Editing High-performance liquid chromatography (section)

===Reversed-phase liquid chromatography (RP-LC)===

[[File:Hplc-perfume-chromatogram.png|thumb|300px|A chromatogram of complex mixture (perfume water) obtained by reversed phase HPLC]]

{{further|Reversed-phase chromatography}}
Reversed phase HPLC (RP-HPLC)<ref>{{Citation |last1=LoBrutto |first1=Rosario |title=Reversed-Phase HPLC |date=2007-01-22 |url=https://onlinelibrary.wiley.com/doi/10.1002/9780470087954.ch4 |work=HPLC for Pharmaceutical Scientists |pages=139–239 |editor-last=Kazakevich |editor-first=Yuri |access-date=2023-10-10 |edition=1 |publisher=Wiley |language=en |doi=10.1002/9780470087954.ch4 |isbn=978-0-471-68162-5 |last2=Kazakevich |first2=Yuri |editor2-last=LoBrutto |editor2-first=Rosario|url-access=subscription }}</ref> is the most widespread mode of chromatography. It has a non-polar stationary phase and an aqueous, moderately polar mobile phase. In the reversed phase methods, the substances are retained in the system the more hydrophobic they are. For the retention of organic materials, the stationary phases, packed inside the columns, are consisted mainly of porous granules of silica gel in various shapes, mainly spherical, at different  diameters (1.5, 2, 3, 5, 7, 10&nbsp;um), with varying pore diameters (60, 100, 150, 300, A), on whose surface are chemically bound various hydrocarbon ligands such as C3, C4, C8, C18. There are also polymeric hydrophobic particles that serve as stationary phases, when solutions at extreme pH are needed, or hybrid silica, polymerized with organic substances. The longer the hydrocarbon ligand on the stationary phase, the longer the sample components can be retained. Most of the current methods of separation of biomedical materials use C-18 type of columns, sometimes called by a trade names such as ODS (octadecylsilane) or RP-18 (Reversed Phase 18).

The most common RP stationary phases are based on a silica support, which is surface-modified by bonding RMe<sub>2</sub>SiCl, where R is a straight chain alkyl group such as C<sub>18</sub>H<sub>37</sub> or C<sub>8</sub>H<sub>17</sub>.

With such stationary phases, retention time is longer for lipophylic molecules, whereas polar molecules elute more readily (emerge early in the analysis). A chromatographer can increase retention times by adding more water to the mobile phase, thereby making the interactions of the hydrophobic analyte with the hydrophobic stationary phase relatively stronger. Similarly, an investigator can decrease retention time by adding more organic solvent to the mobile phase. RP-HPLC is so commonly used among the biologists and life science users, therefore it is often incorrectly referred to as just "HPLC" without further specification. The pharmaceutical industry also regularly employs RP-HPLC to qualify drugs before their release.{{citation needed|date=July 2024}}

RP-HPLC operates on the principle of hydrophobic interactions, which originates from the high symmetry in the dipolar water structure and plays the most important role in all processes in life science. RP-HPLC allows the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand on the stationary phase. This [[solvophobic]] effect is dominated by the force of water for "cavity-reduction" around the analyte and the C<sub>18</sub>-chain versus the complex of both. The energy released in this process is proportional to the [[surface tension]] of the eluent (water: 7.3{{e|−6}}&nbsp;[[Joules|J]]/cm<sup>2</sup>, methanol: 2.2{{e|−6}}&nbsp;J/cm<sup>2</sup>) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol, [[acetonitrile]]) into the mobile phase to reduce the surface tension of water. [[Gradient elution]] uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.

Structural properties of the analyte molecule can play an important role in its retention characteristics. In theory, an analyte with a larger hydrophobic surface area (C–H, C–C, and generally non-polar atomic bonds, such as S-S and others) can be retained longer as it does not interact with the water structure. On the other hand, analytes with higher polar surface area (as a result of the presence of polar groups, such as -OH, -NH<sub>2</sub>, COO<sup>−</sup> or -NH<sub>3</sub><sup>+</sup> in their structure) are less retained, as they are better integrated into water. The interactions with the stationary phase can also affected by steric effects, or exclusion effects, whereby a component of very large molecule may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place. Such surface hindrance typically results in less retention.

Retention time increases with more hydrophobic (non-polar) surface area of the molecules. For example, branched chain compounds can elute more rapidly than their corresponding linear isomers because their overall surface area is lower. Similarly organic compounds with single C–C bonds frequently elute later than those with a C=C or even triple bond, as the double or triple bond makes the molecule more compact than a single C–C bond.

Another important factor is the mobile phase [[pH]] since it can change the hydrophobic character of the ionizable analyte. For this reason most methods use a [[buffering agent]], such as [[sodium phosphate]], to control the pH. Buffers serve multiple purposes: control of pH which affects the ionization state of the ionizable analytes, affect the charge upon the ionizable silica surface of the stationary phase in between the bonded phase linands, and in some cases even act as ion pairing agents to neutralize analyte charge. [[Ammonium formate]] is commonly added in mass spectrometry to improve detection of certain analytes by the formation of analyte-ammonium [[adducts]]. A volatile organic acid such as [[acetic acid]], or most commonly [[formic acid]], is often added to the mobile phase if mass spectrometry is used to analyze the column effluents.

[[Trifluoroacetic acid]] (TFA) as additive to the mobile phase is widely used for complex mixtures of biomedical samples, mostly peptides and proteins, using mostly UV based detectors. They are rarely used in mass spectrometry methods, due to residues it can leave in the detector and solvent delivery system, which interfere with the analysis and detection. However, TFA can be highly effective in improving retention of analytes such as [[carboxylic acids]], in applications utilizing other detectors such as UV-VIS, as it is a fairly strong organic acid. The effects of acids and buffers vary by application but generally improve chromatographic resolution when dealing with ionizable components.

Reversed phase columns are quite difficult to damage compared to normal silica columns, thanks to the shielding effect of the bonded hydrophobic ligands; however, most reversed phase columns consist of alkyl derivatized silica particles, and are prone to hydrolysis of the silica at extreme pH conditions in the mobile phase. Most types of RP columns should ''not'' be used with aqueous [[Base (chemistry)|bases]] as these will hydrolyze the underlying silica particle and dissolve it. There are selected brands of hybrid or enforced silica based particles of RP columns which can be used at extreme pH conditions. The use of extreme acidic conditions is also not recommended, as they also might hydrolyzed as well as corrode the inside walls of the metallic parts of the HPLC equipment.

As a rule, in most cases RP-HPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent. Some biomedical applications require non metallic environment for the optimal separation. For such sensitive cases there is a test for the metal content of a column is to inject a sample which is a [[mixture]] of 2,2'- and 4,4'-[[bipyridine]]. Because the 2,2'-bipy can [[chelate]] the metal, the shape of the peak for the 2,2'-bipy will be distorted (tailed) when [[metal]] [[ion]]s are present on the surface of the [[silica]].{{Citation needed|date=October 2007}}..