page_title: High-performance liquid chromatography
hierarchical_section_title: High-performance liquid chromatography
caption_attribution_description: English: View of a high-performance liquid chromatography (HPLC) system (early 1990's).
context_page_description: High-performance liquid chromatography is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.
  HPLC has been used for manufacturing, legal, research, and medical purposes.
  Chromatography can be described as a mass transfer process involving adsorption. 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, 2–50 μm in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles.
context_section_description: 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 each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.
  HPLC has been 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.  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.), 2–50 μm in size.  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, 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.
  HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the 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 (2–50 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.
  The schematic of an HPLC instrument typically includes a degasser, sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column.  The pumps deliver the desired flow and composition of the mobile phase through the column.  The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components.  A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in an HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase.  Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry.  Most HPLC instruments also have a column oven that allows for adjusting the temperature at which the separation is performed.
caption: An HPLC. From left to right: A pumping device generating a gradient of two different solvents- a steel-enforced column and a detector for measuring the absorbance.
exif: {"Image Make": "Canon", "Image Model": "Canon DIGITAL IXUS 430", "Image Orientation": "Horizontal (normal)", "Image XResolution": "180", "Image YResolution": "180", "Image ResolutionUnit": "Pixels/Inch", "Image DateTime": "2006:03:05 07:12:05", "Image YCbCrPositioning": "Centered", "Image ExifOffset": "196", "Thumbnail Compression": "JPEG (old-style)", "Thumbnail XResolution": "180", "Thumbnail YResolution": "180", "Thumbnail ResolutionUnit": "Pixels/Inch", "Thumbnail JPEGInterchangeFormat": "2548", "Thumbnail JPEGInterchangeFormatLength": "5931", "EXIF ExposureTime": "1/60", "EXIF FNumber": "14/5", "EXIF ExifVersion": "0220", "EXIF DateTimeOriginal": "2006:03:05 07:12:05", "EXIF DateTimeDigitized": "2006:03:05 07:12:05", "EXIF ComponentsConfiguration": "YCbCr", "EXIF CompressedBitsPerPixel": "3", "EXIF ShutterSpeedValue": "189/32", "EXIF ApertureValue": "95/32", "EXIF ExposureBiasValue": "0", "EXIF MaxApertureValue": "95/32", "EXIF MeteringMode": "Pattern", "EXIF Flash": "Flash fired, auto mode, red-eye reduction mode", "EXIF FocalLength": "237/32", "EXIF FlashPixVersion": "0100", "EXIF ColorSpace": "sRGB", "EXIF ExifImageWidth": "2272", "EXIF ExifImageLength": "1704", "Interoperability InteroperabilityIndex": "R98", "Interoperability InteroperabilityVersion": "[48, 49, 48, 48]", "Interoperability RelatedImageWidth": "2272", "Interoperability RelatedImageLength": "1704", "EXIF InteroperabilityOffset": "1860", "EXIF FocalPlaneXResolution": "56800/7", "EXIF FocalPlaneYResolution": "56800/7", "EXIF FocalPlaneResolutionUnit": "2", "EXIF SensingMethod": "One-chip color area", "EXIF FileSource": "Digital Camera", "EXIF CustomRendered": "Normal", "EXIF ExposureMode": "Auto Exposure", "EXIF WhiteBalance": "Auto", "EXIF DigitalZoomRatio": "1", "EXIF SceneCaptureType": "Standard"}