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Why is reversed-phase chromatography used?

Reversed-phase high-performance liquid chromatography has become one of the most popular methods to separate molecules in the pharmaceutical industry. In recent years, reversed-phase chromatography has become the industry standard for precise and reliable proteomics analysis.


What is reversed-phase chromatography? 

Reversed-phase chromatography is a chemical analysis technique used to drive the separation process of a chemical mixture into its basic components. Also known as RPC or hydrophobic chromatography, this pharmaceutical analysis method is distinguished by the use of a hydrophobic stationary phase and a polar mobile phase.

In reversed-phase chromatography, the liquid mobile phase is typically composed of a combination of water and an organic solvent like methanol or acetonitrile. In turn, the stationary phase is non-polar or hydrocarbon-like. 

This method possesses a limited number of stationary phases. However, this is contrasted by the high potential number of liquids and combinations of them employed by the mobile phase. 

Types of reversed-phase chromatography include: 

  • Hydrophobic interaction chromatography (HIC): This chromatographic approach is based on the separation of molecules based on their hydrophobicity. This method possesses the advantage of operating with large molecules with a stationary phase that is mildly hydrophobic. Hydrophobicity can be determined through the silica compound in use.
  • Ion-exchange chromatography (IEC): Chromatographic separation is achieved using electrical charges. Ions with a charge are attracted to their polar opposites, while those with a similar polarity will be separated. A sample with a stronger charge will be attracted to the ionic surface, elongating the time it takes to elute.
  • Size-exclusion chromatography (SEC): This method separates components through a porous stationary phase chosen according to their size. For instance, a combination of polysaccharides and silica can be used for this purpose. 

A smaller particle will completely penetrate through the pores of the stationary phase. Larger molecules will only partially go through, though, as they will be partially stopped by the porous retention mechanisms. 

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How does reversed-phase chromatography work?

Reversed-phase chromatography works by using a hydrophobic stationary phase and a polar mobile phase. This can be achieved by creating a covalent bond between alkyl chains and stationary phase particles. The resulting hydrophobic interactions create a distinction between hydrophobic and hydrophilic molecules.

Hydrophobic molecules in a polar mobile phase tend to absorb the hydrophobic stationary phase, leaving hydrophilic molecules to be eluted first. If a molecule is more hydrophobic, the concentration of organic solvents necessary to elute it will be higher.

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What is the difference between normal and reversed-phase chromatography?

The main difference between both chromatographic performance techniques is based on their use of polar compounds. Normal-phase chromatography is characterized by a very polar stationary phase and a non-polar mobile phase, while reversed-phase chromatography implements a non-polar stationary phase alongside a polar mobile phase.

Normal-phase chromatography has been in use since the 1970s. Back then, liquid chromatography was achieved using silica or alumina resins to create solid support stationary phases. Reversed-phase chromatography is a more recent research method, created using the best high-performance liquid chromatography techniques.

Some of the most important differences between normal-phase chromatography and reversed-phase chromatography are: 

  • Stationary phase: The polar stationary phase used by normal-phase chromatography is mainly composed of pure silica. In contrast, reversed-phase chromatography uses a non-polar stationary phase made up of a modified silica substrate with long hydrophobic chains. Additionally, the stationary phase of normal-phase chromatography may contain a layer of water or protic organic solvent. These substances are absent during reversed-phase chromatography.
  • Mobile phase: A non-polar, non-aqueous solvent, such as chloroform, is used for the mobile phase during normal-phase chromatography. The polar mobile phase of a reversed-phase chromatographic performance is attained mainly through water, methanol, or acetonitrile.
  • Analytes in the mobile phase: Mobile phase conditions in normal-phase chromatography favor non-polar analytes at the beginning of separation. Conversely, polar analytes are favored in reversed-phase chromatography.
  • Types of chromatographic separation: The separation of polar analytes in normal-phase chromatography displays a high retention time in the column, whereas analytes with lower polarity are separated in reversed-phase chromatography.
  • Retention time: The retention time during normal-phase chromatography is increased thanks to a non-polar mobile phase. Reversed-phase chromatography uses a polar mobile phase for the same effect. The reproducibility of the retention time of reversed-phase chromatography is much higher than that of normal-phase chromatography.
  • Elution: To be eluted during the mobile phase of a normal-phase chromatography, analytes must have their polarity increased. By comparison, in reversed-phase chromatography, the analytes can be eluted by decreasing their polarity. 
  • Damage to the column: A reversed-phase column is much more difficult to damage than a normal phase chromatography column.


Similarities between normal and reversed-phase chromatography

  • Similar schematic instrumentation with similar column dimensions.
  • Temperature is an important factor in both methods.
  • Both methods operate under high pressure and separate a small volume of a sample.
  • The delivery of quantitative analysis of the sample components.
  • Both methods may take from a couple of minutes to up to an hour per sample.
  • By increasing the interactions of the analytes with the column, retention times can be increased.
  • Elution is induced by making the polarity of the mobile phase more similar to that of the stationary phase.


How is chromatography used in the pharmaceutical industry?

The most common use of reversed-phase liquid chromatography in the pharmaceutical industry is to run analytic procedures that certify the safety and quality of drugs. Chromatography can be used to perform analysis on biological samples and quality control on both bulk drugs and formulations.


Applications of reversed-phase chromatography 

Analysis of drug components

The separation of drugs and metabolites is fundamental for scientific analysis. Through the use of organic and polar solvents, high-performance liquid chromatography breaks down chemical mixtures so they can be properly studied.

Reversed-phase chromatography is a fast and precise tool to measure the components of chemical mixtures. Good limits of detection and compatibility with a vast range of formats are also major advantages. Moreover, the ability to automate most of the process makes it extremely time-efficient and drastically reduces the possibility of human error.

Isolation of active biomolecules

Active biomolecules possess a large diversity, which complicates their separation into their purest forms. The use of several types of chromatography in conjunction is often implemented to achieve the most accurate results, and reversed-phase chromatography tends to be a fundamental step in that process.

Process purification

The efficient separation of molecules achieved via reversed-phase chromatography can help quantify, identify, and purify samples from a wide variety of sources. Not only is this process useful in analyzing drugs and serums, but it can also effectively examine human, animal, and plant tissue.

Testing for impurities 

Extraction of contaminants

Unfortunately, emerging contaminants such as industrial additives, personal care products, surfactants, plasticizers, and all kinds of human and veterinary pharmaceuticals are periodically introduced in the environment. These substances can have a drastic effect on living organisms, including on common food sources like wheat, oat, barley, and rice. 

The selective extraction of analyte material from the complex organic structure of a substance like wheat can be very strenuous and particular wheat strains must be chosen based on the treatment the plant has received. The use of reversed-phase chromatography simplifies this procedure, helping scientists study contaminants to a higher degree.

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What are the benefits of reversed-phase chromatography?

Reversed-phase chromatography has several advantages over other normal-phase chromatography methods, such as its simplicity and versatility. Moreover, reversed-phase chromatography can be used to handle chemical compounds with a multiplicity in polarity and molecular mass. 


Valentia Analytical Services 

Valentia Analytical provides industry-leading drug development services to any kind of pharmaceutical company that needs to push a new drug into the market. If you’re looking forward to researching, improving, or commercializing a pharmaceutical product, Valentia Analytical has the tools, skillset, and expertise you’re looking for. 

To support the progress of the various phases of drug discovery, the expert minds at Valentia Analytical are ready to perform a wide range of testing services. Furthermore, strong connections with other laboratories provide a holistic CMC package and integral access to data.

Valentia Analytical offers ultra-high/high-performance liquid chromatography services of the following varieties: 

  • Reversed-phase chromatography
  • Ion-exchange chromatography
  • Size-exclusion chromatography
  • Affinity chromatography
  • Hydrophilic interaction chromatography
  • UHPLC with ultraviolet detector
  • UHPLC with mass spectrometry

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