Column Comparison

HPLC column selection by separation modes:

Normal phase chromatography: The column is filled with a polar packing material and the mobile phase is non-polar (such as hexane). Phases available are:
- Unmodified silica
- Amine
- Cyano
- Diol

Reversed-phase chromatography: The column is filled with a non-polar packing material and the mobile phase is polar (mixture of water/acetonitrile for example). In reversed-phase, the hydrophobicity increases with the alkyl chain length (the number of carbons). Available phases are:
- C18
- C8
- C6 (phenyl and cyclohexyl)
- C4
- C1
- Cyano

Ion exchange chromatography: This separation mode is based upon the ionic exchange (IEX) capacity of the stationary surface phase with oppositely charged analytes. This technique is used for ionic or ionisable samples. The mobile phase is an aqueous buffer which pH and ionic strength control the elution time. SiliCycle’s ionic exchange phases are:
- Strong cation exchanger: SCX and SCX-2
- Strong anion exchanger: SAX
- Weak cation exchanger: WCX
- Weak anion exchanger: WAX

Size exclusion chromatography (gel filtration or gel permeation chromatography): The packing material possesses a desired pore diameter adequate for the analyte molecular size. Larger molecules follow the mobile phase without penetrating the packing material porosity and their elution proceeds quickly. Smaller molecules pass through the porosity increasing their elution time. This mode of separation is used for high molecular weight molecules and needs packing materials with pore size of 300Å or more.

Fluorous chromatography:
Fluorous phases have specific interactions with fluorine-containing compounds which allow a separation based mainly on their fluorous content. Their high hydrophobicity makes them suitable alternatives to C18 or C8. SiliCycle’s fluorous phases are:
- Fluorochrom
- Pentafluorophenyl
- Tridecafluoro

Phases Structure Description
silica
The most commonly used normal phase packing material. For separation of polar compounds in non polar solvents (hexane or heptane).
NH2 or WAX
Amino or AminoPropylSilane (APS).
A good substitute for bare silica or alumina in normal phase chromatography. Most retentive for acidic compounds. Can be used as weak anion exchanger in buffered solutions.

Diol
The diol is use in normal phase chromatography. It is less sensitive to water than silica or amino phases. It is the best choice for normal phase chromatography with mobile phase containing water. It is commonly used for aqueous size-exclusion chromatography.
CN
Cyano, Cyanopropylsilane or nitrile
Cyano packing is used in both normal phase and reversed-phase chromatography. It is less retentive in normal-phase chromatography than silica, amine or diol phases. It is sometimes called a deactivated normal phase sorbent.
C18
RP-18, ODS or Octadecylsilane*
The most commonly used reversed-phase sorbent. Highest retention of non-polar analytes. Also used in ion-pairing chromatography, which finds many applications in biomolecule separation.
C8
RP-8 or Octylsilane
Used in reversed-phase chromatography, less hydrophobic than C18 but permits good separation and faster analysis.
C4
Butyl
Lower retention of non-polar compounds than C18 and C8 but useful in ion-pairing chromatography. Has been used to separate large biomolecules.
C1
Trimethylsilane or TMS
Has the lowest degree of hydrophobicity of all reversed-phase. Useful in the separation of large biomolecules that have extensive hydrophobic regions.
Cyclohexyl
Cyclohexylsilane
Intermediate polarity, hydrophobicity comparable to a C4. The presence of the ring provides additional steric hindrance which will affect the separation.
Phenyl
Reversed-phase useful for separation of aromatics by ?-? interactions conjugated to hydrophobic property.
SCX
Strong Cation exchanger
Stationary phase for ion exchange chromatography, commonly employed for basic organic phase separation. Both phenyl- or propyl- phase can be employed.
SCX-2

WCX
Weak cation exchanger or carboxylic acid
Ion exchange stationary phase useful for analysis and purification of basic proteins and peptides
SAX
Strong anion exchanger, TMACl
Ion exchange stationary phase useful for separation and purification of nucleotides, nucleosides, and organic acids.
Fluorochrom
Reversed-phases for fluorinated and non-fluorinated compounds.
Pentafluorophenyl

Tridecafluoro

*For C18 stationary phase, two different bonding types.
**R groups for unreactive groups in monofunctionalization (CH3) and R groups for active groups in polyfunctionalization (-O-Si).

Silica surface possesses active silanols (Si-OH) which permits the modification of the surface chemistry by grafting silane moities. This property enables the control of the surface polarity useful in separation techniques. In HPLC, two types of silanes are grafted over the surface: monochlorodimethylsilane or trichlorosilane.

Monomeric functionalization (Mono)
By grafting a monochlorodimethylsilane, only one bond can be formed with the silica surface. This type of grafting is called monomeric. The dimethyl groups help to protect the surface by steric hindrance, which prevents the material from reaching the highest silane density possible. The residual silanol groups are inhibited by the grafting of a small molecule, trimethylsilane Si(CH3)3, this small reagent is called endcapping. Most of our modified silicas are available endcapped or non-endcapped (ne). Even after end-capping, a small portion of the initial silanols is still present, unable to react due to steric hindrance and hence isolated from the mobile phase and analytes present. This product presents a high stability, batch-to-batch reproducibility, and good hydrophobic properties. The fact that the silane possesses only one bond with the surface makes this phase less stable at low pH, which may lead to silane hydrolysis and consequently leaching. For low pH, the polymeric phase is preferred.

Polymeric functionalization (Poly)
By grafting a trichlorosilane, it is possible to form multiple bonds in three-space direction with the surface and also between silane molecules. This grafting method is called polymeric functionalization. The silica surface is more hydrophobic, has greater stability in strong acidic condition (pH2, 3) and has longer lifetime.