Size Exclusion and Ion-Exchange

Chromatography of Proteins using

the ACQUITY UPLC

H-Class
System
This document provides guidelines for running size exclusion chromatography (SEC) and ion-
exchange (IEX) chromatography of proteins using the ACQUITY UPLC H-Class System. These
guidelines are for applications that essentially use 100% aqueous conditions at relatively high
ionic strength improving system robustness and, in some cases, protecting common protein
separation columns. These instructions are intended for use with a standard ACQUITY H-Class
System.
Note: To be certain you have the latest version of this procedure, please visit the Waters
Service Knowledge Center.
Parts and Supplies
Quaternary Solvent Manager (QSM)
Spare Seals and Preventive Maintenance Kit for the QSM: more frequent replacement of
parts may be required with high ionic strength aqueous buffers than with reversed-phase
eluents; (PM Kit ACQUITY QSM, P/N 201000233).
Titanium solvent bottle filters: required to reduce the possibility of corrosion and leeching
of metals into aqueous buffers. Install on all solvent and wash lines (Titanium Solvent Bottle
Filters [package of 7], P/N 700005378).
Replace the GPV filter with its Titanium counterpart (P/N: 700005419, package of 4).
Replace the standard stainless steel mixer/filter with the titanium equivalent:
700005119 ACQUITY UPLC H-Class Bio Mixer/Filter, Titanium, 100 L
205000737 ACQUITY UPLC H-Class Bio Mixer/Filter, Titanium, 250 L
The optional 250 L mixer is used to replace the standard 100 L mixer in applications
requiring the smoothest baseline with an optical detector. It is particularly recommended
where low UV wavelengths, below about 225 nm, are monitored in the presence of UV-
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*715002909* *Rev.A*

absorbing mobile phases, such as TFA. It is not recommended for MS, but should be used
in such systems when methods are to be used with both MS and UV.
UPLC Flow Restrictor: may be required to increase operating pressure at the solvent
manager without exposing samples or columns to high pressure (UPLC Flow Restrictor, P/N
205000547).
If the QSM has a solvent select valve installed, it is recommended that it be replaced with
the Titanium version (Ti Solvent Select Valve, P/N: 205000722).
Sample Manager (SM)
Preventive Maintenance Kit for Sample Manager; more frequent replacement of parts may be
required with high ionic strength aqueous buffers than with reversed-phase eluents; (PM Kit,
Sample Manager FTN, P/N 201000234).
NOTE: The Preventive Maintenance Kit for the Sample Manager does not include the needle,
loops, and syringes described below.
The standard H-Class needle should be replaced with its MP35N equivalent (P/N
700005421).
The standard H-Class needle should be replaced with its MP35N equivalent (P/N
700005421).
Large sample loops may be required for the large sample injections often typical of ion
exchange. Extension loops increase the volume of the sample that can be drawn and held
for injection. The loop must be matched to the syringe.
Loops
430002591 Kit, Extension Loop, MP35N, 50 L
430002625 Kit, Extension Loop, MP35N, 100 L
430002630 Kit, Extension Loop, MP35N, 250 L
430002585 Kit, Extension Loop, MP35N, 1000 L
Syringes
410001348 Kit, 50 L Syringe
700002570 Kit, 100 L Syringe
410001347 Kit, 250 L Syringe
410001647 Kit, 500 L Syringe
NOTE: It is recommended that you select a syringe size that is at least two times your sample
volume.
715002909, Rev. A
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Column Heater/Manager
The column heater/manager chosen for use with the ACQUITY UPLC system depends on the
length of the columns and the operating temperatures required for the application.
Replace the standard ACQUITY UPLC H-Class active pre-heater(s) (APH) with its MP35N
equivalent (P/N 205000756)
Replace column switching valve pods, as appropriate, with its Titanium equivalent (Ti
Valve Pod Kit, P/N 205000773)
Detector
Some protein samples may exhibit tailing with the standard light-guided flow cells used in the
ACQUITY TUV and PDA detectors. Steel and titanium flow cells are available for both detectors.
205000613 ACQUITY PDA Flow Cell, Titanium, 5mm, 1500 nL
205000612 ACQUITY PDA Flow Cell, Stainless Steel, 5mm, 1500 nL
205000611 ACQUITY TUV Flow Cell, Titanium, 5mm, 1500 nL
205000610 ACQUITY TUV Flow Cell, Stainless Steel, 5mm, 1500 nL
NOTE: Many columns used for protein separations have low pressure limits. Because significant
backpressure is associated with the low dispersion detector cells used in UPLC, measure system
pressure at the intended flow rate. It may be necessary to reduce the flow rate to remain
within column pressure limits.
Columns
700003784 Kit, In-line column filter, Titanium (Designed to protect columns from
particulates)
HPLC IEX Columns and HPLC SEC Columns may require changing screws and ferrules on
the column tubing.
Buffer Preparation and Washes
Recommendations
Filter aqueous buffers before use.
Use ultrapure water.
Use high quality salts when preparing buffered eluents.
Filter salt-containing aqueous buffers through compatible 0.22 M membrane filter.
Degas all solvents.
715002909, Rev. A
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Prevent microbial growth. For information on preventing contamination, refer to
Controlling Contamination in UltraPerformance LC/MS and HPLC/MS Systems, P/N
715001307.
Store solvents in clean glass reservoirs with covers.
Clean laboratory glassware properly.
To prevent accumulation of micro-organisms, clean the buffer reservoirs at 2 to 4 week
intervals.
Use titanium solvent bottle filters for all buffers and wash solutions.
Separation Buffers
The following buffers have been tested with the ACQUITY UPLC H-Class system and provide
reliable operation when the guidelines in this document are fully implemented:
TRIS, phosphate or acetate buffers, 10-20 mM, pH 5-9
Salt concentrations up to 1.5M NaCl
More aggressive tests of pumps have been performed using pH 2 and pH 12 with 1.5M NaCl.
However, the amount of metal ions that may be released under these more extreme conditions
is unknown at this time.
Some buffers may support microbial growth. These must be discarded frequently and the
system should be flushed when new batches of buffer are used. If the system becomes
contaminated, clean it as described in Controlling Contamination in UltraPerformance LC/MS
and HPLC/MS Systems, P/N 715001307.
Needle Wash
The needle wash must dissolve the sample of interest.
Sample diluent is an appropriate needle wash
Initial strength mobile phase is an appropriate needle wash
Sample Manager Wash Solvents
The ACQUITY UPLC H-Class Sample Manager (SM-FTN) has two wash solvent lines labeled
purge and wash. They serve very different purposes and should be placed in reservoirs
containing very different solutions.
Purge Solvent
The purge solvent provides the liquid that fills and flushes the sample syringe. This solution
never comes in contact with the sample and is adjacent to mobile phase within the tubing. The
solution should be chosen to avoid precipitation with mobile phase and to minimize outgassing
that would lead to inaccurate injection volumes. It is convenient and generally recommended
that the purge solvent be the same solution as used for the seal wash, typically 90% water and
10% methanol or acetonitrile.
715002909, Rev. A
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Needle Wash Solvent
The needle wash solution fills the injection/wash port where the injection needle resides during
normal operation. During the injection cycle, this solution is vigorously pumped from bottom to
top through the injection port to ensure that the outside of the sample needle is completely
cleaned of sample to minimize carryover and sample cross-talk.
The selection of the needle wash solution is based primarily on the properties of the sample.
The system itself is compatible with a wide range of possible solvents, but this rinse solution is
chosen based on its ability to dissolve the particular sample. In this sense, the solution is also
compatible with the mobile phase in use. A good first choice in many cases could be the sample
diluent or, alternatively, the initial strength mobile phase. For example, an aqueous-organic
mixture might be chosen for reversed-phase peptide mapping or protein analysis. In contrast,
with ion exchange or size exclusion chromatography of proteins, a buffered aqueous solution of
moderately high ionic strength, or even phosphate-buffered saline would be a reasonable
choice.
When using needle wash solutions containing buffers or salts, some powdery or crystalline
residue may be observed at the top of the injection/wash port. This is normal residue of dried
needle wash solvent at the opening of the cylinder and does not impair operation, but it should
be cleaned away before the accumulation is so large that it interferes with needle movement.
Rinse with water, taking care not to bend or otherwise damage the needle.
The needle wash solution is delivered to the wash station with a pump. When the system is first
used, this pump will pass through a break-in period over several days as it equilibrates to the
chosen solution. The flow may slow over this initial period, but it will stabilize and remain
constant over the working life of the pump.
Storage
When the system is not in use, all the sample manager solvents should be replaced with low
ionic strength solutions that do not allow microbial growth. Generally follow the same guidelines
as for the quaternary solvent manager. Follow the same solvent purge recommendations used
to prime the system at startup.
Seal Wash
Use 90% Water:10% Methanol
Set the instrument method to wash every 6 seconds (default is every 5 minutes). In a day of
operation, the system could use 1000 mL or more of seal wash.
NOTE: Do not use buffers in the QSM seal wash.
Operating Parameters and Recommendations
System Preparation
Flush all solvent and wash lines with water to ensure removal of organic solvents that may
cause precipitation of buffer and salts. For some applications it may be necessary to follow the
full system cleaning procedure. Refer to Controlling Contamination in UltraPerformance LC/MS
and HPLC/MS Systems, P/N 715001307.
715002909, Rev. A
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Injection
Use the loop size and syringe size appropriate for the sample. Note: To prevent the possibility
of leaks, do not use the 50 L titanium loop with HPLC fittings.
Flow Rate
Consistent with pressure limits and flow range of the column. Measure pressure in detector cell
under these conditions to ensure that there is no risk to column or detector cell. It may be
necessary to remove the ACQUITY backpressure regulator for some ion exchange columns with
low pressure limits.
Flow Restrictor
For some IEX configurations, the combination of flow rate and column properties results in very
low system pressure. Waters recommends that the ACQUITY UPLC H-Class QSM operates at
2000 psi or greater. To ensure this condition, the flow restrictor should be installed before the
injector.
Detection
Use the ACQUITY UPLC TUV or PDA detector with the recommended flow cells. See Parts and
Supplies on page 1.
System Care
Diagnostic Tests
Run diagnostic tests every 30 days, or more often, if your chromatography and experience so
indicate.
Quaternary Solvent Manager (QSM)
Test the QSM using the leak test found in the Maintain section of the ACQUITY console. Set the
threshold to 2000 psig above the typical operating pressure for ion exchange and size exclusion
applications. If diagnostics pass, there is no need to change the seals.
NOTE: Both high pressure and low pressure seals may require replacement as often as every
30 days, depending on the conditions used.
Sample Manager (SM-FTN)
Test the SM using the leak test and needle seal readiness test found in the Maintain section of
the ACQUITY Sample Manager console. If diagnostics pass, there is no need to replace syringe
or needle seat respectively.
715002909, Rev. A
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715002909, Rev. A
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Recommendations for Leaving the System Idle
Short term (or overnight):
- Do not stop flow.
- Use 50% A: 50% B at a low flow rate (0.1-0.2mL/min).
- Always observe the column manufacturers recommendations.
Longer term:
- Column: Store according to vendor recommendations.
- System:
1. Remove the column and replace with a union.
2. Place all four buffer lines, sample manager wash solvent and purge solvent lines,
and seal wash line into water.
3. Flush the system with water using System Startup from the ACQUITY Console. Use
5 min for ABCD and seal wash. Use 60sec for wash solvent. Use 30 cycles for
purge solvent.
4. Stop flow.
5. Place all four buffer lines, sample manager wash solvent and purge solvent lines,
and seal wash line into 90% Water: 10% Organic solvent (acetonitrile, methanol,
ethanol, or isopropanol).
6. Flush the system with water using System Startup from the ACQUITY Console. Use
5min for ABCD and seal wash. Use 60 sec for wash solvent. Use 50 cycles for purge
solvent.
7. Stop flow.
- Flush the system every 1 to 2 months with 30% methanol or isopropanol to prevent
microbial growth.
Additional Information
ACQUITY UPLC H-Class System Bookshelf (P/N 715002124)
ACQUITY UPLC Console online help
ACQUITY UPLC System Enhanced Sample Management Capabilities (P/N 715001307)
Controlling Contamination in UltraPerformance LC/MS and HPLC/MS Systems (P/N
715001307)
ACQUITY UPLC System Quick Reference Card (P/N 715002125)