1 / 14
Increased Throughput and Purity of Combinatorial
Libraries Utilizing a Targeted Gradient Profile
Based on Prelimina...
2 / 142 / 14
Challenge
With increasing pressure of a higher sample throughput and
fewer chemists, purification labs in med...
3 / 143 / 14
Objective
This presentation will describe a technique that allows the
analyst to obtain a higher purity and b...
4 / 144 / 14
Methodology
HPLC: Shimadzu Prominence HPLC
Injector SIL-20AC autosampler
Pumping System 2 X LC-20AD gradient ...
5 / 145 / 14
Analytical Sample Data
A random selection of samples was obtained from the lab’s inventory of test
compounds....
6 / 146 / 14
Chromatographic Profile
DatafileName:7compoundmix_7mingradient_1652PM_002.lcd
SampleName:7compoundmix
0.0 0.5...
7 / 147 / 14
Gradient Profile Optimization
After obtaining retention times for each of the individual
compounds, two compo...
8 / 148 / 14
The highlighted
compounds,
Anthracene and
Caffeine, were
used for this
purpose
(Figure 3).
Gradient Profile O...
9 / 149 / 14
Gradient Profile Optimization (continued)
0
10
20
30
40
50
60
70
80
1 1.5 2 2.5 3 3.5 4 4.5 5
observed values...
10 / 1410 / 14
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
0
25000
50000
75000
100000
125000
15000...
11 / 1411 / 14
Optimized Preparative Chromatography
A sample from the original stock solutions was created and run at
thre...
12 / 1412 / 14
DatafileName:BPNapAnth_35to55prep_1236PM_004.lcd
SampleName:BPNapAnth
SampleID:0to10
0.0 0.5 1.0 1.5 2.0 2....
13 / 1413 / 14
Conclusion
After completion of all analysis, preparative separation was greatly
enhanced by the shallow gra...
14 / 1414 / 14
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HPLC Combinatorial Libraries

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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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  • HPLC Combinatorial Libraries

    1. 1. 1 / 14 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
    2. 2. 2 / 142 / 14 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.
    3. 3. 3 / 143 / 14 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.
    4. 4. 4 / 144 / 14 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
    5. 5. 5 / 145 / 14 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.
    6. 6. 6 / 146 / 14 Chromatographic Profile DatafileName:7compoundmix_7mingradient_1652PM_002.lcd SampleName:7compoundmix 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 min 0 100 200 300 400 500 600 700 800 900 mV 0.00 0.25 0.50 0.75 1.00 1.25 psi(x10,000) B.ConcAD2 Caffeine Pappaverine Verapamil ButylParaben Anthraceneimpurity Napthalene Anthracene 0.0 1.0 2.0 3.0 4.0 5.0 6.0 min 200 225 250 275 300 325 350 375 400 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.
    7. 7. 7 / 147 / 14 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.
    8. 8. 8 / 148 / 14 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.
    9. 9. 9 / 149 / 14 Gradient Profile Optimization (continued) 0 10 20 30 40 50 60 70 80 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.
    10. 10. 10 / 1410 / 14 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 0 25000 50000 75000 100000 125000 150000 175000 200000 uV 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
    11. 11. 11 / 1411 / 14 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 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 0 50 100 150 200 250 300 350 400 450 mV 0 10 20 30 40 50 60 70 80 90 % B.ConcAD2 Butylparaben Napthalene Anthracene
    12. 12. 12 / 1412 / 14 DatafileName:BPNapAnth_35to55prep_1236PM_004.lcd SampleName:BPNapAnth SampleID:0to10 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 0 50 100 150 200 250 300 350 400 450 mV 0 10 20 30 40 50 60 70 80 90 % 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 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 -50 0 50 100 150 200 250 300 350 400 450 mV 0 10 20 30 40 50 60 70 80 90 % B.ConcAD2 Butylparaben Naphthalene 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)
    13. 13. 13 / 1413 / 14 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.
    14. 14. 14 / 1414 / 14 Thank you for viewing this presentation. Should you have any questions or require additional information about our research, products or services, please visit our Support page: www.ssi.shimadzu.com/support/ @shimadzussiFollow us on Twitter

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