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![Page 1: Roger Kautz, Principal Research Scientist In Collaboration with Protasis / MRM and Varian Instruments Routine Manual Injection of Trace Samples Our Optimized.](https://reader035.fdocuments.us/reader035/viewer/2022062511/551b5e9d550346dd1a8b660f/html5/thumbnails/1.jpg)
Roger Kautz, Principal Research ScientistIn Collaboration with Protasis / MRM and Varian Instruments
Routine Manual Injection of Trace Samples
Our Optimized LC-MS+NMR Strategy With a CapNMR Probe
The Barnett Institute of Chemical and Biological Analysis
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The Barnett Institute of Chemical and Biological Analysis
LC-Column capacity 100 µg/peak (4 mm column)On-flow NMR sensitivity 30 µg (60 µL flow cell, 15 sec intervals)LC Peak Volume 150 µL (10 sec peak at 1 mL/min)
And LC-MS-NMR had the same problems.
NMR LOD 1 µg overnight (60 µL LC probe) MS LOD 1 ng 1 second
Collect Fractions. Use automated NMR to analyze the fractions. Concentrate fractions and use the most sensitive NMR available µNMR LOD 0.2 µg, 1 hour (1 µL microcoil)
Allocate NMR time intelligently.
Online LC-NMR was a flail
4 sec = 60 µL
5% degradant of interest
Offline LC+NMR Makes Sense:
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Microcoil NMR
Probe Type PreparedSample Volume
Relative Mass Sensitivity1
Observed S/N(Subramanian)
Conventional 5 mm 550 µL 20 µg
3 mm Cryoprobe 3 mm sample tube
160 µL 4 µg 3204
3 mm Cryoprobe 1.7 mm sample tube
40 µL 2 µg 5021
Microcoil Direct Injection
8 µL 2 µg 4098
Microcoil SFA-NMR
2 µL 0.5 µg
The Barnett Institute of Chemical and Biological Analysis
1 Sensitivity radius Solenoid = 3x saddle coil Cold Probe = 4x RT probe Flow Probe is 1 mm smaller than tube probe
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NMR Coil 30 nL
Capillary 30,000 nL
The Barnett Institute of Chemical and Biological Analysis
2 hoursto acquire spectrum
1 minuteto acquire spectrum
NMR Sensitivity is Highly Concentration-DependentA 10-fold increase in concentration reduces the time by 100-fold,
to obtain similar quality data.
Sample Efficiency: “ From Vial to V-observe”
1 L observe
8 L dead
A B C
Kautz, Lacey, Wolters, Webb, Sweedler et al “capillary isotachophoresis”, JACS, 2001
Percent of sample in vial that ultimately produces signal in Vobs 4
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Flow Injection(solvent-filled flowcell)
Direct Injection(air-filled flowcell)
Direct Injection with Chaser
Ways to fill a flow cell
Parabolic Flow
Taylor Dispersion
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MRM Probe(air filled)
200 µm tubing
2 µL solvent“leader”
2 µL sample
Chase solvent
30% sample efficiency
10 µL syringe,Syringe Pump
Direct Injection with Leader and Chaser
Dispers ion of 2 uL sam ple injected into MRM ICG Probe
0.00
20.00
40.00
60.00
80.00
100.00
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Injection Volum e (uL)
Pea
k In
tens
ity
lead_integral
lead_height
sam ple_integral
sam ple_height
chase_integral
chase_height
The Barnett Institute of Chemical and Biological Analysis
filter
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FIA (miscible carrier)
SFA (immiscible carrier)
Zero-Dispersion SFA
Microcoil NMR flow cell (10 uL)1 uL dye, flow injection
Microcoil NMR flow cell (10 uL)1 uL dye, Zero-Dispersion SFA
Sample Wets Capillary Wall
Carrier Wets Capillary Wall
Parabolic Flow
Zero-Dispersion Segmented Flow
Taylor Dispersion
The Barnett Institute of Chemical and Biological Analysis
Behnia & Webb “Perfluorocarbon Plugs” 1998, ; Lacey et. al “Single Bead”, 2001
Curcio & Roeraade “Continuous Flow PCR” (2003); Nord & Karlberg, 1984.
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The Barnett Institute of Chemical and Biological Analysis
0.3 µg/µL beta-methyl-glucoside in D2OSmall Samples Produce Equivalent Spectra 8
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NMR Quality of D2O Plugs in FC 43 Carrier FluidPassing Through MRM ICG Microcoil Probe
0
1
2
3
4
5
6
7
8
-3 -2 -1 0 1 2 3
3 uL lock
3 uL linewidth
1 uL lock
1 uL linewidth
2 uL lock
2 uL linewidth
2 uL lock
2 uL linewidth
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Preparing Trace Sampleswith keeper no keeper no keeperwith keeper
Recovery from FAS-coated glass vial insert. Note DMSO contact angle even as drawn down. (360 µm o.d. capillary)
The Barnett Institute of Chemical and Biological Analysis
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Microcoil NMR Sensitivity Enhanced by SFA
Peaks labeled “D” are seen in similar acquisition of clean solvent (DMSO)
Indirect Carbon NMR Data from 50 ug taxol (HSQC, HMBC)
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1.5 µg Erythromycin gives COSY and TOCSY in 10 hr
The Barnett Institute of Chemical and Biological Analysis
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High Throughput Segmented Flow Microcoil NMR
The Barnett Institute of Chemical and Biological AnalysisKautz et al., J. Combi Chem 7: 14-20. (2005)
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TMSP peak, 2 sec intervals
Start flow,(Well 74 in probe)
Wash PlugDetected Flow Stopped,
Well 75 centered in probe
Detect and Position Sample Plugs
The Barnett Institute of Chemical and Biological Analysis
flow
WashplugSample
plug
Washplug
Sampleplug
Previous Sample
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UV-DAD
LC-MS
Nanosplitter ESI-MS
Culture
Bioactivity
Segmented Flow Loading
Sample Recovery
LC-MS-microNMRNatural Product Identification
Bioactive Fraction
LC Separation
Fraction Collection200 µL Fractions
NMR
Microcoil
Evaporate LC Solvent
Resuspend in 2 uL NMR solvent
The Barnett Institute of Chemical and Biological Analysis
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The NanoSplitter LC-MS Interface
Sampling Flat Region of Parabolic FlowPreserves Chromatographic Resolution.
Sampling 0.1% of LC Flow Makes MS A Non-destructive Method
100-fold Better S/N.
Nano-electropray Avoids Ion Suppression
The Barnett Institute of Chemical and Biological Analysis
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Total Ion Chromatogram
min2 4 6 8 10 12 14
mAU
0
250
500
750
1000
1250
1500
1750
2000
DAD1 C, Sig =210,8 Ref=360,100 (YIQING \ YIQING_092706_5.D)
1.61
8
4.61
1
10.4
37
13.0
49
5.09
1
7.33
2
9.65
7
11.8
78
RT: 0.06 -16.05 SM: 7B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Time (min)
5101520253035404550556065707580859095
100
Rel
ativ
e A
bund
ance
4.42
12.8910.26
9.49
4.93 8.597.185.251.18 7.504.09 6.162.33 2.52 10.65 12.24 13.300.84 14.13 14.55
UV
NMR
LC-MS-NMR
cycloheximide
20 µg
1 µg
.2 µg
(1/15 y-scale)
1.5x y-scale
1x y-scale
X
The Barnett Institute of Chemical and Biological Analysis
1 hr /fraction
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ConclusionsImproving sample efficiency can give several-fold gains in sensitivity. Dry with keeper in low-retention vial; recover in 1-3 uL Minimize dead volume
CapNMR LOD’s (with segmented-flow sample loading) : 50 ng overnight for dereplication (200 ng, 1 hr) 1-2 µg for COSY, TOCSY overnight 20 µg for HSQC 50 µg for HMBC Offline LC-NMR (or LS-MS+NMR) can be recommended Can acquire comprehensive LC-NMR data overnight; Can review LC or MS data to select samples of interest. Could be performed using any automated loading method. Lets chromatographer use his own, validated equipment. Can be done retrospectively. DirectionsData Dependent AcquisitionSample Recovery Better Software; LEAP autosampler The Barnett Institute
of Chemical and Biological Analysis
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Northeastern Yiqing LinCarmelina Freni
Paul Vouros
Barry KargerFrantisec ForetTomas Rejtar
James Waters
Illinois
Jonathan SweedlerAndrew WebbMichael LaceyAndrew Wolters
Protasis / MRM
David Strand
Tim PeckDean OlsonJim Norcross
Varian
Daina AvizonasSteve Smallcombe
Paul Keifer
With Gratitude To:
Flow InjectionCharles Patton
MGHJ. Manuel Perez
ArquleWolfgang GoetzingerJun Zhao
Univ. Illinois ChicagoJimmy Orjala
NIH R01 GM075856-01
The Barnett Institute of Chemical and Biological Analysis
Yiqing Lin
Jimmy Orjala
Paul Vouros
Roger Kautz