in the gel fltration chromatogram shown above, what kind of data would be needed to assess the correct mass of each of the peaks?

Question

BlackTom AI · Accepted Answer

To approach this question, consider what information gel filtration (size-exclusion chromatography) provides and what is required to assign a molecular weight to each peak.
Option 1: 'separate the proteins in the peaks in an SDS PAGE' — While SDS-PAGE can separate proteins by size and help identify components, using SDS-PAGE after the gel filtration run is not directly giving the masses of the peaks in the context of the chromatogram alone. It provides approximate sizes of isolated bands, but it does not link those sizes to the specific elution times without additional calibration steps. This step is often used for verification, not as the primary data needed to assign masses from the chromatogram itself.
Option 2: 'collect data on the mass of known proteins to compare to the experimental sample' — This aligns with standard practice: you run a set of protein standards with known molecular weights through the same gel filtration column to generate a calibration curve (elution volume or retention time vs. logMW). Then you compare the retention times of your sample peaks to the calibration curve to estimate their masses. This provides the necessary reference data to assign approximate masses to each peak.
Option 3: 'separate the proteins in the peaks in a different column' — Using a different column won’t directly yield the masses for the peaks observed in the original gel filtration run. Calibration with standards on the same column is essential; switching columns could introduce different void volumes and pore sizes, leading to incorrect mass estimates. This would not provide the required data to assess peak masses accurately.
Option 4: 'Perform an SDS-PAGE and repeat the experiment' — Repeating with SDS-PAGE again gives size information for individual bands, but it does not establish a direct, quantitative relationship between the chromatographic peak retention and molecular weight on the same calibration basis as a standard curve from the same column. It’s more of a separate technique, not the primary data set needed for assigning masses to the peaks observed in the gel filtration trace.
Putting these together, the core data needed to assign correct masses to the peaks is a set of known-mass standards run on the same column to build a calibration reference, which is described in Option 2.

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in the gel fltration chromatogram shown above, what kind of data would be needed to assess the correct mass of each of the peaks?

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