Mildred R. Bernardo is lead scientist with Steris Corp. 

Mildred R. Bernardo

Stainless steel pipe fittings, clamps, and connectors arranged in industrial. Precision manufacturing, mechanical assembly, and hygienic processing systems for food and pharmaceutical industries. | Image Source: ©Artinun -stock.adobe.com

Type III or Class III rouge (magnetite) appears as a black oxide in water systems and production equipment exposed to sanitization, pasteurization, or steam sterilization conditions. Remediation of type III rouge often requires electropolishing or mechanical polishing of the stainless-steel surface, resulting in significant labour cost, safety risk to operators with confined space entry, downtime of the equipment or system, and a reduction in thickness of the stainless steel. Raman spectroscopy is a non-invasive analytical method that can quickly identify the presence of magnetite (Fe

) in rouge. Based on the identification of magnetite, a detergent additive can be combined with a formulated citric/oxalic or a phosphoric/citric acid blend to transform insoluble magnetite to a more soluble form of iron oxide. This approach to type III rouge remediation utilizing a detergent blend, spray device, and recirculating pump reduces downtime, safety risk, material loss, and overall cost of maintenance.

A common surface in production equipment and piping is 300 series stainless steel. This surface is compatible with most pharmaceutical and biopharmaceutical processes; however, certain conditions can lead to discoloration and/or rouge on the surface. Rouge is a corrosion product on or in the surface of the iron alloy. Rouge is generally classified as Type I–III or Class I–III.

 Type I rouge (migratory rouge) is an iron oxide that is loosely attached to the surface and can be wiped off the surface. The origin of this rouge is often from an upstream source. Type II rouge (in situ rouge) is difficult to wipe off and often contains underlying damage to the surface. Examples of Type II rouge include galvanic, intergranular, pitting and stress corrosion. Common acid blends, such as citric/oxalic or phosphoric/citric solutions, can be effective at removing Type I and II rouge.

 These treatments do not restore original surface roughness. Electro- or mechanical polishing may be required to correct surface damage due to Type II rouge from pitting corrosion. Type III rouge is often associated with high temperature and purity water systems. Type III rouge is challenging to remediate due to the presence of magnetite (Fe

). Magnetite removal often requires aggressive chemicals or electro- or mechanical polishing. An acid detergent with phosphonate can be used alone or in combination with a citric/oxalic or phosphoric/citric acid blend to improve the solubility of magnetite and the removal of Type III rouge.

Understanding when and which acid detergent (or detergent additive) to use in rouge remediation can be difficult. The selection of the acid detergent should consider effectiveness to remove the rouge, substrate compatibility, rinse profile, stability and shelf life, analyzability, toxicity, disposal constraints, personnel safety, toxicity, foaming characteristics, microbial effectiveness, quality of manufacture, long-term availability, quality of manufacture and overall cost.

 Raman spectroscopy can be a powerful tool in identifying the oxide compound(s) present and provide direction to successful remediation. Alternative spectroscopy and microscopy techniques may be applicable for surface characterization and corrosion monitoring, such as X-ray diffraction (XRD) and scanning electrochemical microscopy (SECM).

 Raman uses inelastic light scattering, where the incident light (from a high-intensity laser light source) transfers energy to molecular vibrations, and the scattered light collected at the detector provides structural information relating to the molecule. Each molecule/compound will have a unique spectrum. Magnetite displays a primary peak with Raman spectroscopy between 662 and 670 cm

 A secondary peak is often observed at 532 and 550 cm

. A common roadblock in successful rouge remediation is the presence of magnetite. A quick identification of magnetite in a rouge surface using Raman spectroscopy can be helpful in selecting the right chemical treatment and cleaning parameters.

This article reviews the use of Raman spectroscopy in a series of case studies to quickly identify the iron oxide compound(s), including magnetite and hematite, on the surface of representative endcaps or piping spool pieces. The article also provides a strategy using a detergent additive to remove magnetite without performing costly electro- or mechanical polishing.

A Raman microscope with a 532 nm laser (DXR3, Thermo Scientific) was used to analyze selected sample areas. Areas that visually exhibited the greatest amount of residue were selected and focused on using the confocal microscope prior to analysis. The samples were analyzed as is (without cleaning, modification, etc.) using the following conditions in 

After analysis of the selected sample areas, a spectral library search (Wiley Science Solutions KnowItAll Spectral Library) was performed for each Raman spectrum.

For reference and comparison throughout the following case studies, the Raman spectra of hematite (Fe

Hematite exhibits peaks at approximately 220, 290, 400, 600, and 1300 cm

Figure 2. Raman Spectrum of Magnetite (Fe

A large multinational biopharmaceutical drug manufacturer indicated that visible rouge was unable to be removed from a tank using 10% v/v citric/oxalic acid detergent at 70 to 80 

C. Citric/oxalic and phosphoric/citric acid blends are commonly recommended for rouge removal.

 The nature of the rouge was investigated using Raman spectroscopy to identify the oxide composition and determine the reason for the difficulty in the chemical removal. Raman spectroscopy is an analytical tool that can be used in the laboratory and modified for use on the manufacturing floor.



. The following swab samples of the rouge were analyzed using Raman spectroscopy (See 

TA-9620 Pre-Derouge Swab Bottom Visible Ring

TA-9640 Rouge Observation Top Spot Near Sight Glass

Figure 3. Raman Spectra of Swab Samples. Figure courtesy of the authors.

. A spectral library search (Wiley KnowItAll Spectral Library) was performed for each Raman spectrum. All the residue samples exhibited a best match to a mixture of iron oxide compounds—hematite (Fe

). A representative search result is shown in 

Figure 4. Raman Spectra of Iron Oxide Mixture Reference (Mixture of Fe

) and TA-9720 Bottom of the Tank Outlet. Figure courtesy of the authors.

. All the Raman spectra from the selected swabs were similar to each other and exhibited peaks around 200, 300, 400, 700, and 1300 cm

, which can be attributed to an iron oxide mixture of Fe

. The general recommendation based on the swab analysis by Raman spectroscopy was to increase the concentration of the citric/oxalic agent detergent to 15% v/v and add a detergent additive at 15% v/v with recirculation through a device at 60 to 80

C for 1 to 3 hours to solubilize and remove the rouge containing magnetite (Fe

A large multinational biopharmaceutical blood product drug manufacturer submitted two end caps (one smaller and one larger) for Raman spectroscopy analysis. Both end caps exhibited brown discoloration consistent with rouge (

Figure 5. Small End Cap. Figure courtesy of the authors.

Figure 6. Large End Cap. Figure courtesy of the authors.

With 10x magnification, the small end cap exhibited orange, blue and purple/brown discolored areas. Areas of each colour were selected for Raman analysis to determine if there were any differences in the Raman spectra. Representative areas of analysis for the three different colours observed on the end cap’s surface are marked by a red square in the images below (

Figure 7. Small End Cap – Orange Area (10x mag). Figure courtesy of the authors.

Figure 8. Small End Cap – Blue Area (10x mag). Figure courtesy of the authors.

Figure 9. Small End Cap – Purple/Brown Area (10x mag). Figure courtesy of the authors. 

The three different colored areas of the end cap were analyzed, and the Raman spectra were found to be comparable to each other. A comparison of the spectra is shown in 

Figure 10. Raman Spectra of Analyzed Areas of Small End Cap. Figure courtesy of the authors.

. A magnified image of the residue present on the large end cap is shown in 

. The small area marked by a red square is representative of the area selected for Raman analysis.

Figure 11. Large End Cap – Brown Area (50x mag). Figure courtesy of the authors.

. The Raman spectrum of the brown area from the large end cap is shown in 

Figure 12. Raman Spectrum of Large End Cap. Figure courtesy of the authors.

. The three Raman spectra of the small end cap and the Raman spectrum of the large end cap were comparable to each other, with peaks at approximately 660, 600, 496, 408, 292, and 223 cm

.

When a spectral library search was performed (Wiley Spectroscopy KnowItAll Spectral Library), the residue from the end caps appeared to be a mixture of hematite (Fe

). A comparison of the Raman spectra of the residue from the small and large end caps to the hematite and magnetite Raman spectra is displayed in 

Figure 13. Raman Spectra of End Caps, Magnetite, and Hematite. Figure courtesy of the authors.



The end cap spectra matched well to the hematite spectrum; however, the peak around 665 cm

 had a greater intensity than the hematite reference spectrum, suggesting the presence of magnetite, which has a high intensity peak around 665 cm

 The residue was effectively removed from stainless steel by agitated immersion using a solution of 15% v/v formulated phosphoric/citric detergent plus 15% v/v detergent additive at 80 °C for 24 hours (

Figure 14. Derouge and Passivate End Cap Samples. Figure courtesy of the authors.

A large multinational biopharmaceutical drug product manufacturer submitted pipe samples with suspected rouge for Raman spectroscopy analysis. The pipe samples exhibited a brown discoloration consistent with rouge throughout its outer surface.

. Three different areas on the pipe were analyzed using Raman spectroscopy. The spectra of the three areas were found to be comparable to each other. A comparison is shown below in 

Figure 15. Raman Spectra of Analyzed Areas of Pipe. Figure courtesy of the authors.

. A spectral library search was performed (Wiley Spectroscopy KnowItAll Spectral Library), and the Raman spectrum of the brown discoloration from the pipe had a best match to the spectrum of a mixture of hematite (Fe

). A comparison of the Raman spectrum of the hematite and magnetite mixture and the Raman spectrum of the brown residue from the pipe is shown in 

Figure 16. Raman Spectra of Iron Oxide Mixture and Brown Residue From Pipe. Figure courtesy of the authors.

 The brown discoloration was effectively removed compared to untreated controls from stainless steel by agitated immersion using a solution of 15% v/v formulated phosphoric/citric acid detergent plus 15% v/v formulated detergent additive or a 15% v/v formulated citric/oxalic acid detergent plus 15% v/v formulated detergent additive at 80 °C for 4 hours. The surface was tested with an electrical pen test kit to confirm a passive surface following treatment. Testing with 15% v/v formulated phosphoric/citric acid or the formulated citric/oxalic acid detergent at 80 °C for 24 hours was not successful in removing the discoloration shown in 

 alongside untreated controls (left side) and successfully derouged and passivated pipe samples (right side).

Figure 17. Case Study 3 Heat Exchanger Pipe Samples, Left to Right:

Sample 3, Cleaned with 15% formulated phosphoric/citric acid detergent

Sample 4, Cleaned with 15% formulated citric/oxalic acid detergent

Sample 5, Cleaned with 15% formulated phosphoric/citric acid detergent plus 15% detergent additive

Sample 6, Cleaned with 15% formulated citric/oxalic acid detergent plus 15% detergent additive

A general requirement of pharmaceutical drug manufacturers is that equipment shall be constructed so that surfaces that contact components, in-process materials, or drug products shall not be reactive, additive or absorptive so as not to alter the strength, quality, integrity, identity and purity of the drug product.

 The presence of discoloration or rouge on direct and indirect product surfaces has been cited by regulatory authorities.

Guidelines for Good Manufacturing Practice for Medical Products for Human and Veterinary Use 

(Annex 1) state in section 2.5 that process equipment, utilities, raw materials and premises are potential sources for microbial and particulate contamination. Stainless steel maintenance and cleaning of direct, indirect, and non-direct surface must be considered within any contamination control strategy.

 A risk-based strategy to stainless-steel maintenance utilizing International Council for Harmonisation (ICH) Q9 

 has two primary principles: the evaluation of risk to quality from rouge should be based on scientific knowledge and ultimately linked to the protection of the patient and product, and the level of effort, formality and documentation should be commensurate with the level of risk.

 Stainless steel commonly used in pharmaceutical and biopharmaceutical manufacturing can be discoloured or rouged based on several factors, including the nature of the in-process solution, product, medias, buffers, reagents and water quality.

Through a series of case studies, the authors used Raman spectroscopy to identify the presence of magnetite on various process and utility system samples (swabs, end caps and spool pieces). Type III rouge (magnetite) presents considerable challenges to chemical remediation and stainless steel maintenance programs. Based on the identification of magnetite, a readily available detergent additive can be combined with a formulated citric/oxalic or phosphoric/citric acid detergent commonly used for derouging and passivating stainless steel. The two-detergent approach transforms the insoluble magnetite to a more soluble form of iron oxide for removal without polishing or a secondary passivation step. This science-based approach to type III rouge remediation incorporates a detergent additive, spray device and recirculating pump to reduce equipment downtime, operator safety risk, material loss from polishing and overall cost of maintenance and compliance to current good manufacturing practices.

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Emily Null, MS, scientist; Mildred R. Bernardo, lead scientist; Si Myra Tyson, MS, technical services lab specialist; Dijana Hadziselimovic, technical services laboratory manager; Paul Lopolito, technical services director; all with STERIS Corp.

Mildred R. Bernardo

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