Vocabulary for chromatography column packing

Factor describing the shape of a chromatographic peak. Measured at a defined height of the peak; can be at the base (between tangents drawn) or at 5% or 10% of the actual peak. Peaks are known as tailing when A_s > 1.0 and fronting when A_s < 1.0. Gaussian peaks are defined as A_s = 1.0.

used for the movable end pieces of columns; contains filter, flow distributor, and possibility to connect tubing.

The pressure drop across a column and/or a chromatography system.

The widening of a zone of solute (for example a protein) when passing through a column or a chromatography system. Gives rise to dilution of the solute and reduced resolution. Also often called peak broadening or zone broadening.

Volume of the resin bed that is compressed and locked in place by the adapter.

Specially designed device that could be attached to the system or a stand to hold the column in a vertical position.

Controlled filling of the column hardware with chromatography resin compressed to a certain, predefined extent to achieve a stable particle bed.

The geometrical volume of the column interior.

Depending on the column type and resin, the compression may be performed in two different ways:

Mechanical compression with packing factor:

After applying the settling flow, note the height of the consolidated bed before stopping the flow (after the flow is stopped the bed can slightly expand). The final bed height is calculated by dividing the consolidated bed height with the packing factor (PF):

Final bed height = Consolidated bed height/packing factor (PF).

Set the adapter against the consolidated packed bed, tighten the O-ring, and turn the end cap down until the calculated final bed height is reached.

Flow compression:

After applying the packing flow, the adapter is moved a specific distance (mm) into the packed bed to avoid gap formation.

CF is defined as the settled bed height (bed height measured after settling by gravity, L _settled) divided by the packed bed height (L_packed), that is, CF=L _settled/ L_packed

Bed height measured after settling the resin at a given flow velocity.

The pressure at the critical velocity.

The highest velocity possible to obtain in an open bed. The procedure to determine V_cri is described in Development of column packing methods based on pressure-flow measurements

High efficiency means that the band broadening is low (= sharp peaks will be obtained). It is often given as the number of theoretical plates (N) or as theoretical plates per meter (N/m) in order to be a measure independent of column length used for the experiment.

It plays a central role in the qualification and monitoring of packed bed performance. The desirable high column efficiency gives low band/peak broadening and is an indicator of how well packed the column is before starting purification.

Column efficiency is typically defined in terms of two parameters:

• Peak broadening over the column is described by an equivalent number of theoretical plates.
• Peak symmetry is described by a peak asymmetry factor, A_s.

The procedure  to determine column efficiency is described in each Cytiva resin protocol and is also described in this Column efficiency testing application note.

See Compression

Height Equivalent to a Theoretical Plate (HETP), is a measure of column efficiency denoted as H and is usually reported in millimeter. HETP is calculated from H = L/N, where L=length of column and N= number of theoretical plates. The shorter each theoretical plate of the packed column, the more plates are "contained" in any length of column. This, of course, translates to more plates per meter and higher column efficiency.

See Compression.

A bed that has a liquid head space between the bed surface and the adapter.

The pressure at the critical velocity.

The maximum velocity possible to use in a packed bed.

A bed that is compressed and locked in place by the adapter.

Connects two chromatography columns with each other allowing one of them to be used as packing reservoir.

PF is defined as the consolidated bed height (bed height measured after settling a resin at a given flow velocity, L _conc) divided by the packed bed height (L_packed), that is, PF=L _conc/ L_packed.

The pressure at the packing velocity.

A cylindrical container with a diameter larger than the column which can be connected to the chromatography column when packing the resin.

The preferred term for describing the solution for column packing, as opposed to packing buffer (since a buffer related to pH is not necessarily used for column packing).

A cylindrical tube or column with the same diameter as the column which can be connected to the chromatography column when packing the resin.

The suitable velocity for packing a bed to the correct compression.

The mean diameter of the spherical beads

See Band broadening.

A measure of chromatography peak broadening/column efficiency. Also referred to as number of theoretical plates. The plate number of a column is used to measure column efficiency. The more plates a packed column has, the higher the column efficiency. The plate number is calculated from N = 5.545 × (V_R/W_h)² assuming a Gaussian peak, where V_R = retention (elution) volume and W_h = peak width at half peak-height. V_R and W_h must have the same unit (e.g., mL). V_R may be substituted in the formula with retention time, t_R, but then the unit of W_h must be time.

See also Plate height/height equivalent to a theoretical plate and Reduced plate height. See Efficiency as well.

Height Equivalent to a Theoretical Plate (HETP) is a measure of column efficiency denoted as H and is usually reported in millimeter. HETP is calculated from H = L/N, where L=length of column and N= number of theoretical plates. The shorter each theoretical plate of the packed column, the more plates are "contained" in any length of column. This, of course, translates to more plates per meter and higher column efficiency.

A small volume of a tracer substance (i.e. acetone or NaCl) is added to the liquid flow close to the column inlet and the broadening of this pulse is analyzed when measured as a chromatographic peak at the column outlet. Used to test column efficiency.

Reduced plate height is a normalization of the efficiency based on the average particle diameter (dp) of the resin. This makes it possible to compare efficiency for packed bed between different resins. It is dimensionless and thus convenient parameter for efficiency characterization. This parameter facilitates the comparison of column efficiency irrespective of column length and particle diameter of the resin. The reduced plate height is calculated from h = HETP/dp, where HETP is the Height Equivalent to a Theoretical Plate and dp is the particle diameter of the chromatography resin. Optimal column efficiency typically corresponds to an experimentally determined reduced plate height of h ≤ 3 for the porous resin employed in bioprocess chromatography. This efficiency is achieved when testing a well-packed bed with an optimized set-up of column and system under optimal test conditions.

Bed height measured after setting by gravity.

Resin slurry

Resin particles suspended in liquid.

Slurry concentration

The slurry concentration is used to facilitate the calculation of the amount or resin needed to pack a certain bed height. For successful packing, different resins require different slurry concentrations. To determine the slurry concentration for large columns (approx. > 5L), we recommend that you use the Slurry Concentration Kit. You can also watch our video Column packing tutorial: Determining slurry concentration using a slurry kit. For small columns, other methods involving centrifugation and/or sedimentation are also possible to use.

How to calculate the amount of resin slurry that is needed to pack a column?

Follow the following steps.

Steps	Description of each step	Example: Packing Capto Q in an XK 16/20 column (10 cm bed height)
1. Define the volume to take from the resin bottle (Vs).	The volume of the initial resin slurry1 to take from the bottle is calculated according to the following formula: Vs = (Vc/Cs) × CF where Vs = volume of initial resin slurry in bottle needed (mL) Vc = packed bed column volume Cs = initial slurry concentration of resin in the bottle CF = compression factor stated in instruction for the resin. If not available, use 1.15 1 Most of our resins are supplied as a 75% slurry with an exception for SOURCE resins, which have a slurry concentration of 50%.	According to packing instructions Bed volume: 20.1 mL Packing solution: 20% ethanol with 0.4 M NaCl The recommended slurry concentration to pack an XK 16/20 column with Capto Q to a bed height of 10 cm is between 45 to 55%. Calculation: Vs = (20.1/0.75) × 1.15 = 30.82 mL (≈ 31 mL). So, 31 mL of the initial suspended slurry should be taken from the supplied resin bottle.
2. Wash the initial resin slurry into the packing solution	To ensure that the resin is washed thoroughly into the packing solution before starting the packing, pour Vs into a glass filter and wash it 5 times with 2 column volumes (CV) of packing solution. Gently stir with a plastic spatula between additions.	31 mL of the initial suspended slurry should be directly poured from the bottle into a glass filter. It should be washed 5 times with 2 column volumes (CV) of 20% ethanol with 0.4 M NaCl (the packing solution). In this case this corresponds to 5 × 40.2 mL of packing solution.
3. Adjust the slurry concentration	After the final wash, carefully remove the retained resin from the glass filter and transfer it to a beaker. Adjust the slurry concentration as stated in instruction for the resin. This whole slurry volume is then used when packing the column.	As we are using 31 mL of the 75% initial slurry, it means that 23 mL of resin can be transferred from the glass filter to a beaker. (31 × 75% = 23 mL) To obtain a 50% packing slurry, add 23 mL of 20% ethanol with 0.4 M NaCl (the packing solution) to the beaker and stir gently to obtain a homogenous slurry. This whole packing slurry volume is then used when packing the column.

A measure of chromatography peak broadening/column efficiency. Also referred to as number of theoretical plates. The plate number of a column is used to measure column efficiency. The more plates a packed column has, the higher the column efficiency. The plate number is calculated from N = 5.545 × (V_R/W_h)² assuming a Gaussian peak, where V_R = retention (elution) volume and W_h = peak width at half peak-height. V_R and W_h must have the same unit (e.g., mL). V_R may be substituted in the formula with retention time, t_R, but then the unit of W_h must be time.

See also Plate height/height equivalent to a theoretical plate and Reduced plate height. Also see Efficiency.

Theoretical plate numbers per meter. A high value indicates a well packed bed. Is used for comparing columns packed with resins with the same particle size.

Same as Band broadening.