Editing High-performance liquid chromatography (section)

==Types==

===Partition chromatography===
[[File:HILIC Partition Method Graphic.png|thumb|300px|[[HILIC]] partition technique useful range]]
Partition chromatography was one of the first kinds of chromatography that chemists developed, and is barely used these days.<ref>{{Cite journal| volume = 19| issue = 5| pages = 506–512| last = Ettre| first = C.| title = Milestones in Chromatography: The Birth of Partition Chromatography| journal = LCGC| access-date = 2016-02-26| date = 2001| url = http://images.alfresco.advanstar.com/alfresco_images/pharma/2014/08/22/1598ed6f-5bbe-400b-bc08-ff07d2c59826/article-2090.pdf| archive-date = 2016-03-04| archive-url = https://web.archive.org/web/20160304170852/http://images.alfresco.advanstar.com/alfresco_images/pharma/2014/08/22/1598ed6f-5bbe-400b-bc08-ff07d2c59826/article-2090.pdf| url-status = dead}}</ref> The [[partition coefficient]] principle has been applied in [[paper chromatography]], [[thin layer chromatography]], [[gas phase]] and [[countercurrent chromatography|liquid–liquid separation]] applications. The 1952 [[Nobel Prize]] in chemistry was earned by [[Archer John Porter Martin]] and [[Richard Laurence Millington Synge]] for their development of the technique, which was used for their separation of [[amino acids]].<ref>{{Cite journal| volume = 35| issue = 1–2| pages = 91–121| last1 = Martin| first1 = A J P| last2 = Synge| first2 = R L M| title = Separation of the higher monoamino-acids by counter-current liquid-liquid extraction: the amino-acid composition of wool| journal = Biochemical Journal| date = 1941| pmid = 16747393| doi=10.1042/bj0350091| pmc=1265473}}</ref> Partition chromatography uses a retained solvent, on the surface or within the grains or fibers of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of some [[coulombic]] and/or [[hydrogen donor]] interaction with the stationary phase. Analyte molecules partition between a liquid stationary phase and the eluent. Just as in [[HILIC|hydrophilic interaction chromatography]] (HILIC; a sub-technique within HPLC), this method separates analytes based on differences in their polarity. HILIC most often uses a bonded polar [[Stationary phase (chemistry)|stationary phase]] and a mobile phase made primarily of [[acetonitrile]] with water as the strong component. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences. HILIC bonded phases have the advantage of separating [[acidic]], [[Base (chemistry)|basic]] and neutral solutes in a single chromatographic run.<ref>{{cite book|year=1987 |title=High performance liquid chromatography |url=https://archive.org/details/highperformancel00lind |url-access=registration |publisher=Wiley |author1=Lindsay, S. |author2=Kealey, D. |osti = 7013902}} from review {{cite journal|journal=J. Am. Chem. Soc. |volume=110 |issue=11 |year=1988 |doi=10.1021/ac00162a003|title=Theoretical and experimental foundation for surface-coverage programming in gas–solid chromatography with an adsorbable carrier gas|last1=Hung|first1=L. B.|last2=Parcher|first2=J. F.|last3=Shores|first3=J. C.|last4=Ward|first4=E. H.|pages=1090–1096}}</ref>

The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase) the longer the elution time. The interaction strength depends on the functional groups part of the analyte molecular structure, with more polarized groups (''e.g.'', hydroxyl-) and groups capable of hydrogen bonding inducing more retention. [[Coulombic]] (electrostatic) interactions can also increase retention. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times.{{citation needed|date=July 2024}}

===Normal–phase chromatography===

Normal–phase chromatography was one of the first kinds of HPLC that chemists developed, but has decreased in use over the last decades. Also known as normal-phase HPLC (NP-HPLC), this method separates analytes based on their affinity for a polar stationary surface such as silica; hence it is based on analyte ability to engage in polar interactions (such as [[hydrogen-bonding]] or [[dipole-dipole]] type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (''e.g.'', [[chloroform]]), and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on [[Steric effects|steric factors]]. The effect of steric hindrance on interaction strength allows this method to resolve (separate) [[structural isomer]]s.{{citation needed|date=July 2024}}

The use of more polar solvents in the mobile phase will decrease the retention time of analytes, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. This behavior is somewhat peculiar to normal phase chromatography because it is governed almost exclusively by an adsorptive mechanism (''i.e.'', analytes interact with a solid surface rather than with the solvated layer of a ligand attached to the sorbent surface; see also reversed-phase HPLC below). Adsorption chromatography is still somewhat used for structural isomer separations in both column and thin-layer chromatography formats on activated (dried) silica or alumina supports.{{citation needed|date=July 2024}}

Partition- and NP-HPLC fell out of favor in the 1970s with the development of [[reversed-phase chromatography|reversed-phase]] HPLC because of poor reproducibility of retention times due to the presence of a water or protic organic solvent layer on the surface of the silica or [[alumina]] chromatographic media. This layer changes with any changes in the composition of the mobile phase (''e.g.'', moisture level) causing drifting retention times.{{citation needed|date=July 2024}}

Recently, partition chromatography has become popular again with the development of [[Hilic]] bonded phases which demonstrate improved reproducibility, and due to a better understanding of the range of usefulness of the technique.

===Displacement chromatography===
The use of [[displacement chromatography]] is rather limited, and is mostly used for preparative chromatography. The basic principle is based on a molecule with a high affinity for the chromatography matrix (the displacer) which is used to compete effectively for binding sites, and thus displace all molecules with lesser affinities.<ref>[http://www.sacheminc.com/industries/biotechnology/teaching-tools.html Displacement Chromatography]. Sacheminc.com. Retrieved 2011-06-07. {{webarchive |url=https://web.archive.org/web/20080915113736/http://www.sacheminc.com/industries/biotechnology/teaching-tools.html |date=September 15, 2008 }}</ref>
There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than "peaks". Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentration.{{citation needed|date=July 2024}}

===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}}..

===Size-exclusion chromatography===
{{further|Size-exclusion chromatography}}
'''Size-exclusion chromatography''' ('''SEC''')<ref>{{Citation |last1=Kazakevich |first1=Yuri |title=Size-Exclusion Chromatography |date=2007-01-22 |url=https://onlinelibrary.wiley.com/doi/10.1002/9780470087954.ch6 |work=HPLC for Pharmaceutical Scientists |pages=263–279 |editor-last=Kazakevich |editor-first=Yuri |access-date=2023-10-10 |edition=1 |publisher=Wiley |language=en |doi=10.1002/9780470087954.ch6 |isbn=978-0-471-68162-5 |last2=LoBrutto |first2=Rosario |editor2-last=LoBrutto |editor2-first=Rosario|url-access=subscription }}</ref> separates polymer molecules and [[biomolecule]]s based on differences in their molecular size (actually by a particle's [[Stokes radius]]). The separation process is based on the ability of sample molecules to permeate through the pores of gel spheres, packed inside the column, and is dependent on the relative size of analyte molecules and the respective pore size of the absorbent. The process also relies on the absence of any interactions with the packing material surface.

Two types of SEC are usually termed:

# '''Gel permeation chromatography (GPC)'''—separation of synthetic polymers (aqueous or organic soluble). GPC is a powerful technique for polymer characterization using primarily organic solvents.
# '''Gel filtration chromatography (GFC)'''—separation of water-soluble biopolymers. GFC uses primarily aqueous solvents (typically for aqueous soluble biopolymers, such as proteins, etc.).

The separation principle in SEC is based on the fully, or partially penetrating of the high molecular weight substances of the sample into the porous stationary-phase particles during their transport through column. The mobile-phase eluent is selected in such a way that it totally prevents interactions with the stationary phase's surface. Under these conditions, the smaller the size of the molecule, the more it is able to penetrate inside the pore space and the movement through the column takes longer. On the other hand, the bigger the molecular size, the higher the probability the molecule will not fully penetrate the pores of the stationary phase, and even travel around them, thus, will be eluted earlier. The molecules are separated in order of decreasing molecular weight, with the largest molecules eluting from the column first and smaller molecules eluting later. Molecules larger than the pore size do not enter the pores at all, and elute together as the first peak in the chromatogram and this is called total exclusion volume which defines the exclusion limit for a particular column. Small molecules will permeate fully through the pores of the stationary phase particles and will be eluted last, marking the end of the chromatogram, and may appear as a total penetration marker.

In biomedical sciences it is generally considered as a low resolution chromatography and thus it is often reserved for the final, "polishing" step of the purification. It is also useful for determining the [[tertiary structure]] and [[quaternary structure]] of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works also in a preparative way by trapping the smaller molecules in the pores of a particles. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules: that is, the smaller the molecule, the longer the retention time.

This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weight [[heparin]]s.<ref>{{Cite journal |last1=Mulloy |first1=Barbara |last2=Heath |first2=Alan |last3=Shriver |first3=Zachary |last4=Jameison |first4=Fabian |last5=Al Hakim |first5=Ali |last6=Morris |first6=Tina S. |last7=Szajek |first7=Anita Y. |date=2014-08-01 |title=USP compendial methods for analysis of heparin: chromatographic determination of molecular weight distributions for heparin sodium |url=https://doi.org/10.1007/s00216-014-7940-3 |journal=Analytical and Bioanalytical Chemistry |language=en |volume=406 |issue=20 |pages=4815–4823 |doi=10.1007/s00216-014-7940-3 |pmid=24958344 |hdl=1721.1/104914 |s2cid=492085 |issn=1618-2650|url-access=subscription }}</ref>

===Ion-exchange chromatography===
{{further|Ion-exchange chromatography}}
'''Ion-exchange chromatography''' ('''IEC''') or '''ion chromatography''' ('''IC''')<ref>{{Cite book |last1=Fritz |first1=James S. |url=https://onlinelibrary.wiley.com/doi/book/10.1002/9783527613243 |title=Ion Chromatography |last2=Gjerde |first2=Douglas T. |date=2000-04-25 |publisher=Wiley |isbn=978-3-527-29914-0 |edition=1 |language=en |doi=10.1002/9783527613243}}</ref> is an analytical technique for the separation and determination of ionic solutes in aqueous samples from environmental and industrial origins such as metal industry, industrial waste water, in biological systems, pharmaceutical samples, food, etc. Retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Solute ions charged the same as the ions on the column are repulsed and elute without retention, while solute ions charged oppositely to the charged sites of the column are retained on it. Solute ions that are retained on the column can be eluted from it by changing the mobile phase composition, such as increasing its salt concentration and pH or increasing the column temperature, etc.

Types of ion exchangers include [[polystyrene]] [[resin]]s, [[cellulose]] and [[dextran]] ion exchangers (gels), and controlled-pore glass or porous [[silica gel]]. Polystyrene resins allow cross linkage, which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity. Cellulose and dextran ion exchangers possess larger pore sizes and low charge densities making them suitable for protein separation.

In general, ion exchangers favor the binding of ions of higher charge and smaller radius.

An increase in [[counter ion]] (with respect to the functional groups in resins) concentration reduces the retention time, as it creates a strong competition with the solute ions. A decrease in pH reduces the retention time in cation exchange while an increase in pH reduces the retention time in anion exchange. By lowering the pH of the solvent in a cation exchange column, for instance, more hydrogen ions are available to compete for positions on the anionic stationary phase, thereby eluting weakly bound cations.

This form of chromatography is widely used in the following applications: water purification, preconcentration of trace components, ligand-exchange chromatography, ion-exchange chromatography of proteins, high-pH [[anion-exchange chromatography]] of carbohydrates and oligosaccharides, and others.

===Bioaffinity chromatography===
{{further|Affinity chromatography}}
High performance affinity chromatography (HPAC)<ref>{{Cite journal |last1=Zhang |first1=Chenhua |last2=Rodriguez |first2=Elliott |last3=Bi |first3=Cong |last4=Zheng |first4=Xiwei |last5=Suresh |first5=Doddavenkatana |last6=Suh |first6=Kyungah |last7=Li |first7=Zhao |last8=Elsebaei |first8=Fawzi |last9=Hage |first9=David S. |date=2018 |title=High performance affinity chromatography and related separation methods for the analysis of biological and pharmaceutical agents |journal=Analyst |language=en |volume=143 |issue=2 |pages=374–391 |doi=10.1039/C7AN01469D |issn=1364-5528 |pmc=5768458 |pmid=29200216|bibcode=2018Ana...143..374Z }}</ref> works by passing a sample solution through a column packed with a stationary phase that contains an immobilized biologically active ligand. The ligand is in fact a substrate that has a specific binding affinity for the target molecule in the sample solution. The target molecule binds to the ligand, while the other molecules in the sample solution pass through the column, having little or no retention. The target molecule is then eluted from the column using a suitable elution buffer.

This chromatographic process relies on the capability of the bonded active substances to form stable, specific, and reversible complexes thanks to their biological recognition of certain specific sample components. The formation of these complexes involves the participation of common molecular forces such as the [[Van der Waals interaction]], electrostatic interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites.

=== Aqueous normal-phase chromatography ===
'''Aqueous normal-phase chromatography''' ('''ANP''') is also called '''hydrophilic interaction liquid chromatography''' ('''HILIC''').<ref name=":1">{{Cite journal |last=McCalley |first=David V. |date=2017-11-10 |title=Understanding and manipulating the separation in hydrophilic interaction liquid chromatography |url=https://pubmed.ncbi.nlm.nih.gov/28668366/ |journal=Journal of Chromatography A |volume=1523 |pages=49–71 |doi=10.1016/j.chroma.2017.06.026 |issn=1873-3778 |pmid=28668366}}</ref> This is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). HILIC is used to achieve unique selectivity for hydrophilic compounds,<ref name="doi.org">{{Cite journal |last1=Buszewski |first1=Bogusław |last2=Noga |first2=Sylwia |date=2012 |title=Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique |url=https://doi.org/10.1007/s00216-011-5308-5 |journal=Analytical and Bioanalytical Chemistry |language=en |volume=402 |issue=1 |pages=231–247 |doi=10.1007/s00216-011-5308-5 |issn=1618-2650 |pmc=3249561 |pmid=21879300}}</ref> showing normal phase elution order, using "reversed-phase solvents", i.e., relatively polar mostly non-aqueous solvents in the mobile phase.<ref name=":1" /> Many biological molecules, especially those found in biological fluids, are small polar compounds that do not retain well by reversed phase-HPLC. This has made hydrophilic interaction LC (HILIC) an attractive alternative and useful approach for analysis of polar molecules. Additionally, because HILIC is routinely used with traditional aqueous mixtures with polar organic solvents such as ACN and methanol, it can be easily coupled to MS.<ref name="doi.org"/>