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DATE DUE DATE DUE ' DATE DUE 6/01 c:/CIRC/DateDue.p65-p.15 AN ANALYSIS OF THE CHEMISTRY OF THE SCOTT (RUYBAL) TEST FOR COCAINE. By Tamiika K. Hurst A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 2002 ABSTRACT AN ANALYSIS OF THE CHEMISTRY OF THE SCOTT (RUYBAL) TEST FOR COCAINE. By Tamiika K. Hurst Based on the information found in the literature, the chemical processes that yield a positive field test for cocaine have not been well-defined. Often, due to the composition of these drug samples, interpretation of the results of the field tests is limited. The intent of this work is to explain the mechanistic aspects of the Scott Test for cocaine via UV-Visible spectroscopy, laser desorption mass spectrometry (LDMS), and through related published literature. In this test, an aqueous reagent is reacted with a small amount of the drug sample. If cocaine is present, a blue precipitate is formed. This precipitate will redissolve if treated with HCI. Adding an immiscible organic solvent, chloroform, will cause the chloroform layer to turn blue. This test, with two requirements for a positive response, is more specific than similar tests that only rely on precipitate formation. Data will be presented to verify that the blue color is due to a unique combination of Co”, SCN' and cocaine. A reasonable structure is a neutral complex having the form C02+(SCN')2(cocaine). Structures of the complex will be compared to other known complexes organic molecules with Co”, and also to other compounds that give false-positive results for the cobalt thiocyanate field test for cocaine. Also, chemical similarities and differences between the Scott Test and its many variations will be presented to aid in interpretation of results. Quantitative and qualitative information cannot be gathered from the results of a field test, however the validity of the initial screening procedures is important. The work that has been done, describes the chemical nature of the Scott Test for cocaine, supporting its usefulness and reliability. This thesis is specially dedicated in loving memory of Elizabeth “Boog” Hurst. Granny, I miss you! ~Love Eternal~ iii I HUMBLY THANK: GOD for daily blessings and the knowledge that I could not do this alone, MY FAMILY, all of them, especially my mother, Diane T. Hurst, for being my rock and source of inspiration, BRYAN, for your unconditional love and support, MY MENTORS for praying with and for me, MY FRIENDS, old and new, who allowed me to be part of “the journey.” I hope you had as much fun as I did, NOBCChE for working hard to empower the future, DR. JOHN ALLISON for being a patient and reliable advisor. Your discipline and commitment to science has motivated me to meet the challenges of higher education. DR. JAY SIEGEL for teaching me to trust the guidance I receive, to have faith in my ability to succeed and to accept all that comes my way, THE CHEMISTRY DEPARTMENT STAFF for the morning smiles and afternoon laughs. Thank you! iv TABLE OF CONTENTS LIST OF TABLES ............................................................................... vi LIST OF FIGURES ............................................................................. vii CHAPTER 1 INTRODUCTION 1. Chemical Reactions ............................................................................ 1 11. Spot Tests ....................................................................................... 4 III. Forensic Investigations ........................................................................ 7 CHAPTER 2 VALIDATION STUDIES ...................................................................... 16 CHAPTER 3 EXPERIMENTAL ................................................................................ 1 9 CHAPTER 4 INSTRUMENTATION I. UV-Visible Spectroscopy ................................................................... 20 CHAPTER 5 RESULTS 1. UV-Visible Spectroscopy .................................................................. 22 CHAPTER 6 INSTRUMENTATION ‘ 1. Laser Desorption Mass Spectrometry .................................................... 32 CHAPTER 7 RESULTS CONTINUED 1. Laser Desorption Mass Spectrometry ................................................... 37 H. UV-Visible Spectroscopy .................................................................. 43 CONCLUSION .................................................................................. 53 LIST OF REFERENCES ..................................................................... 6O LIST OF TABLES Table 1. Spectral Colors, wavelengths, crystal field splitting energies, and colors of complexes ............................................................................... 12 vi Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. LIST OF FIGURES Images in this thesis are presented in color. Chemical structure of cocaine-HCI ................................................ 16 UVN IS spectrum of CoClz- 61120 dissolved in H20 ........................... 23 Visible spectra of [Co(H20)6]2+ (Curve A) and [CoCl4]2' (Curve B). . . . . .....24 UV/V IS spectra of aqueous solutions. Peak maxima shift upon addition of SCN Co(H20)62+ ..................................................................... 26 Equilibria for Co”, x'(c1', SCN'), H20 ........................................... 27 Scott reagent protocol ................................................................ 29 UVN IS spectrum of aqueous layer after the addition of cocaine ............. 30 WW IS spectrum of cocaine and Cody 6H20 dissolved in H20 ............ 31 A: A laser pulse irradiates the surface of a sample B: Neutral molecules ions begin to desorb C: Neutral molecules are pumped away; ions are drawn in the mass analyzer .......................................................... 34 Voyager-DE Mass Spectrometer .................................................. 35 Time of flight analysis .............................................................. 36 LD mass spectrum of cocaine in positive ion mode ............................. 38 LD mass spectrum of Co(SCN)2 in negative ion mode ......................... 39 LD mass spectrum of blue solid in positive ion mode .......................... 41 LD mass spectrum of blue solid in negative ion mode ......................... 42 Structure of the organic phase complex that was proposed by Oguri ........ 43 Probable ion pair structure of the complex ...................................... 43 Postulated tetrahedral structure of the blue complex formed in step 2 of the Scott test; R groups represent cocaine molecules .............................. 44 vii Figure 19. Figure 20. Figure 21. Figure 22. Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Analyzing the precipitate. A: Precipitate mixed with fresh water. B: Dissolution of precipitate in CHCl3 ............................................. 46 A: Aqueous layer after addition to cocaine sample. B: Aqueous layer after CHC13 extraction. C: Organic layer .................................... 48 UVN IS absorption spectrum of cocaine dissolved in H20. Peak maximum at 237 nm ............................................................................ 49 UV/V IS absorption spectrum of cocaine dissolved in CHC13. Peak maximum at 243 nm ............................................................... 50 Tetrahedral complex of blue solid once dissolved in organic layer ......... 52 IR absorption spectrum of: A: Blue Solid in KBr B: Cocaine HCl in KBr C: Cocaine in KBr ...................................................... 54 IR absorption spectrum of: A: Blue Solid in KBr B: Cocaine Thiocyanate in KBr ............................................................................... 55 A: SOO-MHz proton-NMR spectrum of cocaine HCl in D20. B: SOO-MHz proton-NMR spectrum cocaine base in CDCl3 ................................ 57 A: SOO-MHz proton-NMR spectrum of blue solid in CDCl3 B: SOO-MHz proton-NMR spectrum of blue solid in CDCl3 after the addition of free cocaine base ........................................................................ 58 viii CHEMICAL REACTIONS Fundamentally, the principles/laws of chemistry are based on change. Changes involving physical properties (i.e. water boiling) of a substance are called physical changes. The molecules present before and after the change are not different, but their arrangement relative to each other is different. How and why these changes occur is understood by studying chemical changes, the process whereby elements (reactants) combine to form new compounds and /or molecules (products). This phenomenon is commonly referred to as a chemical reaction. The results of chemical reactions are what scientist use to develop experiments and form theories about the properties of compounds at a micro and macro level. Is the product formed chemically combined or a physically separable mixture? How does one detect and/or prove the identity of a particular product? Qualitatively, an easily observed and very useful means for proving the identity of a product is by considering the state/phase of the product. At low temperatures, most matter is found in the solid state, however at room temperature a substance can exist as a solid, liquid or gas. The characteristic properties of gases, liquids, and solids include the size of the molecule, its color, whether or not is dissolves in water. Historically, chemical reactions have been developed to respond not only to minimal changes in the identity of a particular species, but to create a way to detect the changes in the presence of other substances. The two main types of chemical reactions are qualitative inorganic and qualitative organic analyses. The objectives of qualitative inorganic analyses are different from qualitative organic analyses; therefore these tests involve different types of reactions. Inorganic analyses are utilized to be able to detect metallic and non-metallic species in aqueous solutions. When many different ions are present in the same aqueous solutions, any test performed to identify one element or compound can interfere with the detection of other species present. For example: Ag+ ions yield a white precipitate when treated with Cl', but ng2+ or Pb2+ ions will react similarly with chloride ions.1 Consequently, the identification of mercury and/or lead ions may be falsely interpreted if silver ions are present in the sample. Although water-soluble organic species exist, the majority of organic compounds react only when dissolved in organic solvents. In addition, most tests for organic materials depend on the participation of certain functional groups in the chemical reaction. Consequently, organic analyses are usually grouped based on: 1) identification or detection of individual organic compounds 2) identification or detection of functional groups and 3) the study of the behavior of non-metallic and metallic elements toward organic and aqueous solvents. Frequently, the two objectives of inorganic and organic analyses are closely related. For instance, experiments dealing with the analytical techniques used to detect acidic and basic organic compounds are characterized by the ability of such compounds to form non-colored/colored and/or insoluble/soluble ionic salts. For example: All hydroxamic acids produce a red or violet color in the presence of weakly acidic ferric chloride solutions. This color reaction is due to the acid —NHOH group that reacts with the ferric ion to form a water-soluble salt complex.2 Whenever the main product of a chemical reaction, when dissolved in an appropriate inorganic or organic solvent, produces a material that can be characterized by its color [intensity] and solubility features, the test is called an exploratory test. Such procedures as the tests for hydroxamic acid and lead ions are examples of exploratory tests. Both positive and negative findings in exploratory tests assist further analytical testing. Exploratory tests provide reliable guidance in the choice of confirmatory tests. The selectivity and sensitivity of these tests often relieve the analyst of the tedious and troublesome steps necessary to development scientific methods. SPOT TESTS Spot tests analyses or field tests are modern terms referring to exploratory tests. The generic term “spot test analysis” is based on chemical reactions whereby the use of a drop of a solvent solution is the essential step. Specific reagents are often organic compounds. The majority of spot test reactions result in the formation of products with distinctive colors. The colored products include colored precipitates, soluble complexes with certain colors, and ionic species with redox and catalytic properties. Precipitation and color reactions, reactions involving solids with solutions and solids with gases and catalytic reactions are grouped as direct tests. These are the most commonly performed spot tests. Degradation, synthetic and masking reactions are characteristic of indirect tests. Spot tests are performed by utilizing one of the following techniques: 1) By adding a drop of a test solution to a reagent on a supporting surface such as glass 2) By placing a drop of test solution on a medium, e.g., filter paper, with slightly soluble sulfides (ZnS); This procedure is not practical in macro analysis because compact materials, in general, react slowly. 3) By mixing a drop of test solution with a small amount of a solid sample. The choice of the technique to be followed is dependent on the nature of the sample and the reagents available. The most essential step in spot test analyses is the actual “spotting” of the reactants, the test solution and the sample. As a result, the usefulness of spot test procedures is derived from the selectivity of reagents used to enhance the sensitivity for detecting both inorganic and organic compounds. The mechanism of some spot tests can be complicated. Therefore, to obtain consistent results, care must be taken when developing spot test protocols. The experience gained from spot test analysis in the examination of various materials has, over the years, proven to be very useful supplements to standard confirmatory tests i.e. mass spectrometry or spectroscopy. Though they lack the sophistication of instrumental methods, spot tests are invaluable to various fields of research like analytical chemistry, biochemistry, environmental science, and the forensic sciences. For instance, in forensic science procedures, like drug analyses, where plant materials and powders that do not appear to by uniform in appearance are encountered, spot tests are, in many cases, the first test employed. The Duquenois-Levine test for marijuana, the Marquis test for heroin [diacetylmorphine] and the Scott test for cocaine are examples of such tests. In biochemistry research, salts of the antibiotic known as Penicillin G can be reacted with an aqueous solution of phosomolybdic acid in boiling water to produce an intense blue colored solution. This blue color proves the presence of Penicillin G.3 The overall objective of spot tests is to provide reference points that will assist further chemical analyses of compounds and mixtures. Chemical spot tests have been found to be very selective and sensitive with limits of detection that are typically 0.001-10 ug depending on the reagent and the analyte.4 It is evident that only larger amounts of materials will respond to less sensitive spot reactions. The identification limit and/or limiting concentration of spot tests, however, can present problems. What determines the test’s specificity, selectivity, and sensitivity? In other words, what factors influence positive and negative results? Some studies suggest the test reagent relative to the sample mixtures and concentration of the ion or complex to be detected is what is the key factor of influencing the usefulness of spot tests. Others propose the experimenter, the person who performs the spot test. Although there is a demand for quick screening technologies that provide accurate results without quantification, a confirmatory test or confirmation is necessary. Such tests are usually performed using analytical techniques like mass spectrometry, chromatography and/or spectroscopy. These techniques are more costly and require more sample manipulation than spot tests, but prevent misinterpretation of spot tests results. FORENSIC INVESTIGATIONS The relationship of the theories and techniques of spot tests that are used for identifying elements or functional groups to the impact that the results of these tests have on further chemical analysis as well as any legal procedures that take place, constitute an essential part of judicial interpretation of spot test results, and ultimately the forensic sciences. Standards for the issuance of search warrants vary widely by the varying interpretations that search and seizure law have generated. These laws are outlined in the 4th amendment. Warrants are sought in a small percentage of police investigations, but drug and property crimes predominate. Prescreening procedures like spot tests, support the reliability of the finding of probable cause to make arrests, broaden the areas that may be searched legally, and influence what additional analyses will be conducted. The value of performing and interpreting the results of a spot test analysis plays a vital role in an overall drug investigation. The intrinsic reliability of spot tests cannot, therefore, be limited to a “class of compounds”, but rather be selective for individual compounds with similar, but different properties. COBALT CHEMISTRY Cobalt is a hard, gray metal with a melting point of 1493 °C and a boiling point of 3100 °C.2 It dissolves slowly in dilute mineral acid, but it is relatively unreactive. Commonly formed ions of cobalt are C02+ and Co3+ with Co3+(aq) being unstable relative to C02+(aq, in acidic solutions.2 Co(II) yields insoluble products such as C03 (5), Co(OH)2 with the same anions as does Ni (11). These compounds are similarly soluble in dilute acids. One major difference between the chemistry of Ni(II) ions and Co(II) ions is that, in solutions containing no complexing agents, the oxidation of Co(II) ions is unfavorable. In the presence of complexing agents such as NH3, which can react with cobalt ions to form stable complexes, the stability of Co” complexes is improved. Water reduces Co3+ to Co2+ at room temperature. This phenomenon is supported by the relative instability of uncomplexed Co” to form simple salts.2 Co(II) on the other hand forms soluble salt compounds in abundance. Some Co(II) ions are distinguished by the formation of colored compounds. For instance, addition of S2' ions to solutions containing Co2+ ion causes a black solid, CoS to be precipitated.2 Reacting N205, a gas, with CoF2, a solid, in solution forms volatile green crystals of Co(NO3)3.2 The color of Co2+ complexes is not only influenced by the anion, but also the degree of hydration. Co (11) ions form an extensive group of simple hydrated salts. Cobaltous hydroxide may be precipitated as a blue, violet, or pink solid. The pink solid is more stable. But, because of its amphoteric properties when dissolved in a concentrated alkali solution, the pink solution will form a blue complex, [Co(OH)4]2'. Another example of aqueous complexes of Co (11) is cobalt (II) chloride. [CO(H20)6]C12 (3, pink) :5 [Co(H20)2]Clz (5, blue) + 4H20 Hydrated salts of Co(II) ions are red or pink and contain octahedrally coordinated Co+2 ions. Coordination chemistry is the study of compounds that are formed by attaching ligands to a central metal ion. Ligands can be any molecule or ion that donates electrons to the metal. Simple coordination complexes are those in which the metal combines with monofunctional ligands in a number equal to the coordination number. A complex may be a cation, an anion, or a neutral molecule. The complexes formed by the interaction between metal ions and polydentate ligands are called chelates. Metal ions simply dissolved in solution will spontaneously complex with water; they form aquo ions. The term, "forming a complex" is really the phenomenon of displacing H20 ligands from a metal. When six ligands surround the central metal, these complexes are octahedral. Tetrahedral complexes only have four ligands attached to the metal. A typical formula might be [ML6]X., or [ML4]y+. As stated previously, varying the number and types of ligands ofien changes the color of the complex. Trivalent Co3+ ions form numerous complexes. All known complexes of Co” are octahedral, with the majority of these complexes containing NH3 and H202 Co3+ ions in aqueous acidic solutions act as reducing agents with some organic compounds.2 The chemistry of Co” complexes is very extensive. For the purpose of this research project, the discussion of cobalt complexes will be limited to those containing Co2+ ions. Divalent, Coy, like Co”, forms complexes that have been well studied over the years. Octahedral and tetrahedral forms of Co2+ are common and well understood. Thus, the aquo ion of Co2+ can be either [Co(HzO)6]2+ or [Co(H20)4]2+. Knowing the mechanism of inorganic reactions in solution is very useful when trying to understand the behavior of metal ions in aqueous solutions. Simply knowing how many H2O molecules will bind directly to the metal is logically a place to begin. It should be noted that, even when a well-defined aquo ion exists, the number of molecules of H20 that will be displaced by other ligands is important. Most aquo ions are labile and will dissociate in the following manner:3 [M(Hzo).]"* —> [M(H20).-.(0H)]‘“"’* + H" Let’s consider the equation: [Co(H20)4]2+ + 2 H20 —' [Co(H20)6]2+ Because there is a small stability difference between [Co(H20)6]2+ and [Co(H2O)4]2+, these complexes are known to exist in equilibrium. Some other tetrahedral complexes of Co2+ ions are those formed with the anions Cl', Br', SCN', and OH'. Co2+ ions form tetrahedral complexes more readily than any other transitional metal ion.3 Some sources consider the stability of a complex from the viewpoint of the formation of a complex rather than how readily they dissociate. I will discuss the stability of complexes based on the ability of the complex to be formed in a chemical reaction. The thermodynamic stability of a complex measures the extent to which this complex will form under equilibrium conditions. The kinetic stability refers to the speed of formation that ultimately leads to a system at equilibrium. The ability of a ligand to complex with metal ions is described by the formation constant, Kr. A large Kf value suggests a product-favored reaction; one in which the ligand is a better Lewis base than is the H20 ligand. 10 The colors of complex ions can be explained using crystal field theory. Crystal field theory assumes that, in a complex, all metal-ligand interactions are electrostatic in nature. The theory deals with the electrostatic effect of a field of ligands on the energies of a metal’s valence shell orbitals. For transitions metals, the valence shell electrons are in the d orbitals. The difference in energy of the valence electrons of the metal that interact with the ligand is called the crystal field splitting energy. The color of a complex results when an electron in the lower d orbital is excited. The photon will be absorbed at some energy, E = hck, where E = A= 10dq. For a given cation, A, is determined by the ligands. The amount of light absorbed at a specific A. is proportional to the concentration of the complex ion. The relationship between crystal field splitting energy and color is represented in Table l.3 The magnitude for A is smaller for tetrahedral complexes than it is for octahedral complexes. The ligands that produce a large value of A are called strong field ligands. Likewise, weak field ligands are described as having a small value of A. ll Color of light lOdq = A (kJ/mol) Wavelength range Complementary absorbed (mm) color of the resulting complex Red 155-195 770-620 Green Orange 195-200 620-600 Blue Yellow 200-025 600-5 80 Violet-blue Green 205-245 580-490 Red Blue 245-260 490-455 Orange Violet 250-305 455-390 Yellow-green Table 1. Spectral Colors, Wavelengths, Crystal Field Splitting Energies, and Colors of Complexes The relative strengths of ligands are summarized in the spectrochemical series relation: CN' > -N02' > ethylenediammine>NH3 > -NCS' > H2O~ C2042' > OH ' > F’ > Cl' > Br ' > I’. The general pattern of this series is useful when understanding the color changes that may occur in a chemical reaction.3 The reaction of aqueous cobalt (II) with a large excess of thiocyanate ions in the presence of mixed solvents has long been used for the detections of cobalt. The cause of the resulting blue solution has made [Co(SCN)4]2', Kr: 1.0 X103, a suitable subject for many research groups.3 This test is named the Vogel reaction, afier its originator.4 The test consists of adding SCN' ions to an aqueous solution, and then adding an organic solvent. Vogel used 1:1 mixture of amyl acetate and amyl ether. The organic solvent must be soluble in water. The organic solvent ions will ultimately replace H2O ligands to 12 form a blue colored solution. The formation of a blue color in the organic solvent indicates the presence of cobalt (II). Many theories have been proposed to understand the nature of the Vogel reaction. Hill and Howells’ 6 proposed that the color change in cobaltous solutions is due to the dehydration of a cobaltous-hexaquo complex ion. In strongly acidic media they suggest that the color of the solution is transformed to blue conforming to the reaction, [CoCl4(H20)2]2' (red): [CoCl4]2' (blue) + 2H20 Bassett and Crouchers’ 7 did not accept this dehydration theory. They claim that the reasoning of Hill and Howell, which was based on the comparison of magnesium and cobalt oxides, is unjustified and that cobalt need not have a coordination number of six. A more recent study by Katzin-Geberts’ 8 seemingly accepted the theory based on the dehydration mechanism. In dilute solutions only the Co(SCN)2, Co(SCN)2, and Co(SCN)42' complex ion species are identified. Very convincing evidence is given in favor of the existence of six coordinate thiocyanate aquo complexes in aqueous solution and four coordinate complexes in organic solvents. These historical research studies however, do not judge the relative stability of these cobalt (11) ions in mixed solvent systems. For example, cobalt-containing water-insoluble complexes will dissolve in chloroform (CHCl3) to give pink, blue, or green solutions. In addition, since the solvent is also a reactant in many reactions, 3 given substance behaving as a base in one solvent can behave as an acid in another. Nevertheless, by the proper choice of acid concentration, anion concentration, organic solvent and valence state of the metal, it is highly probable to effect the extraction and, ultimately, the stability of the metal complex. 13 Although the description of inorganic analyses involving metals primarily deals with samples in aqueous solutions, the term liquid-liquid extraction that will be discussed here implies the use of an aqueous-organic solvent pair. In solvent extractions, one is dealing with immiscible solvents and a solute and/or complex distributed between them. Two important factors to recall are: 1) water has a tendency to solvate ions and 2) metal salts are strong electrolytes, highly soluble in aqueous media. Therefore, in metal extraction systems, some or all of the H20 molecules bound to the cation must be displaced to obtain a species that can be extracted into an organic solvent. The probable candidate to replace H20 molecules is a neutral species. In 1872, Berthelot and Jugfeish9 proposed a distribution theory. It states that a solute will distribute between two immiscible solvents in such a manner that, at equilibrium, the ratio of the concentrations of the solute will be constant between the two phases. The distribution ratio, D, is: Concentration in organic phase/Concentration in aqueous phase Later, when describing liquid-liquid extraction systems, it was the percent complex extracted, %E, that became the focus. Many extraction systems like the one to be discussed later in the work uses chloroform as the organic solvent. Chloroforrn is more dense than the original aqueous phase from which the complex is extracted. The advantages of a two phase (aqueous and organic) solvent system is to minimize losses of the solute to be complexed, especially when the amount to solute to be studied is low or the D value is small or unknown. 14 The specific nature of the solvent-solute interaction obviously differs from one metal extraction system to another. Nevertheless, the fundamentals of every metal complex extraction process are the same. First, there are some interactions of the compound in the aqueous phase such as ionization. This is based on the properties of the complex being studied. Secondly, the compound has to be distributed in the solvent system in a way to form complexes. The relative solubility of the complex in each solvent does not have to be equal. Lastly, some interactions between the complex and organic phase must occur once the complex is extracted. These occurrences may include dissociation of the complex and further reactions with other species. It is now accepted, for instance in the case of cobalt (11) ions, that solvents which can compete strongly with anions such as SCN', octahedral complexes will be formed.10 These species tend not to dissolve in organic liquids. If, on the other hand, the anion competes favorably, tetrahedral complexes will result and hence increase the solubility in the organic phase. What happens if, in the aqueous phase of cobaltous thiocyanate, additional complexing agents are added? Will the complex formed be soluble in both the aqueous and the organic phase? For instance, the results of modern spot tests analysis depend on a particular analyte to form inorganic complexes with metals. If the formation of these complexes is influenced by interferences that may be present, the usefulness of the spot test decreases. 15 VALIDATION STUDIES The chemical behavior of cobaltous thiocyanate in the presence of complexing agents can be explored via the Scott Test, the spot test for the controlled substance, cocaine-HCl (Figure l). The chemical name for cocaine is benzoylmethyl ecognine (C17H21N04). It is an ester of benzoic acid and the amino alcohol, ecgonine. Cocaine is a naturally derived central nervous system stimulant. This bitter, white, odorless, crystalline drug is extracted and refined from the Coca plant. 0 . + O \H CH3 0 Figure 1. Chemical Structure of Cocaine-HCI In simplest terms, in the Scott Test1 ', an aqueous cobalt thiocyanate-HCI glycerine reagent is added to a small amount of a powder mixture suspected to contain cocaine. If cocaine is present, a blue flaky precipitate is formed. Early research reported this to be a presumptive result for the presence of cocaine. Later research studies proposed a modified method that would ultimately improve the test’s overall usefulness.12 This modification involved the addition of chloroform to the aqueous solution containing the blue precipitate to produce a turquoise-to -blue organic phase. As demonstrated in many inorganic analyses there are a number of variables that determine and influence the outcome of chemical reactions. A very recently published article by O’Neal et al. 13 describes this 2-step protocol, as what current laboratories rely on for the 16 preliminary identification of cocaine. However, in that study, over 25 drugs were analyzed. Five of these drugs were reported to react positively to the Scott Test. The presence of pharmacologically active adulterants and inert diluents in illicit drugs, manufactured and distributed in any part of the world, is very common. For cocaine-HCl, this is especially true due to the several possibilities of adulteration and dilution that this drug presents. Diluents such as lactose, talcum powder, and procaine are often used; creating more product and increasing the drug’s dollar value. The determination of cocaine content and adulterants in street samples is important to the forensic sciences, more specifically the typical drug analysis performed in a forensic science laboratory. The presence of illegal drugs is essentially what the analyst must determine. The forensic and legal communities continue to rely on the preliminary results of spot tests for the issuance of search warrants and arrests. Nevertheless, because of the number of additional compounds which have been reported to produce a blue precipitate upon the initial reaction of cocaine with the aqueous cobalt thiocyanate reagent, and a turquoise-to-blue colored organic layer upon addition of chloroform, the lack of specificity of the Scott Test to cocaine is often questioned. In addition, contaminants interfere with the reproducibility and limits of detection of spot test analyses of powder samples suspected to contain cocaine. The intent of this research project is to determine the mechanism by which the Scott Test for cocaine occurs. The initial approach to this research project was to understand how the Scott Test for cocaine was performed. The chemistry of cocaine relative to metals, non-metals and other organic compounds commonly used as diluents in cocaine containing solids was also considered. This investigation required 17 understanding phase transfer reactions, complexes formed in the aqueous and organic solutions, and the reasons for the colors that result in a positive and negative analysis. The analytical instrumentation utilized in this project involved the use of spectroscopy and later analyses included laser desorption mass spectrometry. In this contribution, the value of the Scott Test is described based on a mechanistic understanding of the chemistry of the reactions that take place when a sample suspected to contain cocaine is analyzed by the modified Scott spot test analysis. 18 EXPERIMENTAL Spectroscopic measurements were made using an ATI Unicam UV2 (Cambridge, UK) dual-beam spectrophotometer controlled externally from a computer. Absorption spectra were acquired using quartz cuvettes of 1 cm pathlength. The spectrophotometer was operated under the following conditions: wavelength range, 700-200 nm; scan rate, 120 nm/min; wavelength interval, 0.1 nm; bandwidth, 2.0 nm; smooth,10. The reference samples for each measurement were distilled H20 or spectroscopic grade CHCl3. Each solution was prepared at concentrations between 0.1-1.0%. Laser desorption (LD) mass spectra were recorded on a PerSeptive Biosystems Voyager delayed extraction time-of-flight (TOF) mass spectrometer equipped with a nitrogen laser (337 nm, 3ns pulse). For the negative ion LD spectra reported here, the accelerating voltage was -15 kV, the delay time was 150 nsec, the grid voltage was 94.5% of the accelerating voltage. Typically, 50 laser shots were averaged for each spectrum. For the positive ion spectra reported, the accelerating voltage was 20 kV, all other parameters were not changed. 19 ULTRAVIOLET AND VISIBLE ABSORPTION SPECTROSCOPY Ultraviolet and visible (UV-Vis) absorption spectroscopy is the measurement of the attenuation of a beam of light after it passes through a sample or after reflection from a sample [surface]. Absorption measurements can be based on a single wavelength or over a defined spectral range. Both ultraviolet and visible photons are energetic enough to promote outer shell electrons to higher energy levels. As a result, UV-Vis spectroscopy is very common for the analysis of molecules and inorganic complexes in solution. UV-Vis spectroscopy is useful for characterizing the absorption, transmission, and reflectivity of a variety of solutions and sample types. UV-Vis spectroscopy can also be used for quantitative measurements. Measuring the absorbance at a wavelength and applying the Beer-Lambert Law can determine the concentration of an analyte in solution. Basically, this law is the linear relationship between absorbance and concentration of an absorber (the analyte being measured) of electromagnetic radiation. Experimental measurements are usually measured in terms of transmittance (T). There is an inverse relationship between absorbance (A) and T. Conventional light sources for UV-Vis spectroscopy include tungsten filament lamps for visible measurements and deuterium discharge lamps for UV measurements. Spectrophotometer instruments frequently automatically switch lamps when seaming between the UV and visible regions. The photons of different wavelengths from these continuous light sources are typically dispersed by a monochromator. 20 The most common type of spectrophotometer that is used for the UV-Vis region is the double-beam spectrophotometer. Double beam refers to the feature of having two beams of continuum radiation. Afier leaving the monochromator, the beam is directed onto a beam splitter. One beam is sent through the sample cell containing an analyte in a certain solvent and the other through a reference cell containing only the solvent. The beams then pass through a modulator and are recombined onto the detector. The detectors used in single-detector instruments are mostly photodiodes. Photodiodes are semiconducting devices that convert light (absorption of photons) into electronic signals or voltages. Photodiode detectors can be used to measure and detect light over the entire UV to visible spectrum. 21 RESULTS Various researchers have examined the UV-VIS absorption spectra of inorganic complexes in H20 and a number of non-aqueous solvents. Comprehensive databases of absorption spectra for just cobalt complexes alone have been created. There seems to be little reason to doubt the basics: the red or pink solutions of cobaltous salts are associated with octahedral systems, and the blue solutions with tetrahedral systems. The usefulness of the Scott Test is based on the reagents’ specificity to cocaine, therefore we decided to prove the test’s validity. By first considering a series of qualitative spectroscopic measurements, we will “construct” the reagent, and attempt to understand what is present in the solution at each step. Step 1, CoCl2'6H20 is dissolved in water to produce a 0.18M solution. The solution turns pink. The results of the UV/V IS absorption scan of this pink solution indicated a maximum absorbance of the pink complex at 513nm (Figure 2). The spectrum matches the literature spectrum of hexahydrato cobalt (II) (Figure 3).2 Thus, in a solution of CoCl2°6H20(aq), Co(H20)62+, and Cl' ions are the main species present. The Scott reagent contains, in addition to CoCl2°6H2O, NH4SCN, glycerine and HCl. Preparation of the Scott reagent required a solution of: 6.8 g cobalt chloride hexahydrate, 4.3 g ammonium thiocyanate, 3 drops of glycerine, 4 drops of concentrated [12 N] hydrochloric acid, and IOOmL of H20. When CoCl2°6H2O is dissolved in water the solution is pink. Upon addition of the compounds that make up the Scott reagent, the color of the solution does not change. What are the main species present? Is the reagent composed of Co(H20)62+, Cl', NHX, SCN', H+, and glycerine in water? 22 Absorbance 0.8 0.7 - 0.6 4 0.5 i 0.4 ~ 0.3 . 0.2 2 0.12 350 400 450 500 550 600 Wavelength (nm) Figure 2. UV/VIS Spectrum of CoCl2° 6H20 Dissolved in H20 23 650 R3 '3 6 Molar Absorbance (octahedral) (leipoqanor) oouuqrosqv Jelow o r ‘°° 59° 6:30 Tia? eclzo Wavelength (nm) Figure 3. Visible Spectra of [Co(H20)6]2’ (Curve A) and [CoCLrlz’ (Curve B) 24 The absorption spectrum generated from the Scott reagent, a similarly concentrated and colored system as Co(H20)62+, showed a different absorbance maximum. The peak maximum shifted from 513nm to 475nm (Figure 4). These data are important; a new species is formed. The starting solution was made by dissolving CoCl2-6H20 in water to produce Co(H2O)62+. SCN' and more Cl' were added. The increased levels of Cl' could have created a new complex. Referring back to Figure 3, the visible spectrum to the right (higher 2.) of the hexaquo Co2+ ion is for another system, [CoCl4]2'. This tetrahedral system is a blue colored solution. At high Cl' concentrations, this system would be achieved (Figure 5). However, the Cl' and the SCN' concentrations of the Scott reagent do not favor this product. The pink color suggests that the species is still an octahedral system. The message lies in the molar ratio of Co:SCN. Consider the equilibrium equations for Co2+ in the presence of C1' or SCN' (Figure 5). A series of equilibria, based on the SCN' and Cl' concentrations, could exist. Because the Scott reagent is prepared based on a 1:2 Co:SCN ratio, the most likely complex formed is C02+(H20)4(SCN')2 (Figure 5). Co(Cl)42', Co(SCN‘)42’, Co(SCN)2S2 are examples of blue colored, tetrahedral systems that could be created at higher anion concentrations. What causes Amax to decrease even if the solution color does not change? By replacing 2 H20 molecules with 2 SCN' ligands, the ligand field strength increases. Based on the facts listed in Table 1, the stronger the ligand field strength (the larger A), the smaller the value of km... The new species formed in the Scott reagent increases the ligand field strength but ultimately maintains an octahedral system. 25 0 8 ‘ — COC12.6H20 in H20 ’ Ruybal reagent in H20 0.7 1 km“ = 511 nm g 0.6 d m l 43 0,5 . —- rm... = 474 nm 300 350 400 450 500 550 600 Whvelergth (rm) Figure 4. UVN IS Spectra of Aqueous Solutions Peak Maxima Shift Upon Addition of SCN' to Co(H20)62+ 26 This is a very important concept for this work; SCN' ions are attached directly to the metal and this complex may in fact be what reacts with cocaine. In addition, these preliminary results prove how strongly the ligand content relative to the metal influences the structure, color, and shape of the inorganic complexes. 2+ 2+ Co(H20)6 —> Co(H 0) Octahedral Tetrahedral Con—120),)2+ + x- c.5‘ir-r,o>.,(x-i, + H20 Coz+(X)4 + 3H,o Figure 5. Equilibria for Co“, x’(cr‘, SCN'), H20 The goal is to be able to understand the chemistry of the reactions that take place when the Scott reagent is added to cocaine. These reactions include the aqueous layer, the precipitate that is formed, and the organic layer. Simply, the question is: what is present in the Scott reagent that may complex with [Co(H20)(,]2+ and later, cocaine? Step two, the Scott reagent, a solution containing {C02+(H20)4(SCN')2, Cl', NHX, H+ and glycerine} is added to a pinch (approximately 1 mg) of cocaine (Figure 6). The 27 color of the aqueous layer remains pink. A blue flaky precipitate is formed. To understand what chemical processes take place, we consider both the solution and the solid. The absorption spectrum of the aqueous layer was taken. Again, a different spectrum was generated (Figure 7). The peak maximum equals 51 lnm. C02+(H20)4(SCN')2 is no longer present in the solution. This pink complex is essentially the species Co(H20)62+. This was also confirmed by considering the spectrum of cocaine and CoCl2°6H20 dissolved in H20 (Figure 8). What information can be obtained by analyzing the solution? What is the chemical reaction that takes place, resulting in the formation of the blue solid? What is the blue solid? The blue color indicates that a tetrahedral complex was formed. This complex could be Co(SCN)42', which is blue. However, high SCN' concentrations are needed to form such species. These concentrations are not characteristic of the solutions used in this project. Therefore, Co”, SCN' and cocaine must be present in the solid. After studying the spectroscopy of the aqueous solution of the Scott test, it was realized that the key component is the blue precipitate that was formed. In order to investigate the structure of the colored solid, laser desorption mass spectrometry was used. 28 e 9 A ueous la er —> 5;, —> . “ y 0.025 mol CoCl2'6H20 ' 50 mL CHCI3 0.05 mol NH4SCN ' -- U 50 mL H2O U . Organic layer glycerine ' HCl Figure 6. Scott Reagent Protocol Step 1: Preparation of Scott reagent Step 2: Addition of reagent to sample containing cocaine Step 3: Extraction of blue solid with CHCl3 29 H20 Co” SCN' Cocaine Cl' . H” Glycerol Figure 7. UV/V IS Spectrum of Aqueous Layer After the Addition of Cocaine 30 xm=511nm A 700 Absorbance 3 I _ — i i l ! 2.5 7 2 '4 1.5 ~ 1 0.5 < 0 Y ‘ r /\ 1 200 250 300 350 400 450 500 550 600 Wavelength (nm) Figure 8. UV/V IS Spectrum of Cocaine and CoCl2° 6H20 Dissolved in H20 31 LASER DESORPTION MASS SPECTROMETRY Common to all experiments in mass spectrometry is the creation of gas-phase ions. Electron impact (EI) ionization is the accepted method for creating ions from volatile gas molecules. In El ionization, fast-moving electrons add energy, resulting in ejection of an electron from a neutral molecule to produce a positively-charged ion. Another common ionization technique is chemical ionization (CI). An ion-molecule reaction between the sample molecule and a reagent ion results in a proton transfer to the molecule forming a singly protonated species. Here, the reagent ion is CH5+ or NH4+. These processes are most useful when the gas-phase molecules are formed without decomposition or rearrangement of the sample.14 For non-volatile and therrnally-labile sample molecules, other ionization methods have been developed. These methods include fast atom bombardment (FAB), and laser desorption (LD). The shared feature of these techniques is the rapid addition of energy into a condensed-phase sample, with subsequent generation and release of gas phase ions into the mass analyzer. The mechanisms of energy transfer, volatilization, and ionization involved in FAB, electrospray and LD are still debated. However, the development of these methods has permitted analysis of nonvolatile samples not conducive to conventional mass spectrometry.14 Laser desorption was initially developed for its spatial revolving power. Over the years, as many as 500 articles have been written on the subject of laser desorption/mass spectrometry. In 1980, Conzemius and Capellen published an extensive review of current instrument designs, experimental parameters and applications of laser mass 32 spectrometry. ‘5 Decades later, laser desorption methods have become particularly useful for analyzing substances such as polymers, paints, and inorganic complexes. Due primarily to its usefulness for the analysis of inorganic complexes, laser desorption was one of the analytical tools used in this project. A laser is a device that can deliver a large density of energy into a small area. The energy delivered per unit area, therefore becomes very large. In theory, any laser can be used to cause desorption and ionization as long as it provides enough energy at the right wavelength, in a short space of time, to the sample. In practice, the sorts of lasers used are restricted to a few types. Photon energies corresponding to the UV region of the electromagnetic spectrum (e.g., 337 nm) or the infrared region are used. The laser radiation can be pulsed or continuous. Lasers used in mass spectrometric experiments are always pulsed. The energy delivered from continuous lasers is much less than from pulsed ones. Thus, the irradiated area of the sample for pulsed lasers is higher and, therefore, the energy input is also greater. Every molecule possesses rotational, electronic and vibrational energy. If the sample is a liquid or a gas, it will also have kinetic energy. If the internal energy of a sample is increased (e. g., by heat or radiation), the molecules can equilibrate the energy in such a way that the structure of the molecule remains unchanged. If however, an excess amount of energy is put into the sample in too short of a time span such that the energy cannot be dissipated fast enough, the substances will melt then vaporize. The internal energy of vibration and rotation is transformed into kinetic energy. As a result of the change in rotational, vibrational, and kinetic energy, electronic excitation may be result. Electrons may be removed from molecules to form ions (Figure 9). 33 a) W , 337 nm pulsed laser neutral 1 - O O \ molecules 0 Sample surface Figure 9' a) A Laser Pulse Irradiates the Surface of a Sample b) Neutral Molecules and Ions Begin to Desorb c) Neutral Molecules are Pumped Away; Ions Are Drawn in the Mass Analyzer 34 The ions are examined in the usual way by a mass spectrometer. A typical mass spectrometer includes: Linear 4 detector l | - — o | Ion path I ...... > I Laser path I l l . Flt ht Beam ' tuge gurde wire I ! l l | l l 1 Laser l I Video . I camera Laser — :— y I .A enure d attenuator | ’ Gfound giimun 9d) ‘ I Prism V - . ~ ’ I l Variable-voltage ' ~ _ r.-. Sample grid ' A i loading Sample pjate Main chamber l source chamber Figure 10. Voyager-DE Mass Spectrometer 0 Ion source- Ionizes samples and generates gas phase ions. 0 Analyzer- Separates ions according to individual mass-to-charge (m/z) ratios. 0 Detector- Detects ions 0 Data system- Converts signals into a readable display (Not shown in figure 10). 35 When pulsed lasers are used, time-of-flight (TOF) instruments can record ions over a broad mass range after each pulse. TOF mass spectrometry works on the principle that, if ions are accelerated with the same potential from a fixed point after ionization, at a fixed initial time and allowed to drifi, the ions will separate in space and time according to their mass-to-charge ratios. Mass is a molecular characteristic that can help to identify a molecule. Heavier ions travel (drift) more slowly and thus reach the detector later than the lighter ions (Figure 11). The times required for ions to reach the detector at the opposite end of the flight tube (time-of-flight) are measured. The number of ions reaching the detector at any give time is also measured. This number is referred to the ion abundance or signal intensity. Heavier ions Lighter ions 00 6 CD Intensity 3 Flight Path Lighter ions Heavier ions Figure 11. Time of Flight Analysis 36 LASER DESORPTION RESULTS A systematic investigation of the components of the Scott Test was performed in an attempt to understand the application of laser desorption mass spectrometry to cocaine and inorganic complexes. A number of excellent articles that describe the use of mass spectrometry to detect cocaine are available.16 For electron impact MS experiments involving cocaine, the mass spectrum generated shows peaks at m/z 303 and m/z 182 and m/z 82 representing intact molecular ion and fragment ions of cocaine.16 It was expected that similar mass spectral information might be generated using laser desorption. Little treatment of the samples was needed. In order to determine if the cocaine is present in the precipitate formed in step 2 of the Scott Test, positive ion laser desorption experiments were first performed on free cocaine hydrochloride. The spectrum in Figure 12 shows the results. The peak at m/z 304 represents [cocaine + H]+. The more intense peak at m/z 182 represent a fragment ion. This fragment is CmHmN02+ formed by the loss of the C7H502 side group which occurs during the desorption- ionization process. Thus, in positive ion mode, a LD mass spectrum of cocaine was generated. As anticipated, positive ion mode analyses are not useful when trying to obtain information about negatively charged species. Cobalt thiocyanate complexes are, typically, negatively charged. A saturated solution of cobaltous thiocyanate was analyzed via LD. Figure 13 depicts the laser desorption spectrum of cobalt thiocyanate complexes obtained in negative ion mode. The peaks represent complexes 37 [C 101—I l 61\J()2]+ 182‘ [Cocaine + H]+ 304 g .E‘ .5 D .2. E Q) 9.”. l I l l 130 180 230 280 330 m/z Figure 12. LD Mass Spectrum of Cocaine in Positive lon Mode 38 Relative Intensity Co(SCN)3' 233‘ -S C02(SCN)5' ‘— 407 \A ‘— -S <— e— -S <— ‘— _ - L-l Ll 1.21 1:81: I Am. r 1 I 200 400 600 m/z Figure 13. LD Mass Spectrum of Co(SCN)2 in Negative Ion Mode 39 formed between Co2+ and SCN' ions. These complexes are formed in different stoichiometric ratios. The peak at m/z 407 represents [C02(SCN)5]'. Notably, the prominent peak at m/z 232 is [Co(SCN)3]'. The complexes formed may also include CN' due to the loss of sulfur from SCN' ions. This is seen at m/z 200, Co(SCN)2CN'. Hoping that the spectra generated from the analysis of cocaine and cobalt- thiocyanate mixtures would be similar to what would be obtained for a compound containing C02: -SCN' and cocaine, which we anticipate in the precipitate, we analyzed the precipitate. A portion of the precipitate was first analyzed under conditions similar to those needed to analyze free cocaine. The mass spectra that were generated were comparable to those acquired from the LDMS analysis of cocaine-HCI. This spectrum is illustrated in Figure 14. The negative ion mode spectra were then generated (Figure 15). The presence of cocaine, Coz', and SCN' in the precipitate was confirmed. The two most abundant peaks in Figure 15 are located at m/z 233 and m/z 408. The results generated by analyzing the precipitate prove that all species (cobalt, thiocyanate and cocaine) are present in the blue solid. We showed LDMS spectra for cocaine, Co(SCN)2, and the blue solid. The positive ion spectrum for cocaine was not identical to the spectrum generated from the analysis of the precipitate. LD spectra for cocaine-HCl should be different from cobalt complexes of cocaine. The information provided, did however, prove the presence of cocaine in the solid. Similarly, the spectrum that resulted from the analysis of a saturated solution of Co(SCN)2, was used as a “fingerprint” for determining the presence of Co2+ and SCN'. These results allowed us to prove the presence of cobaltous thiocyanate components of the precipitate when analyzed in negative ion mode. 40 Relative Intensity 0 4444241.... [CroHrrsNoz]+ 182 \ 150 [Cocaine + H]+ ‘x 304 ““411: - 1.1L AL. 11 UL .J m/z 300 Figure 14. LD Mass Spectrum of Blue Solid in Positive Ion Mode 41 Relative Intensity Co(SCN)3' 233‘ 200 300 C02(SCN)5' 407 x 400 00 m/z Figure 15. LD Mass Spectrum of Blue Solid in Negative Ion Mode 42 The structure of the complex has not yet been defined. It could be Co(SCN)2(cocaine)2 (Figure 16, Structure 1). This is a reasonable structure. It had been proposed by 0guri.' '7 The Co:SCN ratio is 1:2. Cocaine acts as a bidentate ligand forming two six-membered rings. However, though a stable complex, this structure is octahedral and should be pink rather than blue. _ _, 0013—3 ocrr,— cl . i‘c“ .- \CH . N 3 Ncs—co—SCN \CHJ CS 5C ~. SC” rr,c rr,c I(IC)\ \_H co H’CO Figure 16. Structure 1. Figure 17. Structure 11. Perhaps the complex is an ion pair, such as [Coz+(cocaine)2](SCN')2 (Figure 17, Structure 11)? This would be a tetrahedral system. However, this complex is not just specific for cocaine. Another ion pair that could form is [C02+(SCN)4](Cocaine-H+)2 (Figure 18, Structure 111). The formation ofthis structure could possibly explain why HCl is needed in the Scott reagent. It is known that the basic form of cocaine will not react positively to the Scott reagent. By making the reagent acidic, basic cocaine, if present, will be transformed into the salt form and ultimately be detected. The Co:SCN ratio is 1:4. 43 . 2_ SCN SCN + + H— R R — H /Co\ SCN SCN Figure 18. Structure III. Postulated Tetrahedral Structure of the Complex Formed in Step 2 of the Scott Test; R groups Represent Cocaine Molecules 44 The basic form of cocaine is not very soluble in H20; 1 g dissolves in 600 mL of H20. In contrast, cocaine°HCl is highly soluble in H20; 1 g dissolves in 0.4 mL of H20. If we then compare the relative solubilities of these compounds in CHC13; 1 g of cocaine dissolves in 0.7 mL of CHCl3 while 1 g of Cocaine-HCl dissolves in 12.5 mL. The addition of HCl is not to create Cocaine-HCl that later will form the blue solid, but to increase the solubility of cocaine in the Scott reagent, an aqueous solution. Eisman has developed a method to analyze cocaine that definitely takes advantage of Structure 111, but an 8M SCN' solution is required.18 Other authors, like Oguri et al., have measured the molar Co:SCN ratio in the solid to be 1:2.l7 Oguri’s research was supported by stoichiometric studies of the isolated precipitate. The colored precipitate was isolated after cocaine was treated with cobaltous thiocyanate, hydrochloric acid and chloroform. The color of the complex of cocaine and cobaltous thiocyanate is believed to be labile and can be readily dissociated in the isolation procedures and recrystallization. Oguri also performed a quantitative determination of cocaine and cobaltous thiocyanate in the chloroform extract. These results indicate that cocaine and cobaltous thiocyanate bind in a 2:1 ratio. Further studies of the Scott reagent and the blue solid in water were completed. The precipitate was isolated and mixed with fresh water. The precipitate, as expected, did not dissolve into the water. Instead, a slightly pink solution with a blue flaky solid was produced. The absorption spectrum of the pink solution (Figure 19) was similar to the spectrum generated for [Co(H20)6]2+. How can this be explained? We suggest that, as the precipitate was isolated from the solution via evaporation of the liquid, a trace 45 Wavelength (nm) im, = 624nm B l v . All Wavelength (nm) CHcr, Figure 19. Analyzing the precipitate A: Precipitate mixed with fresh water B: Dissolution of precipitate in CHC13 46 amount of CoCl2 remained on the solid. This is what dissolves in the water to produce a spectrum with kmax = 511 nm. The rest of the blue solid, the precipitate, remains insoluble in water. This result supports the proposed stucture; a tetrahedral complex composed of cobalt, thiocyanate and cocaine. Step three of the protocol is the extraction of the blue precipitate into chloroform, CHCl3. Addition of CHC13 creates a two-phase solvent system and the precipitate disappears (Figure 6). The lower CHCl3 layer turns blue. Why does CHC13 turn blue? Blue indicates a tetrahedral system. Is the solution made of [Co(SCN)4]2' ions? Upon mixing equal parts of the Scott reagent and chloroform, again, a two-phase system resulted. The chloroform layer remained clear unlike when the cocaine and the precipitate are present, which produces a blue chloroform layer. This again proves the presence of cocaine in the precipitate. The absorption spectrum generated for this lower clear layer highly resembled that of chloroform. The CHCl3 induced changes in the absorption spectra. Two peaks having maxima at 237 nm and 624 nm were formed (Figure 20). To better understand these changes, the absorption spectra of pure cocaine dissolved in water and chloroform were generated. The peak maxima of cocaine dissolved in these solvents are at 237 nm and 243 nm, respectively. (Figures 21-22). In addition, the intensity of the peak at maximum 511 nm decreased (Figure 20). Some material is moved from the aqueous layer into the organic layer. The complex formed at 624 nm is, therefore, a different spectral feature than just cocaine or reagent ions dissolved in chloroform. Ammonium thiocyanate and CoCl2-6H20 are insoluble in chloroform. But, the blue CHC13 layer is not produced 47 300 400 500 coo 700 Wavelength (nm) rm = 51 1 nm B Add CHCI, \ , j A 3(1) 4(1) 5(1) 6(1) 700 H20 W(m CHCI, rm, = 624 nm C ' t I l I A] 200 300 400 500 600 700 W (In) Figure 20. A: Aqueous Layer After Addition to Cocaine Sample B: Aqueous Layer After CHCl3 Extraction C: Organic Layer 48 Absorbance 0.8 '- 0.6 - 0.4 ~ 0.2 . 200 210 220 230 240 250 *Y 260 Wavelength (nm) 270 280 290 300 Figure 21. UV/V IS Absorption Spectrum of Cocaine Dissolved in H20. Peak Maximum at 237 nm. 49 Absorbance 0.9 4 0.8 - 0.7 4 0.6 - 0.5 2 0.4 0.3 ~ 0.2 - 0.12 .1 200 220 240 260 280 300 Wavelength (nm) Figure 22. UV/VIS Absorption Spectrum of Cocaine Dissolved in CHCl3. Peak Maximum at 243 nm. 50 320 unless NH4SCN is added to the Cl2-6H2O aqueous layer. This ultimately proves that cocaine acts as the phase transfer reagent for Co(SCN)2 complexes. The blue organic phase was brought into contact with fresh water. Again a two- phase solvent system was created. The aqueous layer remained clear. To understand the reversibility of the phase transfer properties of the system, the clear aqueous layer was analyzed. The presence of a peak having an absorption maximum at 237 nm was generated. This spectrum (Figure 21) is simply the UV/V IS absorption spectrum of pure cocaine dissolved in water. Free cocaine will partition between the CHCl3 and the H20 layers. However, the tetrahedral complex did not partition between the two solvent layers. Finally, the isolated precipitate was dissolved in CHCl3. The same spectral features at km. = 237 nm and 624 nm were generated (Figure 19). This proves that once the precipitate is formed, the tetrahedral complex detected in the organic layer, is independent of the ions present in the aqueous layer. The proposed structure of the solid is [Co(SCN)2-N,O-eta 2-cocaine]. The complex detected in the organic layer is postulated to be a blue complex that is in fact tetrahedral (Figure 23).19 The peak at 237 nm is consistent with the electronic absorption of the phenol ester of cocaine. This region does not participate in the bonding with cobalt thiocyanate. The presence of cocaine in the organic layer may also be the result of free complex cocaine or dissociation of cocaine from the solid. 51 0C 0a., \CH3 Ncs—‘Co—SCN Figure 23. Tetrahedral Complex of Blue Solid Once Dissolved In Organic Layer 52 OTHER SPECTROSCOPIC STUDIES AND CONCLUSION In this study, the mode by which cobalt complexes with cocaine was explored. The data presented clearly demonstrate the specificity of the Scott Reaction to cocaine. Previous studies of at least 30 drugs and medicines report precipitate-forming reactions in the first step of the Scott protocol.11 This in fact shows that cobaltous thiocyanate is not specific for cocaine. However, the color reaction using chloroform proves the test’s specificity for cocaine. In addition, we performed further analytical studies to support this work. These studies included the use of Infrared Spectroscopy (IR) and Proton Nuclear Magnetic resonance (NMR). IR spectroscopy involves the interaction of infrared radiation with a sample. The sample will absorb the infrared radiation at a specific wavelength. Information obtained from an IR spectrum includes identification of the functional groups present in a molecule. IR spectroscopy can be useful for confirming a structure by direct comparison with a known spectrum. Absorption spectra were recorded on a Nicolet FTIR using the KBr pellet method to prepare samples of the blue solid precipitate. Upon completion of the IR analysis, the spectrum of the blue solid was compared with reference materials of standard cocaine (base) and cocaine hydrochloride.”22 This comparison was not only to confirm the presence of cocaine in the solid, but also to determine if additional peaks were present in the spectrum of the blue precipitate (Figure 24). The IR spectrum of the blue solid showed a strong band at 2050 cm". The peak at 2150 cm'1 is attributable to the IR absorption of 53 Ev— E 0:380 H.U. ..mg 5 C: 05800 “m 5v— 5 2.0m 0:5 N.... go 8.50on 5398.? y: .E... oSwE A788 33855285 com cog com _ ooom comm Ii; r i coon comm {‘ .53 E 05300 ”U M Nd ... ed wee H. 2 3 43.4.? iii .5. e 6: :28 “m 4242424. 5 .5. 5 2cm 2.5 H.... 4. m. we ... o.— .. ed M. .3 .. so . we I. M ed ooueqrosqv ed 54 5.x 5 035525. 05300 ”m 2mm E 30m 03m 2. .«o 828on coca—coma}. 5 .mm oczwfi 9-83 2383:0553 cow 82 com 4 coon comm coon cow m 7 i/\l 2mm 5 065325 :BoU ”m 13.433 /<