Use of Graphite Plate for Homogeneous Sample Preparation Bull. Korean Chem. Soc. 2000, Vol. 21, No. 4 401
Use of Graphite Plate for Homogeneous Sample Preparation in Matrix/Surface-assisted Laser Desorption and Ionization
of Polypropyleneglycol and Polystyrene
JunghwanKim and WeekyungKang*
Department ofChemistry, Soongsil University,Seoul156-743, Korea Received November6, 1999
Matrix/Surface-assisted laser desorption/ionization (M/SALDI) mass spectrometry of polypropylene glycol andpolystyrene, directly deposited ongraphite plate, is demonstrated. Graphite plate is effective both as anen ergytransfermedium and robustsampling support for LDI ofpolymers.Massspectra ofpolymers can be easily obtaineddue to homogeneousdistribution on graphite surface and their ion signals are long-lived bylargeef fectivedesorption volumeenoughto investigate M/SALDI process.
Introduction
Matrix-assistedlaser desorption/ionization (MALDI) time- of-flight(TOF) mass spectrometry is a relatively new analyt ical method1,2 thathas a demonstratedusefulnessinthe anal
ysis ofpolymers.3,4 Compared with conventional methods such as GPC and vapor phase osmometry, it has theadvan tage of highmass accuracy,wide mass range, and rapiddata acquisition.However, despitethe great potentialof MALDI, its capability is limitedby difficultiesin homogeneoussam ple preparation in solid state.5 Most solid matrices suffer from inhomogeneous sampling due to inconsistencies in crystal formation, leadingtolowsignalreproducibility, and rapid destruction, leading to temporal signal degradation.
Forthese reasons, the fast evaporation method6-7 and appli cation of liquid matix8 etc. have been suggested, but these methods stillrequirescrupulous care and show low shot-to- shot stability. In other approaches, studies on various sub strate have beenperformed with etched metal plates,9 self assembledmonolayer10 and hydrophobicmaterials11,12 such as paraffin wax film and poly(tetrafluoroethylene) plate as thesamplesupportstoimprovesampleuniformity.
Graphite plate is a good candidate for sample support.
Graphite itself can be used as a matrix dueto its black-body absorption, andits large and effective surface. Evaporation by smearing sample solution into graphite can prevent the heterogeneous concentration in round rim of sample spot caused bythespreadof solution on the metal surface.Appli cation ofgraphitein MALDIhasbeen triedin powder form mixed with absorbing liquid matrix or with non-absorbing liquid matrix.13,14Thesemixtureswerecapableofproducing high-quality mass spectra ofpeptides, proteins,oligosaccar- ides and synthetic polymers, evenwhenthe liquid matrices had no appreciableabsorptionatthedesorption wavelength (337 nm).However,theuseof a mixtureofgraphitepowder and liquid matrix hasthedisadvantageof being highlysensi tive to the sizeand the mixing ratio ofgraphite particulate and samplemanupulation is not easy. In addition, a mixture ofgraphite powderand liquid matrixcan beused onlyon the horizontalsampleprobe because of its fluidity.
This report describes an easy and simple sample prepara
tion method using graphite plate and application to analysis of polypropyleneglycol (PPG) and poylstyrene (PS), which arethe prototypes ofGPC standards for synthetic polymer.
Though graphiteplate had been usedtoinvestigate the sub strate effect in the laser desorption process of small mole- cules,15 the application as MALDI sample support has not beenreported toour knowledge.
Experiment지 Section
Instrumentation. A home-built0.65 mlinear TOF mass spectrometer was used, a full description of which can be found elsewhere.16 The typical background pressure in the vacuumchamberduringoperationwas 1 x 10-7 Torr, using a 4"-diffisionpump (VHS-4, Varrian) for a sourceregionand a turbo molecular pump (V 250, Varrian) fora drift tube.
Thesample platewas mounted on therepellingplate, which was normally maintained at 15 kV (4.5 kV for PPG2000), and 25 mm fromthe gridless and grounded extraction plate with a 15 mm hole in diameter. A circular (25 mm diameter) dual micro-channel plate detector (Galileo Electro-Optics) wasused todetect ions. The ion signalswere digitized using a LeCroy 9354AL(500MHz)digitaloscilloscope and spec tra weretransferredtoanIBMcompatible PC for masscali bration and data processing. The laser source was a forth harmonics of a Nd:YAGlaser system (Minilite II, Contin uum), with a pulse width of 5 ns and a maximum output energy of5 mJ. The laser beam, atanangleof 45o, was irra
diated on thesampleplate or wasfocused by a quartzlens of 25 cm focallength. The power of the laserwas attenuated using aneutral densityfilter.
Sample preparation. Graphite plate (2 mm thickness) for lubrication and machining purposes was used without any treatment unlessotherwisenoted. Intheexperiments on dependence of sodium salt content, graphite plates were washed in de-ionized water for 1 hour, usinga sonicatorto remove the aqueous-solublemetalsaltscontained and dried in afurnace at 500 oC. All chemicalswere used with no fur
ther purification as purchased from AldrichCo. A PPG solu
402 Bull. Korean Chem. Soc. 2000, Vol. 21, No. 4 Junghwan Kim and Weekyung Kang tion (1 mg/mL in CH3CN) mixed with the NaCl aqueous
solutionwasdeposited on a spotofdithranol (10 mg/mL in THF)onthestainlesssteelplateordirectlyon graphiteplate.
A few卩L of Polystyrene (PS) solution (1 mg/mL in THF) mixedwith a CF3COOAgsolution(10 mg/mL in THF)and a dithranolsolution with equi-volumeratioweredroppedon the stainless plate andgraphiteplate.
ResultsandDiscussion
Figure 1 shows the optical microscope images of sample spots of thePPG2000with thedithranoland NaCl deposited onthe stainless steel plate andgraphite plate. Onthe stain less steelplate,the best sample spotwasobtainedwithdiffi cultyafterseveraltrials,and it appearedhomogeneous to the nakedeyes.However,Figure 1(a)of the opticalmicroscopic view indicates that a number of grains about100pm in size were formedby local aggregationof an analyte,matrix and salts. On the contrary, the rim around spot as well as the large grain samples are not seen on the graphite plate as shownin Figure 1(b). Theparticulatessmaller than 10 pm in the microscope image represent the morphology of the graphite surface,not theirregulardistribution of sample.
Figure2 shows theMALDI-TOFmass spectra of PPG2000 obtained at an extractionvoltageof 4.5 kV. For a MALDI spectrum of PPG2000on thestainless steel plate, it was nec essaryto find “good spots” orto avoid “bad spots”on the
Figure 1. Optical microscope images of sample spots of PPG 2000 deposited on (a) stainless steel plate and (b) graphite plate with dithranol in THF and NaCl in water by layering method.
w c n
은.
<
M
은
，
으
u 으
500 1000 1500 2000 2500 3000 3500
Mass (m/z)
Figure 2. MALDI mass spectrum of PPG 2000. Samples were prepared to same deposition method as explained in Figure 1.
Spectra were averaged of 100 laser shots in sampling rate of 100 M/s of the digital oscilloscope. Symbols, + and x represent 58n+81 and 58n+97 mass series of the fragment ions, respectively.
substrate. This resulted in lowering the spot-to-spot repro ducibility. When the graphite plate was used as a sample support,theionsignalsweremoreintenseandshowed a bet tersignal-to-noiseratio than those of thestainlesssteelplate.
More than anything else, the spectrum was more easily obtainedinanyplace of sample spotregardlessof the irradi
ationposition ofthe laser. The mass series of dominant ion signal observed is 58n+41 (n =20-50), which is consistent with that of the sodiated PPG molecule, HO-(-CH(CH3)- CH2O-)n-H Na+.The calculatednumberaveragedmolecular weight is 2010 ± 10 for both spectra, which is within the errorrange.Thenearlyequalmassresolution, 350 of thetwo spectra, indicates that theroughness of the sample surfaceis not enough toaffectthemassresolution. The graphiteplates used in this studyare for machining andlubrication, not of chemical grade, so they have a large number of various impurities. These impurities of thelow molecularweightare highly congested with hydrogenated carbon cluster peaks fragmented from the graphite surface in the mass region lowerthan 300 amu, which didnot affect the spectraofthe polymer inthis study. The ion signals shown in the mass region of500-1200 amu are interpreted as ions fragmented during ionizationor desorption. They consist of two mass series; oneisthe 58n+ 81 (n= 10-20,marked with +) series and the other is the 58n+97 (n= 10-20, marked with x) series, which arewellconsistentwith the resultsof Donovan under delayed extraction mode.17 The signals of fragments aremoreintense than thoseinDonovan'sresultat 337 nm of laser light, and it seems to be causedby the higherphoton energy of266nmusedas a lightsourceinthepresentstudy.
Figure 3 showsthemassspectraofPPG2000withoutthe matrix, dithranol. These spectraindicate that graphiteeffec tively acts as a photon-absorbingmaterial. Even when only thePPG2000isadsorbedonthegraphite plate untreated,the spectrum with goodresolution is acquired as shown inFig ure 3(b) though the ion signals are less intense compared with Figure 3(a), and its ion peaks correspond to sodiated propyleneglycololigomers. The attatchedsodiumionsseem
Use of Graphite Plate for Homogeneous Sample Preparation Bull. Korean Chem. Soc. 2000, Vol. 21, No. 4 403
(a) PPG 2000:NaCI=1:30
( 으드
】 . 은<
0
은
(DC一
U 으
(d) PPG2000:NaCI=1:0.03 (c) PPG2000 only
17.7 MW/cm2
I
卩
¥**队#*부뿌빼(b) PPG 2000 only
untreated graphite
desalted graphite untreated graphite
desalted graphite
desalted graphite
艸새伸忡忡새』
1000
丽 」 血 e
2000 3000 Mass (m/z)
(
up완
은
<
0
으
」으 으
u으
Figure 4. SALDI mass spectrum of PPG 5000 on graphite plate according to variation of laser fluence. Symbols, +, x and O represent 58n+81, 58n+97 and 58n+65/67 mass series of the fragment ions, respectively.
Figure 3. SALDI mass spectrum of PPG 2000 deposited on graphite plate according to variation of amount of sodium salt at laser fluence of 1.65 MW/cm2 except (b) (17.7 MW/cm2). Spectra were acquired at sampling rate of 500 M/s of the digital oscilloscope.
嚣 55
tohaveoriginatedfromimpurities. To investigatethecation izationeffect of sodium salt, the graphite plates weredesalted indeionized water.Theionsignal was hardly observed when onlythePPG2000withoutsodiumsaltwasdepositedonthe desalted graphite plate as shown in Figure 3(c). As the amount of sodium salt increases, the higher quality mass spectrum is obtained.However, too much sodium saltgives a broadening ofthe ion peak and fragmentation with lower molecularion intensity.
Thisgraphitesurface-assistedionizationeffectwasnamed SALDI(surface-assisted laser desorption/ionization) in the analysis of peptide andproteinbySunneretal.,13 who used 10-150 pm graphite powder. According to Sunner, when using the dry graphite powder without aliquid matrix, the ionintensity was much lowerand very short-lived, andthe mass resolution was poor, thus both a liquid matrix and graphite powder were essential to obtain the SALDI spec
trum. Careful attentionisrequiredintheuse ofpowder,and themixing solution must bemechanically shakento ensure thorough dispersion of the graphite powder in a liquid matrix.14 Onthe contrary, using a graphite plate as photon absorbing material and substrate ensures the robust sam pling. When onlythe graphite platewithout a liquid matrix was usedforSADLIin thisstudy,theionsignalexistedlong enough to investigate the desorption and fragmentation dynamics of PPG under delayed-extraction mode.16 This shows that the plate form has more available effective vol
umetodesorbtheanalyte than thepowder form.
It wasreportedpreviously that itwaspossible to obtain the molecular ionsignals for only lowmolecular weight poly
mers (< 2000 amu.)by direct desorption, where no matrix was utilized.18,19 Figure 4 shows the SALDI spectrum of
PPG 5000 without a matrixobservedatanextractionvoltage of 15 kV with the variation oflaserpulse fluence. A well- resolved massspectrum ofPPG 5000, of which the resolu
tion is ca.600, is obtained at a moderate laserpulse fluence (0.61 MW/cm2), but further increase in the laser fluence results in the loss of mass spectral resolution with a new mass series,58n+ 65 or 67 of fragmentedion.17 This can be attributed tothebroaddistributionof desorption velocity and theinitiation of anotherPPGdegradationchannelatelevated excess energy. A detailed study is required to interpret the powerdependencyof fragmentationof PPG.
Figure 5 is the MALDI spectra of a polystyrene standard (PS2500) at a laser fluence of 8.74 MW/cm2, using the
으( 一으
一
은
<
0SU
으 드
(b) Stainless steel plate
1st 20 shot' 2nd 20 shot' 3rd 20 shot
1000 2000 3000 4000 1000 2000 3000 4000 1000 2000 3000 4000
Mass (m/z)
Figure 5. MALDI mass spectra of polystyrene standard (PS 2500) deposited on (a) the treated graphite plate and (b) stainless steel plate at laser fluence of 8.74 MW/cm2 with dithranol and silver trifluoroactate as matrix and cationization agent, respectively.
404 Bull. Korean Chem. Soc. 2000, Vol. 21, No. 4
treated graphiteplate and stainless steelplate. The oligomer distribution isrepresented by (M+Ag)+ ions. The ion peaks are separated by 104 Da, which is consistent with the -[CH2CH(C6H5)]- repeating unitand the mass of end cap ping groupwascalculated to be 58, indicating -C(CH3)3 and
-H. Polystyrene, which is the prototype of nonpolar syn thetic polymer, poses difficulty for the hard sampling on metal surfacedueto its hydrophobicity.20The MALDI spec trum of PS requires the higher energy fluence compared with PPG, and the high irradiatedpowerof the laser deplete thesampleon the suface or destroythe surfacecomposition.
The ion signals of PS on the stainless steel plate rapidly decrease with an increase in irradiation time on a sample spot and disappear within a few tenshots ofthe laser. How
ever, onthe graphiteplate,the ion signalspersistlongerand show higher spot-to-spot reproducibility. This seems tobe the effects ofthe penetratable surface ofgraphite and the homogeneous sampling on thegraphiteplate.
In summary, graphiteplatewasshown tobeeffective both asanenergy transfer medium and sample support for simple samplepreparation. Homogeneous samplingonthegraphite plate provides for easy acquisition of mass spectrum and generates a long-lived ion signal by large effective desorp
tionvolume. A persistention signal is needed to investigate the M/SALDI process which is not well understood. How
ever, thepossibiltiesofthegeneral application of thegraph ite calls for morestudies ofthevariousmolecules with high molecularweight and thewavelengthdependence.
Acknowledgment. This work wassupported by theKorean Ministryof Education through theresearchFund(1998-015- D00140).
References
Junghwan Kim and We이cyung Kang Chem. 1991, 63, 1193A.
2. Beavis, R. C. Org. Mass Spectrom. 1992, 27, 653.
3. Rashidzadeh, H.; Guo, B. Anal. Chem. 1998, 70, 131.
4. Burkoth, A. K.; Anseth, K. S. Macromolecules 1999, 32, 1438.
5. Allwood, D. A.; Perera, I. K.; Perkins, J.; Dyer, P. E.; Old- ershaw, G. Appl. Surf. Sci. 1996, 103, 231.
6. Vorm, O.; Roepstorff, P; Mann, M. Anal. Chem. 1994, 66, 3281.
7. Kim, S. H.; Shin, C. M.; Yoo, J. S. Rapid Commun. Mass Spectrom. 1998, 12, 701.
8. Williams, J. B.; Guser A. I.; Hercules, D. M. Macro-mole
cules 1996, 29, 8144.
9. Ens, W.; Mao, Y; Mayer, F.; Standing, K. G. Rapid Com
mun. Spectrom. 1991, 5, 117.
10. Brockman, A. H.; Dodd, B. S.; Orlando, R. Anal. Chem.
1997, 69, 4716.
11. Hung, K. C.; Ding, H.; Guo, B. Anal. Chem. 1999, 71, 518.
12. Hung, K. C.; Rashidzadeh, H.; Wang, Y.; Guo, B. Anal.
Chem. 1998, 70, 3088.
13. Sunner, J.; Dratz, E.; Chen, Y.-C. Anal. Chem. 1995, 67, 4335.
14. Dale, M. J.; Knochenmuss, R.; Zenobi, R. Anal. Chem.
1996, 68, 3321.
15. Zhan, Q.; Wright, S. J.; Zenobi, R. J. Am. Mass Spectrom.
1997, 8, 525.
16. Kim, J. H.; Kang, W. K.; Desorption dynamics andfrag- mentation of desorbed molecule/ion from graphite surface underpulsed-delayed extraction, in preparation.
17. Mowat, I. A.; Donovan, R. J.; Maier, R. J. Rapid Com- mun. Mass Spectrom. 1996, 68, 3321.
18. Holm, R.; Karas, M.; Vogt, H. Anal. Chem. 1987, 59, 373.
19. Mattern, D. E.; Hercules, D. M. Anal. Chem. 1985, 57, 2041.
20. Lim, H.; Lee, Y; Han, S.; Yoo, Y; Kim, K.-J. Bull.
Korean Chem. Soc. 1999, 20, 853.
1. Karas, M.; Hillenkamp, F.; Beavis, R. C.; Chait, B. T. Anal.
