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, schram@hsnr.de, Krefeld, Germanyb Shimadzu Europa GmbH, Spectroscopy, shimadzu@shimadzu.eu, 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