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Capillary electrophoresis and related techniques for the analysis of fresh water algal toxins.

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.

Identiferoai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/4868
Date January 1997
CreatorsJohn, Wilson.
ContributorsRaynor, Mark W.
Source SetsSouth African National ETD Portal
LanguageEnglish
Detected LanguageEnglish
TypeThesis

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