Experimental Chemistry II
Experimental Chemistry II CHEM C1260
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mmu immu ipppppr apppwwwwwwwwwwNNNNNN mpwwu Aommxlmm Naomm mmbwwu Aommummb CPLETI39 26216 1 July 2008 Disk Used No of Pages 5 Model SG Chemical Physics Letters xxx 2008 xxxexxx IELSEV I ER Contents lists available at ScienceDirect Chemical Physics Letters journal homepage wwweseviercomocatecpett CHEMICAL PHYSICS LETTERS Intracellular quanti cation by surface enhanced Raman spectroscopy Ali Shamsaie jordan Heim Ahmet Ali Yanik Joseph Irudayaraj Bindley Biosciences Center W Purdue University 225 SLUniversity Street 215 ABE Building West Lafayette IQ 47907 United States ARTICLE INFO ABSTRACT Article history Received 3 March 2008 In nal form 21 June 2008 Available online xxxx Metallic nanoparticles in a cellular environment have a tendency to aggregate which poses a major obstae cle in extending in vivo surface enhanced Ram tion and into quanti cation domain We introduce and demon enable precise quanti cation of exogenous an spectroscopy SERS applications beyond the quali car strate a novel SERS technique that will chemicals in living human cells Effective quanti cation of the local concentrations of a dinitrophenol derivative DAMP based on a normalizing technique is shown by utilizing gold nanoparticle entrapment in the that the quanti cation technique developed lysosomal compartments in human cells We believe here is general and can be extended to different environ ments utilizing different types of nanoparticles beyond the intracellular scheme pro osed p 2008 Published by Elsevier BV Local optical elds of metallic nanoparticles can give rise to en hanced Raman si als that contain highly localized information about the surrounding environment Using this principle it is pos sible to deploy these nanosensors inside living cells and collect informative signals from the intracellular microcosm within sec ondsi Since the original work by Chourpa et a1 there have been few studies on the intracellular applications of SERS spectroscopy In these studies detection of both native cellular constituents an exogenous molecules has been reported 2 5 Major obstacles prevent the extension of such applications beyond the quali cation and into the quanti cation domaini Conventionally SERS signals are highly irreproducible and do not render themselves to accept able quanti cation of target intracellular constituents In addition delivering SERS inducers for example goldsilver nanostructures ints of interest inside cells is another major challenge For effective enhancement effects gold nanoparticles used for SERS studies must be larger than 20 nm in size 671 When such parti cles are used for in vivo studies after passive uptake by the cells gets caught in the endosomallysosomal pathway and is inevitably trapped in thesegompartmentsJaBL Kneipp et all in one of their lat est works have rlghtly considere the lysosmal context of nanopar ticle aggregates when interpreting the SERS bands 91 The only example of a semi quantitative intracellular application of SERS was presented by Talley et all where functionalized silver nanopar ticles were employed as sensors to provide a rough estimate of intracellularthJ lO For t rst tlme we introduce an experimental setup that takes advantage of the cooccurrence of gold nanoparticles and an exogenous chemical ie DAMP inside the lysosomal compart ment of a living human cell for quantifying the local concentration of this chemical in living human cells The focus of our report is not Corresponding author Fax 1 765 496 1115 Email address josephipurdueedu j Irudayaraj 0009726145 7 see front matter 2008 Published by Elsevier BV doi101016jcplett200806064 on SERS quanti cation rather to suggest a simple normalization strategy to quanti 39 a known analyte intracellularlyi In order to perform a consistent calibration we have also developed a novel normalization technique that uses the phononvibronplasmon modes inherent to the nanostructure as an internal standard thus eliminating the need for a second referencing analyte For quanti cation studies we chose a non uorescent weak basic amine a derivative of dinitrophenol DAMP because it has been shown that this chemical selectively accumulates in the cellular compart ments with low internal RH L1 11 Conventionally DAMP is used as a LysoTrackerW probe to investigate the biosynthesis and pathogen esis of lysosomes through secondary uorescent antibody imaging techniquesl12 Fig 1A depicts the structure of DAMP F10 epithelial cells were grown to con uence and incubated overnight with 50 nm gold nanoparticles at a concentration of about 455 1010 particlesml Then the cell monolayer was washed and incubated for 30 min with 30 uM DAMP solution Molecular Probes Carsland CA at 37 C1 The washing step en sured that no ectopic binding iiei outside the cell could have hap pened between gold nanoparticles and DAMP moleculesi Cell monolayers were washed with phosphate buffer saline PBS 714 pH from Sigma Aldrich Inc St Louis MO before Raman measure ments SERS spectra were collected using a confocal Raman micro scope Senterra from Bruker Optics Inc Billerica MA tted with a 60x water immersion objective and a 633 nm laser source whose excitation source was close to the surface plasmon resonance of aggregates or aggregate intermediates The system is tted with a pair of interference edge lters that pass longer wavelength and re ect the laser line For the 633nm laser the cutoff is lt80 cmquot It should be noted that single particle resonance can be observed at N53Onm as particles aggregate the resonance 74 gt v M r quot dihydrochloride Please cite this article in press as A1 Shamsaie et all Chem Phys Lett 2008 doi1011016jicplett12008061064 CPLETI39 26216 1 July 2008 Disk Used No of Pages 5 Model SG 2 A Shamsaie et aL Chemical Physics Letters xxx 2008 mew shifts to higher wavelengths which is desirable not only for reso nance coupling to maximize enhancement but also to minimize the effect of uorescence LEI Laser power was set to 10 mW and the laser spot size was estimated to be close to 2 pm and spec tral resolution was 3 5 m li Collection times were set to losin our experiments and the OPUS software was used to chop the spec tra to reveal Raman bands in the 400 cm to 1800 cm 1 range and baseline correction was done by the rubber band method using a rubberband39 like string icon stretched between the endpoints of the spectrum to provide the spectrum minimal Spectra were col lected both in a random fashion and also speci cally from the dark spots believed to be gold nanoparticle aggregatesi TEM images con rm the entrapment of gold nanoparticles in the endosmal lysosomal compartments Figi IBAand Fig 1C shows a complete spectrum of DAMP with the key peaks that can be used as signa ture for intracellular studies The characteristic band at 837 cm 1 assigned to the ring breathing vibration modes of this molecule was used for quanti cation in this stud i Fig 2A presents the intracellular SERS spectra of DAMP at vary ing concentrations 8 mM and 130 mM of this chemical consistent with the in vitro raman ngerprint for this chemical Fig 1C One important feature consistently observed in our measurements Fig 2A is the Raman shift at lower frequenciesAlOO ZSO cmquot extre mely low to be attributable to any molecular vibrational modes Experiments have also shown that this band exists even in the ab sence of the analyte and in the background spectra collected from bare gold nanoparticles Fig 2B In Fig 2B SERS signals are com pared in the absence shown as background39 spectrum of gold and presence bare gold nanoparticles Our comparison con rms that the secondary peak observed is clearly due to the presence of gold nanoparticles It is also shown that the location of the secondary peak depends on the excitation signal wavelength 633 785 830 nm and the Raman shift increases with decreasing wave length of the excitation frequency as reported in literatureL1415i It is widely accepted that theselowfrequency shifts are due to the vibrational acoustic phonons mode couplings to the electro magnetic signal through the dipoles created by the modulation of the surface polarization charges surface plasmon polaritons The spherical and torsional vibration modes ofa homogeneous elastic sphere has been studied and classi ed according to their symmetry groups Am using spherical harmonic functions Yin by Iamb 46L More recently using symmetry arguments Dulvar has shown that only the symmetric l O and quadrupolar l 2 spheroidal modes are Kamanactivekl i The radial wave vector of the surface modes are roughly at kn rugL where n 01m in an increasing order for xed angular shape 1111 Accordingly peaks are expected at shift SM20c n where S is a constant of the order of unity depending on the symmetry of the mode and VP is the longitudi naltransverse sound velocity lowfrequency shifts observed in our measurements are extremely large for any resonant vibration modes fundamental frequencies of nanoparticles withwithout analytesi For excitations close to the surface plasmon resonances Aem 633 nm the electronic cloud is strongly polarized and may interact strongly with the opticalphonon vibrations leading to a clear lowfrequency Raman peaki This peak is not well inves tigated in the literature in fact in a majority of the SERS studies in biology the spectra below 400 cm 1 is often truncatedignored Alternatively we suggest that one can utilize this band to obtain a quantitative measure of the amount of the metallic nanoparticles or particle aggregates which gives rise to SERSI This could pave the pathway to formulating a simple protocol for intracellular quanti cation using SERS which to this date has been compounded by a lack of control of the formation of consistent nanoparticle aggre gatesi This is a major concern since the measured Raman signal is spatially averaged including hotcold areas with densefew gold particles Therefore it is desirable to adjust the detected Raman sig nals relative to the metallic nanoparticle aggregates involved in the measurement In this article we have used the vibronplasmon couplings to normalize the SERS spectra to prove that the precision of the calibration and predication steps could be improved Both band intensity and band integrals were tried for normalization but the best result was achieved with the band intensitiesi In Fig 2g it is shown that for a xed concentration of DAMP the normal ized SERS signal at the characteristic band 837 cmquot is indepen dent of the location where measurement are taken The behavior of the RIasmon vibron modes with respect to gold nanoparticle concentration was also investigated Experiments ea Intensity 400 600 300 1000 1200 1400 1600 1300 2000 Raman Shift cm391 I Fig L A Chemical structure of DAMP B TEM image magni cation x27500 showing the entrapment of gold nanoparticles in the membrane bound lysosomal t endosomal compartmen s and C Raman spectrum of DAMP showing the ring breathing vibration mode peaks at 837 cm Please cite this article in press as Al Shamsaie et all Chem Phys Lett 2008 doi11OIIOI6jicpletti2008i06064 CPLETI39 26216 No of Pages 5 Model SG 1 July 2008 Disk Used A Shamsaie et ILChemical Physics Letters xxx 2008 xxxrm 3 A B E g a E E a E e r a a 3 5 s x pthDnplasmon coupling 5 g 7 V 397 77 r 77 17 g 337 cm1 um Sn Q 25 mn 5 E background 6 260 460 660 860 1600 1200 1400 163900 who who Raman shift cmU C normallzed to rst peak 05 normalized to second peak 055 05 9 E 045 04 035 D 3 4 Measurement Fig 2 A Samples of the intracellular SERS signals of DAMP collected in vivo B In vitro background spectrum of nanoparticle aggregates without DAMP denoting the presence of the rstsecondary peak at low waveinumbers due to the phononicrplasmonic couplings C The normalization was done by dividing the 837 cm peak intensity with the signal intensity for the rst and the second peak for a xed amount of DAMP concentration a b c c I I 39 Intensity au m o 260 460 660 860 1600 123900 Moo 163900 133900 zo39oo Raman shift cm Fig 3 Three in vitro SERS spectra of DAMP at 130 mM concentration incubated with nanoparticles of three different concentrations a b and c spotted on glass sides of approximately 5 pm2 in crossrsectional area Please cite this article in press as A Shamsaie et al Chem Phys Lett 2008 doi101016jcplett200806064 CPLETI39 26216 1 July 2008 Disk Used No of Pages 5 Model SG 4 A Shamsaie et aL Chemical Physics Letters xxx 2008 mew raman spectra from different surfaces for example plain glass slide gold slide as well as glass and gold slides incubated with gold nanoparticle clearly show that the presence of the plasmon vibron peaks below 400 cm 1 is due to the plasmon modes of the spheri cal particles The variation of the signal intensity with respect to gold nano particle densities collected from 5 pm2 spots deposited on a glass slide is depicted in Pig 3 The signal intensities of the two normal izing peaks N100 cm 1 and N230 cmquot varying in proportion to the gold particle concentration N45 X 102 45 X 105 and 45 X 1010 particlesml respectively denoted by a39 b39 and c39 is demonstrated The ef ciency of this normalization approach can be veri ed by a decrease in the RSD percentage of relative standard deviation of the SERS signal intensities tested over a range of analyte concen tration Pig 4 To quantify the local concentration of DAMP in the vicinity of gold nanoparticle aggregates an in vitro univariate cal A 06 Intensity au 6 Id ibration experimental model was designed To simulate the intra cellular condition the following procedure was followed a 20 ul droplet of 50 nm gold nanoparticle colloidal solution was allowed ry on a glass slide to which different concentrations of DAMP solutions prepared in water or buffer were applied A glass cover slip was placed on the top and SERS signals were collected with the same settings mentioned above SERS spectra of DAMP obtained from this platform contained a high degree of irreproducibility RSD of band intensity gt20 as expected and used for quanti cation e inconsistent nature of nanoparticle aggregation in cells is one of the main cited reasons behind the lack of reproducibility of spectral signals rendering quanti cation impossible Loren et aliLISJQ used a second analyte a self assembled monolayer con sisting of a thiol derivative of Dabcyl tethered to gold colloids as an internal standard to normalize the SERS signals to reduce the varying enhancements originating from the chaotic assemblies of R2 09212 02 E 01 l u 20 4 0 60 80 100 1 o 140 DAMP concentraion mM B 50 El before name 39on 45 r I after normali 4D 7 35 7 30 i Z o a 20 15 g 1o 539 0 y y 130 867 65 325 1625 8125 intracellular DAMP concentration mM H ha 4 A a m nerrea p in the Fig4 erl nn cm band ohm c y u Enui RSD values of SERS intensities n 10 after normalization The hashed pair on right is related to the intracellular data Please cite this article in press as Al Shamsaie et al Chem Phys Lett 2008 doi1101016jcpletti2008i06064 CPLETI39 26216 1 July 2008 Disk Used No of Pages 5 Model 56 A Shamsaie et ILChemical Physics Letters xxx 2008 mew 5 goldnanoparticles18l The use of a linker or a second chemical ngerprint necessitates additional chemical steps and interferes with the ngerprint of the target analytel In addition approaches that utilize a second analyte or a chemical monolayer as an inter ponsible for enhancement to serve as an intrinsic internal standard for in vivo quanti cation of analytes in cells Experiments were conducted in the concentration range be tween 8 mM and 130 mM correlating the Raman intensity depen dent concentration of D MP in 1170 to n acellular concentration of DAMP in the lysosome of MCF10 cells Calibration coef cient improved from 53 not shown to 92 after applying our proposed normalization Fig 4A Here the normalization was done by dividing the 837cm 1 band intensityintegral by the excitation dependent plasmon 180 250 cm or the back scattered light peak observed around 100 cm 1 band Since the plasmon and the back scattered peak are intrinsic to the enhancing metal these appear in the analyte spectra which lie in the ancement range of the metal The direct proportionality of these intrinsic peaks rst peak depicting the back scattering and the second depicting the Plasmon used as a normalizing standar Fig 2C for the same concentration 50 nM of the analyte from eight different measurements show that the normalization is con sistent Fig 2B The random SERS spectra of DAMP from these eight different measurements were surprisingly closeJ10l The same normalization protocol was also applied to the intracellular 39 39 39 39ar i rov ments in the reproducibi ity RSDs were 36 without normalization and 917 with normaliza tion respectively Fig 4B Based on the above approach the local concentration of DAMP surrounding the gold nanoparticle aggre gates right most pair in Fig 4A was estimated to ex44 mM n summary in this s u we have for the rst time demon strated a novel SERS strategy that enablesnon uorescent intracel lular concentration determination in a quantitative manner using the inherent plasmon peak of gold nanoparticles in the spectral We have proposed and experimentally shown a quanti cation method which can be exten ed to SERSbased measurements in different environments with different nanoparticlesl Lower detec tion limits might be achievable using chemicals with a high Raman crosssection eg using a SERRS tagl Acknowledgment This research was conducted at the Physiological Sensing facil ityJat Purdue39s Discovery Park References 1 I Chourpa H Morjani JF Riou M Manfait ngS Lett 397 1 2 K Kneipp t alAppl Spectrosc 56 2 2002 150 3 J Kneipp Kneipp WL Rice K Kneipp Aila Chem 77 8 2005 2381 4 Loren J Engelbrektsson Josefson J Abrahamsson K Abrahamsson pectrochim Acta Part A7 Mol Biomol Spectrosc 61 4 2005 755 5 A Shamsaie M Jonczyk J Sturgis JP Robinson J Irudayaraj JBiomed Opt 2 2 2007 020502 1995 51 n E E B 1 gt 6 K Kneipp H Kneipp J KneippAcc Chem Res 39 7 2006 443 7 A Wei B Kim B Sadtler SL Tripp Chemphyschem 2 12 2001 743 8 ED Chithrani AA Ghazani WCW Chan Nano ken 6 4 2006 662 9 J Kneipp H Kneipp M McLaughlin D Brown Kneipp Nano kett 6 10 2006 2225 10 CE Talley L Jusinski CW Hollars SM Lane T Huser Anal Chem 76 23 2004 7064 11 RGW Anderson JR Falck JL Goldstein Ms Brown Kroc Natl Acad Sci USA 7 Biol Sci 81 15 1984 4838 12 RP Haugland The Handbook Invitrogen Corp Carlsbad 2005 p 580 13 K Faulds R Littleford D Graham G Dent E Smith Chem 76 2004 592 14 DA Weitz TJ Gramila AZ Genack JI Gersten Phys Rev Lett 45 5 1980 15 JI Gersten DA Weitz TJ Gramila AZ Genack Phys Rev B 22 10 1980 4562 16 H Lamb Proc London Math Soc 13 1882 187 17 E Duval Phys Rev B 46 9 1992 5795 L En elbrektsson C Eliasson M Josefson J Abrahamsson K Abrahamsson NanoJitt 42 2004 309 19 A Loren J Engelbre tsson C Eliasson M Josefson J Abrahamsson M Johansson K AbrahamssonAnal Chem 76 24 2004 7391 Please cite this article in press as A1 Shamsaie et all Chem Phys Lett 2008 doi1011016jlcplett12008061064 Manipulation of Protein Charge States and Polarity in Vacuo Min He and Scott A McLuckey Department of Chemistry Purdue University West Lafayette IN Introduction Electrospray ionization coupled with ionion reactions has been demonstrated to be efficient in manipulating charge states of multiply charged polyatomic ions However the onechargestateper step reaction fashion limits the extent the charge state manipulation can reach Furthermore most of the work done so far involves the reduction of charge states of multiply charged macroions while increasing the charge state of a singly charged ion within the mass spectrometer has not been explored as extensively The work presented here demonstrates that by employing multiple electrospray sources both charge states and polarities of macroions can be readily manipulated Particularly a twostep ionion reaction approach that provides means for increasing ion charge states by increasing the number of excess protons is demonstrated4 The success of this work indicates the possibility for application of tandem mass spectrometry on multiplycharged macroions formed initially as predominantly singly charged ions Methods All experiments were performed on modified FinniganITMS quadrupole ion trap mass spectrometers coupled with homemade electrospray sources which allow for the sequential injection and subsequent reaction of oppositely charged ions generated via electrospray ionization Bovine ubiquitin bradykinin and peptide GLSDGEWQQVLNWVGK were sprayed from aqueous 1 acetic acid for positive ions DABAM 32 polypropylenimine dotriacontaamine dendrimer was sprayed out of aqueous 5 acetic acid solution Carboxylateterminated polyamindoamine dendrimer PAMAM and 14 phenylenedipropionic acid PDPA were sprayed out of 2 ammonium hydroxide in the negative mode The homopolymer 539 dAAAAAA339 was prepared in 25uM piperidine and 25 mM imidazole solution Results Manipulation of macroion charge states and polarity can be achieved by both complex formation and proton transfer As an example of the first case the positively charged bovine ubiquitin ions U7H7 were introduced into the trap via one ion source and isolated Then they were allowed to react with doubly deprotonated 13 benzenedisulfonic acid ions CeH48032239which were injected into the trap through another electrospray ion source The charge states were reduced in steps of 2 by complex formation see Figure 1A The reduction in charge states was driven to the limit until the negatively charged complex U4CeH48032H2H39 was formed after four anions were sequentially attached to the protein ions This complex was then isolated By reintroducing positive ubiquitin ions U7H7 into the ion trap and allowing them to react with the negative complexes a new sixbody complex 2U4CeH4SOa2H26H6 was produced However the formation of complexes may not be desirable Charge polarity inversionreversion of protein ions by proton transfer which will result in the formation of uncomplexed protein ions is more desirable Therefore a series of chemical reagents capable of forming dianions in gaseous phase were investigated As an example the reaction between singly charged positive bradykinin ion and doubly deprotonated 14 phenylenedipropionic acid successfully inverted the polarity of the peptide ions and formed negatively charged bradykinin ions This experiment indicated the possibility to invert the polarity of macroions by proton transfer reaction with the novel instrument Further experiments demonstrated that the dendrimer ions were particularly well suited for proton transfer reactions Carboxylateterminated PAMAM dendrimer generation 05 was capable of inverting 1 charged peptide GLSDGEWQQVLNWVGK to 2 charge state On the other hand multiply charged positive ions produced via electrospray of DAB32 dendrimer generation 4 successfully inverted negatively charged homopolymer 539dAAAAAA339 from 2 charge state to 2 charge state By combining these two reagents the goal of increasing the charge state of macroions initially formed as singly charge ions was successfully accomplished This experiment was demonstrated by a twostep charge inversion approach The first step involved accumulation and isolation of singly charged bradykinin ions formed via electrospray Figure 2A A population of anions formed via negative electrospray ionization of PAMAM was then admitted into the ion trap and allowed to react with the bradykinin MH After reaction with the bradykinin MH ions both residual PAMAM anions and bradykinin MH39 ions were observed data not shown In the second step the isolated MH39 bradykinin ions were allowed to react with the cations formed from DAB dendrimer that was introduced into the ion trap through the third ion source The resulting mass spectrum is shown in Figure 28 where the newly formed MH bradykinin cations were observed in addition to the appearance of M2H2 bradykinin cations formed by the twostep charge inversion For comparison the spectrum resulting from the sequence used to acquire spectrum of Figure 2B but without the admission of anions is shown in Figure 2C The position of M2H2 if present is indicated with an arrow A A UHKC H sod1 100 BHl U3HZC H sou1 80 ZHZ H sod12 Abundance arb umt 1200 B UMCSH s iner arb umt Abundance F 3200 ZU HAC HSOKH2 Abundance arb umt 2000 4000 6000 8000 10000 500 600 700 800 900 1000 1100 1200 mz mz Figure 1 Double charge inversionreversion Figure 2 Two ionion charge inversion of protein ions by complex formation steps to form a doubly protonated peptide from a singly protonated peptide Conclusions The manipulation of charge states and polarity of macroions in gas phase by ionion reaction has been demonstrated where both complex formation and proton transfer have been observed By employing the twostep charge inversion approach doubly protonated peptide was readily formed from a singly protonated peptide This experiment indicates the potential to enhance the structural information that can be obtained using ionization techniques that tend to form predominantly singly charged ions from macroions Reference 1 McLuckey SA Reid GE Wells JM Anal Chem 2002 74 336 2 Reid GE Shang H Hogan JM Lee GU McLuckey SA JArnChernSoc 2002 124 7353 3 He M Reid GE Shang H Lee GU McLuckey SA AnaChern 2002 74 4653 3 He M McLuckey SA JArnChernSoc 2003 In Press Acknowledgements The authors acknowledge Mr Chris Doerge of the Amy Facility for Analytical Instrumentation and Dr Ethan Badman for instrument development Research was supported by the National Institutes of Health and US Department of Energy AMERICAN JOURNAL OF INDUSTRIAL MEDICINE 402l 57220 200l CASE REPORT Fatal Unintentional Occupational Poisonings by Hydro uoric Acid in the US David W Blodgett MIl Anthony J Suruda MD MPH and Barbara Insley Crouch PharmD MSPHZ Background Case reports have identi ed hydro uoric acid HF as causing fatal work injury and HF has both local and systemic toxicity Surveillance for HF related mortality is problematic because of the lack of unique coding for this acid in hospital records and vital statistics Methods We identi ed HF related fatal work injuries investigated by the Occupational Safety and Health Administration OSHA for 1984794 from coding of Hazardous Substance Z460 HF and requested case investigation les under the Freedom of Information Act We attempted to identify HFrelated deaths in the US for the same period through literature case reports the Consumer Product Safety Commission and the American Association of Poison Control Centers AAPC C Results For the 1 year period OSHA investigated nine deaths in eight incidents which involved HF Four deaths were from skin contact with concentrated HF and ve deaths involved both skin contact and inhalation of vapor Unsafe work practices were factors in all of the deaths Calcium chloride or gluconate was noted to have been administered to ve of the nine victims Calcium was administered 90 min after exposure to two victims and more than 6 h after exposure to a third We were able to establish that the regional poison control center had been contacted in regard to only one victim For the period 1984794 we were able to identify no additional deaths from CPSC reports one additional death from AAPC C annual reports and four other deaths from case reports in the medical literature Conclusions For the period of this study OSHA records identi ed the greatest number of HF related fatalities The limited information in the records suggest that some victims did not receive appropriate medical care nor was the regional poison center contacted regarding care The full extent of health problems related to HF could be better quanti ed if usual surveillance sources such as vital records included unique coding for this acid Am J Ind Med 4022157220 2001 2001 Wiley Liss Inc KEY WORDS hydro uoric acid poisoning fatalities chemical poisoning work place fatality workplace poisoning INTRODUCTION Deparlmenl of Family 8 Prevenlive Medicine School of Medicine Universily of Ulah Sall Lake Oily Ulah i i i i i i i 2Ulah PuisunOunlrulOenleLOullegeofPharmacy Universilyuf UlahSall Lake Oily Ulah HydIO uonc amd 15 a conoswe Inorgamc amd OunlmclgranlspunsurNIOSHOunlmclgranlnumberT42OOT810426 Himes 1989 It is used in industry for etching glass A W C quot IInIPl il uflllah Sall Lake electronics and semiconductor manufacturing and in the petroleum industry to produce high octane fuels Bertolini Accepled 5 May 2001 1992 It is also a component of some consumer products Oily UT 84qu 0512E mail asurudadfpmulahedu 2001 WileyLiss Inc 216 Blodgett et al such as rust removers Saadi et all 1989 With regard to human toxicity HF is of particular concern because not only does its contact with skin cause local burns but the uoride ions readily penetrate the skin to cause systemic poisoning Skin penetration by HF is retarded by the lipid layer of intact skin and accelerated by abrasions or minor trauma Noonan et all 1994 Death can occur from a splash of as little as 25 of the body surface with concentrated HF Tepperman 1980t Inhaling HF vapors boiling point 68 F leads to pulmonary edema and in high enough concentrations death O Neil 1994t Fluoride ions that penetrate the skin into systemic circulation bind to divalent cations primarily calcium and magnesium leading to electrolyte imbalance cardiac arrhythmia and death Sadove et all 1990 Fluoride also inhibits glycolysis and oxidative phosphoryla tion with the same results DeLauder et all 1994 Contact with HF in concentrations less than 50 does not cause immediate pain so systemic poisoning can begin before the person is aware they have had contact with the acid US Department of Energy 1986 There is little reported f on the A of HF poisoning There is no unique International Classi cation of Diseases ICD9 code for HF poisoning and fatal injuries involving HF would be included under ICD9 983t1 toxic effect of corrosive acids or under coding for burns and so information on HF is not available from vital statistics or hospiml discharge dam Fatalities from HF have been reported as case reports Menchel and Dunn 1984 Mayer and Gross 1985 Manoguerra and Neuman 1986 Chan et all 1987 Mullet et all 1987 Chela et all 1989 Cappell and Simon 1993 Several case reports demiled the circumstances surrounding the 1987 HF release in Texas City Texas Brender et all 1991 Alexeef et all 1993 Dayal et all 1994 For 1985 86 there were four fatalities involving HF reported to the American Associa tion of Poison Control Centers AAPCC three were from ingestion and one from dermal exposure Caravati 1988 Emergency management of cutaneous HF exposure includes removal of contaminated clothing copious irriga tion with water topical application of calciumcontaining gels or quaternary ammonium compounds and intralesional or regional injection of calcium compounds Caravati 1988 Sheridan et all 1995 The unique toxicity of HF led us to attempt to de ne the epidemiology of fatal injury and to examine deaths due to HF poisoning as a result of occupational contact using a variety of sources that allowed identi cation of HF We wish to draw attention to the preventability of these deaths and search for ways to prevent further deaths a 1 METHODS Occupational Safety and Health Administration OSHA investigation summaries for 1984 94 which had been obtained for another study Suruda et all 1999 were available and provided in electronic form by OSHA from the Integrated Management Information Sytems HVIIS database This database contains information on OSHA famlity and catastrophe investigations in 47 smtes for 1984 89 and all 50 smtes for 1990 94 California Washington state and Michigan smte OSHA programs had data les incompatible with the federal system until 1990 and reports from those states were excluded until that year Included in the data were all deaths reported to OSHA for which an investigation was conducted approximately 2000 deaths each year Each report included a eld for a hazardous substance involved in the fatality We rst identi ed all famlity reports containing an entry for substance 1460 HF We then printed out the content of the IMIS investigation summary for each of these incidents identi ed the OSHA of ce which conducted the investigation and the identi ca tion code for the OSHA compliance of cer and requested in writing from the applicable OSHA of ce a copy of the full investigation report we made a written request to the Con sumer Product Safety Commission CPSC for all fatality reports for 1984 94 involving workrelated faml injury from HF that could be identi ed from the CPSC Medical Exa miner le and also from the National Electronic Injury Surveillance System AAPCC maintains the Toxic Exposure Surveillance System TESSt TESS data include reports from the major ity of US Poison Control Centers We reviewed TESS annual reports from 1984 94 for mention of any fatal work injury involving HF RESULTS For the period from 1984 94 OSHA investigated eight incidents with nine deaths due to HF poisoning Seven of the famlities were in manufacturing Table I All victims were males Seven of the nine victims were employed at workplaces which were covered by a collective bargaining agreement with a labor union For the same period the CPSC death certi cate le identi ed three fatal injuries involving HF all of which had also been investigated by OSHA One workrelated poison ing was identi ed from the 1992 AAPCC annual report and has been published previously Litovitz et all 1992 A brief summary of individual cases identi ed from OSHA reports follows Case I Case 1 occurred on 20 August 1984 A 53yearold worker was exposed to HF in the process of using methyl chloroform to unclog a line leading to a storage tank of HF In the process of connecting a hose to the tank valve the TABLE I Distribution of HF Deaths by Industry 198471994 US Industry group Speci c industry Number at deaths Agricultural services Citrusfarming 1 Manufacturing Industrial organic chemicals 2 Industrial inorganic chemicals 1 Petroleum refining 2 Glass and glassware 1 Measuring and controlling devices 1 Services Metals testing 1 Total 9 coupling came loose splashing him with methyl chloroform the cleaning agent and HF from the tank resulting in burns to the arm face and neck covering 30 of his body He was showered and was transported to the plant s rst aid area arriving approximately 30 min following the accident He was transferred to a hospital where he developed seizures and then cardiac arrest dying 7 8 min after arrival Case 2 Case 2 occurred on 20 September 1985 A 51yearold worker entered an area where there had been an HF spill and cleanup efforts were underway The worker entered the spill area with only rubber gloves rubber boots and a self conmined breathing apparatus A sump pump was being used to pump the spilled acid back into a tank for storage As the employee moved the pump to a lower level to continue the process the hose came off of the pump and the worker was sprayed with HF across 40 of his body face and chest and died shortly after A review of pump speci ca tions revealed that this pump was not recommended for use with corrosive materials Case 3 Case 3 occurred on 26 January 1986 A 62yearold worker entered the storage area of another company to retrieve a set of tires stored in an overhead lofts He stepped on a 5 gallon plastic bucket of HF Goelonging to another company to ascend in lieu of using a ladder Upon descending from the loft the lid of the bucket failed The worker was splashed with 70 HF on his right lower leg and foot as well as his posterior thighs The burn was later estimated to have been to 8 of the total body surface The worker was stripped showered for 15 min and immediately mken to the hospital where he was seen approximately 35 min after the exposures Morphine sulfate meperidine and hydroxyzine were given to control excruciating pain Two hours later he was transferred to another hospimlr Upon arrival at the second hospital some laboratory testing was Fatal Hydrofluoric Acid Poisonings 217 done but this did not include calcium determinations Approximately 5 h after the exposure a decision was made to operate and to debride the burned skint However he experienced runs of ventricular brillation and surgery was canceled Serum calcium concentration was measured and was found to be low at 512 mgdL Six hours and twenty minutes after injury calcium gluconate 60 mL 10 cal cium gluconate and calcium chloride 2 g were adminis tered intravenously His condition declined with repeated episodes of ventricular brillation necessitating cardiover sionr Normal calcium levels were obtained 10 h after injury and mainmined thereafter However ventricular brillation eventually became refractory and he died approximately 15 30 min after exposure Case 4 Case 4 occurred on 27 February 1986 A 40yearold worker was replacing a valve attached to a HF line in a water condensers The worker had closed down the two valves on the line and opened a drain line to allow HF in the line to drain out After replacement of the valve the worker opened one of the valves to place it back on line without closing the drain valve The open drain valve released HF into a catch basin splashing the worker across the arms chest face and left leg In addition the HF vaporized and was inhaled by the workers The demils of this worker s medical care were not in the OSHA le other than a notation that autopsy showed pulmonary engorgementr Case 5 Case 5 occurred on 18 October 1988 A29year old worker was transporting 216 gallons of HF in an open 15 L pails The HF was being transported from storage bins to etching tanks where it was used to test airplane parts for aws The pail spilled and the worker was splashed across the chest neck and face with 70 HF This worker was wearing work clothing long rubber gloves and street glassesr He was immediately stripped and bathed in a water bath Inhalation of HF fumes was inferred because he was frothing at the mouth He died en route to the hospimlr Cases 6 and 7 Case 6 and 7 occurred on 7 March 1991 Multiple workers at a petroleum re nery were involved in the same incident and two diedr A 50yearold worker and a 42year old worker were exposed to HF while replacing a pump motor An improper lockout procedure failed to keep HF from owing through the pipes to the pump as it was removed for repairs This resulted in the pressurization of the pumps The pump containment failed releasing several hundred pounds of HF onto the unprotected workersr One 218 Blodgett et al worker was pronounced dead at the scene Another was mken to a local hospital and died shortly thereafter autopsy revealed severe lung injury Case 8 Case 8 occurred on 15 August 1991 A 48yearold worker was working to modify an HF drain line for an experimental incinerator burn The drain line was not properly locked out and the up line valve broke when the line was pressurized This allowed the acid to splash the worker across the head neck shoulders and arms appro ximately 15 of his total body surface He also inhaled HF fumes Immediately following the incident the worker was showered taken to the company rst aid smtion and then to a hospiml emergency department where calcium chloride was administered He died shortly thereafter from cardio respiratory arrestr An autopsy revealed pulmonary edema Case 9 Case 9 occurred on 10 October 1992 A 37yearold worker was in the process of pouring HF acid from a 55 gallon drum into a 3 gallon pail for distribution The worker and a coworker had stacked crates on top of each other as a makeshift work platform in order to raise the work area closer to a ventilation shaftr In the process of pouring the HF the drum struck the bucket and knocked it from the platform to the oor One worker jumped free while the other was splashed with 70 HF to approximately 40 of body surface area The worker who was splashed was wearing only rubber gloves work clothes and goggles Following the incident calcium gluconate gel was applied within 30 min by the company nurse and he was transferred to a local community hospital He was latter transferred to a regional medical centerr At the regional medical center 3 h after injury he developed ventricular brillation Several doses of calcium chloride were delivered along TABLE II Use of CalciumTreatment Appmximate time lmm with cardioversion but he died approximately 4 h after exposure Treatment of the above nine individuals with calcium is summarized in Table 11 In the ve individuals who received calcium therapy in addition to decontamination by wash ing with water the time of administration was noted in four cases In two cases this time was approximately 1 h 30 min and in one case it exceeded 6 h We were able to establish via telephone inquiry that the regional poison control center was contacted regarding the medical care of one of the above cases that was investigated by OSHA Case 8 In addition to the fatalities mentioned above we identi ed four deaths reported in the medical literature that did not appear to match the cases reported by either OSHA CPSC or AAPCCr Two defense workers unloaded a truck containing 70 HF acid were splashed when a hose disconnected and died of HF poisoning Mullet et all 1987 A worker ingested rust remover containing HF mistaking it for water and died of cardiac arrest Manoguerra and Neuman 1986 There was a case report of a worker splashed across the thighs with HF who died of cardiac arrhythmia approximately 17 h following exposure Menchel and Dunn 1984 DISCUSSION These data show the dif culty in determining the exact number of workrelated poisonings from HF in the US during a particular time period We were able to identify several cases but we did not identify all such deaths in 1984 94 using information from AAPCC CPSC and OSHAA In contrast to HF poisoning by carbon monoxide is identi able from hospital records and death certi cates by the ICD9 code 986 If there were a unique ICD code for HF it would facilitate surveillance of poisoning from this acidr Calcium gluclmate Time lmm expasure Gase nu expusure ta death administered VIN ta administratian 1 30 min N 2 3 h 30 min N 3 15h Y 6 h 4 4h Y 1 h 30 min 5 30 min N 6 30 min N 7 2 h 30 min Y Unknown 8 4h Y 15 h 9 4h Y 30 min Some of the OSHA reports suggest that appropriate treatment for HF poisoning was not given in a timely manner A common recommended treatment for HF burns for relieving pain from local injury is application of calcium gluconate after surface decontamination is done by remov ing clothing and washing the affected skin areas with water Blunt 1964 Velart 1983 Pegg et al 1985 Dunn et al 1996 Intravenous administration of calcium gluconate is recommended if systemic poisoning is suspected Calcium chloride is not recommended because it stimulates sensory bers causing pain at the site ofinjection Pegg et al 1985 Regional poison control centers are generally recog nized as community resources which can provide informa tion concerning proper treatment of poisoning including poisoning with HF It would be useful for research purposes to be able to link OSHA reports of workplace poisoning with AAPCC records for the purpose of determining whe ther a regional poison center was consulted regarding care of the injured worker Prevention of HF poisoning involves engineering cont rols to reduce or eliminate exposure proper work practices personal protective equipment and appropriate rst aid and emergency care The danger of work on or in the vicinity of pressurized lines conmining HF should be recognized and appropriate lockouttagout procedures followed When such work is performed personal protective equipment effective in reducing dermal and respiratory exposure in case of a spill should be provided One of the authors provided emergency care to a worker who was splashed by the rupture of a high pressure HF line during servicing of a valve This worker sustained only a small second degree burn on the chin despite a direct splash to the head and the burn responded well to topical calcium gluconate therapy At the time of the splash the worker was wearing a suit including a helmet that was impermeable to corrosive acids and was also equipped with an SCBA At the time of the incident there was a standby worker similarly equipped who was ready to assist with evacuation and decontamination At work sites where HF or solutions containing HF are in use decontamination showers should be readily avail able Personnel should be trained in decontamination and in application of topical calcium gluconate if onsite medical care is not immediately available Appropriate topical therapy would be calcium gluconate gel 25 10 Com mercially available calcium gluconate gels are not approved by the Food and Drug Administration for sale or use in the US but are available from Canadian and European vendors We are aware of one major US manufacturer which stocks tubes of gel from European sources at US work sites and trains worker in its use Calcium gluconate gel can be compounded readily for emergency use by mixing equal amounts of lubricating jelly KY and 10 calcium gluconate solution and applying it to a wound For burns associated with severe pain out of proportion to Fatal Hydrofluoric Acid Poisonings 219 physical evidence such as swelling or blistering local injection of calcium gluconate into the burn area can relieve pain and prevent or delay systemic poisoning Blunt 1964 Velart 1983 Pegg et al 1985 ACKNOWLEDGMENTS We thank Dr Philip Edelman for his review of the manuscript Fatality reports were provided with the kind assistance of Bruce Beveridge Sanford Hamilton and Thomas Tyburski of OSHA Management Data Systems REFERENCES Alexeeff GV Lewis DC Ragle NL 1993 Estimation of potential health effects from acute exposure to hydrogen uoride using a benchmark dose approach Risk Anal 1363 69 Bertolini JC 1992 Hydro uoric acid a review of toxicity J Emerg 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