In order to have a seamless and problem-free scale-up, and to be able to establish an efficient and cost-effective production, one must assure that the large scale column used is packed with the same high efficiency as the column used for the method development. And this can relatively easy be done with today´s DAC columns available on the market!
All method development could normally be performed in analytical or 10 mm ID columns, since this will save solvents, valuable sample, and be less laborious. Any size column should be able to generate the same efficiency as in analytical scale, and hence there is no need to perform the method development in any larger column. However, if scaling up to a very large column size it could be good to have a proof of concept in an intermediate size column, to ensure that the total purification process can be duplicated with the larger equipment. When scaling up to an 80-100 cm ID column one does not want too many surprises!
To pack at a reasonable bed compression speed is an important factor, in order to obtain a highly efficient bed. We have observed that packing at a speed that gives total compression of the bed within 30 seconds to 1 minute, usually generates the highest efficiencies. In order to obtain a high packing speed one should not have too viscous slurry solvents. Pure or neat IPA (isopropanol) could be too viscous, while neat ethanol, or mixtures with IPA and lower viscosity solvents usually work well.
Another critical factor is to ensure that the capacity for compressed air, used to drive the compression, is high enough. The air tubing must be large enough to give a high enough capacity.
A second very critical factor is the frits! The best type of frits is the woven ones, giving a better flow distribution, and facilitates easier cleaning. The sintered type are more easily blocked, could give a bad flow profile, and do not facilitate as easy cleaning as the woven type.
If not using new frits, be sure that the ones used are thoroughly cleaned! A frit that is partly blocked will undoubtedly lead to an uneven flow distribution, causing peak distortion, and hence low efficiency and poor purity/yield in the purification.
Please see the below table for recommended slurry solvents to use for packing DAC columns. Normally it is not as critical to choose the right slurry solvent, as it is when packing analytical columns. However, some guidelines are necessary!
(1:1) → (9:1)
(1:1) → (9:1)
|Silica (60 Å, 100 Å)|
|C4/C8 (100 Å, 300 Å)|
|C18 (100 Å, 300 Å)|
From the desired bed length, and packing density given in the data sheet for the packing material, one can calculate the amount of packing material needed for the column. For making up the slurry it is recommended to use at least 2 liters of slurry solvent per kg of packing. Calculate the amount of solvent by adding the total porosity and the excess for making up the slurry. Total porosity is the porosity of the particles and the interstitial porosity (space in between the particles), and is given in the table below for different Kromasil packings. Make sure that there is some margin, so that all slurry goes into the column!
For making up the slurry a polyethylene beaker with a propeller stirrer can be used, or alternatively, more sophisticated, dedicated apparatuses provided by the DAC column manufacturers. Be sure to pour the silica slowly into the slurry solvents, in order to easily disperse the silica, and stir the slurry for 5-10 minutes, or until it is totally homogeneous.
Before starting the packing make sure that the solvent tubing diameter is large enough. For 5 cm ID columns for example, use at least 2 mm ID (1/8 inch, OD) outlet tubing during the packing procedure, and then change to smaller solvent tubing during chromatography (e.g. 1 mm ID for 5 cm columns). For larger columns the tubing should be scaled according to the diameter.
The slurry is then poured or pumped into the column, depending on size of the column, and how the actual column is constructed. The lid is put on and quickly fastened, so that the silica does not settle too much. The problem with sedimentation of the silica is more pronounced the larger the particles are.
After the lid is put on the compression of the column can start, normally using the highest pressure that the system is rated for, 70-100 bar. The solvent inlet tubing can preferably be plugged during the packing procedure.
The whole process of compressing the bed should take 30 seconds to 1 minute, for a slurry solvent with a viscosity of around 1 cP.
For testing, preferably inject via a loop, and have the delivery system well optimized, ensuring small dead volumes. For recommended test substances see the table below.
|Reversed phase||Bare silica and CelluCoat/AmyCoat|
|Acetophenone, toluene, etc||Nitrobenzene, aromatic esters, etc|
For Kromasil the efficiency obtained in a DAC column should be at least 40 000 pl/m for 10 µm material, corresponding to a reduced plate height h = 2.5. However, significantly higher efficiencies have been obtained for perfectly optimized columns and delivery systems.
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