2 Affinity-Based Methods for the Analysis of Emerging …
39
2017). Common examples are GC or GC coupled with mass spectrometry (GCMS), high-performance liquid chromatography (HPLC or LC), which is often used
in combination with MS (LC-MS) or tandem MS (LC-MS/MS), and enzyme-linked
immunosorbent assays (ELISAs) (Nelson and Hage 2006; García-Córcoles et al.
2019). GC and GC-MS are most readily applied for the analysis of volatile compounds and can provide low detection limits for such analytes, but derivatization
or other pretreatment steps may be required to extend these methods to more polar
targets (Fatta et al. 2007; Comerton et al. 2009; García-Córcoles et al. 2019). LC,
LC-MS, and LC-MS/MS are often employed for the separation and analysis of polar
or thermally labile compounds in water (García-Córcoles et al. 2019). LCLC-MS/MS has been of particular interest for this work and can lead to the analysis
of trace organic contaminants in water at low levels (Comerton et al. 2009; GarcíaCórcoles et al. 2019; Kumar 2016). However, sample pretreatment is still often
needed in LC-MS/MS to minimize matrix effects and to provide suitable selectivity
and detection limits (Comerton et al. 2009). ELISAs employ antibodies and enzyme
labels and can be used as a cost-effective means for the rapid detection of specific
emerging contaminants, such as antibiotics or pesticides (Aga and Thurman 1997).
Some limitations of ELISAs are that they do require antibodies that can bind to the
given target and they are mainly used in a manual format, which is most commonly
employed in screening assays (Aga and Thurman 1997; Nelson and Hage 2006;
Nicolardi et al. 2012).
Affinity chromatography is an alternative approach that has been used with emerging contaminants for both sample pretreatment and analysis in environmental testing (Nelson and Hage 2006). Affinity chromatography and high-performance affinity chromatography (HPAC) are liquid chromatographic methods that employ a
biological-related binding agent, known as the affinity ligand, that is immobilized
onto a solid support and used as a stationary phase (Hage 2006). Figure 2.1 shows a
typical separation scheme that is used in affinity chromatography. The target analyte
and sample are first introduced onto the affinity column in the presence of an application buffer. The analyte is often strongly retained under these conditions, which have
been selected to mimic the natural binding conditions of the analyte and immobilized
agent. Other sample components, which are non-complementary to the immobilized
binding agent, tend to elute under the same conditions as a non-retained peak. After
elution of these non-retained components, the retained analyte is then released by
passing through the column and support an elution buffer that causes release of the
retained analyte, such as by changing the pH of the mobile phase or by adding a
competing agent. The analyte is then measured as it elutes from the column or is
collected for use in a separate analysis method. The original application buffer is
then reapplied to the system. The column and support are allowed at that time to
regenerate prior to the next sample injection (Hage 2006; Hage et al. 2012; Zheng
et al. 2014; Zhang et al. 2018).
There are many types of binding agents and formats that can be used in affinity chromatography. For instance, the binding agent that is used in affinity chromatography may consist of an immobilized antibody, enzyme, transport protein, or
DNA/RNA sequence, among many other possibilities (Hage 2006). The immobilized
39
2017). Common examples are GC or GC coupled with mass spectrometry (GCMS), high-performance liquid chromatography (HPLC or LC), which is often used
in combination with MS (LC-MS) or tandem MS (LC-MS/MS), and enzyme-linked
immunosorbent assays (ELISAs) (Nelson and Hage 2006; García-Córcoles et al.
2019). GC and GC-MS are most readily applied for the analysis of volatile compounds and can provide low detection limits for such analytes, but derivatization
or other pretreatment steps may be required to extend these methods to more polar
targets (Fatta et al. 2007; Comerton et al. 2009; García-Córcoles et al. 2019). LC,
LC-MS, and LC-MS/MS are often employed for the separation and analysis of polar
or thermally labile compounds in water (García-Córcoles et al. 2019). LCLC-MS/MS has been of particular interest for this work and can lead to the analysis
of trace organic contaminants in water at low levels (Comerton et al. 2009; GarcíaCórcoles et al. 2019; Kumar 2016). However, sample pretreatment is still often
needed in LC-MS/MS to minimize matrix effects and to provide suitable selectivity
and detection limits (Comerton et al. 2009). ELISAs employ antibodies and enzyme
labels and can be used as a cost-effective means for the rapid detection of specific
emerging contaminants, such as antibiotics or pesticides (Aga and Thurman 1997).
Some limitations of ELISAs are that they do require antibodies that can bind to the
given target and they are mainly used in a manual format, which is most commonly
employed in screening assays (Aga and Thurman 1997; Nelson and Hage 2006;
Nicolardi et al. 2012).
Affinity chromatography is an alternative approach that has been used with emerging contaminants for both sample pretreatment and analysis in environmental testing (Nelson and Hage 2006). Affinity chromatography and high-performance affinity chromatography (HPAC) are liquid chromatographic methods that employ a
biological-related binding agent, known as the affinity ligand, that is immobilized
onto a solid support and used as a stationary phase (Hage 2006). Figure 2.1 shows a
typical separation scheme that is used in affinity chromatography. The target analyte
and sample are first introduced onto the affinity column in the presence of an application buffer. The analyte is often strongly retained under these conditions, which have
been selected to mimic the natural binding conditions of the analyte and immobilized
agent. Other sample components, which are non-complementary to the immobilized
binding agent, tend to elute under the same conditions as a non-retained peak. After
elution of these non-retained components, the retained analyte is then released by
passing through the column and support an elution buffer that causes release of the
retained analyte, such as by changing the pH of the mobile phase or by adding a
competing agent. The analyte is then measured as it elutes from the column or is
collected for use in a separate analysis method. The original application buffer is
then reapplied to the system. The column and support are allowed at that time to
regenerate prior to the next sample injection (Hage 2006; Hage et al. 2012; Zheng
et al. 2014; Zhang et al. 2018).
There are many types of binding agents and formats that can be used in affinity chromatography. For instance, the binding agent that is used in affinity chromatography may consist of an immobilized antibody, enzyme, transport protein, or
DNA/RNA sequence, among many other possibilities (Hage 2006). The immobilized