With increasing pressure of a higher sample throughput and fewer chemists, purification labs in medicinal chemistry groups need to be more productive now than ever before. This presentation will describe a technique that allows the analyst to obtain a higher purity and better resolution using information from the preliminary analytical screening of these samples prior to purification.

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Increased Throughput and Purity of Combinatorial
Libraries Utilizing a Targeted Gradient Profile
Based on Preliminary Analytical Screening
Todd M. Anderson
Shimadzu Scientific Instruments, Inc., Columbia, MD

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Challenge
With increasing pressure of a higher sample throughput and
fewer chemists, purification labs in medicinal chemistry groups
need to be more productive now than ever before.
Many of these labs have historically utilized a steep low-to-high
organic gradient, as the broad spectrum of compounds that
separate tend to have a wide range of elution profiles. Typically,
a 5 to 95% organic gradient profile is utilized.
Separating tightly resolved compounds and impurities can be
somewhat difficult with these typical gradient profiles. The analyst
is then forced to sacrifice either speed or purity, and ultimately
requires multiple purifications.

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Objective
This presentation will describe a technique that allows the
analyst to obtain a higher purity and better resolution using
information from the preliminary analytical screening of these
samples prior to purification.
 By utilizing the retention time of the compound from preliminary runs, an optimal set
of conditions may be obtained that allows for a greater success rate of separation.
Compared to the standard 5 to 95% elution gradient, a narrow, short gradient profile
can be determined.
 Along with a better separation, the chromatographic time can be shortened, saving
the analyst both instrument time and solvent consumption, while allowing for
purification with a single injection.

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Methodology
HPLC: Shimadzu Prominence HPLC
Injector SIL-20AC autosampler
Pumping System 2 X LC-20AD gradient pumps
Oven CTO-20A
Detector SPD-M20A PDA detector
Mobile Phase
A: H2O with 0.1% TFA
B: Acetonitrile (LC grade)
Software LabSolutions V. 5.54
Column Shimadzu ODS (C-18) 5 um

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Analytical Sample Data
A random selection of samples was obtained from the lab’s inventory of test
compounds. Below is the compound list and their stock concentrations:
1) Saccharin 13.0 mg/ml
2) Caffeine 6.8 mg/ml
3) Papaverine 5.36 mg/ml
4) Verapamil 8.95 mg/ml
5) Butylparaben 14.31 mg/ml
6) Naphthalene 5.78 mg/ml
7) Anthracene 8.0 mg/ml
Individual retentions for these compounds were obtained by: 1) Running an
initial screening of these compounds using a 5 to 95% ACN gradient paired
with an aqueous phase of 0.1% TFA at 1.5 ml/min, and 2) A one-minute
hold at 95% organic and a two-minute re-equilibration back at 5% ACN.

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Chromatographic Profile
DatafileName:7compoundmix_7mingradient_1652PM_002.lcd
SampleName:7compoundmix
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B.ConcAD2
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Pappaverine
Verapamil
ButylParaben
Anthraceneimpurity
Napthalene
Anthracene
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nm
To obtain a single chromatogram with all components, a mixture of
100 ul of each solution was combined and run on the same gradient
profile (Figure 1). Figure 2 shows the PDA contour plot.
Figure 1.
Figure 2.

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Gradient Profile Optimization
After obtaining retention times for each of the individual
compounds, two compounds were chosen, one with a late
elution and one with an early elution, to plot the optimal
organic percent needed to elute those compounds at the
height of the gradient profile.
This should achieve the best resolution and purity for
preparative conditions.

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The highlighted
compounds,
Anthracene and
Caffeine, were
used for this
purpose
(Figure 3).
Gradient Profile Optimization (continued)
Compound RT Opt. Org. % Final Peak RT
Saccharin 0.90 5 4.1
Caffeine 1.40 10 4.27
Papaverine 2.20 30 4.1
Verapamil 2.78 40 4.37
Butylparaben 3.10 45 4.33
Napthalene 3.60 55 4.28
Anthracene 4.20 67 4.27
Arbitrary RT Calculated Org. %
1 2.36
2 22.71
3 43.07
4 63.43
4.5 73.61
Values used to calculate slope of optimal elution
percent.
Figure 3.

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Gradient Profile Optimization (continued)
0
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1 1.5 2 2.5 3 3.5 4 4.5 5
observed values
Calculated retention
A slope was then calculated, and all the compounds were run based
on their predicted optimal elution gradient. Figure 4a compares the
calculated and observed values with little deviation, and Figure 4b
shows an overlay of the optimized chromatograms using LabSolutions
software.
Figure 4a.

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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min
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Data7:Anthracene_47 to 67_1613 PM_001.lcd AD2
Data6:Napthalene_35 to 55_1523 PM_003.lcd AD2
Data5:Butyl Paraben_25 to 45_1523 PM_002.lcd AD2
Data4:Verapamil_20 to 40_1709 PM_005.lcd AD2
Data3:papaverine_10 to 30_1709 PM_003.lcd AD2
Data2:caffeine_2 to 10_1728 PM_004.lcd AD2
Data1:Sacharin_1 to 5_1709 PM_002.lcd AD2
Based on the chromatograms, it was determined that a mixture of
Butylparaben, Naphthalene, and Anthracene (with its impurity peak –
see below) would make the best example to test purification conditions.
Optimized Preparative Chromatography
Figure 4b.
Anthracene impurity
Data 1: Saccharin_1 to 5_1709 PM_002.Icd AD2
Data 2: Caffeine_2 to 10_1728 PM_004.Icd AD2
Data 3: Papaverine_10 to 30_1709 PM_003.Icd AD2
Data 4: Verapamil_20 to 40_1709 PM_005.Icd AD2
Data 5: Butylparaben_25 to 45_1523 PM_002.Icd AD2
Data 6: Napthalene_35 to 55_1523 PM_003.Icd AD2
Data 7: Antharacene_47 to 67_1613 PM_001.Icd AD2

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Optimized Preparative Chromatography
A sample from the original stock solutions was created and run at
three gradient profiles: one below (Figure 5), one at (Figure 6), and one
above optimal conditions for Naphthalene (Figure 7).
Figure 5. A 25 to 45% organic gradient profile, optimized for Butylparaben.
DatafileName:BPNapAnth_25to45_1236PM_003.lcd
SampleName:BPNapAnth
SampleID:0to9
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B.ConcAD2
Butylparaben
Napthalene
Anthracene

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DatafileName:BPNapAnth_35to55prep_1236PM_004.lcd
SampleName:BPNapAnth
SampleID:0to10
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B.ConcAD2
Butylparaben
Naphthalene
Anthracene
Datafile Name:BP Nap Anth_45 to 65_1236 PM_006.lcd
Sample Name:BP Nap Anth
Sample ID:0 to 12
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Anthracene
Figure 6. A 35 to 55 % gradient profile optimized for Naphthalene.
Figure 7. A 45 to 65 % gradient profile optimized for Anthracene.
Optimized Preparative Chromatography (continued)

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Conclusion
After completion of all analysis, preparative separation was greatly
enhanced by the shallow gradient determination.
With the results of the analytical screening, a table could be
generated to indicate optimal gradient ranges for given windows of
analytical retention time. This premise would need to be optimized for
differences in analytical to preparative column performance, as well
as dwell time differences from instrument to instrument. However, it
would allow one analytical instrument to provide data for an entire lab
running multiple preparative instruments, drastically increasing
throughput and purity, while creating a more efficient workflow in the
high-throughput purification lab.

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