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Special Cleaning Procedures
 
As the chart below shows, for most trace metal analyses, plastic is generally "cleaner" or less contaminated than glass or other materials. However, plastic does contain trace levels of certain metals. To minimize potential low-level contamination, remove these metals or leach them from plastic by soaking in 1N HCl and rinsing in distilled water. For extremely precise work, use HCl, followed by soaking in 1N HNO3, and rinsing in distilled water. Soaking time may vary according to individual needs, but plastic should be soaked no longer than 8 hours. If more rigorous cleaning is desired, increase the concentration of acids used. Caution: concentrated nitric acid is a strong oxidizing agent and will embrittle many plastics.

To remove trace organics which contribute to trace metal absorption, clean plastic surfaces with alcohol, alkalies, alcoholic alkalies or chloroform. A final rinse of 1N HCl also minimizes absorption of trace elements.

Polysulfone (PSF), a resin used in NALGENE centrifuge tubes and reusable filterware, is extremely "cleanable.'' The following qualification testing was performed on NALGENE PSF centrifuge tubes at The University of Rhode Island's Graduate School of Oceanography (URI-GSO).

URI-GSO was determining the concentration of trace metals in atmospheric aerosols and seawater from remote marine locations. They were measuring the input to the Pacific Ocean of heavy metals and other trace elements; pesticide, petroleum and plastic residues; other natural and man-made organic materials, as well as compounds causing acid rain. The trace metal concentrations in the samples were extremely low, so any labware used needed to be extremely cleanable to prevent leaching of contaminants from the vessels or filtration equipment into the samples. In addition, the labware could not irreversibly adsorb metals from sample solutions. The labware in question included filter holders, which were used during procedures for extracting various trace metals from the seawater samples.

The following cleaning procedure was used:

  1. One-week soak in 1:1, analytical reagent HCl: deionized water,
  2. Deionized water rinse,
  3. One-week soak in 1:1, analytical reagent HNO3: deionized water,
  4. Deionized water rinse,
  5. One-week soak in 1:1000, quartz redistilled HNO3: deionized water,
  6. Deionized water rinse,
  7. One-week soak in 1:1000, quartz redistilled HNO3: deionized water, and,
  8. Deionized water rinse.

This cleaning procedure was generated from the procedures used at URI-GSO, J.R. Moody and R.M. Lindstrom Analytical Chemistry 49:2264 (1977) and conversations with faculty of the California Institute of Technology.

The following testing procedure was used:

  1. Approximately 25 mL 1N quartz redistilled HNO3 was put into five of the centrifuge tubes.
  2. Approximately 25 mL of 1 part per billion aluminum, copper, lead and zinc in 1 N quartz redistilled HNO3 was put into the other five centrifuge tubes.
  3. The tubes were allowed to stand for one month, and the contents of the tubes were analyzed.

The analyses were performed by atomic absorption spectrophotometry on a Perkin Elmer 5000 equipped with an HGA 5000 utilizing Zeeman background correction. All standards used are traceable back to NBS (National Bureau of Standards).

The results and conclusions were:

  1. The 1N quartz redistilled HNO3 had immeasurably low concentrations of metals (e.g., aluminum, copper and lead 0.1 parts per billion; zinc 0.01 parts per billion). Therefore, once cleaned, polysulfone leaches insignificant amounts of these four metals into solution.
  2. The 1 part per billion aluminum, copper, lead and zinc solutions had, within experimental error, 1 part per billion aluminum, copper, lead and zinc. Therefore, polysulfone does not adsorb these metals from an acidified solution.

The analysis of aluminum, copper, lead and zinc involved a two-step extraction procedure. The first, a "liquid/liquid" extraction with an organic complexing agent, removed copper, lead and zinc. The second employed an iron hydroxide co-precipitation technique. Iron nitrate was added to the seawater and the pH was adjusted. Iron hydroxide and aluminum were filtered from the rest of the mixture. This is where the NALGENE reusable filter holders, molded of PSF, were used.
Summary of Average Element Content of 12 Plastics and Borosilicate Glass (^1)
Material No. of Elements Total Conc., ppm Major Constituents
PS 8 (8 N.D.*) 4 Na, Ti, Al
PSF 16 (12 N.D.*) 17 Na, Fe, Ca
TFE 24 19 Ca, Pb/Fe, Cu
LDPE 18 23 Ca, Cl, K
PC 10 85 Cl, Br, Al
PMP 14 178 Ca, Mg, Zn
FEP 25 241 K, Ca, Mg
PVC-tubing 9 280 Fe, Zn, Sb
PP 21 519 Cl, Mg, Ca
HDPE 22 654 Ca, Zn, Si
ETFE 32 1.007 Cl, Pb, Si
PVC-rigid 7 (11 N.D.) 2.541 Sn, Ca, Mg
Borosilicate Glass 14 497,249 Si, B, Na

* N.D. Not Detected

NOTE: Values listed in the chart above represent typical contents for major constituents. Various NNI grades of plastics may vary from these values.

(^1) Selection and Cleaning of Plastic Containers for Storage of Trace Element Samples, John R. Moody and Richard Lindstrom, ANALYTICAL CHEMISTRY, Vol. 49, Page 2264, December 1977.
 
RNase, an enzyme that breaks down RNA, and DNase, which breaks down DNA, are contaminants that can interfere with nucleotide research. DNase can be destroyed by autoclaving for 15 minutes at 121C or by following any of the procedures listed below. One or more of the following techniques will inhibit or remove RNase from your plastic container. Match the resin code on the bottom of your NALGENE container with the correct technique.

  1. Heat at 180C for at least 8 hours.(^1)
  2. Rinse in chloroform.(^1)
  3. Soak in a 0.1% aqueous solution of diethyl pyrocarbonate(^2) (DEPC) for 2 hours at 37C; rinse several times with sterile (DEPC-treated) water(***); heat to 100C for 15 minutes OR autoclave for 15 minutes at 121C on a liquid/slow exhaust cycle. (Heating or autoclaving will remove DEPC residues.) Note heating variations in the following chart.
  4. Clean equipment with a detergent solution, rinse thoroughly with water and rinse with 95% ethanol to dry. Soak the equipment in a 3% hydrogen peroxide (H2 O2) for 10 minutes at room temperature. Rinse the equipment thoroughly with DEPC-treated water.***
  5. Soak equipment in 0.1N Sodium Hydroxide (NaOH) in 0.1% EDTA in water overnight and then rinse thoroughly with DEPC-treated water.(^3)
RNase Removal Chart - Techniques
Plastic      1        2        3        4        5  
Resin (Heat) (Rinse) (Soak) (Soak) (Soak) Comments
ETFE       X       X       X    
FEP     X       X       X       X       X  
HDPE       X*     X       X       X   Heat to 100C for 20 minutes
LDPE       X*     X       X       X   Heat to 70C for 120 minutes
PC       X*     X*      
PETG         X       X       X   Heat to 60C overnight
PFA     X       X       X       X       X    
PP/PPCO       X*     X       X       X    
PMP       X*     X       X       X    
Barex           X       X    
HIPS         X       X       X   Heat to 70C for 120 minutes
PVC         X       X       X   Heat to 60C overnight
TPE         X       X       X    

* Rinse only, no long-term contact
**Rinse copiously to minimize chemical attack
***DEPC-treated water: Add 0.1% DEPC to water and allow to sit for at least 12 hours at 37C. Then heat the water to 100C for 15 minutes or autoclave at 121C (250F) for 15 minutes.

(^1) Sambrook, J.; Fritsch, E.F.; Maniatis, T.; "Extraction and Purification of RNA''; Molecular Cloning: A Laboratory Manual, Second Edition; 7.3, Cold Spring Harbor Laboratory Press (1989)
(^2) Caution: DEPC is a suspected carcinogen and should be handled with care. DEPC solutions are irritating to the eyes, mucous membranes and skin.
(^3) Titus, David E.; Nucleic Acid Detection, Purification and Labeling; Rapid Isolation of Total RNA; PROMEGA Protocols and Applications Guide, Second Edition; pp. 125-126, 203; Promega Corporation (1991)

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