ONLINE COUPLING OF ROBOT-ASSISTED SINGLE
DROP MICROEXTRACTION AND LIQUID
CHROMATOGRAPHY ANALYSIS
Deyber A. Vargas Medina; Luis Felipe Rodriguez Cabal; Fernando Mauro Lanças;
Álvaro José dos Santos Neto*
São Carlos Institute of chemistry (IQSC/USP -Brazil)
*alvarojsn@iqsc.usp.br
MATERIALS AND METHODS
INTRODUCTION
In this study, single drop microextraction (SDME) and liquid chromatography (LC)
a.
• Experimental conditions
I: Robot assisted in syringe microextraction
were online integrated for the first time in a fully automated setup. SDME is carry
•
A lab made autosampler (Figure 1a) was hyphenated with a HPLC
out by a lab made cartesian robot actuating a 100 µL syringe equipped with a three
instrument, through a NResearch three way solenoid valve
way solenoid microvalve. In the “off” position, the microvalve connect the syringe
poppet. The valve was connected in the endpoint of the syringe thought
barrel to the extraction needle and in the “on” position, the valve allows to transfer
valve.
a dual 1/4-28 female and actuated by a 10 A relay (Songle®) module.
Analytical column
the syringe content to the chromatographic system, through a six-port switching
Waste
c.
b.
•
an additional 10 A relay (Songle®) module.
robot perform the SDME procedure. When the extraction is completed, the “on”
II: on-line injection of the extract
•
position is activated and the robot inject the organic extract into a 2-µL sampling
the fully automation process (Figure 1b-c).
d.
into the analytical column. Finally, the valve returns to the initial position and,
•
while the chromatographic analysis is carried out, the robot clean-up the system
bus module, a SIL-20AC auto sampler, a DGU-20AS degasser, two LC 20-
All method, including the combined action of the robot, valves system and the
Waste
AD pumps, a CTO-20A column oven and a SPD-20A UV-Vis detector,
HPLC triggered, is controlled by an Arduino microcontroller. Triazines were
monitoring at 220 nm.
employed as model compounds to assess the automated developed setup. The
Figure 1. (a) Lab made autosampler;
Figure 2. Schematic representation of the on-line •
A mixture A: Water and B: MeOH/ACN was employed as mobile phase at
(b-c)
coupling of the robot assisted SDME process and
the flow rate of 0.2200 mL min-1. Isocratic elution was programed with
the chromatographic analysis.
54 % of B for 10 min.
Drop
expander;
(d)
HPLC
instrument.
DISCUSIONS
RESULTS
and the maximum fraction of the extracted analyte is limited by the equilibrium
between phases, according with the relation:
𝐾𝐷 𝛹
1+ 𝐾𝐷 𝛹
(1)
Where, E is the fraction of extracted analyte, KD is the distribution constant and Ψ
the phase relation (Vorganic/Vaqueous). On the other hand, reaching equilibrium state
120
120
100
100
80
80
60
60
40
40
20
20
0
0
can be a slowly process, even when small fractions of organic phase are employed.
In this study, in order to obtain an efficient and high-throughput method for the
extraction of triazines via on-line SDME, the effect of the stirring, salt addition,
extraction solvent, extraction drop volume and extraction time was studied (Figure
3).
Using the developed setup, a 60 µL 1-octanol drop, under stirring conditions (600
Stirried
extractions in equilibrium conditions are time-consuming process. The developed
method allow the extraction and on-line analysis of target analites just in 10 min,
the
synchronization
between
the
sample
treatment
and
the
chromatographic analysis.
No-stirried
1-octanol 1-nonanol1-decanol
200000
200000
150000
150000
150000
100000
100000
100000
50000
50000
50000
0
0
0
0
60
40
40
20
20
0
0
30 µL
60 µL
100 µL
200
0 % Sal
c.
2 % Sal
5 % Sal
d.
120
•
Automated
hyphenation
of
SDME
and
analyte
determination
Simazine
80
60
Atrazine
Propazine
4 min
liquid
chromatography allowed the development of a high throughput analytical
method. In the shown case, all the analytical protocol, including, extraction,
separation and analyte detection, was carry out just in 10 min.
•
Future work will be directed towards the use of the developed setup in the
online hyphenation of others microextraction techniques. Hollow fiber liquid
phase microextractions (HF-LPME) or microextractions by packed sorbent
7 min
10 min
400
c.
10
5
0
-5
-10
-15 0
1
2
3 4
Level
propazine
Atrazine
5
6
10
10
5
0
-5
-10
-15 0
1
2
3 4
Level
a.
5
6
5
0
-5
-10
-15 0
calibration linear model.
µg L-1 of each analyte.
• Recovery/Enrichment
Simazine
Atrazine
propazine
2
3 4
Level
5
6
c.
effect; (e) extraction time. Experiments were carried out in direct
immersion static mode using 8.0 mL of ultrapure water spiked with 100
1
b.
Figure 6. Percentage of relative error Vs triazines concentration
of hardware.
for the non weighted
• Analytical performance of the automated on-line
SDME-LC procedure
Analyte
Linearity
(µg L-1)
Intercept
Slope
r2
(µg L-1)
LOD
LOQ
(µg L-1) (µg L-1)
Intraday
Interday
RSD, %
RSD, %
RF
EF
%
Simazine
10-250
-2735.5
760.25
0.9942
2.5
5
13.46
13.67
16.7 12.5
Atrazine
10-250
-2353.2
917.18
0.9933
2.5
5
12.03
11.32
15.2 11.4
Propazine
10-250
-4224.9
888.93
0.9917
5
10
11.13
10.58
18.4 13.8
• Determination of triazines in real samples by
the developed automatic online-SDME
Finally, future work will be directed towards the hyphenation of the developed
sample preparation set up with mass spectrometry instrumentation for the
development of non-separtive analytical methods.
Sample
2.075 4.575 7.075 9.57512.07514.575
Retention time / min
Figure 4. Illustrative chromatogram for the direct injection of a 100 µg
L-1 triazines standard solution (black line) and the extract of the same
São Paulo Research Foundation (FAPESP), grant #2010/19910-9, #2014/03795-0,
#2016/21950-5, and #2017/02147-0.
200
b.
e. solvent; (c) drop volume (d) Salting out
(b) selection of the extraction
(MEPS), could be carry out in the similar setup, without additional requirements
•
0
400
• Residue analysis of the linear models
100
1 min
by
200
Simazine
0
analysis has been carried out for the first time.
0
400
triazines by on-line automated SDME. Concentration Range 5 – 250 ng mL-1.
20
In this study, the automated hyphenation of SDME and liquid chromatography
200000
Figure 5. Plots Chromatographic response Vs concentration for the extraction of the target
Figure 3. Relative extraction efficiency (EE) for the (a) effect of stirring;
•
250000
a.
40
CONCLUSIONS
250000
80
60
y = 888.93x - 4224.9
R² = 0.9917
250000
100
80
propazine
y = 915.4x - 2038.1
R² = 0.9942
y = 760.25x - 2735.5
R² = 0.9942
120
100
Atrazine
Simazine
b.
120
rpm) and not salt addition allowed the efficient extraction of triazines with recovery
factors from 11 to 15 %. Although, equation (1) preconize recoveries around 70 %,
a.
• Linear models (under selected conditions)
X - Relative residue
extraction efficiency. Nevertheless, the LPME techniques are not exhaustive process
• Study of the influencing parameters
X - Relative residues
the sample solution to the organic drop must be controlled to obtain maximum
X - Relative residues
In liquid phase micorextractions (LPME), parameters influencing mass transfer from
allowing
Triazines were employed as model compounds and the analysis was
carry out in a Shimadzu LC system including a CBM-20A communication
Analytical column
and prepare the next extraction.
𝐸=
A lab made adjustable needle endpoint (drop expander) was attached in
the sample reservoir, making compatible the drop volume expansion and
loop, afterwards the six-port valve is switched and the organic extract is injected
performance and analysis of real samples will be presented and discussed.
All the components were controlled by an Arduino UNO microcontroller.
Arduino was communicated with the HPLC thought the events port and
Initially, the LC pumps deliver mobile phase to the analytical column, while the
method development, including study of the influencing variables, analytical
with PTFE
standard solution after automated SDME-HPLLC (green line).
Analytes
Spiked
(µg
L-1)
Mineral water
Coconut water
Peach tea
Found
Recovery
Found
Recovery
Found
Recovery
(µg L-1)
(%)
(µg L-1)
(%)
(µg L-1)
(%)
Simazine
20
17.8 ± 1.1
89
13.3 ± 0.4
67
7.7 ± 1.0
38
Atrazine
20
16.2 ± 1.4
81
12.8 ± 0.6
64
< LQ --
--
Propazine
20
18.2 ± 1.3
91
12.8 ± 0.8
64
5.4 ± 0.9
27