Ultra High Sensitivity NMR: 1mm HTS Triple Resonance Probe

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Todays Lecture
12) Fri, Oct 30 Probes and S/N
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NMR is Very Insensitive
b
The NMR signal comes from a transition between ?
and ? energy states
Bgt0
500 MHz
750 MHz
900 MHz
B0
11.7 T
(Na/Nb)1.00008
17.6 T
?
21.1 T
1.00012
1.00014
Very low starting population!
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Ways to Improve S/N in NMR

• Higher magnetic field strengths S/NB07/4
• Low temperature (not so easy with
  proteins/aqueous samples)
• Hyperpolarized gases
• Dynamic Nuclear Polarization
• Smaller coils S/N1/d
• Cryogenic probes (lower the noise)
• High Temperature Superconducting Probes (higher
  Qmore signal)
• Combination of small coils, cryoprobe, and HTS

Improve Boltzmann term

Improve detection
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Standard NMR Probes Require a Very Large Amount
of Sample

• 1 mM
• 500-600 mL
• 500 nanomoles
• 10 mg of a 20 kDa protein
• 1/2 mg of a 500 Da natural product

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Some common copper-wire probe designs
The basic requirements for a probe are that it
has an electrical conductor oriented to deliver a
magnetic field B1 that is perpendicular to the
static field B0.
Alderman-Grant or Slotted line coil
Saddle coil
Solenoid
Typical inner coil of conventional top-loading
probes
Used by Protasis for microflow probes
Typical decoupling coil
Alderman Grant, JMR 36, 447 (1979)
Hoult Richards, JMR 24, 71 (1976)
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NMR and Mass Spec are the Primary Analytical
Techniques To Identify Natural Products

• Mutacin 1140
• Potent broad spectrum antibiotic discovered at UF
• Structure determined in 2000 by Jim Smith in the
  Edison lab
• Required gt1 mg of material with the best
  commercial instrumentation
• It took gtgt1 month of work to produce and purify
  the sample
• Now being developed by UF spin-off company,
  Oragenics

Smith et al. Eur. J. Biochem. 267, 1-8 (2000)
Smith et al. Biochemistry 42, 10372-10384 (2003)
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NMR Can Provide Covalent Structure of Molecules
Mass spectrometry initially got the thioether
bridges wrong. NOE distances between protons on
both sides of the sulfur atoms established the
correct structure.
Smith et al. Eur. J. Biochem. 267, 1-8 (2000)
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Problem

• NMR is very useful in molecular structure
  identification, but it is very insensitive.
• Therefore, NMR is often the major rate limiting
  step in natural product discovery.
• Mass Spec techniques are much more sensitive and
  amenable to small quantities of sample.

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Signal to Noise

• Q is defined as the frequency of the resonant
  circuit divided by the half power bandwidth
• Q Values
• RT copper) several hundred
• Cryo copper) a few thousand
• YBCO) 10-25 thousand

(Neglecting Sample Loss)
Ps RF power absorbed by sample Pc RF power
absorbed by coil Ta preamp temperature Tc coil
temperature Ts sample temperature
M0 Sample magnetization B1 RF Field h filling
factor Q Qualify factor of coil
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Smaller Coils are More Sensitive
Excellent Commercial Versions Available From
Protasis
60 mL 1 mM IA3
600 mL 0.1 mM IA3
2.5 mm solenoid
5 mm TXI
Li et al., JMR 164, 128-35 (2003) Wang et al.,
JMR 170, 206-12. (2004) Li et al., Mag Reson Chem
44, 255-62 (2006)
Collaboration with Andrew Webb, Penn State
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Small Solenoids can be Multiplexed
8 COSY Spectra Collected Simultaneously
Wang et al., JMR 170, 206-12. (2004)
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Small, cold, and superconducting1-mm HTS Probe

• Triple-resonance
• z-axis gradient
• 600 MHz
• Minimum sample volume 5 mL

Funded by NIH/NCRR P41 to UF Planning/Design
Edison, Bill Brey (NHMFL) Construction/Design
Rich Withers, Rob Nast (Bruker) Testing Jim
Rocca (UF), Edison (UF)
Brey, W., Edison, A.S., Nast, R.E., Rocca, J.,
Saha, S., Withers, R. Design, Construction,
and Validation of a 1-mm Triple-Resonance
High-Temperature-Superconducting Probe for NMR
Journal of Magnetic Resonance 179, 290-3 (2006).
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Overall Probe Design
Approx fill height

• 1-mm sample chamber temp regulated
• HTS coils cooled to 20 K
• Optimized for 1H
• Uses commercial Bruker Cryoplatform
• Sample vertically loaded

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1-mm HTS Probe
Photograph of a 1H coil
Thin coating of Y1Ba2Cu3O7-x (YBCO) on a sapphire
supporting surface. The desired resonance
frequency is obtained by adjusting the number of
turns, introducing gaps, and trimming the ends of
the material with lasers.
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1-mm HTS Has Excellent 1H S/N
S/N per mmole per scan with 10 mM sucrose
20-25x greater S/N per mass than a conventional
5mm NMR probe.
Can measure low nanomole to high picomole amounts
of material e.g. 13C-HMBC on lt30 nanomole
natural abundance
Brey et al. JMR 179, 290-3 (2006).
Olson et al., Anal. Chem. 76, 2966 (2004).
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Very Limited Sample!Molinski Lab, UCSD
H. sanguineus egg masses - Rose of the Sea
HMBC with 1-mm HTS 48 hours
30 mg of isolated sample existed Not enough for
NMR for nearly 2 decades
90-001-1F4F5
HR-ESI-TOFMS m/z 936.4581982 (MNa)
C46H67N5O14Na, ?0.8 mmu 14 mass unit lower than
Kab B
Dalisay, D. S., Rogers, E. W., Edison, A. S., and
Molinski, T. F. (2009) Structure elucidation at
the nanomole scale. 1. Trisoxazole macrolides and
thiazole-containing cyclic peptides from the
nudibranch Hexabranchus sanguineus, J Nat Prod
72, 732-738
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How to make a better 13C detect probe? Part 1
Put the 13C coil on the inside Problem Now 1H
not optimal
Rich Withers, Varian
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How to make a better 13C detect probe? Part 2
Double Tuned HTS Coils
Spiral resonator for low frequencies (e.g. 15N,
2H)
Racetrack resonator for high frequencies
Single substrate with two HTS coils
Bill Brey, NHMFL Rich Withers, Varian
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Possible HTS Coil Layout
1, 2) 1H and 13C 3, 4) 15N and 2H Other
configurations possible Other nuclei possible
Bill Brey, NHMFL
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What About Higher Fields?

• Commercial instruments available to 1GHz
• In 5-10 years, HTS technology should enable gt30T
  NMR magnets
• How will probes work at these fields?

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We wont be using 5-mm standard tubes with high
fields
At high fields, the sample resistance becomes the
limiting factor (e.g. salt effects)
Ps RF power absorbed by sample Pc RF power
absorbed by coil Ta preamp temperature Tc coil
temperature Ts sample temperature
Ps gtgt Pc
This is true even for pure water at gt900 MHz
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We wont be using 5-mm standard tubes with high
fields

• When Ps is much larger than Pc (gt100 mM salt)
• The radius of the tube needs to be small
• Consider tubes with rectangular geometry

Tom de Swiet, Varian
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How Much Will Salt Hurt Cryogenic HTS at 950 MHz?

• An organic solvent should be 11,000.
• Pure water is 7000, best case scenario.
• Volume of 2x2-mm (72 mL)
• Volume of 1x4-mm (148 mL)
• Above 110 mM salt, 2x 2-mm tubes will give better
  S/N than 1x4-mm tube with the same concentration!

Rich Withers, Varian
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How Much Will Salt Hurt Cryogenic HTS at 950 MHz?

• 60 C6D6 ASTM
• Pure water should be over 6000.
• Volume of 2x2.5-mm (137 mL)
• Volume of 1x4-mm (254 mL)
• Above 200 mM salt, 2x 2.5-mm tubes will give
  better S/N than 1x5-mm tube with the same
  concentration!

Rich Withers, Varian
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Practical Considerations for Probe Choices

• 5-mm 1H or triple res for routine 1H detection
  in multi-user setting
• 5-mm broadband for X detection in multi-user
  setting
• 1-mm Protasis for high sensitivity without fuss
  of cryo probe (e.g. if you need to change probes
  often)
• 5-mm standard 1H cryo for 1H detection of
  standard biological nuclei (13C, 15N)
• Smaller cryo (HTS or copper) for more sensitive
  detection (still more specialized)
• Cryo optimized for other nuclei (most sensitive
  but least flexibility)