Synthesis of nanodiamond derivatives carrying amino functions and quantification by a modified kaiser test

Article abstract of DOI:10.3762/bjoc.10.288

Nanodiamonds functionalized with different organic moieties carrying terminal amino groups have been synthesized. These include conjugates generated by Diels-Alder reactions of ortho -quinodimethanes formed in situ from pyrazine and 5,6-dihydrocyclobuta[ d]pyrimidine derivatives. For the quantification of primary amino groups a modified photometric assay based on the Kaiser test has been developed and validated for different types of aminated nanodiamond. The results correspond well to values obtained by thermogravimetry. The method represents an alternative wet-chemical quantification method in cases where other techniques like elemental analysis fail due to unfavourable combustion behaviour of the analyte or other impediments.

Full text of DOI:10.3762/bjoc.10.288

Synthesis of nanodiamond derivatives carrying amino
functions and quantification by a modified Kaiser test
Gerald Jarre1, Steffen Heyer1, Elisabeth Memmel1, Thomas Meinhardt1
and Anke Krueger*1,2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 2729–2737.
1Institute for Organic Chemistry, Julius-Maximilians-Universität
Würzburg, Am Hubland, 97074 Würzburg, Germany and 2Wilhelm
Conrad Röntgen Research Center for Complex Material Systems
(RCCM), Julius-Maximilians-Universität Würzburg, Germany
doi:10.3762/bjoc.10.288
Received: 31 July 2014
Accepted: 24 October 2014
Published: 20 November 2014
Email:
Anke Krueger* - anke.krueger@uni-wuerzburg.de
This article is part of the Thematic Series "Functionalized carbon
nanomaterials".
* Corresponding author
Associate Editor: P. J. Skabara
Keywords:
amino groups; carbon nanomaterials; Diels–Alder reaction; Kaiser
test; nanodiamond; pyrazine
© 2014 Jarre et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Nanodiamonds functionalized with different organic moieties carrying terminal amino groups have been synthesized. These include
conjugates generated by Diels–Alder reactions of ortho-quinodimethanes formed in situ from pyrazine and 5,6-dihydro-
cyclobuta[d]pyrimidine derivatives. For the quantification of primary amino groups a modified photometric assay based on the
Kaiser test has been developed and validated for different types of aminated nanodiamond. The results correspond well to values
obtained by thermogravimetry. The method represents an alternative wet-chemical quantification method in cases where other tech-
niques like elemental analysis fail due to unfavourable combustion behaviour of the analyte or other impediments.
Introduction
Surface bound amino groups are most versatile for the grafting termination with amino groups is typically not complete and the
of larger moieties onto the surface of nanoparticles. Typically, stability of amino groups directly bound to the diamond surface
they are used for the formation of amides using protocols from is limited as they are at least partially replaced by hydroxy
peptide chemistry or in reductive aminations [1,2]. Addi- groups under ambient conditions [4].
tionally, amino groups have an influence on the surface polarity
and dispersion behaviour due to their protic character and the Therefore, in most cases linkers are used in order to establish
possibility to protonate the nitrogen.
stable bonding situations on the surface. These include silox-
anes (e.g., APTMS) [5], alkyl chains connected to the diamond
In the case of diamond the direct amination of the surface has surface by amide or ester bonds [6], and aminomethyl groups
rarely been reported. Sotowa et al. used the photochemical reac- formed by the nucleophilic substitution of tosylated OH groups
tion of ammonia with chlorinated diamond [3]. However, the by cyanide followed by a reduction with LiAlH4 [7]. Except for
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the last case, the linkers are coupled to the diamond surface via the one for benzenic starting materials by in situ generation of
heteroatoms. These sites are more easily cleaved compared to the diene and reaction with surface π-bonds on the nanodia-
carbon–carbon single bonds, e.g., in a physiological environ- mond acting as dienophiles. The successful grafting can be
ment or other aqueous conditions. It is hence of interest to monitored by the appearance of the distinct signal of nitriles in
develop methods that form stable bonds between the diamond the IR spectrum at ~2225 cm−1 (Figure 1). In addition the
surface and the aminated moiety. One commonly used method aromatic vibration modes of C=N and C=C bonds are observed
is the (in situ) formation of aromatic diazonium salts and the at 1613 and 1530 cm−1. The removal of solely adsorbed reagent
reaction of the aryl residue with π-bonds, hydrogenated sites or is ensured by a thorough washing procedure and control of the
various surface groups (in this case via heteroatoms) on the washing solutions using TLC. The functionalized nanodiamond
diamond surface [8-10]. Recently, we have reported an effi- 4 forms very stable colloidal solutions in organic solvents such
cient method for the grafting of organic moieties onto diamond as acetone or dichloromethane with very low particle sizes close
carrying sp2 carbon atoms at its surface using the Diels–Alder to the size of the pristine primary particles. The determination
reaction of ortho-quinodimethanes onto π-bonds on the particle of the surface loading with the pyrazine moieties, however,
surface. By this reaction two C–C single bonds are formed in leads to contradictory results at first sight. In the elemental
one step, thus increasing the stability of the conjugate [11]. We analysis by combustion a surprisingly high surface loading of
were able to show that the aromatic molecules immobilized by 1.56 mmol g−1 is measured whereas the thermogravimetric
this technique can be further modified using conventional syn- analysis yields a value of 0.61 mmol g−1. It appears that nanodi-
thetic chemistry [12]. This enables the utilization of such amond-carrying nitrogen-rich aromatic moieties have a different
diamond conjugates for biomedical applications as the C–C combustion behaviour compared to other diamond derivatives.
bound linker between the nanoparticle and the functional The observed incomplete combustion leads to an underestima-
moiety is not prone to hydrolytic or enzymatic decay. Stable tion of the carbon content and hence overestimated values for
conjugation is an essential prerequisite for applications such as nitrogen and hydrogen (and sulfur, if present). As will be
labelling or targeting.
discussed below, the wet-chemical quantification supports the
value obtained by thermogravimetry and hence one has to
Here we report on the grafting of nitrogen-containing hetero- assume that combustion analysis is not suitable for this type of
cyclic aromatic compounds using the Diels–Alder reaction of nanodiamond derivatives.
suitable starting materials and diamond carrying sufficient
amounts of sp2 carbon in order to allow the cycloaddition to The dicyanopyrazine conjugate 4 can be submitted to a reduc-
occur. The aim was to increase the surface loading with tion of the nitrile groups using borane solution in THF. This
aromatic moieties compared to the benzenic moieties due to the mild yet efficient reaction leads to the complete transformation
modified reactivity of the respective precursors.
of the nitriles to amino groups as evidenced by the disappear-
ance of the IR signals of the CN group. Furthermore, no diffi-
cult to remove side products are formed. The borate generated
by the hydrolysis is easily washed away with water. Again, the
value for the surface loading shows a strong discrepancy
between TGA and elemental analysis. Both methods show the
completely retained surface architecture by comparable values
for the dinitrile 4 and the reduction product 5. However, the
incomplete combustion of the nanodiamond samples leads to
overestimated values of 1.75 mmol g−1 in the case of combus-
tion analysis. The loading determined by TGA (0.60 mmol g−1)
is corroborated by the value obtained in the modified Kaiser test
(see below). The reduction of the nitrile groups has an impor-
tant influence on the colloidal stability of the ND conjugate.
Results and Discussion
The functionalization of nanodiamond with
aromatic moieties carrying amino groups
For the synthesis of a broad variety of aminated nanodiamond
(ND) derivatives, a series of nanodiamond conjugates with
different linker systems was synthesized starting from detona-
tion nanodiamond 1 and thermally annealed nanodiamond 2 as
depicted in Scheme 1. Besides the novel functionalization
methods using pyrazines and cyclobutenes (see below) several
nanodiamond conjugates were prepared for comparison similar
to already reported procedures.
It is known that pyrazine derivatives react with fullerenes in a Whereas the nitrile derivate 4 is soluble in rather nonpolar
cycloaddition reaction [13]. Pyrazine 3 was synthesized from organic solvents, the amino derivate 5 forms stable aqueous
1,4-dibromobuta-2,3-dione and diaminomaleonitrile similar to a colloids with well dispersed primary particles of nanodiamond.
procedure reported by Fukunishi and coworkers [14]. The
resulting 2,3-bis(bromomethyl)-5,6-dicyanopyrazine (3) is a The use of the pyrazine-derived reagents enables the grafting of
precursor for the respective ortho-quinodimethane required for ~0.6 mmol g−1 of the aromatic moieties onto the particle
the Diels–Alder reaction. The reaction proceeds in analogy to surface, possessing 1.2 mmol g−1 of amino groups. Comparing
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Scheme 1: Synthesis of nanodiamond derivatives carrying primary amino groups. a) Δ, b) 18-crown-6, KI, c) BH3·THF, d) 4-aminobenzonitrile,
isopentyl nitrite, e) BH3·THF, f) Δ, 1,2-dichlorobenzene, g) MCPBA, h) NH3, i) BH3·THF, j) APTMS, k) 4-aminobenzyl(N-Boc)amine, isopentyl nitrite,
l) TFA.
this to the typical value of 0.15 mmol g−1 for the Diels–Alder
reaction of the non-functionalized ortho-quinodimethane [11]
proves that a significantly increased grafting is observed using
the pyrazine derivative.
So far, the reported cycloaddition reactions of ortho-quinodi-
methanes leading to the grafting of aromatic rings by
Diels–Alder reaction were based on the 1,4-elimination from
suitable precursors. This step requires reagents such as potas-
sium iodide and 18-crown-6 and hence the removal of the
resulting side products [11]. Another approach to ortho-quino-
dimethanes makes use of the thermal ring opening of
Figure 1: FTIR-spectra of annealed nanodiamond 2 (a), nitrile 4 (b)
cyclobutenes. This system has been successfully used for the
functionalization of fullerenes [15]. The precursor 8 was
synthesized according to Martinez et al. starting from cyclobu-
tanone and methyl thiocyanate [16]. The cyclobutene ring opens
at temperatures around 190 °C and the ortho-quinodimethane is
and amine 5 (c). As can be seen from the disappearance of the CN
signal after the reduction of 4 the nitrile is fully converted into the
aminomethyl group. The C=O signal position is given as reference to
show the absence of respective groups. The peaks marked with an
arrow (CO2*) at 2200–2300 cm−1 are due to carbon dioxide from
ambient air.
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formed in situ. The reaction with the nanodiamond surface control experiment using nanodiamond not functionalized with
occurs simultaneously as soon as the diene is formed. The the pyrimidine proved the reactivity of surface groups of the
solvent dichlorobenzene was chosen not only for its suitable nanodiamond (CH, OH, π-bonds etc. are present on thermally
boiling point but also for the efficient solubilization of the reac- annealed nanodiamond [18]) with MCPBA showing the forma-
tion participants.
tion of oxidized surface groups and an increase of the respec-
tive IR signals for carbonyl groups (see Supporting Information
The ND conjugate 9 shows characteristic signals in the IR spec- File 1 for details). When the mass loss related to the groups
trum (Figure 2), namely the vibrations at 1590, 1530 and generated by this side reaction is subtracted from the overall
1430 cm−1 are caused by the grafted heteroaromatic moieties. result using the results from the control experiment, the values
The spectrum corresponds well to the signals observed for 2,4- for the surface loading for compound 10 measured by combus-
bis(methylthio)pyrimidine [17]. The surface loading was deter- tion analysis (using values for N and S and hence only consid-
mined by TGA and elemental analysis based on the nitrogen ering the heterocycle) and TGA (where the combined mass loss
and sulfur content. All analytical methods yield comparable for the heterocycle and the oxidized groups is measured) are
results around 0.2 mmol g−1.
fully comparable.
The generation of the amino functions is carried out using
gaseous ammonia and hence the isolation and purification of the
reaction product 11 is very simple. As expected the intensity of
the sulfone related IR bands decreases upon amination (but does
not vanish as only the SO2Me group in 2-position is exchanged)
and new signals corresponding to amino functions (1590 and
~3450 cm−1) appear in the spectrum. Furthermore, the sulfur
content of the material is reduced to one half of the original
value. This shows that the reaction occurs with high efficiency
as the remaining sulfur stems from the conserved sulfone group.
From these results it is obvious that the thermal ring opening of
suitable cyclobutenes is another valuable addition to the port-
folio of cycloaddition related surface reactions of nanodiamond.
It allows the efficient immobilization of quite complex organic
moieties without the use of auxiliaries or further reagents. No
problematic side products or waste are formed and the selective
transformation of the different functional groups on the
aromatic ring also allows the orthogonal functionalization with
different moieties in the subsequent grafting steps.
Figure 2: FTIR spectra (left) of compounds 2 (a), 9 (b), 10 (c) and 11
(d). The formation of the sulfone groups in 10 and the reduction of
signal intensity for 11 are clearly visible. In the spectrum of 10 and 11
the carbonyl signals generated by the side reaction of MCPBA with
surface groups are marked with a dotted line (the CO2 signal is due to
ambient air).
Analysis of surface amino groups on
The further reaction depicted in Scheme 1 enables the forma- nanodiamond
tion of an aromatic amine at the 2-position of the ring by the Besides spectroscopic techniques, thermogravimetry and
oxidation of the thioethers and subsequent substitution of the combustion (elemental) analysis are typically applied for the
sulfone by an amino function. Throughout the transformation quantification of surface groups on nanomaterials. In most cases
the surface loading (see TGA data and elemental analysis in the these methods yield reliable results and the obtained values are
experimental part) with the organic moieties remains the same comparable for the different methods. However, it turned out
showing the high stability of the double grafting by covalent that for some nanodiamond derivatives with a high concentra-
C–C bonds. However, during the oxidation of the thioethers to tion of nitrogen in the surface functionalities the discrepancy
the sulfones a side reaction has been observed. MCPBA reacts between surface loadings determined using elemental analysis
not only with the sulfur-containing groups in 9 but also with the and TGA was exceeding the variations brought upon by
annealed diamond surface itself as evidenced by the emergence different measurement methods and experimental deviations.
of IR signals of carbonyl and ether bonds besides the expected As shown in Table 1 the values obtained by combustion
signals at 1320 and 1140 cm−1 for the symmetric stretching analysis were three times higher than those measured by TGA.
mode of the sulfones (see spectra c) and d) in Figure 2). A Reasons for this difference can be the incomplete combustion
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Table 1: Surface loading of nanodiamond-carrying amino groups on different linkers using three independent quantification methods.
sample (sample mass in
Kaiser test /mg)
surface loading (mmol g−1)
Kaiser test
surface loading (mmol g−1)
elemental analysis
surface loading (mmol g−1)
TGAa
5 (0.8)
7 (0.7)
11 (–)
0.60
0.17
–c
1.75b
0.61
0.19
0.23
0.49e
0.32
0.22
0.23 (0.20)d
1.07
13 (1.2)
15 (2.0)
1.05
0.27
0.29
aSurface loading has been calculated from the mass loss step related to the removal of the organic matter. The desorption of water has been ensured
by heating to 120 °C where the desorption step ends, bthis unexpectedly high value is due to the incomplete combustion of this sample type;
caromatic amines cannot be quantified using the Kaiser test as no proton in the α-position is available, the qualitative test was negative as expected;
dvalue based on sulfur (nitrogen) content; ethe thermogravimetric analysis of silanized samples often gives less pronounced steps in the thermogram,
making the quantification less reliable, furthermore the formation of a stable silica shell reduces the observed mass loss.
and soot formation during elemental analysis. As evidenced by test results either [24]. One report on the non-quantitative appli-
black residues in the crucibles used for the combustion this cation of the Kaiser test on ethylene diamine functionalized
hypothesis is reasonable. Another aspect to be taken into nanodiamond has been reported recently [25].
consideration is the accessibility of functional groups. Using
elemental analysis the entire amount of nitrogen in the sample For comparison, the monoaminated diamond conjugates 7, 13
will be determined, including nitrogen in the diamond lattice and 15 have been synthesized (see Supporting Information
(which can be segregated of nitrogen content in the pristine ma- File 1 for details). As it can be seen from the analytical data the
terial after the measurement) [19] and all amino groups surface loading with amino groups for 7 and 15 is about
anywhere in the sample. However, this does not mean that all ~0.2 mmol g−1, a very typical value for this type of reaction.
these amino groups are accessible, e.g., for further coupling The observed surface loading for 13 is higher due to secondary
reactions due to agglomeration or porosity of the material.
reactions of already grafted alkoxy silanes [5].
Furthermore, the cleavage of strongly bound organic moieties In a first series of experiments the assay was carried out on
from the surface will occur only at rather elevated temperature. nanodiamond samples 5 and 15 using the protocol reported by
In this case the thermogravimetric step will be less pronounced Troll et al. [22]. Surprisingly, very low values for the amino
as it coincides with the early sublimation of carbonaceous ma- group loading were obtained for these samples in the first
terial from the diamond surface. The latter sets in at tempera- attempts of wet-chemical analysis (see Supporting Information
tures above 450 °C, where the cleavage and desorption of File 1 for details). However, as 15 did not show a discrepancy
strongly bound species is not yet completed. Hence, this overlay between combustion and thermogravimetric analysis the Kaiser
makes an unambiguous determination of the step temperatures test was apparently yielding false results. To elucidate the origin
in TGA challenging. It is therefore attractive to find other, inde- of the deviation a deeper look into the mechanism of the forma-
pendent methods for the quantitative determination of func- tion of Ruhemann’s Blue is required. An important intermedi-
tional groups on the diamond surface, especially those ate is hydrindantin, which is formed upon reduction using KCN
imparting high nitrogen content, such as amino groups. (see Supporting Information File 1 for a complete reaction
mechanism). Its role in the process is still under discussion, and
A very popular technique for the quantification of primary the rate constant of the reaction depends on the hydrindantin
amino groups is the so-called Kaiser test [20]. It makes use of concentration in a complex manner – emphasizing its role as an
the formation of Ruhemann’s Blue by the reaction of ninhy- important intermediate [26,27]. Moore and coworkers reported
drine with the primary amino groups [21,22]. A major applica- that in the event of insufficient amounts of reducing agent the
tion of this assay is the test on residual amino groups of amino formation of the coloured species is too low and variable [28].
acids in solid-phase supported peptide synthesis. A report on a Although the amount of KCN in the standard protocol is taking
quantitative variation of the Kaiser test was published by Sarin this finding into account we suspected that nanodiamond itself
et al. [23]. As this assay is colorimetric, its execution is very could be adsorbing the intermediate species and thus hampering
convenient. One limitation of the method, though, is the restric- the further proceeding of the reaction. It is well known, that
tion to primary amines. Secondary amines cannot be detected hydrophilic nanodiamond (and samples carrying amino groups
reliably and aromatic and tertiary amines do not yield positive count among these) is prone to unspecific adsorption of organic
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molecules via hydrogen bonding and other interactions [29]. reproducibility and correspond well within the error limits to
Therefore it seems reasonable to assume that the rather low the values obtained by thermogravimetry in all cases except 13
amounts of hydrindantin formed upon the addition of KCN can and with elemental analysis when complete combustion of the
be adsorbed on the diamond surface and hence withdrawn from nanodiamond derivative is achieved. The deviation of the TGA
the reaction mixture. By forming higher amounts of hydrin- results in the case of 13 is most likely due to the incomplete
dantin using increased amounts of the reducing agents and thus removal of the siloxane shell, which can crosslink and form a
increasing the concentration of the free intermediate in the solu- glassy layer around the nanodiamond core upon heating. This
tion we hoped to overcome this issue. By tripling the amount of reduces the apparent surface loading as complete removal is
KCN it was indeed possible to obtain reproducible values for assumed in the calculation. As can be seen from the results, the
the surface loading with NH2 groups that correspond to the modified Kaiser test is a valuable tool for the quantification of
values obtained by other methods. Due to the increased KCN amino groups on nanoparticle surfaces, namely when only low
concentration and the increased formation of the intermediate amounts of sample are available, e.g., when using fluorescent
hydrindantin the reaction mixture shows a characteristic red nanodiamond as the starting material or when the material
colouring at elevated temperatures as already reported by Sarin shows unfavourable properties in other analytical methods.
et al. [23]. Upon cooling, this colour vanishes almost However, care should be taken in the event of strong agglomer-
completely (only a weak residual absorption at 570 nm is ation of the nanoparticles. Only the surface groups accessible by
observed in the UV–vis spectrum, which has to be taken into the reagents will be measured using this wet-chemical method.
account during the quantification). It has to be mentioned that
the addition of 60% ethanol solution should be executed only Conclusion
after the disappearance of the red colour. The back oxidation is In summary, we have established two new functionalization
otherwise hindered and a quantitative determination is rendered methods for the covalent grafting of organic moieties onto
impossible (Figure 3).
nanodiamond using pyrazine and cyclobutene precursors for the
in situ formation of ortho-quinodimethanes. These can be used
as dienes in Diels–Alder reactions with π-bonds on the surface
of nanodiamond. The use of pyrazine 3 results in a significantly
increased grafting rate compared to other aromatic moieties
linked by carbon–carbon bonds. Furthermore, a modified Kaiser
test for the quantitative analysis of primary amino groups on
nanodiamond has been developed and tested with a broad
variety of aminated diamond samples. The modified conditions
ensure the formation of sufficient amounts of the required
hydrindantin intermediate and deliver reproducible and compa-
rable results. The method can be used for other nanoparticles
with similar adsorption properties as well and represents a
useful addition to the wet-chemical analysis of functional
Figure 3: Modified Kaiser test. a) Left: control, right: positive result;
b) left: control, control with premature addition of 60% ethanol solution
(before solution has fully cooled down and colour has vanished).
In order to calibrate the measurement a reference sample for the groups on nanomaterials.
colorimetric assay was used. Using benzylamine at different
Experimental
concentrations in water a standard graph was established for the
new test protocol (with triple amount of KCN and delay before General chemicals and methods
the addition of the ethanol) measuring the extinction of the Detonation diamond has been purchased from Gansu Lingyun
Ruhemann’s Blue in the absorption spectra. Additionally, the Corp. (Lanzhou, China). All other chemicals have been
required reaction time was determined using nanodiamond 5 purchased from Aldrich and Fluka and have been used without
with the same protocol and taking samples of 1 mL at time further purification. Solvents have been dried according to stan-
intervals. After work-up these samples were submitted to the dard procedures.
colorimetric assay and a minimum duration until complete
transformation was found to be in the range of 10 min. Using The following apparatus have been used for the experimental
this optimized assay protocol a series of aminated nanodia- work:
mond samples have been examined (Table 1). Furthermore, the
dicyano derivative 4 and the Boc-protected derivative 14 were Sonication: Ultrasound bath: Bandelin Sonorex Digitec Type
tested and showed a negative test result as expected. The results DT52 (max. 80 W, 35 kHz), centrifuges: Hettich EBA 21 Type
of the Kaiser test for compounds 5, 7, 13 and 15 show a high 1004, FTIR: Perkin-Elmer 1600 Series with ATR equipment
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Beilstein J. Org. Chem. 2014, 10, 2729–2737.
and home-made vacuum cell (KBr pellet). Samples for calibration curve and the reaction time optimization see
measurements using the vacuum cell were heated at Supporting Information File 1.
100–120 °C for 2 h in vacuo in order to remove most of the
Synthesis of aminated nanodiamond
water adsorbed on the diamond surface; UV–vis: Kontron
Instruments Uvikon 943, Thermogravimetry (TGA): Perkin samples
Elmer STA 6000, measurements were performed in high purity Synthesis of 4: A suspension of 250 mg of annealed detonation
nitrogen with a heating rate of 10 °C min−1 (ca. 20 mg of diamond 2 (2 h, 750 °C, vacuum), 1.16 g (6.96 mmol) potas-
sample were used for each analysis); elemental analysis: Euro sium iodide, 520 mg (1.81 mmol) 2,3-bis(bromomethyl)-5,6-
Vector Euro EA 3000 Series; NMR: Bruker AC 250, tube dicyanopyrazine (3) and 660 mg (2.50 mmol) 18-crown-6 in
furnace: Thermolyne Type 21100.
10 mL of abs. toluene was heated for 72 h under reflux and
nitrogen atmosphere. After cooling the diamond particles were
isolated by centrifugation (15000 min−1, 10 min). The precipi-
Reagents for Kaiser test
1.) Buffer pH 5.5: 36 g of sodium acetate was dissolved in tate was washed eight times with acetone, three times with
6.9 mL of conc. acetic acid and distilled water was added until a water, three times with acetone and three times with
volume of 100 mL was reached.
dichloromethane in consecutive dispersion/centrifugation
cycles. 150 mg (60% yield) of a black solid was obtained as the
2.) 5% ninhydrin solution: 5 g of ninhydrin were dissolved in product. Elemental analysis: N: 10.4%, C: 74.1%, H: 1.7%;
100 mL of ethanol.
FTIR (vacuum cell) : 3370, 2923 (CH), 2227 (CN), 1635,
1614, 1529, 1384, 1197, 1110. 842, 696 cm−1; TGA (% weight
3.) KCN pyridine reagent: 2 mL of 0.03 M KCN solution were loss): 120–460 °C: 9.6% (fragment: C8H4N4; 156 g mol−1);
diluted to a volume of 100 mL with pyridine.
Surface loading: 1.56 mmol g−1 (calculated from EA),
0.61 mmol g−1 (calculated from TGA), Kaiser-Test: negative,
4.) Phenol solution: 80 g of phenol were dissolved in 20 mL of zetapotential: not measured as no stable colloidal solution in
ethanol while being gently heated. water was obtained. Particle size: 15 nm (acetone).
5.) Ethanol solution: 60 mL of ethanol was diluted with 40 mL Synthesis of 5: 65 mg of 4 was suspended in 5 mL BH3∙THF
of deionized water.
(1 M) and heated for 20 h under nitrogen atmosphere at 50 °C.
Excess borane was decomposed by adding 2 N HCl. The
diamond particles were isolated by centrifugation (15000 min−1,
10 min). The precipitate was washed six times with a mixture of
Procedure for the quantification of amino
groups on nanodiamond
The nanodiamond carrying amino groups (typically 0.5–2 mg of dioxane/water (9:1), three times with acetone and three times
the powder) was suspended in 1 mL of dist. water. To this with dichloromethane in consecutive dispersion/centrifugation
suspension 1 mL of the buffer solution (no. 1, see above) was cycles. 49 mg (75% yield) of a black solid was obtained as the
added and the mixture was sonicated in an ultrasonic bath for product. Elemental analysis: N: 11.2%, C: 68.4%, H: 3.9%;
15 min. After that, 1 mL of the KCN solution (no. 3) and 1 mL FTIR (vacuum cell) : 3359, 3215, 2924 (CH), 2854 (CH),
phenol solution (no. 4) were added and the suspension was 1620 (NH), 1384, 1261, 1108, 799, 673, 614 cm−1; TGA (%
heated at 120 °C (oil bath temperature) for 10 min, 1 mL of weight loss): 140–300 °C: 10.0% (fragment: C8H12N4;
ninhydrin solution (no. 2, see above) was added and heated for 164 g mol−1); Quantitative Kaiser test: UV: A = 2.2501
another 10 min. The solution was cooled to room temperature (568 nm); Surface loading: 1.75 mmol g−1 (calculated from
within 30 min and 5 mL of the ethanol solution was added. The EA), 0.61 mmol g−1 (calculated from TGA), 0.60 mmol g−1
solid was separated by centrifugation and from the supernatant (Kaiser-Test); zetapotential: 19.9 mV, particle size: 13 nm
a UV-spectrum was recorded.
(water).
The following equation was used for the quantification:
Synthesis of 9: 120 mg of annealed nanodiamond 2 was
suspended in a solution of 120 mg (0.61 mmol) of 8 in 10 mL
1,2-dichlorobenzene and heated to reflux for 20 h under
nitrogen atmosphere. The solid was recovered by centrifuga-
tion and the supernatant was discarded. The solid was washed
surface loading [mmol g−1]
with
A = absorption; X = weight of diamond sample [g]; N = number six times with acetone, five times with water and five times
of amino groups per molecule. The absolute values are obtained with dichloromethane in consecutive dispersion/centrifugation
from the calibration curve measured with benzylamine. For the cycles. After drying at 70 °C for 24 h the product was obtained
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Beilstein J. Org. Chem. 2014, 10, 2729–2737.
as a dark grey solid. Yield: 101 mg (84%). Elemental analysis:
C: 88.9%, H: 1.3%, N: 2.9%, S: 1.4%; FTIR (vacuum cell)
Supporting Information
:
3430 (br), 2934 (s, ν(C-H)), 2871 (s, ν(C-H)), 1589 (w,
ν(C=Carom.)), 1528 (m, ν(C=Carom.)), 1425 (m, ν(C=Carom.)),
1312 (w), 1252 (w), 1110 (m), 860 (w), 810 (w), 677 (m) cm−1;
TGA (% weight loss): 150–540 °C: 4.2% (C8H10N2S2); surface
loading: 0.23 mmol g−1 (calculated from sulfur content in EA),
0.21 mmol g−1 (calculated from nitrogen content in EA),
0.21 mmol g−1 (calculated from TGA); zetapotential: +34.2 mV
(measured in aqueous dispersion).
Supporting Information File 1
Details for calibration of the Kaiser test, test robustness and
optimization of the reaction time, reaction mechanism for
colorimetric assay; synthesis of organic precursor
compounds and further nanodiamond derivatives.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-288-S1.pdf]
Synthesis of 10: 50 mg of functionalized nanodiamond 9 were
suspended in a solution of 170 mg (0.99 mmol) meta-chloroper-
oxybenzoic acid (MCPBA) in 10 mL dry dichloromethane and
stirred for 20 h at room temperature under nitrogen atmosphere.
The solid was separated by centrifugation and the supernatant
was discarded. The solid was washed six times with
dichloromethane, six times with acetone and six times with
water in consecutive dispersion/centrifugation cycles. After
drying at 70 °C for 24 h the product was obtained as a grey
solid. Yield: 40 mg (80%). Elemental analysis: C: 85.9%, H:
1.2%, N: 3.0%, S: 1.3%; FTIR (vacuum cell) : 3433 (br), 2931
(m, ν(C-H)), 1722 (m, ν(C=O)), 1630 (m), 1550 (w), 1405 (w),
1320 (s, ν(C2SO2)), 1211 (w), 1140 (s, ν(C2SO2)), 1046 (m),
968 (m), 770 (m), 670 (m) cm−1; TGA: (% weight loss):
130–520 °C: 7.0% (C8H10N2O4S2) (including 1.4% direct
surface oxidation by MCPBA as demonstrated in a control
experiment); surface loading: 0.21 mmol g−1 (calculated from
sulfur content in EA), 0.23 mmol g−1 (calculated from nitrogen
content in EA), 0.21 mmol g−1 (calculated from corrected TGA
data); zetapotential: +19.8 mV (measured in aqueous disper-
sion).
Acknowledgements
We gratefully acknowledge the financial support by the
Deutsche Forschungsgemeinschaft (DFG) under projects
KR3316/1-2 and FOR1493 (KR3316/6-1), by the European
Commission (project DINAMO, contract number: 245122) and
the Fonds der Chemischen Industrie.
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