Dionex IONPAC NG1 Manual de usuario

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)

©DIONEX Corporation, 1997-2004

Document No. 034024

Revision 06

15 January 2004

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.

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 ULTRAII in Chemical Suppression.............................................................................................................. 13

3.8 Using Pressurized Regenerant Reservoir with the AMMS-ICE II ..................................................................13

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 Test Chromatogram ............................................................................................................................... 15

4.1.2 Separation ofAnions by MPIC with Suppressed Conductivity Detection and Solvent........................................... 16

4.1.3 Separation ofAliphatic SulfonicAcids by Ion-Pairing with Suppressed Conductivity (MPIC).............................. 17

4.1.4 Separation ofAromatic SulfonicAcids Using Ion-Pairing with Suppressed Conductivity ..................................... 18

4.1.5 Separation ofAliphatic Quaternary Ammonium Ions Using Ion-Pairing with Suppressed Conductivity (MPIC) .. 19

4.1.6 Separation ofAlkanolamines 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 ofAliphatic 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 SupportAssemblies ........................................................................................................... 27

5.2 High Background or Noise ................................................................................................................................... 27

5.2.1 Preparation of Eluents.............................................................................................................................................. 27

5.2.2 ContaminatedAnion Trap Column, theATC-3 (4-mm)........................................................................................... 28

5.2.3 Contaminated Guard orAnalytical 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

IonPac NS1 Manual Document No. 034024-06 Page 4 of 35

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.

SECTION 1 - INTRODUCTION

The IonPac®NS1 (10-micron packing) and the IonPac®NS1-5µm (5-micron packing) analytical columns are polymer-based

reversed-phasecolumns fortheanalysis ofionicand nonpolarorganiccompounds. Thepackingmaterial isahighly 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

solventsbutalsoto the full pH range from0to14.Often organic analytes of interest tothechromatographerare 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

resolvingpower and greatersensitivity.The Anion ICEIIMicroMembrane Suppressor (AMMS®-ICEII)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

ionpairreagentandthecolumnpacking.Thisvarieswiththelengthofthehydrophobicportionoftheionpairreagent.Thesecond

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

tosuppresstetraalkylammoniumpairingreagents.Note:theAnionMicroMembraneSuppressor(AMMSIII)cannotbeusedwith

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

withtypicalorganicsolventsupto40%byvolumeusedinreversed-phaseion-pairchromatography.Theexternalwatermodemust

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.

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

IonPac NS1 Manual Document No. 034024-06 Page 6 of 35

SECTION 2 - INSTALLATION

2.1 System Requirements

TheIonPacNS1GuardandanalyticalcolumnscanberunonanyPEEKDionexChromatograph.Dependingupontheapplication,

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

gradientpumpconfiguredforstandardboreoperation.Forisocraticanalysesatflowratesbelow0.5mL/minandgradientanalyses,

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

GradientorstepchangeapplicationsforanionMPICrequireanIonPacAnionTrapColumn(ATC-3(4-mm),P/N037151);cation

MPIC applications require an IonPac Cation Trap Column (CTC-1 (4-mm), P/N 040192). The IonPac Trap Column should be

installedinplaceofthehighpressuregradientmixerbetweenthegradient pumpandtheinjectionvalve.TheIonPacTrapColumn

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. ConnectthegradientpumpdirectlytotheIonPacTrapColumn.Connectawastelinetothetrapcolumnoutletanddirect

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. ConnecttheIonPacTrapColumn,afterflushingitwitheluent,totheeluentlinethatisconnectedtotheinjectionvalve.

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. RegeneratetheATC-3ortheCTC-1.FordetailinformationontheoperationontheATC-3ortheCTC-1,seeDocument

No. 032697 for the ATC-3 and Document No. 034536 for the CTC-1.

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

approximately20%whenaguardcolumnisplacedin-linepriortotheanalyticalcolumn.Aguard is placed prior to the analytical

columntopreventsamplecontaminantsfromelutingontotheanalyticalcolumn.Itiseasiertocleanorreplaceaguardcolumnthan

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

TheAnionSelf-RegeneratingSuppressor(ASRSULTRAII)isusedforeluentsuppressionofMPIC(ion-pairing)eluentsbyusing

theMPICSuppressionModeofoperation.Thissuppressionmodeisacombination oftheAutoSuppressionExternalWaterMode

augmented with a chemical regenerant such as sulfuric acid (H2SO4). The MPIC Suppression Mode uses an applied current and

aconstantsourceofdilutesulfuricacidsolutiondeliveredfromanAutoRegenAccessoryorapressurizedbottlesystem.TheMPIC

SuppressionModereliablyprovidessuppressionoftypicaleluentsforMPICapplicationsusingsuppressedconductivitydetection.

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.

TheCation Self-RegeneratingSuppressor(CSRS ULTRAII)can beusedfor suppressionofMPIC (ion-pairing)eluentsby 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

chemicalregenerantifnecessarysuchasboricacid(H3BO3).WhentheCSRSULTRAIIisoperatinginthismode,itusesanapplied

currentandaconstantsourceof dilute boric acid solution delivered from a pressurized bottle system.Fordetailedinformationon

the operation of the CSRS ULTRA II, see Document No. 031956.

AnAnionorCationSelf-RegeneratingSuppressorshouldbeusedforionpairingapplicationsthatrequiresuppressedconductivity

detection. The ASRS ULTRA II in the MPIC Suppression Mode of operation and the CSRS ULTRA II in the AutoSuppression

ExternalWaterorMPICSuppressionModeofoperationarecompatiblewithaqueousioniceluentsofallconcentrationswithwhich

thecolumnandsystemarecompatibleandwithsolventcontainingeluentsupto40%byvolume.Forapplicationsrequiringsolvent

above40%byvolume,theChemicalSuppressionModeofoperationmustbeused.Aqueousioniceluentscanbeusedinallmodes

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,useanASRSULTRAII,4-mm,(P/N061561)forAnion-MPICortheCSRSULTRAII,4-mm,(P/N061563)forCation-

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 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.

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

ReagentGradeinorganicchemicalsshouldalwaysbeused 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 megohm-

cm.Thedeionizedwatershouldbefreeofionizedimpurities,organics,microorganismsandparticulatematterlargerthan0.2µm.

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

pumpedover thecolumn containsminimally1%solventtoensurethatthehydrophobicsurfacesare“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,solventsanddegassedwatershouldbepremixedinconcentrationsthatallowpropermixingbythegradientpump

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 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.Thesolvents used must be free ofionicimpurities.However, since most manufacturers ofsolventsdonottest for ionic

impurities,itisimportantthatthehighestgradeofsolventsavailablebeused.Currently,severalmanufacturersaremakingultrahigh

purity solvents that are compatible for HPLC and spectrophotometric applications. These ultrahigh purity solvents will usually

ensurethat yourchromatographyis notaffectedby ionicimpurities inthesolvent. AtDionex,we haveobtainedconsistentresults

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 water-

methanol 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 HNO3can 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.

NOTE

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.

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