TOSOH BIOSCIENCE TSKgel HPLC Column User manual

Table of Contents
What to do first 2
Installation 2
Column protection 4
Quality control 4
Operating conditions 6
Troubleshooting 9
Return policies 11
Additional help 11
TSKgel®HPLC Column
Instruction Manual
Important! Do not throw away!
Thank you for purchasing a
TSKgel liquid chromatography column!
ANALYSIS

2
WHAT TO DO FIRST
First, inspect your shipment. Immediately notify Tosoh Bioscience’s customer
service department if your shipment is damaged or incomplete. Make sure you
received the following with your column:
• Data sheet containing a chromatogram, the Quality Control test data and the
conditions under which the QC test was performed (guardcolumns are not
supplied with a chromatogram).
• Operating Conditions and Specifications (OCS) sheet, listing the specifi-
cations for this column type and conditions under which you can expect
optimum column performance.
After you have checked the appearance of your column, test its performance
under the conditions listed in the Data Sheet using the same or similar test com-
pounds. Calculate the performance parameters as discussed under the „Quality
Control“ section of this manual. Compare your results with the specifications.
If your column is not within specifications, please contact Tosoh Bioscience
within 30 days after receiving the column. Returns can only be accepted if the
column is shipped back in the Tosoh Bioscience column box and is accompanied
by the original data sheet and the Tosoh Bioscience column tag (see „Return
Policy“ in this manual).
INSTALLATION
Glass columns
The TSKgel glass column you received requires 1/4·28 fittings for connecting it to
a liquid chromatography (LC) system. For glass columns, we recommend the use
of plastic nuts and ferrules that can be installed without flanging the tubing. You
can also use standard metric M6 nuts and ferrules.
In this case, use 1/6“ Teflon®tubing with a maximum inner diameter (ID) of 0.015“
or 0.4 mm to connect the column to the guardcolumn and the detector. Cut the
tubing at a 90° angle with a tubing cutter designed for use on polymer tubing. Now
put the ferrule on with the tapered end facing the nut. Cut the tubing flush to the
base of the ferrule. Insert this into the column end fitting and hand tighten the nut.
Stainless steel columns
The TSKgel column you received requires 10·32 (1/16“) fittings for connecting
it to any LC system (see Figure 1). We recommend that you make up two short
pieces of 0.01“ (0.25 mm) ID x 1/16“ capillary tubing to connect the column to the
guardcolumn and the detector. To prepare these connectors, use 10·32 (1/16“) nuts
and ferrules, plus the appropriate fittings for connecting the injector and detector.

3
Alternatively, you can use 10·32 (1/16“) fingertight fittings that are available from
chromatography supply houses.
In either case, make sure to insert the capillary tubing fully into the column end
fitting before setting the ferrule by tightening the nut. Repeat to make the connec-
tions at the other end of the 1/16“ capillary tubing.
Mobile phase
Prevent air bubbles from entering the column during its installation, use and stor-
age, since this may cause degradation of column performance through the forma-
tion of channels in the packed bed. Mobile phases must be thoroughly degassed
before use. This can be accomplished by vacuum filtration, helium sparging or
in-line degassing. In addition to degassing the solvent, vacuum filtration will also
prevent small particles from plugging the column frit. You can use 0.20 or 0.45
micron nylon membranes to filter aqueous and aqueous/organic mobile phases.
Sample
If possible, always dissolve your sample in mobile phase or the starting mobile
phase when operating under gradient conditions. Alternatively, try to match the
pH, salt concentration and organic solvent of the sample with those of the mobile
phase and run a test to ensure that no precipitate, suspension or flocculate is
formed. Finally, before making an injection, filter the sample through a 0.20-0.45
micron porosity membrane.
Flow direction
The recommended flow direction through the column is indicated by the arrow
on the tag. Operating the column with the flow in the reverse direction is only
recommended as part of the installation procedure (see next section), cleaning,
or when removing particulates from a clogged frit (see the „Troubleshooting“
section of this manual).
Installing the column
After manufacturing and quality control, the column has been flushed with stor-
age solvent and closed with caps to prevent evaporation of the solvent. As men-
tioned above, it is important to prevent air from entering the column. Remove
the cap from the column inlet side. Solvent should be visible at the inlet fitting (if
not, see below). Before starting the flow, first connect the column to the injector
only. Start the flow. After liquid flows out of the column exit, you can connect the
column to the detector inlet line.

4
If the column inlet fitting appears dry, we recommend that you first disconnect
the bottom cap and hook up the column exit to the injector. Then slowly start the
flow in this reversed flow direction until a few drops of mobile phase exit from the
column. Turn off the flow, let the pressure go to zero, and disconnect the column
from the system. Turn the column around and hook it up so that the flow is now
in the direction of the arrow. Start the flow at a low setting and stop it as soon as
the mobile phase exits from the bottom fitting. Now you can hook up the column
to the detector inlet and increase the flow to the desired setting. Be sure to set the
flow within the recommended range shown on the OCS sheet.
COLUMN PROTECTION
In addition to filtering the sample and the mobile phase, the best way to protect
the separation column is to install a guardcolumn in front of it. This will trap high-
ly adsorptive sample components and residual particulates in the sample, the
mobile phase or from instrument components, such as pump and injector seals.
There are two types of guard products for protecting your TSKgel column. The
most convenient option, which is available for some column types, is to purchase
a prepacked TSKgel guard column or cartridge. Alternatively, you can purchase
a guardgel Kit, which consists of the necessary glass or stainless steel hardware
and packing material to pack your own guard column. Consult the OCS sheet for
the part number of the appropriate guardgel Kit for your separation column. The
kits come with a separate instruction sheet for assembling, packing and installing
the guard column.
Helpful hints about guardcolumn replacement can be found in the
“Troubleshooting“ section of this manual and in the OCS sheets that are shipped
with each TSKgel column and guard column product.
QUALITY CONTROL
The performance of each TSKgel column has been tested under the conditions
described in the enclosed data sheet. Table 1 lists suggested suppliers for stan-
dard compounds to perform the QC test.
Column performance is judged by the following characteristics:
1. Theoretical plates (efficiency)
2. Asymmetry factor
3. Resolution
4. Retention time

5
TABLE 1
Standards for quality control of TSKgel columns
Standard Supplier Catalogue #
New: Protein Standard Mix Sigma-Aldrich 69385
1.5 - 600 kDa
Can be used for all TSKgel G3000SW, G3000SWXL,
SuperSW3000 and QC-PAK GFC-300 columns
p-Aminobenzoic acid Sigma-Aldrich A9878
Bombesin Sigma-Aldrich B4272
Bovine serum albumin Sigma-Aldrich A4503
Chymotrypsin Sigma-Aldrich C7762
Chymotrypsinogen Sigma-Aldrich C4879
Cytidine Sigma-Aldrich C4654
Cytidine-5-monophosphate Sigma-Aldrich C1006
Dopamine.HCl Sigma-Aldrich H8502
Ethylene glycol Fluka 03750
Ferritin Sigma-Aldrich F4503
y-Globulin Sigma-Aldrich G5009
Lysozyme Sigma-Aldrich L6876
d-Mannitol Sigma-Aldrich M4125
Ovalbumin Sigma-Aldrich A2512
Polyethylene oxide (kit) Tosoh Bioscience 05773
Polystyrene (kit) Tosoh Bioscience 06476
Ribonuclease A Sigma-Aldrich R5503
Thyroglobulin Sigma-Aldrich T1001
Trypsin Sigma-Aldrich T7409
Trypsin inhibitor Sigma-Aldrich T9003
Trypsinogen Sigma-Aldrich T1143
Tryptamine. HCl Sigma-Aldrich 246557
Uric acid Sigma-Aldrich U2625
Note: Common chemical standards are not included in this list.
To use a sample component, you must establish baseline data when the column
is new and performing well. After establishing that the column is performing
properly, using standard test probes, calculate the asymmetry factor, theoretical
plates and resolution of one or more of your sample components. Also note the
retention time. This becomes your „baseline test mix“ with which you can later
compare.
Consult Figure 1 to calculate the quality control parameters that were determined
after the column was manufactured.

6
Resolution (Rs)
Efficiency (N)
Ve= elution volume at the peak maximum
W1/2 = width of peak at half height
N = 5.54 (Ve/W1/2)2
W1/2
Ve
½ h
h
V1
V2
Rs= 2(V2- V1)/W2+ W1)
V1, V2= elution
volume of first
and second
component
W1, W2= widths
of peaks 1 and
2 at baseline
W1W2
Injection Time
Asymmetry factor (AF)
ab
AF = b/a
1
/ 10
h
h
AF is measured at 10% of the peak height.
FIGURE 1
OPERATING CONDITIONS
The OCS sheet lists what solvent is in the column; what flow rates and pressure
to use; what the pH limits are; what percent organic solvent you can use; what
the maximum salt concentration is; what the maximum temperature is; and
how to protect, clean and store the column.

7
It also states the specifications for columns of each type. The following para-
graphs provide additional detail about the role of temperature, column cleaning
and storage.
Temperature
All TSKgel columns should be operated above 0°C. They are usually operated at
ambient temperature or at a higher controlled temperature. The maximum tem-
perature your column can tolerate is listed on the OCS sheet.
For all columns the use of elevated temperature may change column efficiency
and resolution, compared to measurements performed at room temperature or
lower. When performing measurements at elevated temperature, we recom-
mend that you do not stop the pump immediately after finishing the experiment.
Instead, continue to pump mobile phase while allowing the column to equilibrate
to room temperature. Otherwise, air may be sucked into the heated column due
to contraction of the solvent.
Cleaning
Occasionally, samples are run that adsorb onto the packing material. When this
occurs, it is time to clean your column. You do not necessarily have to run a
standard test probe to monitor your column. If you have a well-resolved peak,
establish baseline data when the column is new and operating correctly. Then, if
one of the performance characteristics of your column changes by 10% or more,
it is prudent to clean your column.
When you clean the column, there are a few basic rules to follow. These rules
apply regardless of what type of TSKgel column you are running.
1. Clean your column in the reverse flow direction.
2. While cleaning, do not connect the column to the detector.
3. Run the column at half of the maximum recommended flow rate. Take special
care to monitor the pressure, as the cleaning solutions may be of different
viscosities than your normal mobile phase.
4. If you are cleaning with a high or low pH solution, make certain that the rest of
your chromatographic system (pump, pump seals, injector, etc.) is compatible.
Each type of TSKgel column has a recommended set of cleaning solutions specif-
ic to the column. Choose a cleaning solution based upon the column and sample
type. For example, a low pH salt solution will remove basic proteins. Organics will
remove hydrophobic proteins. Chaotropic agents will remove strongly adsorbed
materials (e.g., via hydrogen bonding).
For columns or column types not listed below, please contact Tosoh Bioscience
Technical Service for the appropriate solutions.

8
Cleaning solutions
Generally, 3-5 column volumes (CV) of cleaning solution is sufficient. Rinse well
with 3-5 CV of distilled, deionized water between each solution. Only use chaotropic
agents when neutral salt or organic has not improved performance characteristics.
SW and SWXL, UP-SW, Super SW
1. Concentrated neutral salt (e.g., 0.5 M) at low pH (e.g., pH 3.0)
2. Water soluble organic (MeOH, ACN, EtOH, 10%-20%) in aqueous buffer
PW and PWXL
1. High concentration neutral salt (e.g., 0.5 M-1.0 M) of your normal run buffer
2. Buffered solutions at low pH (e.g., 2-3) or high pH (e.g., 11-12)
3. Water soluble organic (MeOH, ACN, EtOH, 10%-20%) in aqueous buffer
Ion Exchange, SW Type
1. High concentration neutral salt (e.g., 0.5 M-1.0 M) of your normal run buffer
2. Buffered solutions at low pH (e.g., 2-3)
3. Water soluble organic (MeOH, ACN, EtOH, 10%-20%) in aqueous buffer
Ion Exchange, PW Type
1. 0.1 M-0.2 M NaOH
2. 20%-40% aqueous acetic acid
3. Water soluble organic (MeOH, ACN, EtOH, 10%-20%) in aqueous buffer
Hydrophobic Interaction
1. 0.1 M-0.2 M NaOH
2. 20%-40% aqueous acetic acid
Reversed Phase, silica-based
1. Acetonitrile or methanol
2. Gradient from 10%-100% acetonitrile in 0.05% trifluoroacetic acid
Reversed Phase, PW Type
1. Acetonitrile or methanol
2. 0.1 M-0.2 M NaOH
3. 20%-40% aqueous acetic acid
Hydrophilic Interaction, Amide-80
1. 45% Acetonitrile
2. 50 mM phosphate buffer pH 6.0/ acetonitrile 50/50
Choose cleaning solvent 1. to desorb compounds, 2. for ionic compounds
Protein-A Affinity, PW Type
1. Up to 2 mL of 0.1 M NaOH solution
FcR-IIIA Affinity, NPR Type
1. Up to 0.5 - 2 mL of a buffer containing 0.5 mol/L NaCl or 20 % ethanol

9
As a last resort, buffered solutions of SDS (0.1%), urea (8 M) or guanidine (6 M) can be used to clean
the column. We only recommend this method if all other methods fail.
Storage
Consult the OCS sheet for the recommended storage solvent and storage tem-
perature or temperature range for this column.
After your experiments, you can leave the column overnight in the LC system at
low flow rates if you plan to use it the next day. If not, flush the system and the
column with the storage solvent and screw the caps in both ends of the column.
For long-term storage, the column must be protected from the growth of micror-
ganisms by incorporating a
bacteriostat such as 0.05% NaN3or 10%-20% acetonitrile or ethanol into the stor-
age solvent.
Avoid exposing the column to corrosive solvents and gasses and avoid exposure
to direct sunlight.
Rehydration
Dehydration of TSKgel liquid chromatography columns can result from improper
use or during long-term storage, for example, if the plugs are not tightened or
if air inadvertently is pumped into the column during use. It is easier to detect
dehydration in glass columns because the dry packing will appear to pull away
from the column walls. This condition can be remedied by using the following
procedure:
1. Connect the column to your LC system in reverse flow direction.
2. Do not connect the column to the detector.
3. Pump a filtered mobile phase of 20% acetonitrile in distilled, deionized water
over the column at half of the recommended maximum flow rate. Please note
that reversed phase columns require 60% methanol.
4. Continue this procedure until you are confident that the column has been
rehydrated. Rehydration can take several hours, depending on the column
size. Rinse well with distilled, deionized water (3-5 CVs).
5. Connect the column to your LC system in proper flow direction.
6. Equilibrate with your normal mobile phase.
7. Perform the recommended QC tests to ensure that the column is performing
properly.
TROUBLESHOOTING
Instrumentation
Although liquid chromatography is a highly reliable qualitative and quantitative
technique, there are things that can go wrong, such as excessive back pressure,
poor peak shape, or changes in retention or selectivity. The problem is either with
the column, the sample, the mobile phase or the instrumentation. Use a process

10
of elimination to find out where the problem is. Does the problem still occur
when you inject a standard (see Table 1) and another batch of mobile phase?
If so, take the guardcolumn out of the system and again inject standards. If the
problem is still there, then also take out the column. Directly connect the injector
to the detector. If the problem still persists, then you have determined that the
instrumentation is the problem. Otherwise, the column should be investigated.
Column
The useful column lifetime is a function of factors such as: the cleanliness and
composition of the mobile phase and the sample; the flow rate and pressure
used; and the temperature. Refer to the section above about „Cleaning“ cleaning,
however, is not effective when the column is damaged by irreversible sample
adsorption, channeling, exposure of the packing material to excessive heat, par-
ticle fracturing due to freezing of the mobile phase, or large pressure pulsations.
Column problems due to clogging of the top frit and a depression of the packed
bed may be remedied by following the procedures described below. Also take a
look at one of the troubleshooting guides, textbooks or expert programs that are
on the market.
Clogged frit
The inlet frit may become (partially) clogged by particulate matter derived from
the sample, mobile phase or components in your liquid chromatography system.
If completely clogged, this will result in increased column back pressure. The
pressure does not increase in the case of partial clogging of the frit, although this
can result in peak tailing or splitting due to uneven sample distribution.
Backflushing is often successful in cleaning the top frit. Follow these steps: stop
the flow and let the pressure return to zero. Disconnect the column from injector
and detector. Reconnect the column exit to the injector. Do not connect the col-
umn to the detector! Start the flow and flush the column for at least 30 minutes at
half the standard flow rate. Monitor the pressure to be sure it does not exceed the
maximum. Then, install the column again with the flow going in the direction of
the arrow. Establish that the procedure has resulted in a lower pressure drop or
in improved peak shape in the case of partial clogging. If not, clean and/or replace
the end fitting (available from Tosoh Bioscience) as described below.
To clean or replace the end fitting, use a vise to carefully remove the old fitting
from the top of the column, making sure not to disrupt the gel packing at the col-
umn top. Soak the fitting in a detergent solution for 5-10 minutes or sonicate in
6 M nitric acid. If not successful, replace the end fitting with a new one. Follow
the instructions in the section titled „Installing the column“ to displace air from
the column end fitting when reassembling the column.

11
Filling voids
Column packings can sometimes settle, thereby creating a void or depression at
the top of the column. To fill such voids, we recommend that you use
SW- or PW -type top-off gels for gel filtration columns or one of the TSK Guardgel
products or other TSKgel column types.
RETURN POLICY
Tosoh Bioscience is fully committed to quality products and service. TSKgel col-
umn products are accompanied by a chromatogram demonstrating the perfor-
mance of a test mixture on the column and by a sheet that contains information
about the OCS for the column.
Despite our commitment to product quality, columns and resins occasionally
perform differently than expected in a customer’s application. Therefore, we ask
you to inspect your TSKgel column within 30 days of receipt by using the same
conditions we employed to ensure product performance. Let us know within this
30-day period if the product does not meet the specifications on the OCS sheet
and QC document, or as listed in the Inspection Data Sheet.
Subject to prior authorization, we will accept for return all products that do not
perform according to their specifications. If a product is authorized for return for
reasons other than Tosoh Bioscience’s error or because of a product defect, there
will be a restocking charge of 25 Euro or 10% of the list price, whichever is greater.
Tosoh Bioscience warrants only that the product will conform to its chemical
descriptions. TOSOH BIOSCIENCE MAKES NO OTHER WARRANTIES, EXPRESS
OR IMPLIED, AND EXPRESSLY DISCLAIMS ANY IMPLIED WARRANTY OF FITNESS
FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL TOSOH BIOSCIENCE BE
RESPONSIBLE FOR SPECIAL, INCIDENTAL OR CONSEQUENTIAL DAMAGES
WHETHER THE CLAIM IS IN CONTRACT, NEGLIGENCE OR OTHERWISE.
ADDITIONAL HELP
If you require additional technical assistance, call Tosoh Bioscience at:
Technical hotline: [phone] or e-mail: [email protected]
We hope your Tosoh Bioscience TSKgel column will help you
achieve success with your research!
TSKgel is a registered trademark of the Tosoh Corporation.
Teflon is a registered trademark of DuPont.
TSKgel columns, guardgels, and guardgel kits contain media manufactured under one
or more of U.S. patents 4,224,415; 4,256,840; 4,382,124 and 4,501,826. The purchaser is
entitled to use these products under U.S. patent 4,297,220.

Im Leuschnerpark 4, 64347 Griesheim, Germany
Phone: [phone] Fax: [phone]
Order Phone: [phone] Order Fax: [phone]
[email protected], www.tosohbioscience.de M19L12A