ZORBAX Reversed-Phase Columns | Agilent (2024)

FAQs

Are all C18 columns reverse phase? ›

Note that C18, C8 and phenyl are dedicated reversed-phase stationary phases, while CN columns can be used in a reversed-phase mode depending on analyte and mobile phase conditions. Not all C18 columns have identical retention properties.

To clean these columns, reverse them and wash them with three to five column volumes of 100% isopropanol, three to five column volumes of methylene chloride, three to five volumes of isopropanol, and then the original solvent system.

Reverse phase columns are often used with more polar solvents such as water, methanol or acetonitrile. The stationary phase is a nonpolar hydrocarbon, whereas the mobile phase is a polar liquid.

Reversed-phase chromatography has become the most widely-used method for purification and analysis of peptides owing to its powerful resolving capability, reproducibility and recovery [1,2] even at ultramicroanalytical levels. The purification factor achievable with reversed-phase chromatography ranges from 2 to 200.

A C8 and C18 column are almost the same (since they are both reverse phase columns) except that a C18 has a greater retention capacity and lower LOQ. Thus, the switch may not affect your results as long as; 1. The sample matrix does not interfere.

The SDS approach is not recommended if using the column in mass spectrometry applications. Flush the column at half the normal flow rate using 70:30 n-propanol: water or isopropanol:water.

Peak Broadening: Over time, the stationary phase in an HPLC column can settle slightly. Reversing the flow might disturb this settled packing, leading to a slight broadening of chromatographic peaks. Broadened peaks can make it harder to distinguish between closely eluting components.

To switch from reversed- to normal-phase – prime with methanol (or acetonitrile) followed by ethyl acetate and then hexane, all between 50 to 100 mL. For most flash systems, the above solvent switching is a manual operation. With the new Selekt system this process is now automated, which is really convenient.

In reversed-phase chromatography, the mobile phase is very polar – water or mixtures of water with polar, water-miscible solvents – and the stationary phase is nonpolar, often a 18-carbon-long hydrocarbon attached to the surface of silica or closely related materials.

Which solvent is best for column washing? ›

To wash the column usewater-miscible, nonaqueous solvents such as methanol, acetonitrile, or tetrahydrofuran. If you are using a buffered aqueous mobile phase, do not jump immediately to the strong solvent.

C18 is often called the “traditional reverse phase matrix” because it has the highest degree of hydrophobicity. The reason why C-18 is more hydrophobic than other reverse phases is because length of the carbon chains are longer. C8 is used when shorter retention times are desired.

C18 is a type of column used in chromatography and sometime is called inverted phase because the traditional type of column is made of silica which is polar. C18 is a chemically modified silica in which an hydroxyl group was changed with a long carbonic chain and is thus apolar.

In reversed-phase HPLC the order of elution is the opposite of that in a normal-phase separation, with more polar solutes eluting first. Increasing the polarity of the mobile phase leads to longer retention times. Shorter retention times require a mobile phase of lower polarity.

The C18 chromatographic column is on the weaker side of the polarity, but on the issue of steric hindrance, due to the difference in the C chain, the C18 chromatographic column can play a better role and retain better characteristics.

The primary distinction between XTerra RP18 and C18 HPLC columns is rooted in the bonding of the C18 ligand and the existence of an embedded polar group at the base of the ligand, specifically within the XTerra RP18 column.

T3 columns have a lower carbon coverage, and they are less hydrophobic than a normal C18 trifunctional column. The proprietary trifunctional T3 bonding and endcapping technology yields packing materials with superior low-pH stability.

C18 is more non polar stationary phase if compared to C4 , the type of column will affect retention time and order of sample eluation in chromatogram , choice of column depends mainly on experimental trails however , C18 is preferred for less polar samples.