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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.
3.3.2 Deionized Water ........................................................................................................................................................ 9
3.3.5 Base Modifiers......................................................................................................................................................... 10
3.4Preparing 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
3.5Regenerant Preparation for the Self-Regenerating Suppressors ......................................................................11
3.6Regenerant Preparation for the AMMS-ICE II ................................................................................................. 12
3.7Using 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
3.8Using Pressurized Regenerant Reservoir with the AMMS-ICE II ..................................................................13
IonPac NS1 ManualDocument No. 034024-06Page 3 of 35
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
4.2.1 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
5.1High Back Pressure ............................................................................................................................................... 26
5.1.1 Finding the Source of High System Pressure........................................................................................................... 26
5.1.2 Replacing Column Bed Support Assemblies ........................................................................................................... 27
5.2High 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
5.3.1 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
IonPac NS1 ManualDocument No. 034024-06Page 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
ColumnParticleSubstrate
DiameterX-linking
µm%
IonPac NS1 analytical column1055
4 x 250 mm
IonPac NG1 guard column1055
4 x 35 mm
IonPac NS1-5 µm analytical column555
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.
IonPac NS1 ManualDocument No. 034024-06Page 5 of 35
IonPac NS1 ManualDocument No. 034024-06Page 6 of 35
SECTION 2 - INSTALLATION
2.1System 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.
2.2The 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.
2.3The 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.
IonPac NS1 ManualDocument No. 034024-06Page 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.
2.4The 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.
2.5Eluent 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).
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-MPICor 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.
IonPac NS1 ManualDocument No. 034024-06Page 8 of 35
2.7Detector Requirements
See Section 2, “Ion Chromatography System Operation Summary,” for 4-mm system detector, cell and thermal stabilizer
requirements.
IonPac NS1 ManualDocument No. 034024-06Page 9 of 35
SECTION 3 - OPERATION
3.1General Operating Conditions
Sample Volume:4-mm: 10 µL Loop + 0.8 µL Injection valve dead volume
Column:4-mm: IonPacNS1 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
3.2IonPac 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)
3.3Chemical 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.
IonPac NS1 ManualDocument No. 034024-06Page 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.
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.
3.4Preparing 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
IonPac NS1 ManualDocument No. 034024-06Page 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)
3.5Regenerant 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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