Size Exclusion Chromatography- Protein Separation Polyacrylamide Gel Electrophoresis - Lab Report Example

Size Exclusion Chromatography Results To identify protein profiles in the sample mixture, the elutionwas monitored continuously using a UV light detector at a flow rate of 7mL/min. The absorbance of each fraction at 200nm was determined and plotted as shown in figure 1 below. Even though the resolution between the different components is poor, the less distinct peaks suggest an acceptable degree of separation.Figure 1: Elution profile of fractions, maximum absorbance at 200nm is seen in the fifth and eighth fractions.

To determine the molecular weight of the unknown, a standard solution containing a mixture of polymers of known molecular weight was used to calibrate the column. Table 1 below shows the standards and their relative molecular weights incorporated in this analysis. These standards were used to plot the calibration curve.Table 1: List of standards and their Molecular weightsElution fraction of both the standards and unknown samples were run through SDS-PAGE gel electrophoresis and their retention times calculated as shown in the table below.

Table 2: Retention of known standards and unknown.Retention time of standardsRetention Time of unknownsBand no.Retention timeunknown no.Retention time10.19047620.24761920.20952430.59047630.26666740.60761940.36190550.5250.41904860.49523860.53333370.209524The retention times, which is equivalent to elution volume (Ve) of each protein was plotted as a function of the log 10 of the molecular weight of the standards as shown in figure two. This calibration curve was used to estimate the relative molecular weights of the unknown samples.

Figure 2: Calibration curve of the standards plotted using retention time and log10 of the molecular weights of the standardsIn determining the molecular weights, the function in equation one was employed. Log M = b - c Ve (where b and c are constants) (Bernd, 10)Equation 1: Log of molecular weight as a function of elution volume (Ve), the constants b and C were determined from the calibration curve.Using the calibration curve of the standards, constants b and c were substituted to give equation two below. Y= -2.1263x +5.

3719 (where Y =Log M and X=Ve)Equation 2: A substituted function of equation one The Value for X and Y in this function is equivalent to elution volume (Ve) and Log10 of the molecular weight. Substituting Ve with the retention time value, the Log M and molecular weights of the unknown samples were determined as shown on table three below. Table 3: Estimated Log10 M and the Molecular weights of unknown samples.Molecular weight of unknownUnknownLog MMolecular weight24.8453876 70,047 34.1163705 13,073 44.

0799196 12,020 54.266224 18,460 64.3188752 20,839 74.9263895 84,4092. Conclusion Size exclusion chromatography is a HPLC technique that separates molecules based on their size or molecular weight (Bernd, 2). Using this technique the molecular weight of five unknown protein molecules was estimated as shown in table three. However, the validation assay of the technique did not give expected resolution, limits the validity of the results. Clear and distinct peaks on this graph depict a high resolution of the assay and thus credible results.

A number of factors; such as column length, flow rate and technical errors may have influenced the poor resolution of the peaks in the UV protein absorption profile (Hong and Fountain, 5-6). Further optimization experiments may be necessary to overcome these errors and achieve a good resolution. ReferencesHong, Paula and Fountain, Kenneth. Method Development for Size-Exclusion Chromatography of Monoclonal Antibodies and Higher Order Aggregates. Milford: Waters Corporation, 2011. Print.Bernd Trathnigg “Size-exclusion Chromatography of Polymers” Encyclopedia of Analytical Chemistry Ed.

Meyers Chichester: John Wiley & Sons Ltd, 2000. 8008–8034. Print.