Spare parts
Stainless steel bed support kit 20 µm (28-9664-47) and related parts.
Important: The 10 and 20 µm bed supports are not interexchangable. The entire bed support kit, that is the bottom plate and top assembly, must be exchanged.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Bed Support kit AxiChrom 50, 20 µm SS | 28966447 | 2,324.00 USD |
Add to cart
|
Screw M3x16 mm | 28901467 | 33.02 USD |
Add to cart
|
|
Bed support 50 Bottom/20SS | 28963228 | 1,001.00 USD |
Add to cart
|
|
Bed support 50 top/20SS | 28963415 | 1,090.00 USD |
Add to cart
|
Please note:
Item numbers in gray illustrates parts in contact with process stream.
Item numbers are not necessarily in sequential order.
Important: AxiChrom stainless steel and plastic bed supports are not directly interexchangable.
Important: The 10 and 20 µm bed supports are not interexchangable. The entire bed support kit, that is the bottom plate and top assembly, must be exchanged.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
301 | Bed support 50 top/bottom 20mikrom PE | 28988320 | 710.00 USD |
Add to cart
|
301 | Bed support 50 top/bottom 10mikrom UHMWPE | 28988322 | 710.00 USD |
Add to cart
|
401 | Set screw M3x12.5 mm special | 28988329 | 75.54 USD |
Add to cart
|
402 | Distributor 50 top, PE bed support | 28988312 | 702.00 USD |
Add to cart
|
403 | Screw M3x16 mm | 28901467 | 33.02 USD |
Add to cart
|
404 | Distributor 50 top, PE bed support | 28988312 | 702.00 USD |
Add to cart
|
409 | O-ring 28.3x1.78 EPDM | 28963367 | 213.00 USD |
Add to cart
|
412 | Washer 3.2x9x0.8 mm | 28988461 | 30.55 USD |
Add to cart
|
413 | O-ring 2.5 x 1.3 EPDM | 28988459 | 61.50 USD |
Add to cart
|
421 | Snap ring 50 top/bottom, PE bed support | 28988325 | 467.00 USD |
Add to cart
|
422 | Support net 50, PE bed support | 28988334 | 96.66 USD |
Add to cart
|
501 | Adaptor plate AxiChrom 50 | 28987619 | 778.00 USD |
Add to cart
|
502 | Bottom plate | 28988309 | 620.00 USD |
Add to cart
|
604 | O-ring 44.12x2.62 EPDM | 28963385 | 117.74 USD |
Add to cart
|
605 | Scraper 50 UHMWPE | 28963373 | 499.00 USD |
Add to cart
|
606 | O-ring 44.2x3 FPM/FKM | 28963209 | 227.00 USD |
Add to cart
|
Please note:
Item numbers in gray illustrates parts in contact with process stream.
Item numbers are not necessarily in sequential order.
Important: AxiChrom stainless steel and plastic bed supports are not directly interexchangable.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
101 | Stop Plug 5/16" Male | 18111250 | 36.22 USD |
Add to cart
|
114 | O-ring 54x2 mm | 28401771 | 288.00 USD |
Add to cart
|
115 | Tube support 50 | 28901444 | 260.00 USD |
Add to cart
|
116 | Check valve holder | 28401767 | 309.00 USD |
Add to cart
|
117 | Check valve | 18110213 | 119.73 USD |
Add to cart
|
118 | Check valve | 18110213 | 119.73 USD |
Add to cart
|
119 | Washer M6 6.4x12x2 mm | 28901462 | 37.65 USD |
Add to cart
|
201 | Column tube 50/300, Short | 28963237 | 1,955.00 USD |
Add to cart
|
201 | Column tube 50/500 long | 28963243 | 2,219.00 USD |
Add to cart
|
804 | Top bayonet AxiChrom 50 | 28978948 | 1,084.00 USD |
Add to cart
|
805 | Bottom flange for AxiChrom 50 | 28401744 | 455.00 USD |
Add to cart
|
806 | Nut M6 | 18112648 | 40.35 USD |
Add to cart
|
901 | Tie bar short | 28401760 | 178.37 USD |
Add to cart
|
901 | Tie bar long | 28401796 | 192.98 USD |
Add to cart
|
Please note:
Item numbers in gray illustrates parts in contact with process stream.
Item numbers are not necessarily in sequential order.
Important: AxiChrom stainless steel and plastic bed supports are not directly interexchangable.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
101 | Stop Plug 5/16" Male | 18111250 | 36.22 USD |
Add to cart
|
102 | O-ring 15.45x2.8 mm | 28401775 | 48.86 USD |
Add to cart
|
104 | O-ring 10.3x2.4 mm | 28401783 | 25.23 USD |
Add to cart
|
105 | Adaptor rod top 50 | 28401766 | 371.00 USD |
Add to cart
|
106 | Set screw M3x6 | 28401790 | 54.15 USD |
Add to cart
|
107 | Vent valve | 28960875 | 107.61 USD |
Add to cart
|
108 | O-ring 48x2.5 mm | 28401776 | 28.77 USD |
Add to cart
|
109 | Tube connection 5/16 i.d. 1.7 mm fem/fem | 28963186 | 228.00 USD |
Add to cart
|
111 | O-ring 11.1x1.6 mm | 28401778 | 34.24 USD |
Add to cart
|
602 | O-ring 3x1 EPDM | 28963214 | 38.69 USD |
Add to cart
|
802 | Adaptor rod 50 | 28401801 | 755.00 USD |
Add to cart
|
803 | Top plate AxiChrom 50 | 28978940 | 1,085.00 USD |
Add to cart
|
Please note:
Item numbers in gray illustrates parts in contact with process stream.
Item numbers are not necessarily in sequential order.
Important: AxiChrom stainless steel and plastic bed supports are not directly interexchangable.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
101 | Stop Plug 5/16" Male | 18111250 | 36.22 USD |
Add to cart
|
102 | O-ring 15.45x2.8 mm | 28401775 | 48.86 USD |
Add to cart
|
104 | O-ring 10.3x2.4 mm | 28401783 | 25.23 USD |
Add to cart
|
105 | Adaptor rod top 50 | 28401766 | 371.00 USD |
Add to cart
|
106 | Set screw M3x6 | 28401790 | 54.15 USD |
Add to cart
|
107 | Vent valve | 28960875 | 107.61 USD |
Add to cart
|
108 | O-ring 48x2.5 mm | 28401776 | 28.77 USD |
Add to cart
|
109 | Tube connection 5/16 i.d. 1.7 mm fem/fem | 28963186 | 228.00 USD |
Add to cart
|
111 | O-ring 11.1x1.6 mm | 28401778 | 34.24 USD |
Add to cart
|
405 | Tube id 1.7 cpl, PE bed support | 28988332 | 489.00 USD |
Add to cart
|
602 | O-ring 3x1 EPDM | 28963214 | 38.69 USD |
Add to cart
|
802 | Adaptor rod 50 | 28401801 | 755.00 USD |
Add to cart
|
803 | Top plate AxiChrom 50 | 28978940 | 1,085.00 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
123 | Foot for AxiChrom 50 column | 28401794 | 614.00 USD |
Add to cart
|
131 | Stop plug 5/16" | 28981957 | 98.28 USD |
Add to cart
|
Figure 4. Spare parts, Stainless steel bed supports.
Please note:
Item numbers in gray illustrates parts in contact with process stream.
Item numbers are not necessarily in sequential order.
Important: AxiChrom stainless steel and plastic bed supports are not directly interexchangable.
Important: The 10 and 20 µm bed supports are not interexchangable. The entire bed support kit, that is the bottom plate and top assembly, must be exchanged.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
301 | Bed support adaptor 10 µm | 28924562 | 1,553.00 USD |
Add to cart
|
301 | Bed support 50 top/20SS | 28963415 | 1,090.00 USD |
Add to cart
|
302 | Bed support bottom 10 µm | 28401849 | 912.00 USD |
Add to cart
|
302 | Bed support 50 Bottom/20SS | 28963228 | 1,001.00 USD |
Add to cart
|
401 | Screw M3x8 mm | 28901470 | 45.21 USD |
Add to cart
|
403 | Screw M3x16 mm | 28901467 | 33.02 USD |
Add to cart
|
501 | Adaptor plate AxiChrom 50, 20 µm bedsupport | 28963437 | 1,759.00 USD |
Add to cart
|
501 | Adaptor AxiChrom 50 10microm | 28966755 | 1,734.00 USD |
Add to cart
|
502 | Bottom Plate AxiChrom 50, 20 µm Bed Support | 28963077 | 1,703.00 USD |
Add to cart
|
502 | Bottom plate AxiChrom 50, 10 µm bedsupport | 28966775 | 1,734.00 USD |
Add to cart
|
603 | O-ring 28.3x1.78 EPDM | 28963367 | 213.00 USD |
Add to cart
|
604 | O-ring 44.12x2.62 EPDM | 28963385 | 117.74 USD |
Add to cart
|
605 | Scraper 50 UHMWPE | 28963373 | 499.00 USD |
Add to cart
|
606 | O-ring 44.2x3 FPM/FKM | 28963209 | 227.00 USD |
Add to cart
|
607 | O-ring 34.646x1.78 EPDM | 28963194 | 124.27 USD |
Add to cart
|
Bed Support kit AxiChrom 50, 20 µm SS | 28966447 | 2,324.00 USD |
Add to cart
|
|
Bed Support kit AxiChrom 50, 10 µm SS | 28966449 | 2,391.00 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Bayonet tool | 28401390 | 112.88 USD |
Add to cart
|
2 | Hook wrench key | 28907141 | 147.34 USD |
Add to cart
|
3 | Mounting aid for O-rings | 28924749 | 16.96 USD |
Add to cart
|
4 | Centering tool 50 | 28401886 | 361.00 USD |
Add to cart
|
5 | Tool kit small AxiChrom 50–200 | 28936136 | 653.00 USD |
Add to cart
|
6 | Tool Kit Complete for AxiChrom 50–200 Columns | 28944261 | 1,948.00 USD |
Add to cart
|
Stainless steel bed support kit 10 µm (28-9664-49) and related parts.
Important: The 10 and 20 µm bed supports are not interexchangable. The entire bed support kit, that is the bottom plate and top assembly, must be exchanged.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Bed Support kit AxiChrom 50, 10 µm SS | 28966449 | 2,391.00 USD |
Add to cart
|
Bed support bottom 10 µm | 28401849 | 912.00 USD |
Add to cart
|
|
Screw M3x16 mm | 28901467 | 33.02 USD |
Add to cart
|
|
Bed support adaptor 10 µm | 28924562 | 1,553.00 USD |
Add to cart
|
|
Adaptor AxiChrom 50 10microm | 28966755 | 1,734.00 USD |
Add to cart
|
|
Bottom plate AxiChrom 50, 10 µm bedsupport | 28966775 | 1,734.00 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
801 | Screw M4 x 16 | 18110956 | 66.03 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. Please keep a set of each on your site to prevent long downtime. For code numbers refer to the table below.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Tube connection 5/16 i.d. 1.7 mm fem/fem | 28963186 | 228.00 USD |
Add to cart
|
2 | Adaptor tube id 1.7 L=637 | 28963219 | 183.66 USD |
Add to cart
|
3 | Bed support adaptor 10 µm | 28924562 | 1,553.00 USD |
Add to cart
|
4 | Bed support 50 top/20SS | 28963415 | 1,090.00 USD |
Add to cart
|
5 | Bed support bottom 10 µm | 28401849 | 912.00 USD |
Add to cart
|
6 | Bed support 50 Bottom/20SS | 28963228 | 1,001.00 USD |
Add to cart
|
7 | O-ring 3x1 EPDM | 28963214 | 38.69 USD |
Add to cart
|
8 | O-ring 28.3x1.78 EPDM | 28963367 | 213.00 USD |
Add to cart
|
9 | O-ring 44.12x2.62 EPDM | 28963385 | 117.74 USD |
Add to cart
|
10 | Scraper 50 UHMWPE | 28963373 | 499.00 USD |
Add to cart
|
11 | O-ring 44.2x3 FPM/FKM | 28963209 | 227.00 USD |
Add to cart
|
12 | O-ring 34.646x1.78 EPDM | 28963194 | 124.27 USD |
Add to cart
|
13 | Vent valve | 28960875 | 107.61 USD |
Add to cart
|
14 | Check valve | 18110213 | 119.73 USD |
Add to cart
|
15 | Foot for AxiChrom 50 column | 28401794 | 614.00 USD |
Add to cart
|
Accessories
Figure 2. Accessories for packing and running AxiChrom 50 column with ÄKTApilot when column is placed on floor and other accessories.
Please note:
Item numbers are not necessarily in sequential order.
Items 1, 2 and 4 are part of tubing kit.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | O-ring 3 x 1 mm | 11002547 | 122.95 USD |
Add to cart
|
1 | ÄKTApilot O-Ring, 3 × 1 mm | 18116912 | 813.00 USD |
Add to cart
|
1 | Tubing Kit for Connecting AxiChrom 50 and AxiChrom 70 on Floor to ÄKTApilot, i.d. 1.7 mm | 28913613 | 465.00 USD |
Add to cart
|
5 | Valve One Straight Channel, 5/16" | 28907674 | 1,152.00 USD |
Add to cart
|
6 | Vent valve | 28960875 | 107.61 USD |
Add to cart
|
7 | Stop Plug 5/16" Male | 18111250 | 36.22 USD |
Add to cart
|
8 | Pivot Stand for AxiChrom 50/70/100-300 | 28401709 | 1,919.00 USD |
Add to cart
|
9 | Pivot stand for AxiChrom 50/70/100-500 | 28401710 | 1,977.00 USD |
Add to cart
|
10 | Mechanical Locking for AxiChrom 50 | 28401839 | 578.00 USD |
Add to cart
|
11 | Tool kit AxiChrom 50 | 28404777 | 707.00 USD |
Add to cart
|
12 | Union, M6 Female - 5/16" Female | 18116917 | 106.60 USD |
Add to cart
|
13 | Connector, 5/16" Female - 5/16" Female | 18117351 | 272.00 USD |
Add to cart
|
14 | Connector 5/16" Female - M6 Male | 18112776 | 65.20 USD |
Add to cart
|
15 | Union 5/16" female - 1/16" male | 18114208 | 56.92 USD |
Add to cart
|
16 | Domed Nut, M6 | 18245001 | 56.92 USD |
Add to cart
|
17 | Media Stirrer, 40 mm diameter | 28923180 | 455.00 USD |
Add to cart
|
Figure 1. Accessories for packing and running AxiChrom 50 with ÄKTAexplorer when column and system are placed on desk and other accessories.
Please note:
Item numbers are not necessarily in sequential order.
Items 1, 2, 3 and 7 are part of tubing kit.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | O-ring 3 x 1 mm | 11002547 | 122.95 USD |
Add to cart
|
1 | ÄKTApilot O-Ring, 3 × 1 mm | 18116912 | 813.00 USD |
Add to cart
|
1 | Tubing Kit for Connecting AxiChrom 50 on Bench to ÄKTAexplorer | 28905541 | 407.00 USD |
Add to cart
|
8 | Valve SRV-1 | 19214501 | 288.00 USD |
Add to cart
|
9 | Union, M6 Female - 5/16" Male | 18116916 | 125.23 USD |
Add to cart
|
10 | Fingertight Union 1/16" Male/M6 Female | 18111258 | 135.19 USD |
Add to cart
|
11 | Vent valve | 28960875 | 107.61 USD |
Add to cart
|
12 | Stop Plug 5/16" Male | 18111250 | 36.22 USD |
Add to cart
|
13 | Stop plug | 19517001 | 47.19 USD |
Add to cart
|
14 | Pivot Stand for AxiChrom 50/70/100-300 | 28401709 | 1,919.00 USD |
Add to cart
|
15 | Pivot stand for AxiChrom 50/70/100-500 | 28401710 | 1,977.00 USD |
Add to cart
|
16 | Mechanical Locking for AxiChrom 50 | 28401839 | 578.00 USD |
Add to cart
|
17 | Tool kit AxiChrom 50 | 28404777 | 707.00 USD |
Add to cart
|
18 | Union, M6 Female - 5/16" Female | 18116917 | 106.60 USD |
Add to cart
|
19 | Connector, 5/16" Female - 5/16" Female | 18117351 | 272.00 USD |
Add to cart
|
20 | Connector 5/16" Female - M6 Male | 18112776 | 65.20 USD |
Add to cart
|
21 | Union 5/16" female - 1/16" male | 18114208 | 56.92 USD |
Add to cart
|
22 | Domed Nut, M6 | 18245001 | 56.92 USD |
Add to cart
|
23 | Media Stirrer, 40 mm diameter | 28923180 | 455.00 USD |
Add to cart
|
Figure 2. Accessories for packing and running AxiChrom 50 with ÄKTAexplorer when column is placed on floor and other accessories.
Please note:
Item numbers are not necessarily in sequential order.
Items 1, 2, 3 and 7 are part of tubing kit.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | O-ring 3 x 1 mm | 11002547 | 122.95 USD |
Add to cart
|
1 | ÄKTApilot O-Ring, 3 × 1 mm | 18116912 | 813.00 USD |
Add to cart
|
1 | Tubing Kit for Connecting AxiChrom 50 on Floor to ÄKTAexplorer | 28905603 | 405.00 USD |
Add to cart
|
8 | Valve SRV-1 | 19214501 | 288.00 USD |
Add to cart
|
9 | Union, M6 Female - 5/16" Male | 18116916 | 125.23 USD |
Add to cart
|
10 | Fingertight Union 1/16" Male/M6 Female | 18111258 | 135.19 USD |
Add to cart
|
11 | Vent valve | 28960875 | 107.61 USD |
Add to cart
|
12 | Stop Plug 5/16" Male | 18111250 | 36.22 USD |
Add to cart
|
13 | Stop plug | 19517001 | 47.19 USD |
Add to cart
|
14 | Pivot Stand for AxiChrom 50/70/100-300 | 28401709 | 1,919.00 USD |
Add to cart
|
15 | Pivot stand for AxiChrom 50/70/100-500 | 28401710 | 1,977.00 USD |
Add to cart
|
16 | Mechanical Locking for AxiChrom 50 | 28401839 | 578.00 USD |
Add to cart
|
17 | Tool kit AxiChrom 50 | 28404777 | 707.00 USD |
Add to cart
|
18 | Union, M6 Female - 5/16" Female | 18116917 | 106.60 USD |
Add to cart
|
19 | Connector, 5/16" Female - 5/16" Female | 18117351 | 272.00 USD |
Add to cart
|
20 | Connector 5/16" Female - M6 Male | 18112776 | 65.20 USD |
Add to cart
|
21 | Union 5/16" female - 1/16" male | 18114208 | 56.92 USD |
Add to cart
|
22 | Domed Nut, M6 | 18245001 | 56.92 USD |
Add to cart
|
23 | Media Stirrer, 40 mm diameter | 28923180 | 455.00 USD |
Add to cart
|
The illustrations show tubing connections for ÄKTA avant 150.
The illustration to the left refers to Tubing kit, AxiChrom 50 -70 / ÄKTA avant / desk, 28-9888-89.
The illustration to the right refers to Tubing kit, AxiChrom 50 - 70 / ÄKTA avant / floor, 28-9888-92.
Pos. |
28-9888-89 Tubing kit, AxiChrom 50 - 70 / ÄKTA avant / desk |
28-9888-92 Tubing kit, AxiChrom 50 - 70 / ÄKTA avant / floor |
---|---|---|
1 | i.d. 1.7 / L=1200 (x2) | i.d. 1.7 / L=1600 (x2) |
2 | i.d. 1.7 / L=800 (x1) | i.d. 1.7 / L=1200 (x1) |
3 | i.d. 2.9 / L=1600 (x1) | i.d. 2.9 / L=1600 (x1) |
4 | i.d. 1.7 / L=200 (x2) | i.d. 1.7 / L=200 (x2) |
# | Product Name | Product Code | Price | |
---|---|---|---|---|
Tubing kit AxiChrom 50-70 AKTA avant, desk | 28988889 | 610.00 USD |
Add to cart
|
|
Tubing kit AxiChrom 50-70 AKTA avant, floor | 28988892 | 618.00 USD |
Add to cart
|
Item | Comment |
---|---|
1 |
Low Flow Kit, 28-9301-82 (for flow rates 3 l/h-175 l/h). |
2 | Connector 25 mm TC- 5/16" female 18-1169-22. Two connectors needed to connect column to system. |
3 | TC Gasket 25 mm, i.d. 15 mm 44-5492-88. Two gaskets supplied with the flow kit. Two gaskets i.d. 15 mm needed to connect column to system. |
4 | TC Clamp 25 mm, 28-4043-38 (Stainless steel) or 28-4043-36 (Polypropylene). |
5 |
For appropriate tubing i.d. refer to table below. |
6 | Stop Plug 5/16" male 18-1112-50. |
To isolate the column from the system use Stop Plug 5/16" 28-9819-57.
Tubing
Column | Tubing i.d. | Tubing connection |
---|---|---|
AxiChrom 50 | 1.7 mm | 5/16" |
AxiChrom 70/100 | 2.9 mm | 5/16" |
# | Product Name | Product Code | Price | |
---|---|---|---|---|
2 | Connector, 25 mm TC - UNF 5/16" Female | 18116922 | 191.48 USD |
Add to cart
|
3 | TC-Gasket 25 mm i.d. 15 mm | 44549288 | 209.00 USD |
Add to cart
|
4 | TC clamp kit, 25 mm TC | 28404336 | 302.00 USD |
Add to cart
|
4 | TC Clamp kit TC25/SS | 28404338 | 148.09 USD |
Add to cart
|
6 | Stop Plug 5/16" Male | 18111250 | 36.22 USD |
Add to cart
|
Stop plug 5/16" | 28981957 | 98.28 USD |
Add to cart
|
Figure 1. Accessories for packing and running AxiChrom 50 with ÄKTApilot when column is placed on desk and other accessories.
Please note:
Item numbers are not necessarily in sequential order.
Items 1, 2 and 4 are part of tubing kit.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | O-ring 3 x 1 mm | 11002547 | 122.95 USD |
Add to cart
|
1 | ÄKTApilot O-Ring, 3 × 1 mm | 18116912 | 813.00 USD |
Add to cart
|
1 | Tubing Kit for connecting AxiChrom 50 on bench to ÄKTApilot | 28905676 | 401.00 USD |
Add to cart
|
5 | Valve One Straight Channel, 5/16" | 28907674 | 1,152.00 USD |
Add to cart
|
6 | Vent valve | 28960875 | 107.61 USD |
Add to cart
|
7 | Stop Plug 5/16" Male | 18111250 | 36.22 USD |
Add to cart
|
8 | Pivot Stand for AxiChrom 50/70/100-300 | 28401709 | 1,919.00 USD |
Add to cart
|
9 | Pivot stand for AxiChrom 50/70/100-500 | 28401710 | 1,977.00 USD |
Add to cart
|
10 | Mechanical Locking for AxiChrom 50 | 28401839 | 578.00 USD |
Add to cart
|
11 | Tool kit AxiChrom 50 | 28404777 | 707.00 USD |
Add to cart
|
12 | Union, M6 Female - 5/16" Female | 18116917 | 106.60 USD |
Add to cart
|
13 | Connector, 5/16" Female - 5/16" Female | 18117351 | 272.00 USD |
Add to cart
|
14 | Connector 5/16" Female - M6 Male | 18112776 | 65.20 USD |
Add to cart
|
15 | Union 5/16" female - 1/16" male | 18114208 | 56.92 USD |
Add to cart
|
16 | Domed Nut, M6 | 18245001 | 56.92 USD |
Add to cart
|
17 | Media Stirrer, 40 mm diameter | 28923180 | 455.00 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.
Drifting baseline
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. |
Possible cause | Suggested remedy |
---|---|
The air trap buffer was not completely replaced by mobile phase buffer before starting the Intelligent Packing method. |
Change the liquid in the air trap and retest. |
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 |
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. |
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 |
---|---|
Leakage in the tubing between system and column or in the connection between the end cell and adaptor rod tubing. |
Ensure that no connections are leaking. Change gaskets if necessary. |
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 |
---|---|
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 |
---|---|
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 |
---|---|
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. |
Column leakage
Possible cause | Suggested remedy |
---|---|
Missing O-ring |
Assemble the O-ring, according to User Manuals |
Damaged thread on tube connection |
Replace damaged parts, according to the User Manual. |
Possible cause | Suggested remedy |
---|---|
- |
Disassemble the adaptor and top plate and change the O-ring according to User Manual or carefully take off and rinse the O-ring and the O-ring groove. Reassemble. |
Possible cause | Suggested remedy |
---|---|
Damaged O-ring inside the top plate.
|
Replace the damaged O-ring, according to User Manual Preventive action: |
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 |
---|---|
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.
|
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 |
For AxiChrom 50, 70, 140 & 200 columns, BPG columns and STREAMLINE columns - Incorrect torque specifications used during assembly of glass columns |
Use the torque and torque wrench setting specified in the instructions. |
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. |
Possible cause | Suggested remedy |
---|---|
Tubing not compatible with solvents |
Check chemical resistance with the tubing supplier. Preventive action: |
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. |
Back pressure increases during operation
Possible cause | Suggested remedy |
---|---|
Air trapped in the bed supports |
Get rid of the air by wetting and washing the bed supports with 20% ethanol. If necessary disassemble the column according to the instructions. |
Pre-filter blocked |
Check the pre-filter and remove the blockage. |
Protein precipitated on the column |
Clean and regenerate the column and chromatography medium according to instructions. |
Sample and collection vessels at different levels |
Adjust the vessels to approximately the same level. |
Valve flow-paths too narrow |
Check all valves. Replace if necessary with valves that have a correct (wider) inner diameter. |
Valve not fully open |
Check all valves. Open any that is not fully open. |
Auxiliary equipment such as manometers and pumps not working properly |
Check the function of all auxiliary equipment. Repair/replace if necessary. |
Bed packed too densely |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Bent tubing |
Check that the flow path is not restricted. |
Buffer viscosity too high |
Check the viscosity of all buffers. Viscosity is a function of temperature. (Lower temperature gives higher viscosity.) Let low-temperature buffer reach operating temperature before starting the run. |
Column bed supports blocked by protein or medium |
To remove protein: To remove chromatography medium: Preventive action: |
Equipment in general (valves, flow cells, gaskets, etc.) has internal diameters too narrow for the flow rates used. |
Ensure that the internal diameter of all equipment before and after the column is the same as the column inlet and outlet. |
For hydrophobic interaction chromatography - Protein precipitated on the column due to incorrect ionic strength. |
Check and adjust conductivity to the correct value. Clean and regenerate the column and chromatography medium according to instructions. |
Microbial growth in buffers |
Check buffers, especially those with phosphate, for microbial growth. Replace with fresh buffer if necessary. |
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: |
Unusual column appearance
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: |
Possible cause | Suggested remedy |
---|---|
The scraper seal is damaged. |
Change the scraper seal according to user manual |
Possible cause | Suggested remedy |
---|---|
The adaptor is not tight enough. |
Tighten the adaptor to the adaptor rod using a wrench and a hook wrench key, according to User Manual. Preventive action: |
Possible cause | Suggested remedy |
---|---|
One of the ferrules inside the adaptor is loose and does not seal properly. This can be checked by undoing the adaptor rod top and control if the tubing is easy to rotate. If so one of the ferrules is loose. |
Insert a stop plug into the tube connection and push it down to assist sealing. Raise pressure under adapter to see if adapter moves. Follow the instructions in the User Manuals. |
Possible cause | Suggested remedy |
---|---|
Adaptor still in column tube when top plate is removed with bayonet tools |
Reassemble the top plate onto the column and continue to move the adapter upwards by pumping liquid into the process chamber. |
Possible cause | Suggested remedy |
---|---|
Incorrect "centering" of the column tube. |
Place a centering tool into the mouth of the column according to the instructions in the User Manual. |
Possible cause | Suggested remedy |
---|---|
Nuts at the mechanical locking are too tight to loosen by hand |
Ensure that there is no air in the hydraulic chamber. Pump in liquid at a very low flow rate to pressurize the hydraulic chamber and move the adapter down by 1-2 mm. Loosen the nuts. Make sure not to overpressurize the column or the bed. |
Possible cause | Suggested remedy |
---|---|
The column was not correctly leveled. |
|
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 |
---|---|
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 |
---|---|
The adaptor is just above the glass line |
Give the adaptor a very gently push downwards 0.5 - 1 mm. |
The scraper seal is damaged.
|
Change the scraper seal according to user manual. |
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. |
Poor packing evaluation result
On this troubleshooting site, we refer to leading and tailing peaks as defined according to the following figures.
Leading peak | Tailing peak |
Experience shows that the best method of expressing the efficiency of a packed column is in terms of its height equivalent to a theoretical plate (HETP), reduced plate number (h) and its peak asymmetry factor (As).
Possible cause | Suggested remedy |
---|---|
Cracks in the bed or a gap between the chromatography medium and inlet. The column may have been packed too loosely |
It may be possible to only adjust the adaptor. Otherwise, repack the column with increased packing flow rate or pressure. Preventive action: |
Blocked bed support creates zones of different flow velocities |
To remove protein: |
Possible cause | Suggested remedy |
---|---|
Cracks in the bed due to pump pulsation or a too densely packed bed. The cracks are mainly vertical. |
Repack the column with a pulse-free pump or add an air trap and a pressure relief valve. Preventive action: |
A crack due to a torn bed support resulting in a jet stream. |
Replace the bed support according to instructions |
Possible cause | Suggested remedy |
---|---|
Test molecule is repelled by the chromatography medium under the conditions used, e.g. salt for ion exchange chromatography, or chromatography medium in water as running buffer |
Use appropriate test molecule and conditions, e.g. 800 mM salt as sample in 400 mM salt buffer for ion exchange media, or use acetone. |
Possible cause | Suggested remedy |
---|---|
Test molecule interacts with the chromatography medium under the conditions used, e.g. acetone interacts with SOURCE media |
Use appropriate test molecule and conditions, e.g. salt for SOURCE media. |
Possible cause | Suggested remedy |
---|---|
Too large sample volume (gives a broad peak) |
Use a sample volume that is maximum 2% of the column volume. |
Too great a distance between the sample injection and column inlet, or between the column outlet and analyzing instrument |
Shorten these distances (minimizes dilution). |
Large liquid volume in tubing, valves, connectors, etc |
Minimize dilution by reducing the liquid volume. For example, if the inner diameters of the tubing, valves, connectors, etc. are wider than the column inlet and outlet, change them to the same diameter. |
Flow velocity too high or too low |
Flow velocity should be in the range 15-60 cm/h depending on particle size. The smaller the particle, the higher the velocity. |
Possible cause | Suggested remedy |
---|---|
Bed packed too densely |
Repack the column with decreased packing flow rate or pressure (prepare a pressure/flow curve and use the data to define the correct packing flow rate, i.e. approx. 70% of maximum flow (plateau)). |
Possible cause | Suggested remedy |
---|---|
Bed settled too hard during the first step |
Repack the column with decreased packing flow rate for the sedimentation step. |
Possible cause | Suggested remedy |
---|---|
Extra volume in the system, e.g. new tubing (longer, shorter or different diameter) affects peak position and HETP |
Ensure the equipment is the same or identical to your test routine or SOP. |
Possible cause | Suggested remedy |
---|---|
Column not properly equilibrated before packing evaluation |
Re-equilibrate the column. Evaluate the packing again, using recommended methods. |
Possible cause | Suggested remedy |
---|---|
Column not properly equilibrated before packing evaluation |
Re-equilibrate the column. Evaluate the packing again, using recommended methods. |
Possible cause | Suggested remedy |
---|---|
Bed packed too densely or too loosely |
Repack the column. Evaluate the packing afterwards, using recommended methods. For a leading peak and one-step packing: For a leading peak and two-step packing: For a tailing peak and one-step packing: For a tailing peak and two-step packing: Preventive action: |
Possible cause | Suggested remedy |
---|---|
Bed packed too densely. Cracks present at the beginning get worse |
Repack the column. Evaluate the packing again, using recommended methods. One-step packing: Two-step packing: Preventive action: |
Possible cause | Suggested remedy |
---|---|
Bed too loosely packed causing space between chromatography medium and inlet |
Repack the column. Evaluate the packing again, using recommended methods. One-step packing: Two-step packing: Preventive action: |
Possible cause | Suggested remedy |
---|---|
Bed not packed densely enough |
Repack the column with increased packing flow rate or pressure (prepare a pressure/flow curve and use the data to define the correct packing flow rate, i.e. approx. 70% of maximum flow (plateau)). After re-packing the column evaluate the packing. Preventive action: |
Possible cause | Suggested remedy |
---|---|
Bed settled too loosely during the first step |
Repack the column with increased packing flow rate for the sedimentation step. Preventive action: |
Unsatisfactory elution
Possible cause | Suggested remedy |
---|---|
Sample not properly equilibrated |
Equilibrate sample to correct operating conditions (pH, ionic strength, etc.) |
Column not properly equilibrated |
Re-equilibrate the column. |
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’. |
Elution strength too low |
Check and adjust the elution strength. Also check gradient, gradient shape or isocratic step. |
Possible cause | Suggested remedy |
---|---|
Column is bleeding from the previous run |
Check and adjust your cleaning procedure. Clean the equipment and chromatography medium according to instructions |
Target protein not stable under the chosen conditions and partly degrades |
Find better ways to stabilize the protein, e.g. shorten the process time. |
The detergent has formed micelles with the protein, thereby increasing its size and changing its elution position. This effect is especially critical in gel filtration |
Reduce the concentration of detergent to below it´s critical micelle concentration (CMC ) value. |
Possible cause | Suggested remedy |
---|---|
Components in the sample displace the target molecule before elution starts |
Reduce the amount of sample or find more suitable binding conditions. |
Column not properly equilibrated |
Check the pH and conductivity of the effluent before applying the sample. Continue to equilibrate with start buffer if necessary. |
Possible cause | Suggested remedy |
---|---|
Column overloaded |
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 approximately 10 %. |
Flow velocity too high |
Run the separation at a lower flow velocity. This is especially important for adsorbents that bind several substances and where selectivity is low. |
Microbial growth in the column |
Microbial growth rarely occurs in a column during use. However, always take steps to prevent infection of packed columns and solutions. Store the column according to instructions. |
Slope of gradient too steep |
Use a shallower gradient (larger gradient volume) |
Possible cause | Suggested remedy |
---|---|
Buffers have wrong pH, ionic strength or solvent concentration |
Check and adjust the buffer conditions. Calibrate your instruments. |
Possible cause | Suggested remedy |
---|---|
Column overloaded |
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. |
For ion exchange chromatography - Reduced net charge due to complex between the component in the sample and the target molecule. |
Check the composition of the sample. Remove any interfering compounds such as nucleic acids. |
Oppositely –charged detergents or other additives adsorbed on the column. |
Clean the column and chromatography medium according to instructions. |
pH of the sample incorrect |
Check and adjust the pH. Calibrate your pH meter. |
The column not properly equilibrated |
Check the pH and conductivity of the effluent before applying the sample. Continue to equilibrate with start buffer if necessary. |