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色谱聚焦(法)    :  chromatofocusing
Introduction to Chromatofocusing
  • Separates proteins according to differences in their isoelectric point.
  • The technique is highly resolving, concentrating, and very specific.
  • Mono P is available prepacked with 10-µm MonoBeads particles that give high resolution.
Chromatofocusingis a chromatography technique that separates proteins according todifferences in their isoelectric point (pI). The pI of each protein isthe pH at which the protein has zero surface charge. Proteins withdifferent pI can be separated by being passed through achromatofocusing column (packed with a specific medium) while a pHgradient is generated on the column by specifically designed andmatched amphoteric buffers. Proteins elute in order of theirisoelectric points. To achieve consistently high resolution, generationof the linear pH gradient requires an even buffering capacity over theentire pH range used for a separation. Hence the need for specificallydesigned buffers (Polybuffer 74 or Polybuffer 96) and media substitutedwith charged, buffering amines (Mono P).
The figure on this page illustrates the focusing effect which takesplace during a separation and contributes significantly to the highresolution possible with chromatofocusing. In a descending pH gradient,a protein can exist in three charged states: positive, negative, orneutral and is constantly changing its charged state as the pH gradientdevelops and the protein travels through different pH zones on thecolumn. Molecules at the rear of a zone will migrate more rapidly thanthose at the front, gradually forming narrower bands of proteins, eachband comprised of one or more proteins of the same isoelectric point.Thus, during chromatofocusing, proteins with different pIs migrate atdifferent rates down the column as the pH gradient develops,continually binding and dissociating while being focused into narrowbands and finally eluted. The protein with the highest pI elutes firstand the protein with the lowest pI will elute last.
2009-12-2 09:13

Proteins with different pIs separate as they pass through thecolumn. Molecules with the same isoelectric point are focused in narrowbands during the separation.
SELECTION GUIDE - Chromatofocusing
pH RangeMediaEluent
9-6Mono P™Polybuffer 96
7-4Mono P™Polybuffer 74
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