enantiomer ratios (ER) were difficult to interpret due to the non-linear relation between
ERs. Small but significant differences in the enantiomeric signature, which may
demonstrate different enzymatic or enantiomer-selective uptake processes, were difficult to discuss when an extremely high enantiomeric excess was measured or only one
enantiomer was determined (ER ~ 1).
Several authors expressed their concern about these obvious restrictions (Harner
et al. 2000; Hühnerfuss 2000; Hegeman and Laane 2002; Ulrich et al. 2003). As a
result of this discussion, two groups suggested almost simultaneously (De Geus et al.
2000; Harner et al. 2000) the introduction of the term “enantiomeric fractions (EF)”
as a new method for the calculation of enantiomeric signatures (De Geus et al. 2000;
Harner et al. 2000). Therefore, both studies should be credited for the introduction of
this term. This calculation method, where the amount or concentration of the first
eluting enantiomer E
(1) or the (+) enantiomer E
(+) is usually divided by the sum of
the eluting enantiomers: EF ¼ E
(+) /(E
(+) + E
(À) ) today, is the preferred method for the
determination of enantiomeric signatures in environmental studies. For more details,
please read the earlier publications by Harner et al. and De Geus et al., cited above.
7.3 Possible Sources of Error in Enantiomer-Selective
High-Performance Liquid Chromatography
As valid for all technologically advanced high-resolution chromatographic methods,
high- and ultra-high-performance liquid chromatographic (HPLC/UHPLC) methods
are prone to many technique and method-related potential error sources, and thus, a
proper method validation and quality control protocol needs to be established for
quantitative ultra-trace analytical methods in environmental chemistry. The general
performance criteria for method validations are comprehensively summarised in
recent monographs on this topic (Chan 2004; Tuzimski and Sherma 2015). In
general, the validation criteria are as outlined above (van Zoonen et al. 1998;
Asmund et al. 2004; Matsuda 2012).
Enantiomer-selective separation methods in HPLC/UHPLC again add additional
complexity to the already comprehensive quality requirements for ultra-trace quantification of chiral environmental pollutants. Since the determination of the correct
enantiomeric signature is the major goal for the here chosen analytical method, the
correct elution order and structure information of the respective enantiomers need to be
established (see considerations outlined above). Usually, the chromatographic resolution of HPLC methods is considerably lower than for UHPLC method. Thus, retention
windows for target peak covering several seconds (1–3 s) may be expected, and,
therefore, the peak resolution plays a major role in determining the enantiomeric
signature. For a sound resolution of enantiomers, a peak resolution of at least R ¼ 1
should be achieved. Here, the resolution “R” of a chromatographic peak separation is
defined as a quantitative measure for how well two elution peaks are separated in a
chromatographic run. In this case, “R” is defined as the difference in retention times
between the two peaks, divided by the combined widths of the elution peaks (Fig. 7.2).
100
7 Quality Control and Evaluation Criteria for Enantiomer-Selective Separation. . .
ERs. Small but significant differences in the enantiomeric signature, which may
demonstrate different enzymatic or enantiomer-selective uptake processes, were difficult to discuss when an extremely high enantiomeric excess was measured or only one
enantiomer was determined (ER ~ 1).
Several authors expressed their concern about these obvious restrictions (Harner
et al. 2000; Hühnerfuss 2000; Hegeman and Laane 2002; Ulrich et al. 2003). As a
result of this discussion, two groups suggested almost simultaneously (De Geus et al.
2000; Harner et al. 2000) the introduction of the term “enantiomeric fractions (EF)”
as a new method for the calculation of enantiomeric signatures (De Geus et al. 2000;
Harner et al. 2000). Therefore, both studies should be credited for the introduction of
this term. This calculation method, where the amount or concentration of the first
eluting enantiomer E
(1) or the (+) enantiomer E
(+) is usually divided by the sum of
the eluting enantiomers: EF ¼ E
(+) /(E
(+) + E
(À) ) today, is the preferred method for the
determination of enantiomeric signatures in environmental studies. For more details,
please read the earlier publications by Harner et al. and De Geus et al., cited above.
7.3 Possible Sources of Error in Enantiomer-Selective
High-Performance Liquid Chromatography
As valid for all technologically advanced high-resolution chromatographic methods,
high- and ultra-high-performance liquid chromatographic (HPLC/UHPLC) methods
are prone to many technique and method-related potential error sources, and thus, a
proper method validation and quality control protocol needs to be established for
quantitative ultra-trace analytical methods in environmental chemistry. The general
performance criteria for method validations are comprehensively summarised in
recent monographs on this topic (Chan 2004; Tuzimski and Sherma 2015). In
general, the validation criteria are as outlined above (van Zoonen et al. 1998;
Asmund et al. 2004; Matsuda 2012).
Enantiomer-selective separation methods in HPLC/UHPLC again add additional
complexity to the already comprehensive quality requirements for ultra-trace quantification of chiral environmental pollutants. Since the determination of the correct
enantiomeric signature is the major goal for the here chosen analytical method, the
correct elution order and structure information of the respective enantiomers need to be
established (see considerations outlined above). Usually, the chromatographic resolution of HPLC methods is considerably lower than for UHPLC method. Thus, retention
windows for target peak covering several seconds (1–3 s) may be expected, and,
therefore, the peak resolution plays a major role in determining the enantiomeric
signature. For a sound resolution of enantiomers, a peak resolution of at least R ¼ 1
should be achieved. Here, the resolution “R” of a chromatographic peak separation is
defined as a quantitative measure for how well two elution peaks are separated in a
chromatographic run. In this case, “R” is defined as the difference in retention times
between the two peaks, divided by the combined widths of the elution peaks (Fig. 7.2).
100
7 Quality Control and Evaluation Criteria for Enantiomer-Selective Separation. . .