High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC), is an analytical technique used to separate, identify, and quantify components dissolved in a liquid mixture. Other versions of liquid chromatography such as gravity flow or fast protein liquid chromatography (FPLC) utilize column particles typically 50 microns and greater in size. In contrast, HPLC adopts the use of smaller particles ranging from 2 to 50 microns. This allows for a greater surface area exposure to the mobile phase compounds, providing higher separation efficiency and greater resolution. This smaller size, however, produces a greater resistance to flow. To compensate and maintain consistent flow rates, HPLC systems are designed to pump liquids at much higher pressures, ranging from 50 to 400 bar (700 to 6000 psi or 5 to 40 MPa). The high separation power of HPLC lets researchers tackle many analytical questions, such as in biomedical applications of drug analysis, diagnostics, and proteomics. Naturally, this also brings forth many specialized options to consider.

HPLC Systems:

There are a considerable variety of HPLC instruments in the market, many boasting flexible capabilities and useful features. A defining aspect of HPLC, the operating pressure, can reach maximum values of 1,300 bar (130 MPa or 18,800 psi) in high end models. These high pressures can flexibly traverse into the realm of “ultra high performance” liquid chromatography, or UHPLC. Certain instruments are also tailored to the scale of the application- analytical, semi-preparative, preparative, or process. Other HPLC features to keep in mind include:

• Number of column configurations and solvent combinations
  
• Capacity of autosamplers and number of supported microplates and vials
  
• Sample and column incubation temperature range
  
• Sample carryover performance
  
• Customizable module options
  

For the most detailed and up-to-date information, it is best to get a quote and hear directly from the manufacturer. Compare different instruments side by side and receive multiple quotes at once with a simple click from our HPLC systems search tool.

HPLC Columns:

Perhaps the most important consideration in choosing an HPLC column is the mode, or type, of chromatography. This, in turn, is determined by the chemical attributes of the sample, such as polarity, charge, and molecular size. Some of the most common types include:

• Normal-Phase - Also known as NP-HPLC, this is the classical mode of chromatography. A non-polar mobile phase passes through a polar stationary medium, such as silica.
  
• Reversed-Phase - The reverse of normal-phase, in which the mobile phase is aqueous and polar, and the stationary phase is non-polar. Reverse-phase particles are typically coated with carbon chains, such as C18. RP-HPLC is the most common separation mode.
  
• Hydrophilic Interaction (HILIC) - HILIC is a variant of normal phase, in which polar solvent (such as water) is added to the organic mobile phase. Analytes elute in order of increasing polarity.
  
• Hydrophobic Interaction (HIC) - HIC is a variant of reversed-phase in which an aqueous mobile phase interacts with a moderately hydrophobic stationary phase (such as C4). This method is ideal for separating large biomolecules such as proteins.
  
• Ion Exchange - Ion exchange chromatography uses attraction from electrical charges on the surface of stationary phase particles. The retention of sample compounds depends on their inherent charge and the concentration of salt ions in the solution. Cation exchange particles are negatively charged and interact with positive ions; anion exchange particles are positively charged.
  
• Size Exclusion - Size exclusion- and gel-filtration-based chromatography involve stationary particles that are porous, trapping smaller particles inside and letting larger molecules pass through faster. Retention time increases as the size of the compound decreases.
  

Note that increasing column length and decreasing particle size come at a cost of greater solvent consumption, longer run times, and higher backpressure. Discover different kinds of HPLC columns in our chromatography search tool.