Dionex 063000 Manuel utilisateur

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

DNAPac PA200 Document No. 065036 Page 2 of 25

TABLE OF CONTENTS

SECTION 1 - INTRODUCTION ...................................................................................................................4

1.1

DNAPAC PA200 ................................................................................................................................ 4

1.2

BIOLC SYSTEM (WITHOUT COLUMNS) .............................................................................................. 4

1.3

GUARD COLUMN USE ........................................................................................................................ 5

1.4

DNAPAC PA200 ANION EXCHANGE COLUMNS ............................................................................... 5

1.5

DNAPAC COLUMN FAMILY ............................................................................................................... 5

SECTION 2 - OPERATION AND SYSTEM REQUIREMENTS .............................................................. 6

2.1

SYSTEM REQUIREMENTS.................................................................................................................... 6

2.2

SYSTEM OPERATION REQUIREMENTS ................................................................................................ 6

2.3

DNAPAC PA200 COLUMN OPERATIONAL PARAMETERS................................................................. 7

SECTION 3 - PURITY REQUIREMENTS FOR CHEMICALS...............................................................8

3.1

DEIONIZED WATER ............................................................................................................................ 8

3.2

INORGANIC CHEMICALS..................................................................................................................... 8

3.3

SOLVENTS .......................................................................................................................................... 8

SECTION 4 - QUALITY ASSURANCE.......................................................................................................9

4.1

CERTIFICATE OF PERFORMANCE –RESIN BATCH TESTING ............................................................... 9

4.2

PRODUCTION TEST CHROMATOGRAMS ........................................................................................... 10

SECTION 5 - METHODS DEVELOPMENT.............................................................................................11

5.1

SAMPLE CLEANUP ............................................................................................................................ 11

5.2

ELUTION ORDER............................................................................................................................... 11

5.3

EFFECT OF SALT TYPE ON OLIGONUCLEOTIDE ELUTION ................................................................ 11

5.3.1

Eluent Strength ...................................................................................................................... 11

5.3.2

Loading Capacity................................................................................................................... 11

5.4

GRADIENT SLOPE.............................................................................................................................. 12

5.5

EFFECT OF PHAND SOLVENT ON OLIGONUCLEOTIDE CHROMATOGRAPHY .................................... 12

5.5.1

Effect of pH on Hydrogen Bond Interactions ........................................................................ 12

5.5.2 Effect of pH on Retention...................................................................................................... 13

5.5.2

Effect of pH on Retention...................................................................................................... 13

5.5.3

Effect of Solvent on Retention............................................................................................... 13

5.5.4

Effect of pH on Selectivity .................................................................................................... 14

5.6

EFFECT OF TEMPERATURE ON OLIGONUCLEOTIDE RETENTION ...................................................... 15

5.7

EFFECT OF TERMINAL BASE ON SELECTIVITY ................................................................................. 16

5.7.1

Selectivity in Sodium Chloride (NaCl) Gradients. ................................................................ 16

5.7.2

Selectivity in Sodium Perchlorate (NaClO

) Gradients ......................................................... 17

5.8

APPLICATION-SPECIFIC MOBILE PHASE RECOMMENDATIONS ........................................................ 18

5.8.1

For synthetic ODNs where the goal is to evaluate purity. ..................................................... 18

5.8.2

When multiple possible ODNs of similar length in the same solution must be resolved. ..... 18

SECTION 6 - APPLICATIONS................................................................................................................... 19

6.1

DENATURING CONDITIONS FOR CONTROL OF SECONDARY STRUCTURE ........................................ 19

6.2

EFFECT OF HIGH TEMPERATURE AND HIGH PHON COLUMN LIFETIME.......................................... 19

6.3

PHOSPHODIESTER ANALYSIS ........................................................................................................... 20

6.3.1

Sodium Perchlorate Eluent Systems ...................................................................................... 20

6.3.2

Sodium Chloride Eluent Systems .......................................................................................... 21

SECTION 7 - DNAPac™ PA200 RESOURCES.........................................................................................22

DNAPac PA200 Document No. 065036 Page 3 of 25

Section 8 - TROUBLESHOOTING GUIDE ...............................................................................................23

8.1

FINDING THE SOURCE OF HIGH SYSTEM BACK PRESSURE .............................................................. 23

8.2

BACKPRESSURE ON COLUMN HAS INCREASED................................................................................ 23

8.3

DECREASING PEAK RETENTION TIMES ............................................................................................ 23

8.4

DECREASING PEAK EFFICIENCY AND RESOLUTION......................................................................... 23

8.5

POOR PEAK EFFICIENCY AND RESOLUTION ..................................................................................... 24

8.6

UNIDENTIFIED PEAKS APPEAR......................................................................................................... 24

8.7

DECREASED DETECTION SENSITIVITY ............................................................................................. 24

8.8

COLUMN PROBLEMS ........................................................................................................................ 24

8.9

PEAK EFFICIENCY AND RESOLUTION ARE DECREASING ................................................................. 24

8.10

SYSTEM PROBLEMS.......................................................................................................................... 24

8.10.1

High Detection Background Caused by the System .............................................................. 24

8.10.2

No Peaks, Poor Peak Area Reproducibility or Unexpectedly Small Peak Area.................... 25

8.10.3

Incorrect or Variable Retention Times .................................................................................. 25

8.11

COLUMN CLEANUP........................................................................................................................... 25

8.11.1

High Salt Wash to Remove Ionic Components ..................................................................... 25

8.11.2

Organic Solvent Wash to Remove Non-Ionic Components .................................................. 25

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:

•High temperature, pH 8 or below

•High pH (12.4) at 30° or below

Because of the unique pH stability of the packing material, elevated pH conditions can be used to optimize

selectivity for specific oligonucleotides.

The packing material inside the DNAPac PA200 is composed of 130 nm quaternary amine functionalized

MicroBeads™ bound to an 8 µm solvent compatible, non-porous substrate. The non-porous substrate design

provides rapid mass transport resulting in narrow high efficiency peaks. The low column capacity, typical of non-

porous packings, is avoided by agglomerating functionalized MicroBeads to the surface of the substrate particle,

resulting in higher loading capacity than is possible with conventional non-porous materials, and good durability.

This produces a column with oligonucleotide resolution superior to columns using 2 to 3 µm resins.

Resin Characteristics:

Particle Size: 8 µm

Pore Size: non porous

Cross-linking: 55%

Ion exchange capacity: ~40 µeq/column

Latex Characteristics:

Functional Group: quaternary ammonium ion

Latex Diameter: ~130 nm

Latex Cross-link: 5 %

Typical Operating Parameters:

pH range: 4-10 unrestricted eluents

2.5-4 and 10-12.5, (Operation at these pH values require co-ion concentration to be

at least equimolar with hydroxide at high pH or H

at low pH)

Temperature: ≤85°C

Pressure: 3,000 psi

Organic Solvent Limit: 100% acetonitrile or methanol for cleaning

Typical eluents: High purity water (18.2 megohm-cm), sodium chloride, sodium perchlorate, buffers,

sodium acetate and sodium hydroxide

1.2 BioLC System (without Columns)

Table 1: System Components Recommended for DNA Analysis

Basic Gradient System Standard Gradient System

BioLC gradient pump, with degas BioLC gradient pump (degas recommended)

Chromatography oven with injection valve

and regulator assembly Autosampler

Column Oven

Absorbance detector (D

lamp for UV) Absorbance detector (D

lamp for UV)

EO1 eluent organizers EO1 Eluent organizers

DNAPac PA200 Document No. 065036 Page 5 of 25

1.3 Guard Column Use

A guard column is usually placed before the analytical column to prevent contaminants in the sample from eluting

onto the analytical column. The addition of the guard column increases the net column capacity, which translates

into an increase of about 20% in the retention times for isocratic runs. If a guard is added to a system running a

gradient method that was initially developed for an analytical column alone, the analytes will elute slightly later and

usually with slightly better resolution.

1.4 DNAPAC PA200 Anion Exchange Columns

Part Number Product Description

063000 DNAPac PA200, Analytical (4 x 250mm)

062998 DNAPac PA200, Guard (4 x 50mm)

1.5 DNAPac Column Family

There are two varieties of columns in the DNAPac column family. Both columns are non-porous anion exchangers

that provide high-resolution oligonucleotide separations. The choice of column depends upon the goal of the

separation. The DNAPac PA100 consists of a 13 µm substrate particle with 100 nm functionalized MicroBeads.

This column is available in a variety of formats and should be used when higher capacity is required and if scale-up

to semi-preparative scale separations is anticipated.

The DNAPac PA200 consists of an 8 µm substrate particle with 130 nm functionalized MicroBeads. This column

provides higher resolution than the DNAPac PA100. The DNAPac PA200 is operated at a lower flow rate than the

DNAPac PA100, thus less eluent is consumed during a run. In addition, the DNAPac PA200 has been

manufactured to provide greater stability to high pH at elevated temperature, although this combination is not

recommended.

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

Offices.”

DNAPac PA200 Document No. 065036 Page 6 of 25

SECTION 2 - OPERATION AND SYSTEM REQUIREMENTS

2.1 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. The released metals will irreversibly foul the column.

Each of the possible configurations offers multiple sampling options; however, consistently reproducible

quantification and an absence of disturbing artifacts are achieved best using an autosampler and “full loop”

injection mode. Reproducibility of retention time results can be enhanced by regulating the temperature of the

column using a column oven or thermal compartment.

Figure 1 Oligonucleotide System Configuration

2.2 System Operation Requirements

The oligonucleotide analysis systems should be configured with Dionex modules to provide the following

attributes:

a) All components of the fluid path are non-metallic, to eliminate column poisoning.

b) Mobile phase components are kept under helium or nitrogen to minimize out-gassing (bubble formation)

in the detector cell. On-line degassing of eluents may be provided with the eluent degas option on Dionex

pump modules.

c) Accurate reproducible flow and gradient generation at settings between 0.20 and 2.0 mL/min.

d) Minimal contribution to the background signal by contaminants from the system and reagents.

e) Thermostated column compartment for consistent temperature control of the guard and separation

columns.

f) Minimal system volumes (employ low volume unions and minimal tubing length).

•4-mm operation, liquid line inside diameter (I.D.) should be between .007” and 0.01”.

•2mm operation, liquid line inside diameter (I.D.) should be between .003” and 0.005”.

In both operations, PEEK tubing is preferred as it does not contribute to metal leaching.

Absorbance

Detector

Gradient

Pump Autosampler Thermal

Compartment

Data System

DNAPac PA200 Document No. 065036 Page 7 of 25

2.3 DNAPAC PA200 Column Operational Parameters

TABLE 2 Column Operational Parameters

pH = 4-10 (unrestricted eluents)

pH Range:

pH = 2.5- 4, and 10-12.5: Operation at these pH values require co-ion

concentration (e.g., Cl

or ClO

at high pH and Na

or NH

at low

pH) to be at least equimolar with hydroxide at high pH or H

low pH.

Temperature Limit: <85°C

Pressure Limit: 4,000 psi

Organic Solvent Limit: 100% Acetonitrile, or methanol, if required for cleaning.

Chaotrope Limit: 30% formamide, 6 M Urea.

Note: Use of these chaotropes will increase back pressure, and

reduce column lifetime.

Typical Eluents: High purity water (18 megohm-cm), sodium chloride, sodium

perchlorate, buffers, sodium acetate and sodium hydroxide.

Detergent

Compatibility: Nonionic, cationic or zwitterionic detergents.

CAUTION: Do not use anionic detergents. Anionic detergents will bind irreversibly to the column.

DNAPac PA200 Document No. 065036 Page 8 of 25

SECTION 3 - PURITY REQUIREMENTS FOR CHEMICALS

Reliable and reproducible results require eluents that are prepared consistently and are free from impurities.

3.1 Deionized Water

The de-ionized (DI) water, used to prepare eluents, should be Type I reagent grade water with a specific resistance

of 18 megohm-cm. The water should be free from ionized impurities, organics, microorganisms, and particulate

matter. Ultra Violet (UV) treatment in the water purification unit is recommended. Follow the manufacturer’s

instructions regarding the replacement of ion exchange and adsorbent cartridges. All filters used for water

purification must be free from UV absorbing components. Contaminated water in eluents causes high background

signals, gradient artifacts, and even sample degradation due to nucleases arising from microbial contamination.

3.2 Inorganic Chemicals

Inorganic chemicals of reagent grade or better should 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 products will include detailed lot analyses on their labels.

3.3 Solvents

Solvents can be added to the ionic eluents used in DNAPac PA200 columns to modify the ion exchange process.

The solvents used must be free from 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 “ultra high” purity solvents that are compatible with HPLC and spectrophotometric

applications. 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.

When using an ionic eluent with solvent, column generated back pressure will depend on the solvent used, the

concentration of the solvent, the ionic strength of the eluent, and the flow rate applied. The column backpressure

will also vary if the composition of the water-solvent mixture varies. The practical backpressure limit for the

DNAPac PA200 is 4,000 psi (27.6 MPa). The DNAPac PA200 can withstand common HPLC solvents in a

concentration range of 0-100%. Solvents and water should be premixed in concentrations which allow proper

mixing by the gradient pump and to minimize out-gassing. Ensure that all of the inorganic chemicals are soluble in

the highest solvent concentration to be used during the analysis.

Solvent-Water mixtures are usually specified with 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 also

minimize the possibility of out gassing which causes bubble formation in the detector cell. If you choose to mix

eluents containing solvents with those that do not – the eluent degas option for the pump is highly recommended.

As a second choice, pre-degassing the eluents and covering the eluent reservoir with Helium gas to limit gas

dissolution into the eluents will help limit out-gassing.

DNAPac PA200 Document No. 065036 Page 9 of 25

SECTION 4 - QUALITYASSURANCE

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.

4.1 Certificate of Performance – Resin Batch Testing

Each batch of resin used for packing the DNAPac PA200 columns is tested to ensure reliable performance and

resolution. Separations of dT

19-24

with DNAPac PA200 columns, packed with both production and test resins, are

compared. This procedure ensures that resins with the highest quality are used, and produces consistent column

performance.

Eluent 1: 25 mM Tris pH 8

Eluent 2: 25 mM Tris pH 8, 1.25 M NaCl

Flow rate: 1.20 mL/minute

Detection: Absorbance (260 nm)

Injection: 25µL

Storage Solution: Eluent 2

Gradient: Time %1 %2 Comments

0.0 68 32 Equilibration solution

0.1 68 32 Equilibration solution

10.1 53.6 46.4 Gradient ramp 400 – 580 mM NaCl

10.11 35 60 Column wash start

11.11 35 60 Column Wash end

11.2 68 32 Re-Equilibration solution

14.12 68 32 End equilibration

Sample: dT

19-24

1.5 µg each / mL

CHART 1 Certificate of Performance

0.0 4.0 8.0 12.0

260

Time (min)

dT19

dT20

dT21

dT22

dT23

dT24

Flow: 1.20 ml/min

46.4

60.0

46.4

60.0

1.25M NaCl:

32..0 %

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.

The retention time of sulfate is used to measure the capacity of the column. Peak efficiency and peak symmetry of

sulfate are used to measure the packing quality of the column. Retention times and resolution of chloride, nitrate,

and phosphate are used to measure the overall selectivity of the column.

Eluent 1: 1.28mM NaHCO

1.35 mM Na

Flow rate: 1.00 mL/minute

Detection: Conductivity (Suppressed)

Sample: 7 Anion Standard with (1/10 dilution)

Injection Volume: 25 µL

Storage Solution: 25 mM Tris pH 8, 1.25M NaCl

CHART 2 Production Test Chromatogram

0.0 2.0 4.0 6.0 8.0 10.0

Col # 102, 25.0

µS

Time (min)

0.0 2.0 4.0 6.0 8.0 10.0

Col # 102, 25.0

µS

Time (min)

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