paper 112 - ACS: Division of Environmental Chemistry

Preprints of Extended Abstracts Vol. 42 No. 2
ANALYSIS OF INTERMEDIATES OF EDTA BIODEGRADATION BY HPLC
Zhiwen Yuan and Jeanne M. VanBriesen
Carnegie Mellon University, Department of Civil and Environmental Engineering,
Pittsburgh, PA 15213
Email: Jeanne@cmu.edu
Introduction
Ethylendiaminetetraacetate (EDTA) is an anthropogenic chelating agent (see Figure 1)
widely used for its ability to form stable, water soluble complexes with metals. Many
industrial processes and consumer products make use of EDTA (see Figure 2) to
sequester metals and control process chemistry. Due to minimal biodegradation in
natural and engineered systems and the low potential for photodegradation in many
turbid waters, EDTA persists in surface waters at concentrations ranging from 1-100
µmg/L. 1-4 EDTA is generally regarded as nontoxic and is used in food products and
medicines. 5 The effects of low persistent concentrations in the environment are unclear.
EDTA may enhance eutrophication, remobilize heavy metals, and have long-term
toxicological effects on microorganisms. 2
O
OH
O
HO
N
N
OH
O
HO
Figure 1. Structure of Ethylene-diaminetetraacetate (EDTA)
O
GENERAL PAPERS
Organized by
T. Mill
Symposia Papers Presented Before the Division of Environmental Chemistry
American Chemical Society
Boston, MA August 18-22, 2002
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Preprints of Extended Abstracts Vol. 42 No. 2
Water
Agro
Chemicals -
5%
Textiles - 6%
Other - 16%
Treatment - 1%
Paper
Products - 7%
Photo Industry -
17%
Industrial
Detergents -
32%
Household
Detergents -
16%
Figure 2. Uses of EDTA (following Frimmel, 1997 1 )
Biodegradation of EDTA is a potential mode of removal from the environment. DSM
6780, an EDTA degrading organism isolated by Nortemann, 6 initiates EDTA
biodegradation but does not mineralize it. Instead, intermediates are produced that
have significant chelation ability. Intermediates formed in the biodegradation of EDTA
include ethylendiaminetriacetic acid (ED3A), 2-oxo-1,4-piperazinediacetic acid (3KP),
N,N’-ethylenediaminediacetic acid (N,N’-EDDA), iminodiacetic acid (IDA), and
glycine. 7-11 If these intermediates persist, they sequester carbon and electrons,
reducing cell yield and slowing the rate of removal of EDTA from the system.
EDTA intermediates were previously analyzed by Kluner et al 7 with ED3A and 3KP
measured simultaneously and N,N’ EDDA measured on a separate column. Witschel et
al 12 and Sorensen 13 use a method similar to Kluner. Glycine has been measured by
Einarsson et al 14 as an amino acid with HPLC with fluorescence detection. IDA has
been previously measured using fluorescence detection as well. 15 The contribution of
this work was to develop a method for concurrent measurement of ED3A, N, N’EDDA
and 3KP and concurrent measurement of IDA and glycine. The new methods also rely
on a UV detector, which although less sensitive than a fluorescence detector, still
allows detection to 10 -7 M for these intermediates.
Materials and Methods
The HPLC was performed with a Quaternary HP 1050 Pump and HP 1050 Series
Variable Wavelength UV/Visible Detector. The manual injector (Model 7125,
Rheodyne) was equipped with a sample loop of 20ml. Nucleosil 100-5 C18 Column
(Agilent Technologies), 250 mm in length, 4.0 mm in diameter with a particle size of 5
mm, was used for both methods. HP Chemstation software (Reversion A.07.01,
Hewlett-Packard) was used for chromatogram integration and linear regression of
calibration table and curve. The calculation of concentrations was based on peak area.
Because copper complexes of N,N’-EDDA, ED3A and 3KP are very stable, they were
concurrently measured by ion-pair reversed-phased HPLC after complexation with
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Preprints of Extended Abstracts Vol. 42 No. 2
excess copper acetate. The ion-pair reagent is dodecyltrimethyl ammonium bromide.
The chromatography are as following. Mobile phase: 1mM copper acetate, 0.6g/L
dodecyltrimethyl ammonium bromide, 5 µl/l CH3COOH, and finally pH adjusted to 5.4
with NaOH solution. Flow rate: 1.0 ml/min. Detection wavelength: 280nm. Injection
volume: 20 µl. All three intermediates were separated within 7 min.
IDA and glycine were measured as amino acids after derivatization with
9-fluorenylmethyl chloroformate (FMOC-Cl). The reaction of FMOC-Cl with the amino
acids proceeds as follows under basic conditions:
H 2C O C
O
+ H2N C C H
Cl
H
H
O
H 2C O C
O
H
+ H +
O
NH C
H
C H
+ Cl -
The derivatives are highly fluorescent and stable. The fluorescent derivatives with
FMOC-Cl can be monitored by fluorescence detector or ultraviolet (UV) detector. The
separation was carried out by gradient elution under room temperature. The mobile
phase was made by mixing elution buffer solution and acetonitrile-water solution. The
elution buffer is made by adding 3ml/l glacial acetic acid in deionized water and
adjusted to pH 4.3 with NaOH solution. The acetonitrile-water solution contained 70%
acetonitrile (v/v). The gradient elution immediately began after sample injection, and
the eluent varied linearly from 40% acetonitrile-water solution to 70% acetonitrile-water
solution in 8min. The flowrate was 1.2ml and the whole elution ended at 12 min. The
detection wavelength is 280 nm.
Results and Discussion
The representative chromatograph for separation of N,N’ EDDA, ED3A, and 3KP is
shown in Figure 3. This is the first report of concurrent measurement of these EDTA
biodegradation intermediates. The representative chromatogram for separation of IDA
and glycine is shown in Figure 4. This is the first report of concurrent measurement of
these intermediates and the use of UV rather than fluorescence detection.
The detection limits of these five intermediates are all below 10 -7 M, which are low
enough for most applications involving biodegradation of EDTA. The detection limit can
be lowered by increasing the injection volume. These two HPLC methods are simple,
fast, accurate, specific, and applicable to the analysis of these intermediates in
different matrixes.
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Preprints of Extended Abstracts Vol. 42 No. 2
mAU
5
4
3
2
1
0
-1
VWD1 A, Wavelength=280 nm (YUAN\80700003.D)
2.616
Area: 19.5348
N,N'-EDDA
Area: 25.0715
0 1 2 3 4 5 6
ED3A
5.491
5.974
Area: 26.6155
Figure 3. Chromatogram of N,N’-EDDA, ED3A and 3KP (10mM for each compound)
mAU
8
6
4
2
0
VWD1 A, Wavelength=280 nm (YUAN\80600004.D)
4.514
Area: 56.4618
IDA
0 2 4 6 8 10
6.449
Area: 43.5847
glycine
Figure 4. Chromatogram of 10 mM IDA and 10 mM glycine
10.450
3KP
Area: 46.0853
FMOC-OH
References
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289-312.
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Notes: have copy
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