Multidimension Liquid Chromatography

 

 

High Preformance Liquid Chromatography (HPLC) is a technique for fractionating proteins. It takes a seirries of purifying steps to achive a high accuracy. These steps can include precipitation, ultrafiltration, centrifugation, electrophoresis, solvent extractions, and lyophilization.
"Multidimensional HPLC (MDLC) is distinguished from conventional procedure by comprising a specific set of carefully chosen and matched chomatographic separation modes capable of yielding highly purified proteins in a minimum number of steps. Automation is frequently used to join the various stages of separation into a single seamless procedure" (Apffel 2004).

There are two different separation modes used in MDLC: retentive and nonretentive.
"Retentive separation modes, such as reversed-phase and ion-exchange-chromatography (IEC), can retain and concentrate samples under a given set of mobile-phase condiations and selective elute them under continuous or step gradients in mobile-phase composition.
Nonretentive separation modes, such as size exclusion chromatography (SEC), do not retain analytes through interaction with the stationary-phase surfacesand are generally operated under isocratic conditions. Nonretentive modes are generally used in the first dimension because it is often necessary to transfer relitivly larege volumes to the second dimension. If the second dimension is retentive, the proteins in the eluted fractions will be preconcentrated, thus minimizing band bradening and maintaining resolution. A retentive second dimension will also eliminate any extra-column band broadening introduced by connecting tubing, valves, or trapping columns used to automate the multidimensional coupling of separate columns" (Apffel 2004).

There are two different appraches to MDLC: on-line and off-line

"An on-line 2-D LC proteomic analysis workflow. In a typical on-line 2-D LC workflow, a mixture of tryptic peptides are eluted from an SCX column using stepwise isocratic injections of an aqueous salt of increasing concentration. After each injection, the analytes are captured on the C18 enrichment column, washed free of salt and, after a valve switch, back flushed into the C18 analytical column and chromatographed using AcN/H2O gradient with increasing organic content. The eluent from the analytical column this is sprayed directly into the mass spectrometer" (Vollmer 2004).

(Agilent 2004)

"An off-line 2-D LC proteomic analysis workflow. In the off-line workflow, the SCX column is eluted using a linear salt gradient, and equally timed microliter fractions are collected in plate wells using the microfraction collector. After any intervening operations, each fraction, in turn, is injected onto the second-dimension C18 enrichment column by means of an autosampler, and further operations are allowed to proceed in a manner identical to the on-line workflow" (Vollmer 2004).

(Agilent 2004)

 

Sources:

Agilent. 2004. http://www.chem.agilent.com/

Apffel A. 2004. Multidimensional Chromatography of Intact Proteins, Purifying Proteins for Proteomics. 74-98.

Vollmer M., Nagele E., and Moritz R. 2004. On-line and Off-line 2-D LC-ESI MS-MS Methods in Proteomic Analysis, Pharmaceutical Discovery. 42-49.