The solvent used to separate components in a liquid sample for HPLC analysis is called the mobile phase. The mobile phase is delivered to a separation column, otherwise known as the stationary phase, and then to the detector at a stable flow rate controlled by the solvent delivery pump. A certain amount of sample is injected into the column and the compounds contained in the sample are separated. The compounds separated in the column are detected by a detector downstream of the column and each compound is identified and quantified.
Going into more detail, HPLC consists of a variety of components, including a solvent delivery pump, a degassing unit, a sample injector, a column oven, a detector, and a data processor. As for HPLC, the pump delivers the mobile phase at a controlled flow rate a. Air can easily dissolve in the mobile phase under the standard atmospheric pressure in which we live in.
The degassing unit helps prevent this issue by removing air bubbles in the mobile phase b. After the dissolved air has been removed, the mobile phase is delivered to the column. The sample injector then introduces a standard solution or sample solution into the mobile phase c. Temperature fluctuations can affect the separation of compounds in the column.
The column is placed in a column oven to keep the temperature constant d. They are well separated. Technique 3. In this, the most powerful approach, the sample passes through a column or a cartridge device containing appropriate particles [stationary phase].
These particles are called the chromatographic packing material. Solvent [mobile phase] flows through the device. In solid-phase extraction [SPE], the sample is loaded onto the cartridge and the solvent stream carries the sample through the device.
Here the black sample is loaded onto a cartridge. Different solvents are used in each step to create the separation. When the cartridge format is utilized, there are several ways to achieve flow. Gravity or vacuum can be used for columns that are not designed to withstand pressure. In addition, small plastic columns, typically in the shape of syringe barrels, can be filled with packing-material particles and used to perform sample preparation.
This is called solid-phase extraction [SPE]. Here, the chromatographic device, called a cartridge, is used, usually with vacuum-assisted flow, to clean up a very complex sample before it is analyzed further. However, smaller particles have greater resistance to flow, so higher pressures are needed to create the desired solvent flow rate. Pumps and columns designed to withstand high pressure are necessary. When moderate to high pressure is used to flow the solvent through the chromatographic column, the technique is called HPLC.
In the beginning, pumps only had a pressure capability of psi [35 bar]. This was called high pressure liquid chromatography, or HPLC. The early s saw a tremendous leap in technology. These new HPLC instruments could develop up to 6, psi [ bar] of pressure, and incorporated improved injectors, detectors, and columns. HPLC really began to take hold in the mid-to lates. With continued advances in performance during this time [smaller particles, even higher pressure], the acronym HPLC remained the same, but the name was changed to high performance liquid chromatography.
High performance liquid chromatography is now one of the most powerful tools in analytical chemistry. A polar solvent is used, for example, a mixture of water and an alcohol such as methanol. Polar compounds in the mixture will pass more quickly through the column because a strong attraction occurs between the polar solvent and the polar molecules in the mixture.
Non-polar molecules are slowed down on their way through the column. They form varying degrees of attraction with the hydrocarbon groups principally through van der Waals dispersion forces and hydrophobic interactions.
They are also less soluble in the aqueous mobile phase components facilitating their interactions with the hydrocarbon groups. Please note that to comment on an article you must be registered and logged in.
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