Appendix 2 System Cleaning and Passivation Treatment

Transcription

1 DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA ISBNs: (Hardback); (Electronic) 207 Appendix 2 System Cleaning and Passivation Treatment A 2.1 Background Information HPLC separation problems caused by corroding stainless steel surfaces have been reported for the analysis of inorganic ions and proteins. Poor electrochemical detection (detection by oxidation or reduction of the sample peak) has sometimes been shown to be due to metal ion contamination found or released from stainless steel surfaces [1, 2]. While systems free of metal contamination has been needed for some types of HPLC, DNA Chromatography exhibits the highest system requirements needed to be able to reliably separate and detect nucleic acid separations. The surfaces of the system cannot contain metal ions that will either trap the nucleic acids or perform structural or conformational changes to the nucleic acid. Nor can the surfaces release metal ions to travel to another point within the system (the column end inlet frit for example) that will damage the separation [3 6]. Fortunately, it is possible to clean and maintain stainless steel components or other (plastic or metal) materials that contain harmful metal ions [7, 8]. Most manufacturers of HPLC equipment have standard operating procedures on how to clean and passivate their equipment. These original procedures have been further modified to ensure the DNA Chromatography system surfaces are contamination free and stable. Treatment with nitric acid will clean and passivate a stainless steel and make it resistant against further corrosion. The concentrations of nitric acid used for HPLC system passivation vary from 3 to 13 M; however, treatment with 8 M nitric acid seems to be sufficiently rapid and effective for cleaning a DNA Chromatograph. In the cleaning mechanism, nitric acid will remove existing metal ion contamination and some organic contaminants. For example, nitric acid is used to clean organic and metal material on quartz detector windows in UV detectors and it is used to remove surface corrosion of the tubing of the system. This does not mean that this cleaning procedure should be used to clean incompatible components. For example a (rusty prone) stainless steel frit is not a suitable component for a DNA Chromatograph. And a system that has worked well for protein separations for example will not necessarily work for DNA separations. Stainless steel is

2 208 System Cleaning and Passivation Treatment an excellent material for HPLC and is used in systems where high structural strength or reliability is required. However, stainless steel frits (that contain very high surface area and may easily corrode) should be replaced with a titanium or polymer frit material rather than relying on system cleaning to maintain performance. Even then, titanium or polymers may contain metal ion contamination that must be removed. In the passivation mechanism, nitric acid oxidizes the metal surface by producing a very thin oxide layer, which protects the metal from further attack by oxygen, acid, and other influences. Nitric acid will also preferentially remove iron from the surface (or sharp edges prone to corrosion) effectively leaving chromium and nickel metal at relatively higher concentrations at the surface. These metals are less likely than iron to undergo further corrosion. In the procedure described here, a treatment with a chelating reagent is included to ensure complete cleaning and passivation. System treatment should be performed once every 6 months or whenever necessary due to deteriorating column performance (broad, inconsistent or missing peaks). It is a good idea to change piston seals, injection port seals and flow path filter after the passivation. Even a new column could show deteriorating performance. In fact, if performance degradation of a new column occurs quickly, it is a strong sign that the system needs cleaning. CAUTION: Nitric acid can be dangerous. Always wear safety glasses and gloves. The nitric acid effluent should be collected in a separate (empty) waste container. Do not mix concentrated nitric acid with organic solvents of any kind. The operator performing the passivation must be familiar with the basic operation of the DNA Chromatographic system and must know how to remove and install the injection port and seal and the injection needle. Purging and rinsing may have to be done in manual mode with 50% A and 50% B to passivate both reservoir flow paths. The column and inline degassers should be removed from the flow path to prevent harming these components. The detector should also be removed unless there is a need to clean this component. The recommended flow rate for cleaning and rinsing is 2 ml/min. Do not close the door to the column heater or turn off the heater. Passivation should be performed at room temperature. An experiment was performed to show the effect of passivation of a new system that had not yet undergone final cleaning and conditioning. The identical column was used for all four chromatograms. Figures A2.1 and A2.2 show how a contaminated system might perform for a size standard and a mutation standard. In these cases, the peak pattern is unusual and the resolution of some peaks is poor. In other cases (discussed in Chapter 3, the peaks may split into doublets or even disappear. Figures A2.3 and A2.4 show how the separation should look in a clean system. In these cases, the peak patterns are normal (as compared with the standard chromatograms included with the new column) and the peak resolution is excellent. This cleaning and passivation procedure should not to be considered as an exotic rescue attempt but rather as a simple way to prevent premature system failure.

3 Appendix Figure A2.1. Separation of puc 18 Hae III digest size standard on a new DNA Chromatogrampic System before system treatment. Separation conditions are specified as standard test conditions for performing a size based separation for a new DNASepr column. From Transgenomic, Inc. with permission. Figure A2.2. Separation of Dys271 mutation performing a size based separation for a new standard on new DNA Chromatgraphic System before system treatment. Separation conditions DNASepr column. From Transgenomic, Inc. with permission. are specified as standard test conditions for

4 210 System Cleaning and Passivation Treatment Figure A2.3. Separation of puc 18 Hae III digest size standard on a new DNA Chromatogrampic System after system treatment. Separation conditions are specified as standard test conditions for performing a size based separation for a new DNASepr column. From Transgenomic, Inc. with permission. Figure A2.4. Separation of Dys271 mutation standard on new DNA Chromatgraphic System after system treatment. Separation conditions are specified as standard test conditions for performing a size based separation for a new DNASepr column. From Transgenomic, Inc. with permission.

5 Appendix A2.2 Reagents 1. DI Water 2. 8 M Nitric acid (approx. 35 % w/w) Preparation: In 250 ml media bottle, add slowly 70 ml ofconcentrated nitric acid (HNO 3, 69.5 %, d ˆ 1.4 g/ml) to 100 ml ofdi water. The solution will get warm; wait until the acid has reached room temperature before using. 3. Ethylenediamine tetraacetic acid, tetrasodium salt (tetrasodium EDTA), 50mM (Na 4 EDTA) Preparation: Dissolve 22.6 g oftetrasodium EDTA (FW ˆ ) in about ml of DI water (1 L volumetric flask). After all the solids have dissolved dilute with DI water to the 1 L mark. A2.3 Preparation of the System 1. Replace the column with a PEEK TM union. 2. Bypass detector flow cell, with Teflon tubing (0.010 or 0.020ªID), from preheat coil to a new waste container of appropriate length. 3. Rinse entire system with distilled water (all channel reservoirs): purge for 5 minutes and flush for another 30min at 2 ml/min. Check for any leaks! Make sure system is without leaks. Do not close door to column oven or turn off oven. A2.4 Passivation of System 1. Remove the white solvent inlet filter caps from the solvent lines of all channel reservoirs. 2. Fill 8 M nitric acid in appropriate glass bottle (Erlenmeyer flask, media bottle) and insert tubing ends of all channel reservoirs into the acid. 3. Rinse all channel reservoirs with 8 M nitric acid: purge for 5 min; rinse system for another 15 min at 2 ml/min. 4. Replace nitric acid with DI water. Rinse all channel reservoirs DI water: purge for 10min and flush for 90minutes at 2 ml/min. During this time change the DI water at least three times. 5. Check the ph of the effluent with ph paper. Keep rinsing with water until ph i5. 6. Replace the DI water with a 50mM solution of tetrasodium EDTA (Na 4 EDTA). Purge for 5 min and rinse system for 30min at 2 ml/min. Caution: If nitric acid is not rinsed thoroughly, the EDTA could precipitate plugging the system.

6 212 System Cleaning and Passivation Treatment A2.5 Equilibration of System 1. Install new inlet filter caps. 2. Replace Na 4 EDTA solution with water, purge for 5 min and rinse for 15 minutes. 3. Replace water with fresh eluents (A and B), purge for 5 min and rinse for at least 2 hours at 2 ml/min. 4. Install column, equilibrate column with at least one gradient run and test system with puc 18 Hae III digest and Dys271 mutation standard. A2.6 Passivation of Injection Port and Injection Needle It is a good idea to passivate injection port and needle at the same time of system passivation. The same reagents are used for this process. In addition a small glass beaker (100 ml, tall form) and a sonicator (dental cleaner) are necessary. 1. Remove injection port and needle. Remove injection port seal from the port 2. Rinse port and needle with DI water 3. Put needle and port into beaker. 4. Fill beaker with 8 M nitric acid until needle and port are covered 5. Place beaker in sonicator and sonicate for 5 min (under fume hood) 6. Decant nitric acid into recycle bottle. Acid can be reused for passivation. 7. Rinse needle and port several times with DI water. 8. Sonicate needle and port in fresh DI water for 5 minutes. 9. Check ph. If phi4 replace DI water and sonicate 5 minutes. Repeat until phi Replace water with 50 mm Na 4 EDTA solution and sonicate for 5 minutes. 11. Rinse with DI water one more time. 12. Install new injection port seal. 13. Re install needle and port.

7 Appendix References 1. C. Duda and C. S. Brunlett, Amperometric Techniques, in Handbook Instrumental Tech. Anal. Chem. Bioanalytical Systems, F. A. Settler ed., Prentice Hall: Upper Saddle River, NJ, pp , K. E. Collins, C. H. Collins and C. A. Bertran, Stainless steel surfaces in LC systems, Part I corrosion and erosion, LC/GC Mag., 18, 600, D. T. Gjerde, R. M. Haefele, and D. W. Togami, Method for performing polynucleotide separations using liquid chromatography, U. S. Pat. 6,017,457, D. T. Gjerde, R. M. Haefele, and D. W. Togami, System and method for performing polynucleotide separations using liquid chromatography, U. S. Pat. 5,772,889, D. T. Gjerde, R. M. Haefele, and D. W. Togami, Apparatus for performing polynucleotide separations using liquid chromatography, U. S. Pat. 6,030,527, D. T. Gjerde, R. M. Haefele, and D. W. Togami, Liquid chromatography systems for performing polynucleotide separations, U. S. Pat. 5,997,742, K. E. Collins, C. H. Collins and C. A. Bertran, Stainless steel surfaces in LC systems, Part II passivation and practical recommendations, LC/GC Mag., 18, 688, R. Shoup and M. Bogdan, Passivation of liquid chromatography components, LC/GC Mag., 7, 742, 1989.