automated fraction re-analysis does it really make … · automated fraction re-analysis does it...

Chairperson: Ingrid Ginnutt

Automated Fraction Re-Analysis Does it really make sense?

Presented by: Udo Huber

• 1995 PHD in organic chemistry from the University Karlsruhe/Germany• 1996 - 97 Postdoctoral fellow at the University of Hawai’i at Manoa• Since 1997 Application Chemist with HP/Agilent• Since 2000 Senior Application Chemist for the purification system and valve solutions


Contents

• IntroductionWhy purification, why re-analysis?Where is my target compound?WorkflowWhat is purity?

• Is my compound pure enough?Possible errors in automated fraction re-analysisThe right point in the workflow to check the purityOther aspects

• A dedicated system for preparative and analytical workSharing the DAD and MSD


Why purification at all?

min0 2 4 6 8 10 12 14

mAU

0

50

100

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250

R1 X

O

R2 R3 R1

O

R2

R3+

Target compound

Byproduct

ImpuritiesStarting material

• Target compound goes to activity testing.• Therefore it must be 100 % pure.


Why re-analysis?

1. If more then one fraction was collected for the same sample re-analysis has to be done to identify the fractions containing the target compound.

2. Check purity of the target compound to make sure it is pure enough for activity testing.


More then one fraction

Why do I get more then one fraction?⇒ Time- or peak-based fraction collection

To identify the right fractions I need:either standard for the compound ⇒ analytical systemor an MSD

Prep system has no MSDif it had an MSD I didn’t get more then one fraction⇒ analytical system


Strategies for purification on a system equipped with MSD

min0 1 2 3 4 5 6 7

mAU

0

500

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1-Vial

1

1-Vial

21-V

ial 31-V

ial 4

DAD

Peak-based fraction collectionthreshold only (50 mAU)

min0 1 2 3 4 5 6

1-Vial

1

1-Vial

21-V

ial 31-V

ial 4

0500000

100000015000002000000250000030000003500000

MSD

7


Strategies for purification on a system equipped with MSD

Target mass: 241 amuTIC

5.2 5.4 5.6 5.8 6 6.2 6.4 6.6

1-Vial

2

EIC (242 [M+H]+)Fraction containing target compound

20000040000060000080000010000001200000140000016000001800000

min

050000100000150000200000250000300000350000400000

5.2 min5.4 5.6 5.8 6 6.2 6.4 6.6


Purity check – Sample workflow

1.Sample submitted by chemistSolvent often DMSO/DMF

2.Sample purifiedMobile phase water/acetonitrile

3.Fraction takenMobile phase water/acetonitrile


Sample workflow

4.Solvent evaporatedCompound weighed

5.Compound re-dissolvedCertain concentration, solvent often DMSO/DMF

6.Portion taken to activity testing(either DMSO solution, diluted with aqueous buffers or without DMSO)Rest of solution stored


Important questions

• Confirm that target compound is going to activity testing, usually done by MS

• Measure purity of compound going to activity testing, must be higher then 90 - 95 %

Activity testing

A representative portion of the sample going to activity testing must be re-analyzed to check the purity!


What is purity?

min0 1 2 3 4 5

mAU

020406080

100DAD: 254/8 nm (ref. 360/60 nm)

min0 1 2 3 4 5

mAU

020406080

100 DAD: 204/8 nm (ref. 360/60 nm)

min0 1 2 3 40

200000

600000

1000000MSD

Compound did not ionize!

5

min0 1 2 3 4 5

Compound evaporated!mVELSD

0

50

100


Break Number 1


Fraction re-analysis –where in the workflow?

Automated fraction re-analysis:Directly from fraction container (step 3)

• Advantage: High level of automation• Disadvantages: ???


Possible errors in when doing automated fraction re-analysis (1)

23

4

fill

timemin0 1 2 3 4 5 6 7 8 9

mAU

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3500



min0 1 2 3 4 5 6 7 8 9

mAU

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Red

Pink

Yellow

No mixing when vial is filled!

Blue



T = 0 h T = 6 h T = 24 h

Flow = 1 mL/min

Flow = 4.5 mL/min

Flow = 20 mL/min

T = 96 h



Portion drawn for re-analysis. Is this a representative sample?




Sample was submitted in DMSO

Mobile phase is water/ACN



Sample decomposed while evaporated

Dissolved in

DMSOEvaporation

Portion taken to activity testing


Do you do compound confirmation on a no-hit?


What is the solution?

What can I What can I do???do???


Take a second sample from the solution that goes to activity testing!

• Let the purification system do what it was designed for!• Use an analytical system to confirm compound and determine purity.

Portion taken to activity testing



Does this approach solve the problems?

Fraction mixed before re-analysis

Compounds dissolved before re-analysis

Decomposition discovered during re-analysis


Other aspects

• Using a combined inject/collector seriously compromises the pluming of the system and leads to additional broadening and therefore loss of recovery. At […] they see 90% recovery with our system and 60% with others

• You make a very good, in fact the best, analytical system already which is optimised for that specific job. All the labs interested have [Agilent] 1100 [Series] systems available to them for such use.

• A separate analytical system means our expensive Prep system is not tied up doing routine analysis as the real prep runs start to build up in a queue -Not cost effective.

• Performing re-analysis off line means you can specifically select which fractions you would like to re-analyse rather than all collected fractions being re-analysed. This eliminates unneeded analytical runs which are both time consuming and wasteful.


Break Number 2


A single system for purification and analytical work

• Two separated systemsabsolutely no compromises regarding flow pathspreparative AND analytical work in parallel

• Two systems sharing a single MSD and DADcan be operated with ChemStation only (rev. A.10.01), purification software and with Easy Access Plusnot possible to do analytical and preparative work in parallelpreparative autosampler and pumps used for analytical work (delay volume)

Recommended for:Preparative flow rates up to 40 mL/min (1 inch columns)Injection volumes < 900 µLAnalytical flow rates above 0.8 - 1 mL/min (4.6 mm columns)Further details will be described in an Application Note


The Agilent active splitter

HPLC Flow

ActiveSplitter

MS Flow


The Agilent active splitter

• Agilent active splitter provides control of the transfer of material from one flow stream to another.

• Rotor seal includes three selectable aliquot volumes (22 nL, 100 nL, or 300 nL). • Gating frequency (0.25-2.0 Hz) and aliquot volume determines the rate of mass

transfer from source stream to destination stream.


Two systems sharing a single MSD and DAD – preparative work

Splitter on

Waste

Waste

Plug


Two systems sharing a single MSD and DAD – analytical work

Splitter on

Waste

Waste

Plug


Two systems sharing a single MSD and DAD – direct injection

Splitter on

Waste

Waste

Plug


Automated fraction re-analysis –does it really make sense?

• IntroductionWhy re-analysis, where is my target compound and what is purity?

• Possible errors in automated fraction re-analysisConcentration gradientCrystallizationDecompositionHow to avoid those problems?

• A dedicated system for preparative and analytical workSharing the DAD and MSD

automated fraction re-analysis does it really make … · automated fraction re-analysis does it...

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