Στήλες HPLC & UHPLC

Normal phase and hydrophilic interaction liquid chromatography (HILIC)

Normal phase and hydrophilic interaction liquid chromatography (HILIC) are primarily used to separate polar and hydrophilic compounds. In reversed phase mode very polar compounds are often not sufficiently retained in low percent organic, or even in 100% aqueous mobile phase. The order of elution in normal phase is opposite that found in reversed phase 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 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 liquid chromatography.

By using an amide or amino bonded phase column, polar compounds can be retained by a normal phase or hydrophilic interaction chromatography retention mechanism. Typical mobile phases in HILIC are aqueous buffers with organic modifiers – primarily acetonitrile.

In contrast to the retention behavior in reversed phase, in HILIC, solutes will be retained longer when increasing the percent acetonitrile.

Typical applications for HILIC are:

Analysis of polyols, carbohydrates, or vitamins
Characterization of protein glycosylation by fluorescence or mass spectrometric detection
Separation of polar peptides, e.g. after enzymatic digestion of proteins (peptide mapping)
Analysis of polar drugs and separation of drug metabolites LC/MS analysis of polar compounds

Reversed Phase Chromatography (RPLC or RPC)

Reversed Phase Chromatography (RPLC or RPC) is the most efficient of all biomolecule separation techniques. It has been the technique of choice for the analysis of small molar mass compounds in both the pharmaceutical and chemical industries, as well as in biomedical research, since the late 1970s. More recently, RPC has become the accepted tool for the separation of peptides, proteins and other biopolymers, making it largely responsible for the widespread popularity of HPLC as a chromatographic technique.

The opposite of normal phase chromatography, RPC requires a non-polar stationary phase and a mobile phase that consists of a mixture of water and polar-solvent mobile phase. The so-called “hydrophobic effect” is the major driving force for retention in RPC. The hydrophobic effect is related to the non-polar surface area of the solute molecule, which varies as a function of mobile phase composition, while the strength of the hydrophobic bond is proportional to the decrease in molecular surface area when the solute associates with the carbon-based stationary phase. Mobile phase additives, such as trifluoroacetic acid, increase protein hydrophobicity by forming ion pairs that strongly adsorb to the stationary phase. Typically, the mobile phase consists of a mixture of water (buffer) and acetonitrile, methanol or, less common, THF, or 2-propanol. The biological molecules are eluted from the chromatographic support by a change in the polarity of the mobile phase.

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 nonpolar 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.

Affinity Chromatography (AFC)

Affinity Chromatography (AFC) offers the greatest potential specificity and selectivity for the isolation or purification of biomolecules. Almost all biological molecules can be purified on the basis of a specific interaction between their chemical or biological structure and a suitable affinity ligand.

In AFC, the target molecule is specifically and reversibly adsorbed by a complementary ligand and immobilized on a matrix. Examples of a complementary ligand include an inhibitor, substrate analog or cofactor, or an antibody which specifically recognizes the target molecule. The selectivity is often based on spatial recognition, a ‘lock-and-key’ mechanism.

The adsorbed molecule is subsequently eluted either by competitive displacement or a conformation change through a shift in pH or ionic strength. Typical molecular pairs are antigens and antibodies, enzymes and coenzymes, and sugars with lectins.

Purification of several thousand-fold may be obtained due to the high selectivity of the affinity interactions. Although affinity chromatography is not specific, in that no enzyme interacts with only one substrate, it is the most selective method for separating proteins.

Hydrophobic interaction chromatography (HIC)

Hydrophobic interaction chromatography (HIC) is based on non-polar interactions that are induced by high salt mobile phases. Stationary phases are similar to reversed phase chromatography (RPC) but the density of functional groups is lower. 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 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 chromatography. 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.

HIC is used in the biopharmaceutical industry for the analysis of antibody drug conjugates (ADCs) or to determine the aggregate content of monoclonal antibodies.

Reprospher

Your workhorse
Reprospher media are based on a 100 A ultrapure silica and are fully scalable from UPLC (1.9 µm particles) to preparative and process scale applications.
Among the offered phases there are some unique selectivities such as a wide range of aromatic specialty phases, phases with polar selectivities and unique phases for achiral SFC.

ReproSil XR

Premium performance at exceptional value
An allround and fully scalable spherical Silica from 1.5 to 15 µm with an extra high purity.
The metal content is less than 100 ppb.
The silica impresses with its very narrow pore size distribution this make 99 % of the surface accessible for the separation.
The uniform bonded phase coverage translates to symmetrical peaks for acids/bases, and predictable reversed phase selectivity.
Because of its very low metal content it also impressed with a high pH stability.

ReproSil Saphir

Alternative to Luna C18
A low metal content spherical silica media scalable from sub-2-micron to 10 µm particles with high carbon load for small molecules and peptides and wide pore ion exchange media for protein and enzyme analysis and purification.

ReproSil-Pur

Specifically designed for pharmaceutical and biotechnical separations.
A robust modern and all-purpose ultra-high purity spherical silica media scalable from UPLC to prep.
Various pore sizes are available to cover applications for small molecules up to biomolecules.
State-of-the-art surface modification technologies are employed for normal and reversed phase media including shape selective, high carbon load as well as water wettable alkyl phases.

Micro/NanoLC Columns

Miniaturisation of liquid chromatography in combination with mass spectrometry has several advantages including improvements in sensitivity, especially at low concentration levels and dramatically reduced solvent consumption, compared to conventional HPLC or UHPLC. With further method optimisation, run times can also be reduced giving further savings in solvent use or time. To meet the requirements of MicroLC/CapillaryLC/NanoLC YMC offers capillary columns specifically designed to use with the corresponding chromatography systems.

Normal Phase (NP) Columns

Whilst historically it was the earliest form of HPLC, normal phase separations have recently received less attention due to the belief that it is complicated and unpredictable. However, normal phase chromatography is a powerful tool for the separation of positional isomers that are difficult to separate in reversed phase mode. Due to a rigid surface, compared to the more flexible carbon chains of reversed phases, the analytes are influenced by well-defined steric interaction with polar groups. YMC offers columns packed with non-bonded silica or with silica gel modified with polar groups

HILIC Columns

HILIC HPLC/UHPLC columns from YMC are rugged stationary phases which provide improved LC/ESI-MS response, direct SPE solvent compatibility and complementary selectivity to reversed phases. This is important to R&D and drug metabolism scientists since the impurity or metabolite is frequently more polar and present at much lower concentrations than the parent compound. With YMC HILIC columns, these very polar compounds elute later than the higher hydrophobic parent compound, thereby minimizing the MS ion suppression that can occur at the beginning of the chromatogram.

Chiral Columns

YMC offers several solutions for separating chiral compounds with different chiral selectors and different chiral separation mechanisms. The selectors are either coated/immobilised or bonded to the support material which has different pore sizes dependent on the selector. According to the selector chiral chromatography can be performed in NP, RP and/or SFC mode.

Biochromatography Columns

RP Bioseparation,
Ion Exchange (IEX),
Size Exclusion (SEC),
Hydrophobic Interaction (HIC)

Reversed Phase (RP) Columns

YMC’s selection of reversed phase (RP) UHPLC/HPLC columns
YMC-Triart,
YMC-Pack ProFamily,
YMC RP Classics,
Meteoric Core

Application Specific HPLC Columns

Designed for key pharmaceutical and environmental applications, these columns utilize novel and unique chemistries to provide superior resolution with ease of use.

Size Exclusion HPLC Columns

Want high resolution separation of water-soluble polymers? Our size exclusion columns (SEC) are available in tailored pore sizes to cover a broad range of molecular weight.