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Rust Removal Experiments

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Applied and Environmental Microbiology
Aug 2015



Iron oxidation, known as rust formation, causes enormous loss in term of property damages and associated economic risks. Depending on the degree of formation, rust consists of several layers of iron in different oxidation states. The brownish top layer is mostly iron (III) oxide-hydroxide [FeO(OH), Fe(OH)3] while the deepest black layers possess iron oxide (Fe2O3.nH2O). The flaky nature of surface rust meditates diffusion of oxygen and water to inner material sections which can lead to total disintegration of iron mass. As a result, it is desirable to remove rust and protect fresh surface from oxidizers. The common rust removal reagents are mainly based on complex formation of ferric ion with organic and inorganic acids such as citric acid, oxalic acid, and phosphoric acid. Rust removal ability is typically a qualitative observation which makes direct comparison between treatment options cumbersome if not impractical. In our recent work (Ahmadi et al., 2015), we have developed a colorimetric assay to measure ferric concentration in rust removal treatment media using a bacterially-produced siderophore (yersiniabactin) in comparison to a commercial rust removal reagent. In this approach, ferric concentration is correlated to the mass of rust being dissolved in the presence of different removal agents. This assay is based on a modification of the 1, 10-phenanthroline assay (Skoog and West, 1979) to enable detection using a 96-well plate format for higher throughput screening and assessment.

Materials and Reagents

  1. Rusted iron samples
  2. 1,10-phenanthroline (Sigma-Aldrich, catalog number: 131377 )
  3. Hydroxylamine hydrochloride (Sigma-Aldrich, catalog number: 159417 )
  4. Ammonium acetate (Sigma-Aldrich, catalog number: 09689 )
  5. Ammonium iron (II) sulfate hexahydrate (Sigma-Aldrich, catalog number: 09719 )
  6. Oxalic acid (Sigma-Aldrich, catalog number: 241172 )
  7. High pure water (Milli-Q)
  8. 0.3% 1,10-phenanthroline (wt/vol) (see Recipes)
  9. 1% Hydroxylamine hydrochloride (wt/vol) (see Recipes)
  10. 0.1 M Ammonium acetate (see Recipes)
  11. Ammonium iron (II) sulfate hexahydrate standard solution (see Recipes)
  12. 10% Oxalic acid (wt/vol) (see Recipes)


  1. MaxQTM 4000 Benchtop Orbital Shakers (Thermo Fisher Scientific, catalog number: SHKA4000 )
  2. Biotek Synergy 4 microplate reader (http://www.biotek.com/resources/articles/hybrid-plate-reader.html)
  3. (Preferred) Milli-Q (Millipore) water purification system
  4. (Optional) Vertical Band Saw (for machine-generated iron samples)


  1. Rust removal experiment
    1. (Optional) Machine cut the rusted iron samples (~1” x 1”).
    2. Clean the metal surface by washing it with acetone three times (50 ml total), rinse with excess high pure water, and air dry for 20 min.
    3. Insert the cleaned samples in an appropriate container (e.g., 50 ml centrifuge tube).
    4. Add the rust removal agent and then immediately submerge the sample in high pure water. The final volume should be recorded (V1).
    5. Put the tubes in shakers at 100-200 rpm.
    6. Collect 100 μl samples every 30 min for the desired time period (our test periods have ranged from 15 min to 2 h).

  2. Remaining rust removal experiment
    1. Remove the metal sample and rinse it with pure water several times.
    2. Immerse the washed samples in a known volume (V2) of 10% (wt/vol) oxalic acid solution.
    3. Shake at 100-200 rpm for 30 min.
    4. Collect 100 μl of sample.

  3. Iron (II) calibration curve
    1. Add 40 μl of 1, 10-phenanthroline solution (0.3% wt/vol), 40 μl of hydroxylamine hydrochloride solution (1% wt/vol), and 40 μl of ammonium acetate (0.1 M) to each well of a 96 well plate.
    2. Add the iron (II) working solution: 0, 10, 20, 30, 40, 50, and 90 μl to wells containing the above mixture and adjust the final volume to 250 μl with high pure water [the iron(II) concentration will be 0, 1, 2, 3, 4, 5, and 9 mg/L, respectively]. Repeat twice more to produce triplicate curves.
    3. Mix thoroughly via pipetting up and down 3 times. Allow the solutions to stand for 20 min to fully develop color.
    4. Measure the absorbance at 510 nm.
    5. Plot the calibration diagram and fit the best linear curve (Figure 1).

      Figure 1. The developed colorimetric assay. Increasing concentration of Fe (II) from zero to 10 ppm (row A and B) increases the absorbance at 510 nm linearly. Row C includes treatment samples at different time points and remaining rust after treatment with oxalic acid.

  4. Ferric content in rust removal treatment and total rust
    1. Dilute the collected samples to obtain iron ion in a range of 1 to 10 ppm or alternatively add serially diluted samples. We usually dilute our metal samples 100-400 times. Record the dilution factor (D1 for samples and D2 for total rust).
    2. Add 20 μl of diluted samples to the 96 well plate mixture of 1,10-phenanthroline solution (40 μl of 0.3% wt/vol), hydroxylamine hydrochloride solution (40 μl of 1% wt/vol), and ammonium acetate (40 μl of 0.1 M). Adjust the volume to 250 μl per well with high pure water.
    3. Mix thoroughly via pipetting up and down 3 times. Allow the solutions to stand for 20 min to fully develop color.
    4. Measure the absorbance at 510 nm.
    5. Use the calibration curve to calculate the concentration (C1 for samples and C2 for total rust). Consider the dilution of samples in the total well volume  times.

  5. Calculation
    1. Removed rust (mg):

    2. Remaining rust (mg):

    3. Rust removal (%):

Representative data

The following are typical data for a commercial rust remover (Rust-Oleum):

Table 1. Protocol results for a commercial rust remover (Rust-Oleum)

Dilution factor (D1 and D2): 1,250 times; Treatment volume for rust removal experiment (V1): 10 ml; Treatment volume for remaining rust removal experiment (V2): 15 ml.

Figure 2. Visual examples of metal samples treated with rust removal agents. Metal samples before treatment (A), after 2 h of treatment with a commercial rust removal agent (B; Rust-Oleum) and after thirty minutes treatment with 10% oxalic acid (C).


  1. A rusted iron sample was obtained from the University at Buffalo School of Engineering and Applied Sciences machine shop. Alternatively, rusted carbon steel can be prepared by spraying samples with acid (i.e., 100 mM HCl) and allowing subsequent air exposure for three hours.
  2. Instead of using oxalic acid solution (10% wt/vol), it is possible to use Clark’s solution to remove rust from steel. The solution is prepared as 2% Sb2O3 (wt/vol) and 5% SnCl2 (wt/vol) in concentrated HCl (100 ml) at room temperature with constant stirring (Moller et al., 2006). However, we have found using oxalic acid solution to be more convenient and equally suitable.


  1. 0.3% 1,10-phenanthroline (wt/vol)
    Dissolve 30 mg in minimum amount of methanol (~200 µl) and adjust the volume to 10 ml with high pure water
  2. 1% Hydroxylamine hydrochloride (wt/vol)
    Dissolve 100 mg in 10 ml of high pure water
  3. 0.1 M Ammonium acetate
    Dissolve 77.1 mg in 10 ml of high pure water
  4. Ammonium iron (II) sulfate hexahydrate standard solution
    Dissolve 176 mg in 10 ml of high pure water. This will serve as an iron stock solution [2,500 mg/L of iron (II)]. Dilute the stock solution 100x to make a working iron solution [25 mg/L of pure iron (II)].
  5. 10% Oxalic acid (wt/vol)
    Dissolve 10 g of acid in 100 ml of high pure water.
    Note:In all experiments high pure water has been used and unless otherwise stated, the experiments were performed at ambient temperature (~22 °C).


The authors recognize support from the New York State Pollution Prevention Institute, the NSFI-Corps program, and a SUNY-4F grant. The protocol provided here was adapted from work previously published (Ahmadi et al., 2015).


  1. Ahmadi, M. K., Fawaz, S., Jones, C. H., Zhang, G. and Pfeifer, B. A. (2015). Total biosynthesis and diverse applications of the nonribosomal peptide-polyketide siderophore yersiniabactin. Appl Environ Microbiol 81(16): 5290-5298.
  2. Moller, H., Boshoff, E. T. and Froneman, H. (2006). The corrosion behaviour of a low carbon steel in natural and synthetic seawaters. Journal of the South African Institute of Mining and Metallurgy 106(8): 585-592.
  3. Skoog, D. A. and West, D. M. (1979). Fundamentals of analytical chemistry, 2nd ed. Mir. Chapter 29.


铁氧化,被称为生锈,导致财产损失和相关的经济风险的巨大损失。根据形成的程度,锈由几层不同氧化态的铁组成。褐色顶层主要是氧化铁(III) - 氧化物[FeO(OH),Fe(OH)3],而最深的黑色层具有氧化铁(Fe 2 O 3) O 3 nH O)。表面锈蚀的片状性质沉积了氧气和水扩散到内部材料部分,这可能导致铁质的完全分解。因此,希望除去锈和保护新鲜表面免受氧化剂的影响。常见的除锈剂主要基于三价铁离子与有机和无机酸如柠檬酸,草酸和磷酸的络合物形成。除锈能力通常是定性观察,其使得治疗选择之间的直接比较繁琐(如果不是不切实际的话)。在我们最近的工作(Ahmadi等人,2015)中,我们开发了一种比色测定法,用于测量除铁处理介质中的铁浓度,使用细菌产生的铁载体(yersiniabactin)与商业生锈去除试剂。在该方法中,铁浓度与在不同去除剂的存在下溶解的锈的质量相关。该测定基于1,10-菲咯啉测定(Skoog和West,1979)的修饰,以使得能够使用96孔板格式进行更高通量筛选和评估的检测。


  1. 生锈的铁样品
  2. 1,10-菲咯啉(Sigma-Aldrich,目录号:131377)
  3. 盐酸羟胺(Sigma-Aldrich,目录号:159417)
  4. 乙酸铵(Sigma-Aldrich,目录号:09689)
  5. 硫酸铁(II)六水合物(Sigma-Aldrich,目录号:09719)
  6. 草酸(Sigma-Aldrich,目录号:241172)
  7. 高纯水(Milli-Q)
  8. 0.3%1,10-菲咯啉(wt/vol)(参见配方)
  9. 1%盐酸羟胺(wt/vol)(参见配方)
  10. 0.1 M乙酸铵(见配方)
  11. 硫酸铁(II)六水合物标准溶液(参见配方)
  12. 10%草酸(wt/vol)(参见配方)


  1. MaxQ TM 4000台式轨道振动器(Thermo Fisher Scientific,目录号:SHKA4000)
  2. Biotek Synergy 4酶标仪( http://www.biotek.com/resources /articles/hybrid-plate-reader.html
  3. (优选)Milli-Q(Millipore)水净化系统
  4. (可选)垂直带锯(用于机器生成的铁样品)


  1. 除锈实验
    1. (可选)切割生锈的铁样品(约1"x 1")
    2. 清洁 金属表面用丙酮洗涤三次(总共50ml), 用过量的高纯水冲洗,并空气干燥20分钟。
    3. 将清洁的样品放入适当的容器(例如,,50ml离心管)。
    4. 加入除锈剂,然后立即浸没样品 在高纯水中。应记录最终体积(V1)。
    5. 将管在振荡器中以100-200rpm。
    6. 每30分钟收集100微升样品所需的时间(我们的测试周期范围从15分钟到2小时)。

  2. 剩余除锈实验
    1. 取出金属样品,用纯水冲洗几次。
    2. 将已洗涤的样品浸入已知体积(V2)的10%(wt/vol)草酸溶液中。
    3. 在100-200rpm摇动30分钟。
    4. 收集100μl样品。

  3. 铁(II)校准曲线
    1. 加入40μl的1,10-菲咯啉溶液(0.3%wt/vol),40μl 盐酸羟胺溶液(1%wt/vol)和40μl铵 乙酸盐(0.1M)加入96孔板的每个孔中。
    2. 添加铁 (II)工作溶液:0,10,20,30,40,50和90μl至孔中 含有上述混合物并用最终体积调节至250μl 高纯水[铁(II)浓度将为0,1,2,3,4,5, 和9mg/L]。重复两次以产生一式三份 曲线。
    3. 充分混合通过吹吸上下和3次。让溶液静置20分钟以充分显色。
    4. 测量510 nm处的吸光度。
    5. 绘制校准图并拟合最佳线性曲线(图1)。

      图1.开发的比色测定。增加的浓度 ?从0到10ppm(A和B行)的Fe(II)增加了的吸光度 510 nm。行C包括在不同时间的处理样品 点和用草酸处理后的残留锈

  4. 除锈处理中的铁含量和总锈
    1. 稀释收集的样品以获得1至10范围内的铁离子 ppm或者加入连续稀释的样品。我们通常稀释我们的 ?金属样品100-400次。记录稀释因子(D1为样品 ?和D2为总生锈)
    2. 加入20μl稀释的样品到96 孔板的1,10-菲咯啉溶液(40μl的0.3% wt/vol),盐酸羟胺溶液(40μl的1%wt/vol) 乙酸铵(40μl,0.1M)。调整体积到250微升/孔 用高纯水。
    3. 充分混合通过吹吸上下和3次。让溶液静置20分钟以充分显色。
    4. 测量510 nm处的吸光度。
    5. 使用校准曲线计算浓度(C1 样品和C2为总锈)。考虑样品中的稀释 总井容积  次。

  5. 计算
    1. 去除锈(mg):

    2. 剩余锈(mg):

    3. 除锈(%):




稀释因子(D1和D2):1,250 ;除锈实验处理体积(V1):10ml;剩余除锈实验的处理体积(V2):15ml

图2.用除锈剂处理的金属样品的视觉实例。 处理前(A),用商业除锈剂(B; Rust-Oleum)处理2小时后,用10%草酸(C)处理30分钟后的金属样品。


  1. 生锈的铁样品从布法罗工程和应用科学大学机械工厂的大学获得。或者,生锈的碳钢可以通过用酸(即100mM HCl)喷射样品并随后空气暴露3小时来制备。
  2. 代替使用草酸溶液(10%wt/vol),可以使用Clark溶液来除去钢中的锈。将该溶液制备为在浓HCl中的2%Sb 2 O 3(wt/vol)和5%SnCl 2(wt/vol) (100ml),在室温下不断搅拌(Moller等人,2006)。然而,我们发现使用草酸溶液更方便和同样合适


  1. 0.3%1,10-菲咯啉(wt/vol)
  2. 1%盐酸羟胺(wt/vol)
  3. 0.1M乙酸铵
  4. 硫酸铁(II)六水合物标准溶液
  5. 10%草酸(wt/vol)




  1. Ahmadi,M.K.,Fawaz,S.,Jones,C.H.,Zhang,G.and Pfeifer,B.A。(2015)。 非生物合成和非核糖体肽 - 聚酮化物铁载体yersiniabactin的多样化应用。 Appl Environ Microbiol 81(16):5290-5298。
  2. Moller,H.,Boshoff,E.T.和Froneman,H。(2006)。 低碳钢在天然和合成海水中的腐蚀行为。 南非采矿与冶金研究所杂志 106(8):585-592。
  3. Skoog,D.A。和West,D.M。(1979)。分析化学基础,2 nd 。 Mir。第29章。
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引用:Ahmadi, M. K. and Pfeifer, B. A. (2016). Rust Removal Experiments. Bio-protocol 6(7): e1776. DOI: 10.21769/BioProtoc.1776.