Environmental Analysis by On-Line Immunoextraction and Reversed-Phase Liquid Chromatography:  Optimization of the Immunoextraction/RPLC Interface

Abstract

The use of antibodies in HPLC columns for on-line immunoextraction combined with reversed-phase liquid chromatography (RPLC) is of growing interest in environmental and agricultural analysis. This technique is typically performed by using a small RPLC precolumn to capture and concentrate analytes as they elute from the immunoextraction column; however, there is little information on the conditions required for optimizing this interface. This study examined the behavior of this interface by using 2,4-dichlorophenoxyacetic acid (2,4-D) and related herbicides as model analytes. It was found that analyte dissociation from immunoextraction columns followed first-order decay and that the elution of these analytes through the immunoextraction/RPLC interface gave an exponentiallymodified Gaussian profile. Computer simulations were used to see how analyte elution through the interface changed with different dissociation and retention conditions. Several guidelines were developed from this work that could be used for developing and optimizing on-line immunoextraction/RPLC systems for other chemicals of environmental or agricultural interest.

Biointeraction analysis by high-performance affinity chromatography: Kinetic studies of immobilized antibodies

“…The other tested analytes would have retention times ranging from 2.8 min (4-CPA) to 5.5 min (2,4,5-T) under the same conditions. Careful control of the application and wash step could also allow the anti-2,4-D antibody column to be used for the immunoextraction of these agents, as has been demonstrated in recent simulations of immunoaffinity/reversed-phase systems [23] and in past work examining the binding of anti-atrazine immunoaffinity columns for triazine herbicides and their degradation products [10,22]. …”

“…The ability of an antibody to recognize a specific target and bind this with high affinity gives these methods good selectivity and low limits of detection [7–14]. In addition, the use of immobilized antibody supports in combination with other methods (e.g., reversed-phase chromatography) has seen growing use in environmental and biological applications as a means for extracting and concentrating a given group of analytes from complex samples [8,9,15–23]. …”

A naturally occurring, noncanonical GTP aptamer made of simple tandem repeats

“…Elution profiles were fit to the equation F = e - kv where F is the fraction of aptamer remaining on the column, k is the decay constant, and v is the elution volume. Similar approaches have previously been used to model elution profiles in affinity chromatography experiments 50 , 51 . This formula was used to calculate the volume required to elute 50 percent of the aptamer from the column at each GTP concentration.…”

Immunoaffinity Chromatography: An Introduction to Applications and Recent Developments

“…In addition, the elution buffer used in IAC is an aqueous solution, which acts as a weak mobile phase for RP columns. Online immunoextraction coupled with RPLC has been used to measure compounds in food extracts [91], bodily fluids [92–99], cell extracts [100] and environmental samples [74,101]. An example of an HPLC system which allows for the combination of immunoextraction with RPLC is shown in FIGURE 6.…”