" 75-_ m High Throughpu[ LC/MS Separations and Structural Information Achieved with Automated LC/MS Michael P. Balogh, Matthew J. Dilts Waters Chromatography Since the mid-1980s the LC interfaced with a mass spectrometer is found in an increasing number of applications. Waters Integrity system, using the patented Thermabeam particle beam interface, produces electron impact spectra, unlike soft ionization LC/MS techniques such as electrospray or thermospray. Integrity is used where positive compound identification is of primary importance.
Waters Integrity Design Goals Create an instrument: to enable positive compound identification applicable to a broad range of compounds b.that was easy for chromatographers to use and reduced the complexity of acquiring mass information _.with advanced data processing to acquire and process data from PDA and MS into accessible information Waters experience designing and building high quality HPLC systems logically extends to mass specific detection to add positive compound identification to LC separations. Such a detector would have to: - complement chromatographers existing practices - be easy-to-use - and include software that integrates PDA and MS data into accessible information. 2-
Particle Beam LC/MS Applications Fine Chemicals - degradants and synthetic impurities J Pharmaceutical Manufacture - degradants and synthetic impurities Natural Products - screens and confirmation for medicinal chemistry Organic Synthesis -including peptides and oligonulceotides This list represents a few of the current applications areas. Simply put, any compound exhibiting fused rings or double bond structures is a good candidate for El. The majority produce the molecular ion as well as significant fragment ions indicative of the structure of the compound. Methods development benefits from high throughput ability as well as having an abundance of samples to run. The number of runs needed in order to refine the conditions to chromatographically resolve compounds can be time consuming. Automating the permutations and using the PDA software to assess the quality of the separation is efficient. 3
Millennium Sample Information ---_, Intensity m/z -qnec_rum ooo,,oo_,oo i_oo_oooo _ooindex Report 8.00 i 3 2- Mass spectra for: - dehydroabietic acid (MW 300),ooois -!1!'" _'_ (MW 302) - abietic acid 67 157"u": I _ i i ;, 11.oo_I I [[ I.,o,, 2,33o2 0.00 1.00 2.00 100.00 200.00 300.00 Counts m/z Apex _ Lib Search Particle beam LC/MS works by desolvating the particle from the solvent stream before vaporization and ionization in the high vacuum chamber of the mass spectrometer. The result is electron impact produced spectra similar to GC/MS. Abietic acid produces the molecular ion in the bottom frame of this index plot at 302 m/z and the dehydro form produces a molecular ion at 300 m/z in the middle plot. The spectra are quite different even though the structures may appear similar. "t
Library Reference Pr- i Pr - i Me HO 2 C Me Ab_e ic acid H O acid Dehydmeb_c Serial: 44947 CAS Reg No: 514-10-3 Serial: 44597 CAS Reg No: 1740-19-8 MW: 302 Form: C20 H30 02 MW: 300 Form: C20 H28 02 1-I:)henanthmnecatt)oxyl=c ac_l I -Phenanthrenecart_ic acid 1.2.3.4.4a,4b.5,6,10,10a-clecahy_lro -1,4a-dirnethyl-7-(1-rnethylethyl) 1.2.3.4.4a.9.10,10a-octahydro -1,4e-dimethyl-7-(1--methytethyl) The Wiley library contains a number of phenanthrene based compounds based on the results. Many vary only slightly as seen here between the abietic acid and the dehydro form but produce significantly diverse spectra in view of the charge stabilization abilities of the two molecules. f b
D ;:;, Spectrum o_: Index Report oi J... =ii it i " / /I MSresuJtsforan _'_-- i _, ' S;! --.[,..,_,_1tL J I industrialsample. _J I Differentiatesspectra _-:. _ for each peak and _i,,-..._,.,,,. J illustrates resultof low _;',;i."\\\._ ---- ii _ temperatureacquisition (peak 9 minutes). A sample from an industrial process was separated by GC and LC. Only LC produced the fused peak at 9 minutes. Information can be deduced from the spectral differences leading to structural characterization In this case the appearance of the peak at 9 minutes ("tailing") could be due to co-elution or possibly the source temperature since the material is known to be thermally unstable.
L, Overlay 1 075 _ 0.70 _. 0 65 _, Report o.60 _ II I... o.55 _ -_ I 050 I.. Peak at 9 o,, _ ' ",... minutesaligns 0.40 _ I +_,m, o3 +' in PDA and MS -_ traces - +i \! o o2s : degradant was 020 _ not visibleby GC. o.,o? O.O5 /!'_' 0.00 "_. -"_'_ i _ I " _ "_ 000 5,00 0 00 15_.00 IVlin_t_= Pement (0.371nten)... o O MS (120AU)POA An overlay of the PDA and MS ion current traces clearly indicates the alignment. Information is derived directly from the mass spectrometer result, the PDA result and the comparison of the two. If there was a question about the possibility of co-elution on the peak at 9 minutes the PDA purity algorithm for the same peak is invaluable since it indicates the presence of a distinct second entity. Comparison of GC/MS and particle beam spectra matched well for the known components allowing some confidence in deducing the structure of the unknown.
Optimized Mass Spectrometry for Chromatographers Advantage of two independent in-line detectors Low dispersion efficiency without sacrificing automation _-Automation without sacrificing interactive control The Integrity design incorporates low dispersion efficiency at flow rates common to 2mm columns The system tubing routing and other considerations also provide complete sample analysis automation and post-run processing without inhibiting the freedom built into Millenium version 2.1 to develop methods from "scratch".
Waters Integrity System (System photograph) Integrity is designed for low dispersion chromatography and access to functions and controls without sacrificing the single point control of Millennium. Manufacturers have been able to afford some LC control in the past but Waters has developed a truly integrated system.
Step 3 Sample-to-Data Acquisition Order run steps - samples, standards, optimize and validate new methods LC Routines A Column D Sys.Suitability MSRoutines B EasySetup C EasyOptimize Step 4 Post-acquisition Processing - Extract data, integrate/quantitate - Export/report according to method Since this isa multispectralinstrument both LC and MS facilitiesare supportedto tune and validatean Integritysystemfor use. Jl
Step 1 Sample-to-Data LC Preparation - Solvents - Order samples in Autosampler tray Step 2 Specify: - Acquisition and Processing - Export/report formats for data A quick overview of the "Sample-to-Data" path begins as does as analyses with some LC preparation. Step 2 we will take a look at preparing to acquire data. Step 3 and 4 are the actual acquisition options within Millennium and an examination of the results.
Step 1 Sample-to-Data LC Preparation - Solvents - Order samples in Autosampler tray Step 2 Specify: - Acquisition and Processing - Export/report formats for data jz
Die Help TMD... Os.,_ Os..,d._ r_s.d_3 Os, d,..,.4 '_ Channel Name D_ MMle: i 1 I"MD T_ 1lll4_l'llk Iu" I"_" R'_'uH"-"Ii"._J The next three slides will illustrate elements of instrument control that we can build into a given method for the Integrity system. The sytem includes pump, injector and both detectors. Pump control including the optimize function for the mass spectrometer interface.
The mass spectrometer can be run at a number of scan rates and either scan or select ion mode for greater sensitivity. Functions such as commanding the LC stream to waste can also be specified as the default with this screen. i,/
The PDA functions such as scan range and acquisition rate use the same conventions. /
Millennium Method Set Incorporatesall instructionsyou create for use by the software. Ultimately any instructionsfor a given instrument condition including report formats, instructions to export data over a network after analysis, etc are compiled in a Method Set of instructions which the computer looks up for acquisition.
i. Sample-to-Data Step 3 Acquisition Order run steps - samples,standards, optimize and validate new methods LC Routines A Column D Sys. Suitability MS Routines B EasySetup C Easy Optimize Step 4 Post-acquisition Processing - Extractdata,integrate/quantitate - Export/reportaccordingto method j:t"
i The actual acquisition occurs through the QuickSet screen. A number of real time displays and condition monitoring capabilities are present here including: Spectra and wavelength for PDA and MS Interactive ability to change operating conditions on the fly A complete list of activites to be executed in the extended automated format of the Sample Table (Reference listed functions).
Step3 Sample-to-Data Acquisition Order run steps - samples, standards, optimize and validate new methods LC Routines A Column D Sys. Suitability MS Routines B Easy Setup C Easy Optimize Step 4 Post-acquisition Processing - Extractdata, integratelquantitata - Export/reportaccordingto method