Editing Chromatography (section)

==Special techniques==

===Reversed-phase chromatography===
{{main|Reversed-phase chromatography}}
Reversed-phase chromatography (RPC) is any liquid chromatography procedure in which the mobile phase is significantly more polar than the stationary phase. It is so named because in normal-phase liquid chromatography, the mobile phase is significantly less polar than the stationary phase. Hydrophobic molecules in the mobile phase tend to adsorb to the relatively hydrophobic stationary phase. Hydrophilic molecules in the mobile phase will tend to elute first. Separating columns typically comprise a C8 or C18 carbon-chain bonded to a silica particle substrate.

===Hydrophobic interaction chromatography===

Hydrophobic Interaction Chromatography (HIC) is a purification and analytical technique that separates analytes, such as proteins, based on [[Hydrophobic effect|hydrophobic interactions]] between that analyte and the chromatographic matrix. It can provide a non-denaturing orthogonal approach to reversed phase separation, preserving native structures and potentially protein activity.  In hydrophobic interaction chromatography, the matrix material is lightly substituted with hydrophobic groups. These groups can range from methyl, ethyl, propyl, butyl, octyl, or phenyl groups.<ref>{{cite book | vauthors = Ninfa AJ, Ballou DP, Benore M | title = Fundamental Laboratory Approaches for Biochemistry and Biotechnology | location = Hoboken, NJ | publisher = John Wiley | date = 2010 }}</ref> At high salt concentrations, non-polar sidechains on the surface on proteins "interact" with the hydrophobic groups; that is, both types of groups are excluded by the polar solvent (hydrophobic effects are augmented by increased ionic strength). Thus, the sample is applied to the column in a buffer which is highly polar, which drives an association of hydrophobic patches on the analyte with the stationary phase. The eluent is typically an aqueous buffer with decreasing salt concentrations, increasing concentrations of detergent (which disrupts hydrophobic interactions), or changes in pH. Of critical importance is the type of salt used, with more [[kosmotropic]] salts as defined by the [[Hofmeister series]] providing the most water structuring around the molecule and resulting hydrophobic pressure. Ammonium sulfate is frequently used for this purpose. The addition of organic solvents or other less polar constituents may assist in improving resolution.

In general, Hydrophobic Interaction Chromatography (HIC) is advantageous if the sample is sensitive to pH change or harsh solvents typically used in other types of chromatography but not high salt concentrations. Commonly, it is the amount of salt in the buffer which is varied. In 2012, Müller and Franzreb described the effects of temperature on HIC using Bovine Serum Albumin (BSA) with four different types of hydrophobic resin. The study altered temperature as to effect the binding affinity of BSA onto the matrix. It was concluded that cycling temperature from 40 to 10 degrees Celsius would not be adequate to effectively wash all BSA from the matrix but could be very effective if the column would only be used a few times.<ref>{{cite journal | vauthors = Müller TK, Franzreb M | title = Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions | journal = Journal of Chromatography A | volume = 1260 | pages = 88–96 | date = October 2012 | pmid = 22954746 | doi = 10.1016/j.chroma.2012.08.052 }}</ref> Using temperature to effect change allows labs to cut costs on buying salt and saves money.

If high salt concentrations along with temperature fluctuations want to be avoided one can use a more hydrophobic to compete with one's sample to elute it. This so-called salt independent method of HIC showed a direct isolation of Human Immunoglobulin G (IgG) from serum with satisfactory yield and used β-cyclodextrin as a competitor to displace IgG from the matrix.<ref>{{cite journal | vauthors = Ren J, Yao P, Chen J, Jia L | title = Salt-independent hydrophobic displacement chromatography for antibody purification using cyclodextrin as supermolecular displacer | journal = Journal of Chromatography A | volume = 1369 | pages = 98–104 | date = November 2014 | pmid = 25441076 | doi = 10.1016/j.chroma.2014.10.009 }}</ref> This largely opens up the possibility of using HIC with samples which are salt sensitive as we know high salt concentrations precipitate proteins.

=== Hydrodynamic chromatography ===
Hydrodynamic chromatography (HDC) is derived from the observed phenomenon that large droplets move faster than small ones.<ref>{{cite journal | vauthors = Song H, Tice JD, Ismagilov RF | title = A microfluidic system for controlling reaction networks in time | journal = Angewandte Chemie | volume = 42 | issue = 7 | pages = 768–72 | date = February 2003 | pmid = 12596195 | doi = 10.1002/anie.200390203 }}</ref> In a column, this happens because the [[center of mass]] of larger droplets is prevented from being as close to the sides of the column as smaller droplets because of their larger overall size.<ref>{{cite journal|last1=Small|first1=Hamish|last2=Langhorst|first2=Martin A. |date=1982-07-01|title=Hydrodynamic Chromatography|journal=Analytical Chemistry|volume=54|issue=8|pages=892A–898A|doi=10.1021/ac00245a724|issn=0003-2700}}</ref> Larger droplets will elute first from the middle of the column while smaller droplets stick to the sides of the column and elute last. This form of chromatography is useful for separating analytes by [[molar mass]] (or molecular mass), size, shape, and structure when used in conjunction with [[Scattering|light scattering]] detectors, [[viscometer]]s, and [[refractometer]]s.<ref>{{cite journal | vauthors = Brewer AK, Striegel AM | title = Characterizing string-of-pearls colloidal silica by multidetector hydrodynamic chromatography and comparison to multidetector size-exclusion chromatography, off-line multiangle static light scattering, and transmission electron microscopy | journal = Analytical Chemistry | volume = 83 | issue = 8 | pages = 3068–75 | date = April 2011 | pmid = 21428298 | doi = 10.1021/ac103314c }}</ref> The two main types of HDC are open tube and [[Packed bed|packed column]]. Open tube offers rapid separation times for small particles, whereas packed column HDC can increase resolution and is better suited for particles with an average molecular mass larger than <math>10^5</math> [[Dalton (unit)|daltons]].<ref name="Stegeman-1994">{{cite journal|last1=Stegeman|first1=Gerrit.|last2=van Asten|first2=Arian C.|last3=Kraak|first3=Johan C.|last4=Poppe|first4=Hans.|last5=Tijssen|first5=Robert |date=1994|title=Comparison of Resolving Power and Separation Time in Thermal Field-Flow Fractionation, Hydrodynamic Chromatography, and Size-Exclusion Chromatography|journal=Analytical Chemistry|language=en|volume=66|issue=7|pages=1147–1160|doi=10.1021/ac00079a033|issn=0003-2700|url=http://dare.uva.nl/personal/pure/en/publications/comparison-of-resolving-power-and-separation-time-in-thermal-fieldflow-fractionation-hydrodynamic-chromatography-and-sizeexclusion-chromatography(dbf9005b-3cd3-4824-810e-c15cccabf1ce).html}}</ref> HDC differs from other types of chromatography because the separation only takes place in the interstitial volume, which is the volume surrounding and in between particles in a packed column.<ref>{{cite journal|last=Small|first=Hamish|date=1974-07-01|title=Hydrodynamic chromatography a technique for size analysis of colloidal particles|journal=Journal of Colloid and Interface Science|language=en|volume=48|issue=1|pages=147–161|doi=10.1016/0021-9797(74)90337-3|bibcode=1974JCIS...48..147S|issn=0021-9797}}</ref>

HDC shares the same order of elution as [[Size-exclusion chromatography|Size Exclusion Chromatography]] (SEC) but the two processes still vary in many ways.<ref name="Stegeman-1994" /> In a study comparing the two types of separation, Isenberg, Brewer, Côté, and Striegel use both methods for [[polysaccharide]] characterization and conclude that HDC coupled with [[multiangle light scattering]] (MALS) achieves more accurate [[molar mass distribution]] when compared to off-line MALS than SEC in significantly less time.<ref name="Isenberg-2010">{{cite journal | vauthors = Isenberg SL, Brewer AK, Côté GL, Striegel AM | title = Hydrodynamic versus size exclusion chromatography characterization of alternan and comparison to off-line MALS | journal = Biomacromolecules | volume = 11 | issue = 9 | pages = 2505–11 | date = September 2010 | pmid = 20690593 | doi = 10.1021/bm100687b }}</ref> This is largely due to SEC being a more destructive technique because of the pores in the column degrading the analyte during separation, which tends to impact the mass distribution.<ref name="Isenberg-2010" /> However, the main disadvantage of HDC is low [[Resolution (chromatography)|resolution]] of analyte peaks, which makes SEC a more viable option when used with chemicals that are not easily degradable and where rapid elution is not important.<ref name="Striegel-2012">{{cite journal | vauthors = Striegel AM, Brewer AK | title = Hydrodynamic chromatography | journal = Annual Review of Analytical Chemistry | volume = 5 | issue = 1 | pages = 15–34 | date = 2012-07-19 | pmid = 22708902 | doi = 10.1146/annurev-anchem-062011-143107 | bibcode = 2012ARAC....5...15S }}</ref>

HDC plays an especially important role in the field of [[microfluidics]]. The first successful apparatus for HDC-on-a-chip system was proposed by Chmela, et al. in 2002.<ref name="Chmela-2002">{{cite journal | vauthors = Chmela E, Tijssen R, Blom MT, Gardeniers HJ, van den Berg A | title = A chip system for size separation of macromolecules and particles by hydrodynamic chromatography | journal = Analytical Chemistry | volume = 74 | issue = 14 | pages = 3470–5 | date = July 2002 | pmid = 12139056 | doi = 10.1021/ac0256078 | s2cid = 6948037 | url = https://ris.utwente.nl/ws/files/6705845/Chmela02chip.pdf }}</ref> Their design was able to achieve separations using an 80&nbsp;mm long channel on the timescale of 3 minutes for particles with diameters ranging from 26 to 110&nbsp;nm, but the authors expressed a need to improve the retention and [[Dispersion (chemistry)|dispersion]] parameters.<ref name="Chmela-2002" /> In a 2010 publication by Jellema, Markesteijn, Westerweel, and Verpoorte, implementing HDC with a recirculating bidirectional flow resulted in high resolution, size based separation with only a 3&nbsp;mm long channel.<ref>{{cite journal | vauthors = Jellema LJ, Markesteijn AP, Westerweel J, Verpoorte E | title = Tunable hydrodynamic chromatography of microparticles localized in short microchannels | journal = Analytical Chemistry | volume = 82 | issue = 10 | pages = 4027–35 | date = May 2010 | pmid = 20423105 | doi = 10.1021/ac902872d }}</ref> Having such a short channel and high resolution was viewed as especially impressive considering that previous studies used channels that were 80&nbsp;mm in length.<ref name="Chmela-2002" /> For a biological application, in 2007, Huh, et al. proposed a microfluidic sorting device based on HDC and gravity, which was useful for preventing potentially dangerous particles with diameter larger than 6 microns from entering the bloodstream when injecting [[contrast agent]]s in [[Medical ultrasound|ultrasounds]].<ref>{{cite journal | vauthors = Huh D, Bahng JH, Ling Y, Wei HH, Kripfgans OD, Fowlkes JB, Grotberg JB, Takayama S | display-authors = 6 | title = Gravity-driven microfluidic particle sorting device with hydrodynamic separation amplification | journal = Analytical Chemistry | volume = 79 | issue = 4 | pages = 1369–76 | date = February 2007 | pmid = 17297936 | pmc = 2527745 | doi = 10.1021/ac061542n }}</ref> This study also made advances for environmental sustainability in microfluidics due to the lack of outside electronics driving the flow, which came as an advantage of using a gravity based device.[[File:GCxGC-TOFMS Analytical Dept Chemical Faculty GUT Gdansk.jpg |thumb|upright| Two-dimensional chromatograph GCxGC-TOFMS at [[Faculty of Chemistry, Gdańsk University of Technology|Chemical Faculty]] of [[Gdańsk University of Technology|GUT]] [[Gdańsk]], [[Poland]], 2016]]

===Two-dimensional chromatography===
In some cases, the selectivity provided by the use of one column can be insufficient to provide resolution of analytes in complex samples. Two-dimensional chromatography aims to increase the resolution of these peaks by using a second column with different physico-chemical ([[chemical classification]]) properties.<ref name="Prebihalo-2018">{{cite journal | vauthors = Prebihalo SE, Berrier KL, Freye CE, Bahaghighat HD, Moore NR, Pinkerton DK, Synovec RE | title = Multidimensional Gas Chromatography: Advances in Instrumentation, Chemometrics, and Applications | journal = Analytical Chemistry | volume = 90 | issue = 1 | pages = 505–532 | date = January 2018 | pmid = 29088543 | doi = 10.1021/acs.analchem.7b04226 }}</ref><ref name="Stoll-2017">{{cite journal | vauthors = Stoll DR, Carr PW | title = Two-Dimensional Liquid Chromatography: A State of the Art Tutorial | journal = Analytical Chemistry | volume = 89 | issue = 1 | pages = 519–531 | date = January 2017 | pmid = 27935671 | doi = 10.1021/acs.analchem.6b03506 }}</ref> Since the mechanism of retention on this new solid support is different from the first dimensional separation, it can be possible to separate compounds by [[two-dimensional chromatography]] that are indistinguishable by one-dimensional chromatography. Furthermore, the separation on the second dimension occurs faster than the first dimension.<ref name="Prebihalo-2018" /> An example of a TDC separation is where the sample is spotted at one corner of a square plate, developed, air-dried, then rotated by 90° and usually redeveloped in a second solvent system.

Two-dimensional chromatography can be applied to GC or LC separations.<ref name="Prebihalo-2018" /><ref name="Stoll-2017" /> The heart-cutting approach selects a specific region of interest on the first dimension for separation,<ref>{{cite journal | vauthors = Tranchida PQ, Sciarrone D, Dugo P, Mondello L | title = Heart-cutting multidimensional gas chromatography: a review of recent evolution, applications, and future prospects | journal = Analytica Chimica Acta | volume = 716 | pages = 66–75 | date = February 2012 | pmid = 22284880 | doi = 10.1016/j.aca.2011.12.015 | url = http://www.sciencedirect.com/science/article/pii/S0003267011016801 | series = A selection of papers presented at the 12th International Symposium on Extraction Technologies (ExTech 2010) | bibcode = 2012AcAC..716...66T }}</ref> and the comprehensive approach uses all analytes in the second-dimension separation.<ref name="Prebihalo-2018" /><ref name="Stoll-2017" />

===Simulated moving-bed chromatography===
{{further|Simulated moving bed}}
The simulated moving bed (SMB) technique is a variant of high performance liquid chromatography; it is used to separate particles and/or chemical compounds that would be difficult or impossible to resolve otherwise. This increased separation is brought about by a valve-and-column arrangement that is used to lengthen the stationary phase indefinitely.
In the moving bed technique of preparative chromatography the feed entry and the analyte recovery are simultaneous and continuous, but because of practical difficulties with a continuously moving bed, simulated moving bed technique was proposed. In the simulated moving bed technique instead of moving the bed, the sample inlet and the analyte exit positions are moved continuously, giving the impression of a moving bed.
True moving bed chromatography (TMBC) is only a theoretical concept. Its simulation, SMBC is achieved by the use of a multiplicity of columns in series and a complex valve arrangement. This valve arrangement provides for sample and solvent feed and analyte and waste takeoff at appropriate locations of any column, whereby it allows switching at regular intervals the sample entry in one direction, the solvent entry in the opposite direction, whilst changing the analyte and waste takeoff positions appropriately as well.

===Pyrolysis gas chromatography===
[[Pyrolysis–gas chromatography–mass spectrometry]] is a method of chemical analysis in which the sample is heated to decomposition to produce smaller molecules that are separated by gas chromatography and detected using mass spectrometry.

Pyrolysis is the thermal decomposition of materials in an inert atmosphere or a vacuum. The sample is put into direct contact with a platinum wire, or placed in a quartz sample tube, and rapidly heated to 600–1000&nbsp;°C. Depending on the application even higher temperatures are used. Three different heating techniques are used in actual pyrolyzers: Isothermal furnace, inductive heating (Curie point filament), and resistive heating using platinum filaments. Large molecules cleave at their weakest points and produce smaller, more volatile fragments. These fragments can be separated by gas chromatography. Pyrolysis GC chromatograms are typically complex because a wide range of different decomposition products is formed. The data can either be used as fingerprints to prove material identity or the GC/MS data is used to identify individual fragments to obtain structural information. To increase the volatility of polar fragments, various methylating reagents can be added to a sample before pyrolysis.

Besides the usage of dedicated pyrolyzers, pyrolysis GC of solid and liquid samples can be performed directly inside Programmable Temperature Vaporizer (PTV) injectors that provide quick heating (up to 30&nbsp;°C/s) and high maximum temperatures of 600–650&nbsp;°C. This is sufficient for some pyrolysis applications. The main advantage is that no dedicated instrument has to be purchased and pyrolysis can be performed as part of routine GC analysis. In this case, quartz GC inlet liners have to be used. Quantitative data can be acquired, and good results of derivatization inside the PTV injector are published as well.

===Fast protein liquid chromatography===
{{further|Fast protein liquid chromatography}}
Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid (the "mobile phase") and a porous solid (the stationary phase). In FPLC the mobile phase is an aqueous solution, or "buffer". The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked [[agarose]], packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.

===Countercurrent chromatography===
{{further|Countercurrent chromatography}}Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids and the liquid stationary phase is held stagnant by a strong centrifugal force.<ref>{{cite journal |last1=Berthod |first1=Alain |last2=Maryutina |first2=Tatyana |last3=Spivakov |first3=Boris |last4=Shpigun |first4=Oleg |last5=Sutherland |first5=Ian A. |date=2009-01-01 |title=Countercurrent chromatography in analytical chemistry (IUPAC Technical Report) |journal=Pure and Applied Chemistry |language=en |volume=81 |issue=2 |pages=355–387 |doi=10.1351/PAC-REP-08-06-05 |issn=1365-3075|doi-access=free }}</ref>

==== Hydrodynamic countercurrent chromatography (CCC) ====
The operating principle of CCC instrument requires a column consisting of an open tube coiled around a bobbin. The bobbin is rotated in a double-axis gyratory motion (a cardioid), which causes a variable gravity (G) field to act on the column during each rotation. This motion causes the column to see one partitioning step per revolution and components of the sample separate in the column due to their partitioning coefficient between the two [[Miscibility|immiscible]] liquid phases used. There are many types of CCC available today. These include HSCCC (High Speed CCC) and HPCCC (High Performance CCC). HPCCC is the latest and best-performing version of the instrumentation available currently.

==== Centrifugal partition chromatography (CPC) ====
{{Further|Centrifugal partition chromatography}}
In the CPC (centrifugal partition chromatography or hydrostatic countercurrent chromatography) instrument, the column consists of a series of cells interconnected by ducts attached to a rotor. This rotor rotates on its central axis creating the centrifugal field necessary to hold the stationary phase in place. The separation process in CPC is governed solely by the partitioning of solutes between the stationary and mobile phases, which mechanism can be easily described using the partition coefficients (''K<sub>D</sub>'') of solutes. CPC instruments are commercially available for laboratory, pilot, and industrial-scale separations with different sizes of columns ranging from some 10 milliliters to 10 liters in volume.

===Periodic counter-current chromatography===
{{further|Periodic counter-current chromatography}}

In contrast to Counter current chromatography (see above), periodic counter-current chromatography (PCC) uses a solid stationary phase and only a liquid mobile phase. It thus is much more similar to conventional [[affinity chromatography]] than to counter current chromatography. PCC uses multiple columns, which during the loading phase are connected in line. This mode allows for overloading the first column in this series without losing product, which already breaks through the column before the resin is fully saturated. The breakthrough product is captured on the subsequent column(s). In a next step the columns are disconnected from one another. The first column is washed and eluted, while the other column(s) are still being loaded. Once the (initially) first column is re-equilibrated, it is re-introduced to the loading stream, but as last column. The process then continues in a cyclic fashion.

===Chiral chromatography===
Chiral chromatography involves the separation of [[Stereoisomerism|stereoisomers]]. In the case of enantiomers, these have no chemical or physical differences apart from being three-dimensional mirror images. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes. [[Chiral column chromatography|Chiral chromatography HPLC columns]] (with a chiral stationary phase) in both normal and reversed phase are commercially available.

Conventional chromatography are incapable of separating racemic mixtures of enantiomers. However, in some cases ''nonracemic'' mixtures of enantiomers may be separated unexpectedly by conventional liquid chromatography (e.g. HPLC without chiral mobile phase or stationary phase ).<ref>Jürgen Martens, [[Ravi Bhushan|Bhushan, R.]],  Mieczysław Sajewicz, Teresa Kowalska ''[[J. Chromatogr. Sci.]]'' '''2017''',  Vol. 55, 748–749. ({{doi|10.1093/chromsci/bmx031}})</ref><ref>Jürgen Martens, Ravi Bhushan,   ''[[Helv. Chim. Acta]]'' '''2014''',  Vol. 97, 161–187. ({{doi|10.1002/hlca.201300392}})</ref>

===Aqueous normal-phase chromatography===
{{further|Aqueous normal-phase chromatography}}

Aqueous normal-phase (ANP) chromatography is characterized by the elution behavior of classical normal phase mode (i.e. where the mobile phase is significantly less polar than the stationary phase) in which water is one of the mobile phase solvent system components. It is distinguished from hydrophilic interaction liquid chromatography (HILIC) in that the retention mechanism is due to adsorption rather than partitioning.<ref>{{cite journal | vauthors = Kulsing C, Nolvachai Y, Marriott PJ, Boysen RI, Matyska MT, Pesek JJ, Hearn MT | title = Insights into the origin of the separation selectivity with silica hydride adsorbents | journal = The Journal of Physical Chemistry B | volume = 119 | issue = 7 | pages = 3063–9 | date = February 2015 | pmid = 25656442 | doi = 10.1021/jp5103753 }}</ref>