Optimizing column packing by proper determination of slurry concentration

Many factors affect your chromatography purification results, of which selectivity is determined by resin chemistry and choice of buffers. While bead diameter and particle size distribution are the main factors that determine purification efficiency (or band broadening), packed bed geometry is also a strong contributor. Achieving proper bed geometry and consistent column performance for the lifetime of your campaign is why proper column packing is of utmost importance.

Column packing methods

Proper bed geometry is determined by several factors, all of which are predicated on accurate determination of resin slurry concentration. A successful column packing therefore begins with correct measurement of the slurry concentration. Thereafter, the required resin volume can be transferred to an empty column for packing to the target bed height at the correct level of compression. Using correct compression factor (CF) targets delivery of a known amount of resin to a calibrated column volume.

When working with axial compression packing methods, the packing factor (PF) is often used instead of CF. When settling using flow, PF is determined by detection of the height of the consolidated (not yet mechanically compressed) bed. Axial compression is mostly associated with AxiChrom columns but can also be applicable for other columns such as the BPG columns. The difference between the columns is that the packing process is automated for AxiChrom columns and manual for BPG columns.

CF = Lsettled / Lpacked

PF = Lcons / Lpacked

where

Lsettled = bed height measured after settling by gravity (cm).

Lpacked = packed bed height (cm).

Lcons = consolidated bed height, that is, bed height measured after settling the resin at a given linear velocity (cm/hour).
 

A well-packed bed (middle) generates a stable column that offers good resolution. A poorly compressed bed (left) from a non-homogenous slurry generates a less stable bed and an uneven flow through the column. A too high bed compression (right) generates elevated operating pressures, cracked bed, or dry areas in the bed (= wasted resin).

Why accurate slurry concentration is important

To obtain the correct amount of chromatography resin for packing to target bed height or compression, it is important to measure the slurry concentration correctly. Considering packing a 50% slurry to a target bed height of 20 cm at a CF of 1.15, an inaccuracy of ± 2% in the slurry concentration can cause the final packed bed height to vary between 19.2 and 20.8 cm.

Appropriate bed compression and resin slurry concentration ensure:

  • Stable bed, especially at high flow velocities.
  • Reproducible performance between column packings and campaign products.
  • Optimal column resolution (RS) and asymmetry (AS).
  • Consistent process step throughput.

Methods for of slurry concentration determination

Here follows some options.

OPTION 1: SLURRY CONCENTRATION DETERMINATION USING TRICORN COLUMN

Slurry concentration can easily and accurately be determined using the Slurry Concentration Kit. This method uses a Tricorn 10/100 column and resin in water. Slurry is added to the column and allowed to settle.

  1. Column filling: add thoroughly mixed slurry to the column up to the 10 cm mark with a pipette, and then add distilled water until the column is filled.
  2. Resin settling: fill a syringe with distilled water and fit it to the top of the column, press the syringe at 5–10 mL/min until the liquid over the resin bed is clear, and then stop the flow.
  3. Slurry concentration measurement: allow the bed to stabilize for 0–30 min (time for stabilization depends on resin), and then read the bed height.
Slurry concentration determined using the Slurry Concentration Kit from GE Healthcare.

OPTION 2: ALTERNATIVE METHODS FOR DETERMINATION OF SLURRY CONCENTRATION

Alternative 1. Settling by gravity

  1. Settle the resin bed by gravity overnight, remove storage buffer by decanting, and replace with an equal volume of water or 20% EtOH.
  2. Shake or stir the container until the content is homogenous. For larger volumes, use the Media Handling unit.
  3. Immediately fill a calibrated container, such as a 100 mL graduated cylinder, with a representative sample from the resin container and allowed to sit undisturbed overnight or until the resin has completely settled. Perform in triplicate samplings from different locations within the bulk container to normalize local concentration effects.
  4. The percentage of resin in the slurry (% slurry) can now be read directly from the graduation on the cylinder.
     
A graduated cylinder can be used for determination of slurry concentration for gravity-settled beds.
 

Alternative 2. Using a PD-10 column

  1. A 10 mL calibrated syringe barrel can be fitted with a frit (supplied with empty PD-10 columns) that will retain the resin within the syringe.
  2. A calibrated pipette is used to add 10 mL of the homogeneous slurry into the syringe, which is held in an upright position until all liquid has drained off (15–20 min).
  3. Gently tap the side of the syringe barrel three times and wait another 5 min.
  4. The % slurry can now be determined by the volume of drained resin remaining in the syringe minus the volume occupied by the frit (~ 0.25 mL).
     
The % slurry is calculated using the equation: 100 × (volume of drained resin - volume of frit)/10.
 

The slurry volume required for a desired column bed volume (volume of resin settled by gravity, Vgs) can now be calculated using the equation: 100 × Vgs/% slurry. These calculations are performed in triplicate to capture any user error or method variation, using sample from different locations within the resin slurry container to normalize local concentration effects. Use plastic gradient cylinders and pipettes if possible, as resin tends to adhere more to glass than to plastic surfaces. Plastic ware are also more secure from a breakage standpoint than glassware.

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