Introducing Parallel Segmented Flow LC separations to pesticide residue analyses Cecilia Rodrigueza, Arianne Solivena, Andrew Shallikerb, Lucia Parejaa, Andres Pérez-Paradaa*, Horacio Heinzena* aGrupo de
Análisis de Contaminantes Traza (GACT), Universidad de la Republica, URUGUAY for Research on Separation Sciences (ACROSS), Western Sydney University, AUSTRALIA Email:
[email protected] /
[email protected]
bAustralian Centre
INTRODUCTION 4.6 mm i.d.
Active Flow Technology (AFT) refers to a new HPLC column technology, with two modes of operation, either parallel segmented flow (PSF) or curtain flow (CF). In both modes, the AFT column end fitting design allows for the mobile phase traversing the radial central region of the bed, to be isolated from the mobile phase traversing along the column wall region. As such, the mobile phase that carries sample via the radial central region of the column bed is free from column wall effects, and the separation efficiency is much higher than conventional columns. The AFT end fitting has a three piece annular frit with a porous central (inner) zone, separated from an outer peripheral porous zone by an impermeable ring. The endfitting is designed with multiple exit ports, enabling simultaneous multiplexed detection, with the peripheral port directed to waste/a second detector. In the search of a new state of the art separation technique for LC trace residue analyses, a mixture of pesticides was studied via PSF and CONVENTIONAL separations.
22% means virtual 2.1 mm i.d.
To waste or second detector To MS RADIAL SEGMENTATION Experimental peak profiles and HETP plots of Naphthalene for 4.6 x 100mm columns packed with 3um,comparing Conventional vs PSF1.
EXPERIMENTAL Comparisons were made at constant flow to the detector
Tested pesticides: Atrazine – log Kow= 2.7 Imazalil – pH sensitive, log Kow =2.56 Diazinon – log Kow =3.69 Pyraclostrobin – log Kow = 3.99 Pirimiphos- Methyl - log Kow = 3.9
CONVENTIONAL C18 (100 x 4,6 mm id) 3µm – Thermo Scientific
INSTRUMENTAL CONDITIONS Mobile Phase: H2O:MeCN, Gradient Conventional: 5-100 % MeCN in 30 min, 0.6 mL/min. PSF: 5-100 % MeCN in 12 min, 1.5 mL/min 41% to detector.
• Detector: Diode array, 13uL flow cell
Mobile Phase: H2O (0,1%Formic acid): MeCN (0.1%Formic acid) (55:45) Conventional: 0.6 mL/min PSF: 3 min for PSF 1.5 ml/min separation with 43% (0.6 ml/min) to detector ESI sorce at 500ºC, Ion spray voltage 5500 V, Curtin gas: 20psi; Ion source gas 1: 50; Ion Source gas 2: 50
PSF C18 (100 x 4,6 mm id) 3µm – Thermo Scientific
Solvent Citrate QuEChERS extracts of Lemon
RESULTS Atrazine 220 nm
CHROMATOGRAPHIC ISSUES 1400 1200
AcOEt
Conv.
• HPLC Agilent 1200 • Sciex 4000 QTRAP® MS/MS • ESI Turbo V Positive Mode
LC-MS/MS
PSF
N
108939
56043
Res
4,66
5,48
Area
59,47
24,57
Height/P. Width
2727
4626
MS INTERFACING
2,50E+06
AREA vs FLOW RATE TO MS AT SAME CONCENTRATION
Conventional + standard ESI
ATRAZINE
2,00E+06
1000
Conventional + optimized ESI for low flow rates 28% segmentation
1,50E+06
800
Ionization efficiency effect on flow rate to MS
600
Diazinon + Pyraclostrobin 400
Area
mAu a 200 nm
LC-DAD
MS interfacing
CALIBRATION CURVES PERFORMED IN
TIME REDUCED BY HALF
• Thermo Scientific Ultimate 3000
Chromatographic issues
1,00E+06
12 % segmentation
5,00E+05
1400 200
Imazalil
AcOEt
1200
Atrazine
AcOH
AFT
Pirimiphos-Me
Conventional
0,00E+00 0
500 1000 Flow rate to MS (uL/min)
1
1000
1500
-200 0
800
2
4
6
8
10
min
12
14
16
PSF peaks are thinner and taller but with lower area than Conventional
0,5
12 min
Diazinon + Pyraclostrobin
600
0 1
400
Imazalil
200
Atrazine
AcOH
5 10 25 50
1
5
Peak width
Pirimiphos-Me
5
5
AFT 10 25 50 1,20E+05 1,00E+05
0
5
10
15
20
min
25
30
35
30 min
CONCLUSIONS
Atrazine
8,00E+04 6,00E+04 4,00E+04 2,00E+04
The AFT separation diminishes half time of the chromatografic run in gradient separation
High throughput analysis protcols can be developed platforms2
Interfacing with MS employed for trace analysis with separation at higher flow rate. Segmentation ratio can be adjusted to improve sensitivity Under these conditions, the peak area was reduced proportionally to the segmentation but peak height and width improved, particularly at lower concentrations Potential to reduce matrix effect. This is an important issue as the matrix tested was Lemon, a very complex one. Further work is foreseen seeking the sensibility maximization and high throughput analysis implementation
Acknowledgments Marcos Colazzo for technical assistance in LC-MS CSIC I+D Project 685, UdelaR A.S. is a recipient of an ANII PD scholarship.
0,00E+00 0
20
40
1,00E+06 9,00E+05 8,00E+05 7,00E+05 6,00E+05 5,00E+05 4,00E+05 3,00E+05 2,00E+05 1,00E+05 0,00E+00
Atrazine
0
60
AREA
0
HEIGHT
-200
Area
mAu a 200 nm
0
Height
20
Solvent AFT
Matrix matched AFT
Solvent Conventional
Matrix matched Conventional
pka log Kow
Acetamiprid 0,7 0,8
Sensitivity (slope comparison) in terms PSF vs Conv. of: Area - Solvent -53,80% Area - Matrix matched -75,50% Height - Solvent 18,30% Height - Matrix matched 60,70%
References
40 ppb
ppb
Atrazine 1,6 2,7
Pirimicarb 4,53 1,7
PSF vs Conv.
PSF vs Conv.
-58,10% -57,42% 15,90% 13,00%
-60,00% -64,26% 53,50% 49,69%
1.F. Gritti et al., J. Chromatogr. A 1333 (2014) 32-34. 2.R.A. Shalliker, J. Chromatogr. A 1509 (2017) 176-178.
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