Chromaflow 400 mm, automatic nozzle, 300-500 mm, 20 PE

Test equipment:
Pressure gauge range: 0-1.43 x column design pressure
2 x Manual valve, 1-way, 2-port
Packing station

Set up before test
1. Mount the pressure gauge and valve on MPT (the valve after the pressure gauge).
2. Connect the valve to MPB. Use the blind cap to prevent leakage.
3. Connect the column to the Packing station according to the figure above.

4. Prime the column according to the following instructions (a-k)
a. Prepare a tank of packing buffer or water.
b. Set the bottom nozzle to the packing position (mid) and the top nozzle to the unpacking position (extended) making sure that SOT is open.
c. Start pumping through SIB (upward flow) until the column is filled and nearly all air is removed.
d. When the level of liquid is within 10 mm of the top bed support, slow the flow down, keeping the air as one single bubble.
e. When all the air has been removed via SOT, open MPT and retract the top nozzle to its run position.
f. Start the pump again at approximately half its maximum rate.
g. Open MPB and close MPT.

Note: To prevent siphoning, either use valves or ensure that the outlets are positioned above the column.

h. Stop the pump and close MPB.
i. Retract the bottom nozzle to its run position.
j. The flow distributors, bed supports and the column should now be purged of air.
k. With both nozzles retracted, start the packing pump to prime the slurry lines (top or bottom) with packing buffer.

Note: Take great care at this stage. A high flow rate is needed to drive the air out of the packing lines.

Test
Note: Perform the test on both maximum and minimum bed heights.

1.Extend the bottom nozzle to its packing position (mid-position)
2.Set the top nozzle to its run position (fully retracted)
3.Start the packing pump slowly. Then increase the pressure in the column to the test value (1.43 x design pressure).
4.Close the SIB valve on the packing station.
5.Retract the bottom nozzle to its run position.
6.Start to register pressure and time.
7.Wait 20 min and register the pressure fall. It should not exceed 0.5 bar

Chromaflow columns
Specifications
Assembly drawings
Parts list
Spare parts list
Instructions for use
Maintenance guide
EC declaration of conformity
Production records:

Material certificates

Packing station for Chromaflow columns:
Specifications
Assembly drawing
Pneumatic scheme
Pneumatic components list
Process flow scheme
Parts list
Spare parts list
Instructions for use
EC declaration of conformity
Production records:
Valve and control panel test
Pump capacity test
Leakage test
Pressure gauge
Tube welding
Tubing surface finish
Certificate of conformity
Material certificates
Contents list

Surface finish of wetted steel parts: Ra 0.5 µm (electropolished).

The relief pressure must be lower than or equal to the maximum operating pressure.

Chromaflow

Packing stations for Chromaflow columns

Cracks are mainly due to pump pulsation or a too densely or too loosely packed bed.

Possible cause: Pump pulsation
Remedy: Repack the column with a pulse-free pump or add an air trap and a pressure relief valve to the assembly.

Possible cause: Too densely packed bed
Remedy: Repack the column with reduced packing flow rate or pressure.

Possible cause: Too loosely packed bed
Remedy: It may be possible to only adjust the adaptor. Otherwise repack the column with increased packing flow rate or pressure.

How do I correct a gap between the packed bed and lid/adaptor?
It may be possible to only adjust the adaptor. If not, you have to repack the column.

Packed bed columns
Packing and running packed bed columns requires a pump that gives a pulse-free flow. Installing a pulse reducer may also be necessary. For high flow velocities and low pressures up to approximately 3 bar, we recommend lobrotor pumps. For higher pressures, use membrane pumps with three pump heads (pump heads reduce pulsations) or screw pumps, although both these have limited flow capacity


Expanded bed adsorption
Use peristaltic pumps for STREAMLINE and STREAMLINE Direct columns

For more information 

Cleaning-in-place
Cleaning-in-place (CIP) removes precipitated material, strongly bound substances and other contaminants from the column bed without dismantling the column.
Regular CIP of the packed column between production batches ensures proper product quality and maintains expected medium and equipment life. An efficient CIP protocol depends on medium in use and the characteristics of the contaminants. Refer to the instructions for each medium.

Sanitization
Sanitization is the reduction of microbial contamination in the column and related equipment to an acceptable minimum. A specific sanitization protocol should be designed for each process according to the type of contaminants present.

Note: Always ensure that columns, systems and auxiliary components are compatible with the chemicals used.

Packed columns
Equilibrate and store packed columns according to the instructions for the medium.

Systems
Systems must be cleaned and sanitized prior to storage. During storage, keep the system filled with 0.01 M sodium hydroxide or 20% ethanol solution.
Please note that acrylic column tubes can withstand 20% ethanol up to 12 months with maximum pressure of 0.5 bar.

BPG, Chromaflow, FineLINE 70-350 columns and ÄKTAprocess
All wetted parts of plastics/elastomers have been approved according to biological reactivity testing in vivo, USP class VI.

Chromaflow

Column evaluation
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, A_s. 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.


HETP calculation
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
where,
L = Bed height (cm)
N = Number of theoretical plates.

N is defined by the equation:
N = 5.54 (V_e /W_h)²
where,
V_e = Elution volume (ml)
W_h = Peak width at half height (ml)
V_e is measured as the volume passed through the column to the peak maximum.
W_h 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
Acetone	18800	900	2417	4203	0.024

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, A_s, is calculated from the graph above:
A_s = b/a
where,
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.

Chromaflow
Chromaflow columns are available with 10 µm and 20 µm stainless steel bed supports, and a 20 µm plastic bed support. Use 10 µm stainless steel bed supports for beads with average particle diameters less than 70 µm. Otherwise use 20 µm.

Stainless steel bed supports have very high chemical resistance. Use when salt concentrations are low and pH above 5. Use polyethylene for high salt and low pH applications.

Sodium hydroxide is widely accepted for cleaning empty equipment, cleaning packed chromatography columns (cleaning-in-place , CIP), sanitizing and storing chromatography media and systems.

For CIP, sodium hydroxide has been extensively used to remove proteins and nucleic acids from ion exchange, hydrophobic interaction and gel filtration media. Its use with affinity media is restricted due to the limited stability of affinity ligands. If lipids are bound to a protein, an increased concentration of sodium hydroxide may be required for effective cleaning.

Sodium hydroxide is also effective for inactivating most viruses, bacteria, yeasts and endotoxins. It is common practice in industrial manufacturing to save time by adding salt, e.g. sodium chloride, to the sodium hydroxide solution to combine cleaning with sanitization as well. Since sodium hydroxide is a bacteriostat, it is also useful for storage.

The benefits of using sodium hydroxide include efficacy and low cost, plus ease of detection, removal and disposal.

Precautions
Always ensure that chromatography media, columns, systems and auxiliary components are compatible with sodium hydroxide at the concentration, time and temperatures used. Also keep in mind that sodium hydroxide may be corrosive to both metal and skin.

Compatibility with chromatography media
The concentration of sodium hydroxide used for CIP and/or sanitization will often depend on the level of contamination. For chromatography media, the ability to withstand stringent sanitizing conditions depends on the functional groups, attachment chemistries and the stability of the base matrices to alkaline conditions.
Table 4 in Application Note “Use of sodium hydroxide for cleaning and sanitizing chromatography media and systems” lists the general stability of a wide range of media as a function of pH. See more on Application notes: Use of sodium hydroxide for cleaning and sanitizing chromatography media and systems

Chromaflow column and packing station
Connect the column to the packing station as shown in the figure below.



Fill the packing buffer tank with cleaning solution. Then select the following positions:
1. With both top and bottom nozzles in the UNPACK position (fully-extended into the column), configure the packing station as follows:
Inlet (B) - to packing buffer tank with cleaning solution
Outlet (A) - to collection vessel for waste
Outlet (D) - to collection vessel for waste

2. Turn the packing pump on and toggle between the column (SIT) and (SIB). Cleaning solution will spray out from the top and bottom nozzles. Continue this procedure for some minutes.
3. Stop the packing pump and run the unpacking pump until all cleaning solution is removed from the column.
4. Retract the top nozzle to its RUN position (level with the bed support). Run both pumps simultaneously. Stop the pumps when the top bed support appears to be free of beads.
5. If cleaning liquid is still in the column, remove it by extending the top nozzle to its unpack position and using the unpacking pump to empty the column.
6. To clean the mobile phases, select PACK position on the bottom nozzle (mid-position)
7. Select UNPACK position on the top nozzle (fully-extended)
8. Pump liquid via the bottom nozzle. Once the column is full, liquid starts to exit via SOT. Check the column pressure on the packing pressure gauge. It should not rise above the column pressure rating; lower pressure by reducing pump speed.
9. Open MPB and close SOT by retracting the top nozzle when the column interior appears to be free of air. Liquid will now exit via MPB and clean the bottom mobile phase.
10. To clean the top mobile phase, close MPB and open MPT.
11. Stop the pump and close MPB and MPT when both mobile phases are cleaned. Extend the top and bottom nozzle to their unpack position and use the unpacking pump to empty the column.
12. Finally, repeat steps 1 to 9 using 20% ethanol. Then allow the column to drain. The column is now clean and ready to pack or store.

Chromarflow
Complement the valves on the mobile phase with two 3-port, 2-way valves; one at the top and one at the bottom of the column.

Chromaflow Packing station Selection guide

Chromaflow
Various standard exchangeable bed heights are available in the ranges 100-300 mm, 200-400 mm and 300-500 mm. For other bed heights, please contact your Cytiva representative. Note that you must also replace the tube rods when changing the column tube.