The chlorophyll concentration of each chlorophyll
species (fig 3) is equimolar to the magnesium
concentration. All chlorophylls have only one
magnesium central atom. In this way no
expensive and unstable chlorophyll standards are
necessary for quantification.
Sketch of different chlorophyll species.
■Methode Parameters
TAB 1 Methode parameters of coupled HPLC and FAAS.
Parameter
■Sample preparation and enrichment
Surface water samples are homogenized on a
magnetic stirrer and are pressed by a peristaltic
pump through a syringe filter (0,45 µm) (fig 4a).
a) Enrichment of chlorophyll with an automatic
filtering system.
b) Extraction of chlorophyll enriched filters with methanol.
Afterwards the filter is extracted with methanol.
The extract can directly analyzed by the above
described system (fig 4b).
Fig 5 shows a chromatogram of 9 different
chlorophyll species directly extracted from plants
Chromatogram of 9 different chlorophylls extracted
from plants.
■Introduction
Species analysis by coupling chromatography
with atomic spectroscopy is a special cost
intensive part of instrumental analysis. The
atomic spectroscopy used as monitor consumes
a lot of expensive gases. Flame atomic
absorption spectroscopy (FAAS) is a much
cheaper element monitoring technique. Further
on FAAS shows nearly any matrix effect for
element calibration.
The concentration of chlorophyll is an important
characteristic parameter in environmental water
analysis. There is a correlation between
eutrophication and chlorophyll content of surface
waters, analyzed as sum parameter (fig1). The
magnesium analytic of chlorophyll species should
help to get more information about the water
quality.
Sampling: Holthausens Kull in Krefeld Germany
■Measurement System
A Flame atomic absorption spectrometer was
coupled with an HPLC system. Seperation was
done on a C18 reverse phase system (fig 2)
Shimadzu Atom Absorption Spectrometer:
•AA-7000F Dual Atomizer System with FAAS-
7000 flame atomizer
•Acetylen/air burner
Coupling of FAAS with HPLC.
Shimadzu Prominence Series(Modular HPLC):
•LC-20AD/ADXR
•Online-Degrasser DGU-20A
•Autosampler SIL-20A
•Controller CBM-20A
■Method
The chlorophyll species can be distinguished by
their side chains, separated with Shimadzu LC-
20AD/ADXR HPLC and detected by the
magnesium central atoms of chlorophyll with
Shimadzu AA-7000.
Malte Plenkera ,Lars Reindersa, Florian Pahlkea, Uwe Oppermannb, Jürgen Schrama
a Niederrhein University of Applied Sciences, Faculty of Chemistry, [email protected], Krefeld, Germanyb Shimadzu Europa GmbH, Spectroscopy, [email protected], Duisburg, Germany
■Sample preparation and enrichment
Fig 6 (a) shows an extract from chlorella algae
with methanol extracted and measured with
HPLC-FAAS. There are clearly separated peaks.
The first peak at 102 s is free Mg2+ ions, the
second peak at 165 s is chlorophyll b species and
the last peak at 203 s is chlorophyll a species.
Chromatogram of extracted chlorella algae (a)
compared with a surface water sample of Kaiserpark
in Krefeld Germany (b).
Under the condition of our sample preparation in
all our surface water samples only chlorophyll a
species could be detected (fig 6 b).
Tab 2 shows the measured chlorophyll contents
with FAAS compared with the DIN 38412–L16
method.
TAB 2 Chlorophyll contents of different surface waters in
Krefeld Germany analyzed with FAAS and compared with DIN
38412-L16
■Conclusion
A DIN corresponding method was developed and
applied for chlorophyll, concerning species, by
coupling HPLC with FAAS.
The chlorophyll content of surface waters could
be analyzed and eutrophic situations could be
revealed. It was astonishing that only chlorophyll
a could be found in all samples. The limit of
detection of this method is 0,178 µmol/L
chlorophyll.
The quantification can be realized easily by Mg2+
calibration.
So the species analysis of chlorophyll could be
realized in a fast and cheap way.
■Outlook
The present work deals with the couple of thin
layer chromatography with the graphite furnace
atomic absorption spectroscopy (GFAAS) to
determine the surface waters. With this coupling
– useable in field - the cost of analyzes would be
reduced. The limit of detection could be
decreased by using the GFAAS as element
detector. Further research should be done to
determine magnesia containing chlorophylls in
eutrophic surface waters and to look for its
speciation.
Parameter Setting
column 150 mm C18-phase with 4,5 µm particles
flow rate 0,5 ml/min
eluent64,6 Vol-% methanol /30,4 Vol-% ethyl acetate / 5 Vol-% water
gradient isocratic
injection volume 100 µL
duration 10 min
wavelength 285,2 nm
gap width 0,7 nm
background correction
Deuterium background correction
burner height 7,5 mm
gas flow rate 15,0 L/min air2,4 L/min acetylene
Designation Chlorophyll aDIN 38 412 –L16[µg/L]
Chlorophyll aHPLC-FAAS-coupling[µg/L]
Holthausens Kull 11,8 12,1
Kaiserpark 11,1 11,7
Töppersee 31,2 29,3
University atriumpool
5,3 4,9
FIG 1
FIG 2
FIG 3
FIG 4
FIG 6
a) b)
8
FIG 51
2
34 5
6
7
9
Magnesium Species Analysis: Chlorophylls with HPLC-FAAS