What is ACD (At Column Dilution)? - WKB207774

ENVIRONMENT

• MassLynx 4.1
• MassLynx 4.2
• Autopurification system
• FractionLynx

ANSWER

At Column Dilution (ACD) is used to

• Inject relatively large volumes of relatively strong sample diluents which may distort the chromatography in a conventional system.
• Prevent bulk precipitation in the sample loop or in the column itself.

In a conventional system, the sample is dissolved in a strong diluent, such as DMSO, and carried from the injector to the head of the column as a plug sandwiched in a stream of weak solvent, often 95% aqueous. Precipitation might occur at the edges of this plug where the strong sample solvent is diluted with the weak chromatographic solvent. This precipitation might occlude the fluid path and lead to a high-pressure shutdown. In the absence of such precipitation, the sample enters the column, but there will be no retention until the sample plug is diluted with the initial-strength mobile phase in the pores of the column. With larger injections, the volume required to dilute the sample can only be derived by moving a substantial distance along the column. In such cases, the sample is deposited as a broad band that occupies a large fraction of the column volume. Elution of such samples gives incomplete resolution with the overlapping peaks spread over large volumes of eluent and fractions. These problems may be reduced by strictly limiting both the volume and mass of sample injected. The alternative is substantial dilution of the sample with water, or most generally, a weak solvent, to ensure adequate retention. Neither approach is completely satisfactory because throughput and recovery are compromised. The required larger injection volume may be incompatible with the injector and fluidics present in the system.

In an At-Column Dilution system, the system is reconfigured to allow the sample plug to be carried to the head of the column in a stream of strong solvent. At the entrance of the column, this stream is continuously diluted with a stream of aqueous mobile phase. The rate of transfer into the column is so high that precipitation cannot occur. The sample molecules are then adsorbed to the packing material as very narrow bands that can be eluted as well-resolved, small-volume peaks.

ADDITIONAL INFORMATION