High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originated from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc, which have been dissolved into liquid solutions. It relies on high pressure pumps, which deliver mixtures of various solvents, called the mobile phase, which flows through the system, collecting the sample mixture on the way, delivering it into a cylinder, called the column, filled with solid particles, made of adsorbent material, called the stationary phase. Each component in the sample interacts slightly different with the adsorbent material, causing different migration rate for the each component, leading to their separation, as they flow out of the column into a specific detector. The output of the detector is a graph, called Chromatogram. Chromatograms are graphical representations of the signal intensity versus time or volume, showing peaks, which represent components of the sample, each appears in its respective time, called Retention Time, having area proportional to its amount.
HPLC is widely used for manufacturing (e.g., during the production process of pharmaceutical and biological products), legal (e.g., detecting performance enhancement drugs in urine), research (e.g., separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and medical (e.g., detecting vitamin D levels in blood serum) purposes.
Chromatography can be described as a mass transfer process involving adsorption and/or partition. As mentioned, HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made of solid particles (e.g., silica, polymers, etc.), 1.5–50 μm in size, on which various reagents can be bonded. The components of the sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents (e.g., water, buffers, acetonitrile and/or methanol) and is referred to as a "mobile phase". Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination.
Operation
The liquid chromatograph is complex and has sophisticated and delicate technology. In order to properly operate the system. It is also desirable that there should be a minimum basis for understanding of how the device performs the data Processing to avoid incorrect data and distorted results
HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–~1400 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the packed column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller adsorbent particles (1.5–50 μm in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.