Editing High-performance liquid chromatography (section)

==Parameters==
===Theoretical===
The theory of high performance liquid chromatography-HPLC is, at its core,  the same as general chromatography theory.<ref>{{Cite journal |last1=Martin |first1=A. J. P. |last2=Synge |first2=R. L. M. |date=1941-12-01 |title=A new form of chromatogram employing two liquid phases |url=https://portlandpress.com/biochemj/article/35/12/1358/34734/A-new-form-of-chromatogram-employing-two-liquid |journal=Biochemical Journal |language=en |volume=35 |issue=12 |pages=1358–1368 |doi=10.1042/bj0351358 |issn=0306-3283 |pmc=1265645 |pmid=16747422}}</ref> This theory has been used as the basis for ''system-suitability'' tests, as can be seen in the USP Pharmacopeia,<ref>{{Citation |title=〈621〉 CHROMATOGRAPHY |date=2022-12-01 |url=https://www.usp.org/sites/default/files/usp/document/harmonization/gen-chapter/harmonization-november-2021-m99380.pdf |access-date=2025-01-21 |publisher=U.S. Pharmacopeial Convention |doi=10.31003/uspnf_m99380_06_01}}</ref> which are a set of quantitative criteria, which test the suitability of the HPLC system to the required analysis at any step of it.

This relation is also represented as a normalized unit-less factor known as the ''retention factor'', or retention parameter, which is the experimental measurement of the capacity ratio, as shown in the Figure of Performance Criteria as well. t<sub>R</sub> is the retention time of the specific component and t<sub>0</sub> is the time it takes for a non-retained substance to elute through the system without any retention, thus it is called the Void Time.

The ratio between the retention factors, k', of every two adjacent peaks in the chromatogram is used in the evaluation of the degree of separation between them, and is called '''selectivity factor''', α, as shown in the Performance Criteria graph.

The plate count N as a criterion for system efficiency was developed for isocratic conditions, i.e., a constant mobile phase composition throughout the run. In gradient conditions, where the mobile phase changes with time during the chromatographic run, it is more appropriate to use the parameter '''''peak capacity'' ''P''<sub>c</sub>''' as a measure for the system efficiency.<ref>{{Cite journal |last=Wren |first=Stephen A. C. |date=2005-06-15 |title=Peak capacity in gradient ultra performance liquid chromatography (UPLC) |url=https://www.sciencedirect.com/science/article/pii/S0731708505000142 |journal=Journal of Pharmaceutical and Biomedical Analysis |volume=38 |issue=2 |pages=337–343 |doi=10.1016/j.jpba.2004.12.028 |pmid=15925228 |issn=0731-7085|url-access=subscription }}</ref> The definition of ''peak capacity'' in chromatography is the number of peaks that can be separated within a retention window for a specific pre-defined resolution factor, usually ~1. It could also be envisioned as the runtime measured in number of peaks' average widths. The equation is shown in the Figure of the performance criteria. In this equation tg is the gradient time and w(ave) is the average peaks width at the base.

[[File:SST Equations single peak.jpg|alt=Basic Chromatography Equations|thumb|658x658px|The quantitative parameters and equations which determine the extent of performance of the chromatographic system]]
The parameters are largely derived from two sets of chromatographic theory: plate theory (as part of [[partition chromatography]]), and the rate theory of chromatography / ''[[Van Deemter equation]]''. Of course, they can be put in practice through analysis of HPLC chromatograms, although rate theory is considered the more accurate theory.

They are analogous to the calculation of [[retention factor]] for a [[paper chromatography]] separation, but describes how well HPLC separates a mixture into two or more components that are detected as peaks (bands) on a chromatogram. The HPLC parameters are the: efficiency factor(''N''), the retention factor (kappa prime), and the separation factor (alpha). Together the factors are variables in a resolution equation, which describes how well two components' peaks separated or overlapped each other. These parameters are mostly only used for describing HPLC reversed phase and HPLC normal phase separations, since those separations tend to be more subtle than other HPLC modes (''e.g.'', ion exchange and size exclusion).

Void volume is the amount of space in a column that is occupied by solvent. It is the space within the column that is outside of the column's internal packing material. Void volume is measured on a chromatogram as the first component peak detected, which is usually the solvent that was present in the sample mixture; ideally the sample solvent flows through the column without interacting with the column, but is still detectable as distinct from the HPLC solvent. The void volume is used as a correction factor.

Efficiency factor (''N'') practically measures how sharp component peaks on the chromatogram are, as ratio of the component peak's area ("retention time") relative to the width of the peaks at their widest point (at the baseline). Peaks that are tall, sharp, and relatively narrow indicate that separation method efficiently removed a component from a mixture; high efficiency. Efficiency is very dependent upon the HPLC column and the HPLC method used. Efficiency factor is synonymous with plate number, and the 'number of theoretical plates'.

[[Retention factor]] (''kappa prime'') measures how long a component of the mixture stuck to the column, measured by the area under the curve of its peak in a chromatogram (since HPLC chromatograms are a function of time). Each chromatogram peak will have its own retention factor (''e.g.'', ''kappa''<small>1</small> for the retention factor of the first peak). This factor may be corrected for by the void volume of the column.

Separation factor (''alpha'') is a relative comparison on how well two neighboring components of the mixture were separated (''i.e.'', two neighboring bands on a chromatogram). This factor is defined in terms of a ratio of the retention factors of a pair of neighboring chromatogram peaks, and may also be corrected for by the void volume of the column. The greater the separation factor value is over 1.0, the better the separation, until about 2.0 beyond which an HPLC method is probably not needed for separation.
Resolution equations relate the three factors such that high efficiency and separation factors improve the resolution of component peaks in an HPLC separation.

===Internal diameter===
[[File:Nano-LC - (1).jpg|thumb|Tubing on a nano-liquid chromatography (nano-LC) system, used for very low flow capacities]]
The internal diameter (ID) of an HPLC column is an important parameter.<ref>{{Cite journal |last1=Zelenyánszki |first1=Dóra |last2=Felinger |first2=Attila |date=2020-10-01 |title=The Impact of Column Hardware on Efficiency in Liquid Chromatography (LC) |url=https://www.chromatographyonline.com/view/the-impact-of-column-hardware-on-efficiency-in-liquid-chromatography-lc- |journal=LCGC Europe |series=LCGC Europe-10-01-2020 |language=en |volume=33 |issue=10 |pages=498–504}}</ref> It can influence the detection response when reduced due to the reduced lateral diffusion of the solute band. It can also affect the separation selectivity, when flow rate and injection volumes are not scaled down or up proportionally to the smaller or larger diameter used, both in the isocratic and in gradient modes.<ref>{{Cite journal |last=Dolan |first=John |date=2014 |title=LC Method Scaling, Part II: Gradient Separations |url=https://www.chromatographyonline.com/view/lc-method-scaling-part-ii-gradient-separations-0 |journal=LCGC North America |series=LCGC North America-03-01-2014 |language=en |volume=32 |issue=3 |pages=188–193}}</ref> It determines the quantity of analyte that can be loaded onto the column. Larger diameter columns are usually seen in preparative applications, such as the purification of a drug product for later use.<ref>{{Cite journal |last1=Jensen |first1=Ole Elvang |last2=Kidal |first2=Steffen |date=2006-03-01 |title=Using Volumetric Flow to Scaleup Chromatographic Processes |url=https://www.biopharminternational.com/view/using-volumetric-flow-scaleup-chromatographic-processes |journal=BioPharm International |series=BioPharm International-03-01-2006 |language=en |volume=19 |issue=3}}</ref> Low-ID columns have improved sensitivity and lower solvent consumption in the recent ultra-high performance liquid chromatography (UHPLC).<ref name=":2">{{Cite journal |last1=Walter |first1=Thomas H. |last2=Andrews |first2=Richard W. |date=2014 |title=Recent innovations in UHPLC columns and instrumentation |journal=Trends in Analytical Chemistry |volume=63 |pages=14–20 |doi=10.1016/j.trac.2014.07.016 |issn=0165-9936|doi-access=free }}</ref>

Larger ID columns (over 10&nbsp;mm) are used to purify usable amounts of material because of their large loading capacity.

Analytical scale columns (4.6&nbsp;mm) have been the most common type of columns, though narrower columns<ref name=":2" /> are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a [[spectrophotometer|UV-Vis absorbance detector]].

Narrow-bore columns (1–2&nbsp;mm) are used for applications when more sensitivity is desired either with special UV-vis detectors, [[fluorescence]] detection or with other detection methods like [[liquid chromatography-mass spectrometry]]

Capillary columns (under 0.3&nbsp;mm) are used almost exclusively with alternative detection means such as [[mass spectrometry]]. They are usually made from [[fused silica]] capillaries, rather than the stainless steel tubing that larger columns employ.

===Particle size===
Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical [[silicon dioxide|silica]] particles (very small beads). These particles come in a variety of sizes with 5&nbsp;μm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.<ref>Majors, Ronald E.. (2010-09-07) [http://www.lcgceurope.com/lcgceurope/article/articleDetail.jsp?id=333246&pageID=3# Fast and Ultrafast HPLC on sub-2 μm Porous Particles — Where Do We Go From Here? – LC-GC Europe]. Lcgceurope.com. Retrieved 2011-06-07.</ref><ref>{{cite journal|last = Xiang|first = Y. |author2=Liu Y. |author3=Lee M.L.|year=2006|title = Ultrahigh pressure liquid chromatography using elevated temperature| journal = Journal of Chromatography A|pmid = 16376355|volume = 1104|issue = 1–2|pages = 198–202|doi = 10.1016/j.chroma.2005.11.118}}</ref><ref>{{cite journal|last = Horváth|first = Cs. |author2=Preiss B.A. |author3=Lipsky S.R.|year=1967|title = Fast liquid chromatography. Investigation of operating parameters and the separation of nucleotides on pellicular ion exchangers| journal = Analytical Chemistry|pmid = 6073805|volume = 39|issue = 12|pages = 1422–1428|doi = 10.1021/ac60256a003}}</ref>

According to the equations<ref>{{Cite journal|last1=Nguyen|first1=Dao T.-T.|last2=Guillarme|first2=Davy|last3=Rudaz|first3=Serge|last4=Veuthey|first4=Jean-Luc|title=Fast analysis in liquid chromatography using small particle size and high pressure|journal=Journal of Separation Science|year=2006|language=en|volume=29|issue=12|pages=1836–1848|doi=10.1002/jssc.200600189|pmid=16970187|issn=1615-9306|doi-access=free}}</ref> of the column velocity, efficiency and [[backpressure]], reducing the particle diameter by half and keeping the size of the column the same, will double the column velocity and efficiency; but four times increase the backpressure. And the small particles HPLC also can decrease the width broadening.<ref>{{Cite journal|last1=Gritti|first1=Fabrice|last2=Guiochon|first2=Georges|title=The van Deemter equation: Assumptions, limits, and adjustment to modern high performance liquid chromatography|url=https://linkinghub.elsevier.com/retrieve/pii/S0021967313009278|journal=Journal of Chromatography A|year=2013|language=en|volume=1302|pages=1–13|doi=10.1016/j.chroma.2013.06.032|pmid=23838304|url-access=subscription}}</ref> Larger particles are used in preparative HPLC (column diameters 5&nbsp;cm up to >30&nbsp;cm) and for non-HPLC applications such as [[solid-phase extraction]].

===Pore size===
Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.

===Pump pressure===
[[Pump]]s vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible [[volumetric flow rate]]. [[Pressure]] may reach as high as 60&nbsp;MPa (6000&nbsp;[[Pound per square inch|lbf/in<sup>2</sup>]]), or about 600&nbsp;atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2&nbsp;μm). These "ultra high performance liquid chromatography" systems or UHPLCs, which could also be known as ultra high pressure chromatography systems,<ref>{{cite journal | doi=10.1016/j.chroma.2005.11.118 | title=Ultrahigh pressure liquid chromatography using elevated temperature | date=2006 | last1=Xiang | first1=Yanqiao | last2=Liu | first2=Yansheng | last3=Lee | first3=Milton L. | journal=Journal of Chromatography A | volume=1104 | issue=1–2 | pages=198–202 | pmid=16376355 }}</ref> can work at up to 120&nbsp;MPa (17,405&nbsp;lbf/in<sup>2</sup>), or about 1200&nbsp;atmospheres.<ref>[http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1290-infinity-quaternary-pump#specifications  1290 Infinity Quaternary Pump] {{Webarchive|url=https://web.archive.org/web/20151120172747/http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1290-infinity-quaternary-pump#specifications |date=2015-11-20 }}. Agilent</ref> The term "UPLC"<ref>{{cite web|url=http://www.waters.com/waters/en_US/Trademarks/nav.htm?cid=1000238&locale=en_US|title=Trademarks : Waters|last=waters|website=www.waters.com}}</ref> is a trademark of the [[Waters Corporation]], but is sometimes used to refer to the more general technique of UHPLC.

===Detectors===
HPLC detectors fall into two main categories: universal or selective. Universal detectors typically measure a bulk property (''e.g.'', [[refractive index]]) by measuring a difference of a [[physical property]] between the mobile phase and mobile phase with solute while selective detectors measure a solute property (''e.g.'', [[Ultraviolet–visible spectroscopy|UV-Vis absorbance]]) by simply responding to the physical or [[chemical property]] of the solute.<ref>{{Cite book|title=Principles and practice of modern chromatographic methods|last=K.|first=Robards|date=1994|publisher=Elsevier/Academic Press|others=Haddad, P. R., Jackson, P. E.|isbn=9780080571782|location=Amsterdam|oclc=815471219}}</ref> HPLC most commonly uses a [[spectrophotometer|UV-Vis absorbance detector]]; however, a wide range of other [[chromatography detector]]s can be used. A universal detector that complements UV-Vis absorbance detection is the [[charged aerosol detector]] (CAD). A kind of commonly utilized detector includes refractive index detectors, which provide readings by measuring the changes in the refractive index of the eluant as it moves through the flow cell. In certain cases, it is possible to use multiple detectors, for example [[Liquid chromatography–mass spectrometry|LCMS]] normally combines UV-Vis with a mass spectrometer.

When used with an electrochemical detector (ECD) the HPLC-ECD selectively detects [[neurotransmitters]] such as: [[norepinephrine]], [[dopamine]], [[serotonin]], [[glutamate]], GABA, [[acetylcholine]] and others in neurochemical analysis research applications.<ref>{{Cite web|url=https://www.amuzainc.com/eicom-electrochemical-detection-ecd-fundamentals/|title=Electrochemical Detection (ECD) Fundamentals|website=Amuza Inc}}</ref> The HPLC-ECD detects neurotransmitters to the [[femtomolar]] range. Other methods to detect neurotransmitters include liquid chromatography-mass spectrometry, ELISA, or radioimmunoassays.

===Autosamplers===
Large numbers of samples can be automatically injected onto an HPLC system, by the use of HPLC autosamplers. In addition, HPLC autosamplers have an injection volume and technique which is exactly the same for each injection, consequently they provide a high degree of injection volume precision.
It is possible to enable sample stirring within the sampling-chamber, thus promoting homogeneity.<ref>{{cite journal | title=Automated device for continuous stirring while sampling in liquid chromatography systems | journal=Communications Chemistry | volume=3 | pages=180 | year=2020 | doi=10.1038/s42004-020-00427-5| doi-access=free | last1=Markovitch | first1=Omer | last2=Ottelé | first2=Jim | last3=Veldman | first3=Obe | last4=Otto | first4=Sijbren | issue=1 | pmid=36703458 | pmc=9814086 | bibcode=2020CmChe...3..180M }}</ref>