liquid chromatography

 ►Definition:- 

Is a form of liquid chromatography to separate compounds that are dissolved in solution. And one of most widely used techniques for identification, quantification, and purification of mixture of organic compounds 

► Principle:- 

HPLC (High Performance Liquid Chromatography) depends on interaction of sample analytes with the stationary phase (packing) and the mobile phase to effect a separation. Following are explanations of the separation mechanisms commonly used in HPLC. adsorption chromatography the stationary phase, properly speaking, is the liquid-solid interface Molecules arereversibly bound to this surface by dipole-dipole interactions. Since the strength of interaction with the surface is different for different compounds, residence time at the stationary phase varies for different substances thus achieving separation. Liquid-solid adsorption chromatography is most often used for polar, non-ionic organic compounds. Partition chromatography is the fundamental distribution mechanism in liquid-liquid chromatography, i. e. when both mobile phase and stationary phase are liquids. Separation by distribution is based on the relative solubility of the sample in the two phases. In normal phase partition chromatography the stationary phase is more polar than the mobile phase, in reversed phase (RP) chromatography the mobile phase is more polar than the stationary phase. Stationary phases may be either coated on to a support, or they may be chemically bonded to the surface. Normal phase partition chromatography is used for very polar organic compounds, while reversed phase chromatography is commonly used for nonpolar or weakly polar substances. Ionic compounds are often better separated by ion Distribution Chromatography Due to its liquid stationary phase, the ingredients are only separated by their respective polarity, i.e. according to their distribution coefficient KD. Distribution chromatography is also ideal for the concentration of trace quantities. This is more closely related to Flash, Column, and Gas Chromatography. 

►Basic Terms and Definitions… 

1. supporting medium: a solid surface on which the stationary phase is bound or coated 

2. mobile phase: a solvent that flows through the supporting medium 

 * Polar (Normal Phase): Hexane ; dichloromethane; isopropanol; methanol 

* Non-Polar (Reversed Phase) water ; methanol; acetonitrile; tetrahydrofuran (THF) 

3. stationary phase: a layer or coating on the supporting medium that interacts with the analyte 

* Polar (Normal Phase): Silica, alumina

* Non-Polar (Reversed Phase) ODS Silica gel C18, C8 4. 

Chromatogram: 

A graphical or other presentation of detector response, concentration of analyte in the effluent or other quantity used as a measure of effluent concentration versus effluent volume or time. In planar chromatography "chromatogram" may refer to the paper or layer with the separated zones. The chromatogram below illustrates the most important parameters which characterise a separation. These parameters will be explained in the following paragraphs.

► Types of HPLC : 

There are following variants of HPLC, depending upon the phase system (stationary) in the process : 

1. Normal Phase HPLC: This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. Polar samples are thus retained on the polar surface of the column packing longer than less polar materials. 

2. Reverse Phase HPLC: The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained. 

3. Size-exclusion HPLC: The column is filled with material having precisely controlled pore sizes, and the particles are separated according to its their molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. 

4. Ion-Exchange HPLC: The stationary phase has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.Types of HPLC

► Components of HPLC (Instrument) :

1. Solvent Resorvoir : 

Mobile phase contents are contained in a glass resorvoir. The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample. 

2. Pump : 

A pump aspirates the mobile phase from the solvent resorvoir and forces it through the system’s column and detecter. Depending on a number of factors including column dimensions, particle size of the stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be generated. 

3. Sample Injector : 

The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi). 

4. Columns : 

The physical properties of the target molecules (analytes) determine the most suitable HPLC column for a given separation. The molecular characteristics that impact HPLC column selection include hydrophobicity/hydrophilicity, intermolecular forces (particularly dipoledipole), intramolecular forces (ionic), and size. HPLC column separations can often exploit multiple differences in the molecular properties of the target molecules. Generally, the structure and chemistry of the HPLC column packing (stationary phase) determines the analyte elution profile. HPLC column sizes range from capillary to process scale. 

The internal diameter (ID) and volume of a column determine both how much sample can be loaded onto a column and the sensitivity of separation. The column ID can affect the separation profile, particularly when using gradient elution, with smaller IDs yielding increased separation and detection sensitivity. Therefore, for analytical separations there is typically a trade-off between sensitivity and the sample volume loaded onto a column. Many of the types of packing used for gravity or low-pressure chromatography are not able to withstand the high pressures used in an HPLC system. Common packing materials in HPLC columns include silica or hydroxyapatite media and polymeric resins such as polystyrene divinylbenzene. The use of smaller-diameter beads generally results in improved separation sensitivity due to the increased surface area. However, column pressure increases as bead diameter is reduced for a given flow rate, placing a practical lower limit on bead size. Media bead diameters are typically in the range of 1.8–5 μm for an analytical HPLC column.