TSKgel Columns

Reversed Phase Chromatography

This mode of HPLC, often referred to as RPLC or RPC, has been largely responsible for the widespread popularity of HPLC as a chromatographic technique. The opposite of normal phase chromatography, reversed phase liquid chromatography requires a non-polar stationary phase and a polar mobile phase. Typically the mobile phase consists of a mixture of water (buffer) and acetonitrile, methanol, THF, or 2-propanol.

Silica particles are most commonly used as the support, which then is derivatized with octadecylsilane (ODS). Polymer-based supports have been introduced as an alternative to silica-based reversed phase columns, particularly for analyzing basic compounds in their neutral state at high pH.

RPC columns can be applied to the analysis of a wide variety of compounds, ranging from neutral polar and non-polar solutes to acidic, basic, and amphoteric compounds. RPC is also an efficient technique for the analysis of derivatized amino acids, peptides and proteins, although protein structure is not always maintained due to the high concentration of organic solvent required for their elution.

Normal Phase and Hydrophilic Interaction Chromatography

The ‘normal’ or traditional way of doing liquid chromatography, normal phase liquid chromatography (NPC) uses a polar stationary phase and a non-polar mobile phase. The polar surface can either be obtained by derivatizing the support with a ligand containing a polar functional group, or it can be the original (polar) support, as in the case of silica gel. The popularity of bare silica gel declined after the introduction of bonded phases, since bonded phases feature shorter equilibration times and allow the use of gradient elution. Cyano, amino, and diol bonded phases are the most common normal phase column types. Normal phase HPLC columns are chosen for a number of widely used specialty separations including the analysis of sugars on amino columns, tricyclic antidepressants on cyano columns, and several classes of steroids on diol columns.As a rule, the order of elution in normal phase is opposite that found in RPC for the same mixture of compounds. Although non-polar organic mobile phases and a silica stationary phase were used traditionally in normal phase LC, today most normal phase separations are performed with aqueous-organic mobile phases and a more polar-bonded stationary phase. This mode of HPLC is now commonly referred to as HILIC, hydrophilic interaction chromatography.

Size Exclusion Chromatography

This separation mode is based on the discrimination of individual sample components by the pores of the packing material. It is the dominant mode of separation for polymers. Large sample molecules cannot or can only partially penetrate the pores, whereas smaller molecules can access all or a larger number of pores. Thus, large molecules elute from the column first, smaller molecules elute later, and the smallest molecules that can access all the pores elute last from the column. Size exclusion chromatography is the only mode of chromatography that does not involve interaction with a stationary phase by means of adsorption or partitioning of the solutes.Particles prepared from organic polymers have traditionally been the most widely used packing materials for size-exclusion chromatography (SEC). The terms SEC, GFC (gel filtration chromatography) and GPC (gel permeation chromatography) all refer to the same chromatographic technique. In GFC an aqueous mobile phase is used, while an organic mobile phase is employed in GPC. The general term SEC covers both uses.

GFC is popular among biochemists for the isolation of protein fractions or for the removal of aggregates in a final polishing step in biotechnology production. GFC is also frequently used for desalting a (protein) sample solution, often to prepare the sample for elution by another chromatographic mode. GPC plays an important role in the characterization of industrial organic-soluble polymers.

Ion Exchange Chromatography

Ion Exchange chromatography (IEC) is a technique based on the difference in the strength of the interaction between a sample ion and an oppositely charged functional group on the support. The sample ion competes for the functional group with a counter ion that has been added to the mobile phase as a salt. Elution is most often accomplished by increasing the salt concentration over time.Ion exchange chromatography has important industrial applications (including water purification), and is the most common separation mode for protein purification schemes. Biomolecules generally have charged groups on their surfaces, which change with the pH of the solution. In all cases, stationary phases in ion exchange chromatography are chemically attached to the support.

Anion Exchange Chromatography

is performed with either a strong anion-exchange column, containing a quaternary ammonium ion, or with a weak anion-exchanger, having either a tertiary or secondary amine functional group, such as DEAE (diethylaminoethyl). A counter ion, often Cl-, maintains electroneutrality.

Cation Exchange Chromatography

is performed with either a strong cation exchanger, containing a bonded sulfonic acid group, such as sulfopropyl (SP), or with a weak cation exchanger, containing a weak acid such as carboxymethyl (CM). The advantage of strong vs. weak ion exchangers is that the first are charged over a wider pH range. Weak ion-exchangers often provide slightly different selectivity from strong exchangers.

The term ion-chromatography (IC) refers to ion exchange chromatography of inorganic anions and cations using an ion exchange column. Ion chromatography is employed in many industries, e.g., for the analysis of inorganic ions in acid rain and metal ions in plating solutions.

Ion pair chromatography (IPC) represents another means of separating ionic species. In IPC the retention of sample ions is increased by adding to the mobile phase an ionic detergent or salt (ion pair reagent) that can form an ion pair association with an oppositely charged sample ion. The ion pair shields the charge of the sample ion and increases its interaction with the (usually non-polar) stationary phase. This technique is most often practiced with RPLC, hence IP-RPLC. Ion pair chromatography is employed when traditional methods (such as reversed phase LC) do not give sufficient retention, or when the charged sample components interact with the packing material resulting in asymmetrical peaks.

Hydrophobic Interaction Chromatography

In HIC, a weakly non-polar stationary phase is used with an aqueous mobile phase containing a high concentration of a chaotropic salt. The technique is mainly applied to the separation of proteins, which are eluted by gradually reducing the salt concentration. As in reversed phase LC, proteins are retained by interaction with alkyl or aryl functional groups on the packing material. Unlike RPLC, in HIC the density of these functional groups is low and protein molecules are adsorbed on only one or a few sites. Sorption takes place at high salt concentration, and desorption is accomplished by decreasing the salt concentration or by adding a low percentage of organic solvent. Although also based on hydrophobic interactions, selectivity in HIC separations is distinctly different from that in reversed phase LC. Despite the lower peak capacity in HIC compared to RPC, HIC has the advantage that the mobile phase conditions (primarily aqueous) do not usually disrupt higher-order protein structures.

Affinity Chromatography

Affinity chromatography (AFC) is performed on a support that is functionalized with a ligand that shows biological affinity for a particular enzyme or other biological molecule.  Almost all biomolecules can be purified on the basis of specific interaction between their chemical or biological structure and a suitable affinity ligand. Typical molecular pairs are antigens and antibodies, enzymes and coenzymes, and sugars with lectins. Thus, affinity chromatography distinguishes itself from, e.g., reversed phase and ion exchange chromatography, in that a highly specific interaction with the protein of interest is the cause for separation or purification. Although affinity chromatography is not specific, in that no enzyme interacts with only one substrate, it is the most selective method for separating proteins. The selectivity is often based on spatial recognition via a ‘lock-and-key’ mechanism, as in antibody-antigen interactions. Less selective general ligands have affinity for a whole class of proteins, which often require other chromatographic methods for purification of individual members. The term bioselective adsorption has been proposed as an alternative name for affinity chromatography.