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Высокоэффективная жидкостная хроматография
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ВЭЖХ. Слева направо: насосное устройство, создающее градиент двух разных растворителей - колонка, усиленная сталью, и детектор для измерения оптической плотности.
АкронимВЭЖХ
КлассификацияХроматография
Аналитыорганические молекулы
биомолекулы
ионы
полимеры
Другие техники
СвязанныйХроматография
Водная нормально-фазовая хроматография
Гидрофильное взаимодействие Хроматография
Ионообменная хроматография
Эксклюзионная хроматография
Мицеллярная жидкостная хроматография
С переносомЖидкостная хроматография-масс-спектрометрия
Современная автономная ВЭЖХ.
Схематическое изображение установки ВЭЖХ. (1) Резервуары для растворителя, (2) Дегазатор для растворителя, (3) Градиентный клапан, (4) Смесительный сосуд для подачи подвижной фазы, (5) Насос высокого давления, (6) Переключающий клапан в «положении впрыска», ( 6 ') Переключающий клапан в «положении нагрузки», (7) Контур ввода пробы, (8) Предколонка (защитная колонка), (9) Аналитическая колонка, (10) Детектор ( т.е. ИК, УФ), (11) Сбор данных, (12) Сборщик отходов или фракций.

Высокоэффективная жидкостная хроматография ( ВЭЖХ ), ранее называвшаяся жидкостной хроматографией высокого давления , представляет собой метод аналитической химии, используемый для разделения, идентификации и количественного определения каждого компонента в смеси. Он основан на насосах для пропускания жидкого растворителя под давлением, содержащего смесь образцов, через колонку, заполненную твердым адсорбирующим материалом . Каждый компонент в образце немного по-разному взаимодействует с адсорбирующим материалом, вызывая разную скорость потока для разных компонентов и приводя к разделению компонентов при их выходе из колонки.

ВЭЖХ использовалась для производства ( например , в процессе производства фармацевтических и биологических продуктов), легального ( например , для обнаружения лекарств, повышающих эффективность в моче), исследований ( например , разделения компонентов сложного биологического образца или аналогичных синтетических химикатов. друг от друга), так и в медицинских ( например , определение уровня витамина D в сыворотке крови) в целях. [1]

Хроматографию можно описать как процесс массопереноса, включающий адсорбцию . В ВЭЖХ используются насосы для пропускания жидкости под давлением и смеси пробы через колонку, заполненную адсорбентом, что приводит к разделению компонентов пробы. Активный компонент колонки, адсорбент, обычно представляет собой гранулированный материал, состоящий из твердых частиц ( например , кремнезема , полимеров и т. Д.), Размером 2–50 мкм. Компоненты смеси образцов отделены друг от друга из-за разной степени взаимодействия с частицами адсорбента. Жидкость под давлением обычно представляет собой смесь растворителей ( например,, вода, ацетонитрил и / или метанол) и называется «подвижной фазой». Его состав и температура играют важную роль в процессе разделения, влияя на взаимодействия, происходящие между компонентами образца и адсорбентом. Эти взаимодействия имеют физическую природу, такие как гидрофобные (дисперсионные), диполь-дипольные и ионные, чаще всего их сочетание.

ВЭЖХ отличается от традиционной жидкостной хроматографии («низкого давления») тем, что рабочее давление значительно выше (50–350 бар), в то время как обычная жидкостная хроматография обычно полагается на силу тяжести для прохождения подвижной фазы через колонку. Из-за небольшого количества образца, отделяемого при аналитической ВЭЖХ, типичные размеры колонки составляют 2,1–4,6 мм в диаметре и 30–250 мм в длину. Также колонки для ВЭЖХ изготавливаются с более мелкими частицами адсорбента (средний размер частиц 2–50 мкм). Это дает ВЭЖХ превосходную разрешающую способность (способность различать соединения) при разделении смесей, что делает его популярным хроматографическим методом.

Схема прибора для ВЭЖХ обычно включает дегазатор, пробоотборник, насосы и детектор. Пробоотборник переносит смесь пробы в поток подвижной фазы, который переносит ее в колонку. Насосы подают через колонку требуемый поток и состав подвижной фазы. Детектор генерирует сигнал, пропорциональный количеству компонента пробы, выходящего из колонки, что позволяет проводить количественный анализ компонентов пробы. Цифровой микропроцессор и программное обеспечение пользователя управляют прибором ВЭЖХ и обеспечивают анализ данных. Некоторые модели механических насосов в приборе для ВЭЖХ могут смешивать несколько растворителей вместе в соотношениях, изменяющихся во времени, создавая градиент состава.в мобильной фазе. Широко используются различные детекторы, такие как УФ / видимые детекторы , фотодиодные матрицы (КПК) или основанные на масс-спектрометрии . Большинство приборов для ВЭЖХ также имеют термостат колонок, который позволяет регулировать температуру, при которой выполняется разделение.

Операция [ править ]

Смесь проб, подлежащая разделению и анализу, вводится в дискретном небольшом объеме (обычно в микролитрах) в поток подвижной фазы, просачивающейся через колонку. Компоненты образца движутся через колонку с разными скоростями, которые зависят от конкретных физических взаимодействий с адсорбентом (также называемого стационарной фазой). Скорость каждого компонента зависит от его химической природы, от природы неподвижной фазы (колонки) и от состава подвижной фазы. Время, в которое конкретный аналит элюируется (выходит из колонки), называется временем его удерживания. Время удерживания, измеренное при определенных условиях, является отличительной характеристикой данного аналита.

Доступно множество различных типов колонок, заполненных адсорбентами, различающимися по размеру частиц, пористости и химическому составу поверхности. Использование набивочных материалов меньшего размера требует использования более высокого рабочего давления («противодавление») и обычно улучшает хроматографическое разрешение (степень разделения пиков между последовательными аналитами, выходящими из колонки). Частицы сорбента могут быть гидрофобными или полярными по своей природе.

Обычно используемые подвижные фазы включают любую смешивающуюся комбинацию воды с различными органическими растворителями (наиболее распространенными являются ацетонитрил и метанол ). В некоторых методах ВЭЖХ используются безводные подвижные фазы (см. Нормально-фазовую хроматографию ниже). Водный компонент подвижной фазы может содержать кислоты (такие как муравьиная, фосфорная или трифторуксусная кислота.) или соли, чтобы помочь в разделении компонентов пробы. Состав подвижной фазы может поддерживаться постоянным («режим изократического элюирования») или изменяться («режим градиентного элюирования») во время хроматографического анализа. Изократическое элюирование обычно эффективно при разделении компонентов пробы, которые сильно различаются по своему сродству к неподвижной фазе. При градиентном элюировании состав подвижной фазы обычно варьируется от низкой до высокой элюирующей силы. Сила элюирования подвижной фазы отражается временем удерживания аналита с высокой силой элюирования, обеспечивающей быстрое элюирование (= короткое время удерживания). Типичный профиль градиента в обращенно-фазовой хроматографии может начинаться с 5% ацетонитрила (в воде или водном буфере) и линейно прогрессировать до 95% ацетонитрила в течение 5–25 минут.Периоды постоянного состава подвижной фазы могут быть частью любого профиля градиента. Например, состав подвижной фазы может поддерживаться постоянным при 5% ацетонитриле в течение 1–3 мин с последующим линейным изменением до 95% ацетонитрила.

Вращающийся коллектор фракций, собирающий выходные данные ВЭЖХ. Система используется для выделения фракции, содержащей комплекс I, из плазматических мембран E. coli . Для выделения этого количества потребовалось около 50 литров бактерий. [2]

Выбранный состав подвижной фазы зависит от интенсивности взаимодействий между различными компонентами образца («аналиты») и неподвижной фазой ( например , гидрофобные взаимодействия в обращенно-фазовой ВЭЖХ). В зависимости от их сродства к неподвижной и подвижной фазам аналиты распределяются между ними во время процесса разделения, происходящего в колонке. Этот процесс разделения аналогичен тому, который происходит при экстракции жидкость-жидкость, но является непрерывным, а не ступенчатым. В этом примере, используя градиент вода / ацетонитрил, больше гидрофобных компонентов будет элюироваться (выходить из колонки) поздно, когда подвижная фаза станет более концентрированной в ацетонитриле ( то есть в подвижной фазе с более высокой элюирующей силой).

Выбор компонентов подвижной фазы, добавок (таких как соли или кислоты) и условий градиента зависит от природы компонентов колонки и пробы. Часто с пробой проводят серию пробных прогонов, чтобы найти метод ВЭЖХ, который дает адекватное разделение.

История и развитие [ править ]

До ВЭЖХ ученые использовали стандартные методы жидкостной хроматографии. Системы жидкостной хроматографии были в значительной степени неэффективными из-за зависимости расхода растворителей от силы тяжести. Разделение занимало много часов, а иногда и дней. Газовая хроматография (ГХ) в то время была более мощной, чем жидкостная хроматография (ЖХ), однако считалось, что газофазное разделение и анализ очень полярных высокомолекулярных биополимеров были невозможны. [3] ГХ был неэффективен для многих биохимиков из-за термической нестабильности растворенных веществ. [4] В результате были выдвинуты гипотезы об альтернативных методах, которые вскоре приведут к развитию ВЭЖХ.

Следуя основополагающей работе Мартина и Синджа в 1941 году, в 1960-х годах Кэлом Гиддингсом, Йозефом Хубером и другими было предсказано, что жидкостная хроматография может работать в высокоэффективном режиме за счет уменьшения диаметра упаковочных частиц, существенно меньшего, чем типичный жидкостной хроматограф. (и GC) на уровне 150 мкм и с использованием давления для увеличения скорости подвижной фазы. [3] Эти прогнозы подверглись обширным экспериментам и уточнениям на протяжении 60–70-х годов. На ранних этапах разработки были начаты исследования по улучшению ЖК-частиц, и изобретение Zipax, поверхностно пористой частицы, было многообещающим для технологии ВЭЖХ. [5]

1970-е годы принесли много разработок в области аппаратного и измерительного оборудования. Исследователи начали использовать насосы и инжекторы для создания элементарной конструкции системы ВЭЖХ. [6] Насосы-усилители газа были идеальными, потому что они работали при постоянном давлении и не требовали герметичных уплотнений или обратных клапанов для стабильного потока и хорошего количественного определения. [4] В Dupont IPD (подразделение промышленных полимеров) были достигнуты вехи в области аппаратного обеспечения, такие как использование градиентного устройства с низким внутренним объемом, а также замена инжектора перегородки на клапан впрыска петли. [4]

Хотя инструментальные разработки были важны, история ВЭЖХ в первую очередь связана с историей и развитием технологии частиц . [4] После введения частиц пористого слоя наблюдается устойчивая тенденция к уменьшению размера частиц для повышения эффективности. [4] Однако из-за уменьшения размера частиц возникли новые проблемы. Практические недостатки связаны с чрезмерным перепадом давления, необходимым для проталкивания подвижной жидкости через колонну, и трудностью изготовления однородной насадки из чрезвычайно мелких материалов. [7] Каждый раз, когда размер частиц значительно уменьшается, обычно необходимо проводить еще один этап разработки прибора, чтобы справиться с давлением. [4]

Типы [ править ]

Partition chromatography[edit]

HILIC Partition Technique Useful Range

Partition chromatography was one of the first kinds of chromatography that chemists developed.[8] The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and 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.[9] 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 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 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, basic and neutral solutes in a single chromatographic run.[10]

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.

Normal–phase chromatography[edit]

Normal–phase chromatography was one of the first kinds of HPLC that chemists developed. 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 factors. The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers.

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 widely used for structural isomer separations in both column and thin-layer chromatography formats on activated (dried) silica or alumina supports.

Partition- and NP-HPLC fell out of favor in the 1970s with the development of 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.

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[edit]

The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.[11]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.

Reversed-phase chromatography (RPC)[edit]

A chromatogram of complex mixture (perfume water) obtained by reversed phase HPLC
Further information: Reversed-phase chromatography

Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been surface-modified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RP-HPLC to qualify drugs before their release.

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 C18-chain versus the complex of both. The energy released in this process is proportional to the surface tension of the eluent (water: 7.3×10−6 J/cm², methanol: 2.2×10−6 J/cm²) 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 play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C–H, C–C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure. On the other hand, analytes with higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO− or -NH3+ in their structure) are less retained as they are better integrated into water. Such interactions are subject to steric effects in that very large molecules 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 hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C–C bonds elute later than those with a C=C or C–C triple bond, as the double or triple bond is shorter than a single C–C bond.

Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5×10−7 J/cm² per Mol for NaCl, 2.5×10−7 J/cm² per Mol for (NH4)2SO4), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).

Another important factor is the mobile phase pH since it can change the hydrophobic character of the 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, neutralize the charge on the silica surface of the stationary phase and 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 eluant. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is a fairly strong organic acid. The effects of acids and buffers vary by application but generally improve chromatographic resolution.

Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. 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. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good 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 ions are present on the surface of the silica.[citation needed]..

Size-exclusion chromatography[edit]

Further information: size-exclusion chromatography

Size-exclusion chromatography (SEC), also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of molecular size (actually by a particle's Stokes radius). It is generally 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 by trapping these smaller molecules in the pores of a particle. 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 heparins.

Ion-exchange chromatography[edit]

Further information: Ion-exchange chromatography

In ion-exchange chromatography (IC), retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Solute ions of the same charge as the charged sites on the column are excluded from binding, while solute ions of the opposite charge of the charged sites of the column are retained on the column. Solute ions that are retained on the column can be eluted from the column by changing the solvent conditions (e.g., increasing the ion effect of the solvent system by increasing the salt concentration of the solution, increasing the column temperature, changing the pH of the solvent, etc.).

Types of ion exchangers include polystyrene resins, cellulose and dextran ion exchangers (gels), and controlled-pore glass or porous silica. 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. 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[edit]

Further information: Affinity chromatography

This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. 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[edit]

Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reversed-phase solvents.[citation needed]

Isocratic and gradient elution[edit]

At the ARS Natural Products Utilization Research Unit in Oxford, MS., a support scientist (r) extracts plant pigments that will be analyzed by a plant physiologist (l) using an HPLC system.

A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (meaning constant composition). (The example of these the percentage of methanol throughout the procedure will remain constant i.e 10%) The word was coined by Csaba Horvath who was one of the pioneers of HPLC.[citation needed],

The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution.[12] One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from the column. In reversed-phase chromatography, solvent A is often water or an aqueous buffer, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol.

In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks.

A schematic of gradient elution. Increasing mobile phase strength sequentially elutes analytes having varying interaction strength with the stationary phase.

Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times – all according to the desire for optimum separation in minimum time.

In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change – that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.[citation needed]

The driving force in reversed phase chromatography originates in the high order of the water structure. The role of the organic component of the mobile phase is to reduce this high order and thus reduce the retarding strength of the aqueous component.

Parameters[edit]

Theoretical[edit]

HPLC separations have theoretical parameters and equations to describe the separation of components into signal peaks when detected by instrumentation such as by a UV detector or a mass spectrometer. The parameters are largely derived from two sets of chromatagraphic 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., kappa1 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[edit]

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 that influences the detection sensitivity and separation selectivity in gradient elution. It also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low-ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.

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

Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-Vis absorbance detector.

Narrow-bore columns (1–2 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 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[edit]

Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5 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.[13][14][15]

According to the equations[16] 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.[17] Larger particles are used in preparative HPLC (column diameters 5 cm up to >30 cm) and for non-HPLC applications such as solid-phase extraction.

Pore size[edit]

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[edit]

Pumps 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 MPa (6000 lbf/in2), or about 600 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 μm). These "ultra high performance liquid chromatography" systems or UHPLCs, which could also be known as ultra high pressure chromatography systems[18], can work at up to 120 MPa (17,405 lbf/in2), or about 1200 atmospheres.[19] The term "UPLC"[20] is a trademark of the Waters Corporation, but is sometimes used to refer to the more general technique of UHPLC.

Detectors[edit]

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., UV-Vis absorbance) by simply responding to the physical or chemical property of the solute.[21] HPLC most commonly uses a UV-Vis absorbance detector, however, a wide range of other chromatography detectors 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 LCMS normally combines UV-Vis with a mass spectrometer.

Autosamplers[edit]

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.[22]

Applications[edit]

Manufacturing[edit]

HPLC has many applications in both laboratory and clinical science. It is a common technique used in pharmaceutical development, as it is a dependable way to obtain and ensure product purity.[23] While HPLC can produce extremely high quality (pure) products, it is not always the primary method used in the production of bulk drug materials.[24] According to the European pharmacopoeia, HPLC is used in only 15.5% of syntheses.[25] However, it plays a role in 44% of syntheses in the United States pharmacopoeia.[26] This could possibly be due to differences in monetary and time constraints, as HPLC on a large scale can be an expensive technique. An increase in specificity, precision, and accuracy that occurs with HPLC unfortunately corresponds to an increase in cost.

Legal[edit]

This technique is also used for detection of illicit drugs in urine. The most common method of drug detection is an immunoassay.[27] This method is much more convenient. However, convenience comes at the cost of specificity and coverage of a wide range of drugs. As HPLC is a method of determining (and possibly increasing) purity, using HPLC alone in evaluating concentrations of drugs is somewhat insufficient. With this, HPLC in this context is often performed in conjunction with mass spectrometry.[28] Using liquid chromatography instead of gas chromatography in conjunction with MS circumvents the necessity for derivitizing with acetylating or alkylation agents, which can be a burdensome extra step.[29] This technique has been used to detect a variety of agents like doping agents, drug metabolites, glucuronide conjugates, amphetamines, opioids, cocaine, BZDs, ketamine, LSD, cannabis, and pesticides.[30][31] Performing HPLC in conjunction with Mass spectrometry reduces the absolute need for standardizing HPLC experimental runs.

Research[edit]

Similar assays can be performed for research purposes, detecting concentrations of potential clinical candidates like anti-fungal and asthma drugs.[32] This technique is obviously useful in observing multiple species in collected samples, as well, but requires the use of standard solutions when information about species identity is sought out. It is used as a method to confirm results of synthesis reactions, as purity is essential in this type of research. However, mass spectrometry is still the more reliable way to identify species.

Medical[edit]

Medical use of HPLC can include drug analysis, but falls more closely under the category of nutrient analysis. While urine is the most common medium for analyzing drug concentrations, blood serum is the sample collected for most medical analyses with HPLC.[33] Other methods of detection of molecules that are useful for clinical studies have been tested against HPLC, namely immunoassays. In one example of this, competitive protein binding assays (CPBA) and HPLC were compared for sensitivity in detection of vitamin D. Useful for diagnosing vitamin D deficiencies in children, it was found that sensitivity and specificity of this CPBA reached only 40% and 60%, respectively, of the capacity of HPLC.[34] While an expensive tool, the accuracy of HPLC is nearly unparalleled.

See also[edit]

  • History of chromatography
  • Capillary electrochromatography
  • Column chromatography
  • Csaba Horváth
  • Ion chromatography
  • Micellar liquid chromatography

References[edit]

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  31. ^ Pelander, Anna; Ojanperä, Ilkka; Laks, Suvi; Rasanen, Ilpo; Vuori, Erkki (2003-11-01). "Toxicological screening with formula-based metabolite identification by liquid chromatography/time-of-flight mass spectrometry". Analytical Chemistry. 75 (21): 5710–5718. doi:10.1021/ac030162o. PMID 14588010.
  32. ^ Nobilis, Milan; Pour, Milan; Senel, Petr; Pavlík, Jan; Kunes, Jirí; Voprsalová, Marie; Kolárová, Lenka; Holcapek, Michal (2007-06-15). "Metabolic profiling of a potential antifungal drug, 3-(4-bromophenyl)-5-acetoxymethyl-2,5-dihydrofuran-2-one, in mouse urine using high-performance liquid chromatography with UV photodiode-array and mass spectrometric detection". Journal of Chromatography B. 853 (1–2): 10–19. doi:10.1016/j.jchromb.2007.02.045. PMID 17400036.
  33. ^ Sundström, Mira; Pelander, Anna; Angerer, Verena; Hutter, Melanie; Kneisel, Stefan; Ojanperä, Ilkka (2013-10-01). "A high-sensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine". Analytical and Bioanalytical Chemistry. 405 (26): 8463–8474. doi:10.1007/s00216-013-7272-8. PMID 23954996. S2CID 25743579.
  34. ^ Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam (2015-01-01). "Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children". International Journal of Preventive Medicine. 6: 67. doi:10.4103/2008-7802.161069. PMC 4542329. PMID 26330983.

Further reading[edit]

  • L. R. Snyder, J.J. Kirkland, and J. W. Dolan, Introduction to Modern Liquid Chromatography, John Wiley & Sons, New York, 2009.
  • M.W. Dong, Modern HPLC for practicing scientists. Wiley, 2006.
  • L. R. Snyder, J.J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, John Wiley & Sons, New York, 1997.
  • S. Ahuja and H. T. Rasmussen (ed), HPLC Method Development for Pharmaceuticals, Academic Press, 2007.
  • S. Ahuja and M.W. Dong (ed), Handbook of Pharmaceutical Analysis by HPLC, Elsevier/Academic Press, 2005.
  • Y. V. Kazakevich and R. LoBrutto (ed.), HPLC for Pharmaceutical Scientists, Wiley, 2007.
  • U. D. Neue, HPLC Columns: Theory, Technology, and Practice, Wiley-VCH, New York, 1997.
  • M. C. McMaster, HPLC, a practical user's guide, Wiley, 2007.

External links[edit]

  • HPLC Chromatography Principle, Application [Basic Note] – 2020. at Rxlalit.com
  • Liquid Chromatography at Curlie