1. 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

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.

3. 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.

4. 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

5. 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.

6. 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

7. Figure 6.
Zoomed-­‐in
charts
of
the
(20,11)
reflection
of
the
vernadite
reacted
with
different
EPPS
concentration
REFERENCES
1. N.E. Good, G.D. Winget, W. Winter, T.N. Connolly, S. Izawa, R.M.M. Singh.
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)
Interference by MES [2-(4- morpholino)ethanesulfonic acid] and related buffers with
phenolic oxidation by peroxidase. Free Radic Biol Med 43:1322–1327