learn about the principle of reversed-phase chromatography, and also learn stationary phase, mobile phase, and applications of reversed-phase liquid chromatography.
What is the reversed-phase HPLC?
Reversed-phase chromatography is a type of high-performance liquid chromatography, which is used to separate the solutes based on the hydrophobic interactions between the mobile phase and the stationary phase. RP-HPLC is the most widely used HPLC mode, and this mode is exactly the reverse of normal phase chromatography, in which the mobile phase is more polar than the stationary phase. In general, the stationary phase of reversed-phase chromatography is non-polar (silica-C18 column) and a moderately polar mobile aqueous phase.
| The principle of chromatography: · Chromatography is a separation technique in which molecules are contained within a mobile phase that travels through a stationary phase. · Normally, one phase is hydrophilic, and another is lipophilic. · The mixture of sample compounds interacts with these two phases differently. · As per the polarity of the molecules, they interact more or less time with the stationary phase and thus they eluted at different rates. |
The principle of reversed-phase HPLC:
- The principle of reversed-phase chromatography is based on the interaction of molecules with the hydrophobic group.
- Reversed-phase liquid chromatography is an adsorption process that depends on a partitioning mechanism to effect isolation. The stationary phase In RP-HPLC is solid support applied with hydrophobic as well as hydrophilic groups.
- This distribution is based on the medium's binding properties, hydrophobicity of the analytes, and composition of the mobile phase.
- The polarity of a molecule that is similar to the column packing, their retention time is more as they are attracted to the particles more strongly. The polarity molecule that is similar to the mobile phase will preferably be attracted to it and move rapidly from the HPLC column.
The stationary phase of reversed-phase chromatography (RP-HPLC):
The column is the heart of HPLC chromatography, and the mobile phase can be used to control separation retention and selectivity. The reversed phase HPLC column of is filled with silica particles. The particles are usually characterized by particle size and pore size. Particle sizes vary typically from 3 to 50 microns, the 5 um is the most commonly used particle size. The stationary phase of RP-HPLC is typically composed of hydrophobic alkyl chains that interact with the components. C4, C8, and C18 are three chain lengths that are commonly used in the reversed-phase chromatography.
Different types of columns are used in HPLC; however, C18 (Octyldecylsilane) is the most commonly used stationary phase of reversed-phase HPLC. It has 18 carbons that bonded to silica, which is more and a longer carbon chain than other columns used in RP-HPLC. As it has 18 carbon chains, it has a larger surface area which gives more time of interaction between the compounds and bonded phase. This is the major reason it covers a broad range of samples.
There are two types of reversed-phase chromatography column packing are used as follows.
- Silica gel with chemically bonded alkyl-chains
- Resin-based packing
The mobile phase of reversed-phase chromatography (RP-HPLC):
The role of the mobile phase in reversed-phase chromatography is to control the retention and selectivity of neutral and ionized compounds. In many RP-HPLC developed methods use a mixture of miscible organic solvent (methanol/acetonitrile) and water as the mobile phase. The reason for the organic solvents is to keep the polarity on adequate levels of the compound to dissolve in the solvent system and yet high enough to facilitate binding with the stationary phase of the preferred molecule. In some HPLC method developments, ion-pairing agents can be added to the mobile phase. When the compound to be separated is enters the column matrix it is allowed to dissociate from it by further decreasing the polarity by increasing the organic solvent concentration in the solvent system. This process of changing the concentration of organic solvent in the mobile phase to isolate a compound is called a gradient elution.
In some pharmaceutical analyses or research, several drugs are ionizable, i.e. acidic or basic. Therefore, the pH of the mobile phase must be controlled, as it can have a huge impact on the retention of components. Depending on the mobile phase pH, ionizable compounds that exist in ionized or non-ionized forms, and the ionized have significantly less retention than the non-ionized in reversed-phase liquid chromatography.
A buffer is a solution of a weak acid and its conjugate base, or a weak base and its conjugate acid. Significantly, the buffer has a pKa close to the preferred pH because the buffers best control the pH in their pKa. Different types of buffers such as phosphate buffer, acetate buffer, formate buffer, phosphoric acid, and its sodium or potassium salts are the commonly buffers for reversed-phase HPLC. The different solvent such as acetonitrile and methanol is the most commonly used organic solvents in RP-HPLC.
Applications of reversed-phase chromatography (RP-HPLC):
The reversed-phase chromatography is a significant analytical method used to separate the analytes, which is widely used in the analysis of pharmaceuticals, life-science research, forensics, chemical industries, biotechnology, food industries, and clinical diagnostics.
Advantages of reversed-phase HPLC:
- It is the most commonly used technique of all the HPLC method as it isolates a wide variety of samples.
- In RP-HPLC the water can use in the mobile phase with other solvents.
- This separation method can be stable at all acid and basic pH values.
- It is an inexpensive chromatographic technique.
- It gives precise results even with the minimum amount of samples.
The commonly asked questions on HPLC are as follows.
How many types of injection are there in HPLC?
The three types of injectors are used in HPLC such as Rheodyne injector, septum injector, and stop flow injector.
What are the different types of detectors used in HPLC?
UV/VIS detector, photo-diode array (PDA), mass spectrometer, fluorescence, refractive index, electrochemical, electrical conductivity, and light scattering are the types of detector used in HPLC.
What are the types of HPLC chromatography?
There are four types of HPLC such as reversed-phase HPLC, normal phase HPLC, size-exclusion HPLC, and ion-exchange HPLC
How many types of pumps are there in HPLC?
There are three types of pumps reciprocating pump, syringe pump, and pneumatic pump that provides the required pressure and flow rates.
What is isocratic and gradient elution?
There are two types of elution in HPLC i.e. isocratic and gradient. In isocratic elution, a constant composition of the mobile phase is pumped through the column during the whole analysis and in the gradient elution the composition of the mobile phase is steadily changed during the run.
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