Editing High-performance liquid chromatography (section)

== Operation ==
The liquid chromatograph is complex<ref>{{Cite journal |last=Dong |first=Michael |date=2018 |title=Ten Common-Sense Corollaries in Pharmaceutical Analysis by High Performance Liquid Chromatography |url=https://www.chromatographyonline.com/view/ten-common-sense-corollaries-pharmaceutical-analysis-high-performance-liquid-chromatography |journal=LCGC Europe |series=LCGC Europe-08-01-2018 |language=en |volume=31 |issue=8 |pages=432–436}}</ref> and has sophisticated and delicate technology. In order to properly operate the system, there should be a minimum basis for understanding of how the device performs the data processing to avoid incorrect data and distorted results.<ref>{{Cite book |last1=Snyder |first1=Lloyd R. |title=Practical HPLC Method Development |last2=Kirkland |first2=Joseph J. |last3=Glajch |first3=Joseph L. |publisher=John Wiley & Sons |year=2012 |edition=2nd}}</ref><ref>{{Cite book |last=McMaster |first=Marvin C. |title=HPLC: a practical user's guide |date=2007 |publisher=Wiley-Interscience |isbn=978-0-471-75401-5 |edition=2nd |location=Hoboken, NJ}}</ref><ref>{{Cite book |last1=Hanai |first1=Toshihiko |title=HPLC: a practical guide |last2=Hanai |first2=T. |date=1999 |publisher=Royal Society of Chemistry |others=Royal Society of Chemistry |isbn=978-0-85404-515-0 |series=RSC chromatography monographs |location=Cambridge}}</ref>

HPLC is distinguished from traditional ("low pressure") [[Chromatography#Liquid chromatography|liquid chromatography]] because operational pressures are significantly higher (around 50–1400 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the packed column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6&nbsp;mm diameter, and 30–250&nbsp;mm length. Also HPLC columns are made with smaller adsorbent particles (1.5–50 μm in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.{{citation needed|date=May 2024}}

The schematic of an HPLC instrument typically includes solvents' reservoirs, one or more pumps, a solvent-[[degasser]], a sampler, a column, and a detector. The solvents are prepared in advance according to the needs of the separation, they pass through the degasser to remove dissolved gasses, mixed to become the mobile phase, then flow through the sampler, which brings the sample mixture into the mobile phase stream, which then carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the stationary phase inside the column, then directly into a flow-cell inside the detector. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for [[Quantity|quantitative]] analysis of the sample components. The detector also marks the time of emergence, the retention time, which serves for initial identification of the component. More advanced detectors, provide also additional information, specific to the analyte's characteristics, such as [[Ultraviolet–visible spectroscopy|UV-VIS]] spectrum or [[mass spectrum]], which can provide insight on its structural features. These detectors are in common use, such as UV/Vis, [[photodiode]] array (PDA) / [[Chromatography detector|diode array detector]] and [[mass spectrometry]] detector.{{citation needed|date=May 2024}}

A digital [[microprocessor]] and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in an HPLC instrument can mix multiple solvents together at a ratios changing in time, generating a composition [[gradient]] in the mobile phase. Most HPLC instruments also have a column oven that allows for adjusting the temperature at which the separation is performed.{{citation needed|date=May 2024}}

The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column, each at a different velocity, which are a function of specific physical interactions with the adsorbent, the stationary phase. The velocity of each component depends on its chemical nature, on the nature of the stationary phase (inside the column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time, measured under particular conditions, is an identifying characteristic of a given analyte.{{citation needed|date=July 2024}}

Many different types of columns are available, filled with adsorbents varying in particle size, [[porosity]], and surface chemistry. The use of smaller particle size packing materials requires the use of higher operational pressure ("backpressure") and typically improves chromatographic [[resolution (chromatography)|resolution]] (the degree of peak separation between consecutive analytes emerging from the column). Sorbent particles may be ionic, hydrophobic or polar in nature.{{citation needed|date=July 2024}}

The most common mode of liquid chromatography is [[Reversed-phase chromatography|reversed phase]], whereby the mobile phases used, include any miscible combination of water or buffers with various organic solvents (the most common are acetonitrile and methanol). Some HPLC techniques use water-free mobile phases (see [[#Normal–phase chromatography|normal-phase chromatography]] below). The aqueous component of the mobile phase may contain acids (such as formic, phosphoric or [[trifluoroacetic acid]]) or salts to assist in the separation of the sample components. The composition of the mobile phase may be kept constant ("isocratic elution mode") or varied ("gradient elution mode") during the chromatographic analysis. Isocratic elution is typically effective in the separation of simple mixtures. Gradient elution is required for complex mixtures, with varying interactions with the stationary and mobile phases. This is the reason why in gradient elution the composition of the mobile phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times, as the high eluting strength speeds up the elution (resulting in shortening of retention times). For example, a typical gradient profile in reversed phase chromatography for might start at 5% acetonitrile (in water or aqueous buffer) and progress linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition (plateau) may be also part of a gradient profile. For example, the mobile phase composition may be kept constant at 5% acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.{{citation needed|date=July 2024}}

The chosen composition of the mobile phase depends on the intensity of interactions between various sample components ("analytes") and stationary phase (''e.g.'', hydrophobic interactions in reversed-phase HPLC). Depending on their affinity for the stationary and mobile phases, analytes partition between the two during the separation process taking place in the column. This partitioning process is similar to that which occurs during a [[liquid–liquid extraction]] but is continuous, not step-wise.{{citation needed|date=July 2024}}

In the example using a water/acetonitrile gradient, the more hydrophobic components will [[Elution|elute]] (come off the column) later, then, once the mobile phase gets richer in acetonitrile (''i.e.'', in a mobile phase becomes higher eluting solution), their elution speeds up.{{citation needed|date=July 2024}}

The choice of mobile phase components, additives (such as salts or acids) and gradient conditions depends on the nature of the column and sample components. Often a series of trial runs is performed with the sample in order to find the HPLC method which gives adequate separation.{{citation needed|date=July 2024}}