Fundamental SERS Investigation of Pyridine and Its Derivates as a

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Fundamental SERS Investigation of Pyridine and Its Derivates as a Function of Functional Groups, Their Substitution Position and Their Interaction with Silver Nanoparticles Anna Mühlig, Dana Cialla-May, and Juergen Popp J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b09368 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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The Journal of Physical Chemistry

Fundamental SERS Investigation of Pyridine and its Derivates as a Function of Functional Groups, their Substitution Position and their Interaction with Silver Nanoparticles Anna Mühlig a, Dana Cialla-May * a,b, Jürgen Popp a,b a. Leibniz Institute of Photonic Technology (IPHT), Albert-Einstein-Straße 9, 07745 Jena, Germany b. Institute for Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-University Jena, Helmholtzweg 4, 07743 Jena, Germany

ACS Paragon Plus Environment

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ABSTRACT

The Raman and SERS spectra of pyridine, 2-, 3-, and 4-pyridinemethanol and 2-, 3-, and 4picolylamine were comprehensively studied using two different types of silver nanoparticles as SERS substrates, namely citrate reduced and hydroxylamine reduced silver nanoparticles, which are the most commonly used silver colloids. For a robust band assignment, Raman spectra of the investigated molecules were simulated, applying Møller-Plesset perturbation theory in conjunction with the 6 311++G(d, p) basis set. Subsequently, the observed changes in the spectra were interpreted and discussed in detail, taking into account the simulated Raman spectra. Furthermore, differences in the spectral information resulting from the two different analyzed functional groups, the altering of the substitution position and the molecular orientation relative to the metal surface were discussed, and general conclusions for an application of those molecules as Raman reporters were derived.


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INTRODUCTION Pyridine has been the subject of numerous studies applying Raman and IR spectroscopy1-8. In 1974 Fleischmann et al. discovered the surface enhancement effect by bringing pyridine in the vicinity of silver electrodes9. Thereafter, a large effort was made to investigate pyridine-metal complexes in both quantum-mechanical and experimental investigations. Subjects of these efforts included addressing the electro-potential influence on the SERS signal10, the orientation at the metal surface11, the influence of chloride ions present in the solution and other varying parameters12-14. Furthermore, there is significant interest in substituted pyridine derivates, since they are present in many biologically important compounds. The application of pyridine related molecules has attracted considerable attention in recent years due to their key-function as synthetic intermediates, their use as precursors to pharmaceuticals and agrochemicals, and their applications as a solvent.15-16The influence of substituents, e.g., changes in the functional groups17-19 and changes in the substitution position20-21 were investigated. In general, these effects can be compared with mono- and di-substituted benzene derivates because of identical symmetry properties. However, there are few publications dealing with this topic.22-23 In addition to recording the Raman and SERS spectra, the simulation of vibrational spectra is essential to ensure a robust band assignment. Some groups even simulated SERS spectra for various types of pyridine-Ag complexes24-27. These studies are very valuable for the identification of the ratio between the near- and the far-field contributions28. However, to obtain information about the impact of substituents and their position on the SERS signal it is necessary to identify vibrational modes for the different derivates and isomers that belong to the same fundamental vibration. Pyridine and the mono-substituted derivates of this heteroaromatic ring can be used as excellent analyte models in fundamental investigations29, as the vibrational


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spectra are clearly arranged with less overlaying vibrational modes. Furthermore, the molecules are relatively small, making it convenient to apply quantum-mechanical simulations. For a comprehensive interpretation of the SERS spectra the impact of the used nanoparticle cannot be neglected. It has to be considered how the analyte molecules may bind to the metal surface, since the resulting orientation on the surface has an important impact on the relative intensities of peaks caused by in-plane and out-of-plane vibrations following the surface selection rules.30-31 In this contribution the Raman and SERS spectra of pyridine, 2-, 3-, and 4-pyridinemethanol (2-, 3-, 4-pymeth) and 2-, 3-, and 4-picolylamine (2-, 3-, 4-picam) were studied in detail (the optimized structures are depicted in Figure 1) using two different types of silver nanoparticles as SERS substrates.

Figure 1. Energy optimized structures of the molecules under investigation: 4-picolylamine (4picam), 3-picolylamine (3-picam), 2-picolylamine (2-picam), pyridine, 4-pyridinemethanol (4pymeth), 3-pyridinemethanol (3-pymeth) and 2-pyridinemethanol (2-pymeth). Simulated with GUASSIAN09; MP2 level of theory and 6-311++G(d,p) basis set.


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For a robust band assignment, vibrational modes of the investigated molecules were simulated. Differences in the spectral information resulting from the two different analyzed functional groups and the altering of the substitution position were discussed, and general conclusions were derived for mono-substituted pyridines. Furthermore, the two most commonly used silver colloids, e.g., hydroxylamine reduced silver nanoparticles and citrate reduced silver nanoparticles, were investigated; the observed changes in the spectra were interpreted and discussed in detail, taking the simulated vibrational modes into account. To the best of our knowledge, this report describes the first investigation addressing pyridine derivates in such an extensive range. The findings of this study provide advanced information regarding the selection of extrinsic marker molecules, internal standard molecules and sensor molecules. When applying SERS in practice, e.g., in biomedical applications, industrial approaches or food analytics, it is essential to be aware of the influence of substituent groups and their position on the fingerprint profile, the band assignment and the band ratio. Hence, fundamental investigations regarding this topic are required. EXPERIMENTAL AND THEORETICAL METHODS Computational Details Calculations for the structure optimization and simulated vibrational spectra were performed using the quantum mechanical GAUSSIAN09 program package.32 For the optimization of the isolated structures of pyridine and the pyridine derivates, second-order Møller-Plesset perturbation theory33 was used in conjunction with the 6 311++G(d, p) basis set. It has been proven in numerous publications that for a robust band assignment this level of theory is


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sufficient.34-35 Before the vibrational frequencies were calculated, an energetic structure optimization was performed. For all molecules under investigation the vibrational modes were obtained along with their Raman activity. The symmetry of pyridine is C2v, thus, it has 27 fundamental modes of vibration: ΓC2v=10 A1+3 A2+5 B1+9 B2 Of these, the 19 modes of class A1 and B2 are planar and the eight modes belonging to A2 and B1 are non-planar. The optimized structure of the isolated pyridine derivates belong to no symmetry class. Chemicals and reagents Silver nitrate (ACS reagent, ≥99%), hydroxylamine hydrochloride (ReagentPlus, 99%), sodium hydroxide, potassium chloride and the analytes 2-pyridinemethanol, 3-pyridinemethanol, 2-picolylamine, 3-picolylamine, 4-picolylamine were purchased from Sigma-Aldrich. Pyridine and 4-pyridinemethanol were purchased from VWR. Sample preparation The hydroxylamine reduced silver nanoparticles (h-AgNPs) were prepared according to the protocol published by Leopold and Lendl36. Briefly, under vigorous stirring a 0.1 mmol silver nitrate solution was added to a mixture of hydroxylamine hydrochloride (0.15 mmol) and sodium hydroxide (0.3 mmol). The solution turned to a grey-yellow color instantaneously. Stirring was continued for 10 min.


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The protocol applied to generate citrate reduced nanoparticles (c-AGNPs), was published by Lee and Meisel37. Briefly, 10 ml of a 1% sodium citrate solution was added to a boiling 1 mM AgNO3 solution, the solution was kept boiling for ca. 1 h. Activation of the citrate reduced silver colloid was achieved by addition a solution of 1 M potassium chloride. The aqueous solutions of pyridine and its derivates were prepared with distilled water. Experimental details and measurement conditions The UV/Vis absorption spectra were recorded with a Cary5000-UV-Vis-NIR spectrophotometer (Agilent Technologies, Waldbronn, Germany). For the Raman measurements a WITec microscope (WITec GmbH, Ulm, Germany) was used. The excitation source was a continuous-wave diode-pumped solid-state laser (CoboltTM, Solna, Sweden) with a wavelength of 514 nm and a maximum output power of 100 mW. The Raman measurements were performed using quartz cuvettes, focusing the laser beam into the analyte solution applying a 10× microscope objective (Zeiss EC ‘‘Epiplan’’ DIC, 10×, NA=0.4, Oberkochen, Germany). The SERS measurements were recorded using the same Raman microscope in combination with a droplet-based Lab-on-chip (LOC)-SERS device, allowing for a high throughput with a low volume of required sample solution38-43. Thus, a reliable data set of SERS spectra was recorded and a homogenous mixing of silver nanoparticles, aggregation agent KCl and analyte molecules is guaranteed due to the meander channel system. The LOC-SERS device was mounted onto the microscope stage. For the LOC-SERS investigations the laser beam was focused into the microfluidic channel using a microscope objective (Zeiss EC ‘‘Epiplan’’ DIC, 20×, NA=0.4, Oberkochen, Germany). The same objective was used to collect the backscattered light. The incident laser power present on the sample was approximately 5 mW to 25 mW. For all vibrational measurements, a 600 lines per mm grating was employed with a spectral resolution of


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5 cm-1. A thermoelectrically cooled CCD detector (at -70 C°) with 1024 × 127 active pixels and a pixel size of 26 mm × 26 mm was used for the detection of the Raman signal. A detailed description of the LOC-SERS device can be found elsewhere40. The SERS spectra were recorded continuously in the third channel with a laser power of 5 mW to 25 mW (measured before the objective) and an integration time of 1 s. For every analyte a series of 1000 to 3000 spectra was obtained, containing pure droplet SERS spectra, pure mineral oil Raman spectra and mixed spectra. Burning effects are avoided due to the constant flow within the LOCSERS device. RESULTS AND DISCUSSION UV/Vis - Influence of the analytes on the optical characterization of the applied colloid Before the Raman and SERS spectra were recorded, UV/VIS spectra were measured for every analyte mixed with h-AgNPs and c-AgNPs. With the information of the absorbance spectra one can determine whether the analyte molecules are interacting with the colloids. Thus, the analyte solution was mixed with silver nanoparticles, adding a 1 M KCl solution for the c-AgNPs, for the UV/Vis measurements. The absorption spectra are depicted in Figure 2. In the case of c-AgNPs, the surface plasmon resonance (SPR) band is found around 450 nm, whereas in the case of hAgNPs, the SPR mode is at 414 nm. This is attributed to the different size of the nanoparticles for both colloidal solutions.


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Figure 2. Absorbance spectra of all investigated molecules mixed with c-AgNPs and KCl a) and with h-AgNPs b). From bottom to top: 2-picam, 4-picam, 3-picam, pyridine, colloid (h-AgNPs), 2-pymeth, 3-pymeth, 4-pymeth, colloid with KCl, colloid without KCl (both c-AgNPs). Absorbance values are given relative to the maxima of the absorbance of the colloids.

In Figure 2 panel a) the absorbance of c-AgNPs in combination with the analytes is presented. The absorbance changes significantly for all of the investigated analytes, when a 1 M KCl solution was added. Thus, an aggregation of the c-AgNPs was induced, and this is valid for all analytes. The absorbance of the h-AgNPs in combination with the analytes is shown in Figure 2 panel b). Herein, the absorbance shows significant changes for the pyridine derivates with the methylamine functional group (compared with the pure h-AgNP solution). Here, the absorbance of the pyridine derivates, each possessing a hydroxymethyl functional group, is very similar to the absorbance of the pure h-AgNP. The same is valid for pyridine. This observation indicates that the 4-, 3-, and 2-picolylamine molecules are interacting with the colloid, inducing agglomeration of the h-AgNPs. In contrast, in the cases of 4-, 3-, and 2-pyridinemethanol and pyridine apparently no interaction occurs with the h-AgNPs. Thus, for further experiments the SERS measurements with h-AgNPs were carried out only for the isomers of picolylamine with


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the methylamine functional group. Whereas, both isomers of pyridinemethanol as well as isomers of picolylamine were used for the SERS experiments applying c-AgNPs. As a further consequence, the LOC-SERS experiments were conducted by adding 1 M KCl when c-AgNPs are applied as SERS-active substrate. In the case of h-AgNPs, no KCl was used. Detailed band assignment of the vibrational modes Raman and SERS spectra were recorded in the wavenumber range of 1750 to 500 cm-1; the mean spectra of the Raman measurements and the SERS measurements with c-AgNPs are presented in Figure 3. It is obvious that the spectral shape, of both the SERS and the Raman signal is strongly correlated with the substitution position. The SERS and the Raman spectra of the two para-substituted molecules have a very similar spectral shape. The same is valid for the two meta-substituted pyridine derivates. However, the two ortho-substituted derivates show significant differences.


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Figure 3. Mean spectra of Raman and c-SERS measurements from bottom to top: 4-picam, 3picam, 2-picam, pyridine, 2-pymeth, 3-pymeth, 4-pymeth. The spectra were normalized to their highest intensity. The mean Raman spectra are depicted in light gray and the SERS spectra are shown in dark gray (with the SDV under laid). The asterisk marks the fundamental vibration used for the normalization.

The band assignment in Table 1 shows the calculated vibrational wavenumbers, the experimental Raman vibrational modes and the experimental SERS vibrational modes when using c-AgNPs and h-AgNPs, in the investigated wavenumber range (the detailed calculated atomic motions of every fundamental vibration are depicted in SI1). For the notation of the fundamental vibrational modes of pyridine, the notation proposed by Herzberg44 was used. For


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the pyridine derivates, the fundamental vibrational frequencies correlated with the fundamental modes of pyridine are listed in Table 1, respectively. For comparison, the often used Wilson notation is given in parenthesis. Although, it should be mentioned, the Wilson notation was proposed for benzene (D6h symmetry) and can be misleading regarding the class and the assignment of the fundamentals, belonging to the C2v symmetry group45. This is especially true for the two trigonal ring breathing modes ν8 and ν9 (in Wilson notation: 12 and 1, respectively), depicted in Figure 4.

Figure 4. Optimized molecular structure of isolated pyridine based on the MP2/6-311++G(d,p) level of theory and the two total symmetric A1 fundamentals ν9 and ν8.

Both vibrational modes are dominant in the Raman and the SERS spectra of pyridine. Researchers have to consider that these two fundamentals are of the same symmetry class for molecules of the C2v symmetry group. The simulation confirms that both fundamental vibrations are caused by trigonal ring breathing, ν8 including the atoms C2-C4-C6, while ν9 includes the other atoms of the heteroaromatic ring system, i.e., C3-N-C5 (see Figure 4 for a schematic detailing the atom numbering). The motion of the atoms for each of these fundamental vibrations is given in Figure 4. While, both fundamental vibration ν8 and ν9 are caused by trigonal ring


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breathing, the Wilson notation indicates that only the ν8 mode (in Wilson notation 12) is a total symmetric trigonal ring breathing, while the ν9 mode (in Wilson notation 1) is suggested to be a total symmetrical ring breathing mode including all ring atoms similar to the one appearing in benzene. Subsequently, the sometimes stated shift of the miss-assigned “total symmetric ring breathing” mode labeled with 1 in the Wilson notation10 is not a shift at all; however, it is just another vibration. Due to the altered symmetry of mono-substituted heteroaromatic ring structures, either the ν8 mode (e.g., meta-substituted pyridine derivates) or the ν9 mode (e.g., para- and ortho-substituted pyridine derivates) becomes more dominant in the vibrational spectra. The same is valid for di-substituted aromatic ring structures.


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Table 1. Band assignment: from left to right are given the modes in the Herzberg notation (in parenthesis the Wilson notation can be found for comparison), the wavenumbers in cm-1 for 4picam, 3-picam, 2-picam, pyridine, 2-pymeth, 3-pymeth, and 4-pymeth. For every analyte the left column includes the wavenumber values of the simulated normal modes (calc.), the middle column indicates the wavenumber values of the Raman measurements and the left column includes the wavenumber values of the SERS measurements. The wavenumbers for SERS measurements applying c-AgNPs are given in the first row for every analyte, the wavenumbers for SERS measurements applying h-AgNPs (for 4-picam, 3-picam, and 2-picam) are given in the second row.


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Mode 4-picam

ν4 (8a)

3-picam

2-picam

pyridine

2-pymeth

3-pymeth

4-pymeth

calc.

Raman

SERS

calc.

Raman

SERS

calc.

Raman

SERS

calc.

Raman

SERS

calc.

Raman

SERS

calc.

Raman

SERS

calc.

Raman

SERS

1651

1657

1630/

1633

1604

1594/

1615

1578

1564/

1628

1600

1593

1618

1578

1575

1638

1605

1630

1648

1646

1633

1506

1494

1512

1508

1502

1510

1511

1484

1499

1522

1505

1502

1245

1223

1218

--

--

--

1228

1216

1227

1243

1247

1250

1091

1074

1067

1086

1103

1120

1054

1053

1053

1088

1073

1065

1066

1058

1053

-1641

1517

1593

1565

1615/ 1610

ν5 (19a)

1523

1569

1500/

1505

1488

1528 1517

1503

1495/

1507

1500

--

1499/ 1501

1552/ 1541

ν6 (9a)

1258

1224

1210/

1221

1205

1208 ν7 (18a)

1087

1075

1068/

1212/

1306

1304

1229 1053

1054

1065

1050/

1312/ 1315

1076

1085

1047

1055/ 1057

1061

1061

1049/ 1049

ν8 (12)

1235

1210

1210/

1038

1039

1208 ν9 (1)

1008

1012

1013/

1033/

--

--

--

1043

1040

1035

--

--

--

1041

1039

1034

1213

1213

1206

1014

1011

1007/

1007

1008

1006

1012

1011

1003

--

--

--

1008

1011

1013

604

620

623

817

812

844

645

645

661

818

812

810

635

644

653

585

--

--

1031 --

--

--

1006 ν10 (6a)

809

806

813/ 833

1008 810

827

832/

849

825

829

826/ 826

812

802

823/ 803


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ν11 (17a)

--

--

--

--

--

--

--

--

--

861

--

--

--

--

--

ν12 (10a)

937

--

--/

939

965

958/

943

946

970/

840

892

879

890

880

872

934 ν14 (5)

--

--

--

961 915

941

933/

--

--

--

870

879

882

935

--

--

972 --

--

--

884

949

944

913

904

900

--

--

--

--

--

--

927 ν15 (10b)

--

--

--

--

--

--

--

--

--

867

--

922

928

--

--

--

--

--

911

--

--

ν16 (11)

787

797

798/

768

800

798/

765

782

772/

705

--

697

746

761

752

769

805

813

783

--

785

517

--

753

620

635

632

620

614

613

618

672

659

1618

1581

1569

1638

1604

1622

1619

1588

1592

1609

1617

1607

1470

--

1484

1465

1466

1470

1449

1437

1435

1446

1453

1440

1392

--

1404

1404

1365

1378

1402

1372

1384

1390

1380

1382

1381

--

1387

1323

1319

1323

1364

1341

1348

1357

1344

1348

1172

1157

1150

1173

1194

1195

1140

1129

1128

--

--

--

1077

1055

--

1124

1128

1154

1073

1107

1105

1114

--

--

659

658

651

590

--

--

--

--

--

675

686

700

797 ν17 (16b)

630

668

613/

790 639

645

-ν21 (8b)

1607

1601

1605/

1449

1451

1450/

1617

1588

1391

1375

1379/

1461

1467

1364

1341

1351/

1402

1400

--

--

--

1632

1605

1445/

1405/

1365

1366

1385/

1464

1471

1107

1123/

1403

1407

1109

1123

--/

1080

1084

1124 ν27 (6b)

676

676

667/-

1088/

1337

1343

606

621/-

1360/ 1351

1174

1170

1153/ 1161

1115

--

1092 631

1403/ 1404

1126 ν26 (18b)

1471/ 1469

1366 1138

1590/ 1590

1402

1344 ν25 (15)

1576/

701/ --

--

1411 ν24 (3)

700

1574

1449 ν23 (14)

699

--

1599 ν22 (19b)

645/

770

1096/ 1107

630

639

623/-


16

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For a reasonable comparability one has to consider carefully which band to take into account for the normalization. It should be a vibrational mode that is dominant in both the Raman and the SERS spectra. Thus, it seems to be reasonable to choose either the ν8 or the ν9 fundamental mode. Both of these total symmetric trigonal ring breathing modes are very prominent in the pyridine Raman and SERS spectra, centered at 1037 cm-1 and 1007 cm-1, respectively. However, the ν9 mode is not found in the pyridine derivates substituted at the meta-position, 3-picam and 3-pymeth (valid for calculation and experiment, both, Raman and SERS). The Raman and the SERS spectra of these meta-isomers are dominated by the ν8 fundamental mode, centered at 1039 cm-1 and 1034 cm-1 in the Raman and the SERS spectra, respectively. Moreover, the ν8 fundamental is not present in the ortho-substituted pyridine derivates (2-picam and 2-pymeth). The Raman and the SERS spectra of these ortho-isomers are dominated by the ν9 fundamental, centered at 1011 cm-1 and 1005 cm-1 in the Raman and the SERS spectra, respectively. In the Raman and the SERS spectra of the para-substituted derivates (4-picam and 4-pymeth), both fundamental modes (ν9 und ν8) can be found. However, the ν9 vibration is much more dominant relative to the ν8 vibration. Moreover, the changes in the symmetry cause a shift of the peak position of the ν8 vibrational mode. This observed peak shift is confirmed by the simulated wavenumber values. For this reason for pyridine, 2-picam, 2-pymeth, 4-picam and 4-pymeth the ν9 vibrational mode was used as a reference, while for 3-picam and 3-pymeth the ν8 vibrational mode was taken into account. In the following section the influence of the two different AgNPs as well as the substitution position on the SERS spectra will be discussed in detail, analyzing the differences in the relative band intensities and altering peak position.


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Orientation of the analyte molecule in dependency of the applied AgNPs To investigate the influence of the silver nanoparticles on the spectral information, the two most commonly applied colloids (c-AgNPs and h-AgNPs) were used to record SERS spectra using the LOC-SERS device. For 4-picam, the Raman and the SERS spectra applying h-AgNPs and c-AgNPs are presented in Figure 5a), and a very prominent peak centered at 973 cm-1 appears in the SERS spectrum using h-AgNPs, assigned to the CH2 rocking (ρCH2) and the NH2 twisting (τNH2) of the methylamine functional group, substituted at the para-position in 4-picam.

Figure 5. a) Obtained mean spectra of 4-picam, from bottom to top, Raman spectrum, SERS spectrum using c-AgNPs and SERS spectrum using h-AgNPs. The markings in the top of the panel indicate the most prominent changes in the peak intensities, A1, A2 and B1 are the


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symmetry class of the causing fundamental vibration, while the # indicates modes caused by vibrational modes of the functional group. The asterisk indicates the ν9 vibration, used as a reference mode. b) Two different orientations of the 4-picam molecules relative to the metal surface derived from the changes in the SERS spectra using c-AgNPs and h-AgNPs.

Furthermore, other peaks dominated by vibrational features of functional groups located in para-position of the pyridine ring, are stronger enhanced than in the SERS spectrum using cAgNPs, e.g., 1327 cm-1 (τCH2 and τNH2) and 1422 cm-1 (CH2 wagging ωCH2 and τNH2). The peak centered at 1265 cm-1 (originating from the ν6 fundamental mode) and the two peaks centered at 1541 cm-1 (ν5 and CH2 scissoring σCH2) and 1610 cm-1 (ν4 and NH2 scissoring σNH2) are also more strongly enhanced in the SERS spectrum using h-AgNPs than in the SERS spectrum using c-AgNPs. They were assigned to the A1 class, but all three of them have a notably strong contribution of vibrations of the functional group (C-CH2 vibration, CH2 scissoring and NH2 scissoring, respectively). As mentioned above, the peak caused by the trigonal ring breathing ν8 mode is located at 1209 cm-1 for both applied AgNPs. In general, the in-plane modes belonging to the A1 and B2 class are more strongly enhanced in both the SERS spectra (using c-AgNPs and h-AgNPs), than the modes caused by out-of-plane vibrations, e.g., the peak centered at 797 cm-1 (ν16). This observation can be interpreted regarding the orientation of the 4-picam molecules relative to the metal surface. Taking into account the overall spectra of the Raman measurements, the SERS measurements applying the c-AgNPs and the h-AgNPs with their relative intensities and considering the surface selection rules30, 46 the orientation of the analyte molecules relative to the


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metal surface can be derived. The total symmetric in-plane normal modes (A1 symmetry group) are very strongly enhanced. The same is valid for the other in-plane normal modes (B2 symmetry class) while the out-of-plane modes (A2 and B1 symmetry class), are very weak or not enhanced. Thus, the molecular ring plane (zy-plane, for the normal axes, see Figure 1) is oriented perpendicular to the metal surface of both the c-AgNPs and the h-AgNPs. A schematic of both orientations can be found in Figure 5b). In the SERS spectrum applying h-AgNPs the vibrational modes caused by the methylamine functional group are very prominent and much more enhanced than in the case of c-AgNPs. Thus, it is suggested that the 4-picam molecules are preferentially binding through the amine group of the functional group in the para-position to the h-AgNPs. Considering this orientation of the analyte molecules relative to the metal surface (depicted in Figure 5b), right side) shows that the functional group of 4-picam is orientated very close to the metal surface, leading to a stronger enhancement of the vibrational modes for the SERS measurements using the h-AgNPs. However, the y-axis is expected to be tilted relative to the surface because of the steric influence of the hydrogen atoms of the amine functional group. Thus, the heteroaromatic ring system remains quite close to the surface and the fundamental vibrations of the ring structure are strongly enhanced. The weak intensity of the vibrational modes caused by the functional group in the SERS spectrum using c-AgNPs indicates a binding of the molecules and metal surface through the nitrogen atom of the pyridine ring in the case of c-AgNPs. This leads to a perpendicular orientation of the heteroaromatic ring system relative to the metal surface. Following the surface selection rules30 this orientation causes strong enhancements of the inplane vibrational modes, while the out-of-plane modes are not enhanced. Furthermore, in this orientation the functional group has a very large distance from the metal surface. Hence, the


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modes caused by vibrations of the functional group are not enhanced with the same intensity as the fundamental ring vibrations, due to the evanescent electromagnetic field of the metallic nanoparticles. A binding through both nitrogen atoms is not expected, since in this case, peaks caused by the out-of-plane vibrations (A2 and B1 symmetry class) would be observed, which is not found in the obtained SERS spectra. The Raman and the SERS spectra (applying c-AgNPs and h-AgNPs) of 3-picam are given in Figure 6 panel a). In the spectra of this derivate the peaks caused by out-of-plane fundamental vibrations (B1 and A2 symmetry class), e.g., the peaks centered at 800 cm-1 (ν16) and 960 cm-1 (ν12) have the highest intensity in the Raman spectrum, relative to the ν8 vibrational mode. Their intensities are decreasing in the order, Raman > c-SERS > h-SERS. The same is valid for the modes originated from the vibrations of the methylamine functional group in the meta-position of the pyridine ring, e.g., peaks centered at 1194 cm-1 (τCH2 + τNH2) and 1257 cm-1 (ωCH2).


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Figure 6. a) Obtained mean spectra of 3-picam, from bottom to top, Raman spectrum, SERS spectrum using c-AgNPs and SERS spectrum using h-AgNPs. The markings indicate the most prominent changes in the peak intensities. A1, A2 and B1, B2 are the class of the causing fundamental vibration, while the # indicates modes caused by vibrations of the functional group. The asterisk indicates the ν8 vibration used as a reference mode. b) Two different orientations of the 3-picam molecules to the metal surface derived from the changes in the SERS spectra with cAgNPs and h-AgNPs.

The aforementioned observations can be understood, after considering the orientation of the molecule relative to the metal surface. The suggested orientations are presented in Figure 6 panel b). In the case of c-AgNPs the molecule is binding to the metal surface through the nitrogen atom of the pyridine ring. However, the functional group is not strongly sterically hindered in this case; thus, the amine group is also attracted to the silver surface, resulting in a tilted orientation


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of the 3-picam molecule relative to the surface. In contrast, the binding to the h-AgNPs takes place through the nitrogen atom of the amine functional group located in the meta-position. Thus, the molecule is more perpendicular relative to the surface in this situation. Additionally, the carbon atom participating in the fundamental vibration ν8 is in much closer proximity to the surface, compared to the c-SERS orientation, resulting in a very strong enhancement of this certain vibration. This enhancement leads to relatively smaller intensities of the other fundamental vibrations; the same is valid for the vibrations caused by the functional group, although it is positioned closer to the metal surface in this case. The same experiment was realized for 2-picam, and the spectra are shown in Figure 7 panel a). In this case, the c-SERS and h-SERS spectra are almost identical. However, the Raman spectrum is quite different.

Figure 7. a) Obtained mean spectra of 2-picam, from bottom to top, Raman spectrum, SERS spectrum using c-AgNPs and SERS spectrum using h-AgNPs. The markings indicate the most


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prominent changes in the peak intensities, A1 and B1, B2 are the class of the causing fundamental vibration, while the # indicates modes caused by vibrations of the functional group and the asterisk indicates the ν9 vibrational mode, used as a reverence mode. b) Two possible orientations of the 2-picam molecules to the metal surface derived from the changes in the SERS spectra with c- and h-AgNPs.

The peaks centered at 772 cm-1 and 970 cm-1, both caused by out-of-plane vibrations, ν16 and ν12, respectively, are becoming more strongly enhanced in both the c-SERS and the h-SERS spectra, relative to the ν9 vibrational mode centered at 1007 cm-1. Furthermore, peaks centered at 826 cm-1 (ν10), 1049 cm-1 (ν7), 1312 cm-1 (ν6), 1500 cm-1 (ν5) and 1565 cm-1 (ν4) all caused by in-plane vibrations belonging to the A1 symmetry class are strongly enhanced for both types of colloid, for some of them the methylamine functional group located in ortho-position of the pyridine ring has an additional vibrational influence, e.g., peaks centered at 826 cm-1 (ν10 + ωNH2) and 1312 cm-1 (ν6 + τCH2 + τNH2). The peaks centered at 995 cm-1 (τNH2 + ρCH2), 1166 cm-1 (τCH2 + τNH2), 1268 cm-1 (ωCH2), 1430 cm-1 (ωCH2) and 1623 cm-1 (σNH2), all caused by the vibrations of the methylamine group substituted in the ortho-position of the pyridine ring, become strongly enhanced in SERS spectra applying c-AgNPs and h-AgNPs relative to the ν9 vibrational mode. Again, the spectral appearance can be explained by the orientation of the molecules relative to the metal surface (depicted in Figure 7b)). Due to the close vicinity of the two nitrogen atoms, the molecule is orientated almost identically relative to the metal surface, no matter which nitrogen atom is binding preferentially. The two nitrogen atoms are building a type of chelate bridge, which is a well-known phenomenon47.


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Thus, the presented results confirm that in the case of ortho-, meta- and para-substituted picolylamine the colloid has a very high impact. To the c-AgNPs the molecules are preferentially binding through the nitrogen of the heteroaromatic ring system, while using h-AgNPs means that the nitrogen of the functional group is preferentially binding to the metal surface. However, the spectral differences decrease the closer the two nitrogen atoms are located in the molecule. Thus the c-SERS spectrum and the h-SERS spectrum are differing the most in the case of 4-picam, while no significant differences in the spectra could be identified in the case of 2-picam. Influence of different functional groups on the SERS response Next, the impact of different functional groups on the SERS information was considered. For this, the Raman and c-SERS spectra of 4-, 3-, and 2-picam, 4-, 3-, and 2-pymeth and pyridine were investigated, as shown in Figure 8. In the difference spectra it becomes obvious that the Raman spectra for derivates with the two different functional groups, e.g., methylamine and hydroxymethyl, in the same substitution positon are almost identical for the ortho-, meta- and para-positions, respectively. Thus, for the Raman spectra the functional group has almost no impact, but the position of the substituent group changes the Raman spectra significantly, due to the changed symmetry of the substituted molecule.48-50


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Figure 8. Raman and c SERS spectra of 2-,3-, and 4-picam (in the bottom of each plot) and 2-, 3-, and 4-pymeth (in the top of each plot). Each graph presents the spectra of two derivates having different functional groups in the same substitution position and their difference spectra (in the center of each plot). In the left column the Raman spectra are shown, in the right column the c-SERS spectra are depicted. For all measurements the mean spectra are presented with the standard deviation values indicated in gray.

The Raman spectra of the two ortho-substituted derivates have a very similar shape to the Raman spectrum of pyridine. The peaks centered at 1014 cm-1 (2-picam) and 1012 cm-1 (2-


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pymeth) are caused by the total symmetric trigonal ring breathing mode ν9, in excellent agreement with the same vibrational mode in pyridine (centered at 1007 cm-1). However, the peaks centered at 1061 cm-1 (2-picam) and 1058 cm-1 (2-pymeth) do not originate from the same vibration similar to the peak centered at 1040 cm-1 in the Raman spectrum of pyridine. The simulated values reveal that these peaks are caused by the ν7 fundamental mode for the orthosubstituted derivates. Thus, without the information of simulated normal modes it would be very difficult to find the right band assignment in this case. Furthermore, there is no peak located in the region of 1220 cm-1 (ν6 in the Raman spectrum of pyridine), but centered at 1237 cm-1 (2picam) and 1234 cm-1 (2-pymeth), respectively. These peaks are no shifted signals caused by the ν6 fundamental. Both of them are caused by vibrational modes of the functional groups. In 2picam, it is assigned to the ωCH2 vibrational mode, while a combination of the ωCH2 and the δOH vibrational modes is causing the peak in the Raman spectrum of 2-pymeth. Again, it becomes obvious that the simulation of the vibrational modes is essential in order to make a correct and valid band assignment. The situation changes dramatically regarding the SERS spectra of 2picam and 2-pymeth. The modes caused by fundamental vibrations of the A1 and B2 symmetry class and the modes caused by the functional groups are strongly enhanced compared to the ν9 mode. Subsequently, the orientation of the molecule relative to the metal surface, as discussed above for the influence of the AgNPs on the SERS spectrum of 2-picam is also valid for 2pymeth. The two SERS spectra strongly differ over the whole spectral range under investigation. The most prominent changes arise from vibrations of the hydroxymethyl group of 2-pymeth, 1323 cm-1 (δOH), 1510 cm-1 (ν5 + ωCH2 + δOH), 1618 cm-1 (ν21 + νC-CH2 + δOH) and the methylamine group of 2-picam, 995 cm-1 (τCH2 + ωNH2), 1224 cm-1 (νC-CH2) and 1404 cm-1 (ν23 + τCH2). Generally, the contributions of the hydroxymethyl functional group are more strongly


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enhanced than the ones of methylamine. This indicates a stronger interaction of the hydroxymethyl functional group with the metal surface of the c-AgNPs compared to the methylamine functional group. However, the simultaneous interaction of the nitrogen atom of the heteroaromatic ring system and the functional group causes a very strong enhancement of the vibrational modes of these groups. Thus, in the SERS spectra, the differentiation between the two different pyridine derivates becomes possible. The most prominent changes in the Raman spectrum of 3-pymeth compared to the Raman spectrum of pyridine are the modes centered at 837 cm-1 (ν10 + ωCH2), 1196 cm-1 (δOH) and 1216 cm-1 (ν6 + δOH + ωCH2). While the changes in the Raman spectrum of 3-picam are the peaks centered at 1054 cm-1 (ν7 + ωNH2) and 1653 cm-1 (σNH2). For both meta-substituted derivates, no peak appears that is caused by the ν9 fundamental mode. Regarding the SERS spectra of the two meta-substituted molecules one can still identify some changes in the spectra that arise due to the functional group. For the hydroxymethyl substituted derivate, an enhancement of the peak centered at 1384 cm-1 (ν23 + τCH2) can be observed. The peak marking 3-picam is centered at 1594 cm-1 (ν4 +σNH2). However, it has to be mentioned that the changes in both spectra are not very strongly pronounced, and it could be hard to identify the correct chemical structure, regarding the functional group, with the help of the Raman or the SERS spectra. For both derivates, the peak intensities in the SERS spectra are decreasing compared to the Raman spectra (relative to the very dominant ν8 vibrational mode). In the case of the para-substituted pyridine derivates, 4-pymeth and 4-picam, the most prominent changes in the Raman spectra (compared to the Raman spectrum of pyridine) are the peaks centered at 812 cm-1 (ν10+ ωCH2 + δO-H) for 4-pymeth and 797 cm-1 (ν16 + τNH2), 1083 cm-1


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(ν7 + ωCH2) and 1341 cm-1 (ν24 + τCH2) for 4-picam. Furthermore, the peak centered at 1212 cm-1 is caused by the ν8 fundamental mode. In the Raman spectrum of pyridine the ν8 fundamental mode causes a peak centered at 1040 cm-1. However, the differences between the Raman spectra of the para-substituted derivates and the Raman spectrum of pyridine originate from the symmetric changes due to the substitution position. Thus, no significant signals of the functional groups are observed. For both molecules, the overall shape of the spectrum changes significantly from Raman to SERS, but the differences in the spectra between the two different derivates are only very small in both the Raman and the SERS. In the SERS spectra of 4-pymeth and 4-picam, the changes are the peaks centered at 1440 cm-1 (4-pymeth: ν22 + ωCH2 + δOH) and 1633 cm-1 (4-pymeth: ν5). However, this A1 mode is divided into a duplet for 4-picam (regarding the simulated vibrational modes), therefore, the reason for this difference is the scissoring of the amine (σNH2) group of 4picam causing this shift. Furthermore, the peaks centered at 1605 cm-1 (ν21 + τCH2) and 613 cm-1 (ν17 + ωCH2) are slightly shifted in 4-picam. In general, the changes between the derivates, having different functional groups in the same substitution position are very small for the meta- and para-substituted pyridine derivates investigated in this study. For the ortho-substituted derivates the Raman spectra are also very similar, but the SERS spectra show very significant differences. The observations confirm the suggested orientation of the analyte molecules relative to the metal surface of c-AgNPs. Both the hydroxymethyl and the methylamine substituted pyridine derivates are binding to the metal surface preferentially through the nitrogen atom of the pyridine ring. Depending on the substitution position of the functional group, the signal intensity of peaks


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caused by its vibrational modes are strongly enhanced, e.g., for the ortho- position. Due to the sterically close proximity of the functional group and the nitrogen atom of the heteroaromatic ring system, a simultaneous interaction with the metal surface becomes possible. For the metaand the para-substituted derivates, the sterically hindrance leads to a very weak or no enhancement of the peaks caused by the vibrational modes of the functional groups compared to the fundamental ring vibrations. CONCLUSIONS The SERS spectra of 4-, 3- and 2-picam were recorded with c-AgNPs and h-AgNPs, and the changes in the spectral information of both SERS measurements for each isomer were discussed, confirming that in the case of ortho-, meta- and para-substituted picolylamine, the colloid has a very high impact. The SERS spectra using c-AgNPs and h-AgNPs differ, especially in the case of 4-picam. The reason for the very prominent difference is the changed orientation of the 4picam molecules relative to the metal surface. For c-AgNPs, the analyte molecules are binding to the metal surface preferentially through the nitrogen atom of the pyridine ring. When applying hAgNPs, the binding occurs preferentially between the nitrogen atom of the methylamine functional group and the metal surface. For 3-picam significant changes in the relative intensity ratios were recognized between the SERS spectra using c-AgNPs and h-AgNPs, also arising from the relative orientation of the molecules to the metal surface. The preferred binding positions are the same for 3-picam and 4-picam. However, the relative orientation of the molecules to the surface differs significantly regarding the functional groups. For 3-picam the interaction between the nitrogen of the functional group becomes possible because the steric barrier becomes smaller. Considering 2-picam, no significant changes between the SERS spectra applying c-AgNPs and h-AgNPs were observed. Thus, the molecules have the same orientation


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relative to the surface for this isomer. The nitrogen atoms of the pyridine ring and the functional group are interacting simultaneously with the metal surface. The spectral differences are decreasing more, the closer the two nitrogen atoms are located in the molecule. Thus, the SERS spectra using c-AgNPs and h-AgNPs are differing the most in the case of 4-picam, for 3-picam some slight changes in intensity are denoted, while no significant differences in the SERS spectra using c-AgNPs and h-AgNPs cloud be identified in the case of 2-picam. Furthermore, the impact of the substitution group was investigated using two different functional groups as a substituent, e.g., hydroxymethyl and methylamine, in the three different substitution positions (ortho, meta and para). It was observed that the vibrations of the functional group are strongly enhanced if the functional group is close to the surface, either for steric reasons or because of attractions between atoms of the functional group and the metal surface. Thus, in the SERS spectra of 2-picam and 2-pymeth, the vibrational information of the functional groups becomes very prominent. However, no significant differences are observed in the Raman spectra of these two pyridine derivates, respectively. In this investigation, it becomes obvious that the SERS spectra are influenced by various parameters. Considering the type of the used SERS substrate and the substitution position of the functional group makes it possible to change the SERS spectra and apply these derivates as SERS marker molecules. Hence, the choice of marker and sensor molecules should be made with careful consideration of the functional groups and their position in the molecule. By manipulation of the substitution position spectrally silent regions can be generated, or existing silent regions can be utilized to detect a certain isomer and achieve additional information. Moreover, the observed influence of the substitution position as well as the applied SERS-active colloid on the SERS spectral profile will be applicable to other molecule classes. The


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investigations in this contribution make obvious, that it is essential in all types of SERS applications in life science to pay careful attention to the applied metal nanoparticles, the functional groups and their substitution position. SUPPORTING INFORMATION Figure SI1. Atomic motion for the individual fundamental vibrational modes, simulated with the program package GAUSSIAN09 applying second order Møller-Plesset perturbation theory in conjunction with the 6 311++G(d, p) basis set. ACKNOWLEGMENTS Funding of the research project JBCI 2.0 (03IPT513Y- Unternehmen Region, InnoProfile Transfer) from the Federal Ministry of Education and Research, Germany (BMBF) is gratefully acknowledged. The authors thank Dr. Thomas Henkel for providing the microfluidic chips and Dr. Anne März for constructive discussions and support in the present manuscript. CORRESPONDING AUTHOR [email protected] REFERENCES 1. Corrsin, L.; Fax, B. J.; Lord, R. C., The Vibrational Spectra of Pyridine and Pyridine-D5. J Chem Phys 1953, 21 (7), 1170-1176. 2. Long, D. A.; Murfin, F. S.; Hales, J. L.; Kynaston, W., Spectroscopic and Thermodynamic Studies of Pyridine Compounds .1. Infra-Red and Raman Spectra of Pyridine and the Alpha-Picolines, Beta-Picolines and Gamma-Picolines. T Faraday Soc 1957, 53 (9), 1171-1180. 3. Long, D. A.; Murfin, F. S.; Thomas, E. L., Spectroscopic and Thermodynamic Studies of Pyridine Compounds .2. Normal Co-Ordinate Calculations for Pyridine and Pyridine-D5. T Faraday Soc 1963, 59 (481), 12-&.


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4. Kline, C. H. J.; Turkevich, J., The Vibrational Spectrum of Pyridine and the Thermodynamic Properties of Pyridine Vapors. J Chem Phys 1944, 12, 300-309. 5. Wilmshurst, J. K.; Bernstein, H. J., The Vibrational Spectra of Pyridine, Pyridine-4-D, Pyridine-2,6-D2, and Pyridine-3,5-D2. Can J Chem 1957, 35 (10), 1183-1194. 6. Berezin, V. I., Calculation and Interpretation of the Vibrational Spectra of Pyridine and Deuteropyridines - Planar Vibrations. Opt Spektrosk+ 1963, 15 (3), 310-314. 7. Ten, G. N.; Kadrov, D. M.; Berezin, M. K.; Baranov, V. I., Calculation and Interpretation of Vibronic Absorption and Fluorescence Spectra of the First Electronic n pi* Transitions of Pyridine and Pyrimidine. Opt Spectrosc+ 2014, 117 (5), 713-721. 8. Castellu.E; Sbrana, G.; Verderam.Fd, Infrared Spectra of Crystalline and Matrix Isolated Pyridine and Pyridine-D5. J Chem Phys 1969, 51 (9), 3762-&. 9. Fleischmann, M.; Hendra, P. J.; Mcquilla.Aj, Raman-Spectra of Pyridine Adsorbed at a Silver Electrode. Chem Phys Lett 1974, 26 (2), 163-166. 10. Wen, R.; Fang, Y., Adsorption of Pyridine Carboxylic Acids on Silver Surface Investigated by Potential-Dependent SERS. Vib Spectrosc 2005, 39 (1), 106-113. 11. Choi, S. H.; Park, H. G., Surface-Enhanced Raman Scattering (SERS) Spectra of Sodium Benzoate and 4-Picoline in Ag Colloids Prepared by Gamma-Irradiation. Appl Surf Sci 2005, 243 (1-4), 76-81. 12. Yamada, H.; Nagata, H.; Toba, K.; Nakao, Y., Charge-Transfer Band and SERS Mechanism for the Pyridine-Ag System. Surf Sci 1987, 182 (1-2), 269-286. 13. Zuo, C.; Jagodzinski, P. W., Surface-Enhanced Raman Scattering of Pyridine Using Different Metals: Differences and Explanation Based on the Selective Formation of AlphaPyridyl on Metal Surfaces. J Phys Chem B 2005, 109 (5), 1788-1793. 14. Arenas, J. F.; Tocon, I. L.; Otero, J. C.; Marcos, J. I., Charge Transfer Processes in Surface-Enhanced Raman Scattering. Franck-Condon Active Vibrations of Pyridine. J Phys Chem-Us 1996, 100 (22), 9254-9261. 15. Bantz, K. C.; Meyer, A. F.; Wittenberg, N. J.; Im, H.; Kurtulus, O.; Lee, S. H.; Lindquist, N. C.; Oh, S. H.; Haynes, C. L., Recent Progress in SERS Biosensing. Phys Chem Chem Phys 2011, 13 (24), 11551-11567. 16. Wang, Y. L.; Schlücker, S., Rational Design and Synthesis of SERS Labels. Analyst 2013, 138 (8), 2224-2238. 17. Arenas, J. F.; Montanez, M. A.; Otero, J. C.; Marcos, J. I., Surface Enhanced RamanSpectra of 2-Cyanopyridine and Picolinamide. J Mol Struct 1993, 293, 341-344. 18. Osaki, T.; Yoshikawa, T.; Satoh, Y.; Shimada, R., Adsorption of Pyridine, GammaPicoline and Isonicotinonitrile on Ag Colloidal Particles Studied by Surface-Enhanced Raman Scattering Spectroscopy. J Raman Spectrosc 2005, 36 (3), 199-207. 19. Ikezawa, Y.; Saito, H.; Matsui, K.; Toda, G., A Study of the Competitive Adsorption of Pyridine and Monosubstituted Pyridines on a Silver Electrode by the SERS Method. Surf Sci 1986, 176 (3), 603-609. 20. Bunding, K. A.; Lombardi, J. R.; Birke, R. L., Surface Enhanced Raman-Spectra of Methylpyridines. Chem Phys 1980, 49 (1), 53-58. 21. Kobayashi, M.; Imai, M., SERS Intensities of Pyridine and Its Derivatives Adsorbed on Ag Electrodes. Surf Sci 1985, 158 (1-3), 275-285. 22. Aroca, R. F.; Alvarez-Puebla, R. A.; Pieczonka, N.; Sanchez-Cortez, S.; Garcia-Ramos, J. V., Surface-Enhanced Raman Scattering on Colloidal Nanostructures. Adv Colloid Interfac 2005, 116 (1-3), 45-61.


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Fundamental SERS Investigation of Pyridine and Its Derivates as a

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