CMLD Core C

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CMLD Core C

• Gerald Lushington
• David VanderVelde
• Todd Williams
• Ben Neuenswander

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Core C Resources
Preparative Chromatography Mass Directed
Fractionation
NMR Automation
LC/MS Purity and Exact Mass
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Introduction

• Goal
• Engage client pool to work with Ben
• Content
• Introduce equipment
• Case Studies
• Suggest a sample flow
• Start feedback session

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Preparative Chromatography

• Reverse phase purification of up to 40 mg per
  shot
• Automatic fraction collection based on Mass or UV
  trigger
• Analytical circuit provides LC/MS data on crude
  and purified compounds

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Preparative ChromatographyDye Separation
Mixture of dyes with four different molecular
weights
Chromatographic resolution of dyes
Fractions collected with purified dyes
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Preparative ChromatographyCritical Issues

• HPLC gradient development
• Sample Solution
• Fraction triggering logic
• Evaporating fractions

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Preparative ChromatographyHPLC Gradient
Development

• Use Log P as predictor of necessary organic
  elution strength

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Preparative ChromatographyHPLC Gradient
Development

• Advantages to analytical LC/MS
• Can run in parallel with prep unit
• Accommodates four different solvents
• A Water
• B Acetonitrile
• C AcetonitrileIPA
• D AcetonitrileTHF

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Preparative ChromatographySample Solution
UV Plot

• Sample dissolved in 30/70 MeCl2/MeOH
• Sample dissolved in 95/5 ACN/H2O

Single Ion Plot
UV Plot
Single Ion Plot
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Preparative ChromatographyFraction Triggering
Logic

• Problem 1 MS detection is extremely sensitive
  and the signal saturates quickly.
• Problem 2 Electrospray degrades at high sample
  concentration

UV Plot
Single Ion Plot
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Preparative ChromatographyFraction Triggering
Logic
UV plot

• Fraction collection using single mass trigger
• Fraction collection using UV/Mass trigger

Single ion plot
UV plot
Single ion plot
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Preparative ChromatographyThe Sample Dilutor

• Starts with 800ul of sample solution and
  transforms it into 2-7ml of collected fraction

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Preparative ChromatographyEvaporation

• Savant SpeedVac
• GeneVac

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NMR Automation

• Bruker 400
• Instrument can be used in both tube and plate
  mode
• 96 well plate autosampler (Bruker BEST system)
  with flow probe

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NMR AutomationIssues Encountered to Date

• High cost of deuterated solvent used for washing
  and sample pushing, as well as sample prep
• Carryover of wash/push solvent if protio solvent
  is used
• Solution implementation of solvent suppression
  strategy which required
• Repairing broken glassware in the probe in order
  to get optimal line shape
• Optimizing sample positioning

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NMR AutomationSolvent Suppression

• No solvent suppression (protio solvent remaining
  from wash step)

CHCl3
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NMR AutomationSolvent Suppression
With presaturation 12C solvent peak suppressed,
13C satellites still present
Satellites
Suppressed peak
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NMR AutomationFlow No D
With presaturation/carbon-13 satellite decoupling
No Satellite
Suppressed peak
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NMR AutomationSensitivity and Sample Volume
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NMR AutomationOptions to Increase
Sensitivity/Decrease Sample Volume

• We currently have a 125 ul flow cell and withdraw
  about 375 ul of solvent from the plate
• Bruker also sells 60 and 30 ul flow cells

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LC/MS

• Exact mass determination on TOF
• HPLC analysis with UV and/or MS

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LC/MSTOF vs. Quadrupole
10,000 resolution
5000 resolution
ESI tof 1) exact mass confirmation 2) relative
quantitation (homologs)
Quadrupole for high concentrations And toggle
fractionation
Quadrupole resolution
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LC/MSWhy exact mass
theoretical
Narrow Formula For C0-100 H 3-74 O0-4 N 0-4 Mass
118 next formula 34 ppm Mass
500 5 compounds within 5 ppm Mass 750.4
626 compounds within 5 ppm
measured
Uncorrected 11 mDa or 33 ppm low
To correct add calibration IS or use lock spray
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LC/MSWhy exact mass
Single Ion Plot 256.1 Da

• Fraction collection was triggered off of mass
  256.1
• Analysis of fraction on TOF
• Target Mass 256.0529 Da
• Found Mass 256.1779 Da
• Difference 488 ppm

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LC/MSTypical Electrospray Spectrum
1mg in 500 ul 400 ul to NMR
Chemists Ideal MS spectrum
MH
Remainder diluted 100 ug/ml or 100 ng/ul
10 CHCl3 in MeOH
Can still dilute 1/10
Real Electrospray spectrum
MNa
MK
2MH(K,Na)
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Initial experience examples of good
ones However 6 of 54 have mischief
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Mischief !
Non-chlorinated analog
Target MH
? No Cl
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Sample FlowTentative Scheme
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Sample FlowTentative Scheme
To analytical
Aliquot for MS Screening
To Prep
Crude Sample
(if needed)
Bulk transferred to autosampler vials
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Sample FlowTentative Scheme
Prep C-graphy
Bulk in autosampler vials
Fractions in Culture tubes
1-2mg to analytical
1-2 mg placed in plate
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Sample FlowQuestions to Resolve

• What will drive the decision to purify samples?
• How will an analytical aliquot make its way into
  a 96 well plate?
• What data will be used to assign a purity value?
  (MS, UV, NMR)
• What is an appropriate purity level?