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– Introduction

Thermo Scientific

Product Manual for CarboPac PA200 Columns

Page 7 of 37

065734-01

For Research Use Only. Not for use in diagnostic procedures.

Introduction

1.1

CarboPac PA200

The CarboPac PA200 column is the latest addition to the CarboPac family of columns for

carbohydrate separations. This column has been specially developed to provide high resolution

separations of charged and neutral oligosaccharides and is the recommended column for these

applications. The CarboPac PA200 columns are packed with a hydrophobic, polymeric, pellicular

anion exchange resin stable over the range of pH 0–14. This unique pH-stability of the packing

material allows the use of eluent compositions that are conducive to anodic oxidation of

carbohydrates at gold electrodes.

Resin Characteristics:

Particle Size:

5.5 µm

Pore

Size:microporous (< 10 Å)

Cross-linking:

55%

Ion exchange capacity (1mm):

4.0 µeq per column

Ion exchange capacity (0.4mm):

0.64 µeq per column

Latex Characteristics:

Functional

Group:quaternary ammonium ion

Latex Diameter:

43 nm

Latex Cross-linking:

Typical Operating Parameters:

pH range:

0–14

Temperature Limit:

4–60 °C

Pressure Limit:

4500 psi

Organic Solvent Limit:

100% compatible

Typical eluents:

potassium hydroxide, potassium methane

sulfonate

Содержание CarboPac PA200

Страница 1: ...For Research Use Only Not for use in diagnostic procedures 6 CarboPac PA200 Columns For Dual EGC ICS 6000 065734 Revision 01 January 2018 User Manual ...

Страница 2: ...y Not for use in diagnostic procedures Product Manual For CP PA 200 Dual EGC ICS 6000 CarboPac PA200 Guard Column CarboPac PA 200 0 4 x 50 mm Item 302864 CarboPac PA 200 1 x 50 mm Item 302862 CarboPac PA200 Analytical Column CarboPac PA 200 0 4 x 250 mm Item 302863 CarboPac PA 200 1 x 250 mm Item 302861 ...

Страница 3: ...ation in this document is for reference purposes only System configurations and specifications in this document supersede all previous information received by the purchaser Thermo Fisher Scientific Inc makes no representations that this document is complete accurate or error free and assumes no responsibility and will not be liable for any errors omissions damage or loss that might result from any...

Страница 4: ...ury Indicates a potentially hazardous situation which if not avoided could result in damage to equipment Indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury Also used to identify a situation or practice that may seriously damage the instrument but will not cause injury Indicates information of general interest IMPORTANT Highlights information nec...

Страница 5: ...ystem Start up 11 2 6 1 ICS 6000 Dual EG Start Up Procedure for CarboPac PA200 1 mm Column 12 2 6 2 ICS 6000 Dual EG Start Up Procedure for CarboPac PA200 0 4 mm Column 13 2 6 3 Verification of Column Cleanliness 15 3 Operation 16 3 1 Dionex CarboPac PA200 column Operational Parameters 16 3 1 1 The Best Operational Guidelines 16 3 1 2 Initial Check List 16 3 2 Purity Requirements and Preparation o...

Страница 6: ...y 29 5 3 2 Shortened Retention Times 30 5 3 3 Loss of Resolution for early eluting peaks 31 5 3 4 Spurious Peaks 31 5 3 5 No Peaks Poor Peak Area Reproducibility or too Small Peak Areas 31 5 3 6 Large Baseline Dip in the Chromatogram 32 5 3 7 Unidentified Peaks Appear with Expected Analyte Peaks 32 5 3 8 Decreased Detection Sensitivity 32 5 3 9 Excessive Gradient Rise 33 5 4 Reconditioning or Repl...

Страница 7: ...drophobic polymeric pellicular anion exchange resin stable over the range of pH 0 14 This unique pH stability of the packing material allows the use of eluent compositions that are conducive to anodic oxidation of carbohydrates at gold electrodes Resin Characteristics Particle Size 5 5 µm Pore Size microporous 10 Å Cross linking 55 Ion exchange capacity 1mm 4 0 µeq per column Ion exchange capacity...

Страница 8: ...igh pressure applications the use of high pressure fittings is recommended 2 1 The Dionex High Pressure Ion Chromatography Systems A minimum of a Dionex High Pressure Ion Chromatography System HPIC is recommended due to the higher backpressures generated at typical operational flow rates Systems should have the capability to operate up to at least 5000 psi Standard IC systems with an upper limit o...

Страница 9: ... below in Figure 1 the flow diagram for a capillary system is shown in Figure 2 Figure 1 Plumbing Schematic for Electrolytic Generation of Potassium Methanesulfonate Eluents Using a Dionex EGC 400 KOH and Dionex EGC 400 MSA Cartridge in an RFIC EG System EGC 400 MSA Cartridge Pump H2O EGC 400 MSA Degasser Injector Guard Column Analytical Column Vacuum Pump KEY 1 Degas Eluent In 2 Degas Eluent Out ...

Страница 10: ... wear gloves when handling electrodes Never touch the electrode surface To install a disposable working electrode and reference electrode pH Ag AgCl or PdH refer to Product Manual for Disposable Electrodes Doc No 065040 ICS 5000 Ion Chromatography System Manual Doc No 065342 and User s Compendium for Electrochemical Detection Doc No 065340 In addition the quadruple waveform must be used for carboh...

Страница 11: ...he Dionex CarboPac PA200 Analytical Column Retention times will increase by approximately 20 when a guard column is placed in line before the analytical column under isocratic conditions A guard column is utilized to prevent sample contaminants from eluting onto the analytical column It is easier to clean or replace a guard column than an analytical column Replacing the Dionex CarboPac PA200 Guard...

Страница 12: ... 400 MSA cartridge for 15 minutes at the flow rate of 1 mL min prime mode F Connect the OUTLET port of the Dionex EGC 400 MSA cartridge to a PEEK backpressure tubing Item 22181 20031 Direct the backpressure tubing to waste G Set the pump flow rate to 0 1 mL min H On the E Panel under Eluent Generator Tab set the Eluent Polarity to Acidic Eluents Set MSAprog to 100 mM Turn on the Dionex EGC power I...

Страница 13: ...lumn to waste EE With EGC power OFF turn on the pump and pump DI water at the flow rate of 0 063 mL min for 15 min FF With Eluent Polarity set to Basic Eluents set KMSAprog and KOHprog to the desired concentrations for the applications Turn on the Dionex EGC power for 30 min GG Connect the OUTLET of the column to the CELL IN of the ED cell and direct the CELL OUT to waste HH Confirm that the pH is...

Страница 14: ...ity to Basic Eluents Set KOHprog to 100 mM Turn on the Dionex EGC power T Condition the Dionex EGC KOH Capillary for 30 min U On the E Panel turn the Dionex EGC power OFF and keep the pump running at 0 02 mL min for 10 min V Turn off the pump W Remove the PEEK backpressure tubing from the EGC OUT port of the Dionex EGC KOH Capillary cartridge X Connect the EGC OUT port of the Dionex EGC KOH Capill...

Страница 15: ...in 2 6 3 Verification of Column Cleanliness Once the columns are equilibrated inject a system suitability standard such as the column s QAR standard to establish the performance of the column at start up This chromatogram can then be referred to when troubleshooting your system Once you obtain the expected chromatographic performance you are ready to proceed to running your application Thermo Scie...

Страница 16: ...inutes 3 1 1 2 NEVER Proceed to a next installation step if the previous step has failed Start an installation with any of the check list items below missing Use communal filtration units or filters made of unknown or unsuitable e g cellulose derivatives materials Use MeOH or other organic solvents as rinse fluid in the autosampler Use only water replaced daily or use sampler wash bottles blankete...

Страница 17: ... mm Hg that does not deform the plastic eluent container Degas the reservoir for 5 10 minutes while sonicating Cap each bottle and minimize the length of time the bottle is opened to the atmosphere Vacuum filtration through 0 2 μm Nylon filters Fisher Cat 0974046 is a good alternative to vacuum degassing under sonication and is sufficient for the majority of cases On line eluent and EG produced el...

Страница 18: ...operated non stop for months at a time If the system has been shut down for more than 1 2 days the system should be re started using the following procedure applied to both analytical and capillary systems 3 5 1 System Re Start Procedure for Dionex CarboPac PA200 1 mm Column A B C Set the flow rate to 0 1 mL min Turn the pump and EGC power on With Eluent Polarity set to Basic Eluents set KMSAprog ...

Страница 19: ...t degasser to waste H Set the flow rate to 0 02 mL min and turn the pump on for 10 min Keep EGC power off I Turn the pump off J Connect the ELUENT OUT of the Dionex RFIC Capillary Eluent Degasser Item 22181 60202 to 2 port of the injection valve K Install the CarboPac PA200 0 4 x 50 mm guard column and the CarboPac PA200 0 4 x 250 mm separator column to 3 port of the injection valve Direct the OUT...

Страница 20: ...nkage isomerism The disialylated biantennary peaks are eluted before the trisialylated triantennary peaks which are eluted before the tetrasialylated tetraantennary peaks In addition within each grouping the 2 6 isomer is eluted before and well resolved from the 2 3 isomer The Dionex OligoStandard Sialylalted N Linked Alditols Item 043164 contains 25 nmol oligosaccharides purified from bovine fetu...

Страница 21: ... 80 min 15 mM KMSA in 136 mM KOH Flow Rate CarboPac PA200 3 mm 0 5 mL min CarboPac PA200 1 mm 0 063 mL min CarboPac PA200 0 4 mm 0 010 mL min Detection CarboPac PA200 3 mm PAD Au on PTFE 2 mil gasket Ag AgCl ref CarboPac PA200 1 mm PAD Au on PTFE 1 mil gasket Ag AgCl ref CarboPac PA200 0 4 mm PAD Au on PTFE 1 mil gasket Ag AgCl ref Samples 50 µmol L fetuin oligosaccharide alditol standard Figure 3...

Страница 22: ...m column is for comparison only to demonstrate the comparable performance of the CarboPac PA200 1 mm and 0 4 mm columns with the ease of use Columns CarboPac PA200 3 mm guard separator CarboPac PA200 1 mm guard separator CarboPac PA200 0 4 mm guard separator Gradient CarboPac PA200 3 mm 0 45 min 100 430 mM NaOAc in 100 mM NaOH 45 50 min 430 mM NaOAc in 100 mM NaOH 50 65 min 100 mM NaOAc in 100 mM ...

Страница 23: ...tions Thermo Scientific Product Manual for CarboPac PA200 Columns Page 23 of 37 065734 01 For Research Use Only Not for use in diagnostic procedures Figure 4 Inulin Profiles CarboPac PA200 3 mm vs 1 mm vs 0 4 mm ...

Страница 24: ...ith manually prepared sodium acetate sodium hydroxide eluents The illustration of the application on the CarboPac PA200 3 mm column is for comparison only to demonstrate the comparable performance of the CarboPac PA200 1 mm and 0 4 mm columns with the ease of use Columns CarboPac PA200 3 mm guard separator CarboPac PA200 1 mm guard separator CarboPac PA200 0 4 mm guard separator Gradient CarboPac ...

Страница 25: ... 0 45 0 50 0 55 0 60 0 65 0 70 0 100 50 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 min nC DP1 DP2 DP3 DP4 DP13 0 4 mm 0 0 5 0 10 0 15 0 20 0 25 0 30 0 35 0 40 0 45 0 50 0 55 0 60 0 20 0 21 0 22 0 23 0 24 0 25 0 26 0 27 0 28 0 29 0 30 0 31 0 32 0 33 0 34 0 35 0 36 0 37 0 min nC DP5 DP6 DP7 DP8 DP9 DP10 DP4 DP11 DP12 Wash step DP13 DP1 DP3 3 mm Zoomed in 0 0 5 0 10 0 15 0 20 0 25 0 30 ...

Страница 26: ...R for the column If the total system pressure is much higher than expected it is advisable to determine the cause of the high system pressure A Make sure that the pump is set to the correct eluent flow rate Higher than recommended eluent flow rates will cause higher pressure If necessary measure the pump flow rate by collecting the DI H2O eluent for a specified time at operating pressure and measu...

Страница 27: ...y D Place a new bed support assembly provided with each 1 mm column 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 Drop the bed support assembly into the end fitting making sure that the bed support assembly is centered at the bottom of the end fitting Wrap the end fitting gen...

Страница 28: ...m system the Dionex RFIC Capillary EGC MSA Degasser Item 22181 60211 and the Dionex RFIC Capillary Eluent Degasser Item 22181 60202 for the 0 4 mm system D Column contamination Remove the column set from the system first and replace it with a length of 0 001 i d PEEK tubing generating a pressure drop over 2000 psi If the background reading improves after the column is removed from the system go to...

Страница 29: ...entrations constant and adjust the gradient time This is the simplest way to compensate for total system differences if resolution is the problem B Change the initial and or final eluent concentration and adjust the gradient time This approach requires more time to develop and more knowledge in methods development work Its advantage is that it allows a method to be tailored for a particular applic...

Страница 30: ...n the deionized water used for eluents or components of the sample matrix Be especially careful to make sure that the recommended chemicals are used The deionized water should have a specific resistance of 18 2 megohm cm D Diluting the eluent will improve peak resolution but will also increase the retention times If a 10 dilution of the eluent is not sufficient to obtain the desired peak resolutio...

Страница 31: ...mn is used with a weak eluent system the retention times for the analytes will then decrease and spurious inefficient broad peaks may show up at unexpected times Clean the column as indicated in Column Care For assistance contact Technical Support for Dionex Products In the U S call 1 800 346 6390 Outside the U S call the nearest Thermo Fisher Scientific office B The injection valve may need maint...

Страница 32: ...he following solutions in the auto sampler and inject in sequence Use 25 µL full loop injections 1 1 M NaOH 2 Deionized water 3 IPA 4 Deionized water 5 1 M HCl 6 Deionized water 5 3 8 Decreased Detection Sensitivity Always confirm the loss of response by performing at least one injection of the system suitability standard mix as described in Section 4 1 This is to make sure that a decreased level ...

Страница 33: ...rd analysis This will keep the column conditioned free from carbonate buildup and ready for analysis A Make sure the gradient rise is not caused by the system and or detector cell B Set column temperature to 40 C and wash the guard and column with 100 mM KMSA 100 mM KOH for at least four hours preferably overnight Run a blank gradient at 30 C and if necessary repeat the cleanup with 100 mM KMSA 10...

Страница 34: ...ed in the QAR 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 If chromatographic efficiency or resolution is poorer than the QAR see Sections 5 3 Poor Resolution and Section 5 3 1 Loss of Column Efficiency IMPORTANT W...

Страница 35: ...umn 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 B Wash the Dionex CarboPac PA200 with deionized water 18 2 megohm cm for about 30 minutes and then clean with 200 mM MSA for one to...

Страница 36: ... Quality Assurance Report QAR Thermo Scientific Product Manual for CarboPac PA200 Columns Page 36 of 37 065734 01 For Research Use Only Not for use in diagnostic procedures Appendix B Quality Assurance Report QAR ...

Страница 37: ...Appendix B Quality Assurance Report QAR Thermo Scientific Product Manual for CarboPac PA200 Columns Page 37 of 37 065734 01 For Research Use Only Not for use in diagnostic procedures ...

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