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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 ScreeningTodd M. AndersonShimadzu 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 HPLCInjector SIL-20AC autosampler
Pumping System
2 X LC-20AD gradient pumps
Oven CTO-20A
Detector SPD-M20A PDA detector
Mobile PhaseA: H2O with 0.1% TFAB: 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
Datafile Name:7 compound mix_7 min gradient_1652 PM_002.lcdSample Name:7 compound mix
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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)
1 1.5 2 2.5 3 3.5 4 4.5 50
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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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Data7:Anthracene_47 to 67_1613 PM_001.lcd AD2Data6:Napthalene_35 to 55_1523 PM_003.lcd AD2Data5:Butyl Paraben_25 to 45_1523 PM_002.lcd AD2Data4:Verapamil_20 to 40_1709 PM_005.lcd AD2Data3:papaverine_10 to 30_1709 PM_003.lcd AD2Data2:caffeine_2 to 10_1728 PM_004.lcd AD2Data1: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 AD2Data 2: Caffeine_2 to 10_1728 PM_004.Icd AD2Data 3: Papaverine_10 to 30_1709 PM_003.Icd AD2Data 4: Verapamil_20 to 40_1709 PM_005.Icd AD2Data 5: Butylparaben_25 to 45_1523 PM_002.Icd AD2Data 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.
Datafile Name:BP Nap Anth_25 to 45_1236 PM_003.lcdSample Name:BP Nap AnthSample ID:0 to 9
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Datafile Name:BP Nap Anth_35 to 55 prep_1236 PM_004.lcdSample Name:BP Nap AnthSample ID:0 to 10
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Datafile Name:BP Nap Anth_45 to 65_1236 PM_006.lcdSample Name:BP Nap AnthSample ID:0 to 12
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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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