Reversed-phase chromatography

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Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. [1] [2] [3] The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are. [4]

Contents

The factors affecting the retention and separation of solutes in the reversed phase chromatographic system are as follows:

a. The chemical nature of the stationary phase, i.e., the ligands bonded on its surface, as well as their bonding density, namely the extent of their coverage.

b. The composition of the mobile phase. Type of the bulk solvents whose mixtures affect the polarity of the mobile phase, hence the name modifier for a solvent added to affect the polarity of the mobile phase.

C. Additives, such as buffers, affect the pH of the mobile phase, which affect the ionization state of the solutes and their polarity.

In order to retain the organic components in mixtures, the stationary phases, packed within columns, consist of a hydrophobic substrates, bonded to the surface of porous silica-gel particles in various geometries (spheric, irregular), at different diameters (sub-2, 3, 5, 7, 10 um), with varying pore diameters (60, 100, 150, 300, A).   The particle's surface is covered by chemically bonded hydrocarbons, such as C3, C4, C8, C18 and more. The longer the hydrocarbon associated with the stationary phase, the longer the sample components will be retained. Some stationary phases are also made of hydrophobic polymeric particles, or hybridized silica-organic groups particles, for method in which mobile phases at extreme pH are used. Most current methods of separation of biomedical materials use C-18 columns, sometimes called by trade names, such as ODS (octadecylsilane) or RP-18. 

The mobile phases are mixtures of water and polar organic solvents, the vast majority of which are methanol and acetonitrile.  These mixtures usually contain various additives such as buffers (acetate, phosphate, citrate), surfactants (alkyl amines or alkyl sulfonates) and special additives (EDTA). The goal of using supplements of one kind or another is to increase efficiency, selectivity, and control solute retention. 

Stationary phases

Idealized cartoon of silica gel before and after treatment with octadecyltrichlorosilane ( .mw-parser-output .template-chem2-su{display:inline-block;font-size:80%;line-height:1;vertical-align:-0.35em}.mw-parser-output .template-chem2-su>span{display:block;text-align:left}.mw-parser-output sub.template-chem2-sub{font-size:80%;vertical-align:-0.35em}.mw-parser-output sup.template-chem2-sup{font-size:80%;vertical-align:0.65em} C18H37SiCl3. Most silanol groups (red) are converted to hydrophobic alkylsiloxy groups. ODS-silica.svg
Idealized cartoon of silica gel before and after treatment with octadecyltrichlorosilane (C18H37SiCl3. Most silanol groups (red) are converted to hydrophobic alkylsiloxy groups.

The history and evolution of reversed phase stationary phases in described in detail in an article by Majors, Dolan, Carr and Snyder. [6]

In the 1970s, most liquid chromatography runs were performed using solid particles as the stationary phases, made of unmodified silica gel or alumina. This type of technique is now referred to as normal-phase chromatography. Since the stationary phase is hydrophilic in this technique, and the mobile phase is non-polar (consisting of organic solvents such as hexane and heptane), biomolecules with hydrophilic properties in the sample adsorb to the stationary phase strongly. Moreover, they were not dissolved easily in the mobile phase solvents. At the same time hydrophobic molecules experience less affinity to the polar stationary phase, and elute through it early with not enough retention. This was the reasons why during the 1970s the silica based particles were treated with hydrocarbons, immobilized or bonded on their surface, and the mobile phases were switched to aqueous and polar in nature, to accommodate biomedical substances.

The use of a hydrophobic stationary phase and polar mobile phases is essentially the reverse of normal phase chromatography, since the polarity of the mobile and stationary phases have been inverted – hence the term reversed-phase chromatography. [7] [8] As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydrophilic molecules in the sample pass through the column and are eluted first. [7] [9] Hydrophobic molecules can be eluted from the column by decreasing the polarity of the mobile phase using an organic (non-polar) solvent, which reduces hydrophobic interactions. The more hydrophobic the molecule, the more strongly it will bind to the stationary phase, and the higher the concentration of organic solvent that will be required to elute the molecule.

Many of the mathematical parameters of the theory of chromatography and experimental considerations used in other chromatographic methods apply to RP-LC as well (for example, the selectivity factor, chromatographic resolution, plate count, etc. It can be used for the separation of a wide variety of molecules. It is typically used for separation of proteins, [10] because the organic solvents used in normal-phase chromatography can denature many proteins.

Today, RP-LC is a frequently used analytical technique. There are huge variety of stationary phases available for use in RP-LC, allowing great flexibility in the development of the separation methods. [11] [12]

Silica-based stationary phases

Silica gel particles are commonly used as a stationary phase in high-performance liquid chromatography (HPLC) for several reasons, [13] [14] including:

  1. High surface area: Silica gel particles have a high surface area, allowing direct interactions with solutes or after bonding of variety of ligands for versatile interactions with the sample molecules, leading to better separations.
  2. Chemical and thermal stability and inertness: [15] Silica gel is chemically stable, as it usually does not react with either the solvents of the mobile phase nor the compounds being separated, resulting in accurate, repeatable and reliable analyses.
  3. Wide applicability: [16] Silica gel is versatile and can be modified with various functional groups, making it suitable for a wide range of analytes and applications.
  4. Efficient separation: The unique properties of silica gel particles, combined with their high surface area and controlled average particle diameter pore size, [17] facilitate efficient and precise separation of compounds in HPLC.
  5. Reproducibility: Silica gel particles can offer high batch-to-batch reproducibility, which is crucial for consistent and reliable HPLC analyses throughout decades.
  6. Particle diameter and pore size control: [18] [19] Silica gel can be engineered to have specific pore sizes, enabling precise control over separation based on molecular size.
  7. Cost-effectiveness: Silica is the most abundant element on earth, hence its gel is a cost-effective choice for HPLC applications, making it widely adopted in laboratories.

The United States Pharmacopoeia (USP) has classified HPLC columns by L# types. [20] The most popular column in this classification is an octadecyl carbon chain (C18)-bonded silica (USP classification L1). [21] This is followed by C8-bonded silica (L7), pure silica (L3), cyano-bonded silica (CN) (L10) and phenyl-bonded silica (L11). Note that C18, C8 and phenyl are dedicated reversed-phase stationary phases, while CN columns can be used in a reversed-phase mode depending on analyte and mobile phase conditions. Not all C18 columns have identical retention properties. Surface functionalization of silica can be performed in a monomeric or a polymeric reaction with different short-chain organosilanes used in a second step to cover remaining silanol groups (end-capping). While the overall retention mechanism remains the same, subtle differences in the surface chemistries of different stationary phases will lead to changes in selectivity.

Modern columns have different polarity depending on the ligand bonded to the stationary phase. PFP is pentafluorphenyl. CN is cyano. NH2 is amino. ODS is octadecyl or C18. ODCN is a mixed mode column consisting of C18 and nitrile. [22]

Recent developments in chromatographic supports and instrumentation for liquid chromatography (LC) facilitate rapid and highly efficient separations, using various stationary phases geometries. [23] Various analytical strategies have been proposed, such as the use of silica-based monolithic supports, elevated mobile phase temperatures, and columns packed with sub-3 μm superficially porous particles (fused or solid core) [24] or with sub-2 μm fully porous particles for use in ultra-high-pressure LC systems (UHPLC). [25]

Mobile phases

A comprehensive article on the modern trends and best practices of mobile phase selection in reversed-phase chromatography was published by Boyes and Dong. [26] A mobile phase in reversed-phase chromatograpy consists of mixtures of water or aqueous buffers, to which organic solvents are added, to elute analytes from a reversed-phase column in a selective manner. [7] [27] The added organic solvents must be miscible with water, and the two most common organic solvents used are acetonitrile and methanol. Other solvents can also be used such as ethanol or 2-propanol (isopropyl alcohol) and tetrahydrofuran (THF). The organic solvent is called also a modifier, since it is added to the aqueous solution in the mobile phase in order to modify the polarity of the mobile phase. Water is the most polar solvent in the reversed phase mobile phase; therefore, lowering the polarity of the mobile phase by adding modifiers enhances its elution strength. The two most widely used organic modifiers are acetonitrile and methanol, although acetonitrile is the more popular choice. Isopropanol (2-propanol) can also be used, because of its strong eluting properties, but its use is limited by its high viscosity, which results in higher backpressures. Both acetonitrile and methanol are less viscous than isopropanol, although a mixture of 50:50 percent of methanol:water is also very viscous and causes high backpressures.

All three solvents are essentially UV transparent. This is a crucial property for common reversed phase chromatography since sample components are typically detected by UV detectors. Acetonitrile is more transparent than the others in low UV wavelengths range, therefore it is used almost exclusively when separating molecules with weak or no chromophores (UV-VIS absorbing groups), such as peptides. Most peptides only absorb at low wavelengths in the ultra-violet spectrum (typically less than 225 nm) and acetonitrile provides much lower background absorbance at low wavelengths than the other common solvents.

The pH of the mobile phase can have an important role on the retention of an analyte and can change the selectivity of certain analytes. [28] [29] For samples containing solutes with ionized functional groups, such as amines, carboxyls, phosphates, phosphonates, sulfates, and sulfonates, the ionization of these groups can be controlled using mobile phase buffers. [30]

For example, carboxylic groups in solutes become increasingly negatively charged as the pH of the mobile phase rises above their pKa, hence the whole molecule becomes more polar and less retained on the a-polar stationary phase. In this case, raising the pH of the phase mobile above 4–5 = pH (which is the typical pKa range for carboxylic groups) increases their ionization, hence decreases their retention. Conversely, using a mobile phase at a pH lower than 4 [31] will increase their retention, because it will decrease their ionization degree, rendering them less polar.

The same considerations apply to substances containing basic functional groups, such as amines, whose pKa ranges are around 8 and above, are retained more, as the pH of the mobile phase increases, approaching 8 and above, because they are less ionized, hence less polar. However, in the case of high pH mobile phases, most of the traditional silica gel based Reversed Phase columns are generally limited for use with mobile phases at pH 8 and above, therefore, control over the retention of amines in this range is limited. [32]

The choice of buffer type is an important factor in RP-LC method development, as it can affect the retention, selectivity, and resolution of the analytes of interest. [26] When selecting a buffer for RP-HPLC, there are a number of factors to consider, including:

Some of the most common buffers used in RP-HPLC include: [34]

Charged analytes can be separated on a reversed-phase column by the use of ion-pairing (also called ion-interaction). This technique is known as reversed-phase ion-pairing chromatography. [35]

Elution can be performed isocratically (the water-solvent composition does not change during the separation process) or by using a solution gradient (the water-solvent composition changes during the separation process, usually by decreasing the polarity).

See also

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

<span class="mw-page-title-main">High-performance liquid chromatography</span> Technique in analytical chemistry

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc, which have been dissolved into liquid solutions.

Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC), that separates high molecular weight or colloidal analytes on the basis of size or diameter, typically in organic solvents. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964. The proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.

<span class="mw-page-title-main">Paper chromatography</span> Separation of coloured chemicals on paper

Paper chromatography is an analytical method used to separate coloured chemicals or substances. It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).

<span class="mw-page-title-main">Column chromatography</span> Method to isolate a compound in a mixture

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.

<span class="mw-page-title-main">Ion chromatography</span> Separates ions and polar molecules

Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.

<span class="mw-page-title-main">Thin-layer chromatography</span> Technique used to separate non-volatile mixtures

Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures.

<span class="mw-page-title-main">Liquid chromatography–mass spectrometry</span> Analytical chemistry technique

Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography – MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify each separated component. MS is not only sensitive, but provides selective detection, relieving the need for complete chromatographic separation. LC–MS is also appropriate for metabolomics because of its good coverage of a wide range of chemicals. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC–MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. Since the early 2000s, LC–MS has also begun to be used in clinical applications.

<span class="mw-page-title-main">Solid-phase extraction</span> Process to separate compounds by properties

Solid-phase extraction (SPE) is a solid-liquid extractive technique, by which compounds that are dissolved or suspended in a liquid mixture are separated, isolated or purified, from other compounds in this mixture, according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue.

Chiral column chromatography is a variant of column chromatography that is employed for the separation of chiral compounds, i.e. enantiomers, in mixtures such as racemates or related compounds. The chiral stationary phase (CSP) is made of a support, usually silica based, on which a chiral reagent or a macromolecule with numerous chiral centers is bonded or immobilized.

<span class="mw-page-title-main">Hydrophilic interaction chromatography</span> Type of chromatography

Hydrophilic interaction chromatography is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC); however, SFC typically utilizes carbon dioxide as the mobile phase. Therefore, the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state whereby bulk liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. The idea of liquid and gas properties convergence was first envisioned by Giddings.

Micellar liquid chromatography (MLC) is a form of reversed phase liquid chromatography that uses an aqueous micellar solutions as the mobile phase.

Aqueous normal-phase chromatography (ANP) is a chromatographic technique that involves the mobile phase compositions and polarities between reversed-phase chromatography (RP) and normal-phase chromatography (NP), while the stationary phases are polar.

<span class="mw-page-title-main">Elution</span> Extraction of a material by washing with a solvent

In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent: washing of loaded ion-exchange resins to remove captured ions, or eluting proteins or other biopolymers from a gel electrophoresis or chromatography column.

A monolithic HPLC column, or monolithic column, is a column used in high-performance liquid chromatography (HPLC). The internal structure of the monolithic column is created in such a way that many channels form inside the column. The material inside the column which separates the channels can be porous and functionalized. In contrast, most HPLC configurations use particulate packed columns; in these configurations, tiny beads of an inert substance, typically a modified silica, are used inside the column. Monolithic columns can be broken down into two categories, silica-based and polymer-based monoliths. Silica-based monoliths are known for their efficiency in separating smaller molecules while, polymer-based are known for separating large protein molecules.

<span class="mw-page-title-main">Countercurrent chromatography</span>

Countercurrent chromatography is a form of liquid–liquid chromatography that uses a liquid stationary phase that is held in place by inertia of the molecules composing the stationary phase accelerating toward the center of a centrifuge due to centripetal force and is used to separate, identify, and quantify the chemical components of a mixture. In its broadest sense, countercurrent chromatography encompasses a collection of related liquid chromatography techniques that employ two immiscible liquid phases without a solid support. The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube or a series of chambers connected with channels, which contains both phases. The resulting dynamic mixing and settling action allows the components to be separated by their respective solubilities in the two phases. A wide variety of two-phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation.

<span class="mw-page-title-main">Capillary electrochromatography</span> Method of separating components of a mixture via electro-osmosis

In chemical analysis, capillary electrochromatography (CEC) is a chromatographic technique in which the mobile phase is driven through the chromatographic bed by electro-osmosis. Capillary electrochromatography is a combination of two analytical techniques, high-performance liquid chromatography and capillary electrophoresis. Capillary electrophoresis aims to separate analytes on the basis of their mass-to-charge ratio by passing a high voltage across ends of a capillary tube, which is filled with the analyte. High-performance liquid chromatography separates analytes by passing them, under high pressure, through a column filled with stationary phase. The interactions between the analytes and the stationary phase and mobile phase lead to the separation of the analytes. In capillary electrochromatography capillaries, packed with HPLC stationary phase, are subjected to a high voltage. Separation is achieved by electrophoretic migration of solutes and differential partitioning.

Thermoresponsive polymers can be used as stationary phase in liquid chromatography. Here, the polarity of the stationary phase can be varied by temperature changes, altering the power of separation without changing the column or solvent composition. Thermally related benefits of gas chromatography can now be applied to classes of compounds that are restricted to liquid chromatography due to their thermolability. In place of solvent gradient elution, thermoresponsive polymers allow the use of temperature gradients under purely aqueous isocratic conditions. The versatility of the system is controlled not only through changing temperature, but through the addition of modifying moieties that allow for a choice of enhanced hydrophobic interaction, or by introducing the prospect of electrostatic interaction. These developments have already introduced major improvements to the fields of hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, and affinity chromatography separations as well as pseudo-solid phase extractions.

Chiral analysis refers to the quantification of component enantiomers of racemic drug substances or pharmaceutical compounds. Other synonyms commonly used include enantiomer analysis, enantiomeric analysis, and enantioselective analysis. Chiral analysis includes all analytical procedures focused on the characterization of the properties of chiral drugs. Chiral analysis is usually performed with chiral separation methods where the enantiomers are separated on an analytical scale and simultaneously assayed for each enantiomer.

References

  1. ↑ IUPAC , Compendium of Chemical Terminology , 2nd ed. (the "Gold Book") (1997). Online corrected version: (2006–) " reversed-phase chromatography ". doi : 10.1351/goldbook.R05376
  2. ↑ Žuvela, Petar; Skoczylas, Magdalena; Jay Liu, J.; Ba̧Czek, Tomasz; Kaliszan, Roman; Wong, Ming Wah; Buszewski, Bogusław; Héberger, K. (2019). "Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography". Chemical Reviews. 119 (6): 3674–3729. doi:10.1021/acs.chemrev.8b00246. PMID   30604951. S2CID   58631771.
  3. ↑ Dorsey, John G.; Dill, Ken A. (1989). "The molecular mechanism of retention in reversed-phase liquid chromatography". Chemical Reviews. 89 (2): 331–346. doi:10.1021/cr00092a005.
  4. ↑ Ganesh, V.; Poorna Basuri, P.; Sahini, K.; Nalini, C.N. (2023). "Retention behaviour of analytes in reversed‐phase high‐performance liquid chromatography—A review". Biomedical Chromatography. 37 (7): e5482. doi:10.1002/bmc.5482. ISSN   0269-3879. PMID   35962484. S2CID   251540223.
  5. ↑ Pape, Peter G. (2017). "Silylating Agents". Kirk-Othmer Encyclopedia of Chemical Technology. pp. 1–15. doi:10.1002/0471238961.1909122516011605.a01.pub3. ISBN   9780471238966.
  6. ↑ Majors, Ronald; Dolan, John; Carr, Peter; Snyder, Lloyd (2010). "New Horizons in Reversed-Phase Chromatography". LCGC North America. LCGC North America-06-01-2010. 28 (6): 418–430.
  7. 1 2 3 Akul Mehta (December 27, 2012). "Principle of Reversed-Phase Chromatography HPLC/UPLC (with Animation)". PharmaXChange. Retrieved 10 January 2013.
  8. ↑ I Molnár and C Horváth (September 1976). "Reverse-phase chromatography of polar biological substances: separation of catechol compounds by high-performance liquid chromatography". Clinical Chemistry. 22 (9): 1497–1502. doi: 10.1093/clinchem/22.9.1497 . PMID   8221 . Retrieved 10 January 2013.
  9. ↑ (Clinical Biochemistry, T.W.Hrubey, 54)
  10. ↑ Evans, David R.H.; Romero, Jonathan K.; Westoby, Matthew (2009), "Chapter 9 Concentration of Proteins and Removal of Solutes", Guide to Protein Purification, 2nd Edition, Methods in Enzymology, vol. 463, Elsevier, pp. 97–120, doi:10.1016/s0076-6879(09)63009-3, ISBN   978-0-12-374536-1, PMID   19892169 , retrieved 2022-10-13
  11. ↑ Žuvela, Petar; Skoczylas, Magdalena; Jay Liu, J.; Ba̧czek, Tomasz; Kaliszan, Roman; Wong, Ming Wah; Buszewski, Bogusław (2019). "Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography". Chemical Reviews. 119 (6): 3674–3729. doi:10.1021/acs.chemrev.8b00246. ISSN   0009-2665. PMID   30604951. S2CID   58631771.
  12. ↑ Rusli, Handajaya; Putri, Rindia M.; Alni, Anita (2022). "Recent Developments of Liquid Chromatography Stationary Phases for Compound Separation: From Proteins to Small Organic Compounds". Molecules. 27 (3): 907. doi: 10.3390/molecules27030907 . ISSN   1420-3049. PMC   8840574 . PMID   35164170.
  13. ↑ "Advantages of Silica Gels for HPLC Packing Applications - AGC Chemicals". 2020-11-10. Retrieved 2023-10-17.
  14. ↑ Qiu, Hongdeng; Liang, Xiaojing; Sun, Min; Jiang, Shengxiang (2011). "Development of silica-based stationary phases for high-performance liquid chromatography". Analytical and Bioanalytical Chemistry. 399 (10): 3307–3322. doi:10.1007/s00216-010-4611-x. ISSN   1618-2650. PMID   21221544. S2CID   40721088.
  15. ↑ Claessens, H. A.; van Straten, M. A. (2004). "Review on the chemical and thermal stability of stationary phases for reversed-phase liquid chromatography". Journal of Chromatography A. The Stationary Phase and Chromatographic Retention: Honoring J.J. Kirkland. 1060 (1): 23–41. doi:10.1016/j.chroma.2004.08.098. ISSN   0021-9673.
  16. ↑ Žuvela, Petar; Skoczylas, Magdalena; Jay Liu, J.; Ba̧czek, Tomasz; Kaliszan, Roman; Wong, Ming Wah; Buszewski, Bogusław (2019). "Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography". Chemical Reviews. 119 (6): 3674–3729. doi:10.1021/acs.chemrev.8b00246. ISSN   0009-2665. PMID   30604951. S2CID   58631771.
  17. ↑ Cheong, Won Jo (2014). "Porous Silica Particles As Chromatographic Separation Media: A Review". Bulletin of the Korean Chemical Society. 35 (12): 3465–3474. doi: 10.5012/bkcs.2014.35.12.3465 . ISSN   0253-2964.
  18. ↑ "HPLC Column - Pore Sizes and Particle Diameters". www.crawfordscientific.com. Retrieved 2023-10-17.
  19. ↑ "Pore Size vs. Particle Size in HPLC Columns". Chrom Tech, Inc. Retrieved 2023-10-17.
  20. ↑ "USP L Column Listing - HPLC Columns & Bulk Media". SMT. Retrieved 2023-10-17.
  21. ↑ USP Chromatographic Reagents 2007-2008: Used in USP-NF and Pharmacopeial Forum. United States Pharmacopeia. 2007.
  22. ↑ Žuvela, Petar; Skoczylas, Magdalena; Jay Liu, J.; Ba̧czek, Tomasz; Kaliszan, Roman; Wong, Ming Wah; Buszewski, Bogusław (2019-03-27). "Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography". Chemical Reviews. 119 (6): 3674–3729. doi:10.1021/acs.chemrev.8b00246. ISSN   0009-2665. PMID   30604951. S2CID   58631771.
  23. ↑ Guillarme, Davy; Ruta, Josephine; Rudaz, Serge; Veuthey, Jean-Luc (2010). "New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches". Analytical and Bioanalytical Chemistry. 397 (3): 1069–1082. doi:10.1007/s00216-009-3305-8. ISSN   1618-2642. PMID   19998028. S2CID   21218108.
  24. ↑ Fekete, Szabolcs; Olh, Erzsbet; Fekete, Jen (2012). "Fast liquid chromatography: The domination of core?shell and very fine particles". Journal of Chromatography A. 1228: 57–71. doi:10.1016/j.chroma.2011.09.050. PMID   21982449.
  25. ↑ Fekete, Szabolcs; Ganzler, Katalin; Fekete, Jenő (2011). "Efficiency of the new sub-2μm core–shell (Kinetex™) column in practice, applied for small and large molecule separation". Journal of Pharmaceutical and Biomedical Analysis. 54 (3): 482–490. doi:10.1016/j.jpba.2010.09.021. PMID   20940092.
  26. 1 2 Boyes, Barry; Dong, Michael (2018). "Modern Trends and Best Practices in Mobile-Phase Selection in Reversed-Phase Chromatography". LCGC North America. LCGC North America-10-01-2018. 36 (10): 752–768.
  27. ↑ Boyes, Barry; Dong, Michael (2018). "Modern Trends and Best Practices in Mobile-Phase Selection in Reversed-Phase Chromatography". LCGC North America. LCGC North America-10-01-2018. 36 (10): 752–768.
  28. ↑ Heyrman, Aimee N.; Henry, Richard A. (1999). "Importance of Controlling Mobile Phase pH in Reversed Phase HPLC". Keystone Technical Bulletin.
  29. ↑ Schoenmakers, Peter J.; van Molle, Sylvie; Hayes, Carmel M. G.; Uunk, Louis G. M. (1991). "Effects of pH in reversed-phase liquid chromatography". Analytica Chimica Acta. 250: 1–19. doi:10.1016/0003-2670(91)85058-Z. ISSN   0003-2670.
  30. ↑ Dolan, John (2017-01-01). "Back to Basics: The Role of pH in Retention and Selectivity". LCGC North America. LCGC North America-01-01-2017. 35 (1): 22–28.
  31. ↑ "How does an acid pH affect reversed-phase chromatography separations?". www.biotage.com. Retrieved 2023-10-18.
  32. ↑ Kirkland, J. J.; van Straten, M. A.; Claessens, H. A. (1995). "High pH mobile phase effects on silica-based reversed-phase high-performance liquid chromatographic columns". Journal of Chromatography A. 18th International Symposium On Column Liquid Chromatography Part I. 691 (1): 3–19. doi:10.1016/0021-9673(94)00631-I. ISSN   0021-9673. S2CID   53646741.
  33. ↑ Dolan, John. "A Guide to HPLC and LC-MS Buffer Selection" (PDF).
  34. ↑ V. Agrahari; M. Bajpai; S. Nanda (2013). "Essential Concepts of Mobile Phase Selection for Reversed Phase HPLC". Research J. Pharm. And Tech. 6 (5): 459–464.
  35. ↑ Gilar, Martin; Fountain, Kenneth J.; Budman, Yeva; Neue, Uwe D.; Yardley, Kurt R.; Rainville, Paul D.; Russell II, Reb J.; Gebler, John C. (2002). "Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction". Journal of Chromatography A. 958 (1): 167–182. doi:10.1016/S0021-9673(02)00306-0. ISSN   0021-9673. PMID   12134814.
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