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Application of Solvent-free Sample Preparation Methods for
MALDI-TOFMS to Organometallic and Coordination Compounds
Laura Hughes, Mark F. Wyatt, Bridget K. Stein and A. Gareth Brenton
EPSRC National Mass Spectrometry Service Centre (NMSSC), Swansea University, Swansea, SA2 8PP, U.K.
Results – VIIIacac3 Complex with DCTB Matrix
Samples and matrices were mixed in molar ratios of 1:50, 1:75, 1:100,
1:200, 1:500 and 1:1000.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOFMS) is a very important and successful analytical technique for a
wide variety of samples. Generally, sample preparation involves placing a
droplet of mixed matrix/sample solution onto a sample target, and then allowing
the solvent to evaporate. This technique relies on sample and matrix
combinations that are soluble in the same low boiling point solvent or miscible
solvents. However, a significant number of samples submitted to the NMSSC
are insoluble or only soluble in high boiling point solvents, e.g. DMSO.
Additionally, some samples are unstable in solution. The development of
solvent-free preparation methods is clearly beneficial to the NMSSC and the
wider scientific community. Previous work in this area has focussed mainly on
synthetic polymers,1-3 but has also been applied to biochemical samples.4,5
1. Pressed disc: Sample/matrix mixture was pressed into a disc utilising a
die and press apparatus normally used to press KBr discs for IR
measurements. 10 tonnes of pressure were applied. Discs were fixed onto
the sample plate with adhesive tape.
2. Pressed disc with KBr: KBr was added to the mixtures in molar ratios of
2:1, 1:1, 0.5:1, 0.25:1 and 0.1:1, with respect to the matrix.
3. Suspension in non-solvent: Sample/matrix mixture was suspended in 0.5
mL of water by using a vortex mixer. 1 μL was then placed onto the sample
plate via pipette, and the solvent allowed to evaporate.
4. Dabbing: For pestle and mortar preparations, an aliquot of the mixture
was transferred to the sample plate and lightly flattened with a microspatula.
For ball milled samples, either the above would occur, or the mixture-coated
ball was extracted from the vial with tweezers, and tapped on the plate to
transfer mixture, which was then lightly flattened with a microspatula.
5. Double-sided adhesive tape: Tape was fixed to the plate and the
sample/matrix mixture was spread onto it with a microspatula. The plate
was then brought upright and tapped on the bench to remove excess mixture.
The following solvent-free mixing methods were studied:
1. Pestle and mortar.1
2. Steel ball milling.6
MetIIIacac3
(Met = V, Cr) complexes, were
2 (Met = Fe, Zn) and
purchased from Sigma-Aldrich (U.K.), have been analysed previously.7
•DCTB and 7,7,8,8-tetracyanoquinodimethane (TCNQ) charge-transfer
matrices were purchased from Fluka (U.K.).
•HPLC-grade water purchased from Fisher (U.K.).
•MALDI-TOFMS measurements obtained with a Voyager DE-STR instrument
(Applied Biosystems, U.S.A.).
•Whirlimixer vortex mixer (Fisher, U.K.) was used for milling and creating
suspensions.
•3 mm steel balls were purchased from Wheelies Cycles (Swansea, U.K.).
•Discs were pressed using a C-30 Press (Research and Industrial Instruments
Company, London, U.K.).
•Potassium bromide (KBr) was purchased from Sigma-Aldrich (U.K.).
Intensity (%)
•MetIIacac
VIIIacac3
100
80
60
40
20
0
150
The following methods of sample deposition were also studied:
1. Pressed disc.1
2. Pressed disc with KBr.1
3. Suspension in non-solvent.
4. Dabbing.
5. Double-sided adhesive tape.
Materials and Instrumentation
100
80
60
40
20
0
346.0
100
80
60
40
20
0
346.0
(a)
349.0
347.2
348.4
348.2
349.6
100
80
60
40
20
0
150
100
80
60
40
20
0
150
100
80
60
40
20
0
150
3397.3
(a) Solution prep.
370
348.4
260
250.2
350.3
347.2
348.4
349.6
370
348.2
387.2
370
348.4
480
590
349.1
347.2
3397.3 100
80
60
40
20
0
0
346.0
352.0
348.4
349.6
348.4
0
352.0
350.8
(b)
9.1E+3
349.4
347.2
2.7E+4 100
80
60
40
20
0
0
352.0
346.0
348.4
349.6
348.4
480
590
0
700
2.7E+4
(c) Pressed KBr
disc prep.
500.5
480
590
0
700
1.3E+4
370
480
Mass (m/z)
590
•Proposed structure for solid9 was proven by crystallography.
•Radical molecular ion was not observed with solution preparation (a), but
observed peaks could be fragments, including apparent ½M+˙.
•The Woollins group proposed that the sample was degrading in solution.
•Results for solvent-free preparation method (b) were similar to those observed
with solution preparation.
1.3E+4
(d)
349.4
347.2
348.4
349.6
350.8
0
352.0
Case Study 1 – Insoluble Compound
•Insoluble ferrocene compound8 observed as extremely low intensity radical
molecular ion, with fragments or impurities also observed.
100
90
80
70
60
50
40
30
20
10
0
1000.0
1251.3
HgOAc
AcOHg
AcOHg
Fe
HgOAc
HgOAc
HgOAc
HgOAc
AcOHg
AcOHg
1600.2
2200.4
100
Theoretical
80
Pattern M+˙
60 (1E+4 FWHM)
40
20
0
2756.0
2762.8
2769.6
M
849.9
851.9
852.9
847.9
847
850
853
Theoretical
Pattern ½M+˙ 100
(1E+4 FWHM)
854.9
0
856
859
4944.1
Mass (m/z)
<<STA85WOO(ASMS)_0001>> Voyager Spec #1=>AdvBC(64,0.5,0.1)=>NF0.7[BP = 851.9, 4944]
849.9 851.8
Observed Data 4944.1
853.9
847.9
847
855.8
850
1220
853
0
859
856
1374.8
1480
0
2000
1.8E+4
1740
Fe
Fe
EtO
P
EtO
S
P
Hg
S
S
S
S
S
Hg
S
P
S
OEt
P
Fe
OEt
Fe
1376.8
1220
1480
1740
0
2000
Mass (m/z)
Conclusions and Future Work
•Several mixing and deposition methods were investigated; ‘ball mill and dab’
(BMD) method considered best overall, for ease of application and quality of
results.
•Single steel ball was adequate, and DCTB was easier to handle than TCNQ.
•Case study 1: method was applied to completely insoluble mercury-ferrocene
compound. Extremely low intensity radical molecular ion was observed.
•Case study 2: method was applied to compounds with poor solution stability.
Radical molecular ion was not observed, and results were similar to those
obtained in solution.
•Further work required to refine method in order to improve mixture
homogeneity, data quality and reproducibility.
References
2800.6
3400.8
2771.8
2776.8
2779.8
2783.8
2776.4
2783.2
( / )
0
4001.0
100
0
2790.0
Voyager Spec #1=>AdvBC(32,0.5,0.1)=>NF0.7[BP = 616.4, 8027]
100
Observed Data
80
2766.0
60
40
20
0
2756.0
2762.8
2769.6
851.8
100
90 (a) 849.9 100
50
80
0
70
844
60
50
853.9 100
40
50
30
0
844
20
855.8
10
0
700
960
852.0
100
850.0
90 (b)
80
70
60
50
848.0
40
30
20
856.0
10
0
700
960
HgOAc
2512.9
2254.9 2774.9
2769.9
0
700
Case Study 2 – Solvent-degradable Compound
0
352.0
350.8
•The ball milling method of Hanton and Parees uses 2 balls, but 1 appears to
be sufficient.
•Comparable data was acquired for each method of solids mixing, regardless
of the method of application onto the samples plate. However, ball milling
is quicker, cleaner and less labour intensive.
•The pressed discs were very fragile, making handling difficult.
•KBr was added to the mixtures in order to improve the structural integrity
of the pressed discs. Additionally, promoting the formation of [M+K]+
species was sought, and these species were observed.
•DCTB considered better matrix, due to TCNQ discs being more fragile and
milled TCNQ mixtures adhering to both ball and vial.
•Radical molecular ions were not observed by the suspension and adhesive
tape methods.
•Cleanest, highest resolution data obtained with the dabbing method.
•Similar trends were noted in the data obtained for the other acac complexes.
•The method used affected mass accuracy.
0
700
9.1E+3
(d) Dabbing with
ball prep.
250.3
260
350.8
100
Observations
371.4
371.2
260
(c)
100
80
60
40
20
0
346.0
Theoretical
Pattern
(1E+4 FWHM)
Mass (m/z)
(b) Pressed disc
prep.
249.5
235.4
350.8
349.2
348.1
Complex with DCTB Matrix
348.0
260
for
348.0
Experimental - Deposition of Mixture onto Plate
Results –
Current Investigation
Comparison of the
species
acquired
VIII(acac)3.
Intensity (%)
Introduction
1. Pestle and mortar: Portions of sample and matrix were ground until the
mixture was judged by eye to be homogeneous, which was approximately 3
minutes.
2. Steel ball milling: Portions of sample and matrix were added to a plastic,
cone-shaped, sample vial. A 3 mm steel ball was added and the mixture
ground by agitating the vial with a vortex mixer until judged by eye to be
homogeneous, which was approximately 1 minute.
M+˙
Intensity (%)
Experimental – Solids Mixing
•Recent research has focussed on the use of MALDI-TOFMS to characterize
organometallic/coordination/highly conjugated compounds.
•2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB)
matrix is very effective for these compounds, with radical ions observed.
•Solvent-free preparation is required for such compounds that are insoluble,
soluble in non-volatile solvents, or unstable in solution.
•First-row transition metal acetylacetonate (Met(II)acac2 and Met(III)acac3)
complexes were used to develop a working method.
•Several mixing and deposition methods were investigated; the ‘ball mill and
dab’ (BMD) method was considered best overall.
•Case study 1: BMD method was applied to completely insoluble mercuryferrocene compound. Radical molecular ion was observed.
•Case study 2: BMD method was applied to compounds with poor solution
stability. Radical molecular ion was not observed.
Intensity (%)
Overview
197.4
2774.9
2777.0
2776.4
Mass (m/z)
2783.2
0
2790.0
1. Skelton, R.; Dubois, F.; Zenobi, R. Anal. Chem. 2000, 72, 1707-1710.
2. Marie, A.; Fournier, F.; Tabet, J. C. Anal. Chem. 2000, 72, 5106-5114.
3. Trimpin, S.; Rouhanipour, A.; Az, R.; Räder, H. J.; Müllen, K. Rapid
Commun. Mass Spectrom. 2001, 15, 1364-1373.
4. Wang, M. Z.; Fitzgerald, M. C. Anal. Chem. 2001, 73, 625-631.
5. Trimpin, S.; Deinzer, M. L. J. Am. Soc. Mass Spectrom. 2005, 16, 542-547.
6. Hanton, S. D.; Parees, D. M. Proceedings of the 52nd ASMS Conference,
Nashville TN, 2004.
7. Wyatt, M. F.; Havard, S.; Stein, B. K.; Brenton, A. G. Proceedings of the
52nd ASMS Conference, Nashville TN, 2004.
8. Sample submitted to the NMSSC by M. Parry and I. R. Butler, University of
Wales Bangor, U.K.
9. Sample submitted to the NMSSC by I. P. Gray and J. D. Woollins, University
of St. Andrews, U.K.