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Slides download at: http://boilthisdown.org/?p=1713
ACS WEBINARS How Chemistry is keeping Your Food Safe - Application of ultra high pressure liquid chromatography in a Food Safety Laboratory
Speaker: Walter Hammack Florida Department of Agriculture Moderator: Joe Romano Waters Corporation Please submit your questions for the speaker via the Chat Panel in the GoToWebinar
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ACS Webinars Today’s episode is made possible by the following sponsors.
Slides download at: http://boilthisdown.org/?p=1713
Walter Hammack • Florida Department of Agriculture and Consumer Services, Division of Food Safety Chemical Residue Laboratory •
[email protected] • 850-617-7514
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Section Responsibilities • • • •
Pesticide Residue Enforcement - State Pesticide Data Program – USDA Antibiotic Residue Program – State Food Emergency Response Network- FERN State/FDA/USDA
OVERVIEW • What is Ultra High Pressure/Performance Liquid Chromatography (UHPLC) • Advantage of UHPLC over HPLC • What type of equipment is needed • Applications in Food Safety/Defense
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What is UHPLC? • UHPLC refers to a system of technologies that allows for faster, and/or, higher resolution liquid chromatographic separations • First commercial instruments available in 2004 from Waters Corporation (UPLCtm) • Industry acronym: UHPLC • Systems that are characterized by the ability to operate at pressures up to and above 15,000 PSI (1000 Bar)
Characteristics of UHPLC Systems • Routine use of sub-2 micron particle columns • Higher operating pressures (15,000 to 18,000 PSI) • Lower delay volumes (80 -125 ul) • Faster Autosampler cycle times (30s Low pressure mixing Peak widths of 20 - 30 seconds
15000 PSI limit 1.7 micron 125 ul delay volume Cycle time = < 15s High pressure mixing Peak widths of 1 – 6 seconds
Carbendazim Name 1 B2815_R1_01_5LOQ_43_09_03E
RT
Area
18:14:57
31-Mar-09
18:36:09
3 B2815_R1_01_37397_Mtx
31-Mar-09
18:57:21
31-Mar-09
19:18:33
5 B2815_R1_01_36636
31-Mar-09
19:39:45
6 B2815_R1_01_36637
31-Mar-09
20:00:58
7 B2815_R1_01_36638
31-Mar-09
20:22:10
8 B2815_R1_01_36639
31-Mar-09
20:43:22
9 B2815_R1_01_36640
31-Mar-09
21:04:34
10 B2815_R1_01_36641
31-Mar-09
21:25:46
11 B2815_R1_01_36642
31-Mar-09
21:46:58
31-Mar-09
22:08:10
13 B2815_R1_01_36645
31-Mar-09
22:29:23
14 B2815_R1_01_36646
31-Mar-09
22:50:36
12 B2815_R1_01_36644
3.5878
266668
Acq.Time 31-Mar-09
4 B2815_R1_01_37398_Spk
3.5878
Acq.Date
2 B2815_R1_01_37396_Rgt
3.5878
110982
45816.3
15 B2815_R1_01_HALFLOQ_43_09_01E
3.5878
24006.9
31-Mar-09
23:11:47
16 B2815_R1_01_1LOQ_43_09_02E
3.5947
51761.7
31-Mar-09
23:33:00
17 B2815_R1_02_5LOQ_43_09_03E
3.5947
251840
31-Mar-09
23:54:12
18 B2815_R1_01_10LOQ_43_09_04E
3.5947
526681
1-Apr-09
0:15:25
19 B2815_R1_01_20LOQ_43_09_05E
3.5878
979322
1-Apr-09
0:36:38
20 B2815_R1_01_50LOQ_43_09_06E
3.5876
2274295
1-Apr-09
0:57:50
21 B2815_R1_01_Blank
1-Apr-09
1:19:02
22 B2815_R1_02_Blank
1-Apr-09
1:40:15
23 B2815_R1_01_36647
1-Apr-09
2:01:28
24 B2815_R1_01_36648
1-Apr-09
2:22:41
25 B2815_R1_01_36649
1-Apr-09
2:43:54
26 B2815_R1_01_36650
1-Apr-09
3:05:07
27 B2815_R1_01_36651
1-Apr-09
3:26:20
28 B2815_R1_01_36652
1-Apr-09
3:47:33
29 B2815_R1_01_36653
1-Apr-09
4:08:46
30 B2815_R1_01_36663
1-Apr-09
4:29:59
31 B2815_R1_01_36664
1-Apr-09
4:51:12
32 B2815_R1_01_36665
1-Apr-09
5:12:25
33 B2815_R1_01_36666
1-Apr-09
5:33:38
34 B2815_R1_01_36667
1-Apr-09
5:54:51
35 B2815_R1_02_37398_Spk
3.5947
113288
1-Apr-09
6:16:03
36 B2815_R1_03_5LOQ_43_09_03E
3.5878
267443
1-Apr-09
6:37:15
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Detector Systems Compatible with UHPLC Systems • PDA
• • • • •
FLD Triple quadrupole MS Ion Trap MS Time-of-Flight (TOF) MS FTMS
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Beware Extra Column Volume with narrow bore columns • Extra-column volume is the volume in the system between the injector and the detector, excluding the column. • Injectors on UHPLC systems are optimized by the manufacturer to minimize this volume • Connects made by users in the lab to the column and to the detector can be a source of significant excess volume in the system and must be minimized for good chromatography
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Tailing problem - Pyraclostrobin
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Tailing problem solved
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UHPLC for Food Safety/Defense ?
• Resolution for peak capacity • Speed for high throughput applications • Combinations of both
Chromatographic Resolution
• Very high peak capacity can be obtained with runs of 20 -80 minutes • Peak capacity is often critical to identification of unknown contaminants in complex chromatograms
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DIMETHOMORPH– Waters 2695 (low pressure mixing) UNRESOLVED -32 MIN GRADIENT
DIMETHOMORPH– Agilent 1100 (high pressure mixing) PARTIAL RESOLUTION – 32 MIN GRADIENT
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DIMETHOMORPH – Acquity UHPLC COMPLETELY RESOLVED – 20 MIN GRADIENT
OXAMYL – ALDICARB SULFONE High Pressure Binary – UNRESOLVED – 32 MIN GRADIENT
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OXAMYL – ALDICARB SULFONE UHPLC – baseline resolution- 2.0 x 250 , 1.8u , 20 MIN GRADIENT
Toxin at m/z 276 in Beer
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BEER High Pressure Binary - 2.0 x 150 , 1.7u (80 MIN)
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UHPLC 40 min method
Chromatographic Speed
• Sub-minute gradient runs • Very important for target analysis of emergency samples (10,000) • Only the fastest detectors can acquire data • Cycle time of autosampler is now a factor in sample throughput – 10,000 injections requires 125 hours at 45 seconds per injection cycle • Every additional second of injection and analysis requires 2.8 hours of additional run time for 10,000 samples.
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Examples
• Pesticides in fresh produce • Drug residues in fish tissue • Melamine in food products
UHPLC – 2.0 x 150 mm, 1.7 micron – 32 minute
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DIMETHOMORPH – UHPLC – 2.0 x 150 mm, 1.7 micron 8 MIN GRADIENT
Acquity – 1.0 x 50 mm – 4 minute
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4 min gradient – ACN/H20 Time
%A
%B
0
Flowml/min 0.3
95
5
2
0.3
5
95
2.5
0.3
5
95
2.6
0.3
95
5
4.0
0.3
95
5
Carbendazim – Tomato -100 ppb
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Carbendazim – Spinach -10 ppb
UHPLC – 1.0 x 50 mm – 2 minute
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12.5 Minute Gradient – Fish Tissue UHPLC 1.0 x 100 mm C-18 Time
%A
%B
0
Flow ml/min 0.12
95
5
1
0.12
95
5
6.2
0.12
5
95
8.0
0.12
5
95
8.1
0.12
95
5
12.5
0.12
95
5
Flounder – Matrix
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Flounder – Spike – 5ppb
Flounder – Spike – 5ppb-ms^3
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Melamine – HILIC – 1.7 micron – 5.5 minutes
Conclusions - UHPLC • Ultra high pressure systems offer significantly greater capabilities than standard pressure systems and will ultimately become the “standard” HPLC system • Run time are routinely improved by factors of 2x to 5x with little difficulty • Very high resolving power can be achieved with a combination of shallow gradients, small particles, and long columns • Detector systems require very high acquisition rates to take full advantage of UHPLC speed
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Q&A SESSION How Chemistry is keeping Your Food Safe - Application of ultra high pressure liquid chromatography in a Food Safety Laboratory
Speaker: Walter Hammack Florida Department of Agriculture Moderator: Joe Romano Waters Corporation Please submit your questions for the speaker via the Chat Panel in the GoToWebinar Slides download at: http://boilthisdown.org/?p=1713
Upcoming Event: April 8, 2010. 2-3pm ET “Creating a Successful Career in Public Policy and the Chemical Industry – Lessons Not Taught in Classrooms”
ACS Webinars: Your Career Matters! Series Connecting you with subject experts and global thought leaders in chemical sciences, management, and business. To Register: http://boilthisdown.org/?page_id=385
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ACS Webinars Today’s episode is made possible by the following sponsors.
Slides download at: http://boilthisdown.org/?p=1713
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