A method to pack Agilent Prep C18 Media on Load & Lock Column

Transcription

1 A method to pack Agilent Prep C18 Media on Load & Lock Column Application Note Author Liqin Chen, Rong An Life Science and Chemical Analysis Agilent Technologies Shanghai, China Abstract The successful use of large diameter columns in preparative high performance liquid chromatography requires both high quality of media and appropriate column hardware. Agilent Technologies brings together its Agilent Prep C18 10 µm media and high performance Load & Lock column to provide such a solution. This article discusses how to pack the Agilent Prep C18 10 µm media on Load & Lock column to get a stable column bed, no less than 30,000 plates/meter of column efficiency is achieved.

2 Introduction DAC technology and Agilent bench-top 1in., in., and 3in. Load & Lock columns Dynamic Axial Compression (DAC) is a feature of preparative and process chromatography columns whereby a movable piston constantly applies a controlled pressure on the chromatographic bed to ensure optimum packing density and chromatographic performance. Maximum plate numbers are generated because this design prevents the formation of voids in the column bed. Because the columns are selfpacked, any type of packing material can be used, including small particle-size (10 µm) media and the bed length of a DAC column can be adjusted by introducing different amount of packing material. The Agilent high performance bench-top Load & Lock columns and mobile packing station provide the ideal hardware for ease of use with high performance. It is designed for packing various inner diameters (1in., in., and 3 in. ID) columns and can be performed at either dynamic or static locked axial compression (DAC or SAC) mode. With DAC mode, the packed bed is constantly compressed while being used; with SAC mode, the column is compressed by a plunger held in position with a locking mechanism. A method to pack Agilent Prep C18 10 µm Media on Agilent bench top in. Load & Lock Column To combine the Load & Lock column hardware with Agilent Prep C18 media, a packing method for Agilent Prep C18 10 µm media on Load & Lock columns is developed. Normally, five steps are included in a packing and unpacking process of a DAC column: 1. Preparing slurry. Transferring Slurry 3. Compressing and packing column 4. Keeping pressure during use (DAC mode. If SAC mode, the column piston is locked after compressing and hydraulic pressure is released) 5. Unpacking the column. Following figure1 shows the process: Agilent Prep C18 bulk media In addition to convenient pre-packed columns, Agilent Prep LC also provides C18 and bare silica 10 µm bulk media. It is designed for high loadability to purify a diverse range of products. It s offering high carbon load and large surface area and this allows the maximum amount of sample that can be placed onto the column at both low and high ph, which helps increase product yield and system throughput. Specifications of the Agilent Prep C18 10 µm bulk media are shown in table1 as below: Table1. Specifications of the Agilent Prep C18 10 µm bulk media Figure 1. Packing and unpacking a DAC column Bonded Surface Temp. Pore Size ph Range Endcapped Carbon Phase area Limits Load C Å 400 m /g 60 C Single 4% The detail procedure for packing and unpacking the Agilent Prep C18 material on the Load & Lock in. column by using the related packing station is described as below.

3 1. Preparation of the packing slurry 1.1 Calculating the amount of media Based on the column ID and required column length to be packed, calculate the required column volume, Vcol, ml: ID Vcol = Π. bedlength. In this experiment, in. (5cm) column is used and 5 cm of column length is required, so the required column volume is: Vcol = 5 *3. 14* 5= 490ml (1) Then the amount of media (m) can be calculated by the following formula: m = Vcol ρ () As a matter of experience, the packed bed density (ρ) for C18 media is around 0.6 g/ml. So the anticipative amount of media which is needed to achieve 5 cm bed length on in. column is: m= 490*0.6= 94gram 1. Preparing the slurry A slurry of the packing material must be prepared in an appropriate solvent, and the most recommended slurry solvent for packing silica based media is Iso Propanol (IPA). And the concentration of stationary phase in solvent is generally in the ratio of 1g of stationary phase in ml of slurry solvent. In this experiment, the slurry is prepared by dispersing the 94gram of Agilent Prep C18 10µm media slowly to 600ml IPA solvent and stirring manually at the same time (to avoid breaking the media particle, never use a magnetic stirrer). Then shake the slurry gently to get a homogenous solution. Degas the slurry right before use by ultrasonicating for approximately five minutes. The packing slurry is now ready for use.. Transferring the slurry Before transferring the slurry into the column, rinse the piston seals with IPA, load the piston head assembly into the bottom of Load & Lock column and plug the column inlet. Assembly of the in. Load & Lock column is shown in figure as below. Then pour the homogenous slurry quickly into the assembled column, fix the outlet flange to the top of column and place the column outlet line into a waste container. The packing process should be performed as fast as possible to avoid media settling. Outlet flang Column body Piston head assembly Column inlet Compression spacer Lock collar Safety stop in. L&L column on packing Standalone in. L&L column Figure. Assembly of the in. Load & Lock column 3

4 3. Compressing and packing column A piston pressure of around 1000 psi is used for the column packing. Because the ratio of hydraulic pressure to piston (or mechanical) pressure for in. Load & Lock column is 1.5:1.0, to reach 1000 psi of piston pressure, a 1500 psi of hydraulic pressure is set up. Compression process includes two stages: In the first stage, air is pushed out from the column. And in the second stage, the packing material in the column is actually compressed. Leave the bed under pressure until there s not packing solvent out and the hydraulic pump stops working completely. After column packing finished, lock the locking collar and measure the column bed length. In this experiment, 94 gram of Agilent Prep C18 10 µm media is packed first under 1000 psi and a.5 cm of bed length in in. Load & Lock column is gotten. The real packing density of Agilent Prep C18 10 µm media calculated by the above formula (1) and () is g/l. Accordingly, to get 5 cm of column bed length, approximately 330gram of dry media is needed. Following table shows the column bed length results achieved in this experiment by packing 94 and 330 gram of dry Agilent Prep C18 10 µm media: The packed column is now ready for use. Before running real sample, test the column efficiency first. To run a column efficiency test, connect the Agilent prep C18 10µm column to prep HPLC system and flush the column with 80:0 ACN:HO (v/v) at flow rate 50 ml/min until the column is fully equilibrated. Conditions for column efficiency test in this experiment are shown below: Conditions: Columns: Agilent Prep C18 10 µm L&L column with 5 cm of column bed Mobile phases: 80:0 ACN:HO (v/v) Flow rate: 100 ml/min Sample: ~1%Toluene Injection: ~500 µl UV detector: 54 nm Chromatogram of column efficiency test is shown in the following figure 3 and 9650 plates are gotten at a 5 cm column bed. By this token, no less than 30,000 plates/meter can be achieved by using the packing procedure above. Table. Packing different amount of Agilent Prep C18 10 µm media on in. Load & Lock column Inner diameter in. ID Packing pressure 1000 psi Amount of dry media 94 gram 330 gram Column bed length.5 cm 5 cm Figure 3. Column efficiency test on 5 cm Agilent Prep C18 L&L column The efficiency test of the column can also be determined by using your procedure. 4

5 4. Keeping pressure during use Once the column is packed, it can either be used under Dynamic Axial Compression (DAC) mode or Static Axial Compression (SAC) mode. Under DAC mode, the hydraulic pressure is constantly provided during use. To hear the pump cycle once or twice during use is typical in DAC mode. This indicates that the column bed is slowly reorganizing and stabilizing. Accordingly, the piston moves upward a short distance to eliminate the void. Under SAC mode, the column is compressed by a locking collar to maintain the stability of the chromatographic bed, the hydraulic pressure is released, and the packed column can be removed from the hydraulic group and be used dependently. 5. Unpacking the column When the column pressure becomes too high or column efficiency becomes low, it is necessary to unpack the column. Before unpacking the column, flush the column bed by packing solvent (in this experiment by IPA) to make the media be ready for next packing. When unpacking the column, it must be fixed on the hydraulic group again. Remove the flange which is on the top of column, push the packing material out of the column by moving up the piston. Conclusion In this application note we develop a packing method for the Agilent Prep C18 10 µm media on Load & Lock in. column to get a column with efficiency no less than 30,000 Plates/meters. The Agilent Prep C18 10 µm media shows sufficient mechanical strength to be used under the pressure of the column packing operation. By combing the Load & Lock column hardware with the Agilent Prep C18 10 µm media, Agilent provides a perfect scalable solution from analysis (pre-packed analytical column available) to preparative chromatography. References 1. Laboratory Scale Load & Lock Columns and Mobile Packing Station 1in., in., and 3in.ID, Agilent Technologies User manual, 010. Agilent PLRP-S Media and Load & Lock Columns, Agilent Technologies Brochure, 011, publication number EN 3. PLRP-S Polymeric Reverse Phase Media, PLRP-S Media User Guide Replace the part of packing material in the column head with fresh media if it s contaminated. If the column pressure is still high after repacking column by using clean frits, it indicates that some of packing material is broken, and the fine particles need to be removed by mixing the media with special solvent, settling and sucking up the supernatant part. Or if it doesn t work, use new media directly. 5

6 Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc., 01 Printed in the USA October, EN