Bonded Phases

Octadecylsilane (C18) is the most widely used RP stationary phase. The 18-carbon alkyl chain is covalently bonded to the silica surface via siloxane linkage. Retention is driven by hydrophobic interactions between analyte and ligand. Variants include standard C18, base-deactivated (BDS), and high-carbon-load versions.

A C18 phase specifically designed for highly aqueous or 100 % aqueous mobile phases. An embedded polar group prevents the hydrophobic collapse of the alkyl chains that occurs with standard C18 under purely aqueous conditions. Essential for retaining highly polar analytes in RP mode.

A 12-carbon alkyl chain bonded to silica. Slightly less hydrophobic than C18, offering an intermediate selectivity. Useful for analytes that are over-retained on C18 or for faster method development cycles.

An 8-carbon alkyl chain bonded to silica. Moderately hydrophobic — approximately half the carbon load of C18. Provides faster elution of strongly retained compounds and is commonly used for basic pharmaceuticals, steroids, and lipids.

A 6-carbon alkyl chain offering low RP retention, intermediate between C4 and C8. Used for moderately polar compounds or as an alternative selectivity tool in method development.

A short 4-carbon alkyl chain. Low hydrophobicity makes C4 ideal for large biomolecules (proteins, antibodies) that would be irreversibly retained on longer-chain phases. Standard phase in protein and biosimilar analysis (wide-pore silica, 300 Å).

Very short alkyl chain (2-carbon) bonded to silica — among the weakest RP retentions. Useful for extremely polar compounds or for combined NP/RP selectivity.

The shortest alkyl modification: a single methyl group (trimethylsilane). Minimal RP retention — often used as a silanol-masking agent or for very polar hydrophilic analytes. Also known as SAS (short alkyl silane).

A C18 phase prepared via a 'diametric' bonding process resulting in a uniquely dense and ordered monolayer. Provides high shape selectivity, distinguishing between geometric isomers and rigid planar vs. non-planar molecules.

Phenyl groups bonded to silica via a propyl or other spacer. Offers π–π interaction in addition to hydrophobic retention, giving unique selectivity for aromatic analytes compared to alkyl phases. Especially useful for separating structural isomers of aromatic compounds.

Two linked phenyl rings provide stronger π–π interaction than a single phenyl group. Enhanced selectivity for polycyclic aromatic compounds and planar molecules. Often used as a complementary phase to C18 in orthogonal screening.

A phenyl group attached via a 6-carbon (hexyl) spacer. The longer spacer provides both hydrophobic and aromatic retention mechanisms, combining C6-like RP retention with phenyl selectivity. Good peak shape for bases.

A phenyl ring with all five hydrogens replaced by fluorine, bonded via a propyl spacer. The strong electron-withdrawing fluorines create a highly polarisable surface capable of π–π, charge-transfer, and dipole interactions. Unique selectivity for halogenated compounds, metabolites, and isomers that co-elute on C18.

A fluorinated C8 chain. The fluorine atoms reduce the polarisability of the alkyl chain compared to a standard C8, resulting in altered selectivity particularly for halogenated analytes and polar compounds.

Unmodified porous silica. Retention is governed by polar interactions (hydrogen bonding, dipole) between analytes and surface silanol groups (Si-OH). Used in classical normal-phase chromatography (NP-HPLC) with non-polar organic mobile phases. Also used as base material for SEC columns.

Dihydroxypropane groups bonded to silica. Less polar than bare silica, reducing irreversible adsorption of polar analytes. Used in both NP and aqueous size-exclusion chromatography (SEC) for biomolecules. Also suitable as a HILIC phase for polar analytes.

Aminopropylsilane bonded to silica. Functions as a weak anion exchanger at low pH and as a normal-phase sorbent at intermediate polarity. Widely used for carbohydrate, sugar, and nucleotide separations. Note: NH2 phases can react with aldehydes and ketones (Schiff base formation).

Nitrile (cyano) group bonded via a propyl spacer. Intermediate polarity — applicable in both RP and NP modes. Provides unique selectivity complementary to C18 for polar and moderately hydrophobic analytes. Weaker RP retention than C8.

Hydrophilic Interaction Liquid Chromatography. Polar stationary phase (bare silica or zwitterionic) used with high-organic (typically acetonitrile-rich) mobile phases. Retains hydrophilic and polar compounds that are unretained in RP. Complementary selectivity to RP — ideal for metabolomics, amino acids, and nucleotides.

A zwitterionic sulfobetaine ligand densely bonded to silica, carrying permanent positive (ammonium) and negative (sulfonate) charges in a 1:1 ratio. Net charge is zero. Provides HILIC retention without the anion- or cation-exchange side effects seen on NH2 or bare silica phases. Excellent batch-to-batch reproducibility for polar analytes.

Quaternary ammonium groups (permanently positively charged) bonded to silica. Retains anionic analytes (nucleotides, organic acids, phosphorylated compounds) via electrostatic interactions. Salt gradient or pH gradient used for elution. Remains fully ionized across the entire working pH range.

Sulfopropyl or sulfonyl groups (permanently negatively charged) bonded to silica. Retains cationic analytes (basic pharmaceuticals, amino acids, peptides) via electrostatic interactions. Elution with increasing salt concentration or pH. Fully ionized and pH-independent within the silica pH range.

A silica surface functionalized with both amino and cyano groups. Provides a dual-character NP sorbent with moderate polarity. Used for simultaneous separation of mixed-polarity analytes in normal-phase mode.

Silica coated with a polybutadiene-maleic acid copolymer layer. Provides weak cation-exchange capacity via carboxylic acid groups. Used in ion chromatography and for separation of inorganic cations and small organic ions.

An amide (–CO–NH–) group is inserted into the alkyl chain between the silica surface and the hydrophobic tail. The polar amide group interacts with the mobile phase water layer, reducing silanol activity and improving peak shape for basic compounds. Also provides unique selectivity for polar analytes and works under 100 % aqueous conditions.

Combines a long-chain RP ligand (C18 or C8) with amino groups on the same silica surface. The amino groups act as a weak anion exchanger at low to neutral pH. Allows separation of basic compounds without ion-pair reagents or buffered mobile phases.

A C18 phase with embedded cyano groups. Combines RP hydrophobic retention with the polar interaction character of CN, providing intermediate selectivity between pure C18 and CN phases.

A C18 chain combined with carboxyl (–COOH) groups providing weak cation-exchange capacity. At low pH the phase behaves as standard RP-C18; at higher pH the carboxyl groups ionize and add cation-exchange retention. Useful for simultaneous separation of acidic and basic analytes.

A C8 reversed-phase chain combined with strong ion-exchange groups (SAX or SCX) on the same silica surface. Provides simultaneous hydrophobic and electrostatic retention — useful for separating complex mixtures of charged and neutral analytes in a single run.

An ether or carbamate polar group is embedded within the C18 or C8 alkyl chain. This 'wettability' modification prevents phase collapse in highly aqueous mobile phases and adds a polar interaction dimension. Retains polar analytes that are not retained on standard C18 under RP conditions.

A long alkyl chain (C23) with an integrated positively charged 'basic spacer' (BS) — a secondary amine group embedded within the chain. Provides reversed-phase retention combined with anion-exchange character. Particularly useful for separating anionic compounds (nucleotides, oligonucleotides) under RP conditions without ion-pair reagents.

Porous silica particles with controlled pore size distribution, surface-modified (e.g. with diol groups) to reduce non-specific adsorption. Separation is based solely on hydrodynamic volume: larger molecules elute first (excluded from pores), smaller molecules penetrate deeper and elute later. Used for molecular weight determination of proteins, peptides, and polysaccharides in aqueous mobile phases.

Amylose tris-arylcarbamate derivatives coated or immobilized on silica. The helical amylose backbone forms inclusion complexes with analyte enantiomers via hydrogen bonding, π–π interactions, and steric recognition within the chiral cavity. Broad application range for racemate resolution.

Cellulose tris-arylcarbamate or tris-arylbenzoate derivatives coated or immobilized on silica. The ribbon-like cellulose backbone provides a different chiral recognition environment to amylose phases. Chiral discrimination via H-bonding and inclusion. Multiple substituent patterns cover a broad range of racemates.

Native or hydroxypropyl-modified β-cyclodextrin covalently bonded to silica. The toroidal cyclodextrin cavity (∼6.5 Å inner diameter) forms inclusion complexes with hydrophobic regions of the analyte while the outer hydroxyl rim provides H-bond interactions. Chiral recognition occurs inside the cavity. Works in RP, NP, and polar organic modes.

Teicoplanin antibiotic bonded to silica via multiple covalent bonds. Contains four macrocyclic rings, a sugar moiety, and multiple H-bonding, ionic, π–π, and steric interaction sites. Provides chiral recognition through a 'basket-shaped' binding pocket. Suitable for amino acids, small peptides, and pharmaceutical racemates.

Human serum albumin immobilized on silica. HSA is a globular plasma protein with specific drug-binding pockets (Sudlow sites I and II). Chiral recognition is based on differential binding affinity of enantiomers within the protein binding sites. Particularly relevant for pharmaceutical compounds that are plasma protein-bound in vivo.

N-(3,5-dinitrobenzoyl)-L- or D-phenylglycine covalently bonded to aminopropyl silica. 'Brush-type' chiral phase relying on the three-point interaction model: the chiral selector makes at least three simultaneous interactions with one enantiomer (π–π, H-bonding, steric). Works in NP mode (hexane/IPA). Classic Pirkle-type phase with high thermal stability.

Amino acid-derived Pirkle-type brush phases. N-(3,5-dinitrobenzoyl)-L-leucine provides chiral discrimination for benzodiazepines and other NP compounds. L-proline (Davankov-type) uses ligand-exchange with a copper-ion mobile phase for chiral amino acid separations.

L-hydroxyproline covalently bonded to silica, forming chiral complexes with Cu²⁺ ions in the mobile phase (typically 2–10 mM copper sulfate). Chiral recognition occurs through differential stability of diastereomeric ternary Cu²⁺ complexes. Selective for underivatized amino acids and hydroxy acids.