How Does Gel Chromatography Separate Molecules?
Chromatography is a method for separating a mixture in the laboratory. The mixture is dissolved in a mobile phase fluid (gas, solvent, water, etc.) that transports it through a structure (column, capillary tube, plate, or sheet) on which a stationary phase material is fixed. The stationary process has different affinities for each of the mixture's constituents. Depending on their interactions with the stationary phase's surface sites, various molecules remain in the stationary phase for longer or shorter periods of time. As a result, they differentiate since they move at various apparent velocities in the mobile fluid.
This article will study Molecular exclusion chromatography, gel exclusion chromatography, and application of gel chromatography in detail.
Molecular Exclusion Chromatography/ Exclusion Chromatography
Size exclusion chromatography SEC, also known as molecular sieve chromatography, is a chromatographic process that separates molecules in solution based on their size and, in some cases, molecular weight. Large molecules or macromolecular complexes, such as proteins and industrial polymers, are commonly used. Gel-filtration chromatography is used where an aqueous solution is used to move the sample through the column, as opposed to gel permeation chromatography, which is used when an organic solvent is used as a mobile phase.
Gel Exclusion Chromatography
Gel permeation chromatography (GPC) is a form of size exclusion chromatography (SEC) that uses organic solvents to separate analytes based on their size. Polymer analysis is a popular application of this technique. SEC was first developed as a technique by Lathe and Ruthven in 1955. The term gel permeation chromatography was coined by J.C. Moore of the Dow Chemical Company, who researched the technique in 1964 and licensed the patented column technology to Waters Corporation, which commercialized it in 1964.
Principle of Gel Exclusion Chromatography
The analytes are separated by GPC based on their size or hydrodynamic volume (radius of gyration). Other separation methods, on the other hand, rely on chemical or physical interactions to distinguish analytes. Porous beads packed in a column are used to separate the particles.
Since smaller analytes can penetrate pores more quickly, they spend more time in them and hence have a longer retention time. Since these smaller molecules spend more time in the column, they elute later. Larger analytes, on the other hand, spend little or no time in the pores and are easily eluted. A number of molecular weights can be divided in each column.
Analytes that are too large will not be retained; on the other hand, analytes that are too small will be entirely retained. Analytes that are not retained are eluted with the free volume outside of the particles (Vo), while those that are completely retained are eluted with the volume of solvent held in the pores. The following equation can be used to calculate the total volume, where Vg is the volume of the polymer gel and Vt is the total volume:
Vt = Vg + Vi + Vo
Methods of Gel Filtration Chromatography
Almost all gel permeation chromatography is done in chromatography columns. The experimental model is very similar to that of other liquid chromatography techniques. Samples are dissolved in a suitable solvent, which in the case of GPC is usually organic, and then filtered before being injected onto a column. The column is where a multi-component mixture is separated. The use of a pump ensures a steady supply of fresh eluent to the column. A detector is needed because most analytes are not visible to the naked eye. To obtain additional information about the polymer sample, several detectors are often used.
The stationary process for GPC is gel. In order to apply a gel to a specific separation, the pore size of the gel must be carefully monitored. The absence of ionizing groups and low affinity for the substances to be separated in a given solvent are also desirable properties of the gel-forming agent.
Microporous packing material is used to fill the GPC column. The gel is poured into the column known as the gel filtration column.
The eluent (mobile phase) should be a good solvent for the polymer, allowing the polymer to have a high detector response and wetting the packing surface. Tetrahydrofuran is the most common element in GPC polymers that dissolve at room temperature (THF).
Piston or peristaltic pumps are the two types of pumps available for uniform distribution of relatively small liquid volumes for GPC.
In GPC, a detector will continuously monitor the polymer concentration by weight in the eluting solvent. There are several different types of detectors, which can be classified into two groups. UV absorption, differential refractometer (DRI) or refractive index (RI) detectors, infrared (IR) absorption, and density detectors are the first types of concentration-sensitive detectors.
Application of Gel Filtration Chromatography
GPC is often used to determine the relative molecular weight of polymer samples as well as molecular weight distribution. The molecular volume and shape function, as determined by the intrinsic viscosity, are what GPC truly measures. This relative data can be used to calculate molecular weights within 5% precision if comparable criteria are used. To calibrate the GPC, polystyrene standards with disparities of less than 1.2 are commonly used.
Disadvantages of Gel Permeation chromatography
GPC, on the other hand, has several drawbacks. First, the number of peaks that can be resolved within the short time frame of a GPC run is small. Furthermore, for a satisfactory resolution of peaks, GPC as a technique needs at least a 10% difference in molecular weight. When it comes to polymers, the molecular masses of most of the chains are too close together for the GPC separation to produce anything other than large peaks. Another downside of GPC for polymers is that it needs filtration prior to use to prevent dust and other particulates from destroying the columns and interfering with the detectors.
Did You Know?
SEC is a low-resolution chromatography since it has a hard time distinguishing between similar organisms, so it's usually used as the last step in purification. Since it can be carried out in native solution conditions while maintaining macromolecular interactions, the technique can be used to determine the quaternary structure of purified proteins with slow exchange times. Since SEC tests the hydrodynamic volume (not the molecular weight), it can detect protein tertiary structure, allowing folded and unfolded forms of the same protein to be distinguished.
SEC may be used to determine the size and polydispersity of a synthesized polymer, or the ability to determine the size distribution of polymer molecules. If known-size standards have been run previously, a calibration curve can be developed to determine the sizes of polymer molecules of interest in the solvent of choice (often THF). Techniques like light scattering and/or viscometry can be used online with SEC to produce absolute molecular weights that don't require calibration with known molecular weight standards.
FAQs on Gel Chromatography Explained: Principles & Uses
1. What is gel chromatography and what is its main principle?
Gel chromatography, also known as size-exclusion chromatography, is a lab technique used to separate molecules based on their size. The principle is simple: the column is packed with porous beads. Large molecules are too big to enter these pores, so they travel around the beads and exit the column quickly. Smaller molecules enter the pores, taking a longer, more complex path, so they exit the column last.
2. Why do larger molecules come out of the column first in this technique?
Larger molecules elute first because they are excluded from the tiny pores of the gel beads in the column. This means they have a much shorter path to travel, flowing only in the liquid space between the beads. In contrast, smaller molecules take a detour by entering the pores, which significantly lengthens their journey and causes them to elute later.
3. What are some real-world applications of gel chromatography?
Gel chromatography is very important in biology and chemistry. Its main uses include:
- Purifying proteins: Separating a specific protein from a complex mixture.
- Desalting: Removing small molecules like salt from a protein sample.
- Estimating molecular weight: Helping scientists determine the size of a protein or polymer.
- Analysing polymers: Studying the size distribution in a sample of synthetic polymers.
4. What is the difference between gel filtration and gel permeation chromatography?
The key difference is the type of solvent used. When the solvent is water-based (aqueous), the process is called gel filtration and is typically used for biological molecules like proteins. When an organic solvent is used, the process is called gel permeation, which is common for separating and analysing synthetic polymers.
5. How does the pore size of the gel affect how well the molecules are separated?
The pore size of the gel is the most critical factor for successful separation. If the pores are too small for your molecules, everything will be excluded and elute together. If the pores are too large, almost all molecules will enter them and elute together. The ideal gel has a pore size range that allows smaller molecules to enter while excluding the larger ones, ensuring they travel at different speeds and separate effectively.
6. What are the essential components of a gel chromatography setup?
A standard gel chromatography setup requires a few basic parts:
- A column: A tube, usually made of glass or plastic, to hold the gel.
- Stationary phase: The porous gel beads (like Sephadex) that fill the column.
- Mobile phase: The liquid buffer or solvent that flows through the column and carries the sample.
- A detector: A device at the end of the column to detect molecules as they come out, often a UV spectrophotometer.
7. Can this method be used to find out the molecular weight of a substance?
Yes, gel chromatography is often used to estimate the molecular weight of proteins and polymers. To do this, you first run several molecules of known molecular weights through the column and plot their elution volumes. This creates a calibration curve. You can then run your unknown sample, measure its elution volume, and use the curve to estimate its molecular weight based on where it falls on the plot.
8. What are the main disadvantages of using gel chromatography?
While useful, this technique has a few limitations. First, it offers limited resolution, which means it can be difficult to separate molecules that have very similar sizes. Second, because it often requires slow flow rates for good separation, the process can be time-consuming. Finally, there is a chance that the sample can become diluted as it passes through the column.
