Transcript Preparation for the Analysis of Selenocyanate from the Broth Cultures of Selenium-Resistant Bacteria using Solid-Phase Extraction and Capillary Electrophoresis J.D.

Preparation for the Analysis of
Selenocyanate from the Broth
Cultures of Selenium-Resistant
Bacteria using Solid-Phase
Extraction and Capillary
Electrophoresis
J.D. Fox and T.G. Chasteen
Background (the old news)
• Our interest is in selenium-resistant
microorganisms.
– Pseudomonas fluorescens K27
– Escherichia coli 1VH
– Bacillus sp.
– 130404
• These bacteria grow in the presence of
toxic selenium species.
• Many can even bioprocess said species.
Background cont.
• A substantial amount of the selenium in
solution is reduced to elemental selenium.
• This can be seen as a blood-red
precipitate.
Chemical Species of Interest
• Oxyanions of selenium:
– Selenite
– Selenate
• The current research focuses on
selenocyante
• But where did it start?
Relative Toxicities
• Growth experiments were carried out to
determine the relative toxicity of each of
the selenium species
• For E. coli 1VH:
– 10 mM Selenate: 24.5% reduction in SGR
– 10 mM Selenite: 45.8% reduction in SGR
– 10 mM Selenocyanate: 31.3% reduction in
SGR
Headspace Sampling
• Part of the bioreduction process involves
methylating Se to create several different
volatile species that are out-gassed by the
bacteria [1].
• The headspace of E. coli 1VH was
sampled using solid-phase microextraction
and examined via gas chromatography
with fluorine-induced sulfur
chemiluminescence detection.
Headspace Sample
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MeSH – Methanethiol
DMDS – Dimethyldisulfide
DMSeS – Dimethylselenenylsulfide
DMDSe – Dimethyldiselenide
DMTS – Dimethyltrisulfide
DMSeDS - Dimethylselenodisulfide
DMDSeS - Dimethyldiselenosulfide
Current
• The current research is focusing on the
species produced between the initial
amendment and the aforementioned
products.
• Previously, the real-time conversion of
selenate to selenite was observed via the
use of capillary electrophoresis [2].
Current cont.
• Recently, analysis of the sterile-filtered
broths of these bacteria were evaluated
using ion chromatography with inductively
coupled plasma mass spectrometry.
• One important find was the presence of
selenocyanate in the broth of a culture that
had been amended with selenate.
IC-ICP/MS* Results
IC-ICP/MS determined
concentrations in ppm Se (SD)
Se O32Se O42SeCN0
0
Sterile LB growth me dium
IC-ICP/MS determined
concentrations
2-2-in ppm Se (SD)Se O32Se
SeCN
3 O4
0
0
Sterile LB + 1 mM Se O
Sterile LB growth me dium
92.4
2.8
0.058
Sterile LB + 1 mM Se O42-
0.30
25.4
0.064
24.2 (4) 2.2 (0.04)
0.037 (.003)
Bacte rium 130404 + 1 mM Se O32-
2Bacte
rium
130404
+
1
mM
Se
O
3
Bacte rium 130404 + 1 mM Se O
0.67 (0.08) 39.4 (6.4)
0.172 (0.01)
24
Bacte rium 130404 + 1 mM Se O42-
92.4
2.8
0.058
0.30
25.4
0.064
24.2 (4) 2.2 (0.04)
0.037 (.003)
0.67 (0.08) 39.4 (6.4)
0.172 (0.01)
0
Sterile LB + 1 mM Se O32-
Sterile LB + 1 mM Se O42-
0
*IC-ICP/MS analysis by Applied Speciation and Consulting, LLC, Tukwilla, WA
The Goal
• The goal of this research is to develop a
method to monitor the production of
selenocyante in bacterial cultures
amended with selenate.
• Because of the low concentrations of
selenocyanate found in previous
experiments, solid-phase extraction will be
employed to preconcentrate analytes.
CE and Standard Prep
• The run buffer for the CE was a mixture of 15 mM
potassium dihydrogen phosphate and 3 mM
tetradecyltrimethylammonium bromide (TTAB).
• The pH of this solution was adjusted to 10.5 with a 1.0
mM NaOH solution.
• Selenocyanate standards were prepared in HPLC grade
water.
• All standards, samples and wash fluids were filtered with
0.2 micron syringe filters before being placed in the CE.
Solid-Phase Extraction
• Aminopropyl Isolute SPE cartridges from International
Sorbent Technologies were used.
• The cartridges were first solvated with 10 mL of 15 mM
potassium dihydrogen phosphate in a 50/50 mix of
methanol and water.
• The sample (10 mL of 1.0 mM selenocyanate) was then
run through the cartridge at approximately 10 mL/min.
• Finally, the sample was eluted with 2 mL of a solution of
sodium hydroxide with a pH of 11.8.
CE Conditions
• The capillary was kept at 25 degrees Celsius for each
run.
• Sample injection was accomplished with 0.5 psi pressure
injection for 5 seconds.
• Finally a -25 KV potential was run across the capillary for
five minutes to establish and maintain the electroosmotic
flow (EOF).
SeCN in the CE
40
40
P/ACE MDQ-200 nm
5.0 mM SeCN
Migration Time
Area
Width
20
20
10
10
0
0
mAU
30
mAU
30
-10
0.0
-10
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Minutes
Figure 1: 5.0 mM Selenocyanate
4.0
4.5
5.0
Standards
Selenocyante Standards
900000
800000
Peak Area
700000
600000
500000
400000
300000
y = 82415x - 9477.6
200000
2
R = 0.9948
100000
0
0
2
4
6
8
Conc. (mM)
Figure 2: Standard Curve for Selenocyanate
10
12
SPE Sample
P/ACE MDQ-200 nm
Elution 1
20
Migration Time
Area
Width
10
10
0
0
-10
-10
-20
-20
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Minutes
Figure 3: Sample Extracted via SPE. Concentration: 2.27 mM
5.0
mAU
mAU
20
At This Point…
• Using solid-phase extraction, selenocyanate
concentration has been successfully raised 2.27x.
• But this is just selenocyanate dissolved in water and this
is a far cry from extracting selenocyanate from complex
bacterial media.
• So several components need to be tested:
– NaCl
– Yeast Extract
– Peptone C
Testing NaCl
• The next series of experiments will be designed to test
whether or not the different components of the bacterial
medium will be preferentially adsorbed by the SPE
cartridge.
• A sample containing 1.0 mM selenocyanate and
85.6 mM NaCl was extracted using SPE.
• It was then run under the same conditions as the
previous samples.
SeCN and NaCl
100
80
P/ACE MDQ-200 nm
1.0 mM SeCN + NaCl Extract
100
Migration Time
Area
Width
80
40
40
20
20
0
0
mAU
60
mAU
60
-20
-20
-40
-40
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Minutes
Figure 4: NaCl and SeCN Extraction (SeCN conc. = 0.39 mM)
5.0
NaCl
• SeCN is being retained on the SPE cartridge to the
extent that the concentration of the eluted sample is
increased.
• NaCl is also retained by the cartridge, but more
experiments are needed to determine whether or not it
binds preferentially to the solid phase.
• A refinement to the extraction process will be required in
order to increase the overall extraction yield.
LB Medium with Selenium
• Next, the LB medium will be prepared with 1.0 mM
selenate and selenocyanate.
• To test interference from selenate, NaCl will be omitted
from the sample.
• The same extraction technique will be used.
Extraction with Various
Amendments
Conclusions So Far
• Extraction of selenocyanate becomes problematic in the
presence of NaCl
• The presence of selenate, however, doesn’t seem to
interfere with SPE
• When sample is extracted in a sample medium without
NaCl, selenocyanate is retained on the solid phase
Acknowledgements
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Dr. Thomas Chasteen
Bala Krishna Pathem
SHSU Chemistry Department
The Robert A. Welch Foundation
References
1.
2.
Challenger, F (1945) Chem. Rev. 36:315-361.
Pathem BK, Pradenas GA, Castro ME, Vásquez CC, Chasteen
TG (2007) Anal. Biochem. 364:138-144.