As cyanobacteria (also known as blue green algae) produce a range of cyclic peptides which
are highly toxic, capillary electrophoresis and associated techniques have been investigated
to assess their applicability for toxin monitoring in the water bodies of kwaZulu Natal,
South Africa. Capillary electrophoresis (CE) is a technique in which charged molecules can
be efficiently separated in a buffer solution within a capillary tube under the influence of a
strong electric field. Two CE modes, namely capillary zone electrophoresis (CZE) and
micellar electrokinetic capillary chromatography (MECC) were initially evaluated using a
laboratory-built CE instrument. The former mode lacked selectivity due to the similar
charge to size ratio of the algal toxins. However, with the latter mode, incorporation of a
surfactant (sodium dodecyl sulphate) into the buffer, produced sufficient resolution between
components. Parameters including surfactant concentration, buffer ionic strength, buffer
pH and operating voltage were systematically optimized to separate the four algal toxins
under investigation (microcystin YR, microcystin LR, microcystin RR and nodularin). The
optimum separation conditions were: 30 mM borax, 9 mM sodium dodecyl sulphate, pH
9.18, 30 kV applied voltage, 10 s hydrodynamic injection, 70 cm x 50 Ilm Ld. bare fused
silica capillary (LEFF 40 cm) and UV detection at 238 nm. Under these conditions, typical
detection limits were in the low ng/IlL range (14.13 ng/IlL for microcystin LR to 29.85
ng/ILL for nodularin).
The MECC method was evaluated in terms of migration time precision, efficiency and
resolution, peak area and normalised peak area precision. Standard deviation values for
retention times acquired using replicate electrokinetic injections ranged from 0.018 to 0.054
and 0.069 to 0.148 for hydrodynamic injections. Normalised peak area precision for
replicate hydrodynamic injections were in the range 84 to 97 % RSD, while improved %
RSD values of 11.5 to 18.7 were achieved for electrokinetic injections. Due to poor
precision resulting from the lack of automation on the laboratory built CE system, poor
correlation between increasing concentration and a corresponding change in normalised peak
areas were achieved. The MECC method developed was applied to the analysis of an algal
scum extract to illustrate the technique. A general problem with CE is that it suffers from poor detection sensitivity. Hence in this
study, alternative injection modes, sample concentration strategies and alternative detection
techniques were investigated in an attempt to improve detection limits for algal toxins.
Using optimized electrokinetic injection conditions, detection limits were five to ten times
better than those obtained with hydrodynamic injections. On-line sample concentration
methods were partially successful. Field amplified back and forth MECC in which analyte
injected in the entire column volume and subsequently focused in a narrow band by
manipulating the electric field, resulted in an enormous sensitivity enhancement that ranged
from 197 times for microcystin RR to 777 times for microcystin YR when compared to
hydrodynamic injections. Field amplified sample stacking (FASI) was ineffective for toxin
preconcentration, while electro-extraction produced detection limits ranging from 0.27
ng/J.tL for microcystin YR to 1.08 ng/J.tL for microcystin RR. Solid phase extraction, in
which analytes are first trapped and concentrated on HPLC material in a cartridge and then
eluted in a more concentrated form for injection, was found to be practical only in the offline
mode. A concentration detection limit of less than 0.002 ng/J.tL was obtained.
Attempts with on-line solid phase extraction failed due to problems associated with coupling
the cartridge with the separation capillary. Finally, laser induced fluorescence (LIF)
detection was investigated as an alternative to UV detection. Unfortunately, the algal toxins
were not amenable to LIF detection because tagging with the fluorescent moiety, fluorescein
isothiocyanate (FITC), was prevented by the stereochemistry of these cyclic peptides.
A comparative study between HPLC and MECC revealed that the former displayed poor
efficiency peaks and long analysis times for toxin analysis. However HPLC was superior in
terms of retention time precision (0.12 to 0.64 % RSD) and area precision (1.78 to 2.86 %
RSD). Mass detection limits for MECC (0.0142 to 0.0603 ng) were far superior to those
achieved by HPLC (0.55 to 1.025 ng). In addition to HPLC and MECC, a preliminary
investigation of micro-high performance liquid chromatography (J.tHPLC) and capillary
electrochromatography (CEC) for the analysis of algal toxins was made using 50 J.tm Ld.
capillary columns packed in-house, with reverse phase HPLC packing material. / Thesis (M.Sc.)-University of Natal, 1997.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/4868 |
| Date | January 1997 |
| Creators | John, Wilson. |
| Contributors | Raynor, Mark W. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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