In Situ Determination of Colloidal Gold Concentrations with UV−VisSpectroscopy: Limitations and PerspectivesThomas Hendel, Maria Wuithschick, Frieder Kettemann, Alexander Birnbaum, Klaus Rademann,*and Jorg Polte*

Institut fur Chemie, Humboldt-Universitat zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany

*S Supporting Information

ABSTRACT: This paper studies the UV−vis absorbance ofcolloidal gold nanoparticles at 400 nm and validates it as amethod to determine Au(0) concentrations in colloidal goldsolutions. The method is shown to be valid with restrictionsdepending on the investigated system. The uncertainty of thedetermined Au(0) concentration can be up to 30%. Thisdeviation is the result of the combined influence of parameterssuch as particle size, surface modification, or oxidation state.However, quantifying the influence of these parametersenables a much more precise Au(0) determination for specificsystems. As an example, the reduction process of the well-known Turkevich method was monitored and the Au(0) concentrationwas determined with a deviation of less than 5%. Hence, a simple, fast, easy, and cheap in situ method for Au(0) determination isdemonstrated that has in the presence of other gold species such as Au(III) an unprecedented accuracy.

Metal nanoparticles have become an excelling researchfield with a broad variety of applications.1,2 Among

them, gold nanoparticles (GNPs) play an outstanding role,representing one of the most synthesized and intensely studiedmaterials.3,4 Synthetic pathways are manifold, resulting inGNPs of different sizes, shapes and surface functionalities5−7

that are applied in photonics,8 surface-enhanced Ramanspectroscopy,9 catalysis,10,11 and biomedicine.12 The morphol-ogy and size distribution of GNPs have been studied using avariety of analytical techniques, such as electron micros-copy,13,14 scanning probe microscopy,15,16 dynamic lightscattering (DLS),17 mass spectroscopy,18,19 small-angle X-rayscattering (SAXS),20,21 and X-ray diffraction (XRD).22

However, an exact determination of the Au(0) concentrationin a GNP solution remains a major issue.The Au(0) concentration represents important information

of a GNP solution. Its exact determination is crucial forbiological applications and their related toxicological dis-cussions.23,24 Moreover, for investigations of GNP formationmechanisms or various GNP surface modifications and particleassembling procedures, accurate Au(0) concentration values arerequired.25,26

The optimal determination of Au(0) concentrations wouldbe a fast and easy procedure that requires only standardlaboratory equipment and can be carried out in situ. Differentanalytical techniques have been applied to determine the Au(0)concentration in GNP solutions.27,28

Very low detection limits can be achieved with inductivelycoupled plasma mass spectroscopy (ICP-MS) analysis,29 forwhich GNP solutions can be directly injected into the torch30

or dissolved previously in aqua regia.31

From SAXS measurements of GNPs, the volume fractionmeasured in absolute scale can be converted into a molarconcentration of Au(0).20,32 However, this evaluation is onlysuitable for noninteracting nanoparticles and for commonSAXS setups for which nanoparticle size should not exceed20 nm in radius.X-ray absorption near-edge structure spectroscopy (XANES)

delivers information about absorption edge positions and thusoxidation states. It can be used to calculate the ratio of Au(0)and Au(III) in solution. From this ratio, the evolution of molarAu(0) concentration during a GNP synthesis can beinvestigated if the initial concentration of the gold precursoris known and a complete reduction is assured.25,33 Thedrawback of XANES is its relatively long measurement time,ranging from several minutes to hours. A further approach forX-ray absorption spectroscopy (XAFS) with time resolutions inthe millisecond range is to apply a time-resolved energydispersive XAFS technique (DXAFS).34,35 For DXAFS experi-ments, X-rays are polychromatized with a bent crystal, leadingto an angle-dependent primary beam. X-rays in the requiredenergy range are monitored simultaneously by a position-sensitive detector.36 Nevertheless, X-ray absorption spectros-copy demands synchrotron radiation.Voltammetric methods have been applied to determine the

Au(0) concentration in GNP solutions with very low detectionlimits and relatively high robustness against organic moleculeson the GNPs.37−40

Received: June 2, 2014Accepted: October 7, 2014Published: October 7, 2014

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From atomic spectroscopy such as atomic absorptionspectroscopy (AAS)23,41 or optical emission spectroscopy(OES),42−44 elemental gold concentrations can be obtained.Typically, GNP solutions are dissolved in strong acids beforebeing analyzed.A powerful technique for the elemental analysis in GNP

solutions is neutron activation analysis (NAA).41 The crucialdrawback is the complex instrumentation, since a researchreactor as neutron source is needed for results with highaccuracy.In contrast, UV−vis spectroscopy represents a relatively

cheap and easy characterization technique that is commonlyapplied in research and industrial laboratories. GNP solutionsexhibit a characteristic UV−vis extinction spectra due to thepresence of a localized surface plasmon resonance (LSPR)signal in the visible part of the spectrum.26,45 It was reported inliterature that the size of GNPs and the molar concentration ofAu(0) can be extracted directly from UV−vis spectra. Theapproaches use either the position of the LSPR as well as theextinction at this wavelength46,47 or the ratio of extinctions atthe wavelength of the LSPR and at 450 nm (ALSPR/A450).

48 Inour opinion, the wavelength and the extinction at the LSPRshould only be used for comparison of size and concentrationbetween morphologically and chemically similar GNPsolutions, because the position and absorbance of the LSPRare strongly influenced by further parameters, such as shape andsize distribution,49,50 the dielectric surroundings, or the particlesurface chemistry.49,51

Measurement of UV−vis absorbance at a wavelength of400 nm (Abs400) could enable an accurate determination ofAu(0) concentration, since this wavelength is located betweentwo decisive spectral regions. Below 400 nm, the absorbance isincreasingly influenced by organic substances. Commonstabilizers for GNPs are colorless and thus do not show anabsorption at 400 nm. In contrast, the Abs400 of dye-functionalized GNPs52 might not be suitable for a goldconcentration determination. Above 400 nm, the absorbanceis increasingly influenced by the LSPR of the GNPs. Thedipolar LSPR mode of GNPs located at around 520 nm mightcontribute to the Abs400 but only for the smallest GNPs andonly to a very small extent.53,54 LSPRs with higher modes (e.g.,quadrupoles) are located at around 560 nm but contribute tothe spectra only for GNPs with diameters above 100 nm, whichare not within the scope of this publication.55 Thus, it can beassumed that at 400 nm both organic molecules and LSPRshave only a minor effect on the absorbance of GNP solutions.The photon energy at 400 nm (3.1 eV) is in the range of

interband transition energies from 5d to 6sp in bulk gold.56,57

Moreover, the band structure of bulk gold reveals that directinterband transitions at room temperature are allowed at 3.1 eV(i.e., transitions with preserved k-vector between states belowand above the Fermi level).56,57 Thus, the Abs400 would scalelinearly with the Au(0) concentration and could enable itsdirect determination if the band structure and transitionprobability does not change with nanoparticle size, surfacechemistry, etc. Obviously, for nanoparticles or clusters this israther speculative. Thus, the Abs400 as a measure for Au(0)concentrations in colloidal solutions needs to be validated,which to the best of our knowledge has not been described yet.However, UV−vis spectra of GNPs with equal Au(0)concentration are normalized at 400 nm occasionally and ingeneral without further explanation.44,55,58−62 It has to be noted

that in these publications, the GNPs have different sizes, shapes,and surface modifications.This paper evaluates the determination of the absolute Au(0)

concentration in aqueous GNP solution applying the Abs400. Atfirst, the general applicability of that method was studied.Subsequently, the influences of significant characteristics ofcolloidal GNP solutions on the Abs400 were investigated. Thisincludes particle size, surface modifications, and oxidation state.For size-dependent investigations, two common GNP

syntheses were used to obtain a wide size range. GNPs withmean radii of 1−2.5 nm were synthesized by reduction ofHAuCl4 with NaBH4 (GNPBH4).

21 The classical Turkevichmethod (HAuCl4 reduction with sodium citrate at elevatedtemperature) with and without subsequent seeded growthresults in GNPs with mean radii between 5 and 28 nm(GNPcit).

14,63 Surface modification was carried out withcommon stabilizing agents [i.e., poly(vinylpyrrolidone) (PVP)and Pluronic F-127] and thiol ligands [i.e., mercaptosuccinicacid (MSA) and 1-dodecanethiol (DT) in toluene]. To validatethe selectivity of the Abs400 for Au(0) concentrations, mixturesof GNP with gold precursor HAuCl4 [i.e., Au(III)] wereinvestigated. The interpretation of the resulting spectrademands an analysis of the pH-dependent HAuCl4 absorptionand the precursor interaction with GNPs.

■ EXPERIMENTAL DETAILSMaterials. Tetrachloroauric acid (HAuCl4·3H2O, 99.9%),

sodium citrate (C6H5O7Na3·2H2O, 99%), sodium borohydride(NaBH4, 99.99%), Pluronic F-127 (ethylene oxide−propyleneoxide block copolymer), and DT (C12H26S, 98%) werepurchased from Sigma-Aldrich. PVP was supplied by AlfaAesar (MW 10 kDa, 40 kDa) and Acros (MW 58 kDa). MSA(C4H6O4S, 98%) was supplied by Alfa Aesar. Aqueous solutionswere prepared using ultrapure water (18.2 MΩ·cm, Millipore).

Characterization. UV−vis measurements were carried outusing an Evolution 220 spectrophotometer (Thermo Scien-tific). Time-resolved UV−vis spectra of the GNPBH4 wererecorded using a USB2000 spectrometer (Ocean Optics)including an ILX511B CCD detector (Sony). A DT-MINI-2-GS deuterium/tungsten halogen lamp (Ocean Optics) wasconnected via fiber optics.The pH values were measured with an HI 2211 pH meter

equipped with a glass electrode (Hanna Instruments).The particle size distribution of final colloidal solutions were

investigated using a lab-scale SAXS instrument (SAXSess,Anton Paar GmbH). The final colloidal solutions wereextracted from the batch mix and inserted in a flow cell. APVP solution was added to the colloids to prevent aggregationduring the measurement.64 The scattering curves of thecolloidal solutions were analyzed by assuming spherical shape,a homogeneous electron density, and a Schulz−Zimm sizedistribution. For details about the analysis of SAXS curves, seeour former publications.64,65

Colloidal Synthesis. Syntheses of GNPBH4 were adaptedfrom a procedure reported by Polte et al.21 The GNPcit weresynthesized on the basis of the procedure described byTurkevich et al.63 According to the literature, a variation ofthe mean radii of GNPcit was achieved.

66 The preparation oflarger GNPcit followed a seeded-growth approach previouslyreported.14 Details for all colloidal syntheses can be found inthe Supporting Information.

Time-Resolved Measurements. For time-resolved UV−vis measurements during the synthesis of the GNPBH4, a

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stopped-flow setup was applied. Equal flow rates of a HAuCl4solution (0.5 mM) and freshly prepared NaBH4 solution(2 mM) were obtained by applying an inhouse-built syringepump. The two solutions were injected into an inhouse-built Y-shaped mixer (PTFE), and the homogeneous reaction flow wasconnected to a flow cell (d = 10 mm, 176.700-QS, Hellma).When the reaction flow was stopped, UV−vis data acquisitionwas started in the flow cell. The dead time between the mixerand flow cell was 280 ms.According to literature for in situ SAXS measurements of

GNPcit synthesis, time-resolved UV−vis measurements werecarried out at 75 °C.25,33 Aliquots were taken every 30 or 60 sand immediately cooled using an ice bath. UV−vis spectra weremeasured as soon as the samples reached room temperature.Modification of the GNPs. For the functionalization with

steric stabilizers, stock solutions of different PVPs (10, 40, and58 kDa) and of Pluronic F-127 were prepared withconcentrations of 50, 100, and 250 mg/L. Equal volumes ofGNP and stabilizer solutions were mixed rapidly. As areference, equal volumes of water and the GNP solutionwere mixed.For functionalization with MSA, equal volumes of as-

prepared GNPBH4 and aqueous MSA solution (1 mM) weremixed rapidly. The spectrum of MSA-capped GNPs has beendoubled for comparison with spectra of as-prepared GNPBH4.For the phase transfer of GNPs from water to toluene, equal

volumes of an as-prepared GNPBH4 solution and a solution ofDT in toluene (2.5 mM) were added to a glass vial. Themixture was vortexed for 30 s and stored overnight to ensurecomplete phase separation.For the water or HAuCl4 addition experiments, small

portions of the respective solutions were mixed with theGNP solution in a cuvette equipped with a micro stirring barand UV−vis measurements were carried out subsequently.

■ RESULTS AND DISCUSSION

The principal applicability of the Abs400 to determine Au(0)concentrations is verified by comparing six final colloidalsolutions from two different synthetic procedures (reduction ofHAuCl4 with NaBH4 and with sodium citrate). Theconcentration of HAuCl4 was varied (0.125, 0.25, and 0.5 M)for both synthetic procedures. In each case, the concentrationof the reducing agent was adjusted proportionally to ensure fullprecursor reduction. The syntheses result in GNP solutionswith different Au(0) concentrations and particle size distribu-tions. The mean radii determined from SAXS measurementsand the corresponding Abs400 are displayed in Table 1.In the investigated concentration range, the Abs400 shows a

linear behavior both for the GNPBH4 and GNPcit. The resultsindicate that the Abs400 can be used to determine Au(0)concentrations (Figure 1).

However, comparing the Abs400 of both syntheses at equalconcentrations, those of the GNPcit are constantly higher. Laterin this contribution, this will be attributed to the influence ofGNP radius on Abs400.To meet the criteria for a precise determination of the Abs400

of colloidal gold solutions, possible error sources of thesynthetic procedures and the accuracy of lab instrumentationswere checked carefully.The syntheses for GNPBH4 and GNPcit differ significantly in

their preparation details. Several precautions were taken toavoid preparative mistakes resulting in a deviation of finalAu(0) concentrations in solution and thus a deviation of theAbs400. In general, the synthesis of GNPBH4 occurs on a timescale of a few seconds and is performed at room temperature.Thus, solvent evaporation is negligible. GNPBH4 with radiilarger than 1.5 nm were obtained by short-period heating,whereby solvent evaporation was prevented by sealing thereaction vessel. Contamination of synthesis equipment (e.g.,stirrer or reaction vessel) was not observed.In comparison, the synthesis of GNPcit is a relatively slow

process carried out at elevated temperatures within severalminutes up to 0.5 h. Therefore, solvent evaporation becomes arelevant issue for this synthesis. To avoid evaporation duringGNPcit syntheses, reflux condensers were used for all syntheses.The accuracy of our instrumentation was checked by simple

dilution experiments. Small portions of water were added tofinal GNPBH4 and GNPcit solution and subsequently measuredwith UV−vis. The results are displayed in Figure S1 of theSupporting Information. The resulting plots of Abs400 showgood linearity for both syntheses, with a mean deviation of lessthan 0.4% and a maximum deviation of 1.3%. As a result, GNPsolutions can be diluted with water in a wide concentrationrange without any nonlinear effects.Hence, the instrumentation that was used as well as the lab

operations met the necessary accuracy.Influence of Particle Radius on Abs400. The influence of

the GNP radius on the Abs400 was investigated with colloidalsolutions having a constant Au(0) concentration of 0.25 mM.GNPs with different radii were obtained from NaBH4 andcitrate reduction as well as from a citrate-based seeded-growthprocedure.14,21,63 Mean radii and polydispersities weredetermined by SAXS measurements (see Table S1 in theSupporting Information). The final GNPBH4 radii are in a rangefrom 1.5 to 2.5 nm. The citrate reduction results in GNPcit withradii between 6.5 and 11.3 nm. The mean radius of GNPcit was

Table 1. Abs400 and Mean Radii Determined from SAXSData for the Syntheses of GNPBH4 and GNPcit with DifferentAu(0) Concentrations

Abs400mean radius, nm(polydispersity, %)

Au(0) conc., mM GNPBH4 GNPcit GNPBH4 GNPcit

0.125 0.268 0.284 2.0 (20) 12.7 (10)0.25 0.546 0.573 1.5 (20) 9.9 (10)0.5 1.091 1.192 1.6 (20) 5.3 (10)

Figure 1. Dependence of the Abs400 on a varied precursorconcentration both for the GNPBH4 (black) and the GNPcit (red).

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further increased to 28 nm using the seeded-growth approachas described by Polte et al.14

The final GNPBH4 have radii higher than 1.5 nm. Never-theless, the Abs400 of smaller particles can be measured duringtheir growth, since the reduction with NaBH4 assures a fullconversion to Au(0) within the first 100 ms. Thus, the Abs400values of the smallest particles (mean radii between 1 and 1.3nm) were derived from time-resolved UV−vis measurementsduring the GNPBH4 synthesis, and the size was taken fromprevious SAXS measurements of the same synthesis.21

In a previous work, the evolution of the radius during theNaBH4 reduction was studied via an in situ SAXS setup(Figure 2a). Briefly, in the first milliseconds of the reactionsmall gold clusters with radii of approximately 0.8 nm areformed that subsequently grow to a final radius of 1.7 nm. Thevolume fraction of the GNPs reaches a constant value withinthe first 100 ms, indicating that the reduction of the goldprecursor is already completed at this point, which was alsoproved by XANES measurements.Time-resolved UV−vis measurements of the GNPBH4

synthesis were conducted with a stopped-flow setup to accessinformation on the absorbance of sub-1.5 nm GNPs existent insolution during the first seconds of the synthesis. Thecorresponding particle size was correlated from time-resolvedSAXS measurements.21 A selection of spectra is displayed inFigure 2b. The Abs400 decreases during the reaction, while acharacteristic LSPR at around 500 nm evolves.In Figure 2c, the Abs400 is shown as a function of reaction

time. The absorbance increases in the beginning of the reactionand decreases to a constant value after about 3 s. The decreaseof Abs400 in the first 3 s is about 10%. The higher Abs400 in thebeginning of the reaction can be attributed to the absorbance of

small clusters at 400 nm.67,68 Further discussions on theabsorbance of gold clusters are included in a later section of thispublication.Figure 2d summarizes the dependence of the Abs400 on the

particle radius for all investigated GNPBH4 and GNPcit with aAu(0) concentration of 0.25 mM. The particles’ mean radiirange from 1.1 to 28 nm. A representative selection of the UV−vis spectra can be found in Figure S2 of the SupportingInformation. The Abs400 is strongly size-dependent and can bedivided into four size ranges. The first range (denoted as I inFigure 2d) corresponds to gold particles with mean radii in thesub-1.5 nm regime, for which the Abs400 was obtained fromtime-resolved UV−vis measurements. The Abs400 decreasesfrom around 0.6 to a constant value of around 0.525. As alreadystated, the increased Abs400 can be attributed to the presence ofsub-1 nm gold clusters. These clusters can still be existent insolution, since the Abs400 was determined during the particlegrowth. The second size range (denoted as II in Figure 2d) isrepresented by the final GNPBH4 for which the Abs400 is slightlyincreased to around 0.55. This increase continues withincreasing particle size, including the size ranges of the finalGNPcit (denoted as III in Figure 2d) and the GNPcit from theseeded growth preparation (denoted as IV in Figure 2d). As aresult, the maximum relative deviation of the Abs400 is morethan 27%.To ensure that the deviation of Abs400 is mainly affected by

particle size and not a result of borate or citrate stabilization,the influence of NaBH4 and sodium citrate on the Abs400 wasinvestigated. For this purpose, NaBH4 was added to the GNPcitand citrate to the GNPBH4. Although, a complete ligandexchange cannot be assured, it can be assumed that citrate isable to exchange borate on the GNPBH4. Borate and citrate are

Figure 2. (a) Results from in situ SAXS measurements of the synthesis of GNPBH4.21 (b) UV−vis spectra of the time-resolved measurements during

the NaBH4 reduction. (c) Abs400 as a function of reaction time for the synthesis of the GNPBH4. (d) Dependence of the Abs400 on the particle radiusfor all GNPBH4 and GNPcit. Note that the x-axis is broken for the purpose of clarity.

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weakly bound ligands attracted by electrostatic forces and caneasily be exchanged by covalently binding phosphane or thiolligands.69 Citrate in turn is able to exchange dicarboxy acetoneon the particle surface70 and also dominates when GNPs areproduced by reduction with NaBH4 in the presence of sodiumcitrate.48,70 According to UV−vis measurements (see Figure S3in the Supporting Information), the citrate addition results in aslightly higher deviation (2.6%) than the borate addition(0.3%), which indicates that citrate exchanges borate as surfaceligand. Nevertheless, the strong increase of the Abs400 in Figure2d is attributed predominantly to the GNPs size, whereascitrate and borate have only minor influences.Influence of Surface Functionalization on the Abs400.

Steric Stabilizers. Steric stabilizers are commonly used toincrease the colloidal stability and to obtain long-time stabilityof nanoparticles.71,72 Their influence on the Abs400 wasinvestigated regarding (i) the type of steric stabilizer, (ii) themolecular weight, (iii) the stabilizer concentration, and (iv) themean radius of the functionalized GNPs.Polyvinylpyrrolidone (PVP) with different molecular weights

(10, 40, and 58 kDa) and Pluronic F-127 were added indifferent concentrations to as-synthesized GNPs with differentparticle mean radii (samples r4−r7, r9, r11, r13 and r16−r18 inTable S1 in the Supporting Information). The UV−vis spectraare shown in Figure 3. Pure stabilizer solutions (without GNPs)do not show a detectable absorbance contribution in thespectral range from 300 to 800 nm (see Figure S4 inSupporting Information). For all investigated solutions, theuse of steric stabilizers increases the Abs400 considerably.Moreover, the LSPR is slightly shifted to longer wavelengths,while the shape of the spectra is not altered (Figure 3a−d).Figure 3e shows the Abs400 of GNPBH4 (mean radius 1.5 nm)

and GNPcit (mean radius 8.7 nm) before and after

functionalization. Upon addition of the stabilizer, the Abs400increases significantly, whereby PVP has a higher impact thanPluronic F-127. A relevant influence of the PVPs’ molecularweight on the Abs400 was not found.In Figure 3f, relative deviations of Abs400 after functionaliza-

tion related to the as-prepared GNPs vs the particle meanradius are shown. The deviation is found to be significantlyhigher for smaller particles. The typical deviation for the GNPcitis in the range between 1 and 6%, whereas it increases to over20% for the GNPBH4. For the GNPcit, the deviations both forPluronic F-127 and the PVPs are in the same range.The particle radii remain constant upon addition of polymer,

which was proven by SAXS measurements. Thus, it can beexcluded that the increased absorbance is caused by aggregationor growth of the GNPs. Mean radii of all functionalized GNPsas well as the related deviations of the Abs400 can be found inTable S2 in the Supporting Information. The data include avariation of stabilizer concentration, which shows that anincreased stabilizer concentration also slightly increases thedeviation of the Abs400.

Thiols and Transfer of GNPs to Organic Solvents. GNPscan also be stabilized by small molecules that adsorb on a goldsurface through strong covalent binding. Thiols are among themost studied molecular stabilizers. They can be deprotonatedto form thiolates, which bind to the nanoparticle surface by astrong Au−S bond. A large number of synthetic procedures forthiolate-stabilized GNPs is available.7,26,69

Thiolate-capped GNPs can be dissolved in various solventsdepending on the chemical structure of the thiol. Water-solublethiol-capped GNPs can be prepared by functionalization withshort-chain polar thiols. Solubility in nonpolar solvents can beachieved by long-chain alkanethiols.The influence of thiol functionalization on the Abs400 for

GNPs in water and organic solvents was investigated by acomparison of standard GNPBH4, MSA-functionalized GNPBH4,and DT-capped GNPs in toluene. Water-soluble thiolate-capped GNPs were prepared upon addition of an aqueoussolution of MSA to as-prepared GNPBH4.

44,73 GNP in organicsolvent were prepared by transferring GNPBH4 to DT intoluene via simple extraction (see Experimental Details formore information). The completeness of the phase transferwith DT was checked by measuring the UV−vis spectrum ofthe corresponding water phase. A measurable absorbance wasnot detected (Figure 4, waterDT). Also, aggregation at theinterface or glass vial was not observed.UV−vis spectra of as-prepared GNPBH4 and thiol-function-

alized particles are displayed in Figure 4. The Abs400 values withtheir relative deviations from the as-prepared GNPBH4 as well asparticle mean radii and polydispersities derived from SAXSanalysis are displayed in Table 2.The modification of the colloidal GNP solution with thiols

causes a notable change in the spectra for both aqueous andorganic solutions. It can be attributed to a significant interactionof the covalently bound thiols with the electronic system of theGNPs. The overall absorbance increases and the LSPR isbroadened, damped, and slightly shifted to higher wavelengths,as known from the literature.74 The Abs400 increasessignificantly for both the MSA- (11.4%) and the DT-cappedGNPs (16.9%), which might be a result of the LSPRbroadening or the changed electron band structure. Theparticle mean radius after addition of MSA remains constantwhile being slightly higher upon phase transfer with DT (seeTable 2). The thiol functionalization experiment shows that

Figure 3. UV−vis absorbance spectra of (a) GNPBH4 and (b) GNPcitbefore and after functionalization with steric stabilizers. (c and d) Thesame spectra in a smaller wavelength range. (e) Abs400 of puresemidilute GNPs and GNPs after the functionalization with stericstabilizers and (f) the relative deviation at 400 nm vs nanoparticle size.

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covalently bound molecules have a strong impact on the GNPsspectra and in particular on the Abs400. Therefore, generalstatements for the influence of surface modifications on theAbs400 can hardly be made.Influence of HAuCl4 on the Abs400. In the previous

sections, the Abs400 for colloidal solutions after the completereduction of the gold precursor is discussed. The Abs400 wasinvestigated as a measure for the Au(0) concentration in theabsence of any ionic gold species. In this section, the Abs400 ofgold complexes and its influence on the absorbance of GNPsare discussed.HAuCl4 is the most common precursor for GNP syntheses in

aqueous solution. Its absorption behavior, as well as itsinteraction with GNPs in solution, is an important andcomplex aspect. It can provide information on reductionprocesses that are necessary to understand growth mechanismsof GNPs.An aqueous solution of HAuCl4 has a yellow color as a result

of absorption of AuCl4−x(OH)x− complexes in the UV−vis

region. The absorption of the different complexes includes aspectral contribution at 400 nm (see Figure 5a). Moreover, thepH-dependent equilibrium of AuCl4−x(OH)x

− complexes needsto be considered, since HAuCl4 is an acid and lowers the pHvalue with increasing concentration. In the literature, it is well-described that chloride ions are gradually exchanged againsthydroxy ions, with increasing pH value favoring the formationof hydroxy complexes [e.g., AuCl3(OH)

−, AuCl2(OH)2−, ...].

These complexes exhibit a lower contribution to Abs400compared to AuCl4

−.75 As a rule of thumb, the contributionof gold precursor to the Abs400 decreases with increasing pH

value since the AuCl4− percentage of all gold complexes

decreases. This effect can be investigated by a simple dilutionexperiment of HAuCl4 solution with water (Figure 5a,b). Athigh HAuCl4 concentrations (1−10 mM), the pH value isbetween 2 and 3 and almost only AuCl4

− is present in solution.Consequently, in this concentration range the Abs400 is linear(see Figure 5b). At concentrations typical for most syntheses(below 1 mM) the pH is above 3. Thus, other gold complexesare also present [in particular AuCl3(OH)

− at this pH range],which results in a nonlinear decrease of the Abs400 withdecreasing HAuCl4 concentration (see the inset in Figure 5b).Besides this nonlinear absorption effect, the dilution

experiment reveals that Abs400 is much more sensitive toAu(0) than to Au(III). For all concentrations, Au(0) has amuch higher absorption than the respective Au(III). The Abs400of a GNP solution [i.e., Au(0)] with a concentration of0.25 mM is at around 0.55, whereas a HAuCl4 solution [i.e.,Au(III)] with the same gold concentration is at around 0.02.However, the influence of ionic gold complexes on the Abs400

of GNPs remains unclear. Thus, final GNPs were mixed withHAuCl4 in different percentages retaining a constant total gold

Figure 4. UV−vis spectra for as-prepared and thiol-functionalizedGNPBH4. The water phase after extraction with DT in toluene does notshow an absorption (waterDT). Inset: Photograph of the phase transferexperiment. A solution of DT in toluene (left, top) was added to aGNPBH4 solution (left, bottom). After phase transfer, the uppertoluene phase contains the extracted GNPs, while the lower waterphase is colorless (right).

Table 2. Abs400, the Deviation Related to As-preparedGNPBH4, and Particle Mean Radii and PolydispersitiesDetermined from SAXS for As-prepared GNPs and GNPsafter Thiol Functionalization

GNPBH4 sample Abs400 deviation, %mean radius, nm(polydispersity, %)

as prepared 0.559 1.5 (20)MSA 0.622 11.4 1.5 (20)DT/toluene 0.653 16.9 2.7 (10)

Figure 5. (a) UV−vis absorption spectra of HAuCl4 solutions dilutedwith water. (b) The Abs400 related to the concentration of HAuCl4 athigh concentration values and (inset) at lower concentrations.Changes in the UV−vis spectra of (c) GNPBH4 and (d) GNPcitupon the addition of a HAuCl4 solution. (e and f) The values arecompared to the corresponding dilution of the particle solutions withwater (linear line), and the following deviations are highlighted (g andh).

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concentration of 0.5 mM. In fact, GNPBH4 and GNPcit solutionswere diluted with different amounts of HAuCl4 solutions. BothGNP solutions were synthesized with an initial Au(0)concentration of 0.5 mM to cover a wider concentrationrange upon dilution with HAuCl4. The addition of HAuCl4causes a change of the LSPR at around 520 nm both for theGNPBH4 (Figure 5c) and the GNPcit (Figure 5d). The LSPR isbroadened and shifted towards lower energies. This shift is notcaused by aggregation and growth. It was previously shown bySAXS measurements that particle sizes remain constant uponaddition of HAuCl4 to GNP solutions.14

The evolution of the Abs400 in dependence of HAuCl4addition is illustrated in Figure 5e for GNPBH4 and in Figure5f for GNPcit. The addition of HAuCl4 is represented by themolar fraction of GNPs (x-axis) calculated from the initialsample volume and the resulting volume upon HAuCl4addition. For instance, at a molar fraction of 0.5 the investigatedsolution consists of 0.25 mM Au(0) (the GNPs) and 0.25 mMAu(III) (HAuCl4). The linear line in Figure 5e,f is the fitting ofthe Abs400 values from water addition to the respective GNPs(see Figure S1 in the Supporting Information). It serves as acomparison for Abs400 of the GNP solutions upon addition ofHAuCl4 represented by the open circles in Figure 5e,f. Asexpected, the Abs400 decreases for both additions. However,values from HAuCl4 addition differ significantly from thesimple water dilution. The relative deviations are displayed inFigure 5g,h. The deviation increases exponentially to over 26%for the GNPBH4 and 13% for the GNPcit. The deviations canmainly be attributed to the increased spectral contribution ofHAuCl4 to the Abs400, which is in accordance with the HAuCl4dilution experiment with water (see Figure 5a). Thisinterpretation is also confirmed by the appearance of thecharacteristic absorption band of the AuCl4

− complex at about295 nm.75 The higher deviations for GNPBH4 compared toGNPcit are most likely caused by the stronger decrease of thepH value upon HAuCl4 addition, since citrate acts as a pHbuffer. pH values as well as Abs400 of all prepared dilutions canbe found in Table S3 of the Supporting Information.A further characteristic is found for the dilution of GNPBH4

with HAuCl4 at molar fractions above 0.8 (i.e., only smallportions of HAuCl4 added), for which the deviation reachesnegative values. It can be assumed that a red shift of the LSPR(as shown in Figure 5c) is responsible for this effect whichmight reduce the spectral contribution of the LSPR to theAbs400.Abs400 as a Method To Determine Au(0) Concen-

trations in Colloidal Gold Solutions. The Abs400 of colloidalgold solutions is located between two decisive spectral regions.Below 400 nm organic substances influence the absorbance,and above 400 nm it is influenced by the LSPR of the GNPs.The principle capability of the Abs400 to determine Au(0)concentrations is attributed to the allowed interband transitionsat 3.1 eV. Interband transitions are characteristics of nano-particles with electronic band structure (i.e., bulklike nano-particles). Actually, only gold and copper have interbandtransitions with preserved k-vector in the visible spectrum,making them the only metals with specific colors for the humaneye.57,76−79

In this contribution, it is shown that the Abs400 is in principlecapable of determining Au(0) concentrations of colloidal goldsolutions, but is essentially influenced by a variety of parameters(e.g., particle size, surface modifications). Indeed, it is notsurprising that band structure and transition probability

changes for sub-20 nm particles, resulting in a size dependenceof the Abs400 (see Figure 2d). Concerning the smallest GNPswith particle diameters of around 1 nm, the metal to insulatortransition occurs gradually (i.e., for which small metallic clustersdevelop a band structure and at which size the valence electronscan be treated as electron gas) and needs to be taken intoaccount. In this size regime of molecular-like clusters, theAbs400 can obviously not be attributed to interband transitionsand can therefore most likely not be used as a measure forAu(0) concentrations. The exact cluster size for the metal toinsulator transition can hardly be given for any metal as it evendepends on the criterion for metallicity.80 Nevertheless, theabsorbance of molecular-like metal clusters can be well-understood with recent contributions from Harbich and co-workers. They were able to precisely determine the absorptionof size-selected noble metal clusters in a rare gas matrix at lowtemperatures starting from the single atom.81−83 As expected,the gold atom does not absorb in the visible range and inparticular at 3.1 eV.81 The first absorption peak is around theFermi energy at 5.5 eV (∼225 nm). However, gold clusterslarger than the dimer already absorb at 3.1 eV, but theabsorbance is different for each cluster size (Aun=2−9). As aconsequence, absorbance in the visible spectrum of thesemetallic clusters cannot be used for a reliable determination ofAu(0) concentrations. As a consequence, the Au(0) concen-tration in GNP solutions that contain substantial fractions ofgold clusters (e.g., as part of the inherent size distribution) canhardly be determined using Abs400. The different electronicstructures of metal clusters might be the reason for therelatively large Abs400 deviation between the smallest GNPBH4measured during the growth (mean radius around 1 nm) andthe final GNPBH4 of that synthesis (mean radius around1.5 nm) in Figure 2c.In addition to particle size, the surface chemistry can also

have an influence on band structure and transition probabilityand therefore on Abs400. As a result, the Abs400 of gold colloidschanges due to added ligands or surface oxidation. Thus,changing the surface chemistry by adding steric stabilizers (PVPand Pluronic F-127) or thiols can cause relatively large Abs400deviations.Indeed, the Abs400 deviation determined for the different

particle sizes or ligands might also be affected by a change ofthe dielectric function of the surrounding medium.9,26,84 Bothare known to affect the LSPR. Thus, the contribution of theLSPR to the Abs400 might not be as minimal as previouslyassumed.Regardless, for systems with similar particle sizes and similar

chemistry (e.g., the same stabilizing agent), the Au(0)concentrations can be determined reliably via Abs400. Theaccuracy and detection limit of Abs400 were determined (seeFigure S5 and related discussions in the SupportingInformation) to allow a practical comparison with the above-mentioned techniques of quantitative gold concentrationdetermination (Table S4 in the Supporting Informa-tion).37−39,41,43,85−94 Abs400 is found to be highly accurate,while some techniques can obtain lower detection limits.However, a general extinction coefficient at 400 nm for GNP

solutions cannot be stated. It rather has to be referred to certaincolloidal GNPs. In Table 3, extinction coefficients are presentedas a rough estimation that can be used to determine the Au(0)concentration. For the investigated solutions, the extinctioncoefficient ranges from 2.14 to 2.74 L·mol−1·cm−1. Extinction

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coefficients of all investigated GNP solutions can be found inthe Supporting Information (see Table S2).As previously mentioned, only gold and copper have

interband transitions in the visible spectrum. Consequently,the Abs400 could in principle be applied to determine Cu(0)concentrations for colloidal copper, since the plasmonresonance of copper is at around 550 nm.95,96 Although notwithin the scope of this contribution, a first experiment wasconducted with copper nanoparticles synthesized by reductionof Cu(NO3)2 with NaBH4 (for details see Figure S6 and relateddiscussions in the Supporting Information).The results reveal that for colloidal copper the Abs400 is not

particularly suitable for a determination of Cu(0) concen-tration. In fact, absorbance below 400 nm (e.g., at 300 nm)seem to be more suitable, but at these wavelengths organicstabilizers may contribute to the absorbance. Thus, the resultsof this work concerning colloidal gold cannot be adapted tocolloidal copper in a simple manner.Time-Resolved UV−Vis Measurements of the GNPcit

Synthesis. Time-resolved measurement of the Abs400 can beused to gain precise information on reduction rates in theGNPcit synthesis. It is found that the AuCl4

− complexcontributes to the Abs400. Nevertheless, in the relevantHAuCl4 concentration range between 0.125 and 0.5 mM,common for the synthesis of GNPs, the Abs400 shows onlyminor deviations (<5%) compared to water addition. More-over, the Abs400 of GNPcit also deviates less than 5% betweenparticles with radii less than 10 nm.Exemplarily, this is shown for a GNPcit synthesis carried out

at a moderate reaction temperature (75 °C). Aliquots ofreaction mixture were taken every 30−60 s and UV−vis spectrawere measured. The kinetic results of the Abs400 evolution areshown in Figure 6a and are compared to the monitoring of thevolume fraction of Au(0) via SAXS analysis (Figure 6b).33 Theresulting curves are similar in shape and the relative timepattern of the single reaction phases. According to theliterature, three phases for the GNP formation mechanismare found.25,33 Briefly, a rapid metal cluster formation and theircoalescence into bigger particles occur within the first minutes(1). The second phase is a relatively slow diffusion growth with

ongoing consumption of gold precursor (2). In the third phase,a fast particle growth to the final particle size takes place bycomplete reduction of the remaining precursor (3).For the UV−vis measurement, the reaction volume is

relatively small (500 mL) and the reaction temperature canbe controlled with good accuracy. The total duration of thereaction (thus the duration of its single reaction phases) isslightly different between the syntheses measured with UV−visand SAXS. The longer reaction duration in the time-resolvedSAXS measurement is most likely a consequence of therelatively big reaction volume (3 L). Thus, the reactiontemperature might have been a few degrees lower than 75 °C.It has to be noted that such a difference in the reactiontemperature can influence the duration of the single reactionphases but in principle does not influence the GNP formationmechanism.The time-resolved measurement of the Abs400 presents a fast

and powerful tool to obtain reduction progress data during aGNP synthesis. The technique can be carried out with standardlaboratory equipment and offers higher control of the syntheticparameters.Hence, a simple, fast, easy, and cheap in situ method for

Au(0) determination is demonstrated. Moreover, in thepresence of other gold species such as Au(III), the Abs400method has to the best of our knowledge an unprecedentedaccuracy compared to expensive and laborious methods such asXANES.

■ CONCLUSIONSThis paper validates the determination of Au(0) concentrationsin GNP solutions via UV−vis measurements at 400 nm. Themethod is shown to be valid with restrictions depending on theinvestigated system. The extinction coefficient of the hereininvestigated gold colloids ranges from 2.14 to 2.74L·mol−1·cm−1. As a result, the uncertainty of the determinedAu(0) concentration can be up to 30%. This deviation is theresult of a combined influence of parameters such as particlesize, surface modification, or oxidation state.However, quantifying the influences of these parameter

enables a much more precise Au(0) determination for specificsystems. As an example, the reduction process of the well-known Turkevich method was monitored with a deviation ofthe Au(0) concentration of less than 5%.Hence, measuring the Abs400 is a simple, easy, cheap, and, for

specific systems, very precise method for selective Au(0)determinations. In particular, the measurement can beconducted in situ with a high time resolution.

■ ASSOCIATED CONTENT*S Supporting InformationFigures S1−S6, Tables S1−S4, and additional text providingdetails from GNP syntheses, GNP dilution with water, sizedistributions from SAXS data of GNPs, UV−vis spectra ofGNPs and of steric stabilizers, Abs400 and extinction coefficientsof prepared solutions, accuracy and detection limit of Abs400and comparison with other analytic techniques, experimentaldetails of copper nanoparticle syntheses, and UV−visabsorbance data of copper nanoparticles. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*K.R. e-mail: klaus.rademann@chemie.hu-berlin.de.

Table 3. Ranges of the Estimated Extinction Coefficients forAs-prepared GNPs and GNPs with Steric Stabilization

ε400, L·mol−1·cm−1

sample/mean radii, nm as prepared PVP stabilized Plu F-127 stabilized

GNPBH4/1.5−2.5 2.14−2.23 2.43−2.65 2.34−2.46GNPcit/5−10 2.29−2.38 2.38−2.43 2.36−2.41GNPcit/10−20 2.38−2.57 2.45−2.51 2.44−2.52GNPcit/20−28 2.63−2.68 2.68−2.71 2.70−2.74

Figure 6. (a) Time-resolved UV−vis measurements of the Abs400 forthe GNPcit synthesis. (b) The results are compared to the volumefraction derived from SAXS measurements.33

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*J.P. e-mail: joerg.polte@hu-berlin.de.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSJ.P. acknowledges generous funding by the DeutscheForschungsgemeinschaft within project PO 1744/1-1. M.W.also acknowledges financial support by the Fonds derChemischen Industrie. F.K. acknowledges support by theIMPRS “Functional Interfaces in Physics and Chemistry”.

■ ABBREVIATIONS USEDGNPs gold nanoparticlesLSPR localized surface plasmon resonanceAbs400 UV−vis absorbance at a wavelength of 400 nmGNPBH4 gold nanoparticles synthesized by reduction with

NaBH4GNPcit gold nanoparticles synthesized by reduction with

sodium citrateMSA mercaptosuccinic acidDT 1-dodecanethiol

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