FAQ
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
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 |
Overall height
(m) |
Footprint
(m x m) |
Total weight (dry) (kg)
|
Adaptor weight (kg)
|
Min. overhead height for maintenance (m)
|
Max. operating pressure (bar)
|
Chromaflow 400/100-300 |
1.57
|
0.70x 0.70 |
230
|
78
|
2.00
|
3
|
Chromaflow 400/200-400 |
1.67
|
0.70x0.70 |
238
|
78
|
2.10
|
3
|
Chromaflow 400/300-500 |
1.77
|
0.70x 0.70 |
244
|
78
|
2.20
|
3
|
Chromaflow 600/100-300 |
1.57
|
0.80 x 0.80 |
375
|
143
|
2.16
|
3
|
Chromaflow 600/200-400 |
1.67
|
0.80 x 0.80 |
385
|
143
|
2.26
|
3
|
Chromaflow 600/300-500 |
1.77
|
0.80 x 0.80 |
393
|
143
|
2.36
|
3
|
Chromaflow 800/100-300 |
1.57
|
1.0 x 1.0 |
610
|
250
|
2.31
|
3
|
Chromaflow 800/200-400 |
1.67
|
1.0 x 1.0 |
627
|
250
|
2.41
|
3
|
Chromaflow 800/300-500 |
1.77
|
1.0 x 1.0 |
638
|
250
|
2.51
|
3
|
Chromaflow 1000/100-300 |
1.57
|
1.2 x 1.2 |
930
|
377
|
2.46
|
3
|
Chromaflow 1000/200-400 |
1.67
|
1.2 x 1.2 |
953
|
377
|
2.56
|
3
|
Chromaflow 1000/300-500 |
1.77
|
1.2 x 1.2 |
972
|
377
|
2.66
|
3
|
Packing stations for Chromaflow columns
Packing station | Overall height (m) |
Footprint (m x m) |
Total weight (dry) (kg) |
Pack50 | 1.18 | 0.81 x 0.72 | 115 |
Pack100 | 1.18 | 0.81 x 0.72 | 130 |
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.
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
Surface finish of wetted steel parts: Ra 0.5 µm (electropolished).
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
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.
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.
Chromaflow Packing station Selection guide
Packing Station |
Min. L/min | Max. L/min |
Pack50 |
10
|
50
|
Pack100 |
30
|
100
|
Pack200 |
60
|
200
|
Pack400 |
100
|
400
|
Column |
Inner diameter of tubing (mm) | Column | Inner diameter of tubing (mm) |
Chromaflow 400 | 19 | Chromaflow 400 SFP | For mobile phase i.d. 10-14 mm. Otherwise i.d. 19 mm. |
Chromaflow 600 | 19 | Chromaflow 800 | 19 |
Chromaflow 1000 | 19 |
-
|
-
|
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.
The relief pressure must be lower than or equal to the maximum operating pressure.
Column | Max. operating pressure (bar) | Column | Max. operating pressure (bar) |
Chromaflow 400 | 3 | Chromaflow 400 SFP | 3 |
Chromaflow 600 | 3 | Chromaflow 800 | 3 |
Chromaflow 1000 | 3 |
-
|
-
|
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.
Chromaflow columns
Specifications
Assembly drawings
Parts list
Spare parts list
Instructions for use
Maintenance guide
EC declaration of conformity
Production records:
- Hydrostatic pressure test
- Final inspection report
- Surface finish and electropolishing
- Welding documentation
- Certificate of conformity
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
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, 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.
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 (Ve /Wh)2
where,
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 | |
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, As, is calculated from the graph above:
As = 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
Column |
Min. bed height (cm)
|
Max. bed
height (cm) |
Min. bed volume (L)
|
Max. bed volume (L)
|
Chromaflow 400/100-300 |
100
|
300
|
13.1
|
38.3
|
Chromaflow 400/200-400 |
200
|
400
|
25.6
|
50.8
|
Chromaflow 400/300-500 |
300
|
500
|
38.3
|
63.3
|
Chromaflow 600/100-300 |
100
|
300
|
29.7
|
86.3
|
Chromaflow 600/200-400 |
200
|
400
|
57.9
|
114
|
Chromaflow 600/300-500 |
300
|
500
|
86.3
|
142
|
Chromaflow 800/100-300 |
100
|
300
|
53.3
|
154
|
Chromaflow 800/200-400 |
200
|
400
|
104
|
204
|
Chromaflow 800/300-500 |
300
|
500
|
154
|
254
|
Chromaflow 1000/100-300 |
100
|
300
|
84.3
|
241
|
Chromaflow 1000/200-400 |
200
|
400
|
163
|
320
|
Chromaflow 1000/300-500 |
300
|
500
|
241
|
399
|
Accessories
Figure. Running Chromaflow 1000 Columns.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Tubing, 900 mm, 25 mm TC, i.d.10 mm | 18101262 | 393.00 USD |
Add to cart
|
1 | Tubing, 1400 mm, 25 mm TC, i.d.10 mm | 18101263 | 413.00 USD |
Add to cart
|
1 | Tubing, 1700 mm, 25 mm TC, i.d. 10 mm | 18101264 | 441.00 USD |
Add to cart
|
1 | Tubing, 2000 mm, 25 mm TC, i.d. 10 mm | 18101287 | 507.00 USD |
Add to cart
|
1 | Tubing, 750 mm, 25 mm TC, i.d. 14 mm | 18102728 | 425.00 USD |
Add to cart
|
1 | Tubing, 1800 mm, 25 mm TC, i.d. 14 mm | 18102729 | 535.00 USD |
Add to cart
|
1 | Tubing with Sanitary Fittings, 50 cm, i.d. 9.5 mm, 25 mm TC | 28405373 | 514.00 USD |
Add to cart
|
1 | Tubing with Sanitary Fittings, 80 cm, i.d. 9.5 mm, 25 mm TC | 28405375 | 573.00 USD |
Add to cart
|
1 | Tubing with Sanitary Fittings, 1 m, i.d. 9.5 mm, 25 mm TC | 28405376 | 578.00 USD |
Add to cart
|
1 | Tubing with Sanitary Fittings, 2 m, i.d. 9.5 mm, 25 mm TC | 28405377 | 774.00 USD |
Add to cart
|
1 | Tubing with Sanitary Fittings, 4 m, i.d. 9.5 mm, 25 mm TC | 28405378 | 1,018.00 USD |
Add to cart
|
2 | Tubing with sanitary fittings, L=90 cm, i.d.19 mm, 51 mm TC | 28404230 | 539.00 USD |
Add to cart
|
2 | Tubing with sanitary fittings, L=200 cm, i.d.19 mm, 51 mm TC | 28404232 | 4,088.00 USD |
Add to cart
|
2 | Tubing with Sanitary Fittings, 400 cm, i.d.19 mm, 51 mm TC | 28404233 | 1,525.00 USD |
Add to cart
|
2 | Tubing with sanitary fittings, L=140 cm, i.d.19 mm, 51 mm TC | 28404235 | 647.00 USD |
Add to cart
|
4 | Gasket, 25 mm, i.d. 10 mm | 18101240 | 113.99 USD |
Add to cart
|
4 | TC-Gasket 25 mm i.d. 10 mm | 18103579 | 34.15 USD |
Add to cart
|
5 | Gasket 51 mm | 28966886 | 91.28 USD |
Add to cart
|
5 | Gasket, 51 mm, i.d. 22 mm | 44551203 | 102.54 USD |
Add to cart
|
7 | TC Clamp 25mm SS | 18100131 | 47.13 USD |
Add to cart
|
7 | Clamps 25 mm | 44056801 | 191.20 USD |
Add to cart
|
8 | Clamp, 50 mm TC, SS | 44713401 | 11.38 USD |
Add to cart
|
9 | Valve 2-way, 3-port, i.d. 22 mm, 51 mm TC | 44158301 | 2,568.00 USD |
Add to cart
|
10 | Blind Flange, 51 mm TC | 44713501 | 109.71 USD |
Add to cart
|
11 | Pressure sensor i.d. 22 mm, 51 mm TC | 28934324 | 5,687.00 USD |
Add to cart
|
12 | Safety valve, 3 bar, 51 mm TC | 18573801 | 3,803.00 USD |
Add to cart
|
14 | Castors assembly kit, Chromaflow 800-1000 | 18117152 | 4,066.00 USD |
Add to cart
|
15 | Nozzle Piping, Chromaflow 1000, i.d. 1", 51 mm TC | 18117207 | 3,694.00 USD |
Add to cart
|
Figure. Packing and unpacking the Chromaflow 1000 Columns with a packing station.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
2 | Tubing, 900 mm, 25 mm TC, i.d.10 mm | 18101262 | 393.00 USD |
Add to cart
|
2 | Tubing, 1400 mm, 25 mm TC, i.d.10 mm | 18101263 | 413.00 USD |
Add to cart
|
2 | Tubing, 1700 mm, 25 mm TC, i.d. 10 mm | 18101264 | 441.00 USD |
Add to cart
|
2 | Tubing, 2000 mm, 25 mm TC, i.d. 10 mm | 18101287 | 507.00 USD |
Add to cart
|
2 | Tubing, 750 mm, 25 mm TC, i.d. 14 mm | 18102728 | 425.00 USD |
Add to cart
|
2 | Tubing, 1800 mm, 25 mm TC, i.d. 14 mm | 18102729 | 535.00 USD |
Add to cart
|
2 | Tubing with Sanitary Fittings, 50 cm, i.d. 9.5 mm, 25 mm TC | 28405373 | 514.00 USD |
Add to cart
|
2 | Tubing with Sanitary Fittings, 80 cm, i.d. 9.5 mm, 25 mm TC | 28405375 | 573.00 USD |
Add to cart
|
2 | Tubing with Sanitary Fittings, 1 m, i.d. 9.5 mm, 25 mm TC | 28405376 | 578.00 USD |
Add to cart
|
2 | Tubing with Sanitary Fittings, 2 m, i.d. 9.5 mm, 25 mm TC | 28405377 | 774.00 USD |
Add to cart
|
2 | Tubing with Sanitary Fittings, 4 m, i.d. 9.5 mm, 25 mm TC | 28405378 | 1,018.00 USD |
Add to cart
|
3 | Tubing with sanitary fittings, L=90 cm, i.d.19 mm, 51 mm TC | 28404230 | 539.00 USD |
Add to cart
|
3 | Tubing with sanitary fittings, L=200 cm, i.d.19 mm, 51 mm TC | 28404232 | 4,088.00 USD |
Add to cart
|
3 | Tubing with Sanitary Fittings, 400 cm, i.d.19 mm, 51 mm TC | 28404233 | 1,525.00 USD |
Add to cart
|
3 | Tubing with sanitary fittings, L=140 cm, i.d.19 mm, 51 mm TC | 28404235 | 647.00 USD |
Add to cart
|
3 | Tubing with Sanitary Fittings, 50 cm, i.d. 19.1 mm, 50 mm TC | 28405379 | 2,315.00 USD |
Add to cart
|
3 | Tubing with Sanitary Fittings, 80 cm, i.d. 19.1 mm, 50 mm TC | 28405380 | 2,547.00 USD |
Add to cart
|
3 | Tubing with Sanitary Fittings, 1 m, i.d. 19.1 mm, 50 mm TC | 28405381 | 2,663.00 USD |
Add to cart
|
3 | Tubing with Sanitary Fittings, 2 m, i.d. 19.1 mm, 50 mm TC | 28405382 | 3,472.00 USD |
Add to cart
|
3 | Tubing with Sanitary Fittings, 4 m, i.d. 19.1 mm, 50 mm TC | 28405383 | 4,399.00 USD |
Add to cart
|
5 | Gasket, 25 mm, i.d. 10 mm | 18101240 | 113.99 USD |
Add to cart
|
5 | TC-Gasket 25 mm i.d. 10 mm | 18103579 | 34.15 USD |
Add to cart
|
6 | Gasket 51 mm | 28966886 | 91.28 USD |
Add to cart
|
6 | Gasket, 51 mm, i.d. 22 mm | 44551203 | 102.54 USD |
Add to cart
|
8 | TC Clamp 25mm SS | 18100131 | 47.13 USD |
Add to cart
|
8 | Clamps 25 mm | 44056801 | 191.20 USD |
Add to cart
|
9 | Clamp, 50 mm TC, SS | 44713401 | 11.38 USD |
Add to cart
|
10 | Valve 2-way, 3-port, i.d. 22 mm, 51 mm TC | 44158301 | 2,568.00 USD |
Add to cart
|
11 | Pressure sensor i.d. 22 mm, 51 mm TC | 28934324 | 5,687.00 USD |
Add to cart
|
12 | Safety valve, 3 bar, 51 mm TC | 18573801 | 3,803.00 USD |
Add to cart
|
14 | Castors assembly kit, Chromaflow 800-1000 | 18117152 | 4,066.00 USD |
Add to cart
|
15 | Nozzle Piping, Chromaflow 1000, i.d. 1", 51 mm TC | 18117207 | 3,694.00 USD |
Add to cart
|
Spare parts
Item numbers with a white background are not available as standard spare parts.
Please note that item numbers are not necessarily in sequential order.
Tools are not present in the figure.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Seal kit, nozzle man/auto | 18116104 | 552.00 USD |
Add to cart
|
1 | Nozzle tip, with O-rings mounted | 18116106 | 1,392.00 USD |
Add to cart
|
2 | Nozzle tip, with O-rings mounted | 18116106 | 1,392.00 USD |
Add to cart
|
7 | Tool, Bed support retaining nut | 18116110 | 1,683.00 USD |
Add to cart
|
8 | Mounting Tool, Nozzle tip seal | 18116175 | 293.00 USD |
Add to cart
|
10 | Tool, Bed support nut | 18116178 | 658.00 USD |
Add to cart
|
Item numbers with a white background are not available as standard spare parts.
Please note that item numbers are not necessarily in sequential order.
Tools are not present in the figure.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
7 | Tool, Bed support retaining nut | 18116110 | 1,683.00 USD |
Add to cart
|
8 | Mounting Tool, Nozzle tip seal | 18116175 | 293.00 USD |
Add to cart
|
10 | Tool, Bed support nut | 18116178 | 658.00 USD |
Add to cart
|
11 | Bed support nut | 18116177 | 408.00 USD |
Add to cart
|
12 | Seal kit, CF 1000 S10 and S20 | 28981074 | 4,992.00 USD |
Add to cart
|
14 | Locking rod, Chromaflow 1000, M24 | 18116250 | 390.00 USD |
Add to cart
|
15 | Locking rod nut, M24 | 18116415 | 41.67 USD |
Add to cart
|
16 | Bed support bottom Chromaflow 1000,10 µm complete | 28942705 | 32,200.00 USD |
Add to cart
|
17 | Bed support adaptor Chromaflow 1000,10 µm complete | 28942706 | 30,900.00 USD |
Add to cart
|
18 | Bed support bottom Chromaflow 1000, 20 µm complete | 28942708 | 29,500.00 USD |
Add to cart
|
19 | Bed support adaptor Chromaflow 1000, 20 µm complete | 28942709 | 28,200.00 USD |
Add to cart
|
22 | Column tube 1000/100-300 | 18116119 | 72,300.00 USD |
Add to cart
|
22 | Column tube 1000/200-400 | 18116120 | 76,300.00 USD |
Add to cart
|
22 | Column tube 1000/300-500 | 18116121 | 82,200.00 USD |
Add to cart
|
O-rings should be checked regularly for wear and replaced when needed or at least annually. Worn O-rings may not seal properly.
We recommend you to keep a set of nozzle seals and column seals on your site, for code no refer to table below.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Seal kit, CF 1000 S10 and S20 | 28981074 | 4,992.00 USD |
Add to cart
|
2 | Seal kit, nozzle man/auto | 18116104 | 552.00 USD |
Add to cart
|
3 | Nozzle tip, with O-rings mounted | 18116106 | 1,392.00 USD |
Add to cart
|
4 | Bed support retaining nut | 18116107 | 661.00 USD |
Add to cart
|
5 | Flushing kit sanitary barrier, flushing tube unit | 18116108 | 821.00 USD |
Add to cart
|
Item numbers with a white background are not available as standard spare parts.
Please note that item numbers are not necessarily in sequential order.
Tools are not present in the figure.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
3 | Bed support retaining nut | 18116107 | 661.00 USD |
Add to cart
|
4 | Flushing kit sanitary barrier, flushing tube unit | 18116108 | 821.00 USD |
Add to cart
|
5 | Clamp nut, insert M24 | 18116413 | 1,047.00 USD |
Add to cart
|
7 | Tool, Bed support retaining nut | 18116110 | 1,683.00 USD |
Add to cart
|
8 | Mounting Tool, Nozzle tip seal | 18116175 | 293.00 USD |
Add to cart
|
10 | Tool, Bed support nut | 18116178 | 658.00 USD |
Add to cart
|
12 | Seal kit, CF 1000 S10 and S20 | 28981074 | 4,992.00 USD |
Add to cart
|
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
General advice to achieve good performance
Before packing or running the column please make sure that
- the tubing, valves, gaskets etc. have the same inner diameter as the column in and outlets and tubing is not longer than needed.
- the bed supports have a porosity appropriate for your chromatography medium.
- the column has been correctly assembled, verified by passing a leakage test.
- the chromatography medium and equipment are equilibrated in the packing buffer and to ambient temperature of packing environment.
- that the pump is free from pulsations.
- no pressure peaks appear.
- that you have enough slurry volume, taken in account the compression factor of the medium.
- packing is performed at optimal flow velocity.
- the column meets pressure requirement for selected medium and packing procedure.
After completing the packing procedure please evaluate the efficiency of the column in terms of the height equivalent to a theoretical plate (HETP), reduced plate number (h) and the peak asymmetry factor (As).
Between batches it is recommended to perform cleaning-in-place (CIP) to remove precipitated material, strongly bound substances and other contaminants from the column bed without dismantling the column. Regular CIP is needed to ensure proper product quality and expected life time of medium and equipment.
Many issues that face chromatographers are common to all types of columns, while other issues are quite specific.
Select the appropriate equipment.
Worrying peak shape
Possible cause | Suggested remedy |
---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Protein precipitated on the bed supports and/or in the bed. |
Clean and regenerate the column and chromatography medium according to instructions. |
Possible cause | Suggested remedy |
---|---|
Column over-loaded |
Decrease the amount of sample or increase the amount of chromatography medium. I.e. increase the margin of safety. We recommend a margin of safety of approximately 10 %. |
Possible cause | Suggested remedy |
---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Protein precipitated on the bed supports and/or in the bed |
Clean and regenerate the column and chromatography medium according to instructions. |
Possible cause | Suggested remedy |
---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
For reversed phase chromatography - Organic solvents have denatured the protein. |
Use a higher flow velocity and/or an adapted gradient to shorten residence time on the column and thus minimize exposure. |
Gradient slope or isocratic step too shallow |
Increase steepness of gradient or isocratic step. |
Protein precipitated on the bed supports and/or in the bed |
Clean and regenerate the column and chromatography medium according to instructions |
Possible cause | Suggested remedy |
---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Protein precipitated on the bed supports and/or in the bed |
Clean and regenerate the column and chromatography medium according to instructions |
Possible cause | Suggested remedy |
---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Protein precipitated on the bed supports and/or in the bed |
Clean and regenerate the column and chromatography medium according to instructions. |
Column leakage
Possible cause | Suggested remedy |
---|---|
Screws securing the nozzle to the stainless steel support plate not correctly tightened, or O-rings worn out |
Check that the screws are correctly tightened. If the leak persists, check the O-rings. |
Possible cause | Suggested remedy |
---|---|
Bed support torn |
Disassemble the column according to the Chromaflow maintenance guide and check the bed support. |
Bed support incorrectly assembled |
Check that the bed support is correctly fastened to the adaptor/bottom unit. Check the bed support-retaining device in the center. |
Possible cause | Suggested remedy |
---|---|
Gaskets worn out. |
Gaskets lose flexibility with time and need to be replaced regularly. Inspect and replace if necessary and at least annually. |
Connectors poorly positioned or not tightened |
Check the connectors. |
Connectors not compatible with solvents |
Check chemical resistance with the connector supplier. |
Connectors not compatible with each other |
Check compatibility. Please see the connector guides. |
Possible cause | Suggested remedy |
---|---|
End-piece, O-rings and/or flange not properly positioned with respect to the tube |
Disassemble the column and check the position of the end-piece, O-rings and flange. Assemble the column and perform leakage tests according to instructions. |
O-rings and gaskets not compatible with the solvents |
Most standard O-rings and gaskets are made of EPDM (Ethylene propylene diene), which changes characteristics when exposed to some organic chemicals. For repetitive long-term use, use |
O-rings worn out |
O-rings loose their flexibility with time and need to be replaced regularly. Disassemble the column and inspect the O-rings. Replace if necessary and at least annually. Assemble the column and test for leakage according to instructions. Preventive action: |
Possible cause | Suggested remedy |
---|---|
Tubing not compatible with solvents |
Check chemical resistance with the tubing supplier. |
Strange peaks
Possible cause | Suggested remedy |
---|---|
Leakage of non-eluted protein due to poor cleaning-in-place |
Clean and regenerate the column and chromatography medium according to instructions. |
Unwashed buffer tanks |
Clean the tanks and prepare fresh solutions. |
Possible cause | Suggested remedy |
---|---|
Air inadvertently injected with the sample. |
Try to remove air bubbles by washing extensively with solution at high flow velocity. Run in upward flow direction. |
Column and/or solution subjected to sudden temperature changes |
Equilibrate the column and solutions to working temperature before use. |
Sample not completely dissolved in buffer |
Ensure that the sample is completely dissolved before applying it to the column. |
System effects, valve-switching, dead volumes, etc. cause uneven buffer velocity |
Minimize if possible. Accept the remaining effects. |
Poor reproducibility
Possible cause | Suggested remedy |
---|---|
Column bleeding from previous run |
Check and adjust your cleaning procedure. Clean the equipment and chromatography medium according to instructions. |
Column clogged with denaturated proteins and/or lipids |
Clean and regenerate the column and chromatography medium according to instructions. |
For reversed phase and hydrophobic interaction chromatography - Changes in ambient temperature affect retention times. |
Keep the temperature constant. |
Incomplete equilibration of the column |
Check pH and conductivity of the effluent before applying the sample. Continue to equilibrate if necessary. |
Incorrect pH and/or ionic strength of the solutions. |
Check pH and conductivity and adjust if necessary. Calibrate your conductivity and pH meters. |
Larger sample mass load applied compared with earlier runs |
Keep mass of sample constant when repeating runs. (High protein concentration can cause protein interaction, resulting in change of elution profile.) |
Possible cause | Suggested remedy |
---|---|
Altered stationary phase properties |
Confirm by running a known sample. If the change is persistent, replace the chromatography medium. |
Possible cause | Suggested remedy |
---|---|
Bound substances not removed during cleaning |
Clean the chromatography medium according to instructions. |
Ligand partially degraded |
Replace the chromatography medium. |
Possible cause | Suggested remedy |
---|---|
Insufficient column regeneration |
Prolong the regeneration. |
Column not properly equilibrated |
Check the pH and the conductivity of the effluent before applying the sample. Continue to equilibrate with start buffer if necessary. |
Possible cause | Suggested remedy |
---|---|
Protein properties change with concentrations |
Dilute or concentrate the sample to minimize effects. |
Proteins precipitate at high concentration. |
Reduce sample concentration and/or binding capacity. |
Possible cause | Suggested remedy |
---|---|
For affinity, hydrophobic interaction and ion exchange chromatography - Continuous build-up of contaminants has altered the selectivity of the chromatography medium. |
Clean the chromatography medium according to instructions. |
For reversed phase chromatography - Strongly retained contaminants accumulate on the column and saturate active sites on the stationary phase. |
Clean and regenerate the column and chromatography medium according to instructions. |
Unusual column appearance
Possible cause | Suggested remedy |
---|---|
Column and chromatography medium not correctly cleaned |
Clean the column and chromatography medium according to instructions. Replace the bed supports with new ones if necessary. |
Column not stored under bacteriostatic conditions |
Include a bacteriostatic agent in the storage solution, e.g. 20% ethanol or 10 mM NaOH. |
Connectors not correctly tightened |
Check and tighten if necessary. |
Top adaptor sealing loose and/or supernatant above the seal no longer contains 20% ethanol. |
Always ensure that the seal is tight and that the supernatant above the seal contains 20% ethanol. |
Possible cause | Suggested remedy |
---|---|
Bed support damaged or incorrectly assembled allowing chromatography medium particles to leave the column |
|
Buffer conditions deviate with regard to temperature, conductivity, viscosity, content of organic solvent (reduces surface tension) or other factor |
Check the buffers and choose more suitable conditions. |
Increased resistance to flow due to blocked bed support compressing the packed bed |
Clean or change the bed support. Disassemble the column and replace the support according to instructions. Preventive action: |
Poorly packed bed (not sufficiently compressed during packing) |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Possible cause | Suggested remedy |
---|---|
Buffer volume of the air trap inadequate |
The column must generally be repacked. However, you can remove a small amount of air trapped between the column top bed support and the top adaptor head by pumping a solution with a temperature a few degrees centigrade higher than the chromatography medium through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior to use. Evaluate the packing using recommended methods. If the results are poor, refer to the symptom `Poor packing evaluation´ Preventive action: Check that the size of the air trap corresponds to the flow velocity. Also check that the buffer volume in the air trap is adequate. |
Buffers and column at different temperatures |
The column must generally be repacked. However, you can remove a small amount of air trapped between the column top bed support and the top adaptor head by pumping a solution with a temperature a few degrees centigrade higher than the chromatography medium through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior to use. Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ` Poor packing evaluation´ Preventive action: Equilibrate column and solutions to the working temperature before use. |
Connectors not correctly tightened or not compatible with each other |
The column must generally be repacked. However, you can remove a small amount of air trapped between the column top bed support and the top adaptor head by pumping a solution with a temperature a few degrees centigrade higher than the chromatography medium through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior to use. Evaluate the packing using recommended methods. If the results are poor, refer to the symptom "Poor packing evaluation" Preventive action: Tighten connectors prior to use. Also check that they are compatible with each other. Please see the connector guides. |
The column operates at room temperature after having been stored in a cold room |
Allow thermal equilibration before use. |
Valves that should be closed are not shut tightly |
The column must generally be repacked. However, you can remove a small amount of air trapped between the column top bed support and the top adaptor head by pumping a solution with a temperature a few degrees centigrade higher than the chromatography medium through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior to use. Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. Preventive action: |
Poor product recovery
Possible cause | Suggested remedy |
---|---|
Irreversible adsorption to matrix |
First try to modify the elution conditions. If this doesn’t help, clean and re-generate the column and chromatography medium according to instructions. |
Possible cause | Suggested remedy |
---|---|
Tertiary structure disrupted |
Shorten the residence time on the column. Use higher flow velocities to minimize exposure. |
Possible cause | Suggested remedy |
---|---|
Target molecules transferred from previous run due to poor cleaning between runs. |
Evaluate your cleaning procedure. |
Inhibiting component removed during purification |
Happens some times. Take into consideration and try to avoid. |
Different assay conditions used before and after chromatography |
Use the same conditions for all assays. |
Possible cause | Suggested remedy |
---|---|
A co-factor used to define recovery has been removed, thus causing lower activity |
Check the integrity of the enzyme or enzyme/co-factor complex. |
Column not equilibrated properly |
Check the pH and the conductivity of the effluent before applying the sample to make sure the column is properly equilibrated. Continue to equilibrate with start buffer if necessary. |
Column over-loaded |
Decrease the amount of sample or increase the amount of chromatography medium. I.e. increase the margin of safety. We recommend a margin of safety of approximately 10 %. |
For ion exchange chromatography - Ionic strength of the sample too high or pH incorrect |
Check and adjust the ionic strength and/or pH. Calibrate your conductivity and pH meters. |
Incorrect buffer pH |
Check and adjust the pH. Calibrate your pH meter. |
Oppositely-charged detergents or other additives adsorbed to the column |
Clean the column and chromatography medium according to instructions |
Protein precipitated on the column |
If the protein is initially solubilized with special additives, ensure that these are present during chromatography. Also check pH and ionic strength. |
Too low salt concentration in the elution buffer |
Check and adjust elution buffer composition. |
Drifting baseline
Possible cause | Suggested remedy |
---|---|
Unwashed buffer vessels were refilled with fresh solutions |
Prepare new solutions. Make sure that vessels/containers used to prepare and store solutions are clean. If you use plastic containers, ensure that additives do not dissolve in solution. |
Solution not in equilibrium with the chromatography medium |
Continue to flush the system until equilibrium is established. |
For ion exchange chromatography - Buffering capacity of buffer is too low. Dissociated ions bind to the charged groups of the chromatography medium. |
Make sure that the buffering capacity is sufficient. |
Column bleeding from previous run. |
Check and adjust your cleaning procedure. Clean the equipment and chromatography medium according to instructions |
Possible cause | Suggested remedy |
---|---|
At a certain salt concentration, the detergent forms micelles, resulting in a sharp increase in UV absorption. |
Work above the critical micelle concentration (CMC) or change the gradient so that the increase in UV absorption does not occur while the samples are eluting. |