Analyzing Mild- and Ultra-Mild Sliding Wear of Metallic Materials by Transmission Electron Microscopy

Abstract

Any understanding of tribological behavior is connected to sound analyses of the wear appearances, which render insight into the acting wear mechanisms and their sub-mechanisms. Today one important method to analyze wear appearances at high resolution is transmission electron microscopy (TEM) being invented by Knoll and Ruska in the early 1930th in Berlin (Knoll in Z fuer Phys 78:318–339, 1932 [1]).

Attachments

1.1 Preparation of Samples

This chapter exemplifies processes to prepare samples for TEM analyses of near-surface and subsurface areas of worn specimens and parts of metallic alloys.

1.1.1 Specimens from the Strain Gradient of Austenitic or Ferritic Metal Alloys

For the preparation of cross sections the method developed by Büscher et al. [63] or Hahn [64] can be used. Here, two corresponding segments of body and counterbody were taken and glued onto each other’s articulating surface by means of a suitable adhesive (Epoxy G1, Gatan, Munich, Germany). The sample was fixed by a slotted pipe with a diameter of 2.5 mm and positioned in a brass tube of 3 mm diameter. The tube was cut to slices of 400 μm thickness. The cross section of one slice precisely exposed the segments of cups and head lying on each other separated by a small gap of hardened epoxide adhesive. Using grinding, dimple grinding (Model 656, Gatan, Munich, Germany) and ion milling (PIPS 691, Gatan, Munich, Germany) the sample was thinned to the desired thickness of about 40 nm. Afterward the specimens were investigated by means of transmission electron microscopy (TEM 400 Phillips, Eindhoven, Netherlands). In order to observe chemical changes in the uppermost surface layers, a high resolution TEM (Tecnai F20 Phillips, Eindhoven, Netherlands) with EDS and electron energy loss spectroscopy (EELS) was used.

1.1.2 Specimens from the Worn Surface of Martensitic Steels by FIB [39]

Sample preparation by means of focused ion beam (FIB) , was performed using a dual beam FIB/SEM system (Helios NanoLab 600, FEI, Eindhoven, Netherlands). The cross-section procedure contained several steps. First, a protective Pt-layer was deposited at the area of interest on the sample surface. After this, bulk material was removed in a wedge-shaped trench by Ga+ ions, on one side of the area. Using decreasing energies of the ion beam, the sidewall was polished at a small glancing angle in subsequent polishing steps. The processing parameters are given in Table 2.3. Micrographs of the cross-sections were obtained in SE mode at 2 kV accelerating voltage. Microstructural features emerge due to electron channeling contrast (ECC).

Table 2.3 FIB cross sectioning parameters

1.1.3 Specimens from the Worn Surface of Martensitic Steels by Ion Milling

TEM cross-section samples of the wear tracks were prepared using an ion polishing system (EM-09100IS, Jeol, Akishima, Japan). Therefore, small samples parallel to the sliding direction were cut, as pictured in Fig. 2.4 (1–4) and a silicon wafer was applied to the surface as described in 4.2. Afterwards, the samples were ground with 1200 grit SiC paper to a thickness of 100 μm. Using an ion slicer, the cross-sections were then polished on both sides with Ar+ ions, while a thin ridge of the sample was masked by a metal foil. The process parameters are shown in Table 2.4. In order to gain electron transparency, an additional ion-milling process was necessary (Fig. 2.26). Therefore, the specimens were successively thinned on both sides using an ion-mill (PIPSII Model No. 695, Gatan, USA). The ion-mill was operated at accelerating voltages between 5–0.5 kV and gun angles between 5° and 10°.

Table 2.4 Ion slicer parameter

Fig. 2.26

Cross section preparation out of a wear track. 1 Cutting, 2 Glueing a Si-Wafer on top of the wear track 3 grinding down to a thickness of about 100 µm, 4 ion polishing by IS, 5 final thinning by PIPS from [65]

1.1.4 Wear Particle Preparation from Lubricant (Gear Oil)

Particle Isolation

• (1) Centrifuge lubricant samples and carefully remove supernatants (centrifugation time depends on the viscosity of the medium). Do not touch the pellet at the bottom of the tubes.

• (2) Resuspend and wash particles with cyclohexane, use shaker (Vortex-Genie-2, Scientific Industries, Bohemia, NY, USA) and ultrasonic cleaner.

• (3) Centrifuge for 15 min at 20,000 g.

• (4) Carefully discard supernatants. Do not touch the pellet at the bottom of the tubes!

• (5) Resuspend and wash particles with acetone, use shaker and ultrasonic cleaner.

• (6) Centrifuge for 15 min at 20,000 × g.

• (7) Carefully discard supernatants. Do not touch the pellet at the bottom of the tubes!

• (8) Resuspend and wash particles with isopropanol, use shaker and ultrasonic cleaner.

• (9) Centrifuge for 15 min at 20,000 × g.

• (10) Carefully discard supernatants. Do not touch the pellet at the bottom of the tubes!

  if necessary repeat steps 2–8.

• (11) Store particles in isopropanol or ethanol at 4 °C.

Cyclohexane and acetone are used in order to dissolve the lubricant. These solvents (particularly cyclohexane) should not be stored in the tubes for too long since they can damage the tubes! They would also damage the carbon film on the Cu-grids. Therefore, it is necessary to suspend the particles in a less aggressive solvent, such as isopropanol or ethanol, before applying them on Cu-grids.

Particle embedding

1st day:

• (1) Centrifuge for 15 min at 20,000 × g.

• (2) Prepare acetone/epoxy mixture

  Mix epoxy and hardener at a ratio of 100:50 (Epoxy 3000 Quick, Cloeren Technology GmbH, Wegberg, Germany)

  Add 100% acetone at a ratio of 1:1.

• (3) Carefully discard supernatants. Do not touch the pellet at the bottom of the tubes! Add 0.5 ml of the acetone/epoxy mixture.

• (4) Place tubes in a rotator (Thermomixer compact, Eppendorf, Hamburg, Germany) for 24 h.

2nd day:

• (1) Centrifuge for 15 min at 20,000 × g.

• (2) Place tubes, with open lids, under vacuum for 1 h in order to remove the acetone.

• (3) Place tubes, with open lids, at 80 °C for 24 h for polymerization.

3rd day:

Remove tubes to obtain solid pieces of resin with particles embedded at the bottom.

Section with diamond blade to a thickness of approx. 90 nm.

1.1.5 Wear Particle Preparation from Biomedical Lubricant (Bovine Calf Serum)

The protein rich testing fluid (bovine serum) from both tests containing the wear particles was stored at −20 °C. Proteins disturb the image contrast, increase the degree of agglomeration and inhibit the identification of single particles in SEM and TEM. Compared to rather inert polyethylene particles, the isolation of metallic wear particles requires a more sophisticated protocol using enzymatic digestion to remove organic components, especially proteins, without damaging the particles. Particles in the present study were isolated following the protocol developed by Catelas et al. [55, 66] but with minor changes. This protocol was shown to minimize particle damage. It consists of several washing steps as well as incubation steps with two different enzymes, papain and proteinase K. Papain is a cysteine protease enzyme. Its main mechanism of action is destruction of peptide bond. Proteinase K is a broad-spectrum serine protease. This enzyme is known for its protein denaturing abilities, especially in presence of reagents like etylenediaminetetraacetic acid (EDTA). The amount of testing fluid retrieved from the laboratory tests ranged from 60 ml (hip simulator tests) to 300 ml (sliding wear test rig). Fluid samples were aliquoted into 40 ml tubes and centrifuged at 18,000 × g for 15 min using a Sorvall RC-5B superspeed centrifuge (particles from the different aliquots of the same testing fluid sample were recombined after the first overnight incubation). Supernatants were discarded except 1.5 ml in order to resuspend the pellets and transfer them in microcentrifuge tubes. All centrifugations were performed for 10 min at 18,000 × g. First, the pellets were resuspended in 2.5% sodium dodecyl sulfate (SDS) (v/v distilled water) and boiled for 10 min. This was followed by one wash in 80 % acetone and three washes in 1 ml of 250 mMol sodium phosphate buffer solution (PBS) containing 25 mMol EDTA at pH 7.4. After 30 s of sonification, papain (3.2 Units in 1 ml PBS/EDTA) was added. The samples were then incubated overnight on an Eppendorf thermo-mixer at 65 °C under slight motion. After the incubation, pellets from aliquots of the same initial fluid sample were combined in microcentrifuge tubes. Pellets were then resuspended in 2.5% SDS, boiled again for 10 min and then washed twice in 1 ml of 50 mM Tris-HCl pH 7.6. Before adding proteinase K (3 Units in 1 ml Tris-HCl), the pellets were sonicated for 30 s. A second overnight incubation was conducted at 55 °C under slight motion. After the incubation, the samples were boiled again in 2.5% SDS. The pellets were then washed once in 1 ml of 50 mMol Tris-HCl, once in 3 % SDS (v/v 80% acetone) and once in distilled, deionized water. At the end of the isolation protocol, the pelleted particles were stored in 100% ethanol at 4 °C. In a few cases, the pellets still contained denatured organic components. For those cases, the second overnight incubation was repeated. This may have been due to the use of 40 ml initial aliquots instead of 15 ml. Particles were then embedded in epoxy resin for TEM analysis. Prior to embedding, the particles were centrifuged for 20 min at 18,000 × g. The supernatant was removed and the particles were resuspended in a solution of 0.5 ml acetone and 0.5 ml liquid epoxy resin (Epoxy 3000, Cloeren Technology GmbH, Wegberg, Germany). Microcentrifuge tubes were then placed on a thermomixer overnight under slight motion at room temperature to allow pellet infiltration by the resin. The tubes were further centrifuged for 20 min at 18,000 × g and degassed for 1 h to evaporate the acetone. One ml of epoxy resin was added and the tubes were placed in a vacuum chamber for 1 h to remove potential air bubbles and trace of remaining acetone. The samples were then placed in an oven at 60 °C for one hour to harden the epoxy resin. Once hardened, the transparent epoxy cone at the tip of the tubes showed the embedded particles. Approximately 100 nm thick sections were cut using a diamond knife and placed on carbon film coated copper grid nets (formvar carbon-film S162-4, Plano GmbH, Wetzlar, Germany) for TEM analysis.