different preparations for the two samples. In the case of H-2500, red blood cells are centrifuged out of plasma, placed in corpuscles paste that causes them to burst, and then re-centrifuged. The H-2625 samples are prepared from washed, lysed, and dialyzed erythrocytes1.

The procedure used in this project is GEC, which separates proteins according to their different weights. The setup consists of a column packed with Sepharose CL-6B, an agarose gel. Three general properties of the gel column are: porosity, exclusion limit, and fractionation range. The porosity depends on cross-linking of agarose. The exclusion limit is the molecular mass at which the smallest molecule cannot pass through the gel matrix. The fractionation range for globular proteins is 10,000- 4,000,000D2.

The mathematical technique used to integrate the elution curves for the mass balance is the Simpson’s Rule. This formula takes into account the curvature of the graph by approximating a general curve by a parabola. The integration scheme is given by the equation3:

ò¦(x)dx @ (h/3)(Δt)( ¦o + 4¦1 + 2¦2 + 4¦3 + ... +2¦2m-2 + 4¦2m-1 + ¦2m) (1)

Materials and Apparatus

·  1 X 50 cm glass column, filled with CL-6B Sepharose 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.

·  Test Compounds:

o  Bovine Serum Albumin; MW= 77,000

o  “Original” Bovine Hemoglobin, Product code H-2500; MW= 64,500

o  “More refined” Bovine Hemoglobin, Product code H-2625

·  LabView program called “Chrom.vi” in BE309 folder.

Methods, Protocols, and Procedures

Optimizing Mass Balance Procedures

Mass balance procedure modifications will be implemented using albumin and the more refined hemoglobin sample. First, the volume injected in the column will be determined. To achieve this, water is injected into the loading apparatus. Instead of pumping the water into the column, the water is collected into a graduated cylinder. Air pressure of a syringe is used rather than the pump to drive the water out of the tubing. An electric balance will determine the mass of water collected, which is the volume that would be injected into the column. Several trials will be performed. Second, the flow rate through the column will be determined by dividing a known volume of solution eluting out of the waste tube by the elapsed time. Next, the time interval at which data will be recorded will be decreased to determine its effect in calculating percentage yield. Finally, integration on the elution curves will be done using the Simpson’s rule. The extinction coefficient of protein is measured by injecting different concentrations of the protein solution directly into the mass spectrophotometer and then taking the slope of the resulting absorbance vs. concentration plot. Five solutions of varying concentration will be prepared and then their respective absorbance will be determined using the mass spectrophotometer on the column. A plot of Absorbance vs. Concentration will be used to determine the coefficient needed for the mass balance calculations.

An analysis of the original hemoglobin sample is performed to determine the molecular nature of the small peak seen in the elution curves. A solution of the original hemoglobin solution will run through the column based on the modified mass balance procedure and mass balance calculations are done on the elution curve. In addition, a molar extinction coefficient will be determined for the small peak. Finally, the original hemoglobin solution will be fractionated. First, the small peak will exit the column and this is collected. Next, the large peak will exit the column and it will be collected separately. The two fractions will then be put back into the column separately to obtain the respective elution curves. The curves will be studied to determine some information about the small peak.

Results

Mass balance of both bovine serum albumin and bovine hemoglobin is determined by:

% Recovery = MassOUT / MassIN *100% (2)

The amount of mass going into the column is determined by:

MassIN = Concentration of solution * Injected Volume (3)

The concentration of the solution is measured in g/ml. The injection volume is found to be 0.324 ml + 0.622%.

The amount of mass coming out of the column is determined by the following where ε is the extinction coefficient of the protein:

MassOUT = (Area under absorbance vs. time curve) * flow rate / ε (4)

The flow rate is found to be 1.86 ml/min + 0.72%. The extinction coefficient for albumin is found to be 76.60 AU*ml/g + 4.50%. Below is an absorbance vs. concentration plot for albumin.

Figure 1: Absorbance vs. Concentration for Albumin

Two different methods of integration are considered, the trapezoid method and the Simpson’s rule method. The trapezoid method is given by:

(5)

The Simpson’s rule method is:

Area = 1/3 * Δt * (A0 + 4A1 + 2A2 + 4A3 + 2A4 + … + 2An-2 + 4An-1 + An) (6)

with n being an even number and A the absorbance value at each time interval. The following graph shows various curves graphically similar to the elution curves obtained in the experiment.

Figure 2: Modeling Elution Curve to a Mathematical Function

Table 1 below shows the result obtained by using both methods on these mathematical functions. It is important to note that the Simpson’s rule method can overestimate the percentage yield in area because of its nature of parabolic fitting. However, Simpson’s rule is found to be more accurate in all of the cases and therefore it is used in determining the mass balance calculations.

Table 1: Comparison of Trapezoid and Simpson’s Rule

A representative elution curve for albumin is shown below. The elution curves for the other two trials are similar.

Figure 3: Elution Curve for Albumin

The average % recovery for albumin is found to be 100.54% + 5.81% uncertainty. The standard deviation among the three trials is 1.03%.

The uncertainty in each of the measurements for albumin is presented in Table 2. The total uncertainty is the sum of the individual uncertainty and is found to be 5.81%. The percent uncertainty in the integration method is assumed to be 0% based on the result in Table 2.

Table 2: Breakdown of Experimental Uncertainties of Mass Balance
% Uncertainty
Solution preparation / 0.1%
Injection volume / 0.57%
Flow rate / 0.64%
Extinction coefficient / 4.5%
Integration method / ~0%

A calibration function to determine the molecular weight of a compound based on its elution time is made by graphing V/Vo versus Log(MW). Figure 4 and Eq. 7 show this function. Data obtained is based on a previously done project.

Figure 4: Calibration Function Relating Molecular Weight with Elution Time

V/Vo = -0.9915 log(MW) + 7.0185 (7)

Figure 5 below displays the elution curve for one of the three trials of the more refined hemoglobin. The other two trials performed yielded curves with similar features. Using the calibration equation shown in Eq. 7, the molecular weight associated with the small peak is 924,200 ± 21,100 (%SD = 2.28%) while the molecular weight associated with the large peak is 68,200 ± 1,300 (%SD = 1.93%).

Figure 5: Elution Curve for More Refined Hemoglobin

Figure 6 shows the absorbance versus concentration curve for the more refined hemoglobin. The extinction coefficient is determined to be 269.13 ± 17.38 AU*mL/g. The 95% percent confidence interval is 6.46%. Therefore, the mass balance calculation uncertainty is determined to be 7.80%.

Figure 6: Absorbance vs. Concentration for More Refined Hemoglobin

When integrating the small peak and the large peak of the more refined Hb elution curve separately, the percent area occupied by the small peak compared to the total area under the curve is 0.7 ± 0.4%. When performing a mass balance for the more refined hemoglobin trials, the content of the small peak is assumed to have the same extinction coefficient as hemoglobin. The percent recovery values are displayed in Table 3. If the small peak is ignored in the mass balance calculations, the percent recoveries will be decreased by less than 1%.

Table 3: Percent Recovery values for the More Refined Hemoglobin
% Recovery

Trial 1

/ 104.44%
Trial 2 / 98.03%
Trial 3 / 93.05%
Average ± SD / 98.51 ± 5.71% (%SD = 5.80%)

Only one trial of the original hemoglobin solution is performed. The elution curve is shown in Figure 7.

Figure 7: Elution Curve for Original Hemoglobin

Using the Equation 7, the molecular weight associated with the small peak is 966,200 while the molecular weight associated with the large peak is 67,200. The percent area occupied by the small peak compared to the total area under the curve is 10.8%. The uncertainty of the mass balance calculation is the same of that for the more refined hemoglobin (7.80%). Again, if the extinction coefficient of the small peak is assumed to be the same as the large peak, the mass balance for this run is calculated to be 110.21%. This percent recovery exceeds the acceptable range of percent recovery values (100 ± 7.80%).

The extinction coefficient is estimated based on the following equation:

(8)

Table 4 displays possible values of the extinction coefficient of the small peak based on assumed percent recovery values. It is noted that the lower limit of the percent recovery (100% - 7.80% = 92.20%) could not be reached, even when ignoring the contribution of the small peak.

Table 4: Estimation of the Extinction Coefficient of the Small Peak
Conditions / Extinction Coefficient of Small Peak
Assuming 100% Recovery / 1911.77 AU*mL/g
Assuming 107.8% Recovery (Upper limit) / 337.67 AU*mL/g
Assuming 98.33% Recovery / ¥

The contents of the small peak and the large peak are fractionated as described in the methods. The two solutions are placed back into the GEC. Figure 8 and 9 displays the elution curve for the small peak solution and the large peak solution respectively.