Can you explain what the influence of using reversed column in analysis using HPLC?
Dear Agus:
Reverse-Phase High Performance Liquid Chromatography (RPLC) is the most commonly used mode of HPLC nowadays.
Which is the most relevant difference between RPLC and Normal-Phase Liquid Chromatography? Mainly the nature of the stationary phase (unpolar in RPLC and polar in normal phase liquid chromatography) and consequently the nature of the mobile phase. Whereas RPLC uses solvents with high dielectric constants (also called relative permittivity) and high or medium dipole moment (acetonitrile, water...), normal phase chromatography uses solvents with low dielectric constants and low dipole moment (hexane, heptane, cyclohexane...). On the other hand, whereas RPLC uses silica derivatized with C8, C18...chains; Normal-Phase Liquid Chromatography uses polar stationary phases (for exemple, silica).
Interactions between stationary phase and target analyte are usually (considering columns of similar dimensions and similar flow rates and backpressures) much stronger in RPLC than in Normal-Phase Liquid Chromatography because of the difference between de dielectric constant of stationary phase and mobile phase is higher in RPLC than in Normal-Phase Liquid Chromatography. Note that compounds elute in order of medium polarizability rather than in order of polarity.
Instantaneous alignement of mobile phase dipoles on the stationary phase surface is thought to happen in RPLC thanks mainly to the high backpressures developed inside the column. This spontaneous alignment of dipoles would be possible on inter-phase surfaces of very different dielectric constant (water-air; water-ACN/C18 surfaces...) where breaking of symmetry occurs. This alignement of mobile phase dipoles on the dielectric surface of the stationary phase originates an strong electric field. On the other hand, the higher the difference of dielectric constants between the mobile phase and the stationary phase, the higher the polarization of the dielectric surface.
The surface polarized interacts with polarizable target analytes (i.e. naphthalene,pyrene or benzo(ghi)perylene) in such a way that the retention depends on the medium polarizabilityof the target compound. The higher is the medium polarizability of the compound retained the higher is its retention time. This strong relationship between medium polarizabilities of target analytes and retention times, leads us to the conclusion that electric forces must be involved in the RPLC mechanisms and even in other HPLC modes.
This article contains information about the RPLC retention mechanisms.
Best,
Auréa.
http://ccaasmag.org/chem_2011/vol1/Andrade-Eiroa%20_HPLC_polarization.pdf
Not Reversed Phase-HPLC, I know it. I'm asking about using reversed column. Normally we should use a column based on flow direction of column. Using reversed column makes the pressure higher than normal. Based on experience, when peak was broadening or tailing, analyst usually reverse the flow of column direction, and it made better shape of peak. Anyone knows the mechanism this phenomenon?
Agus, do you mean you want to back-flush or reverse-flush the analytical column (reversal column)?
In that case, from my point of view, this is not recommended. Usually resolution gets worse after back-flushing the column. Some authors state that it is possible to reverse-flush silica-based columns without dammaging them, but I do not agree.
Back-flushing the column is usually applied when the column is thought to be clogged. The assumption is that particulate from your sample, mobile phase, or injector has clogged the inlet frit. In this case, back-flushing the column might displace/remove this material, causing the pressure to go back to normal.
Best,
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