Chromatography is the technique by which components or solutes from a mixture are separated depending on the comparative amount of each solute that has been dispersed between a moving fluid stream, called the mobile phase, and a stationary phase. The stationary phase is usually a solid or a liquid whereas the mobile phase can either be a liquid or a gas.
Liquid chromatography (LC) extracts molecules in a liquid mobile phase using a stationary phase i.e. a solid or an immiscible liquid. Liquid chromatography is used for analytical or preparative applications.
In column liquid chromatography, the mobile phase along with the solutes to be separated passes through the column, and components in the mixture interrelate to varying degrees with the stationary phase. The mobile phase comprising the components of interest is separated depending on the different physicochemical interactions between the two phases.
Based on the physical state of stationary/mobile phase combinations the technique of chromatography is classified. Liquid-solid column chromatography, one of the most popular chromatographic techniques, uses a liquid mobile phase that slowly filters down through the solid stationary phase, bringing the separated components with it.
Types of Liquid Chromatography Methods
Liquid chromatography methods are named according to the mechanism involved:
• Liquid-Solid Chromatography (Adsorption Chromatography):
The separation apparatus in LSC is based on the struggle of the constituents of the mixture sample for the active sites on a solid stationary phase such as silica gel.
• Liquid-Liquid Chromatography (Partition Chromatography):
The stationary solid surface is coated with another liquid (stationary phase) which is immiscible in the solvent (mobile phase). Partitioning of the sample between two phases interrupts or retains some components more than the others to effect partitioning.
• Gel Permeation Chromatography (Exclusion Chromatography):
Gel permeation chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are capable of infusing into more pores and are, therefore, reserved longer than large molecules.
• Normal-Phase Chromatography
In normal phase chromatography stationary phase is polar in nature while the mobile phase is non-polar, meaning the polar analyte interacts more with the stationary phase hence increasing its retention time inside the column.
• Reverse-Phase Chromatography
In reversed-phase chromatography, the stationary phase is non-polar whereas the mobile phase is polar in nature. If the analyte is polar, it will interrelate less with the stationary phase hence decreasing its retention time inside the column.
Instrumentation and Working
The mixture is injected at the injection port. The mobile phase carries it forward through the packed column where it separates into its components. The detector detects the separated analytes and the recorder, usually, a computer records this information.
Following are the components of the liquid chromatography technique:
• Injection site: The sample (mixture) here doesn’t have mobility hence it requires a mobile phase to carry it forward. The injection site is where the sample (mixture) is introduced in the column. The mobile phase carries the sample mixture forward through the column.
• Packed column: The columns are commonly made from stainless steel but thick glass, polymers such as poly-ether-ethel-ketone, a combination of stainless steel and glass, or an amalgamation of stainless steel and polymers are also used. Typical LC analytical columns range from 3 to 25 cm long with a diameter of 1 to 5 mm.
The columns are commonly straight unlike that of Gas Chromatography columns. Particles used for packing columns have a characteristic diameter range of 5 to 10 µm. Liquid chromatographic columns show an increase in efficiency when the diameter of the particles used for packing inside the column decreases.
• Detectors: It detects constituents of the mixture that are being eluted off the chromatography column. In 1942, Tiselius and Claesson reported the first effective inline liquid chromatography (LC) detector which was a refractive index detector. The conductivity detector was described by Martin and Randall in the year 1951. Other detectors used are UV detector, fluorescence detector, mass spectrometer, and electrochemical detector.
Retention Time: The total time taken by the particular compound to travel to the detector, through the column is called retention time. This time is initiated at the moment the sample is injected till the point at which the display shows a maximum peak height for the compound being run. Different compounds show diverse retention times.
For a particular compound, the retention time will vary depending on the following factors:
• Pressure applied (this affects the flow rate of the solvent)
• Nature of the stationary phase (implies particle size as well as the material by which the stationary phase is made up of)
• Solvent composition
• Column temperature
• This means that these conditions have to be vigilantly restricted if using retention time as a method of identifying compounds.
Column Efficiency and Selectivity:
The Van Deemter equation;
HETP= A+ Cµ
A is a constant which signifies the various probable paths that can be taken by the analyte through the stationary phase; it decreases if the packing of the column is kept as small as possible. B denotes a constant used to describe the longitudinal diffusion taking place in a system. C is a constant that describes the rate of adsorption and desorption of the analyte on the stationary phase. So flow rate, µ must be optimized accordingly. If the flow rate is too low, the longitudinal diffusion factor will increase significantly, which will increase plate height.
When the flow rate is low, the analyte rests in the column for a longer time and hence there exists a longitudinal diffusion which poses a significant problem. If the flow rate is too high, the mass transfer term (Cµ) will increase and reduce column efficiency. When the flow rate is high, the analyte gets adsorbed to the stationary phase resulting in some of the sample lagging behind which further results in band broadening.
• It is used to determine impurities in pharmaceutical raw materials and formulated products.
• Often used as a basic identity check on pharmaceuticals raw materials.
• Potentially useful in cleaning validation, this is part of the manufacture of pharmaceuticals.