Question

Gel-filtration chromatography is a useful method for removing salts, such as ammonium sulfate, from protein solutions. Describe how such a separation is accomplished.

Short answer

Gel-filtration chromatography separates molecules based on size. Proteins elute first followed by salts due to differences in their ability to enter the pores of the beads.

Step-by-step solution

- Understanding Gel-Filtration Chromatography

Gel-filtration chromatography separates molecules based on size. It uses a column packed with porous beads, allowing smaller molecules to enter the pores while larger molecules pass between beads and elute more quickly.

- Preparing the Solution

Prepare a solution containing the protein and the salt (in this case, ammonium sulfate). Make sure the solution is well-mixed before loading it onto the column.

- Loading the Column

Carefully load the prepared solution onto the top of the chromatography column. Ensure that the solution is applied evenly to avoid disturbing the separation process.

- Elution Process

Elute the column with a suitable buffer. As the solution passes through the column, larger protein molecules will travel faster and elute first because they do not enter the pores of the beads. Smaller salt molecules will enter the pores and elute more slowly.

- Collecting Fractions

Collect the eluate in fractions. Since the proteins elute faster, the fractions collected first will contain mostly proteins, while fractions collected later will contain the salts.

- Analysis

Analyze the collected fractions to confirm the separation. Protein presence can be determined using appropriate assays or spectrophotometry, while salt content can be measured through conductivity or specific chemical tests.

Key concepts

Molecular Size Separation

Gel-filtration chromatography is a technique that separates molecules based on size. This method uses a porous medium inside the chromatography column. When the solution passes through, larger molecules navigate around the pores and exit the column faster. Smaller molecules enter the pores and travel slower. This characteristic exploitation allows for efficient separation between different molecular sizes.
In gel-filtration, large protein molecules bypass the porous beads while smaller salt molecules enter these pores and are delayed. This process effectively segregates molecules of various sizes.

Chromatography Column

A chromatography column is essential in gel-filtration chromatography. It's filled with tiny, porous beads. The column acts as a sieve ensuring the proper separation of molecules.
When a protein solution mixed with ammonium sulfate is introduced, the column ensures the separation based on molecular size. It's important to load the solution carefully to avoid disrupting the separation process. The elution buffer then facilitates the movement of molecules through the column, where size-dependent separation occurs.
This column separates large proteins and smaller salts as the solution travels through it.

Protein Purification

Protein purification is one of the essential uses of gel-filtration chromatography. When dealing with mixed solutions containing proteins and salts, it's crucial to separate and purify proteins for further analysis or use.
During the elution process, proteins, due to their larger size, flow through the column faster. They bypass the beads and collect in the early fractions. This enables isolation of your desired protein from contaminants or salts.
The purity of collected proteins can be confirmed by specific assays, ensuring the right proteins are separated and collected effectively.

Ammonium Sulfate Removal

Ammonium sulfate is often used in protein precipitation and needs to be removed for pure protein preparation. Gel-filtration chromatography assists in this removal.
In the column, due to its smaller size, ammonium sulfate will enter into the pores of the beads, slowing its journey through the column. Consequently, it elutes after the larger protein molecules.
By collecting the eluate in fractions, the first fractions will mainly contain proteins, whereas later fractions will have the ammonium sulfate, ensuring its efficient removal.

Eluate Fractions Collection

During gel-filtration chromatography, the solution passing through the column is collected in fractions. This fractionation is crucial for separating the proteins from salts.
Larger molecules like proteins will emerge in the early eluate fractions due to their faster traversal through the column. Smaller molecules, such as ammonium sulfate, are delayed within the column beads and collected in later fractions.
After collection, each fraction can be analyzed to confirm the presence of proteins or salts. Proper fraction collection ensures pure protein isolation and effective salt removal.