final version of lc-gc article to be published in 2011

FEBRUARY 2011 LCGC NORTH AMERICA VOLUME 29 NUMBER 2 1www.chromatographyonline.com

MS – THE PRACTICAL ART

In the days before

electrospray and the

rapid growth of mass

spectrometry as a premier

tool for life sciences thanks

to John Fenn’s efforts,

petroleum, polymers, and

environmental interests

were the focus and vacuum

gas-phase techniques

prevailed. Where is the

practitioners’ interest in

solving problems today

in the chemical industry?

What tools do they employ

with good success and

which ones do they look

forward to — or wish they

had?

Michael P. BaloghMS—The Practical Art Editor

Mass spectrometry (MS) has been a cornerstone technol-ogy of the chemical indus-

tries including petroleum research and related commercial product manufactur-ing in polymers. The American Society for Mass Spectrometry (ASMS) will celebrate its 60th anniversary next year. Although christened under its current name in 1969 it began as “ASTM Com-mittee E-14” in 1952. The work of John Fenn and others that brought us elec-trospray ionization (ESI) and heralded the modern era where biological use of MS predominates was not seen until the 1980s. Before the 1980s gas-phase inter-ests predominated and techniques were commonly performed in vacuum as opposed to atmosphere today and many times as a set experiment using a solids probe rather than a flowing or chro-matographic serial sample introduction.

Where is the interest among practi-tioners today? What generates the most problem solving interest today in the chemical industry? What tools do they employ with good success and which ones do they look forward to — or wish they had? I had an opportunity to speak with Colin Moore, Fellow and Technol-ogy Leader in Mass Spectrometry at Chemtura Corporation (Middlebury, Connecticut) about the types of ana-lytical problems that his group is asked to solve and what they learned in the process of solving them; this discussion evolved into a short tutorial on chemical industry practice.

A UK native, Moore worked for Shell Research for seven years doing analytical work on agrochemicals and simultaneously became a graduate of the Royal Society of Chemistry. Post-graduate studies at the University of

Problem Solving in the Chemical Industry

In Memoriam: John B. Fenn and Uwe D. NeueAs I was writing this month’s column I learned of the passing of two important people in the world of analytical science within a week of each other. John Fenn, who died on Dec 10, 2010, was a profes-sor of chemistry first at Yale and then at Virginia Commonwealth University. He won a share of the Nobel Prize in 2002. Uwe Neue, who received his Ph.D. at Saarbrucken and went on to become well recognized as an authority in chro-matography, died December 3, 2010.

Fenn’s name has become almost syn-onymous with the practice of electrospray MS. Reference to his name and work appeared in this column frequently over the past eight years. Being able to say one had at some time spoken with Fenn was not an uncommon claim because he was freely available it seemed to any and all. When I had a chance years ago I com-mented to him how, because he seemed so freely available and generous with his time, his Nobel-inspired fame must at times be a burden. With genuine mod-esty he replied that there are many who deserve recognition for their work and he was fortunate to have been singled out. We have lost a true innovator and pioneer.

The work of my Waters colleague Uwe in chromatography was well known and widely respected. Aside from his scholarly efforts, books, and numerous publications, he also played a significant role in helping to establish the Conference on Small Mol-ecule Science. Since the first year Uwe at times chaired or organized what is now a most popular recurring session examining the theoretical underpinnings of applied chromatography. The session and discus-sion workshops feature some of the great names as participants thanks to Uwe.

2 LCGC NORTH AMERICA VOLUME 29 NUMBER 2 FEBRUARY 2011 www.chromatographyonline.com

Southampton were followed by three years at Warwick University and a Ph.D. with Professor Keith Jennings. He joined Uniroyal (now Chemtura Corporation) in 1994 as a member of the MS group, becoming manager in 1997 and Research Fellow in 2002. He has 20 publications in peer-reviewed journals and 24 posters and presenta-tions at conferences to his credit.

A Simplified OverviewSample analysis in Moore’s laboratory gen-erally falls into one of the following areas:

• Confirmation of the chemical struc-ture of the main components and identification of impurities to help synthetic chemists improve the syn-thesis.

• Identification of minor components in a product that shouldn’t be there (for example, color bodies).

• Identification of additives in a polymer or oil (for example, antioxidants in engine oil).For highly pure samples analysis by

nuclear magnetic resonance (NMR) spectroscopy is probably the best way

to obtain detailed structural informa-tion and therefore most of the samples that mass spectrometrists are asked to analyze are mixtures. The analyst’s first decision therefore is to determine which separation technique will be used: gas chromatography (GC), liquid chromatography (LC), gel permeation chromatography (GPC), solid-phase microextraction (SPME) or some type of liquid–liquid extraction. For example, if an engine oil sample in methylene chloride is shaken with methanol then the phenolic and aminic antioxidants will be concentrated in the methanol. GC–MS analysis of the methanol extract makes it easy to iden-tify the major antioxidants in the oils (Figures 1 and 2).

An interesting aspect of identifying unknowns that, according to Moore, “I’ve not seen in the literature,” stems from making use of both electron ionization (EI) and ESI spectra of an unknown since the fragmentation pat-terns can be “complementary” (see the EI and ESI spectra in Figure 3).

In the EI spectrum the first major loss is a C10 alkyl radical whereas in the ESI spectrum it is loss of the C10 alcohol. For those interested in a quick overview on mass spectra a recent column was devoted to that topic (1) that, in addi-tion, highlights James Little’s insights from his experiences solving problems at Eastman Chemical in Tennessee.

A significant difference in analytical practice between the pharmaceutical and specialty chemical industries is the level of dependence of the latter on GC–MS. Today the pharmaceuti-cal world favors LC–MS. For those interested in the aspects of how LC–MS became what we think of as open access in the pharmaceutical world, I chronicled the insights of a few prac-titioners on how LC–MS transitioned from a relatively obscure novelty to become the beneficial tool we know today (2). A recent article also exam-ined the ongoing need for develop-ment in MS ionization and related technologies to support work in areas where “electrospray just isn’t enough” to do the job (3). According to a survey among practitioners in various disci-plines, those identified with the chemi-cal materials industry arguably rely on

Figure 1: GC–MS response for an oil sample (upper is a methanolic extract; lower is the oil sample dissolved in methylene chloride). (Courtesy of Colin Moore, Chemtura Corporation.)

Figure 2: EI spectrum of peak at 13.82 min in the methanol extract shown in Figure 1. (Courtesy of Colin Moore, Chemtura Corporation.)


GC three times more than LC. A quick look around Moore’s laboratory sup-ports the observation.

One issue that all laboratories need to address is productivity. Two of Moore’s GC–MS systems, an Agilent 5970 (Agilent Technologies, Palo Alto, California) and a Waters GCT (Waters Corporation, Milford, Massachusetts), are fitted with two GC columns in the injection port using a two-hole fer-

rule. One column is connected to the mass spectrometer and the other to an ancillary detection system such as f lame ionization detection (FID). As Moore explained “ as soon as you tell somebody what an unknown peak is in a GC–MS trace, the next question is usually, how much [of it] is there? Col-lecting the FID data in parallel with the GC–MS data allows us to give our customers semiquantitative data as well

as MS identification in a single experi-ment.” Such a practice is analogous to LC–MS data acquisition with an in-line UV detector.

Moore’s future interests include installing a nitrogen–phosphorus detection (NPD) system as well as FID, on one GC system because many of the antioxidants that Chemtura makes are amines. Amines as a class are often easily ionized by ESI. Yet in some cases the analytes are just not amenable to the technique. Insufficient polar-ity, the inability to capture a proton, or perhaps excessive volatility that precludes transport and separation by condensed phase means in an LC could all contribute to ESI failure. Certainly due to its popularity great strides have occurred in recent years to increase the sensitivity, resolution, and overall utility of LC–ESI-MS instruments. Today techniques such as atmospheric pressure gas chromatography (APGC) and atmospheric solids analysis probes (ASAP), analogous to the vacuum sol-ids probes used for years in GC–MS, are viable without sacrificing perfor-mance, which was not the case years ago (3,4).

Data HandlingThe ability of software-driven appli-cations to amass increasingly refined data streams has unleashed a data handling problem that crosses into all practices and disciplines. So much so that handling complex data has become a recurring workshop topic at the Conference on Small Molecule Science (CoSMoS) in recent years (www.CoSMoScience.org).

Moore’s laboratory uses MassLynx mass spectrometry software (Waters Corporation) to process its MS data. For GC–MS data, workers export the files using the NetCDF converter option in ChemStation software in the three Agilent systems (Agilent Tech-nologies) and then convert the files to the MassLynx format using the Waters DBridge program. NetCDF does not produce a file for the FID system that can be read by MassLynx, and therefore processing the FID trace has to be done using ChemStation. On the GCT sys-tem the FID trace is recorded as analog data by MassLynx that can be processed

Figure 3: The complementary diagnostic nature of EI and ESI spectra of the same compound. (Courtesy of Colin Moore, Chemtura Corporation.)

Figure 4: Mass spectrum of the color body (inset) and its UV spectrum. (Courtesy of Colin Moore, Chemtura Corporation.)


with the MS data. The retention time differences between the two traces can be time aligned in the chromatogram plots (the peaks come out earlier in the MS data because the vacuum system of the MS increases the helium flow rate in the column connected to the mass spectrometer).

Understanding the Problem and Designing the ExperimentAs an analytical problem identifying color bodies in polymers is neither trivial nor obvious. In a 2005 paper (5), Moore shows there is often more than one way to solve a problem using mass spectrometry. Analysis by LC–MS with an inline photodiode-array (PDA) detector, is probably the best way to identify a new color body. Techniques like time-of-flight secondary ionization mass spectrometry (TOF SIMS) can be used to quickly confirm that the color body on fresh samples is the same as on previously determined samples.

Moore’s 2005 work detailed the analysis of a yellow discoloration on the surface of a compounded eth-ylene–propylene–diene monomer (EPDM) rubber sample. Surface discoloration of a polymer can result from various phenomena, including contamination, component migra-tion, oxidation, and other chemical reactions. Components rising to the surface can give rise to “bloom,” a process in which one component of a polymer mixture (usually not a poly-mer) undergoes phase separation and migration to an external surface of the mixture, according to the IUPAC definition. Protective waxes can be beneficial, but thiazoles (mercapto-benzothiazole) leading to discoloration of the product are undesirable. For example, oxidation of antioxidants can form color bodies (that is, phe-nolic antioxidants can form quinone methides) (6). The first step in iden-tifying a color body is to separate it from the polymer often by washing the surface with a suitable solvent.

The washings result in a complex mixture of the color bodies, additives, and other surface contaminants. Iden-tification of the colored components requires further separation of the mixture, analysis of the separated com-ponents, and the ability to ascertain which of the components are colored. The combination of LC–MS-MS with an inline PDA detector is able to

do the complete analysis in a single experiment. Moore recalls in 1994 when he joined Uniroyal Chemical, identifying a color body often involved pooling fractions from multiple LC runs to acquire enough material for the particle beam LC–MS system (an early 1990s rather short-lived tech-nique that, although not very sensitive, produced EI spectra from typical LC-amenable analytes not volatile enough for GC–MS). The much greater sen-sitivity of ESI sources and TOF mass spectrometers has made the process much quicker and easier, illustrating a central point in mixture analysis: the importance of matching the separation technique with the sensitivity of the final analysis technique.

Matching aspects of the analytical technique is not as simple as it sounds. When light reflects off a colored substance, the reflected light has the complementary color to the wavelength or wavelengths absorbed. Yellow light covers the wavelength range 570–585 nm, but the complementary color to yel-low is indigo over the range 420–430 nm. So when processing the LC–MS data, Moore looked for a component with strong absorbtion over that range of wavelengths. He found one compo-nent that yielded the UV and LC–MS spectra shown in Figure 4. Note that the color body is the neutral Cu(II) dibutyl dithiocarbamate, but for it to be detected by the LC–MS system it must be oxidized in the electrospray process to the positively charged Cu(III) com-pound. A quick review of spectral color properties can be found at www2.chem-istry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm.

Color bodies are often polar mol-ecules, which means that they are easy to ionize in an ESI source. For some time now, as the instruments capabilities improved, accurate mass MS has been recognized as an effi-cient means of enhancing separation of closely related chemical species. As Moore points out, when “the electro-spray spectrum contains few (if any) fragment ions identifying unknowns requires that either an LC–MS-MS spectrum is acquired and/or exact mass measurements [7] are performed to get the elemental formula of the

Figure 5: Deuterated (ND3) CI and EI spectrum of 4,4′-methylenedianiline. (Courtesy of Colin Moore, Chemtura Corporation.)

Figure 6: Potential alkyl group positions. (Courtesy of Colin Moore, Chemtura Corporation.)


pseudo-molecular ion.” An inline UV detector is sometimes an overlooked adjunct as well in LC–MS. Here the color of the offending samples of course indicates distinct chromophoric benefits. Components separated by the high performance liquid chroma-tography (HPLC) column absorb at the appropriate wavelength to give the observed discoloration with the added

advantage of well-characterized algo-rithms employed by PDA detectors to distinguish differences in homologous components on the samples that the unaided eye cannot.

Moore noted another frequently overlooked element in the analytical chemist’s trade that bears mentioning: Structure elucidation is made much easier if the analyst has a thorough knowledge of the sample chemistry and its history.

He also points out that “imag-ing mass spectrometry is a powerful technique for mapping the concentra-tion of a compound on the surface of a matrix.” The technique applies in many fields, including the analysis of inorganic materials, polymers, and biological materials. An early publica-tion discusses TOF-SIMS analysis in which a TOF system measures sec-ondary ions produced by bombarding a surface with high-energy particles (8). TOF-SIMS has been used to detect light stabilizers (9) and antioxi-

dants (10) on the surface of a polymer as well as to characterize the bulk polymer (11).

In the few years since Moore pub-lished his work, a number of techniques operating by various mechanisms on or near the surface of a material (as opposed to techniques requir-ing analytes of interest be in solution — desorption electrospray ionization [DESI], direct analysis in real time [DART], ASAP and a few others) have been examined in some detail in this column (4,12,13).

DESI can be used in combination with chemical reactions to improve the selectivity and sensitivity of the analysis. Moore’s studies with Keith Jennings and attending presentations by Graham Cooks and John Beynon that emphasized the utility of chemi-cal reactions in MS encouraged him to attempt using novel chemical ioniza-tion (CI) reagent gases to help solve problems. “Many antioxidants are alkylated aromatic amines and there-fore a paper by Buchanan [14] was of great interest”, he says. As shown by the spectra in Figure 5, the technique not only gives the number of aminic protons in the molecular ion, but it also helps identify fragment ions. The m/z 106 ion in the EI spectrum becomes m/z 108 in the CI data because of the NH2 group.

Moore has also updated a method first reported by Morgan and col-leagues (15) for analyzing zinc dial-kyldithiophosphate (ZDDP) in engine oils. Engine oils are complex mixtures of base oils and performance enhancing multifunctional additives, like ZDDP. They are excellent antiwear agents and effective oxidation and corrosion inhibitors (Figure 6).

The original work used negative ion CI to produce chloride ion adducts of the oil without any prior separa-tion. Moore has used an atmospheric pressure chemical ionization (APCI) source and a mobile phase contain-ing methylene chloride to give similar results.

Note that the mass spectrum in Fig-ure 7 yields two complementary pieces of information about the ZDDP sample and gives the molecular weight of any phenolic antioxidants in the

Figure 7: Chloride ion APCI spectrum of an engine oil sample dissolved in methylene chloride. (Courtesy of Colin Moore, Chemtura Corporation.)

Table I: Understanding the comparative diagnostic value of the ZDDP spectrum*

L– [ZnL2Cl]–

Totalm/z R1 or R3 R2 or R4 m/z R1, R2 R1, R4

241 4 4581 4, 4 4, 4 16

609 4, 4 4, 6 18

269 6 4637 4, 6 4, 6 20

665 4, 6 6, 6 22

297 6 6 693 6, 6 6, 6 24

*Courtesy of Colin Moore, Chemtura Corporation.

Figure 8: Generic structure for PAMAs. (Courtesy of Colin Moore, Chemtura Corporation.)


oil. The phenolic antioxidant present in the oil is evident by the response at m/z 389 (M–H ion) and at m/z 425 (M+Cl ion).

The chloride adduct pseudomolecular ion [ZnL2Cl]– permits calculating the total number of carbon atoms in the four alkyl groups (R1+R2+R3+R4). The ligand ions, L–, tell us if the ZDDP was prepared by blending individual ZDDPs or was produced using a mix-ture of alcohols. Table I illustrates how we can deduce that a mixture of C4 and C6 alcohols were used to prepare the ZDDP.

Though effective for determining carbon chain length of the R groups, the chloride adduction technique does not show whether the chains are linear or branched. However, collision-induced dissociation (CID)–ion mobility spec-trometry (IMS)–CID data may hold the key to solve that puzzle (16).

If the chromatographic conditions are not ideal for interfacing with MS or if other analytical techniques are going to be used to assist in the identification then LC fraction collection may be the best methodology. Polyalkylmethac-rylates (PAMAs) are used as viscosity modifiers in oils (Figure 8). Moore has used fraction collection from a GPC system (Figure 9), then pyrolysis GC–MS (Figure 10) and IR analysis to identify PAMAs. If R1 and R2 are likely either H or methyl and R3 is one of a mixture of alkanes, pyrolysis of this type of polymer gives two series of frag-ment ions: alkenes and alkyl methac-rylates. Thus if R3 is C12 then one gets dodec-1-ene and dodecyl methacrylate (Figure 11).

Future DevelopmentsMoore visited Graham Cooks at Purdue to try using a DESI source to detect the color body (17). Simply spraying the yellow polymer with acetonitrile indeed gave a small signal for the Cu dibutyl dithiocarbamate. Nevertheless, the signal was enhanced when the oxidizing agent I2 was added to the DESI spray solvent.

An extension of the thermal inves-tigations coming back into favor may in the not-too-distant future provide yet another chapter for these studies by combining thermal MS capabilities

Figure 11: Acquired EI spectrum(top) and best library match for the peak at 11.79 min found in the oil extract (Figure 10). (Courtesy of Colin Moore, Chemtura Corporation.)

Figure 10: Pyrolysis GC–MS at 550 °C of a PAMA standard (top) and the high mass component from the oil. (Courtesy of Colin Moore, Chemtura Corporation.)

Figure 9: GPC trace for oil sample. 99.5% of the sample yields an Mp of 564 and 0.5% having an Mp of 8630 (Mp, molecular weight, as reported by WatersGPC). (Courtesy of Colin Moore and John Mannello, Chemtura Corporation.)


with the surface information derived by atomic force microscopy (AFM) (Figure 12).

AFM uses a sharp-tipped probe, often only 2 μm long and less than 100 Å in diameter, located on the free end of a cantilever, which is brought close to a sample surface. Forces between the tip and the sample sur-face cause the cantilever to def lect. The def lection of the tip is measured as it is scanned or changes position relative to the sample generating a surface topographic map. Most com-monly tip def lection is a result of interatomic (van der Waals) forces. A good reference for those interested in the topic can be found at http://inv-see.asu.edu/nmodules/spmmod/.

Coupling AFM with MS has given rise to an interest termed “molecular cartography” by Gary Van Berkel (Oak Ridge National Laboratory, Oak Ridge, Tennessee). Their work was presented at the 2010 Conference on Small Molecule Science (Portland, Oregon) by Olga Ovchinnikova and can be downloaded at www.CoSMoScience.org (18).

Ovchinnikova points out currently available techniques usually “face a

trade-off between spatial resolution and chemical information.” Combining spa-tial resolution, using a heated scanning AFM probe to thermally desorb material from a surface, they then draw the sam-ple into either an ESI or APCI source adding chemical information from the sample surface. The authors refer to the technique as atmospheric-pressure hybrid proximal probe topography chemical imaging. The AFM tip plays a dual role being used for the thermal desorption creating ions for MS analysis while obtaining topographic images of that same surface. Initial results dem-onstrate the viability of this technique for automated chemical interrogation of caffeine thin films with ~250-nm spatial resolution in the thermal desorp-tion process. Lower resolution proximal probe thermal desorption chemical imaging results of different classes of compounds amenable to this technique including explosives, herbicides, phar-maceuticals, and dyes. The authors anticipate this analytical tool “will have broad application for determining the nanoscale spatial distribution of target molecules in plant and animal tissue and material junctions” (19).

Acknowledgments The wisdom readers benefit from in this column often is a distillation from many years of endeavor by people like Colin Moore, and as he recognizes “at the end of the day it’s people that solve problems and I’m very fortunate to work with a very talented group of people in the Analytical Services department at Chemtura.”

References(1) M.P. Balogh, LCGC North America 28(2),

122 (2010).(2) M.P. Balogh, LCGC North America 27(6),



368 (2007).(5) C. Moore and P. McKeown, J. Am. Soc.

Mass Spectrom. 16, 295–301 (2005).(6) J. Pospíšil, W.-D. Habicher, J. Pilar, S.

Nešpurek, J. Kuthan, G.-O. Piringer, and H. Zweifel, J. Polym. Degrad. Stab. 77, 531 (2002).

(7) M. Maizels and W.L. Budde, Anal. Chem. 73, 5436 (2001).

(8) M.L. Pacholski, and N. Winograd, Chem. Rev. 99, 2977 (1999).

Figure 12: Nanoscale physical and chemical imaging of plant growth regulators using proximal probe thermal desorption MS. (Courtesy of Olga Ovchinnikova and Gary Van Berkel, Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory.)


For more information on this topic, please visit

www.chromatographyonline.com/balogh

Michael P. Balogh “MS — The PracticalArt” Editor MichaelP. Balogh is principalscientist, MS technol-ogy development,at Waters Corp. (Mil-ford, Massachusetts); a former adjunct professor and visiting scientist at Roger Williams University (Bristol, Rhode Island); cofounder and current president of the Soci-ety for Small Molecule Science (CoSMoS) and a member of LCGC’s editorial advisory board.

Visit ChromAcademy on LCGC’s Homepagewww.chromacademy.com

(9) F. Andrawes, T. Valcarcel, G. Haacke, and J. Brinen, Anal. Chem. 70, 3762 (1998).

(10) M.J. Walzak, N.S. McIntyre, T. Prater, S. Kaberline, and B.A. Graham, Anal. Chem. 71, 1428 (1999).

(11) D. Briggs, I.W. Fletcher, S. Reichlmaier, L.J. Agulo-Sanchez, and R.D. Short, Surf. Inter-face Anal. 24, 419 (1996).

(12) M.P. Balogh, LCGC North America 24(1), 46 (2006).

(13) M.P. Balogh, LCGC North America 25(12), 1184 (2007).

(14) M.V. Buchanan, Anal. Chem. 54(3), 570–574 (1982).

(15) R.P. Morgan, C.A. Gilchrist, K.R. Jen-nings, and I.K. Gregor., Int. J. Mass Spec-trom. Ion Phys. 46, 309 (1983).

(16) C. Moore and A. Alexander, “The Iden-tification of Engine Oil Additives Using Chloride Ion Addition IMS-LCMS/MS,”

presented at the 58th ASMS Conference on Mass Spectrometry and Allied Topics, Salt Lake City, Utah, 2010.

(17) M. Nef liu, R.G. Cooks, and C. Moore, J. Am. Soc. Mass Spectrom. 17, 1091–1095 (2006).

(18) O.S. Ovchinnikova and G.J. Van Berkel, “Molecular Cartography: Moving Towards Combined Topographical and Chemical Imaging using AFM and Mass Spectrom-etry,” presented at CoSMoS 2010, Portland, Oregon, September 25, 2010.

(19) O.S. Ovchinnikova and G.J. Van Berkel, “Molecular Surface Sampling and Chemi-cal Imaging Using Proximal Probe Thermal Desorption/Secondary Ionization Mass Spectrometry,” https://external-portal.ornl.gov/doi/abs/10.1021/ac102766w. Publica-tion Date (Web): December 15, 2010 (Arti-cle) DOI: 10.1021/ac102766w.

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