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Ciclo de Palestras da Graduação -‐ IQSC-‐USP
IQSC-‐USP-‐ 30/10/2017
Uma visão pessoal a respeito da evolução das técnicas
cromatográficas
Fernando M. Lanças Universidade de São Paulo
InsHtuto de Química de São Carlos
µ-GC µ-LC
Softwares (Aquisição, Tratamento, Simulação,...
• SPME • SBSE • µ-SFE
Colunas Capilares (GC, LC, SFC.
CEC)
Interfaces µ-LC/MS
Sistemas Hifenados
Fitoterápicos
Meio Ambiente
Proteômica Fármacos Química Forense
Combustíveis
Alimentos e Bebidas
Grupo de Cromatografia do IQSC-‐USP: Projetos Atuais
µ-SFC
It is not important for the scien<st whether his own theory proves the right one in the end.
Our experiments are not carried out to decide whether we are right, but to gain new knowledge. It is for knowledge sake that we plow and sow. It is not inglorious at all to have erred in theories and hypotheses. Our hypotheses are intended for the present rather than for the future. They are indispensable for us in the explana<on of the secured facts, to enliven and to mobilize them and above all, to blaze a trail into unknown regions towards new discoveries.
R. WillstaTer Willard J. Gibbs Medal Address Chicago, IL, September 14,1933(24)
Cromatografia de Adsorção: A Vida e Obra de Michael TsweL
“For almost every inven<on, one can find persons who worked earlier and more or less carried out similar inves<ga<ons but who are s<ll not considered as the inventors of given techniques, processes, or machinery. Usually, the person recognized as the real inventor not only u<lized or described a phenomenon but also could interpret it and apply it knowingly for some purpose”.
Leslie S. ETre, “The Development of Chromatography”, Anal. Chem. 43 (1971) 20A.
• Em Ciência, o crédito é dado à pessoa que convence o mundo, e não à pessoa à qual a idéia ocorreu pela primeira vez Sir William Osler
12/07/1849 – 29/12/1919 “Pai” da Medicina Moderna
Work If metal electrodes are exposed to light, electrical sparks between them occur more readily. For this "photoelectric effect" to occur, the light waves must be above a certain frequency, however. According to physics theory, the light's intensity should be cri<cal. In one of several epoch-‐making studies beginning in 1905, Albert Einstein explained that light consists of quanta -‐ "packets" with fixed energies corresponding to certain frequencies. One such light quantum, a photon, must have a certain minimum frequency before it can liberate an electron.
…”Experiment has decided for the second alterna<ve. That the decision could be made so soon and so definitely was due not to the proving of the energy distribu<on law of heat radia<on, s<ll less to the special deriva<on of that law devised by me, but rather should it be aLributed to the restless forward thrus<ng work of those research workers who used the quantum of ac<on to help them in their own inves<ga<ons and experiments. The first impact in this field was made by A. Einstein who, on the one hand, pointed out that the introducHon of the energy quanta, determined by the quantum of acHon, appeared suitable for obtaining a simple explanaHon for a series of noteworthy observaHons during the acHon of light”
O período “pré-‐Tswe;”
² Bíblia e escritos egipcios: remoção de contaminantes por filtração em
areia e de excesso de sal por contato com certas folhas
² Alquimistas: desHlação, extração com solventes e outros
² Século XVI: preparação de vinho branco a parHr de Hnto por efeitos
hoje considerados adsorção.
² Século XIX: sólidos para fracionamento de líquidos, fracionamento em
papel e outros (ex. F.F. Runge em 1834).
-‐ O final do século XIX e a contribuição de D.T. Day [ U.S. Geological Survey]:
Colunas de “fuller’s earth” (silicato de alumínio e magnésio) para fracionamento do petróleo.
First Interna<onal Petroleum Congress in Paris, 1897. 43rd mee<ng of the U S . Geological Society in Kashington, D.C., 1900. -‐ Interpretou incorretamente o fenômeno osico-‐químico que ocorria e deu o
nome de “capillary diffusion process”.
-‐ O fenômeno foi explicado corretamente por Michael TsweT, o qual deu ao mesmo o nome de CROMATOGRAFIA.
Michael Semenovich TsweT
• 14-‐maio-‐1872, nasceu em AsH, Piemonte, Itália, filho de Simeon TsweT, russo, e Maria Dorosza, italiana.
• Cresceu e estudou na Suiça. • 1896: na Suiça, defendeu o Ph.D.; mudou para a Rússia. • 1901, depois de empregos menores, conseguiu um cargo na Universidade de
Warsaw (a Polônia fazia parte da Russia).
• 1901-‐1915 foi assistente, associado e Htular na School of Veterinary Medicine, and the InsHtute of Technology of Warsaw até o início da Primeira Gerra.
• 1915 deixou Varsóvia, antes do avanço das tropas alemãs, indo para Nizhnii
Novgorod e fundando o InsHtuto Agronômico de Gorkii.
M. S. Tswett
Physikalisch-chemische Studien Über das Chlorophyll. Die Adsorptionen. M. TsweT, Ber. Dtsch. Botan. Ges. 24, 316-‐326 (1906)
Adsorptionsanalyse und chromatographische Methode. Anwendung auf die Chemie des Chlorophylls. M. TsweT, Ber. Dtsch. Botan. Ges. 24, 384-‐392 (1906)
Warsaw University in 1902.
Capa do livro de M.S. TsweT, publicado em 1910, apresentado como sua tese de doutorado na Rússia Imperial.
Sistema uHlizado por TsweT em 1906. L.ELre, LC•GC Europe September 2003
• 1917 adoeceu e ficou algum tempo em tratamento nos Cáucasos, sendo indicado para a cátedra de botânica na University of Jurjeff *.
• * [nome da cidade na época; em alemão Dorpat. Depois da guerra esta região se tornou a Estonia, e o nome da cidade mudou para Tartu. Quando em 1940 a Estonia se tornou parte da Russia (the Soviet Union),o nome foi manHdo.]
• 1918: a Universidade foi evacuada para Voronezh antes do avanço das tropas alemãs. Inicialmente TsweT havia decidido ficar mas no úlHmo momento seguiu o grupo da Universidade. Sua saúde já estava bastante deteriorada.
• 1919: Faleceu em Voronezh em 26 de junho, ao que tudo indica de problemas
coronários.
• *** Tswe; foi enterrado em um cemitério do monastério de Alekseev, o
qual foi destruido durante a Segunda Gerra Mundial; sua tumba jamais foi localizada depois disso.
Em Genebra, na época da defesa do doutorado 1896.
Em São Petersburgo, na época da defesa do mestrado em 1901.
Em Varsóvia, 1911.
O Renascimento: 1931
R. Kuhn and E. Lederer, Ber. Deut. Chem. Ges. 64, 1349-‐1357 (1931). R. Kuhn, A. Winterstein, E. Lederer, Hoppe Seyler’s Z. Physiol. Chem., 197, 141-‐60 (1931).
R. Kuhn (direita) and E. Lederer; Heidelberg, 1963
Duas xantofilas separadas pela primeira vez por Kuhn, Winterstein, and Lederer em 1930-‐1931, iniciando o renascimento da cromatografia líquida de adsorção
“No other discovery has exerted as great an influence and widened the field of inves<ga<on of the organic chemist as much as TsweL’s chromatographic adsorpt i o n a n a l y s i s . R e s e a r c h i n t h e f i e l d of v i t a m i n e s , h o r mones, carotenoids and numerous other natural compounds could never have progressed so rapidly and achieved such great results if it had not been for this new method, which has also disclosed the enormous variety of closely related compounds in nature”.
E. Lederer and M. Lederer, “Chromatography-‐A Review of Principles and ApplicaHons,”. Preface, Elsevier, Amsterdam, The Netherlands. 1955.
Paul Karrer, the great organic chemist and co-‐winner of the 1937 Nobel prize in chemistry, already had given the following appraisal of chromatography in his plenary lecture at the 1947 Congress of the InternaHonal Union of Pure and Applied Chemistry in London:
In pracHcally his whole working life, TsweT dealt with invesHgaHons related to chlorophyll. Toward the end of the 19th century, many scienHsts showed interest in the pigments occurring in the leaves of plants, but there was no real way to separate them from each other and to check their idenHty in different plants. Since these substances are very labile, one could not be sure whether material obtained through chemical manipulaHon really corresponded to the form exisHng in the living plant.
TsweT's approach was different ; he was looking for a physical method which would permit the separaHon of these pigments from each other and from closely related compounds that others felt to represent the same substances. For he was convinced that chlorophyll, as isolated by other researchers, was not a single sub-‐ stance.
In his work, he systemaHcally checked a large number of solvents capable of extracHng the pigments from vegetable maTer, and more than 100 solid substances capable of selecHve retardaHon of the individual pigments through adsorpHon, and he also deducted a number of important rules for the ad-‐ sorpHon phenomenon.
Os trabalhos “mais relevantes”
Descreve os fundamentos da Cromatografia, sem uHlizar este nome.
• 1903: Primeiro trabalho onde os dados preliminares foram resumidos: "On a New Category of Adsorp<on Phenomena and Their Applica<on to Biochemical Analysis"
Apresentado em 21 de março, 1903, March 21, na Biological SecHon of the Warsaw Society of Natural Sciences. Publicado (em russo) nos Anais da Sociedade.
• 1906: dois trabalhos, em russo:
“Physico-‐Chemical Studies of Chlorophyll. The Adsorp<ons” “Adsorp<on Analysis and the Chromatographic Method.
Applica<on in the Chemistry of Chlorophyll”
Descreve o processo cromatográfico em detalhes no primeiro trabalho:
“When a petroleum ether solu<on is filtered through a column of an adsorbent (I use mainly calcium carbonate which is tamped firmly into a narrow glass tube), then the pigments are resolved, according to the ad-‐ sorp<on sequence, from top to boLom, into various colored zones, since the more strongly adsorbed pigments displace the more weakly adsorbed ones, and force them farther downward. This separa<on becomes prac<cally complete when, aqer the pigment solu<on has flowed through, a stream of pure solvent is passed through the adsorbing column. Like light rays in the spectrum, the different components of a pigment mixture, obeying a law, are resolved on the calcium carbonate column and then can be qualita<vely and quan<ta<vely determined. I call such a prepara<on a chromatogram and the corresponding method the chromatographic method.”
Representação da Cromatografia Líquida “Clássica”
Amostra Coluna de Vidro
Adsorvente
Solvente
Componentes da amostra separados
Fluxo gravitacional,
sem pressão
Cromatografia Líquida de Alta Eficiência
Descarte
Solvente
Injetor
Cromatografia Líquida “moderna”
Cromatografia: Chroma (cor)+ graphe (escrita) ??? Talvez não. TsweT: “It is self-‐evident that the adsorpHon phenomena described are not restricted to the chlorophyll pigments, and one must assume that all kinds of colored and colorless chemical compounds are subject to the same laws.” Portanto, talvez cromatografia não signifique escrevendo com cores mas:
Em russo, o sobrenome TsweT (esta é a grafia em alemão; em inglês Tsvet) escreve-‐se da mesma forma que a palavra “cor”. Portanto, cromatografia pode querer significar “a escrita de TsweT”.
Leslie S. ETre, “The Development of Chromatography”, Anal. Chem. 43 (1971) 20A.
TsweT x Day TsweT did not invent adsorpHon; this had been described well before him and also explained from the theoreHcal point of view (e.g., in the books of W. Ostwald from 1891-‐95). Also, scienHsts before him had separated plant pigments by selecHve soluHon. TsweT’s merits are in the generalizaHon of the technique as an analyHcal method and in the detailed invesHgaHons of the role of various adsorbents and solvents. As pointed out by Zechmeister (1),
“TsweT’s achievement is superior to Day’s in two respects. First, he recognized and correctly interpreted chromatographic processes; and second, he devised a useful laboratory method that includes as an important feature the development of chromatograms by pure solvents. The distances between the individual zones are thus increased and complete resoluHons advised within minutes.”
Cedalion standing on the shoulders of Orion from “Blind Orion Searching for the Rising Sun” by Nicolas Poussin,k 1658. Oil on canvas; 46 7/8 x 72 in. (119.1 x 182.9 cm). Metropolitan Museum of Art.
nanos gigantum humeris insidentes "discovering truth by building on previous discoveries"
MarYn e a Cromatografia de ParYção
Archer John Porter MarYn Born: 1 March 1910 Died: 28 July 2002
1941: A.J.P. Mar<n, Synge -‐ Cromatografia de ParYção.
1944: Consden, Gordon, Mar<n -‐ Cromatografia em Papel.
1952: Mar<n, James -‐ Cromatografia Gasosa.
A.J.P. MarHn and R.L.M. Synge, Biochem. J. 35, 1358-‐68 (1941) R. Consden, A.H. Gordon and A.J.P. MarHn, Biochem. J. 38, 224-‐32 (1944) A.T. James and A.J.P. MarHn, Biochem. J. 50, 679-‐90(1952)
O Prêmio Nobel 1952
A.J.P. Martin recebendo o Prêmio Nobel de Química de 1952 do Rei Gustavo VI da Suécia
R.L.M. Synge recebendo o Prêmio Nobel de Química de 1952 do Rei Gustavo VI da Suécia
hTp://nobelprize.org/nobel_prizes/chemistry/laureates/1952/marHn-‐lecture.pdf
The mobile phase need not be a liquid but may be a vapour. We show below that the efficiency of contact between the phases (theoreHcal plates per unit length of column) is far greater in the chromatogram than in ordinary disHllaHon or extracHon columns. Very refined separaHons of volaHle substances should therefore be possible in a column in which permanent gas is made to flow over gel impregnated with a non-‐volaHle solvent in which the substances to be separated approximately obey Raoult's law. When differences of volaHlity are too small to permit of ready separaHon by these means, advantage may be taken in some cases of deviaHon from Raoult's law, as in azeotropic disHllaHon. Possibly the method may also be found to be of use in the separaHon of isotopes ,e.g. of nitrogen isotopes, by passing ammonia gas over gel impregnated with ammonium sulphate soluHon.
MarHn AJP, Synge RLM. A new form of chromatogram employing two liquid phases: 1. A theory of chromatography. 2. ApplicaHon to the micro-‐determinaHon of the higher mono amino-‐acids in proteins. Biochemical Journal. 1941;35(12):1358-‐1368.
Consden R, Gordon AH, MarHn AJP. The idenHficaHon of lower pepHdes in complex mixtures. Biochemical Journal. 1947;41(4):590-‐596.
Archer John Porter MarYn Born: 1 March 1910 Died: 28 July 2002
As raízes da Cromatografia Bi-‐Dimensional (2-‐D): TLC-‐TLC
Two-‐dimensional chromatogram of a wool hydrolysate (l8Oug.) on Whatman no.1 sheet. Hydrolysate applied at circle. Run with collidine for 3 days in direcHon AB, then in direcHon AC with phenol for 27hr. in an atmosphere of cos gas and NH3 (produced from a 0.3% NH3 soluHon)
Diagram showing method of cu�ng of a two-‐dimensional chromatogram. (a) and (b) represent duplicate sheets, the soluHon to be analyzed being applied at the open circles. The arrows indicate the direcHon of development of the chromatograms in the two solvents. In the guide chromatogram(a), amino-‐acids are shown as black spots and pepHdes as shaded spots. A�er treatment with dilute ninhydrin, the duplicate sheet(b) shows faint spots(some-‐may be barely visible) and is cut as shown by the doTed lines.
“on-‐Line 2D GC” (GC-‐GC ): 1958
Anal. Chem., 1958, 30 (1), pp 32–35
Dr. Loyd R. Snyder
Fig. 6. SeparaHon of stabilized Pla�ormate on Squalane-‐modified Pelletex. T=100oC, He @ 40 mL/min
Fig. 7. Chromatogram for C5, C6, C7 and C8 fracHons obtained by Gas-‐Liquid Chromatography.
Anal. Chem., 1958, 30 (1), pp 32–35
Primeira publicação sobre LC mulHdimensional (LC-‐LC): 1978
Dr. Roland W. Frei 1936-‐1989
The complex chromatograms that are obtained with such a senna glycoside extract when only one dimension is used are shown in Fig. 5 for the GPC run and Fig. 6 for the step-‐gradient RPC run.
Reversed-‐phase chromatograms of seven fracYons from the GPC run as obtained with the apparatus shown in Fig. 4 by using a step gradient are presented in Fig. 7. The value of the two-‐dimensional approach is clear: the originally very complex chromatograms (Figs. 5 and 6) are now subdivided into a set of seven simpler and beTer resolved chromatograms.
• 1906: Mikhail Semenovich TsweL -‐ Cromatografia Líquida de Adsorção (pigmentos em plantas).
• 1941: A.J.P. Mar<n, Synge -‐ Cromatografia de ParHção.
• 1944: Consden, Gordon, Mar<n -‐ Cromatografia em Papel.
• 1952: Mar<n, James -‐ Cromatografia Gasosa, GC.
• 1968: Kirkland, Hueber, Horvath – Cromatografia Líquida (HPLC).
• 1982: Novotny, Lee – Cromatografia com Fluido SupercríHco (SFC).
Histórico da Cromatografia
Milos Novotny, Daniel Armstrong e Milton Lee no Brasil em 2006
A B C
H HEPT dpD k
kdfD
gas
liq
= = + ++
× ×22 8
12 2
2
λγ
µ πµ
( ) .
Van Deemter EquaHon (1956)
µ =LtM
Developed originally for packed GC, this equaHon was later extended do LC and SFC
van Deemter JJ, Zuiderweg FJ and Klinkenberg A (1956). "Longitudinal diffusion and resistance to mass transfer as causes of non ideality in chromatography” Chem. Eng. Sc. 5: 271–289.
Superficially Porous columns (core shell)
1964: C.Horváth and I. Halasz described the concept of porous-‐layer bead (PLB) column packing”
Analy<cal Chemistry 1964;36(7):1178–1186.
1967: Czaba Horvath developed the first pelicullar materials for ion exchange, introducing modern packing materials that contributed to the development of HPLC. This material were made from glass beads coated with a thin layer of an organic polymer. The rigid part (“core”) allowed operaHons at elevated pressures but the columns were not efficient due to the poor mass transfer properHes of the staHonary phase
This was later improved by Kirkland (1969 on) and others.
Czaba Horváth Born:25 January 1930 Died:13 April 2004
Golay EquaHon for open tubular GC (1956)
H = plate height u = mobile phase velocity, k ’ = capacity factor of the solute, Dm and Ds are diffusion coefficients of the solute in the mobile and staHonary phase respecHvely, df is the layer thickness of the staHonary phase, dc is the diameter of the channel available for the mobile phase (equal to the I.D. of the capillary minus 2df).
Marcel Golay ca. 1960
Marcel Jules Edward Golay
Born: May 3, 1902 Died: April 27, 1989
Colunas capilares empacotadas operando em pressões ultra elevadas (U-‐HPLC)
James Jorgenson
(A) 19,000 psi 66 cm x 30 µm i.d. column
MacNair, J.E., et al. Anal. Chem. 1997, 69, 983-989.
(B) 59,000 psi 52 cm x 30 µm i.d. column
Outras condições: 1.5 µm nonporous ODS silica
10% ACN/90% water (0.1% TFA)
GC x SFC x LC: Why?
“There are no theoretical boundaries
between GC, LC and SFC, and these
distinctions are arbitrary, artificial and
counterproductive”
J. C. Giddings, Dynamics of Chromatography,
Marcel Dekker, NY, 1965
Instrumental setup of the capillary column switching LC-‐ ESI-‐MS/MS system.
INSTRUMENTAL SETUP
Simultaneous analysis of 10 pharmaceuHcal compounds
Um enfoque Universal: Separação Mul<dimensional
totalmente automaHzada
On-‐line approaches for determinaYon of residues and contaminants in complex samples Bruno H. Fumes, Mariane A. Andrade, Maraíssa S. Franco , Fernando M. Lanças
J. Sep. Sci. 2017, 40, 183–202 .
DeterminaHon of Ochratoxin A (OTA) in wine by “in-‐tube SPME-‐HPLC-‐MS/MS”
Mariane A. Andrade, Fernando M. Lanças; J. Chromatogr. A, 1493 (2017) 41-‐48.
Mariane A. Andrade, Fernando M. Lanças
Maximum allowed concentra<on of OTA in wine is equal to 2 µg/kg 1, 2
1) Commission regulaHon (EC) nº 1881/2006 of 19 december 2006 se�ng maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, v. 49, p. 5-‐24, 2006;
2) Agência Nacional de Vigilância Sanitária (ANVISA). Resolução -‐ rdc nº -‐ 7, de 18 de fevereiro de 2011 Dispõe sobre limites máximos tolerados (LMT) para micotoxinas em alimentos.
p-‐in tube-‐SPME set up
Details of the in-‐tube SPME capillary column
General scheme of the employed in-‐tube SPME system
Mariane A. Andrade, Fernando M. Lanças; J. Chromatogr. A, 1493 (2017) 41-‐48.
Analysis of OTA in wine samples at ppt levels
Red trace: spiked wine sample ([OTA] = 0.2 µg L-1) Black trace: unspiked wine sample
Maximum allowed concentra<on of OTA in wine is equal to 2 µg/kg 1, 2
LOQ = 0.05 µg L-‐1 ~ 50ppt
Mariane A. Andrade, Fernando M. Lanças; J. Chromatogr. A, 1493 (2017) 41-‐48.
Meire Ribeiro da Silva, Ole K. Brandtzaega, Tore Vehusa, Fernando M. Lancas , Steven R. Wilson, Elsa Lundanes
Fig. 1. SchemaHc illustraHon of the automaHc filtraHon/filter back-‐flushing system with A mulH-‐channel polymer layer open tubular (mPLOT) solid phase extracHon (SPE) and open tubular (OT) analyHcal column. In posiHon 1, the sample is loaded through a stainless steel filter and the hydrophobic compounds are trapped on the mPLOT SPE. In posiHon 2, the stainless steel filter is being back-‐flushed (making it ready for a new injecHon) while the hydrophobic compounds are eluted off the mPLOT SPE, onto the OT separaHon column and further detected by mass spectrometer (MS).
Scanning electron micrograph of the op<cal fiber (top) and the PS-‐OD-‐DVB mPLOT (boLom) used as extrac<on column.
Fig.2. Chromatographic performance when injecHng 1!L of 10pg/!L of sulfamethoxazole(SMZ)(m/z254−>m/z92,m/z108) and N4-‐acetylsulfamethoxazole(n-‐SMZ)(m/z 296 −> m/z 92, m/z 108) into a 50 !m × 5 cm self-‐packed Accucore C18 (top), 50 !m × 5 cm poly(styrene-‐co-‐octadecene-‐co-‐divinylbenzene) (PS-‐OD-‐DVB) monolithic (middle) and a (126 × 8 !m) × 5 cm A mulH-‐channel polymer layer open tubular (mPLOT) solid phase extracHon (SPE) column (boTom) coupled to a 10 !m × 2 m poly(styrene-‐co-‐ divinylbenzene) (PS-‐DVB) open tubular (OT) separaHon column. Analytes were detected by a triple quadrupole MS in MS/MS mode. tR is retenHon Hme and w0.5 is the peak width at half the peak height.
Fase ST-‐DVB
Fase ST-‐DVB
Colunas capilares tubulares abertas -‐ 2017 (a) Si-‐ODS [WCOT]; (b) Si-‐ST-‐DVB [PLOT] Vivane L. Leal e Fernando Lanças; resultados não publicados
Fase ODS
Fase ODS
TsweT, Einstein e John Fenn. O que apresentam em comum???
Albert Einstein 1879-‐1955
John B. Fenn 1917-‐2010
Michael Tswe; 1872-‐1919
CaracterísHcas da carreira: 1. Tumultuada por mudanças frequentes; interefencia de guerras. 2. Carreira cien�fica curta (1901-‐1915) -‐ Varsóvia. 3. Trabalhos mais relevantes entre 1903-‐1906. 4. Publicou os principais trabalhos com ~ 30 anos [1903-‐1906]. 5. Foi bastante combaHdo e não teve reconhecimento em vida.
Michael Tswe;
Albert Einstein 1879-‐1955
CaracterísHcas da carreira: 1. Tumultuada por mudanças frequentes; interferencia de guerras. 2. Carreira cien�fica curta (1905-‐1932) – Suiça/Alemanha. 3. Trabalhos mais relevantes com 26 anos [1905]. 4. Foi bastante reconhecido em vida.
A. Einstein em New York, 1921
Com Charlie Chaplin, 1931
California, 1933 – ano em que se mudou para USA
Concerto na Alemanha, 1930
John B. Fenn
CaracterísHcas da carreira: 1. Tranquila até se aposentar. 2. Carreira cien�fica longa (faleceu com 93 anos). USA. 3. Trabalhos mais relevantes após os 70 anos [Nobel com 85 anos]. 4. Foi bastante reconhecido e controverso em vida.
"John B. Fenn -‐ Facts". Nobelprize.org. Nobel Media AB 2014. Web. 29 Oct 2017. <hTp://www.nobelprize.org/nobel_prizes/chemistry/laureates/2002/fenn-‐facts.html>
Never a very conscienHous student of the literature, I didn’t see Dole’s paper when it appeared, but not long therea�er it was brought to Sandy Lipsky’s aTenHon. A mass spectrometrist as well as a chromatographer, Sandy was very much intrigued by Dole’s ideas because they seemed to offer a way of using mass spectrometry to obtain precise values of molecular weight for large biomolecules like proteins.
Malcom Dole 1903-‐1990
However, from the perspecHve of history’s hindsight one can argue that the ability of intense fields at the surface of a Hny charged droplet, which play a vital role in Dole’s ESI, is simply an obvious extension of the discoveries by Mueller and Beckey on the nature of field ionizaHon at a sharp Hp, i.e., a surface with a very small radius of curvature. Maybe so, but it is also quite clear that nobody recognized these possibiliHes at the Hme, and that Dole arrived at the ESI approach by a logic that depended in no way on the field ionizaHon ideas of Mueller and Beckey. In other words Dole was a true pioneer in that his ESI ideas were breaking brand new ground.
Diagram of Electrospray IonizaYon. (1) Under high voltage, the Taylor Cone emits
a jet of liquid drops (2) The solvent from the droplets progressively
evaporates, leaving them more and more charged
(3) When the charge exceeds the Rayleigh limit the droplet explosively dissociates, leaving a stream of charged ions
In sum, Malcolm’s Dole’s paper in 1968* was the seed which, a�er an extended period of germinaHon, ulHmately blossomed into electrospray ionizaHon— ESI—one of the two “so�” ionizaHon methods that have made the precision, sensiHvity, and elegance of mass spectrometry readily available for the study of biomolecules and their reacHons. The second of those two “so�” ionizaHon methods is so-‐called matrix-‐ assisted laser desorpHon ionizaHon (MALDI), which was introduced by Karas and Hillenkamp in 1988 (see ref. 15) at almost the same Hme that ESI had begun to show its muscle. MALDI had also had a long germinaHon period but was born of very different ancestry. A brief history of each of these techniques is summarized in the next two secHons of this report. MALDI’s story is told first even though ESI’s was the first to begin.
*M. Dole, L.L. Mach, R.L. Hines, R.C. Mobley, L.P. Ferguson, M.B. Alice. Molecular beams of macroions. J Chem Phys 1968;49:2240–2249.
In 1969 at the age of 66, just two years before mandatory reHrement at Northwestern, Dole accepted an offer to become Welch Professor of Chemistry at Baylor University in Waco, Texas where he was assured there would be no mandatory reHrement as there was at Northwestern. At Baylor he conHnued his electrospray experiments using mobility measurements rather than mass analysis to characterize the ions. In retrospect I would have been wise to follow Dole’s example and leave Yale before I became subject to mandatory re<rement in 1987, the last year that it was allowed and just as the electrospray research was becoming most interes<ng. For the next six years Yale bureaucrats did everything they could to prevent that research from conHnuing, though they happily allowed the grant money to be spent and eagerly pocketed all the funds allowed in the grants for indirect expense. The most unfortunate consequence was that for the last five of those years Yale refused to provide the necessary space to install a new triple quadrupole mass spectrometer, which had not only been funded by NIH but which Yale had agreed to provide in several research proposals for which funds had been granted! That instrument didn’t get used unHl I brought it to VCU in 1994 when I le� Yale. I o�en wonder how much more we might have accomplished had we been able to use it during those five lost years.
Fenn's first electrospray ionizaHon source coupled to a single quadrupole mass spectrometer
Lawsuit Fenn's work with electrospray ionizaHon was at the center of a lawsuit pi�ng him against hisalma mater and former employer, Yale University. His iniHal dispute with the university began in 1987, when he turned 70 -‐ Yale's mandatory reHrement age. Per university policy, Fenn was made an emeritus professor, which resulted in a reducHon to his lab space. Emeritus professors at Yale are sHll provided with an office, but cannot conduct their own research, nor manage their own labs. In 1989, when Yale University inquired about the progress and potenHal about his electrospray work, he downplayed its potenHal scienHfic and commercial value. Fenn believed he had the rights to the invenHon under the Bayh-‐Dole Act. Fenn patented the technology on his own, and sold licensing rights to a company he partly owned -‐ AnalyHca of Branford. In 1993, a private company seeking to license the use of electrospray technology traced its invenHon to Yale, when the university discovered that Fenn held the patent. Yale's policy regarding patents generated by faculty or students requires that a percentage of any royalHes generated from the patent are used by the university to fund future research. They do not claim the rights to patents that are produced away from university faciliHes or not related to the researcher's "designated acHviHes.” Fenn claimed that he owned the technology because the work was completed a�er he had been forced to downsize at the university's mandatory reHrement age.
Yale University entered into its own licensing agreement with a private company, leading Fenn to file a lawsuit against the school in 1996. Yale countersued, requesHng damages and reassignment of the patent. The two parHes did not reach an out of court seTlement, despite repeated aTempts at mediaHon. In 2005, U.S. District Judge Christopher Droney ruled against Fenn, awarding Yale $545,000 in royalHes and $500,000 in legal fees. Judge Droney was criHcal of Fenn, saying "Dr. Fenn only obtained the patent through fraud, civil the�, and breach of fiduciary duty.” Evidence presented in the case indicated that Fenn had served on panels at Yale University that reviewed the insHtuHon's policy on intellectual property. A spokesperson for Yale said, "We are pleased by the result in this case and, in parHcular, by the court's vindicaHon of the Yale patent policy.” The ruling, and Yale's response produced a mixed reacHon from some of Fenn's colleagues and former students, who wrote a leTer to the Yale Daily News staHng, "'VindicaHng the Yale patent policy' is a poor excuse for treaHng a Nobel Laureate with a 68-‐year associaHon with and dedicated service to the University, in such a contempHble manner.”
TsweT, Einstein e John Fenn. O que apresentam em comum???
Albert Einstein 1879-‐1955
John B. Fenn 1917-‐2010
Michael Tswe; 1872-‐1919
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