Related Manuals for Dionex 063000
Summary of Contents for Dionex 063000
- Page 1 DNAPac PA200 Document No. 065036 Page 1 of 25 PRODUCT MANUAL DNAPAC PA200 ANALYTICAL COLUMN (4 x 250mm, P/N 063000) DNAPAC PA200 GUARD COLUMN (4 x 50mm, P/N 062998) ©DIONEX Corporation Document No. 065036 Revision 01 20 August 2004...
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Page 2: Table Of Contents
DNAPac PA200 Document No. 065036 Page 2 of 25 TABLE OF CONTENTS SECTION 1 - INTRODUCTION ........................4 DNAP PA200 ..........................4 LC S ) ....................4 YSTEM WITHOUT OLUMNS ........................5 UARD OLUMN DNAPAC PA200 A ................5 NION XCHANGE OLUMNS DNAP... - Page 3 DNAPac PA200 Document No. 065036 Page 3 of 25 Section 8 - TROUBLESHOOTING GUIDE ....................23 .............. 23 INDING THE OURCE OF YSTEM RESSURE ................23 ACKPRESSURE ON OLUMN NCREASED .................... 23 ECREASING ETENTION IMES ................. 23 ECREASING FFICIENCY AND ESOLUTION ..................
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Page 4: Section 1 - Introduction
DNAPac PA200 Document No. 065036 Page 4 of 25 SECTION 1 - INTRODUCTION 1.1 DNAPac PA200 The DNAPac PA200 is a pellicular anion exchange column designed specifically to provide high-resolution separations of single stranded nucleic acids. The DNAPac PA200 provides n, n-1 resolution over a wide range of oligomer lengths and can perform separations under a variety of denaturing conditions: •... -
Page 5: Guard Column Use
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 listed in “DIONEX Worldwide... -
Page 6: Section 2 - Operation And System Requirements
Oligonucleotide separations with the DNAPac PA200 columns are optimized for use with NON-METALLIC systems, such as the Dionex BioLC. The key issue is that the eluent flow path from reservoir to detector is metal- free, because the salts used for oligonucleotide elution attack the metallic components of metallic pumps and tubing. -
Page 7: Dnapac Pa200 Column Operational Parameters
DNAPac PA200 Document No. 065036 Page 7 of 25 2.3 DNAPAC PA200 Column Operational Parameters TABLE 2 Column Operational Parameters = 4-10 (unrestricted eluents) = 2.5- 4, and 10-12.5: Operation at these pH values require co-ion pH Range: concentration (e.g., Cl or ClO at high pH and Na or NH... -
Page 8: Section 3 - Purity Requirements For Chemicals
These “ultra high” purity solvents will usually be of sufficient purity to 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 or Optima Solvents by Fischer Scientific. -
Page 9: Section 4 - Quality Assurance
DNAPac PA200 Document No. 065036 Page 9 of 25 SECTION 4 - QUALITY ASSURANCE The chromatograms in this section were obtained using a calibrated system that meets the operational parameters listed in Section 2. Different systems will differ slightly in performance due to slight variations in column sets, system void volumes, liquid sweep-out times, different component volumes, and laboratory temperature. -
Page 10: Production Test Chromatograms
DNAPac PA200 Document No. 065036 Page 10 of 25 4.2 Production Test Chromatograms To guarantee that all DNAPac PA200 analytical columns meet high quality and reproducible performance specification standards, all columns undergo the following production control test. Because gradient separation is not an accurate test for determining column capacity and packing quality, an isocratic separation of seven inorganic anions is employed to measure individual column performance utilizing a sodium carbonate, bicarbonate eluent. -
Page 11: Section 5 - Methods Development
DNAPac PA200 Document No. 065036 Page 11 of 25 SECTION 5 - METHODS DEVELOPMENT 5.1 Sample Cleanup This table lists some sample preparation and matrix removal guidelines, for oligonucleotide samples, prior to injection onto the DNAPac PA200 column. TABLE 3 Sample Preparation and Matrix Removal Guidelines Matrix Effect Possible Removal... -
Page 12: Gradient Slope
DNAPac PA200 Document No. 065036 Page 12 of 25 5.4 Gradient slope Phosphodiester oligonucleotides generally exhibit good peak shape when the gradient slope is ~15 mM/mL (NaCl) or ~5 mM/mL (NaClO ). Higher values will generally result in shorter run times, but result in poorer resolution. Conversely lower values may produce improved resolution, but also require longer run times. -
Page 13: Effect Of Ph On Retention
DNAPac PA200 Document No. 065036 Page 13 of 25 5.5.2 Effect of pH on Retention The next chart illustrates the influence of pH on oligonucleotide retention. An oligonucleotide with base composition of G was eluted with a gradient of NaCl over 30 minutes at pH 6.5 to 12. Between pH 9 and 11, a substantial increase in retention is observed. -
Page 14: Effect Of Ph On Selectivity
DNAPac PA200 Document No. 065036 Page 14 of 25 5.5.4 Effect of pH on Selectivity The figure below illustrates the influence of pH on oligonucleotide selectivity. Here the elution patterns of two 23-base oligonucleotides differing only in their 5’ and 3’ terminal bases are compared between pH 9 and pH The top trace for each pair of chromatograms has an additional T at the 5’... -
Page 15: Effect Of Temperature On Oligonucleotide Retention
Elevated temperature is often used to limit or eliminate Watson-Crick, and poly-G hydrogen bonding within, and between oligonucleotides that have self-complementary sequences. NOTE: Dionex does NOT recommend combining the use of elevated temperatures with high pH elution systems. Such conditions will accelerate degradation of the DNAPac PA200 stationary phase. -
Page 16: Effect Of Terminal Base On Selectivity
DNAPac PA200 Document No. 065036 Page 16 of 25 5.7 Effect of Terminal Base on Selectivity 5.7.1 Selectivity in Sodium Chloride (NaCl) Gradients The influence of the 5’ and 3’ terminal base on retention is shown below for NaCl eluents. These examples consist of mixed-base oligonucleotide (ODN) 25 mers with identical sequence except for the 3’... - Page 17 DNAPac PA200 Document No. 065036 Page 17 of 25 5.7.2 Selectivity in Sodium Perchlorate (NaClO ) Gradients When NaCl eluent is replaced with NaClO (see below), the retention differences are less pronounced, and the effect of pH on retention is also reduced. However, all of the ODNs with 3’ base substitutions are again resolved, and those with 5’...
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Page 18: Application-Specific Mobile Phase Recommendations
DNAPac PA200 Document No. 065036 Page 18 of 25 5.8 Application-Specific Mobile Phase Recommendations From the observations detailed in the preceding sections, the following suggestions can be made: 5.8.1 Eluent Systems Minimizing Base-Specific Retention For synthetic ODNs where the goal is to evaluate purity, determine the coupling efficiency, or purify the full- length component from “n-1”... -
Page 19: Section 6 - Applications
DNAPac PA200 Document No. 065036 Page 19 of 25 SECTION 6 - APPLICATIONS 6.1 Denaturing Conditions for Control of Secondary Structure Single-stranded nucleic acids may contain inter-, and/or intra-, strand hydrogen bonding. Such interactions, if sufficiently strong, result in spurious peaks and a general inability to distinguish between the oligonucleotide components in the sample. -
Page 20: Phosphodiester Analysis
DNAPac PA200 Document No. 065036 Page 20 of 25 6.3 Phosphodiester Analysis 6.3.1 Sodium Perchlorate Eluent Systems The following separation represents a good starting guideline for developing sodium perchlorate (NaClO based methods for longer oligonucleotides. In this example, phosphorylated deoxycytosine oligomers, 19 – 24 bases long, were injected onto a DNAPac PA200 column and eluted according to the conditions listed below. -
Page 21: Sodium Chloride Eluent Systems
DNAPac PA200 Document No. 065036 Page 21 of 25 6.3.2 Sodium Chloride Eluent Systems The following separation represents a good starting guideline for developing sodium chloride (NaCl) based methods for longer oligonucleotides. In this example, deoxythymidine oligomers, 19 – 24 bases long, were injected onto a DNAPac PA200 column and eluted according to the conditions listed below. -
Page 22: Section 7 - Dnapac™ Pa200 Resources
Presented at the Tides 2004 conference in Las Vegas NV, April 25-29, 2004. Authors: J. R. Thayer, Victor Barreto, Christopher Pohl, Dionex Corporation, Sunnyvale, CA, USA b) Control of oligonucleotide resolution on a new strong anion-exchange column: Optimization using pH and eluent composition. -
Page 23: Document No
Metal fouling can cause increased backpressure, loss of efficiency, and changes in selectivity on DNAPac columns. To eliminate metal fouling, Dionex strongly recommends the use of inert “PEEK-based” pumping systems (e.g., Dionex GP50), PEEK tubing, and connected fittings. 8.3 Decreasing Peak Retention Times Eluent contaminants may be decreasing the capacity of the column. -
Page 24: Esolution
Verify that 0.01” ID or smaller tubing is installed for all connections between injector and detector (for Dionex tubing, black = 0.010”, red = 0.005”, and yellow = 0.003” ID. For Upchurch/Scivex tubing, yellow = 0.007” ID) c) Verify that the shortest possible length of 0.010”... -
Page 25: No Peaks, Poor Peak Area Reproducibility Or Unexpectedly Small Peak Area
DNAPac PA200 Document No. 065036 Page 25 of 25 8.10.2 No Peaks, Poor Peak Area Reproducibility or Unexpectedly Small Peak Area. a) Check the position and filling levels of sample vials in the autosampler. b) Check injector needle-height setting. c) Check each line of the schedule for proper injector parameters. Employ full loop methods if other injection modes (partial loop fill) do not provide acceptable reproducibility.
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