|Column||Maximum operation pressure (bar)|
|FineLINE Pilot 35||20|
Recommended net porosity for Capto adhere, Capto DEAE, Capto MMC, Capto Q, Capto Viral Q and Capto S: 20 µm
|Column||Compression factor||Slurry concentration (%)||Bed height (cm)||Flow velocity (cm/h)|
|Chromaflow 400||1.1||50 - 60||20 - 45||20 to settle the bed|
|Chromaflow 600||1.1||50 - 60||20 - 45||20 to settle the bed|
|Chromaflow 800||1.1||50 - 60||20 - 45||20 to settle the bed|
|Chromaflow 1000||1.1||50 - 60||20 - 45||20 to settle the bed|
BPG. Approximative values for compression factor, packing factor, slurry concentration and flow velocity
|Column||Compression factor*||Packin factor**||Slurry concentration (%)||Bed height (cm)||Flow velocity (cm/h)|
|50 - 60||20 - 45||30 to settle the bed|
* Compression factor = Gravity settled bed height / Packed bed height
** Packing factor = Consolidated bed height at 30-60 cm/h / Packed bed height
|23 µm nets|
|Adaptor net Code no: 18-1103-08|
|End piece net Code no: 18-9252-01|
|23 µm nets|
|Adaptor net Code no: 18-1113-01|
|End piece net Code no: 18-1113-00|
|23 µm nets|
|Adaptor net Code no: 18-9253-01|
|End piece net Code no: 18-9254-01|
|23 µm nets|
|Adaptor net Code no: 18-1012-54|
|End piece net Code no: 18-1012-34|
Recommended flow velocity intervals in the table below are for packing the column. The intervals are given in reverse order to indicate that the high flow velocity can be used for shorter beds whereas higher beds only allow lower flow velocity, due to the higher pressure drop across the bed.
|Column||Compression factor||Slurry concentration (%)||Bed height (cm)||Flow velocity (cm/h)|
|XK 16||1.1||50 - 60||20 - 30||1200 - 900|
|XK 16||1.1||50 - 60||30 - 40||900 - 600|
|XK 16||1.1||50 - 60||40 - 45||600 - 450|
|XK 26||1.1||50 - 60||20 - 30||1200 - 900|
|XK 26||1.1||50 - 60||30 - 40||900 - 600|
|XK 26||1.1||50 - 60||40 - 45||600 - 450|
|XK 50||1.1||50 - 60||20 - 30||1200 - 900|
|XK 50||1.1||50 - 60||30 - 40||900 - 600|
|XK 50||1.1||50 - 60||40 - 45||600 - 450|
Packing buffer: Water, ethanol or NaCl
Please read the Application Note: Methods for packing Capto MMC in production-scale columns in Related Documents.
The same packing technique is valid for Capto adhere and Capto MMC.
Packing small-scale AxiChrom columns
AxiChrom™ 50, 70, 100, 140, and 200 columns are designed to simplify column handling and maintenance during process development.
- Preprogrammed, verified packing methods that include Capto™, MabSelect™ media
- Reproducible, predictable and scalable results.
- Rotating pivot stand for easy emptying, maintenance and safer operation
- Proven sanitary design with construction materials recognized for use in GMP environments.
- Operates with ÄKTAexplorer™ and ÄKTApilot™ with UNICORN™ v 5.01 (and later).
AxiChrom columns are designed to simplify column handling and maintenance. Packing is facilitated by the Intelligent Packing methodology in the UNICORN software. The preprogrammed, verified packing methods shorten start-up time, eliminate errors and pack the column with optimal bed compression.
The pivot design allows the column tube to be tilted for convenient manual emptying as well as easy access to parts for maintenance.
Scalable and predictable performance over the entire AxiChrom platform is assured through the use of computational fluid dynamics in designing the liquid distribution system.
The AxiChrom column is sanitizable, and materials of construction are suitable for use in regulated environments. Comprehensive documentation, including IQ/OQ documents are available for all column sizes.
Packing large-scale AxiChrom columns
Large-scale AxiChrom™ columns are supported with Intelligent Packing and intuitive handling via the AxiChrom Master.The novel, swing-out-design facilitates maintenance and provides a safer working enivironment.
- Preprogrammed verified packing methods that include Capto™ and MabSelect™ media
- Reproducible and predictable results
- Swing-out, hoist-free design for easy maintenance and safer operation
- Proven, sanitary design meets stringent cGMP production standards
- Key steps guided by AxiChrom Master
Intelligent Packing provides preprogrammed, verified axial compression packing methods that promote accurate and reproducible results. Intuitive handling supports preparation and maintenance procedures. Both are controlled by the AxiChrom Master, a separate unit with a touch-screen interface that guides the operator through critical steps for priming, packing, unpacking, and maintenance.
Packing large-scale AxiChrom columns (Video below)
Easy access to all column parts for maintenance in-situ is facilitated by the swing-out, hoist-free, column tube design.
The distribution system design has been developed using analytical methods and modern computational fluid dynamic modeling tools to ensure reproducible and predictable results over the entire range of scales.
Materials of construction meet the ASME Bioprocessing Equipment Standard for biopharmaceutical manufacture.
AxiChrom columns are supplied with comprehensive documentation to facilitate set-up, installation, and operation.
The efficiency of a column depends on how well it is packed. A poorly packed column gives rise to uneven flow, resulting in zone broadening and reduced resolution. It is thus important to have a method by which the column can be tested before it is put into operation. Such a method should be simple, quantitative and should not introduce contaminating materials. It is also an advantage if the same method can be used to monitor column performance over its working life, so that it is easy to determine when the medium should be re-packed or replaced.
Avoid methods that use colored compounds such as Blue Dextran. They do not meet the above criteria and cannot be used with ion exchange and affinity chromatography media.
Experience has shown that the best method of expressing the efficiency of a column is in terms of the height equivalent to a theoretical plate, HETP, reduced plate number, h, and the peak asymmetry factor, As. These values can be determined easily by applying a NaCl or acetone solution, to the column (see below).
It is important that the column is properly equilibrated ( >2 column volumes) before evaluating the packing. Ideally, run three test runs to see whether the values are stable. If an initially poor result improves during a later test, the reason can be that the column was not properly equilibrated. To check that the bed is stable, run the column at 70% packing pressure for 20 hours and test it again.
Note that pressure spikes may cause poor packing (cracking). If this happens, fit an air trap and a pressure relief valve between the pump and column. Locate the pressure relief valve between the air trap and the column.
Choice of test sample for columns
The most appropriate material for column testing is, of course, the sample that is to be run in the application, but this is not always practical or economical. As an alternative, a solution of either NaCl or acetone will give a good indication of the column packing quality. The eluate is monitored by measuring conductivity or UV absorption, and the resulting elution profile is used to calculate the HETP value.
The advantages of using NaCl are that it is readily available and can be used safely to test all columns. One disadvantage is that NaCl may interact with the medium matrix, especially ion exchanger matrices, and thus give erroneous results.
Acetone, in contrast, does not interact with the matrix and is detected by UV absorption at 280 nm. Alternatively, you can increase the running buffer concentration 10-fold and use it as test solution.
The figure below shows a UV trace for acetone in a typical BPG column application and gives calculated HETP and As values.
The sample volume should be approximately 1% of the total bed volume and the concentration 1.0% v/v NaCl, or equivalent when using stronger buffer. Alternatively, use 1.0% v/v acetone. The flow velocity should be between 10 and 30 cm/h depending on the bead size of the chromatography medium. The high flow velocity could be used for small beads whereas large beads only allow low flow velocity. To avoid diluting the sample, apply it as close to the column inlet as possible. If an airtrap is included in the system, by-pass it during sample application to avoid back-mixing. Calculate the HETP value from the conductivity (or UV) curve as follows:
HETP, in its simplest terms, is expressed as:
HETP = L/N
L = Bed height (cm)
N = Number of theoretical plates.
N is defined by the equation:
N = 5.54 (Ve /Wh)2
Ve = Elution volume (ml)
Wh = Peak width at half height (ml)
Ve is measured as the volume passed through the column to the peak maximum.
Wh is measured as the peak width at half-peak height.
From the example in the figure, the HETP value can be calculated from the chromatogram as follows:
|Ve (ml)||Wh (ml)||N||N/m||HETP cm|
Well-packed columns have low HETP values. However, it is only possible to compare columns that have been packed with the same type of media and that have been tested under identical conditions.
As a general rule-of-thumb, a good HETP value is approximately two to four times the mean bead diameter of the medium in question, provided that the sample does not interact with the medium.
In practice, the correlation between HETP and column performance can only be assessed by the column operator. Once this has been established, a standard can be set to judge the acceptability of a column packing.
For example, the column operator may know from experience that a column packed with Sephadex G-25 gel filtration medium with HETP values above 0.05 cm does not give the required separation. Consequently, the operator will set this value as the maximum permissible i.e. the minimum acceptable quality.
Reduced plate number
Definition of reduced plate number: h = HETP/dp
h = reduced plate number.
HETP = above described height equivalent to a theoretical plate.
dp = mean particle diameter of the chromatography medium beads.
The reduced plate number should be in the range of 2-4 times the mean particle diameter of the chromatography medium beads.
Peak asymmetry factor calculation
The peak asymmetry factor should be as close as possible to 1, and the shape of the peak should be as symmetrical as possible. This is usually the case for gel filtration media, but for certain ion exchange and affinity media, the shape may be asymmetrical due to interaction with the media. A change in peak shape is usually the first indication of column deterioration.
The peak asymmetry factor, As, is calculated from the graph above:
As = b/a
a = distance from peak apex to 10% of the peak height on the ascending side of the peak
b = distance from peak apex to 10% of the peak height on the descending part of the peak
Note: Measuring HETP, h and As values is the best way to judge the condition of the packed column. A packed column can look good, but still need repacking for optimal performance. Always check the column after packing and regularly between runs to ensure best performance.
20 µm net
Stainless steel or plastic
For code no and detailed information see the spare part section.
|10 µm nets (5 pieces/pack) Code no: 18-8761-01|
|10 µm nets (5 pieces/pack) Code no: 18-8760-01|
|10 µm nets (5 pieces/pack) Code no: 18-8759-01|
Constant flow packing.
Packing buffer: 20% ethanol
Packing Capto adhere in a XK 16/20 column
Glass filter funnel
Amount of medium
The amount of Capto adhere medium needed can be calculated by: column cross sectional area (cm2) x bed height (cm) x compression factor (settled medium bed height/packed medium bed height). The compression factor is 1.05-1.15 for Capto adhere in XK columns (the higher factor is valid for lower beds).
Washing the medium
Equilibrate all materials to room temperature. Mount the glass filter funnel onto the filtering flask. Pour the medium into the funnel and wash with approximately 5-10 ml 20% ethanol per ml medium.
Preparing the packing slurry
The slurry concentration should be 40-60% in 20% ethanol, measured in a measuring cylinder after settling overnight.
XK 16/20 is used for 10 cm bed height and XK 16/40 for 8-35 cm bed height with two flow adaptors. A packing reservoir can be used to obtain a lower slurry concentration.
An ÄKTAdesign™ 100 system or a stand-alone pump that can deliver 40 ml/min is used. The pump filter unit and flow restrictor should be removed due to the high flow velocity used in the column packing in order to decrease the system backpressure. Mount a pressure gauge at the inlet of the column to make sure that the pressure does not exceed the operating pressure limit of the column.
XK 16/20 packing procedure
To pack the column, use 20% ethanol and proceed as follows:
1. Wet the bottom filter with 20% ethanol, with the aid of a syringe mounted on the outlet tubing. After the filter is wetted mount a stop screw on the outlet.
2. Mount the bottom piece onto the column tube.
3. Wet the adapter filter with 20% ethanol, with the aid of a syringe mounted on the inlet tubing. After the filter is wetted mount a stop screw on the inlet.
4. Keep the adapter in a beaker with 20% ethanol.
5. Mount the column vertically on a stand.
6. Fill the glass tube with medium slurry up to the upper edge of the glass tube.
7. Mount the adapter in the upper part of the glass tube with no air trapped below the filter.
8. Tighten the sealing-ring and connect the column inlet to the system outlet.
9. Open the bottom outlet of the column
10. Pack the column at 1200 cm/h (40 ml/min, XK 16) for 10 min.
Note: Do not exceed the operating pressure limit for the columns (5 bar for XK 16 and XK 26).
11. If a packing reservoir is used: Stop the pump and mount a stop screw on the outlet. Disconnect the packing reservoir and repeat step 7-10.
12. Mark the bed height on the glass tube with a pen.
13. Stop the pump and mount a stop screw on the outlet.
14. Adjust the adapter quickly down to approximately 1 cm above the medium surface with the o-ring loose.
15. Tighten the o-ring and adjust the adaptor down to the mark and further 15 mm into the medium bed.
Note: The compression of the bed is 15 mm independent of bed height.