Page 1
PRODUCT MANUAL
IONPAC® NG1 GUARD COLUMN
(4 x 50 mm, P/N 039567)
IONPAC® NS1 ANALYTICAL COLUMN
(4 x 250 mm, P/N 035321)
IONPAC® NS1-5µm ANALYTICAL COLUMN
(4 x 150 mm, P/N 059568)
QUICKSTART STEPS AND LINKS
Click blue text below to get started.
1. The standard test eluent for the IonPac NS1 is 3 mM Tetrabutylammonium hydroxide/28%
Acetonitrile. See Section 3.4, “Preparing Eluents with Solvents”. Make the required stock
and working solutions for eluents. See Section 3, “Operation,” for details. Note operation
precautions and chemical purity requirements.
2. Run the Production Test Chromatogram as a system check. See Section 4.1.1, “Production
Test Chromatogram,” for details.
3. See Section 4, “Applications” for example applications.
4. See “Column Care” for column cleanup and long-term storage recommendations.
©DIONEX Corporation, 1997-2004
Document No. 034024
Revision 06
15 January 2004
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IonPac NS1 Manual Document No. 034024-06 Page 2 of 35
TABLE OF CONTENTS
SECTION 1 - INTRODUCTION ...................................................................................................... 4
SECTION 2 - INSTALLATION........................................................................................................6
2.1 System Requirements .............................................................................................................................................. 6
2.2 The System Injection Loop, 10 - 15 µL ............................................................................................................... 6
2.3 The IonPac Anion Trap Column ............................................................................................................................ 6
2.4 The IonPac NG1 Guard Column ........................................................................................................................... 7
2.5 Eluent Storage.......................................................................................................................................................... 7
2.6 Anion Self-Regenerating Suppressor Requirements............................................................................................ 7
2.7 Detector Requirements ............................................................................................................................................ 8
SECTION 3 - OPERATION .............................................................................................................. 9
3.1 General Operating Conditions ............................................................................................................................... 9
3.2 IonPac NS1 Operation Precautions ....................................................................................................................... 9
3.3 Chemical Purity Requirements .............................................................................................................................. 9
3.3.1 Inorganic Chemicals .................................................................................................................................................. 9
3.3.2 Deionized Water ........................................................................................................................................................ 9
3.3.3 Solvents...................................................................................................................................................................... 9
3.3.4 Acid Modifiers......................................................................................................................................................... 10
3.3.5 Base Modifiers......................................................................................................................................................... 10
3.4 Preparing Eluents that Contain Solvents............................................................................................................ 10
3.4.1 Eluents for Reversed-Phase and Ion Suppression.....................................................................................................11
3.4.2 Eluents for Gradients ................................................................................................................................................11
3.4.3 Eluents for Mobile Phase Ion Chromatography (MPIC) ..........................................................................................11
3.5 Regenerant Preparation for the Self-Regenerating Suppressors ......................................................................11
3.6 Regenerant Preparation for the AMMS-ICE II ................................................................................................. 12
3.7 Using AutoRegen and Eluents Containing Solvents .......................................................................................... 12
3.7.1 ASRS ULTRA II in MPIC Suppression or Chemical Suppression Mode ............................................................... 12
3.7.2 CSRS UL TR A II in Chemical Suppression.............................................................................................................. 13
3.8 Using Pressurized Regenerant Reservoir with the AMMS-ICE II ..................................................................13
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IonPac NS1 Manual Document No. 034024-06 Page 3 of 35
SECTION 4 - APPLICATIONS ...................................................................................................... 14
4.1 Ion Pair Chromatography .................................................................................................................................... 14
4.1.1 Production T est Chromatogram ............................................................................................................................... 15
4.1.2 Separation of Anions by MPIC with Suppressed Conductivity Detection and Solvent........................................... 16
4.1.3 Separation of Aliphatic Sulfonic Acids by Ion-Pairing with Suppressed Conductivity (MPIC).............................. 17
4.1.4 Separation of Aromatic Sulfonic Acids Using Ion-Pairing with Suppressed Conductivity ..................................... 18
4.1.5 Separation of Aliphatic Quaternary Ammonium Ions Using Ion-Pairing with Suppressed Conductivity (MPIC).. 19
4.1.6 Separation of Alkanolamines Using Ion-Pairing with Suppressed Conductivity (MPIC) ....................................... 20
4.2 Ion Suppression Chromatography....................................................................................................................... 21
4.2.1 Water Soluble Vitamins by Ion Pairing / Ion Suppression Chromatography........................................................... 22
4.2.2 Water Soluble Vitamins by Ion Suppression / Ion Pair Chromatography ................................................................ 22
4.2.3 Separation of Benzoic Acid and o-nitrobenzoic Acid .............................................................................................. 23
4.2.4 Gradient Separation of Aliphatic Carboxylic Acids................................................................................................. 24
SECTION 5 - TROUBLESHOOTING .......................................................................................... 25
5.1 High Back Pressure ............................................................................................................................................... 26
5.1.1 Finding the Source of High System Pressure........................................................................................................... 26
5.1.2 Replacing Column Bed Support Assemblies ........................................................................................................... 27
5.2 High Background or Noise ................................................................................................................................... 27
5.2.1 Preparation of Eluents.............................................................................................................................................. 27
5.2.2 Contaminated Anion T rap Column, the A TC-3 (4-mm)........................................................................................... 28
5.2.3 Contaminated Guard or Analytical Column............................................................................................................. 28
5.2.4 Contaminated Hardware .......................................................................................................................................... 28
5.2.5 Contaminated Suppressor ....................................................................................................................................... 28
5.3 Poor Peak Resolution ............................................................................................................................................ 29
5.3.1 Loss of Column Efficiency ...................................................................................................................................... 29
5.3.2 Poor Resolution Due to Shortened Retention Times ............................................................................................... 29
5.3.3 Loss of Front End Resolution .................................................................................................................................. 30
5.4 Spurious Peaks....................................................................................................................................................... 30
5.5 Small Analyte Peak Areas ..................................................................................................................................... 31
APPENDIX A - QUALITY ASSURANCE REPORT.................................................................... 32
A.1 IonPac NS1 10µm .................................................................................................................................................. 32
A.2 IonPac NS1 5µm .................................................................................................................................................... 33
APPENDIX B - COLUMN CARE .................................................................................................. 34
B.1 Recommended Operation Pressures .................................................................................................................... 34
B.2 Column Start-Up ................................................................................................................................................... 34
B.3 Column Storage ..................................................................................................................................................... 34
B.4 Column Cleanup .................................................................................................................................................... 34
B.4.1 Choosing the Appropriate Cleanup Solution ........................................................................................................... 35
B.4.2 Column Cleanup Procedure ..................................................................................................................................... 35
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IonPac NS1 Manual Document No. 034024-06 Page 4 of 35
SECTION 1 - INTRODUCTION
The IonPac® NS1 (10-micron packing) and the IonPac® NS1-5µm (5-micron packing) analytical columns are polymer-based
reversed-phase columns for the analysis of ionic and nonpolar organic compounds. The packing material is a highly cross-linked,
macroporous copolymer with a very high hydrophobic surface area. The 5-micron version provides higher efficiencies than the
10-micron version. A great advantage of polymer-based packings is their chemical inertness not only to commonly used HPLC
solvents but also to the full pH range from 0 to 14. Often organic analytes of interest to the chromatographer are ionized at neutral
pH. Ion suppression chromatography can often be used to great advantage to control the adsorption of ionizable molecules to the
column packing through eluent pH adjustments and therefore control their resulting retention times. This translates to more
resolving power and greater sensitivity. The Anion ICE II MicroMembrane Suppressor (AMMS® -ICE II) is a high-capacity, low
void volume dynamic eluent suppressor designed for use with ion exclusion and ion suppression separation modes of ion
chromatography.
Many analytes of interest are neither ionizable nor UV detectable. Organic and inorganic anions and cations which are not UV
detectable can be analyzed using Mobile Phase Ion Chromatography (MPIC®) coupled with suppressed conductivity detection.
In these analyses, ionic analytes are complexed in the mobile phase with an ion pair reagent. Separations are achieved through a
two fold mechanism. The first consideration is the degree of adsorption that takes place between the hydrophobic portion of the
ion pair reagent and the column packing. This varies with the length of the hydrophobic portion of the ion pair reagent. The second
consideration is the stability of the ion pair complex between the ion pair reagent and the analyte ion. This can be varied by the
addition of salts to the eluent.
The suppressor for Anion-MPIC is the Anion Self-Regenerating Suppressor
®
ULTRA II (ASRS ULTRA II), which is designed
to suppress tetraalkylammonium pairing reagents. Note: the Anion MicroMembrane Suppressor (AMMS III) cannot be used with
MPIC. The suppressor for Cation-MPIC is the Cation Self-Regenerating Suppressor® ULTRA II (CSRS ULTRA II), which can
also be used to suppress conventional cation-exchange eluents. Both the ASRS ULTRA II and CSRS ULTRA II are compatible
with typical organic solvents up to 40% by volume used in reversed-phase ion-pair chromatography. The external water mode must
be used for eluents containing organic solvent.
This manual assumes that you are familiar with the installation and operation of the Dionex Ion Chromatograph (IC). If you do
not understand the operation of the system, take the time to familiarize yourself with the various system components before
beginning an analysis.
Table 1
IonPac NS1/NG1 Packing Specifications
Column Particle Substrate
Diameter X-linking
µm %
IonPac NS1 analytical column 10 55
4 x 250 mm
IonPac NG1 guard column 10 55
4 x 35 mm
IonPac NS1-5 µm analytical column 5 55
4 x 150 mm
Always remember that assistance is available for any problem that may be encountered during the shipment or
operation of Dionex instrumentation and columns through the Dionex North America Technical Call Center at
1-800-DIONEX-0 (1-800-346-6390) or through any of the Dionex Offices.
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IonPac NS1 Manual Document No. 034024-06 Page 5 of 35
Table 2
NS1/NG1 Operating Parameters
Standard Maximum
Column Typical Back Pressure Flow Rate Flow Rate
psi (MPa) mL/min mL/min
NS1 Analytical 900 (6.21) - 1,400 (9.66) 1.0 3.0
NG1 Guard 200 (1.38) - 300 (2.07) 1.0 3.0
NS1 + NG1 columns 1,100 (7.59) - 1,700 (11.73) 1.0 3.0
NS1-5 µm analytical 1,700 (11.73) - 2,500 (17.24) 1.0 1.5
NG1 Guard 200 (1.38) - 300 (2.07) 1.0 1.5
NS1-5 µm + NG1 columns 1,900 (13.11) - 2,800 (19.31) 1.0 1.5
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IonPac NS1 Manual Document No. 034024-06 Page 6 of 35
SECTION 2 - INSTALLATION
2.1 System Requirements
The IonPac NS1 Guard and analytical columns can be run on any PEEK Dionex Chromatograph. Depending upon the application,
the system should be equipped with either a conductivity detector and an ASRS ULTRA II for MPIC applications or a UV/Vis
detector for reversed-phase and ion pair applications. Gradient or isocratic methods should be performed on a system having a
gradient pump configured for standard bore operation. For isocratic analyses at flow rates below 0.5 mL/min and gradient analyses,
a gradient pump (1/16" pistons) must be employed.
2.2 The System Injection Loop, 10 - 15 µL
For most applications on a 4-mm analytical system, a 10–50 µL injection loop will be sufficient. Dionex recommends that a 10
µL injection loop be used to avoid overloading the IonPac NS1 4-mm analytical column. Generally, do not inject more than 10
nanomoles (100–200 ppm) of any one analyte onto the 4-mm analytical column. Injecting larger volumes of samples can result
in overloading the column which can affect the detection linearity. This phenomenon will be more prevalent at higher
concentrations of the analytes of interest.
2.3 The IonPac Anion Trap Column
Gradient or step change applications for anion MPIC require an IonPac Anion Trap Column (ATC-3 (4-mm), P/N 037151); cation
MPIC applications require an IonPac Cation Trap Column (CTC-1 (4-mm), P/N 040192). The IonPac Trap Column should be
installed in place of the high pressure gradient mixer between the gradient pump and the injection valve. The IonPac Trap Column
is filled with high capacity ion exchange resin which helps to minimize the baseline shift caused by increasing ionic contaminant
levels in the eluent as the ionic concentration of the eluent is increased over the course of the gradient analysis.
To install the IonPac Trap Column, complete the following steps:
A. Remove the gradient mixer. It is installed between the gradient pump pressure transducer and the injection valve.
B. Connect the gradient pump directly to the IonPac Trap Column. Connect a waste line to the trap column outlet and direct
the line to a waste container.
C. If you are doing anion MPIC regenerate the ATC-3. Use 200 mL of 200 mM NaOH at a flow rate of 2.0 mL/min. Note
that the guard and analytical column are out of line.
D. Rinse the IonPac Trap Column with 30 mL of the strongest eluent that will be used during the gradient analysis.
E. Connect the IonPac Trap Column, after flushing it with eluent, to the eluent line that is connected to the injection valve.
At the end of each operating day, the IonPac Trap Column should be regenerated to remove any impurities that may have
accumulated on it.
A. Disconnect the ATC-3 or the CTC-1. It should be installed before the injection valve.
B. Direct the outlet of the ATC-3 or the CTC-1 to a separate waste container.
C. Regenerate the ATC-3 or the CTC-1. For detail information on the operation on the ATC-3 or the CTC-1, see Document
No. 032697 for the ATC-3 and Document No. 034536 for the CTC-1.
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IonPac NS1 Manual Document No. 034024-06 Page 7 of 35
On the next day, prior to the use of the chromatographic system, rinse the IonPac Trap Column. This will help with equilibration
times.
A. Rinse the TC-1 with 30 mL of the strongest eluent used in the gradient analysis.
B. Reconnect the IonPac Trap Column, after flushing it with eluent, to the eluent line that is connected to the injection
valve.
2.4 The IonPac NG1 Guard Column
An IonPac NG1 Guard Column is normally used with the IonPac NS1 analytical column. Retention times will increase by
approximately 20% when a guard column is placed in-line prior to the analytical column. A guard is placed prior to the analytical
column to prevent sample contaminants from eluting onto the analytical column. It is easier to clean or replace a guard column than
it is an analytical column. Replacing the NG1 Guard Column at the first sign of peak efficiency loss or decreased retention time
will prolong the life of the NS1 analytical column.
2.5 Eluent Storage
IonPac NS1 columns are designed to be used with Tetrabutylammonium hydroxide eluent systems. Storage under a helium
atmosphere ensures contamination free operation and proper pump performance (nitrogen can be used if eluents do not contain
solvents).
2.6 Anion Self-Regenerating Suppressor Requirements
The Anion Self-Regenerating Suppressor (ASRS ULTRA II) is used for eluent suppression of MPIC (ion-pairing) eluents by using
the MPIC Suppression Mode of operation. This suppression mode is a combination of the AutoSuppression External Water Mode
augmented with a chemical regenerant such as sulfuric acid (H2SO4). The MPIC Suppression Mode uses an applied current and
a constant source of dilute sulfuric acid solution delivered from an AutoRegen Accessory or a pressurized bottle system. The MPIC
Suppression Mode reliably provides suppression of typical eluents for MPIC applications using suppressed conductivity detection.
The ion pair reagents, such as tetrabutylammonium hydroxide (TBAOH), are used in concentrations ranging typically from 1.0
to 5.0 mM. For detailed information on the operation of the ASRS ULTRA II, see Document No. 031956.
The Cation Self-Regenerating Suppressor (CSRS ULTRA II) can be used for suppression of MPIC (ion-pairing) eluents by using
the AutoSuppression External Water Mode of operation or the MPIC Suppression Mode depending on the specific MPIC
application. The MPIC Suppression Mode is a combination of the AutoSuppression External Water Mode augmented with a
chemical regenerant if necessary such as boric acid (H3BO3). When the CSRS ULTRA II is operating in this mode, it uses an applied
current and a constant source of dilute boric acid solution delivered from a pressurized bottle system. For detailed information on
the operation of the CSRS ULTRA II, see Document No. 031956.
An Anion or Cation Self-Regenerating Suppressor should be used for ion pairing applications that require suppressed conductivity
detection. The ASRS ULTRA II in the MPIC Suppression Mode of operation and the CSRS ULTRA II in the AutoSuppression
External Water or MPIC Suppression Mode of operation are compatible with aqueous ionic eluents of all concentrations with which
the column and system are compatible and with solvent containing eluents up to 40% by volume. For applications requiring solvent
above 40% by volume, the Chemical Suppression Mode of operation must be used. Aqueous ionic eluents can be used in all modes
of operation.
NOTE
When using eluents containing solvent above 40% by volume, the ASRS ULTRA II or CSRS ULTRA II should
be used in the Chemical Suppression Mode.
If you are installing an IonPac NS1 4-mm analytical column for ion pairing chromatography with suppressed conductivity
detection, use an ASRS ULTRA II, 4-mm, (P/N 061561) for Anion-MPIC or the CSRS ULTRA II, 4-mm, (P/N 061563) for Cation-
MPIC.
The AMMS-ICE II (P/N 037107) is used with Ion Exclusion and Ion Suppression Modes of Ion Chromatography. For detailed
information on the operation of the AMMS-ICE II, see Document No. 032661.
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IonPac NS1 Manual Document No. 034024-06 Page 8 of 35
2.7 Detector Requirements
See Section 2, “Ion Chromatography System Operation Summary,” for 4-mm system detector, cell and thermal stabilizer
requirements.
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IonPac NS1 Manual Document No. 034024-06 Page 9 of 35
SECTION 3 - OPERATION
3.1 General Operating Conditions
Sample Volume: 4-mm: 10 µL Loop + 0.8 µL Injection valve dead volume
Column: 4-mm: IonPac NS1 4-mm analytical column + IonPac NG1 4-mm guard column
Eluent: 1–5 mM ion-pairing reagent with 1–100% solvent
Eluent Flow Rate: 1.0 mL/min
Detector: UV or Suppressed Conductivity
Storage Solution: Eluent
3.2 IonPac NS1 Operation Precautions
CAUTIONS
Filter and Degas Eluents
Filter Samples
Eluent pH between 0 and 14
Sample pH between 0 and 14
3.0 mL/min Maximum Flow Rate for 4-mm Columns
Maximum Operating Pressure = 4,000 psi (27.57 MPa)
3.3 Chemical Purity Requirements
Obtaining reliable, consistent and accurate results requires eluents that are free from ionic impurities. Chemicals, solvents and
deionized water used to prepare eluents must be of the highest purity available. Low trace impurities and low particle levels in
eluents also help to protect your reversed-phase columns and system components. Dionex cannot guarantee proper column
performance when the quality of the chemicals, solvents and water used to prepare eluents has been compromised.
3.3.1 Inorganic Chemicals
Reagent Grade inorganic chemicals should always be used to prepare ionic eluents. Whenever possible, inorganic chemicals that
meet or surpass the latest American Chemical Society standard for purity should be used. These inorganic chemicals will detail
the purity by having an actual lot analysis on each label.
3.3.2 Deionized Water
The deionized water used to prepare eluents should be Type I Reagent Grade Water with a specific resistance of 18.2 megohmcm. The deionized water should be free of ionized impurities, organics, microorganisms and particulate matter larger than 0.2 µm.
Bottled HPLC-Grade Water (with the exception of Burdick & Jackson) should not be used since most bottled water contains an
unacceptable level of ionic impurities.
3.3.3 Solvents
CAUTION
The IonPac NS1 and NS1-5µm column packings are spherical, highly cross-linked polymeric materials having
very large hydrophobic surface areas. It is essential that these columns are operated so that the eluent being
pumped over the column contains minimally 1% solvent to ensure that the hydrophobic surfaces are “wetted” and
maximum column performance is maintained.
The IonPac NS1 and NS1-5µm analytical columns can withstand all common HPLC solvents in a concentration range of 1% to
100%. However, solvents and degassed water should be premixed in concentrations that allow proper mixing by the gradient pump
and minimize outgassing. It is therefore more practical to say that the these columns have an operational organic solvent
concentration range of 1 to 95% to ensure proper chromatographic system performance.
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IonPac NS1 Manual Document No. 034024-06 Page 10 of 35
Avoid creating high viscosity pressure fronts that may disrupt the column packing when the eluent solvent is changed. To
do this, equilibrate the column for approximately 10 minutes with an eluent containing only 5% of the current solvent type (e.g.,
methanol). Exchange this eluent for an eluent with 5% of the new solvent type (e.g., acetonitrile) and then equilibrate the column
and allow the system to stabilize (approximately 10 minutes). Next, run a 15-minute gradient from 5% of the new solvent type to
the highest percentage that will be used during the new analysis protocol.
Solvents can be added to the ionic eluents used with IonPac NS1 columns to modify the ion exchange process or improve sample
solubility. The solvents used must be free of ionic impurities. However, since most manufacturers of solvents do not test for ionic
impurities, it is important that the highest grade of solvents available be used. Currently, several manufacturers are making ultrahigh
purity solvents that are compatible for HPLC and spectrophotometric applications. These ultrahigh purity solvents will usually
ensure that your chromatography is not affected by ionic impurities in the solvent. At Dionex, we have obtained consistent results
using High Purity Solvents manufactured by Burdick and Jackson and Optima® Solvents by Fisher Scientific.
When using a solvent in an ionic eluent, column generated back pressures will depend on the solvent used, concentration of the
solvent, the ionic strength of the eluent and the flow rate used. The column back pressure will vary as the composition of watermethanol and water-acetonitrile mixture varies. The practical back pressure limit for the IonPac NS1 columns is 4,000 psi (27.57
MPa).
The IonPac NS1 can withstand common HPLC solvents in a concentration range of 1–100%. Solvents and water should be
premixed in concentrations which allow proper mixing by the gradient pump and to minimize outgassing. Ensure that all of the
inorganic chemicals are soluble in the highest solvent concentration to be used during the analysis.
Table 3
HPLC Solvents for Use with IonPac NS1 Columns
Solvent Maximum Operating Concentration
Acetonitrile 100%
Methanol 100%
2-Propanol 100%
Tetrahydrofuran 20%
3.3.4 Acid Modifiers
Mineral acids such as HCl, H2SO4, and HNO3 can be used at concentrations as high as 1.0 N to acidify eluents.
3.3.5 Base Modifiers
Bases such as NaOH, KOH, and NH4OH can be used up to 1.0 N to alkalify eluents.
3.4 Preparing Eluents that Contain Solvents
When mixing solvents with water remember to mix solvent with water on a volume to volume basis. If a procedure requires an
eluent of 90% acetonitrile, prepare the eluent by adding 900 mL of acetonitrile to an eluent reservoir. Then add 100 mL of
deionized water or eluent concentrate to the acetonitrile in the reservoir. Using this procedure to mix solvents with water will
ensure that a consistent true volume/volume eluent is obtained. Premixing water with solvent will minimize the possibility of
outgassing.
When purging or degassing eluents containing solvents, do not purge or degas the eluent excessively since it is
possible that a volatile solvent can be “boiled” off from the solution.
NOTE
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IonPac NS1 Manual Document No. 034024-06 Page 11 of 35
Always degas and store all eluents in glass or plastic eluent bottles pressurized with helium. Only helium can be used to purge and
degas ionic eluents containing solvents, since nitrogen is soluble in solvent containing eluents.
Acetonitrile (ACN) hydrolyzes to ammonia and acetate when left exposed to basic solutions. To prevent eluent contamination
from acetonitrile hydrolysis, always add acetonitrile to basic aqueous eluents by proportioning the acetonitrile into the basic eluent
with the gradient pump. Keep the acetonitrile in a separate eluent bottle containing only acetonitrile and water. Never add the
acetonitrile directly to the basic carbonate or hydroxide eluent bottle.
3.4.1 Eluents for Reversed-Phase and Ion Suppression
A typical eluent for a reversed-phase application will contain 10% to 70% solvent. Where ion suppression is advantageous, 0.02
to 10.0 mM mineral or organic acid or base is used. If ion pair reagents are used in specific reversed-phase applications, they
typically are used at concentrations ranging from 1.0 to 10.0 mM. The chromatographic benefits of ion pair reagent concentrations
above 10 mM are generally not significant.
3.4.2 Eluents for Gradients
Gradient applications are straightforward as long as solvents and water are premixed in concentrations that allow mixing by the
gradient pump to give the required gradient ramp for your chromatography. For example, if you want to build a solvent gradient
from 10% solvent to 90% solvent, make the following eluents:
Eluent A: 10% solvent/90% water
Eluent B: 90% solvent/10% water
Then, by programming the gradient pump properly, you can go from 100% Eluent A to 100% Eluent B in a prescribed time. This
will avoid outgassing and refractive index problems associated with mixing neat solvents with water.
3.4.3 Eluents for Mobile Phase Ion Chromatography (MPIC)
All of the quaternary ammonium and sulfonic acid ion pair reagents can be used at any concentration for practical chromatographic
purposes. Buffering these reagents for column stability is not necessary.
Typical eluents for MPIC applications using suppressed conductivity detection are very similar to those used for reversed-phase
separations with respect to eluent enhancements with acids and bases, solvents and ion pair reagents. The ion pair reagents are used
in concentrations ranging from 1.0 to 5.0 mM. At higher concentrations, they may be difficult to suppress during conductivity
measurements.
The following Ion Pair Reagents are available from Dionex:
P/N 035360 Tetrabutylammonium hydroxide, 0.1 M TBAOH (MPIC-AR1)
P/N 035363 Tetrapropylammonium hydroxide, 0.1 M TPAOH (MPIC-AR2)
P/N 035361 Hexanesulfonic acid, 0.1 M HSA (MPIC-CR1)
P/N 035362 Octanesulfonic acid, 0.1 M OSA (MPIC-CR2)
3.5 Regenerant Preparation for the Self-Regenerating Suppressors
The ASRS ULTRA II when operated in the MPIC Suppression Mode requires a dilute sulfuric acid solution as the regenerant. For
detailed information on the operation of the ASRS ULTRA II, see Document No. 031956.
The CSRS ULTRA II when operated in the AutoSuppression External Water Mode requires the use of water with a specific
resistance of 10 megohm-cm or greater as the regenerant. For detailed information on the operation of the CSRS ULTRA II, see
Document No. 031956.
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IonPac NS1 Manual Document No. 034024-06 Page 12 of 35
3.6 Regenerant Preparation for the AMMS-ICE II
5 mM tetrabutylammonium hydroxide (TBAOH) is the recommended regenerant for use with the Anion-ICE II MicroMembrane
Suppressor (AMMS-ICE II). Tetramethylammonium hydroxide or potassium hydroxide may be used as alternate regenerants, but
cause higher background conductivity and therefore compromise total system performance. For ease of preparation and guaranteed
purity, use Dionex Cation Regenerant Solution (P/N 039602). For detailed information on the operation of the AMMS-ICE II, see
Document No. 032661.
3.7 Using AutoRegen and Eluents Containing Solvents
When performing Mobile Phase Ion Chromatography (MPIC) using an Anion MicroMembrane Suppressor for Mobile Phase Ion
Chromatography in the analysis of anions with ion pair reagents (AR1 or AR2) or a Cation MicroMembrane Suppressor in the
analysis of cations with ion pair reagents (CR1 or CR2) and a Conductivity Detector Module, Dionex recommends using an
AutoRegen Accessory (P/N 039594). Typical regenerant flow rates required in MPIC analyses varies from 5 to 15 mL/min.
The use of an AutoRegen Accessory saves regenerant preparation time and reduces regenerant consumption and waste. However,
when using an AutoRegen Accessory to continuously remove contaminants from the regenerant and restore the regenerant to the
correct state, it is necessary to replace the regenerant on a regular basis. How often the regenerant is replaced will depend on the
application and the concentration of the solvent in the eluent. Minimally, the regenerant should be replaced once a week. It is not
necessary to change the AutoRegen Regenerant Cartridge until it is completely expended.
Solvent, much like the sodium ions, passes through the membrane of the MicroMembrane Suppressor from the eluent to the
regenerant stream. The solvent and the sodium ions are carried from the suppressor to the regenerant reservoir by means of the
regenerant stream. As the recycled regenerant is pumped through the AutoRegen Regenerant Cartridge, the sodium ions are
exchanged for hydrogen ions. However, the solvent is not removed from the recycled regenerant and continues to accumulate in
the recycled regenerant stream. Eventually the concentration of solvent in the recycled regenerant can cause the background
conductivity to increase which can result in a noisy background. Most solvents have no affect on the cartridge lifetime.
In all cases, the ionic strength of the eluent determines the lifetime of the AutoRegen Regenerant Cartridge. However, if the eluent
contains acetonitrile and is used with a cation AutoRegen Regenerant Cartridge in the AutoRegen Accessory, the acetonitrile will
decompose to acetate and ammonium in the strong basic environment of the cartridge. Acetate ions will exchange onto the Cation
AutoRegen Regenerant Cartridge and severely limit its lifetime. Solvents other than acetonitrile have no effect on the cartridge
lifetime.
CAUTION
Acetonitrile decomposes to ammonium acetate when subjected to basic solutions such as the 0.1 M
tetrabutylammonium hydroxide (TBAOH) used as the regenerant solution in the AutoRegen Cation Regenerant
Cartridge. The acetate ion exchanges onto the AutoRegen Cation Regenerant Cartridge. If high levels of
acetonitrile are used in the eluents, the cartridge can become expended within a few hours. A pressurized
regenerant delivery system should be used as an alterative.
When replacing the recycled regenerant, the first 200 mL of the regenerant should be pumped to waste before recycling of the
regenerant is started. Utilizing AutoRegen in this manner will allow the use of high regenerant flow rates with the minimum of
consumption and waste.
3.7.1 ASRS ULTRA II in MPIC Suppression or Chemical Suppression Mode
To save regenerant preparation time, consumption, and waste, it is recommended that the AutoRegen Accessory (115 V ac version,
P/N 039594; 230 V ac version, P/N 039608) be purchased. The AutoRegen Accessory should be equipped with an Anion
AutoRegen Regenerant Cartridge (P/N 039564). For detailed information on the operation of the AutoRegen Accessory, see
Document No. 032853. For detailed information on the use of the AutoRegen Regenerant Cartridge, see Document No. 032852.
However, when using an AutoRegen Accessory, it is necessary to replace the regenerant on a regular basis. How often the
regenerant is replaced will depend on the application and the concentration of solvent in the eluent. Minimally, the regenerant
should be replaced once a week. It is not necessary to change the AutoRegen Regenerant Cartridge until it is completely expended.
Solvent, much like the TBA + ions, passes through the membranes of the ASRS ULTRA II suppressor from the eluent into the
regenerant stream. The solvent and the TBA + ions are carried from the suppressor to the regenerant reservoir by means of the
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IonPac NS1 Manual Document No. 034024-06 Page 13 of 35
regenerant stream. As the recycled regenerant is pumped through the AutoRegen Regenerant Cartridge, the TBA + ions are
exchanged for hydronium ions. However, the solvent is not removed from the recycled regenerant and continues to accumulate
in the recycled regenerant stream. Eventually the concentration of solvent in the recycled regenerant can cause the background
conductivity to increase which can result in a noisy background. Most solvents have no affect on the cartridge lifetime. In all cases
the ionic strength of the eluent determines the lifetime of the AutoRegen Regenerant Cartridge.
For ease of use and guaranteed high purity, use Dionex Anion Regenerant Concentrate (P/N 037164, 039601) to make regenerant
for the ASRS ULTRA II.
3.7.2 CSRS ULTRA II in Chemical Suppression
For ease of use and guaranteed high purity, use Dionex Cation Regenerant Concentrate (P/N 039602) to make regenerant for the
CSRS ULTRA II.
CAUTION
Acetonitrile is not compatible with the CSRS ULTRA II in the Chemical Suppression Mode when using an
AutoRegen Accessory unit. The acetonitrile diffuses into the TBAOH regenerant, concentrates during recirculation
and eventually hydrolyzes to acetate and ammonia, rapidly depleting the capacity of the AutoRegen Cation
Regenerant Cartridge. If acetonitrile is used as an eluent component, a pressurized regenerant delivery bottle
must be used instead of the AutoRegen Accessory.
3.8 Using Pressurized Regenerant Reservoir with the AMMS-ICE II
The operation of the AMMS-ICE II requires a constant flow of the regenerant over the membrane, in a direction that is
countercurrent to the flow of the eluent.
For the best signal to noise ratio and overall performance, Dionex recommends that the Cation Regenerant Solution (P/N 039602)
be pressurized with nitrogen or helium. Do not use air.
NOTE
Use nitrogen or helium to pressurize the Regenerant Reservoir.
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IonPac NS1 Manual Document No. 034024-06 Page 14 of 35
SECTION 4 - APPLICATIONS
Before attempting any of the following example applications, take the time to ensure that your system is properly configured.
Ensure that all of the eluents have been made from high purity reagents and deionized water. All water used in the preparation
of eluents should be degassed, deionized water. For chemical purity requirements, see Section 3.3, “Chemical Purity Requirements.”
After running synthetic standards to calibrate your system, you may find that real sample matrices foul your columns. For this reason
it is always advisable to use a guard column to protect the analytical column. If column performance deteriorates and it has been
determined that the guard or the analytical column has been fouled, refer to the column cleanup protocols in “Column Care.”
4.1 Ion Pair Chromatography
There are a number of mechanistic hypotheses for ion pair chromatography. In all cases they involve an eluent reagent that
contains a hydrophobic portion and an ionic portion on one molecule. These ion pair reagents can be anionic or cationic. Here
is a partial list of typical ion pair reagents:
A. Cationic: Tetramethyl, ethyl, propyl and butyl ammonium salts of the general form:
R
+
RNR
R
Counter anions for these quaternary amines are chloride, bromide, phosphate and hydroxide.
B. Anionic: Alkyl sulfonates of varying chain length such as pentane, hexane, heptane and octane sulfonates of the general
form:
CH3(CH2)nSO
-
3
Counter cations can be hydrogen ion, sodium ion and potassium ion.
From an ion exchange point of view, it may be said that the hydrophobic part of the ion pair molecule associates with the
hydrophobic surface of the stationary phase. This creates an ion exchange surface that is in dynamic equilibrium with the mobile
phase. The ion exchange capacity can be increased with a higher concentration of ion pair reagent in the eluent.
Inorganic and organic ions can have remarkable selectivity with this technique. The quaternary amine salt reagents make a loosely
defined anion exchange stationary phase. The sulfonates create a similar cation exchange stationary phase. In both cases, the ion
pair reagent itself can act as a mobile phase “pusher” for the analytes.
The following chromatograms and conditions illustrate the versatility of this method.
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IonPac NS1 Manual Document No. 034024-06 Page 15 of 35
4.1.1 Production Test Chromatogram
Isocratic elution of anions on the IonPac NS1 analytical column has been optimized utilizing tetrabutylammonium ion as the ionpairing agent and acetonitrile as the organic modifier. Using these eluent conditions, highly retained anions can be eluted from the
hydrophobic packing of the IonPac NS1. To guarantee that all IonPac NS1 analytical columns meet high quality and reproducible
performance specification standards, all columns undergo the following production control test.
Column: IonPac NS1 (10 µm)
Eluent: 3 mM Tetrabutylammonium hydroxide / 28% Acetonitrile
Flow Rate: 1.0 mL/min
Inj. Volume: 10 µL
Detection: Suppressed conductivity, ASRS ULTRA II
Regenerant: 10 mN Sulfuric acid
Peaks mg/L
1. Propanesulfonate 15
2. Iodide 15
3. Thiocyanate 15
4. Hexanesulfonate 50
where 1 mg/L= 1 ppm
AutoSuppression® MPIC Mode
Figure 1
Production Test Chromatogram
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IonPac NS1 Manual Document No. 034024-06 Page 16 of 35
4.1.2 Separation of Anions by MPIC with Suppressed Conductivity Detection and Solvent
Ion-pairing can separate monovalent anions, such as chlorate and nitrate, which differ in hydration energy. Because ion pairing
has low selectivity for higher valency ions, selected monovalent and higher valency ions can be eluted isocratically on the IonPac
NS1 analytical column. Example B illustrates the use of a higher solvent concentration to elute hydrophobic anions such as
thiosulfate and perchlorate.
Column: IonPac NS1 (10 µm
Eluent: See Chromatogram
Flow Rate: 1.0 mL/min.
Injection Loop: 50 µL
Detection: Suppressed conductivity, ASRS ULTRA II
Regenerant: 10 mN H
AutoSuppression MPIC Mode
2SO4
Peaks mg/L
1. Fluoride 5
2. Chloride 1
3. Nitrite 2
4. Bromide 2
5. Nitrate 2
6. Sulfate 2
7. Phosphate 3
8. Thiosulfate 5
9. Thiocyanate 5
10. Perchlorate 10
where 1 mg/L= 1 ppm
0.25
S
µ
1
2
0
05
Eluent A
1mM TBAOH, 1mM
Na2CO3, 10% ACN
3
4
5
6
7
0.25
µ
S
2
8
1
5
4
6
Eluent B
1mM TBAOH, 1mM
Na2CO3, 20% ACN
9
10
0
10
Minutes
15
20
05
10 15 20 25 30
Minutes
Figure 2
Separation of Anions by Ion-Pairing (MPIC) with
Suppressed Conductivity and the Effect of Solvent
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IonPac NS1 Manual Document No. 034024-06 Page 17 of 35
4.1.3 Separation of Aliphatic Sulfonic Acids by Ion-Pairing with Suppressed Conductivity (MPIC)
Ion-pairing can provide excellent selectivity for surface active analytes such as surfactants. The separation of aliphatic sulfonic
acids on the IonPac NS1 analytical column can be achieved using tetrabutylammonium ion as the ion-pairing agent with
conductivity detection. The eluent is suppressed with an ASRS ULTRA II augmented with 10 mN sulfuric acid regenerant.
Aliphatic sulfonic acids are UV transparent, but well detected by suppressed conductivity.
Column: IonPac NS1 (10 µm)
Eluent: 2 mM Tetrabutylammoniumhydroxide,
Flow Rate: 1.0 mL/min
Injection Loop: 50 µL
Detection: Suppressed conductivity, ASRS ULTRA II
Regenerant: 10 mN Sulfuric acid
Peaks: mg/L
(as the acid forms)
1. Methanesulfonic acid 5.0
2. 1-Propanesulfonic acid 8.6
3. 1-Butanesulfonicacid 8.7
4. 1-Hexanesulfonicacid 8.8
5. 1-Heptanesulfonic acid 8.9
6. 1-Octanesulfonicacid 8.9
7. 1-Decanesulfonicacid 9.1
where 1 mg/L= 1 ppm
24% to 48% Acetonitrile in 10 min
AutoSuppression MPIC Mode
Separation of Aliphatic Sulfonic Acids by Ion-Pairing with Suppressed Conductivity (MPIC)
Figure 3
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IonPac NS1 Manual Document No. 034024-06 Page 18 of 35
4.1.4 Separation of Aromatic Sulfonic Acids Using Ion-Pairing with Suppressed Conductivity
The separation of aromatic sulfonic acids is easily obtained by ion-pairing. The eluent system is identical to that used for the
separation of aliphatic sulfonic acids.
Column: IonPac NS1 (10 µm)
Eluent: 2 mM Tetrabutylammonium hydroxide,
Flow Rate: 1.0 mL/min
Injection Loop: 50 µL
Detection: Suppressed conductivity, ASRS ULTRA II
Regenerant: 10 mN Sulfuric acid
24% to 48% Acetonitrile in 10 min
AutoSuppression MPIC Mode
Peaks mg/L
(as the acid forms)
1. Benzenesulfonic acid 10.0
2. Toluenesulfonic acid 8.0
3. o-, m-, p-Xylenesulfonic acids 9.0
where 1 mg/L= 1 ppm
Figure 4
Separation of Aromatic Sulfonic Acids Using
Ion-Pairing with Suppressed Conductivity
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IonPac NS1 Manual Document No. 034024-06 Page 19 of 35
4.1.5 Separation of Aliphatic Quaternary Ammonium Ions Using Ion-Pairing with Suppressed Conductivity
(MPIC)
The separation of a series of aliphatic quaternary ammonium ions can be obtained by using an anionic ion-pair reagent such as
nonafluoropentanoic acid. The eluent is suppressed with a CSRS ULTRA II operated in the external water mode. The separation
uses an acetonitrile gradient from 20% to 80%* in 10 minutes. As the concentration of acetonitrile increases, a decrease in the
background conductivity is observed.
Column: IonPac NS1 (10 µm)
Eluent: 2 mM Nonafluoropentanoic acid, 20% to 80%* Acetonitrile in 10 min
Flow Rate: 1.0 mL/min
Injection Loop: 25 µL
Detection: Suppressed conductivity, CSRS ULTRA II
* Chemical Suppression is recommended for eluents containing solvents above 40% by volume
Peaks mg/L
(as the chloride salts)
1. Tetrapropylammonium 25
2. Tributylmethylammonium 50
3. Decyltrimethylammonium 25
4. Tetrabutylammonium 50
5. Dodecyltrimethylammonium 50
6. Tetrapentylammonium 100
7. Hexadecyltrimethylammonium 100
where 1 mg/L= 1 ppm
Separation of Aliphatic Quaternary Ammonium Ions
Using Ion-Pairing with Suppressed Conductivity (MPIC)
Figure 5
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IonPac NS1 Manual Document No. 034024-06 Page 20 of 35
4.1.6 Separation of Alkanolamines Using Ion-Pairing with Suppressed Conductivity (MPIC)
The separation of alkanolamines is also obtained with the use of nonafluoropentanoate ion as the ion-pairing agent. The eluent
is suppressed with a CSRS ULTRA II, using 10 mN boric acid as regenerant. The boric acid is added to increase the ionization
of the ethanolamines, thereby increasing the conductivity. The borate counter ion displaces the hydroxide counter ion from the
fully ionized alkali and alkaline earth metals which reduces the observed conductance for these ions.
Column: IonPac NS1 (10 µm)
Eluent: 2 mM Nonafluoropentanoicacid / 2% Acetonitrile
Flow Rate: 1.0 mL/min
Inj.Volume: 25 µL
Detection: Suppressed conductivity, CSRS ULTRA II
Regenerant: 10 mN Boric acid
AutoSuppression MPIC Mode
Peaks: mg/L
1. Lithium 0.5
2. Sodium 2.0
3. Ammonium 5.0
4. Potassium 5.0
5. Monoethanolamine 25.0
6. Diethanolamine 50.0
7. Triethanolamine 100.0
8. Calcium 5.0
9. Magnesium 2.5
where 1 mg/L= 1 ppm
Ion-Pairing with Suppressed Conductivity (MPIC)
Figure 6
Separation of Alkanolamines Using
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IonPac NS1 Manual Document No. 034024-06 Page 21 of 35
4.2 Ion Suppression Chromatography
This chromatographic technique takes advantage of the effect of pH on the dissociation constants for the for acidic and basic
organic species. Depending upon the pKa of an organic ionic molecule, the extent of ionization can be controlled by pH.
Because the IonPac NS1 10-µm and NS1-5µm column packings have hydrophobic surfaces, a non-ionic or neutral organic
molecule will have a greater affinity for it. Therefore, retention and selectivity can be enhanced by suppressing ionization at either
end of the pH scale depending upon the acidic and/or basic nature of the molecule. (see figures 8 and 9).
Acidic molecules such as alkyl or aryl carboxylic acids can be protonated at low pH's (<4). Basic molecules can be deprotonated
at high pH's (>9).
-
O
H +
Anionic: R-C=O R-C=O
pH < 4
OH -
Cationic: R-NH
+
3
pH > 9
OH
R-NH2 + H20
Ionic Neutral
Additionally, selectivity can be effected by working within one pH unit of the pKa of a particular organic species such as
substituted benzoic acids, (see Figure 10).
The difference in the pKa's of o-nitrobenzoic acid and benzoic acid was utilized to increase resolution of the compounds.
At the higher pH of 2.52 in the “after” chromatogram compared to pH 2.30 in the “before” chromatogram, the resolution of onitrobenzoic acid and benzoic acid is almost complete. The pKa of o-nitrobenzoic acid is 2.18. As the pH is raised to pH 2.52,
the o-nitrobenzoic acid becomes more ionic and moves to shorter retention times compared to the benzoic acid which has a much
higher pKa of 4.19. The benzoic acid is completely protonated in both eluents and therefore its affinity for the column does not
change as rapidly as the o-nitrobenzoic acid affinity.
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IonPac NS1 Manual Document No. 034024-06 Page 22 of 35
4.2.1 Water Soluble Vitamins by Ion Pairing / Ion Suppression Chromatography
Column: IonPac NS1-5µm
Eluent Conditions: 12 to 48% Acetonitile in 35 minutes
Flow Rate: 1.0 mL/min
Detection: UV 254 nm
Peaks
1. Ascorbic acid
2. Nicotinic acid
3. Nicotinamide
4. Riboflavin
5. Pyridoxine
6. PABA
7. Folic acid
8. Pyridoxamine
9. Thiamine
10. Cyanocobalamin
Injection: 5 nmoL each
5 mM Octane Sulfonic Acid
Water Soluble Vitamins by
Ion Pairing / Ion Suppression Chromatography
4.2.2 Water Soluble Vitamins by Ion Suppression / Ion Pair Chromatography
Column: IonPac NS1-5µm
Eluent Conditions: 12 to 56% Acetonitile
Flow Rate: 1.0 mL/min
Detection: Suppressed Conductivity
Peaks
1. Ascorbic acid
2. Nicotinic acid
3. Nicotinamide
4. Riboflavin
5. Pyridoxine
6. PABA
7. Folic acid
8. Pyridoxamine
9. Thiamine
10. Cyanocobalamin
Injection: 2 nmoL each
Figure 7
0 to 35 minutes
5 mM TBAOH
Figure 8
Water Soluble Vitamins by
Ion Suppression / Ion Pair Chromatography
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IonPac NS1 Manual Document No. 034024-06 Page 23 of 35
4.2.3 Separation of Benzoic Acid and o-nitrobenzoic Acid
1
BEFORE AFTER
4
Peaks
1. p-Aminobenzoic acid
2. o-Nitrobenzoic acid
3. Benzoic acid
4. p-Nitrobenzoic acid
2, 3
1
4
2
048
12 16
Separation of Benzoic Acid and o-Nitrobenzoic Acid
Using Ion Suppression Chromatography
Eluent: 50 mM HCl / 32% Acetonitrile
Detection: UV 254 nm, 0.1 AUFS
Figure 9
3
04812
Eluent: 3 mM HCl / 32% Acetonitrile
Detection: UV 254 nm, 0.1 AUFS
16
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IonPac NS1 Manual Document No. 034024-06 Page 24 of 35
4.2.4 Gradient Separation of Aliphatic Carboxylic Acids
Ion-suppression chromatography uses an acidic eluent that suppresses ionization of the analytes, allowing separation of weak
acids by using a reversed-phase column such as the IonPac NS1. The AMMS-ICE II suppressor regenerant for this ion suppression
application is potassium hydroxide.
Column: IonPac NS1
Flow Rate: 1.0 mL/min
Temperature: 42 °C
Detection: Suppressed Conductivity
Regenerant: 2.5 mM Potassium hydroxide
Eluent 1: 24% ACN, 6% MeOH, 0.03 mM HCl
Eluent 2: 60% ACN, 24% MeOH, 0.05 mM HCl
Gradient: 0-100% E2 in 20 min.
AMMS-ICE II Suppressor
1
ICE
Region
7
2
6
5
3
4
8
9
12
11
10
13
14
Peaks
1. Butyric
2. Pentanoic
3. Hexanoic
4. Heptanoic
5. Octanoic
6. Nonanoic
7. Decanoic
8. Dodecanoic
9. Tetradecanoic
10. Linolenic
11. Linoleic
12. Hexadecanoic
13. Oleic
14. Octadecanoic
Gradient Separation of Aliphatic Carboxylic Acids
Baseline
20040
Minutes
Figure 10
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IonPac NS1 Manual Document No. 034024-06 Page 25 of 35
SECTION 5 - TROUBLESHOOTING
The purpose of the Troubleshooting Guide is to help you solve operating problems that may arise while using IonPac NS1
columns. For more information on problems that originate with the Ion Chromatograph (IC) or the suppressor, refer to the
Troubleshooting Guide in the appropriate operator’s manual. If you cannot solve the problem on your own, contact the Dionex
North America Technical Call Center at 1-800-DIONEX-0 (1-800-346-6390).
Table 4
NS1/NG1 Troubleshooting Summary
Observation Cause Action Reference Section
High Back Pressure
Plugged Column Bed Supports Replace Bed Supports 5.1.2
Other System Modules Disconnect, Replace System Module Manual
High Background
Conductivity
Contaminated Columns Clean Column 5.2.2, 5.2.3
Contaminated ASRS or AMMS Clean Suppressor 5.2.5
Contaminated Hardware Clean Component
Poor Resolution
Column Headspace Replace Column 5.3.1.B
Short Retention Times
Flow Rate Too fast Recalibrate Pump 5.3.2.A
Bad Eluents Remake Eluents 5.3.2.B
Column Contamination Clean Column 5.3.2.C, 5.3.2.D
Poor Front End Resolution
Column Overloading Reduce Sample Size 5.3.3.B, 2.2
Sluggish Injection Valve Service Valve 5.3.3.C
Large Sy stem V oid Volumes Replum b S ystem 5.3.3.D
Spurious Peaks
Sluggish Injection Valve Service Valve 5.4.C
Unknown Isolate Blocked
Component
Bad Eluents Remake Eluents 5.2, 5.2.1
Poor Efficiency Due to Large
System Void Volumes
Unequilibrated System Lengthen First Eluent
Bad Eluents Remake Eluents 5.3.3.A
Sample Contamination Pretreat Samples 5.4.A, 5.4.B
Replumb System 5.3.1.A, 5.3.3.D
Time before Inject
5.1.1
5.3.3.C
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IonPac NS1 Manual Document No. 034024-06 Page 26 of 35
5.1 High Back Pressure
5.1.1 Finding the Source of High System Pressure
Total system pressure when using the IonPac NG1 Guard and IonPac NS1 analytical columns at 1.0 mL/min should be less than
1,600 psi (11.03 MPa) when using the eluent used to generate the test chromatogram. Total system pressure when using the IonPac
NG1 Guard and IonPac NS1 analytical columns at 2.0 mL/min should also be less than 1,600 psi (11.03 MPa) when using the eluent
used to generate the test chromatogram. If the system pressure is higher than 1,600 psi (11.03 MPa), it is advisable to find out what
is causing the high system pressure.
The system should be used with a High-Pressure In-Line Filter (P/N 035331) for eluents. The filter should be positioned
between the gradient pump pressure transducer and the injection valve. Since the liquid lines on the gradient pump have 10-32
ferrule/bolt fittings and the High-Pressure In-Line Filter has one male 1/4-28 fitting and one female 1/4-28 port, it is necessary
to install two adaptor assemblies on the filter. On the end of the filter with the 1/4-28 male fitting, place a 1/4-28 to 10-32 union
(P/N 042806). On the end of the filter with the 1/4-28 female port place a 1/4-28 male to 10-32 female port adaptor assembly (P/
N 043291). Make sure you have a High-Pressure In-Line Filter in place and that it is not contaminated.
A. Make sure that the pump is set to the correct eluent flow rate. Higher than recommended eluent flow rates will cause
higher pressure. Measure the pump flow rate if necessary with a stop watch and graduated cylinder.
B. Find out what part of the system is causing the high pressure. It could be a piece of tubing that has plugged or whose
walls are collapsed, an injection valve with a plugged port, a column with particulates plugging the bed support, a
plugged High-Pressure In-Line Filter, the suppressor, or the detector cell.
To find out which part of the chromatographic system is causing the problem, disconnect the pump eluent line from the injection
valve and turn the pump on. Watch the pressure; it should not exceed 50 psi (0.34 MPa). Continue adding the system’s components
(injection valve, column(s), suppressor, and detector) one by one, while watching the system pressure. The pressure should increase
up to a maximum of 1,500 psi (10.34 MPa) at a flow rate of 2.0 mL/min when the column(s) are connected. The suppressor may
add up to 100 psi (0.69 MPa). No other components should add more than 100 psi (0.69 MPa) of pressure. Refer to the appropriate
manual for cleanup or replacement of the problem component.
Table 5
Typical NS1/NG1 Operating Back Pressures
Standard
Column Typical Back Pressure Flow Rate
psi (MPa) mL/min
IonPac NS1 analytical 900 (6.21) - 1,400 (9.66) 1.0
IonPac NG1 guard 200 (1.38) - 300 (2.07) 1.0
IonPac NS1 + NG1 columns 1,100 (7.59) - 1,700 (11.73) 1.0
IonPac NS1-5 µm analytical 1,700 (11.73) - 2,500 (17.24) 1.0
IonPac NG1 guard 200 (1.38) - 300 (2.07) 1.0
IonPac NS1-5 µm + NG1 columns 1,900 (13.11) - 2,800 (19.31) 1.0
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IonPac NS1 Manual Document No. 034024-06 Page 27 of 35
5.1.2 Replacing Column Bed Support Assemblies
If the column inlet bed support is determined to be the cause of the high back pressure, it should be replaced. To change
the inlet bed support assembly, refer to the following instructions, using one of the two spare inlet bed support assemblies included
in the Ship Kit.
A. Disconnect the column from the system.
B. Carefully unscrew the inlet (top) column fitting using two open end wrenches.
C. Remove the old bed support. Turn the end fitting over and tap it against a benchtop or other hard, flat surface to
remove the bed support and seal assembly. If the bed support must be pried out of the end fitting, use a sharp pointed
object such as a pair of tweezers, but be careful that you DO NOT SCRATCH THE WALLS OF THE END
FITTING. Discard the old bed support assembly.
D. Place a new bed support assembly into the end fitting. Make sure that the end of the column tube is clean and free
of any particulate matter so that it will properly seal against the bed support assembly. Use the end of the column to
carefully start the bed support assembly into the end fitting.
IonPac NS1 Column
4-mm
Part (P/N)
Analytical Column (10µm) 035321
Analytical Column (5µm) 039568
Guard Column 039567
Bed Support Assembly 042955
End Fitting 052809
CAUTION
If the column tube end is not clean when inserted into the end fitting, particulate matter may obstruct a proper
seal between the end of the column tube and the bed support assembly. If this is the case, additional tightening
may not seal the column but instead damage the column tube or the end fitting.
E. Screw the end fitting back onto the column. Tighten it fingertight, then an additional 1/4 turn (25 in. x lb). Tighten
further only if leaks are observed.
F. Reconnect the column to the system and resume operation.
NOTE
Replace the outlet bed support ONLY if high pressure persists after replacement of the inlet fitting.
5.2 High Background or Noise
5.2.1 Preparation of Eluents
A. Make sure that all eluents and regenerants are made correctly. Were the proper precautions taken to prepare the
sodium hydroxide eluent? If carbonate was present in the eluent, the Anion Trap column will eventually be spent and
the background level will increase.
B. Make sure that the eluents are made from chemicals with the recommended purity.
C. Make sure that the deionized water used to prepare the reagents has a specific resistance of 18.2 megohm-cm.
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IonPac NS1 Manual Document No. 034024-06 Page 28 of 35
5.2.2 Contaminated Anion Trap Column, the ATC-3 (4-mm)
When doing gradient analysis, ensure that the Anion Trap Column, the ATC-3 (2-mm) or the ATC-3 (4-mm) has been
installed correctly. If it has not, install one as directed in Section 2.3, “The Anion Trap Column,” and watch the background
conductivity. If the background conductivity is now low, this means that the ATC is trapping contaminants from the eluent. The
eluents probably have too many impurities (see items A–C above).
Determine if the ATC is the source of high background conductivity. Remove the ATC. If the background conductivity remains
high, then the ATC is not the problem. If the background conductivity decreases, the ATC is the source of the high background
conductivity.
A. Disconnect either the ATC-3 (4-mm) from the injection valve and direct the outlet to waste.
B. Flush the ATC with 200 mL of 35 mM NaOH or the highest NaOH concentration used in the application. Use
a flow rate of 2.0 mL/min on a 4-mm system.
C. Equilibrate the ATC with the strongest eluent used during the gradient run. Use a flow rate of 2.0 mL/min on a
4-mm system.
D. If the problem persists, replace the ATC.
5.2.3 Contaminated Guard or Analytical Column
Remove the IonPac NG1 Guard and IonPac NS1 analytical columns from the system. If the background conductivity decreases,
then one (or both) of these columns is (or are) the cause of the high background conductivity, clean the column as instructed in
Appendix B - Column Care.
5.2.4 Contaminated Hardware
To eliminate the hardware as the source of the high background conductivity, bypass the SRS suppressor and pump deionized water
with a specific resistance of 18.2 megohm-cm through the system. The background conductivity should be less than 2 µS. If it is
not, check the detector/conductivity cell calibration by injecting deionized water directly into it. See the appropriate manual for
details.
5.2.5 Contaminated Suppressor
Assume that the SRS or the AMMS-ICE is causing the problem if the above items have been checked and the problem
persists.
A. Check the regenerant flow rate at the REGEN OUT port of the SRS or the AMMS-ICE. For the example isocratic
applications, this flow rate should be 3–5 mL/min in the Chemical Suppression Mode of operation.
B. Check the eluent flow rate. For most applications, the eluent flow rate for 2-mm applications should be 0.50 mL/min
and for 4-mm applications, it should be 2.0 mL/min. Refer to the Self-Regenerating Suppressor Product Manual
(Document No. 031956) or the Anion-II MicroMembrane Suppressor-ICE Product Manual (Document No. 032661)
to ensure that the eluent is within suppressible limits.
C. Test both the suppressor and the Anion AutoRegen Regenerant Cartridge for contamination if you are using an
AutoRegen Accessory with the ASRS ULTRA II (in the MPIC or Chemical Suppression Mode) or the CSRS (in the
Chemical Suppression Mode). Prepare fresh regenerant solution.
1. Clean or replace your SRS or AMMS-ICE if the background conductivity is high after preparing fresh
regenerant and bypassing the Anion AutoRegen Regenerant Cartridge. Refer to the “Self-Regenerating
Suppressor Product Manual” (Document No. 031956) or the “Anion-ICE MicroMembrane Suppressor II Product
Manual” (Document No. 032661) for assistance.
Page 29
IonPac NS1 Manual Document No. 034024-06 Page 29 of 35
2. Test the Anion AutoRegen Regenerant Cartridge to see if it is expended. If the background conductivity is low
when freshly prepared regenerant is run through the ASRS or CSRS without an AutoRegen Accessory in-line then
the cartridge is expended. Connect a freshly prepared regenerant to the Anion AutoRegen Regenerant Cartridge.
Pump approximately 200 mL of regenerant through the Anion AutoRegen Regenerant Cartridge to waste before
recycling the regenerant back to the regenerant reservoir.
If the background conductivity is high after placing the AutoRegen Accessory in-line, you probably need to replace
the Anion AutoRegen Regenerant Cartridge (P/N 039564). Refer to the “AutoRegen Regenerant Cartridge Refill
Product Manual” (Document No. 032852) for assistance.
5.3 Poor Peak Resolution
Poor peak resolution can be due any or all of the following factors.
5.3.1 Loss of Column Efficiency
A. Ensure that system void volumes have been minimized. Extra-column system effects can result in sample band
dispersion and decreasing peak efficiencies. Make sure you are using PEEK tubing with an i.d. of no greater than 0.010"
to make all eluent liquid line connections between the injection valve and the detector cell inlet on 4-mm systems. Make
all tubing lengths are as short as possible. Check for leaks.
B. Check to see if headspace has developed in the guard or analytical column (e.g., due to improper use of the column
such as submitting it to high pressures). Remove the column’s top end fitting (see Section 5.1.2, “Replacing Column
Bed Support Assemblies”). If the resin does not fill the column body all the way to the top, it means that the resin bed
has collapsed, creating a headspace. The column must be replaced.
5.3.2 Poor Resolution Due to Shortened Retention Times
Even with adequate system and column efficiency, resolution of peaks will be compromised if analytes elute too fast.
A. Check the eluent flow rate. If it is different than the flow rate specified by the analytical protocol, recalibrate the pump.
Measure the eluent flow rate after the column using a stopwatch and graduated cylinder.
B. Check to see if the eluent compositions and concentrations are correct.
For isocratic analysis, an eluent that is too strong will cause the peaks to elute faster. Prepare fresh eluent. If you are
using a gradient pump to proportion the final eluent from concentrated eluents in two or three different eluent reservoirs,
the composition of the final eluent may not be accurate enough for the application. Use one reservoir containing the
correct eluent composition to see if this is the problem. This may be a problem when one of the proportioned eluents
is less than 5%.
For gradient analysis, remake the eluents or adjust the times in the gradient program to obtain the required peak
resolutions.
C. Column contamination can lead to a loss of column capacity because fewer of the anion exchange sites will be
available for the sample ions. Polyvalent anions or metal ions might be concentrating on the column. Refer to Appendix
B - Column Care, for recommended column cleanup procedures.
Possible sources of column contamination are impurities in chemicals, in the deionized water or from the sample matrix
being used. Be especially careful to make sure that the recommended chemicals are used. The deionized water should
have a specific resistance of at least 18.2 megohm-cm .
D. Diluting the eluent will improve peak resolution, but will also increase the analytes’ retention times. If a 10% dilution
of the eluent is not sufficient to obtain the desired peak resolution, or if the resulting increase in retention times is
unacceptable, clean the column (“Appendix B - Column Care”).
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IonPac NS1 Manual Document No. 034024-06 Page 30 of 35
After cleaning the column, reinstall it in the system and let it equilibrate with eluent for about 30 minutes. The column
is equilibrated when consecutive injections of the standard give reproducible retention times. The original column
capacity should be restored by this treatment, since the contaminants should be eluted from the column. If you need
assistance in solving resolution problems, contact the nearest Dionex Office.
5.3.3 Loss of Front End Resolution
If poor resolutions and efficiencies are observed for the very early eluting peaks near the system void volume
compared to the later eluting peaks, check the following:
A. Improper eluent concentration may be the problem. Remake the eluent as required for your application. Ensure that
the water and chemicals used are of the required purity.
B. Column overloading may be the problem. Reduce the amount of sample ions being injected onto the analytical
column by either diluting the sample or injecting a smaller volume onto the column.
C. The column may not be equilibrated to the first eluent. Increase the amount of time that the first eluent runs through
the columns before injection.
D. Sluggish operation of the injection valve may be the problem. Check the air pressure and make sure there are no
gas leaks or partially plugged port faces. Refer to the valve manual for instructions.
E. Improperly swept out volumes anywhere in the system prior to the guard and analytical columns may be the
problem. Swap components, one at a time, in the system prior to the analytical column and test for front-end resolution
after every system change.
5.4 Spurious Peaks
A. The column may be contaminated. If the samples contain an appreciable level of polyvalent ions and the column is
used with a weak eluent system, polyvalent anions may contaminate the analytical column. The retention times for the
analytes will then decrease and spurious, inefficient (broad) peaks can show up at unexpected times. Clean the column
as indicated in “Appendix B - Column Care.”
B. If you need assistance in determining the best way to clean strongly retained solutes in your specific sample matrix from
the IonPac NS1 columns, contact the nearest Dionex Office.
C. The injection valve may be creating a baseline disturbance. This baseline upset can show up as a peak of varying
size and shape. It will happen when the injection valve needs to be cleaned or retorqued (see valve manual). Check to
see that there are no restrictions in the tubing connected to the valve. Also check the valve port faces for blockage and
replace them if necessary. Refer to the Valve Manual for troubleshooting and service procedures. Small baseline
disturbances at the beginning or at the end of the chromatogram can be overlooked as long as they do not interfere with
the quantification of the peaks of interest.
If cleaning and retorquing the valve does not help, replace the valve. Use a Dionex High Pressure Injection Valve (P/
N 037142) or a Dionex High Pressure Inert Valve (P/N 037143) as required.
For DX-300 systems equipped with a Rheodyne Microinjection Valve, Model 9126 (Dionex P/N 044697), consult the
accompanying manual for service instructions. See Section 2.2, “The Injection Loop,” for injection valve and loop
requirements for 4-mm operation.
Page 31
IonPac NS1 Manual Document No. 034024-06 Page 31 of 35
5.5 Small Analyte Peak Areas
Assuming that the suppressor is the cause of this problem, small analyte peak areas are a result of running eluent through the
suppressor with the power off while using the ASRS ULTRA II in the MPIC Suppression Mode or the CSRS ULTRA II in the
AutoSuppression External Water Mode. The problem may also occur in any suppressor while using it in the Chemical Suppression
Mode by not running regenerant through the suppressor chambers.
A. Disconnect the eluent line from the analytical column attached to the ELUENT IN port of the Self-Regenerating
Suppressor (SRS) at the analytical column end of the line. Direct this line to a separate waste beaker.
B. Disconnect the eluent line from the ELUENT OUT port of the Self-Regenerating Suppressor (SRS) to the detector
conductivity cell at the suppressor end of the line.
C. Install a 10-32 Luer adaptor fitting with a plastic syringe in the ELUENT OUT port of the Self-Regenerating
Suppressor (SRS) and inject 5 mL of 0.5 N H2SO4 through the ASRS ULTRA II or 5 mL of 0.5 M NaOH in the reverse
direction to normal flow so that the waste comes out of the ELUENT IN port.
D. Reconnect the eluent lines from the ELUENT IN port of the Self-Regenerating Suppressor (SRS) to the analytical
column and from the ELUENT OUT port of the Self-Regenerating Suppressor (SRS) to the conductivity detector cell
line.
E. Establish the regenerant flow through the suppressor, turn on the power and begin pumping eluent.
NOTE
If you are operating in the Chemical Suppression Mode, all you have to do is reestablish acid or base regenerant
flow through the suppressor and begin pumping eluent. Allow the system to equilibrate before beginning analysis.
Power is not used in this mode of operation.
Page 32
IonPac® NS1 10µ
Analytical (4 x 250 mm)
Product No. 035321
Serial No. : 6139 Pressure (PSI) : 1250 Date : 8/1/00 7:09:11 AM
6139
4.00
3.00
3
2.00
1
2
4
µS
1.00
0
-1.00
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0
Peak Information : Found Components
Peak
No.
Retention
Time
Name
Eluent:
28% Acetonitrile
3.0 mM Tetrabutylammonium hydroxide
Flow Rate:
Detection:
AutoSuppression
1.0 mL/min
Suppressed Conductivity
ASRS®-ULTRA
®
External Water Mode
with 10mN Sulfuric Acid
Range:
Background Conductivity:
Injection Volume:
(mg/L)
10 µSFS
Efficiency
3-15 µS
10 µL
Asymmetry
(10%)
Resolution
1
2
3
4
File Name : C:\PEAKNET\DATA\EXAMPLES\35321 NS1 10U 4MM_A012.DXD
4.42
7.05
9.98
12.90
Propanesulfonate
Iodide
Thiocyanate (SCN)
Hexanesulfonate
15.0
15.0
15.0
50.0
4057
6447
5278
4835
1.0
1.2
0.9
1.2
8.39
6.52
4.53
n/a
Page 33
IonPac® NS1 5µ
Analytical (4 x 150 mm)
Product No. 039568
Serial No. : #10 Pressure (PSI) : 1930 Date : 2/21/01 9:13:47 AM
#10
5.00
4.00
3
3.00
2.00
µS
1
2
4
1.00
0
-1.00
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Peak Information : Found Components
Peak
No.
Retention
Time
Name
Eluent:
26/% Acetonitrile
4.0 mM Tetrabutylammonium hydroxide
Flow Rate:
Detection:
AutoSuppression
1.0 mL/min
Suppressed Conductivity
ASRS®-ULTRA
®
External Water Mode
with 10 mN Sulfuric Acid
Range:
Background Conductivity:
Injection Volume:
(mg/L)
10 µSFS
Efficiency
3-15 µS
10 µL
Asymmetry
(10%)
Resolution
1
2
3
4
File Name : C:\PEAKNET\DATA\EXAMPLES\39568 NS1 5U 4MM_A004.DXD
3.02
5.07
7.48
10.40
Propanesulfonate
Iodide
Thiocyanate
Hexanesulfonate
15.0
15.0
15.0
50.0
3704
4921
5386
4223
1.6
1.5
1.6
1.8
8.44
6.96
5.58
n/a
Page 34
IonPac NS1 Manual Document No. 034024-06 Page 34 of 35
APPENDIX B - COLUMN CARE
B.1 Recommended Operation Pressures
The maximum recommended operating pressure for IonPac NS1 columns is 4,000 psi (27.57 MPa). Operating a column above
its recommended pressure limit can cause irreversible loss of column performance.
B.2 Column Start-Up
The IonPac NS1-5µm is tested with, and shipped in, 26% acetonitrile/4.0 mM tetrabutylammonium hydroxide. The IonPac NS110µm is tested with, and shipped in, 28% acetonitrile/3.0 mM tetrabutylammonium hydroxide. The column should be thoroughly
washed with 80% acetonitrile/deionized water until the background is below 1 µS before switching to other eluents.
Prepare the eluent shown on the test chromatogram, install the column in the chromatography module and test the column
performance under the conditions described in the test chromatogram. Continue making injections of the test standard until
consecutive injections of the standard give reproducible retention times. Equilibration is complete when consecutive injections
of the standard give reproducible retention times.
B.3 Column Storage
For short-term storage, the strongest eluent in use can be used as the storage solution.
For long-term storage, the strongest eluent in use should be used as the storage solution. Flush the column for a minimum of 10
minutes with the storage solution. Cap both ends securely, using the plugs supplied with the column.
B.4 Column Cleanup
The following column cleanup protocols have been divided into three general isocratic protocols to remove acid-soluble, basesoluble or organic contaminants. They can be combined into one gradient protocol if desired but the following precautions should
be observed.
Always ensure that the cleanup protocol used does not switch between eluents which may create high pressure eluent interface
zones in the column. High pressure zones can disrupt the uniformity of the packing of the column bed and irreversibly damage
the performance of the column. High pressure zones in the column can be created by pumping successive eluents through the
column that are not miscible, that have eluent components in one eluent that will precipitate out in the other eluent or by using
an acid eluent followed by a base eluent with may create a neutralization pressure band. The precipitation of the salts in solvents
during column rinses can result in very high pressure zones. High viscosity mixing zones can be created between two eluents
having solvents with a very high energy of mixing.
When in doubt, always include short column rinse steps to reduce the solvent content of the eluent to 5% levels and the ionic
strength of the eluent to 50 mM levels to avoid creating high pressure zones in the column that may disrupt the uniformity of the
column packing.
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IonPac NS1 Manual Document No. 034024-06 Page 35 of 35
B.4.1 Choosing the Appropriate Cleanup Solution
A. Organic solvents can be used alone if the contamination is nonionic and hydrophobic. The degree of nonpolar character
of the solvent should be increased as the degree of hydrophobicity of the contamination within the range of acceptable
solvents listed in Table 4, HPLC Solvents for Use with IonPac NS1 Columns.
B. Concentrated acid solutions such as 1 to 3 M HCl can be used with compatible organic solvents to remove
contamination that is ionic and organic. The acid suppresses ionization and ion exchange interactions of the
contamination with the resin. The organic solvent then removes the subsequent nonionic and hydrophobic contamination.
See Section B.4 above.
A frequently used cleanup solution is 200 mM HCl in 80% acetonitrile. This solution must be made immediately before
use because the acetonitrile will decompose in the acid solution during long term storage.
C. Regardless of the cleanup solution chosen, use the following cleanup procedure in Section B.6, “Column Cleanup
Procedure,” to clean the IonPac NG1 and IonPac NS1.
B.4.2 Column Cleanup Procedure
A. Prepare a 500 mL solution of the appropriate cleanup solution using the guidelines in Section B.4.1, “Choosing the
Appropriate Cleanup Solution.”
B. Disconnect the Suppressor from the IonPac NS1 analytical column. If your system is configured with both a guard
column and an analytical column, reverse the order of the guard and analytical column in the eluent flow path. Double
check that the eluent flows in the direction designated on each of the column labels. Direct the effluent from the outlet
line of the NG1 Guard Column to a separate waste container.
CAUTION
When cleaning an analytical column and a guard column in series, ensure that the guard column is placed after
the analytical column in the eluent flow path. Contaminants that have accumulated on the guard column can be
eluted onto the analytical column and irreversibly damage it. If in doubt, clean each column separately.
C. Set the pump flow rate to 0.50 mL/min (2-mm systems) or 2.0 mL/min (4-mm systems).
D. Pump the cleanup solution through the column for 60 minutes.
E. Reconnect the Suppressor to the IonPac NS1 analytical column. Place the guard column in line between the injection
valve and the analytical column if your system was originally configured with a guard column.
H. Equilibrate the column(s) with eluent before resuming normal operation.
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