INTRODUCTION In order to control the pH value in our main manuscript, Mn atom exchange between aqueous Mn(II) and vernadite (δ-Mn(IV)O2 ), for the purpose of testing the behaviors and reaction rate in different pH, we used different ways to maintain the pH at a certain value we wanted to test. The pH value dropped during the atom exchange reaction; also, the presence of CO2 would lower the pH as well. First, we used 0.1M NaOH to titrate the solution few times to maintain the pH at the certain range. The second and the main method we used was adding a Good’s buffer into the solution to control the pH at around 7.5. Theoretically, an ideal buffer should not interfere chemical and physical properties of the testing substances while maintaining the pH. However, we found that some of the Good’s buffers (e.g. HEPES, MOPS, EPPS) would cause reduction of the MnO2(s) substrate that might account for ~30% of the reductant in the system. To address this problem and find an ideal, usable buffer, we tested the reactivity of five different Good’s buffers which might cause partial reduction of the MnO2(s). Therefore, we would apply the XRD analysis to test if there is a structural modification of vernadite (δ-Mn(IV)O2 ) due to the reduction reactions by the buffers. For this purpose, we set up parallel samples; one is just the MnO2 substrate as the controlled sample, and the others are the MnO2 substrate with the target buffers. Besides, we also tested MOPS and EPPS in different concentration individually to see if there is any associated difference. The results can provide inventory data of the reactivity between vernadite and these five Good’s buffers for a ubiquitous use of a high sensitive geochemical or biochemical experiment. MATERIAL AND METHODS Good’s Buffer

Good et al. purposed twelve of new buffers in 1966 for use in biological experiment. These buffers had some important properties such as the pKa value between 6 to 8, water

Buffer Name IUPAC Name Chemical Formula

pKa

HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

C8H18N2O4S

7.5

BES N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid

C6H15NO5S

7.1

MOPSO 3-Morpholino-2-hydroxypropanesulfonic acid

C7H15NO5S

6.9

EPPS 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid

C9H20N2O4S

8.0

MOPS 3-morpholinopropane-1-sulfonic acid

C7H15NO4S

7.2

Table 1. The chemical characteristic of Good’s buffers in this experiment

1

Test of δ- MnO2 Reactivity with Good’s Buffers

Apollo Yue-Nung, Lin

2

 

soluble, minimum salt effects, well-behaved cation interactions, and supposedly low affinities for biologically important metal ions1, 2, and most of them are zwitterinoic compounds (i.e. a compound with a positive and a negative at different locations within it). Therefore, Good’s buffers have been widely used in geochemical and biochemical laboratories for years. However, it’s found that some of the Good’s buffers may have interferences on plants3; furthermore, it’s been tested that Good’s buffers can also react with some metal ions to form complexes2. In our main manuscript, we tried to use Good’s buffers to control the decreasing pH of the solutions due to the redox reaction between Mn(II) and Mn(IV) that would release protons (Equation 1). Therefore, we tried to find out if there’s any redox reaction between five of the Good’s buffers and vernadite (δ-Mn(IV)O2 ) that might interfere the results of the main experiment. The five buffers which we tested were HEPES, BES, MOPSO, EPPS, and MOPS (Table 1). Mn

2+(aq) + Mn(IV)O2 (s) + 2 H2O → 2 δ -Mn(III)OOH(s) + 2 H

+(aq) (Eq. 1)

Sample Preparation

(a.) Vernadite (δ-Mn(IV)O2 ) Suspensions: First, we added 0.41µg of NaMnO4(s) and 4.89 mL of 1M NaOH into 94.70 mL DI water. Second, we added 0.952g of Mn(NO3)2(s) in 11mL DI water. Next, NaMnO4 solution was titrated by the Mn(NO3)2 solution at a rate of 200µL/min for 50 minutes (i.e. 10mL of Mn(NO3)2(aq) was added). We filtered the solution and collected the mud-like MnO2 after 2 hours settling, and the MnO2 was added into 200mL of DI water to get the vernadite suspensions.

(b.) Buffers with Vernadite powders: Certain weight of the buffer was added into the background 100 mL, 0.1M NaCl(aq) electrolyte to reach the concentration of 20mM solution. Next, we added certain amount of 0.1M NaOH(aq) to bring the pH value up to around 7.3 before 200µL of vernadite suspensions were added. The solution was settled for fully reacted for at least 5 days, and we did the filtration and washed out the remaining salt to get the dried powders of each buffer with vernadite after 2 days.

X-ray crystallography (XRD) Analysis As we mentioned above and will have further discussion later, the crystal structure of vernadite might be modified due to the redox reactions. Thus, to test if there is any crystal structure modification in vernadite by Good’s buffers due to redox reactions. We implemented the XRD test on the dried powders we prepared; for XRD analysis, the dried powders were loaded onto low background sample holders and analyzed on a Bruker D8 Advance diffractometer using Ni- filtered Cu K radiation and a LynxEye XE detector.

RESULTS AND DISCUSSIONS

Structural modification of Vernadite(δ-Mn(IV)O2 )

An unexpected discovery, the partial reduction of vernadite by HEPE, was found in our main manuscript. Therefore, we tried to test if there's other redox reaction between vernadite and other Good's buffers. There’re few differences in the XRD result between vernadite substrate and its substrate with each buffers. The XRD pattern (Fig.1) shows that there’re two additional peaks in four of five hydrated vernadite+buffers we tested (i.e. 20mM of EPPS, MOPSO, HEPES, and MOPS). It’s been proved brinessite (Mn(IV)O2) has four main reflections at ~2.4, ~1.4~7.1, and ~ 3.6 Å. But the hydrated vernadite (δ-Mn(IV)O2 )showed only two peaks at d-spacings ~2.4 and ~1.4 Å which were from the (20,11) and (31,02) reflections . It’s because the verndaite structure is rather ordered4 that shows only two diffraction peaks. Bricker (1965) considered the δ-Mn(IV)O2 as a two-dimensional particles5. The vernadite structure can be deemed as a double hexagonal layer close packed oxygen ions and water4.

Figure  1.  Powder X-ray diffraction (XRD) patterns of the the δ-MnO2 materials hydrated in 0.1 M NaCl background electrolyte at pH 7.5 in the absence or presence of 20 mM Good's buffers.

As just mentioned, two additional diffraction peaks at 7.1 and 3.6 Å are shown in the XRD pattern (Fig.1) due to the partial reduction and/or structural modification of hydrated vernadite by these buffers except BES. The presence of new peaks at 7.1 and 3.6 Å in the oxidized vernadite demonstrates the increasing staking order along the c-axis. Increased ordering of sheet stacking along c is attributed to migration of Mn(II) and/or Mn(III) cations into the interlayer region, where they displace weakly held Na+

as charge balancing cations, and mediate a higher degree of oriented sheet stacking through their relatively strong interactions with the phyllomanganate surface. Besides, the (20,11) reflection of the partial reduction samples showed distinct splittings or bulges (Fig.2), which means a change in layer sheet symmetry from hexagonal to orthogonal or triclinic by the presence of Mn(III) and/or Mn(III) due to redox.

Redox between Mn(IV) and Good’s buffers The structural modification indicated the reduction of Mn(IV) to Mn(III) by the 20mM of EPPS, MOPSO,HEPES, and MOPS. Some of the Good’s buffers are known as redutants to certain metal as well. Previous studies indicated that Cu(II) and Fe(III) could oxidize nitrogen-containing compounds such as HEPES and PIPES. Curiously, previous studies pointed out a common buffer, “Tris” ((HOCH2)3CNH2), which was known to be capable of complexing metal ions, failed to be oxidized by Cu(II). Wang and Sayre (1989) suggested that Cu(II) could be reduced by the tertiary amine in HEPES6. They claimed all tertiary amine buffers should be avoided in a case if any metal ion complex with a redox potential over 0.6V vs NHE which will have ability to oxidize tertiary amine buffers. In our experiment, the redox potential of MnO2(s) is 1.23V vs NHE7 which means Mn(IV) could oxidize the tertiary amine in the buffers. Besides, the inertness of Tris can be explained in term of it being a primary rather than tertiary amine6. However, in our experiment, we observed that BES, a tertiary amine-containing compound, was not able

Figure  2.  Zoomed-­‐in  charts  of  the  (20,11)  reflection  of  the  original  hydrated  vernadite  and  20mM-­‐buffer-­‐reacted  vernadtite  

Figure  3.  The  structure  of  the  tested  Good's  buffer  

to reduce Mn(IV). If the tertiary amine has an capability to reduce metal ion complex with Eo > 0.6V, BES should have been oxidized by Mn(IV) in our experiment. The inertness of BES is probably related to the molecular geometry of these buffers. As we can see in the Figure 3, both MOPS and MOPSO have morpholine rings, and EPPS and HEPE contain piperdine rings. These nitrogen-containing-ring compounds which are structurally related might have similar redox mechanism with Mn(IV). It’s reported that structurally related buffers or compounds might have similar redox reaction with H2O2 or peroxidase8-9. Therefore, EPPS, MOPSO, HEPES, and MOPS might have similar mechanism to react with hydrate vernadite. BES is an acyclic and tertiary amine compound without any cyclic structure that might affect the redox reaction with MnO2 or other metal complex. Otherwise, change of temperature, pH, electrolyte, concentration of BES, etc. might trigger the redox reaction with MnO2 to take place. XRD results of MOPS and EPPS in different concentration We also tested vernadite with MOPS and EPPS in different concentration to see if the partial reduction is affected by the concentration of buffers. The results (Fig.4-6) showed that the partial reduction was distinctive with both buffers of the concentration of 20, 15, and 10mM. These two buffers as reductants are quit surplus to react with the relatively lower concentration vernadite. However, the reflection of ~7.1, and ~ 3.6 Å peaks are not obvious while the concentration of MOPS and EPPS ≤1mM which means the partial reduction is not significant while the buffer concentration is low. There might be not significant Mn(III) to attract the phyllomanganate surface together. Interestingly, the count rate of peaks of a certain buff in different concentration also shows an unexpected pattern. In Figure 4, the count rates of ~7.1, and ~ 3.6 Å peaks are not proportional to the concentration of EPPS. The highest count rates are shown in the 15mM EPPS+ vernadite substrate, and they are decreasing with increasing and decreasing EPPS concentration. Fist, we expected the 20mM EPPS+ vernadite sample should have the highest counts on ~7.1, and ~ 3.6 Å peaks, but 15mM EPPS+ vernadite showed the highest counts on these two peaks. At lower concentration, the stacking order may be less due to the less presence of Mn(III) in the interlayer to bind the sheets together. The stacking order should have been increasing with increasing EPPS concentration (i.e. more Mn(III) is produced). However, the decreased counts in the 20mM EPPS+ vernadite sample indicated the stacking order decreased again. The decreasing stacking order might due to the decreasing charge on the phyllomanganate surface which might be caused by the additional Mn(III) presence in the vacancy sites. Additional Mn(III) got into the vacancy sites might lower the total negative charge of the phyllomanganate surface; therefore, the electrostatic force between Mn(III) and Mn(IV)O2 sheets might decrease that could decrease the stacking order of the sheets. We can also see the similar trend in the result of MOPS+ vernadite samples. Nevertheless, the SEM analysis is needed to take a better look at the specific morphology of the mineral structure. There are few conclusions of this work: (i) two additional peaks and the splittings showed on the XRD patterns indicate the structural modification from partial reduction of vernadite. (ii) The inertness of BES may be due to its molecular geometry. (iii) The stacking orders are not proportional to the concentration of buffers.

Figure 4. Powder X-ray diffraction (XRD) patterns of the the δ-MnO2 materials hydrated in 0.1 M NaCl background electrolyte at pH 7.5 in the presence of 20, 15, 10,5, and 1mM EPPS

Figure 5. Powder X-ray diffraction (XRD) patterns of the the δ-MnO2 materials hydrated in 0.1 M NaCl background electrolyte at pH 7.5 in the presence of 20, 15, 10,5, and 1mM MOPS

Figure 6.  Zoomed-­‐in  charts  of  the  (20,11)  reflection  of  the  vernadite  reacted  with  different  EPPS  concentration

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Biochemistry, 5 (1966), pp. 467–477. 2. R. Nakon, C.R. Krishnamoorthy. Science, 221 (1983), pp. 749–750 3. C.J. Baker et al. / Free Radical Biology & Medicine 43 (2007) 1322–1327 4. Chukhrov, F.V. (1980) Manganese minerals in clays: A Review, Clay and Clay

Minerals, vol. 28, No. 5, 346-354 5. Bricker, O. (1965) Some stability relationships in the system Mn-O2-H2O at 25˚C

and one atmosphere total pressure: American Mineralogist, v. 50, p. 1296–1354 6. Wang, F., Sayre, L.M. (1989) Oxidation of Tertiary Amine Buffers by Copper(II),

Inorganic Chemistry, Vol.28, No.2, 169-170 7. Gordon Aylward & Tristan Findlay (2008). "SI Chemical Data", 6th edition (John

Wiley & Sons, Australia), ISBN 978-0-470-81638-7 8. Zaho, G., Chasteen, N.D.,Oxidation of Good’s buffers by hydrogen peroxide, Anal.

Biochem. 349 (2006) 262–267 9. Baker CJ, Mock NM, Roberts DP, Deahl KL, Hapeman CJ, Schmidt WF et al (2007)

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