18 Elution Chromatography*
Separation science is the science of isolating some or all of the individual chemical components of a mixture.
Chromatography, which encompasses several important methods in separation science, refers to the separation of mixtures by passing a sample-containing mobile phase over a stationary phase.
Elution is a concept at the core of modern chromatography: a mobile phase continuously or sequentially washes over an immiscible stationary phase. Different chemical species are separated on the basis of a different balance of interactions between a stationary phase and mobile phase, such that each chemical species (ideally) travels the length of the stationary phase at a different rate. Various types of interactions are used to effect separations based on different physicochemical properties.
In practice, the stationary phase usually comprises particles packed within a cylindrical column. Mobile phase flows through this packing, either by gravity or by applied pressure (e.g. pumping). After being loaded onto the column at the same time (as a mixture), different species exit the column (i.e. elute) after the flow of different volumes of mobile phase and thus at different times.
Separation Mechanisms
Adsorption Chromatography (Normal Phase, NP). The stationary phase is a polar solid upon which analytes adsorb. Silica particles are a common example, as are silica particles functionalized with diol, amino, or cyano groups. Polar analytes will adsorb to these polar surfaces. The mobile phase is a moderately polar to non-polar solvent. Analytes are polar or moderately polar. Separation of analytes is based on differences in polarity, where less polar analytes tend to elute first.
Partition Chromatography (Reverse Phase, RP). The stationary phase is a non-polar film on a solid support (sometimes called a bonded liquid phase). Octadecyl (C18) chains chemically bonded on silica are a common example. Mobile phases are moderately polar to polar solvents. Analytes are non-polar to moderately polar. Separation of analytes is based on differences in polarity, where more polar analytes tend to elute first.
Hydrophilic Interaction Chromatography (HILIC). This method uses a polar stationary phase (e.g. silica or a bonded film) with mobile phases that are 2–40% aqueous with a miscible polar solvent as the balance. (The method is also called “aqueous normal phase chromatography.”) The mobile and stationary phase combinations yield a thin and semi-static layer of water adsorbed on the stationary phase. Charged, polar, and moderately polar analytes partition between this water-rich layer and the mobile phase. Hydrogen bonding and ionic interactions also contribute to separations. Polar and charged analytes elute last.
Hydrophobic Interaction Chromatography (HIC). This method is used with proteins. While generally hydrophilic, proteins often have hydrophobic surface regions that can adsorb on weakly non-polar stationary phases. The preference of a protein for the stationary phase depends on the intrinsic hydrophobicity of the protein, on the ionic strength of the aqueous mobile phase (hydrophobic regions are less effectively solvated at higher ionic strength), and on the identity of the ions in the mobile phase (some ions increase protein solubility in water whereas others decrease it). After initial binding to the stationary phase, proteins are eluted in order of increasing hydrophobicity by progressively decreasing the salt concentration in the mobile phase.
Size-Exclusion Chromatography (SEC). Stationary phases are porous particles. An interstitial fraction of the mobile phase fills the volume outside the particles and the remaining fraction fills the volume inside the pores. The number of pores that a molecule can access is determined by its size and the size distribution of the pores. Molecules of different size thus experience different total mobile phase volumes and are separated, with larger molecules retained less and eluting first. This approach is also called gel filtration/permeation chromatography (GFC/GPC). The mobile phase can be aqueous or an organic solvent depending on the type of analyte and stationary phase material.
Ion-Exchange chromatography (IEC). The stationary phase is a resin with bound acidic groups (e.g. carboxylic acid or sulfonic acid for cation exchange) or basic groups (e.g. tertiary or quaternary amines for anion exchange). When ionized, these groups have electrostatic interactions with ions of the opposite charge. Charged species are separated based on differing strengths of interaction with the stationary phase. Analytes with larger net charge, smaller hydrated radius, and great polarizability are more strongly retained and elute later. The mobile phase is an aqueous buffer with controlled pH. IEC can be used to separate multiple species of a given net charge, or to selectively separate a particular ion (or type of ion) from other species.
Affinity Chromatography. The stationary phase is modified with a functional group (e.g. boronic acid, Ni2+-nitrilotriacetic acid) or an immobilized species (e.g. lectin, antibody, protein ligand) that has highly selective and reversible binding interactions with a particular analyte or class of analytes. Affinity chromatography is most commonly used to separate the desired analyte(s) from a background of other species. The initial mobile phase is usually an aqueous solution to wash out unretained species. The subsequent mobile phase is an aqueous solution that contains something that either competes for the analyte binding sites on the stationary phase or otherwise disrupts the affinity interaction. In some cases, washes of increasing stringency can separate analytes that have differing but non-zero affinities for the stationary phase. Affinity chromatography is often followed by another form of separation (e.g. size-exclusion) to isolate the analyte from whatever broke its affinity interaction.
Connections
- This chapter does not have strong connections with the prior chapters. It does have a strong connection with introductory chemistry: all of the separation mechanisms, except for SEC, leverage chemical equilibria between the stationary phase and mobile phase (see also Ch. 19).
Post-Reading Questions
- Make a table with two columns. Match the types of chromatography described above with the following simplified mechanisms of separation: differences in polarity, differences in size, differences in charge, selective binding.
- Match the following examples of analytes in mixtures with one of the following chromatography methods: ion-exchange, RP partition, size-exclusion. Also predict the order of elution.
- Mixture: Alanine, aspartic acid, phenylalanine, serine (all derivatized at their N-terminus with phenylisothiocyanate)
- Mixture: chloride, phosphate, sulfate
- Mixture: IgG (150 kDa), myoglobin (17 kDa), thyroglobulin (660 kDa)
Topic Learning Objectives
The chapter is a primer for the following learning objectives, which will be covered in lecture and/or with additional assigned reading:
- Understand the concept of elution.
- Understand how more retention on the stationary phase leads to longer elution times.
- For each type of chromatography:
- Be able to give examples of stationary phases.
- Be able to provide general descriptions and give examples of mobile phases.
- Be able to provide general descriptions of the class(es) of analytes that can be separated.
- Predict the elution order for a given set of analytes.