Application of Vibrational Spectroscopy in ... - Semantic Scholar

22 downloads 0 Views 256KB Size Report
quantification of calcium oxalate crystalline phases by infrared spectrophotometry", Clinica chimica. Acta, 298(1-2): 1-11. 3. Menon, M. and M.I. Resnick, 2002.
Journal of Applied Sciences Research, 3(5): 387-391, 2007 © 2007, INSInet Publication

Application of Vibrational Spectroscopy in Identification of the Composition of the Urinary Stones 1

1

Safaa K.H. Khalil and 2 Mohamad A. Azooz

Spectroscopy Department, Physics Division, National Research Center, Giza, Egypt. 2 Urology Department, Students’ Hospital of Cairo University.

Abstract: The main objective of this study was the application of vibrational spectroscopic techniques (FT-IR and FT -Raman) to identify the chemical composition of the urinary stones, since they offer high specific chemical information, rapid and reliable methods. Urinary stones are common throughout the world. Identification of the chemical composition of the urinary stones provides a precise diagnosis of the cause of lithogenesis and the consequence of appropriate therapeutic regimen and prophylaxis against recurrences. Fifty urinary stones were extracted by different urological interventions for chemical identification by using FT-IR and FT-Raman spectroscopic methods. According to the spectral results, the stones were classified into six groups. The chemical composition of these groups and their statistical existence were calcium oxalate monohydrate (whewellite) 40%, ammonium magnesium phosphate (struvite) 24%, uric acid 12%, mixture of calcium oxalate monohydrate with tricalcium phosphate (W hitlockite) 12%, mixture of tricalcium phosphate with calcium carbonate 8% and lastly cystine stones 4%. In conclusion, Raman spectroscopy can be recommended as a new accurate and reliable tool for chemical identification of urinary stones. However, the extensive spectroscopic study performed in this experimental work can be extended and developed to build a Raman spectral library of all commonly occurring urinary calculi as well as the already existing FT-IR library for urinary stones. Key words: Urinary stones, FT-Raman spectroscopy, FT-IR spectroscopy INTRODUCTION

related to changes in dipole moment of the molecule. In Raman spectroscopy, a Raman shift is correlated with the polarizability of the molecule. Thus, certain vibrations have strong absorption in IR spectroscopy and weak vibrations in Raman and vice versa. IR spectroscopy is based on the interaction of the IR light and the molecules in the spectral region from 4000 to 400 cm -1 , offering advantages in measuring time and information content but often requires significant sample preparation [5 ]. The use of IR spectroscopy increased from 8% in 1980 to 81% in 2005 [1 ]. Raman spectroscopy combines highly specific chemical information with minimum preparation and rapid analysis. It uses a strong monochromatic light source such as laser, to move molecules of the sample into an excited virtual state. The molecules instanteously relax to the ground state, emitting the incident radiation in a process referred to scattering. Most of the radiation is elastically scattered (the frequency of the emitted radiation is exactly at the frequency of the incident laser light) and small percentage is inelastically scattered, shifted away from the excitation laser therapy. The degree of the shift

Urinary stones are common throughout the world. Stone analysis is of great importance to the therapy and metaphylaxis of residual and recurrent stones [1 ]. The diagnostic usefulness of information regarding the chemical composition of urinary stones has been recognized since 1950s and has significantly improved during last years. So it is now possible to correlate the results of every analysis with the appropriate diagnosis and therapeutic regimen [2 ]. Analysis of urinary stones by infrared (IR) spectroscopy and other techniques such as chemical methods, x-ray diffraction, polarization microscopy and thermo analytical procedures have been widely investigated [3 ], while Raman spectroscopy for urinary stones identification still a virgin area of study. Raman spectroscopy has been used to study the presence of struvite in urine [4 ]. Raman and IR spectroscopies are complementary techniques; both measure the fundamental molecular vibrations but the selection rules for activity are very different. In IR spectroscopy, absorption of radiation is

Corresponding Author:

Safaa K.H. Khalil, Spectroscopy Department, Physics Division, National Research Center, El-Bohoth street, Dokki, Giza, Egypt. P.O 12311. Email: [email protected] 387

J. Appl. Sci. Res., 3(5): 387-391, 2007 from the excitation laser frequency, called Raman shift, is related to the energy of the fundamental molecular vibrations of the molecules in a sample [5 -7 ]. However to examine the molecular vibrations to the full, both IR and Raman spectroscopies should be used together. This paper reports the full vibrational spectra of the urinary stones using the vibrational spectroscopic techniques FT-IR and FT-Raman as a conventional reference method and a new promising accurate method, respectively. M ATERIAL AND M ETHODS Fifty urinary stones were obtained by variable surgical interventions throughout the urinary tracts of the patients. The stones were washed with distilled water, air dried and pulverized into fine particles (particle size range 90-125 µm). Each stone sample was examined by Fourier Transform-Infrared (FT-IR) and Fourier Transform-Raman spectroscopy techniques. The samples were prepared in KBr pellets and a Jasco FT/IR-430 spectrometer, operated at absorbance mode and resolution 4 cm -1 , was used for recording the infrared absorption spectra. The FT -Raman spectra were measured by a spectrometer Nexus 670 FT-IRFT-Raman, Nicolet, USA. The excitation source in the FT-Raman module is Neodymium-Yttrium-AluminumGarnet (Nd:YAG) laser, which emits continuous wave laser energy at a wavelength of 1064 nm, with a maximum power level of approximately 1.5W at the sample. The installed air-cooled detector is In GaAs. The beam splitter is XT-KBr. The used sample configuration is 180º reflective with fully motorized sample position adjustment, with an NMR-tube sample holder. The stones powder samples were filled in the NMR glass tubes, 5 mm internal diameter, and the spectra were collected at resolution 8 cm -1 and 132 scans in the spectral range 3701-98cm -1 .

Fig. 1: FT -Raman and FT -IR Spectra of Calcium Oxalate monohydrates stone.

RESULTS AND DISCUSSIONS Results: Fifty common urinary stones were analyzed in this study using vibrational spectroscopic techniques (Raman and Infrared). T he spectra were categorized into six groups according to their spectral patterns. The Raman and IR spectra of each group were presented together in one figure for comparison purpose. Figures (1-5) exhibited the spectra of the five groups in the spectral regions 2000 -100 cm -1 and 2000-400 cm -1 of the Raman and IR spectra, respectively. Figure (6) showed the complete spectral region starting from 4000 cm -1 because it has significant bands of the amino groups in cystine stones. Careful investigation of the IR spectra in figures (1-6) revealed that they were consistent with those

Fig. 2: FT-Raman and FT-IR Spectra of Magnesium ammonium phosphate stone.

388

J. Appl. Sci. Res., 3(5): 387-391, 2007

Fig. 3: FT-Raman and FT-IR Spectra of Uric Acid.

Fig. 5: FT-Raman and FT-IR Spectra of Carbonateapatite stone.

Fig. 4: FT -Raman and FT -IR Spectra of Calcium oxalate/Calcium Phosphate mixed stone.

Fig. 6: FT-Raman and FT-IR Spectra of Cystine.

389

J. Appl. Sci. Res., 3(5): 387-391, 2007 reported in literature and could be attributed to the following chemical compositions:

appeared at 1584 cm -1 and C=O stretching vibrations at 1337 and 1297 cm -1 .

1.

Discussion: Knowledge of the composition of the stones has helped to determine the underlying causes of stone disease, in an attempt to prevent its recurrence, and this had a direct impact on the choice of treatment [8 ]. Numerous standard chemical and physical techniques have been applied for the determination of the urinary stone composition. New frontiers have been used to determine stone composition as high resolution helical computed tomography (CT) [9 ] and microarea x-ray diffractometry and liquid chromatography-mass spectrometry (LC-MS) [1 0 ]. During the last three decades, the use of IR spectroscopy is increasing [1 ]. The application of IR and Raman microscopy to biomedical analysis including calcium and crystalline materials were also addressed [1 1 ]. Stones commonly encountered in urology practice are calcium oxalate, calcium phosphate, mixed calcium oxalate and calcium phosphate, struvite and to less extent, stones composed of uric acid and rarely cystine stone [3 ]. This study included 40% calcium oxalate monohydrate stones and 12% calcium oxalate mixed with tricalcium phosphate stones. Identification of the oxalate was done by two sharp strong bands in the Raman spectrum at 1490 and 1464 cm -1 specific to c=o stretching vibration with a specific oxalate monohydrate band at 207 cm -1 which is due to Ca-O ring stretching or bending modes. This is in agreement with Frost and W eier [1 2 , 1 3]. Calcium phosphate of the whitlockite stones were detected by the strong Raman peak of P-O stretching vibration mode at 964 cm -1 and two additional bands at 585 and 431 cm -1 , while in the struvite stone, the phosphates were identified in the Raman spectrum by strongest vibrational mode peak for P-O bond of the (PO 4 ) -3 group at 947 cm -1 and some additional peaks with variable intensities at 565, 404, 299 and 186 cm -1 . This is in accordance with Frost and his colleagues [4 ]. In this study, specific identifiable peaks of uric acid stones were similar to the data reported by Ohmacht [1 4 ]. The Raman and IR spectra of stones in group (5) showed the main vibrational bands characteristic of a carbonate apatite structure which were more or less near to the results of Bohic and Ohmacht [1 4 , 1 5] . The recorded spectra of group (6) were identical to the reported spectra of cystine which were noticed in other series [1 6 -1 8 ]. The spectra obtained in this work illustrated several important aspects of Raman, in particular in relation to IR spectroscopy. Firstly the Raman spectra are “fingerprints” which are in the same way as IR spectra, they may be potentially useful for identification of stone composition from one to another. Secondly,

2. 3. 4.

5. 6.

Calcium oxalate monohydrate (whewellite) 40% (20 stones). Magnesium ammonium phosphate (struvite) 24%, (12 stones). Uric acid 12% (6 stones). M ixture of calcium oxalate monohydrate (whewellite) and tricalcium phosphate (whitlockite) 12% (6 stones). Carbonate containing calcium phosphate (carbonate apatite, dahllite) 8% (4 stones). Cystine 4% (2 stones).

The oxalates were characterized by two sharp strong bands in the Raman spectrum at 1490 and 1464 cm -1 specific to C=O stretching vibration (Fig. 1). There was also a specific oxalate monohydrate band at 207 cm -1 which was due to calcium-oxygen (Ca-o) ring stretching or bending modes. The phosphates were identified in the Raman spectrum of the magnesium ammonium phosphates (Fig. 2) by the strongest vibrational mode peak for P-O bond of the (PO 4 ) -3 groups at 947 cm -1 and some additional peaks with variable intensities at 565, 404, 299 and 186Cm -1 . Uric acid (Fig. 3) had specific identifiable peaks in IR spectrum that occur at 710, 750, 780, 1590 and 1675cm -1 . In group (4) the stones were of mixed composition. Beside the oxalates peaks, the calcium phosphates of the mineral whitlockite (Fig. 4) were identified by the strong Raman peak of P-O stretching vibration mode at 964 cm -1 and two additional bands at 585 and 431 cm -1 . The Raman and IR spectra of stones in group (5) showed the main vibrational bands characteristic of a carbonate apatite structure. The spectra (Fig. 5) were dominated by a very strong band at 964cm -1 and 1035 cm -1 , respectively which assigned to the symmetrical stretching mode of the phosphate group. The Raman spectra displayed other Raman active phosphate vibrational peaks at 431 and 584 cm -1 which were due to O-P-O symmetric and asymmetric bending mode, respectively. The carbonate vibrational mode showed a peak at 1077 cm -1 . The IR spectra revealed the indicative bands of the carbonate at 1470, 1430, 870 and 760 cm -1 . The Raman Spectra of cystine stones (Fig. 6) were readily distinguished from the other stones by a very strong band at 499 cm -1 of the disulfide bridge S-S stretching modes. There were also two strong bands at 2967 and 2916 cm -1 assigned to CH 2 groups. The IR spectra of cystine exhibited the specific bands of the protein, C=O symmetric and asymmetric stretching vibrations located at 1400 and 1620 cm -1 , respectively. The NH 2 scissors band 390

J. Appl. Sci. Res., 3(5): 387-391, 2007 molecular vibrations normally give rise to both IR and Raman bands but their intensities very different, and the prominent group frequencies familiar to the IR users do not appear reliably in the Raman spectrum and vice versa. As a consequence it should be emphasized that both IR and Raman spectra should be acquired whenever the full molecular vibrational analysis is required. T he third point is the individual features of the Raman spectra may be used to indicate the presence of specific chemical groups as with IR. Lastly, Raman spectroscopy has the advantage that bands below 400 cm -1 are readily obtained. This is important to the study of oxalates as the (M-O) stretching and (O-M-O) bending modes may be determined. In conclusion, Raman spectroscopy can be recommended as a new accurate, reliable and rapid tool for chemical identification of urinary stones. However, the extensive spectroscopic study performed in this experimental work can be extended and developed to build a Raman spectral library of all commonly occurring urinary stones as well as the already existing FT-IR library for urinary stones.

9.

10.

11.

12.

13.

REFERENCES 1. 2.

3.

4.

5.

6. 7.

8.

Sahubert, G., 2006. " Stone analysis". Urol. Res. Feb., 14: 1-5. Estepa, L.M, P. Levillain, B. Lacour, M. Daudon, August 2000. "Advantage of zero-crossing-point first derivative sp ectro p ho tom etry for the quantification of calcium oxalate crystalline phases by infrared spectrophotometry", Clinica chimica Acta, 298(1-2): 1-11. Menon, M. and M.I. Resnick, 2002. "Urinary lithiasis: etiology, diagnosis, and medical management", in W alsh PC, Retik AB, Vaughan ED, W ein A.J. (eds) "Campbell's urology", 8 th ed W .B Saunders Co. Philadelphia Chapt, 96: 3229-3305. Frost, R.L., M.L. W eier, W .N. Martens, D.A. Henry, S.J. Mills, 2005. " Raman spectroscopy of newberyite, hannayite and struvite", Spectrochim Acta Mol Biomol Spectrosc, 62(1-3): 181-8. Pavia, D.L., G.M. Lampman, G.S. Kriz, 1996. "Introduction to spectroscopy", second Edition, Saunders colleague publishing. M cCreery, R.L, 2000. "Raman spectroscopy for chemical analysis", John wiley and Sons. Inc. W artewing, S., 2003. "IR and Raman spectroscopy fundamental processing", wiley-VCH, verlag GmbH and CO. KCoa A, W einheim. Cytron, S.E., S. Kravchick, BEN-AMI Sela, E. Shulzinger, I. Vasserman, Y. Raichlin and A. Katzir, 2003. "Fiberoptic Infrared spectroscopy: A Novel tool for the analysis of urine and urinary salts in situ and in real time", Urology, 61(1): 231-235.

14.

15.

16.

17.

18.

391

Zarse, L.A., J.A. McAteer, A.J. Sommer, S.C. Kim, R.F. Paterson, E.K. Hatt, J.E. Lingeman, A.P. Evan, J.C. W illiams, Jr, 2004. "Helical computed tomography accurately reports urinary stone composition using attenuation values: in vitro verification using high–resolution micro-computed tomography calibrated to fourier transform infrared microspectroscopy", Urology, May 63(5): 828-833. Kaneko, K., T. Yamanobe, M. Onoda, K. Mawatari, K. Nakagomi, S. Fujimori, 2005. "Analysis of urinary calculi obtained from a patient with idiopathic hypouricemia using micro area x-ray diffractometry and LC-MS", Urol. Res., Dec., 33(6): 415-421. Kalasinsky, V.F., 1996. "Biomedical applications of infrared and Raman microscopy", Applied spectrosc. Rev, Aug., 31(3): 193-249. Frost, R.L. and M .L. W eier, 2003. "Thermal treatment of weddellite- a Raman and infrared emission spectroscopic study", Thermochimica Acta, 406: 221-232. W eier, M.L. and R.L. Frost, 2004. "Thermal treatment of whewellite- a thermal analysis and Raman spectroscopic study", Thermochimica Acta, 409: 79-85. Ohmacht, R., 1976. "Analysis of renal tract calculi by the spektromom 2000 infrared spectrophotometer", Hungarian Scientific. instruments, 38. Bohic, S., C. Rey, A. Legrand, H. Sfihi, R. Rohanizadeh, C. Martel, A. Barbier and G. Daculsi, 2000. "Characterization of the trabecular rate bone mineral; effect of ovarlectomy and bis phosphonate treatment", Bone, April, 26(4): 341-348. Nakamura, K., S. Era, X . Ozaki, M. Sogami, T. Hayashi, M. Murakami, 1997. "Conformational changes in seventeen cystine disulfide bridges of bovine serum albumin proved by Raman spectroscopy", FEBs letters, 417(3): 375-378. Thamann, T.J., 1999. “Vibrational spectroscopic assignment of the disulfide bridges recombinant bovine growth hormone and growth hormone analogs”. Spectrochimica Acta Part A, Molecular and Biomolecular spectroscopy, 55(7-8): 1661-1666. Long, D.A., 1977. "Raman spectroscopy", McGraw-Hill, New York.