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Reversed Phase Chromatography (RP)
In contrast to NP Chromatography, in Reversed Phase Chromatography the stationary phase is non-polar and the mobile phase moderately polar. Molecules which are more non-polar in nature have a longer retention time and polar molecules elute more rapidly. One common stationary phase is silica which has been treated with a straight chain alkyl group. The mobile phase is most commonly a mix of buffer and water and methanol or acetonitrile. RP Chromatography knows a number of stationary phases.
The most common phases are C18 or ODS (Octadecyl) respectively. But also C8 (Octyl) and C4 (Butyl) phases are often used when the C18 phase is too hydrophobic.
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C18, C8 & C4
C* shows the number of carbon atoms in the chain that is bonded to the stationary phase (eg. C18 = 18 carbon atoms). These chains are attached to the surface like the bristle to a brush. The density of the carbon chains bonded to the stationary phase varies from phase to phase and is usually epressed in % by the producer. Most commonly the density is between 13 - 16 %.
Endcapping
The silanol groups that are left over are endcapped. Polar groups can also be endcapped in order to give a hydrophobic phase a polar character. Another effect of polar endcapping is that such a phase can be used with 100 % water wihout destroing the carbon chains.
Stationary Phase
The most commonly used stationary phase is silica. Silica however corrodes in an acid sorrounding and in a highly alkaline surrounding the carbon chains are detached. This reduces the pH range of a silica phase and often it is between 2.0 - 7.5.
Polymer Phase
A good aternative are polymer based columns (RSpak, Asahipak). These columns have polymer as stationary phase and are very robust and durabel in both alkaline and acid sourroundings.
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Polymer Phase
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for the seperation of:
Aliphatic Alkohols, Amino Acid, Cyclodextrine, Protein, Peptide, Oligosaccharide, Drugs
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for the seperation of:
Amino Acid, Protein, Peptide
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Silica Phase
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The HALO column allows fast seperation as in UPLC but with "normal" pressure below 400 bar and with regular HPLC systems.
In order to allow faster seperations, columns are packed with smaller particles. In smaller particles the diffusion path into and out of the particle is shorter and peakbroadening is reduced. This allows faster mobile phase flow rates which reduces analysis time. Unfortunately back pressure is massive, so that modern fast columns with 1.7 µm particles make special UPLC systems necessary which are expensive as regards purchase price and operation.
The HALOcolumn is based on Fused Core Technology. A layer of porous 0.5 µm particles is fused to a solid 1.7 µm silica core. The shorter diffusion path of HALO particles reduces axial dispersion of solutes and minimizes peak broadening. A Halo particle has only a 0.5 µm diffusion path compared to the approximately1.5 µm diffusion path of a 3 µm totally porous particle. Thus the HALO column generates more seperation power and can either be used for faster analysis or more seperation power.
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further Silica Phases
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The multi-talent for RP Chromatography
for the seperation of:
alkaline and acid substances, Peptide, Protein, antibiotics, watersoluble vitamins
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like Monitor but slightly different selectivity
for the seperation of:
Antibiotics, Barbituraet, Chemo and other drugs, Protein, Peptide, acid and alkaline substances
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the specialist for critical seperations
vor the seperation of:
Amino Acid, alkaline substances, vitamins soluble in fat, Metabolite, Peptid Mapping, Drugs, acid substances
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for the seperation of:
fatty acids, Complexing Agent, Protein, Peptide, Soft Drink Analysis, Tricyklic Antidepressiva, Vitamins, etc.
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© infochroma ag, Sumpfstrasse 3, CH-6300 Zug; Tel: +41 41 748 50 60, info@infochroma.ch
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