Effective Scaling of Preparative Separations Using Axial Compression Columns

Transcription

1 Effective Scaling of Preparative Separations Using Axial Compression Columns Peter Rahn, Gareth Friedlander, Graham Osborn, and Emmet Welch Phenomenex, Inc., 411 Madrid Ave., Torrance, CA USA

2 Introduction Combinatorial chemistry has led to the development of high-throughput preparative chromatography as the standard practice in drug discovery laboratories. The demand for more compounds with higher purity has promoted the widespread adoption of shorter columns ( mm), operating on open-access systems with generic gradients at very high flow rates. We have previously shown that the major problem with the traditional slurry packing technology occurs after the column is packed. During the necessary disassembly of the slurry packed column from the packing station, the packing pressure must be released from the column allowing the bed to relax or decompress and the media extrudes from the column. Media extrusion (expansion) occurs inside the packing bomb and continues when the column is removed from the packing hardware (Figure 1). Bed expansion is inherent in all slurry packed columns. Although the column is capped as quickly as possible this media extrusion causes: 1) Disruption of the packed bed 2) Reduced packing density 3) Non-uniform packed column with a lower density at the column inlet

3 Figure 1. Inherent Limitations of Conventional Slurry Packing Packed bed disturbed After sedimentation, column is disassembled from slurry chamber and capped (as quickly as possible) Packing density reduced Non-uniform density Lower density at the uncapped end Inherent in all slurry packed columns Few Seconds High pressure solvent forces sedimentation of the slurry. Connection must be removed to install endfitting. During disassembly the bed relaxes and extrudes from column

4 Figure 2. Axia Packing Process for 21.2, 30, and 50 mm Diameter Columns Packing piston Media Added Hydraulic Piston Compresses Bed The packing piston head is integrated into the column and locked by the piston retainers, so the pressure is never released. Media packed under ideal pressure

5 Figure 2. Axia Packing Process for 21.2, 30, and 50 mm Diameter Columns Column Bed Formed Endfitting Attached Media is never allowed to relax, eliminating voids and dramatically improving reproducibility, column-to-column. Retainer Sleeve Attached Finished Column Media Density and Uniformity Maintained Bed Not Disturbed

6 Figure 3. Axia Packing Density Enhancement Each media s bed density is tuned to achieve a specific packing density based on mechanical strength, optimum efficiency and peak asymmetry. Axia technology eliminates post-packing manipulation and bed disruption, resulting in higher density packed beds compared to conventional slurry packed columns. Media is not crushed during the packing or sealing of the column. Axia columns provide true optimization of bed density for chromatographic performance. The Axia packing process is completely automated. Multiple sensors and linear encoders measure and record all process parameters for every column and provide feedback control ensuring columns are packed the same each time. Density (%) Packed Bed Density Comparison of 50 mm Diameter Columns Slurry Packed Axia Packed

7 Figure 4. Axia Packed 50 mm Diameter Columns Improved Performance and Reproducibility Improved process control yields less column-to-column variation (% RSD) for Axia packed columns Efficiency % RSD decreased 4x and asymmetry decreased 6x illustrating better process control due to automation and less technician manipulations For notoriously difficult media, the overall yield has dramatically improved Similar improvements for other media in both 50 and 100 mm length columns are obtained Reproducible Column-to-Column Efficiency Average Efficiency (N) for three 10 µm media in 50 x 50 mm columns Reproducible Column-to-Column Peak Asymmetry Average Peak Asymmetry for three 10 µm media in 50 x 50 mm columns 60,000 4 % %RSD 1.25 %RSD Plates/Meter 45,000 30, % Peak Asymmetry % 3 % 15, Conventional Slurry Packing Axia Packed Hydraulic Piston Compression Packing 1.00 Conventional Slurry Packing Axia Packed Hydraulic Piston Compression Packing

8 Figure 5. Scalability of Axia Design to 50 mm Diameter Same excellent chromatographic performance achieved on 4.6, 21.2, 30 and 50 mm diameter columns. Although resolution between propranolol and diphenhydramine is minimal (R=1.8), each diameter Axia packed column performed well with no performance loss as the column diameter increased min C. D. A. 50 x 4.6 mm Luna 5 µm C18(2) Column 87,120 plates/m σ= µg load 2 µl injection C. B. A. 50 x 30 mm Luna 5 µm C18(2) Axia Packed Column 83,235 plates/m σ= µg load 80 µl injection Conditions Mobile Phase: A: 0.5 % TFA in Water B: 0.5 % TFA in Acetonitrile Gradient: 95:5 (A/B) to 5:95 (A/B) over 4 min Flow Rate: 1.5 ml/min on 4.6 mm column 30 ml/min on 21.2 mm column 60 ml/min on 30 mm column 150 ml/min on 50 mm column Detection: 254 nm Sample Diluent: DMSO B. D. Sample: 1. Propranolol 2. Diphenhydramine 50 x 21.2 mm Luna 5 µm C18(2) Axia Packed Column 84,120 plates/m σ= µg load 40 µl injection 50 x 50 mm Luna 5 µm C18(2) Axia Packed Column 88,280 plates/m σ= µg load 40 µl injection

9 Figure 6. Figure 6a. Purification of 78 mg in 1250 µl DMSO Mixture on 50 x 50 mm Luna C18(2) 5 µm Column 2000 Figure 6c. Fraction 1 from 50 x 50 mm Purification (Propranolol) re-chromatographed on the 4.6 mm column. FRACTIONS:1 FRACTIONS:2 FRACTIONS:3 FRACTIONS:4 FRACTIONS:5 FRACTIONS:6 mau Yield Purity 90 % > 99 % min Figure 6b. Analytical Separation of Propranolol (1) and Diphenhydramine (2) on 50 x 4.6 mm Luna C18(2) 5 µm Column mau Figure 6d. Fraction 3 from 50 x 50 mm Purification (Diphenhydramine) re-chromatographed on the 4.6 mm column. Yield Purity 85 % > 95 % min min min

10 Table 1. Performance Comparison for 10 µm Luna C18(2) Axia Packed Columns Dimension (mm) 100 x x x 50 Average plates/m 56,960 56,280 57,550 Average asymmetry Figure 7. Suzuki Reaction O Br O OEt + O B(OH) 2 (Ph 3 P) 2 PdCl 2 Na 2 CO 3 x H 2 O 1,2-DME-H 2 O-EtOH O OEt Ethyl Trans-4-bromo cinnamate 4-Methoxyphenyl boronic acid Product

11 Figure 8. Same High Performance for 4.6, 21.2, 30, and 50 mm Diameter Columns Figure 8a. Suzuki Reaction Mixture Separated on a 50 x 4.6 mm Luna C18(2) 5 µm Column min Figure 8b. Suzuki Reaction Mixture Separated on an Axia Packed 50 x 21.2 mm Luna C18(2) 5 µm Column Column: 50 x 21.2 mm Flow Rate: 30 ml/min S.F. = 20x min

12 Figure 8. Same High Performance for 4.6, 21.2, 30, and 50 mm Diameter Columns Figure 8c. Suzuki Reaction Mixture Separated on an Axia Packed 50 X 30 mm Luna C18(2) 5 µm Column Column: 50 x 30 mm Flow Rate: 60 ml/min S.F. = 40x min Figure 8d. Suzuki Reaction Mixture Separated on an Axia Packed 50 X 50 mm Luna C18(2) 5 µm Column 1000 Column: 50 x 50 mm Flow Rate: 150 ml/min S.F. = 118x min

13 Figure 9. Direct Scale Up Benefits with Axia Design Axia Packed 21.2, 30mm and 50mm diameter columns provide same purification capability and performance 50 x 21.2 mm, Luna 5 µm C18(2) Axia Packed Sample Load: 16 mg in 250 µl DMSO SF = 20 X for 4.6 to 21.2 mm 50 x 30 mm, Luna 5 µm C18(2) Axia Packed Sample Load: 32 mg in 500 µl DMSO SF = 40 X for 4.6 to 30 mm 50 x 50 mm, Luna 5 µm C18(2) Axia Packed Sample Load: 78 mg in 1250 µl DMSO Gradient: 5 min, 5 % to 95 % Acetonitrile, 0.5 % TFA Flow Rate: 30 ml/min on 21.2 mm column, 60 ml/min on 30 mm column, and 50 ml/min on 50 mm column Detection: 254 nm Sample: 1. Propranolol 2. Diphenhydramine SF = 118 X for 4.6 to 50 mm

14 Figure 10. Separation Capability in Longer Axia Columns 32 mg on 250 x 21.2 mm Length Column Minutes 32 mg on 100 x 21.2 mm Length Column Same Load Minutes Flow rate: Gradient: Detection: Compounds: 30 ml/min on 21.2 mm 10 min on 100 mm length column 25 min on 250 mm length column 5 % to 95 % Acetonitrile, 0.5 % TFA 254 nm 1. Propranolol 2. Diphenhydramine 3X Load Minutes Load increases directly proportional to column length but separation time also increases 96 mg on 250 x 21.2 mm Length Column

15 Results and Discussion The solution to produce mechanically stable preparative columns to withstand high flow rates and viscous DMSO injections is to ensure the packing pressure on the media is maintained after the column is packed and never released. This solution resulted in a new, patent pending microprocessor controlled process. This new Axia technology was awarded the R&D 100 award for one of the best innovations in The same Axia packing technology initially developed for 21.2 and 30 mm diameter columns has now been expanded to produce the larger 50 mm diameter columns. These larger diameter columns allow chemists to quickly purify larger quantities of intermediate compounds on their standard preparative HPLC systems without sacrificing resolution or purity. eliminates the possibility of column voiding or channeling as a source of column failure. The typical packing density improvement achieved for the 50 mm diameter Axia packed columns is shown in Figure 3. The Axia technology provides dramatic improvements in performance and column-to-column consistency (% RSD): 1. Compared to traditional slurry packed columns the efficiency of an Axia packed column is 20 % greater for the three media shown (Figure 4). 2. Column-to-column efficiency variation (% RSD) is reduced from 18 % to 4 % providing chemists consistent performance independent of media bonding chemistry. 3. Peak symmetry variation was reduced from 17 % to 3 % RSD. Adapting the axial compression packing technology with the high level of process control has improved the manufacturing process for these 50 mm diameter preparative columns. Single bed compression is achieved using the new patent pending detachable, lockable piston system. This unique force-transfer design allows the piston to be locked in place and detached from the hydraulic ram after the end of the column is fixed. Compression force is maintained on the column bed and never released or relaxed. The media is not disturbed or allowed to expand after compression (Figure 2). The reproducible Axia packing process produces higher performance 21.2, 30 and 50 mm diameter columns. Similar performance improvement results are also obtained for the 100 mm length columns (Table 1). With this new technology the 50 mm diameter column bed densities are increased and maintained at the mechanical strength limit of the chromatographic media whether the media is silica-based or a softer less rigid polymer-based media. Controlled dense packing

16 Results and Discussion To illustrate the similar preparative capability of the 21.2, 30 and 50 mm diameter Axia packed columns, generic gradient conditions were utilized to separate propranolol and diphenhydramine using water, acetonitrile and TFA modifier. Figure 5 shows the Axia packed preparative column performance is identical and independent of the column diameter. The separations were directly scaled-up from the analytical column, 15.6 mg on the 21.2 mm, 30.2 mg on the 30 mm and 78 mg on the 50 mm diameter column. Fractions from loading 78 mg of the mixture on the 50 x 50 mm Axia column were automatically collected on the Gilson 845Z preparative HPLC system and re-injected to verify that highly pure fractions could be collected (Figure 6). Figure 6a shows the expanded chromatogram for the 78 mg preparative purification on the 50 x 50 mm Axia packed Luna C18(2) 5 µm column. Figure 6b is the initial mixture separated on a 4.6 mm diameter column and Figure 6c and 6d are the chromatograms generated after collecting and analyzing the fractions from the 50 x 50 mm Axia packed column to verify the fraction s purity. For propranolol (first peak) 90 % of the mass was recovered at almost 100 % purity and 85 % of the mass of diphenhydramine (second peak) was greater than 95 % pure. To further illustrate the preparative power and scalability of the Axia packed columns, a Suzuki reaction mixture was purified. The Suzuki reaction was performed with ethyl trans-4-bromo cinnamate and 4-methoxyphenyl boronic acid using a palladium catalyst (Figure 7). The reaction mixture was dissolved in 100 % DMSO, filtered and loaded onto the preparative column. Purification was performed using a generic 5 minute gradient from 5 to 95 % acetonitrile and 0.5 % TFA in both the acetonitrile and water. After initially separating the reaction mixture on a 50 x 4.6 mm column Luna C18(2) 5 µm column, the purification was directly scaled-up to the larger diameter Axia packed columns. All three columns performed the same separation using scale factors (S.F.) from the 4.6 mm column of 20X for the 21.2 mm, 40X for the 30 mm and 118X for the 50 mm diameter column (Figure 8). The second option to increase sample load is to increase the column length requiring adjustments to the chromatographic conditions. Since backpressure is directly proportional to column length, as the column length increases the backpressure also increases - (P= L2/L1). In the following example to maintain the same flow rate when the column length increased from 100 mm to 250 mm the backpressure increased by 2.5X. The second adjustment required when changing column length is that the gradient time is increased proportional to the column length to maintain the same gradient slope for both columns. The gradient change is calculated using the following formula-gt=l2/l1. In Figure 12, the 100 mm column separation was achieved with a 10-minute gradient and the 250 mm column required a 25-minute gradient. Even for a separation that has only an a = 1.1 the sample loaded onto the 250 mm length column was 2.5-3X the load on the 100 mm column. There was no performance or resolution loss with the longer Axia packed column, and the compounds could be purified to the same percent yield and purity value.

17 Conclusion The results achieved with the Axia preparative columns now provide the medicinal chemist the ability to directly scale-up from the 4.6 mm analytical column to a 21.2, 30 or 50 mm diameter preparative column that operate on standard preparative HPLC systems without a loss of performance as the column diameter increases. The absolute load depends on the sample solubility and complexity of the separation and with typical loads these columns yield mg of purified product per run maximizing the chemist s throughput. This work also proves that Axia preparative columns have the same high efficiency as the analytical scale columns and the performance is independent of the column diameter providing the flexibility to scale to larger diameter columns with no loss in resolving power. Axia is a trademark of Phenomenex, Inc. Luna is a registered trademark of Phenomenex, Inc Phenomenex, Inc. All rights reserved. 50 mm 30 mm 21.2 mm 5126_L_3