Reversed phase liquid chromatography (RPLC or RPC) has been 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 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.
Brochure -- Reversed Phase and HILIC TSK-GEL HPLC Columns 
Silica-based Columns |
Methacrylate-based Columns |
The silica-based TSK-GEL reversed phase column products consist of twelve stationary phases over 5 key product families. Each TSK-GEL silica column features:
|
The polymeric-based TSK-GEL reversed phase columns are synthesized from polymethacrylate polymers in a range of pore and particle sizes. Advantages of polymethacrylate particles include:
- Hydrophilic backbone to improve recovery and reduce secondary interactions
- pH stable from 1 to 12
- Compatibility with organic solvents eliminates swelling
|
|
Silica-based Reversed Phase Columns
The silica-based TSK-GEL reversed phase product line consists of eleven stationary phases designed for the analysis of low molecular weight analytes such as:
- pharmaceutical drugs
- forensic compounds
- derivatized amino acids
- carbohydrates
- steroids, lipids, fatty acids
Depending on their molecular weight, peptides may be analyzed with the same silica-based RPC columns, while proteins require a larger pore size column (200-300 angstrom ).
TSK-GEL silica packings are spherical with uniform pore sizes of 100, 140, 150, or 250 angstrom bonded with a monomeric or polymeric layer of octadecyl, octyl, cyano, trimethylsilyl or phenyl groups. Several of the stationary phases are subsequently capped with trimethylsilyl groups by a proprietary method that deactivates the unreacted but accessible silanol groups.
Silica Based Columns – Overview
|
80Å Monomeric
RP Silica |
2µm Super Series
RP Silica |
Specialty
RP Silica |
This latest generation of highly pure 100Å pore size silica is the foundation for sophisticated bonding chemistries.
These are the preferred columns for demanding separations of polar (ODS-100V) and small MW basic compounds (ODS-100Z). |
The 100Å pore size makes this column type ideal for samples smaller than 6,000 Da molecular weight. This product line features exhaustively endcapped 80Ts columns for basic solutes and the moderately endcapped 80Tm columns for peptide separations. |
The “Super Series” provides higher efficiency and improved resolution over conventional columns packed with 3 and 5μm particles.
Pore size of 140Å makes the “Super Series” columns most suitable for samples with molecular weight below 10,000 Da molecular weight. |
TSK-GEL specialty RP silica columns were developed to provide optimum separation for:
|
|
Polymer-based Reversed Phase Columns
Polymethacrylate-based reversed phase columns are available in a range of pore and particle sizes. Although often not as efficient and less robust than silica-based RPC columns, key advantages of polymer-based columns are:
- Wider pH range (1 to 12) allows many basic compounds to be analyzed in their uncharged form, thus reducing secondary adsorption and improving peakshape.
- Improved recovery for peptides and proteins due to reduced secondary interactions
|
Highlights:
- Polymer-based RPC columns are chemically stable from pH 2-12, allowing operation at basic pH where silica-based columns have limited chemical stability.
- Polymer-based TSK-GEL RPC columns can be cleaned and endotoxin removed by using either strong acid or base.
- Polymer-based TSK-GEL RPC columns are available packed with nonporous resins (NPR) or with porous resins of various sizes. The proper column to use is selected based on sample MW or application.
|
