2.2 Satisfacción turística
2.2.4 Satisfacción de la infraestructura
Introduction
As outlined previously, the matrix is a low molecular weight compound used in MALDI analysis for the protection of the sample, but also as a promoter of soft gas-phase transfer and ionisation of the sample. Ideally its absorption maximum is very close to the wavelength of the laser light. It enables soft ionisation of the sample by absorbing the majority of the energy from the laser, ionising and desorbing into the gas phase. The sample of interest will also desorb into the gas phase, and it is within this plume that matrix – sample interactions will ionise the sample. Generally ionisation is by protonation or deprotonation, however, some matrices work by electron transfer.
Unfortunately there is not a single matrix material that is suitable for every compound analysed with MALDI. For different analytes the interaction with a given matrix material may be different and structural features may afford a particular way of ion formation. Consequently, the features of the matrix and the analyte must complement each other. There are, however, some matrices whose usage is fairly prevalent. These include:
• 2,5-dihydroxybenzoic acid, DHB
• α-cyano-4-hydroxycinnamic acid, α-HCCA
• 3,5-dimethoxy-4-hydroxycinnamic acid, Sinapinic acid
• 9-nitroanthracene, 9-NA
Prior to the use of DCTB, the MALDI analysis of fullerenes and their derivatives was typically performed employing 9-NA as the matrix. DCTB was first used as a matrix for various fullerene derivatives in 19991, 2. DCTB led to more abundantly observed molecular ions in both positive-ion and negative-ion mode and the degree of fragmentation was markedly reduced.
DCTB
DCTB was first studied seriously in 2000, by comparing it to the traditional fullerene matrix 9- NA, and the universally used matrix, DHB3. Using the substituted fullerene: bis(4-methoxy-
phenyl)methano[60]fullerene, positive-ion and negative-ion mode spectra were obtained. The matrices and samples were mixed in 100/1 to 1000/1 molecular ratios. In negative-ion mode, clean spectra with a dominant molecular ion peak were obtained. In positive-ion mode, the molecular ion peak was also the most intense signal, however, there was also adduct formation of the type [M+H]+, [M+Na]+ and [M+K]+; and loss of a methyl group – [M- CH3]+.
When the other matrices were used, however, there was fragmentation of the sample and the most intense peaks in the spectra resulted from the matrices.
A more in depth study of suitable matrices for fullerenes and their derivatives appeared in 2001, with 14 different matrices, including DCTB being tested4. These matrices were:
• 9-NA
• DHB
• Sudan Orange G
• α-HCCA
• Sinapinic Acid
• 5-Methoxysalicylic Acid, MSA
• 2-(4-hydroxyphenylazo)benzoic Acid, HABA
• DCTB
• 6 related β-carboline alkaloids:
• Norharmane • Harmane • Harmine • Harmol • Harmalol • Harmaline
This study was aimed at finding a replacement matrix for the fullerene standard 9-NA, as this compound has the following drawbacks:
• It easily transfers oxygen to the analyte, which is problematic if one is not sure about the oxygen content of the analyte.
The matrices behaved differently depending on the polarity of the instrument. In positive-ion mode, only two of the matrices produced an intact molecular ion peak – these were DCTB and 9-NA.
In negative-ion mode, only 3 of the possible candidates did not produce an intact molecular ion – harmalol, MSA, and HABA. Of the remaining 11 matrices only 3 gave an abundant ion signal – DCTB, norharmane, and harmane. Of these 3, only DCTB produced a clean spectrum with very little fragmentation.
It appeared from this study that DCTB gave the best performance as a fullerene matrix for both ion modes.
From this and previous studies DCTB was identified as an electron transfer matrix, as there was no protonation/deprotonation of the sample and only the radical ions of the analyte were observed. This was confirmed by a study into the thermochemistry of DCTB5.
If DCTB does indeed ionise the samples via electron transfer, then the following reactivities can be expected:
In positive-ion mode:
∙+ ! → + !∙ This reaction will occur provided IEDCTB > IEA.
In negative-ion mode:
#∙+ ! → + !#∙ This reaction will occur provided EADCTB < EAA.
The vertical ionisation energy of DCTB was obtained by photo-electron spectroscopy as 8.54 ± 0.05 eV, which is in excellent agreement with the calculated value of 8.47 eV (AM1 calculations). The electron affinity has only been obtained so far by quantum chemical calculations to lie between 2.31 eV and 2.0 eV, depending on the method used.
Using these values, DCTB was used as a matrix for the following samples:
• Phenanthrene: IE = 7.9 eV
• Anthracene: EA = 530 ± 5 meV
• C60F46/48: IE = 12 eV, EA = 4.06 ± 0.25 eV
Using the calculated values for the IE and EA of DCTB, it is possible to predict the appearance of the spectra for the aforementioned analytes:
Phenanthrene:
IEphenanthrene < IEDCTB Molecular ions should be detected in positive-ion mode MALDI
Anthracene:
EAanthracene < EADCTB Molecular ions should not be detected in negative-ion mode MALDI
C60F46/48:
IEC60F46/48 > IEDCTB Molecular ions should not be detected in positive-ion mode MALDI
EAC60F46/48 > EADCTB Molecular ions should be detected in negative-ion mode MALDI
The actual experiments performed reflected the above predictions, as tabulated below:
Phenanthrene Positive LDI No M+
Positive MALDI Intense M+
Anthracene Negative LDI M-
Negative MALDI No M-
C60F46/48 Positive LDI No M
+
Positive MALDI No M+
Negative LDI M-, fragmentation Negative MALDI M-, low degree of fragmentation Table 5.1: Results of using DCTB as a matrix for various compounds
As a result it was shown that MALDI analysis with DCTB proceeds in fact within the boundaries of this thermodynamic frame provided by the respective ionisation energies and electron affinities.
Pencil Lead
Graphite has been used in various forms as a matrix for MALDI; however, it is typically very difficult to apply to the target plate as well as to achieve an even reproducible surface coverage. Graphite particles were combined in solution with more common matrix materials and such suspensions were used for MALDI analysis in an attempt to alleviate the application problems.
Graphite has been shown to be an effective matrix material for various analytes, such as peptides, proteins and polymers, so it is therefore desirable to find an easy way of using graphite as a MALDI matrix.
Pencil lead consists of graphite mixed with clay and varying levels of oils and waxes. It is produced in different levels of hardness and blackness - ranging from 9H, which is the very hardest and creates a very light grey mark upon paper, to 9B, which is very soft and will leave a very dark mark upon paper. Generally the softer the pencil, the more graphite it contains. There are many reasons why pencil lead would be a desirable matrix – its usage is safe, cheap and quick and easy to prepare – one would just have to draw on the target plate. Also, there are no solvent compatibility issues, and the hydrophobic surface tends to help in producing a sweet spot: as the sample solution is applied, it will dry in one place. Also, there are benefits from the additives in pencils – the wax appears to help with ensuring an even reproducible coverage of graphite on the surface.
Pencil lead was first used as a matrix in 2006, in the analysis of various samples including peptides such as substance P, polymers, actinide metals such as uranium, and terfanadine6. The results were impressive, in particular, as a matrix for Uranium. The laser power could be reduced from 100% for LDI, to 50% with pencil lead, and a better signal-to-noise ratio was achieved. There was also complete matrix suppression, so that no unwanted matrix signals had to be considered.
In cases where the matrix was not suppressed, it was found that the matrix produced carbon cluster peaks in the low mass range, ranging from C9 to C24, which could then be used for
calibration.
Interestingly enough, when used as a matrix for Substance P, fragment ions were produced which were adducted by sodium and potassium. This demonstrated that the pencil lead had partially protected the sample and aided the ion formation, since if the fragments had been formed as a direct result of laser irradiation, they would be lacking these adducts.
For this study a range of pencil grades were tested, ranging from 4H to 6B. It was found that 6B gave the best performance and this was assumed to be caused by it having the highest graphite content of all the pencils tested. Moreover, the softness of the pencil, which led to easier application and a more even coverage, appeared to offer a better reproducibility. A follow up study tested a wider range of pencils on a selection of 50 small molecules, which included peptides, polymers, steroids and sugars. This included such molecules as α- cyclodextrin, atropine, caffeine, cocaine, disopyramide, and haloperidol. This study led to
slightly differing results. Here, the 2B pencil was found to give the best results for the range of samples tested7.
Of the 50 molecules tested, pencil lead did not work as a matrix for six of them. This study also found that the 6B pencil gave only poor ionisation, possibly as a result of too much graphite being deposited on the surface.
Of course the differences could also have arisen from the different brands of pencil used, as the additives differ both in what is added and the amounts used. However, in both studies a wide range of pencil brands were employed as matrices and were tested on all the different samples.
Pencil lead was considered in further detail as a matrix for polymers in a study looking specifically at silyl hydride functionalised polystyrenes, which are particularly sensitive to the preparation methods commonly used in MALDI for polymers8.
The common method for MALDI analysis of polymers is to use dithranol as the matrix, with silver trifluoroacetate (AgTFA) as an additive. If this method is used for the derivatised polystyrenes, then the silane moiety is oxidised.
Mass spectra of non-functionalised polystyrene were obtained using a 6B pencil as a matrix, with and without AgTFA as an additive. The sample was successfully ionised and sufficiently protected. With the AgTFA additive present, the peaks were the most intense of all the spectra obtained, with a good signal-to-noise ratio, with the silver adducted peaks the most intense of all. Without the AgTFA additive, the most intense peaks present were sodiated. It was found that using AgTFA and pencil lead as the matrix, better spectra were obtained than for the typical method of dithranol/AgTFA.
To apply this to the silyl hydride functionalised polystyrene, spectra were obtained and compared using as a matrix either a 6B pencil, or dithranol, with the additives lithium trifluoroacetate (LiTFA) and AgTFA, respectively.
It was important to find conditions under which the integrity of the end groups could be preserved, as they easily underwent side reactions using dithranol/AgTFA.
It was found that using pencil lead and LiTFA, there were no side reactions, and the only adducts formed were lithium adducts.
In summary, pencil lead is looking promising as a new matrix with a broad range of applications. In the present investigation, several fullerene derivatives were tested with the use of pencil lead in order to test the suitability of it as a matrix material for this class of compounds. Comparison was made with DCTB as the benchmark matrix and samples were chosen which would allow easy cationisation by the pencil lead matrix.
Experimental
The fullerene derivatives studied in this chapter are below. (Note the abbreviations underneath the structures, which were used throughout to characterise the sample)
Figure 5.1: Structure of “Isopropyl Mono”
Figure 5.2: Structure of “Mono”
Figure 5.3: Structure of “Bis”
Figure 5.4: Structure of “Tris”
O O O O CH3 C H3 CH3 C H3 O O O O CH3 CH3 O O O O CH3 CH3 O O O O C H3 C H3 O O O O CH3 CH3 O O O O C H3 C H3 O O O O CH3 CH3
These compounds were isomerically pure. Since these compounds are already several years old they showed slight signs of ox
present investigation.
Some of the open cage fullerenes from chapter 3 were also studied. Here the following isomers were of interest:
Figure 5.5: Structures of 2.1 (above left) and 2.2 (above right)
Figure 5.6: Structures of 2.3 (above left) and 2.4 (above right)
These compounds were isomerically pure. Since these compounds are already several years old they showed slight signs of oxidation. This, however, was of no relevance to the
Some of the open cage fullerenes from chapter 3 were also studied. Here the following isomers were of interest:
Figure 5.5: Structures of 2.1 (above left) and 2.2 (above right)
Figure 5.6: Structures of 2.3 (above left) and 2.4 (above right)
These compounds were isomerically pure. Since these compounds are already several idation. This, however, was of no relevance to the
Some of the open cage fullerenes from chapter 3 were also studied. Here the following
Figure 5.5: Structures of 2.1 (above left) and 2.2 (above right)
Figure 5.7: Structures of 2.5 (above left) and 2.6 (above right)
A typical sample preparation method involved dissolving the sample in toluene at a 1 mg/ml concentration. A Whirlimixer w
DCTB was prepared in a 10 mg/ml solution, which was added to the sample as required, typically in a 1:50 sample:DCTB molar ratio.
6B and 8B Staedtler branded Mars Lumograph pencils were used throughout and were applied individually on the target spots. A Grafix branded HB pencil
pressure equivalent to writing was used to transfer pencil lead to the target plate, except in the experiments where the pressure used to apply was harder or lighter, in an
increase and decrease the amount of pencil lead on the target plate. The entire slide was held in an air stream to remove any loose pencil lead prior to sample application.
The sample solutions were applied one drop at a time using a 1 allowed to dry between applications. Typically 5
spot.
For the DCTB experiments, the DCTB solution and sample were mixed and applied together and typically 5 µl of this solution was used per target spot.
Figure 5.7: Structures of 2.5 (above left) and 2.6 (above right)
A typical sample preparation method involved dissolving the sample in toluene at a 1 mg/ml concentration. A Whirlimixer was used to ensure complete dissolution.
DCTB was prepared in a 10 mg/ml solution, which was added to the sample as required, typically in a 1:50 sample:DCTB molar ratio.
6B and 8B Staedtler branded Mars Lumograph pencils were used throughout and were ed individually on the target spots. A Grafix branded HB pencil
pressure equivalent to writing was used to transfer pencil lead to the target plate, except in the experiments where the pressure used to apply was harder or lighter, in an
increase and decrease the amount of pencil lead on the target plate. The entire slide was held in an air stream to remove any loose pencil lead prior to sample application.
The sample solutions were applied one drop at a time using a 1-10 µl Bar
allowed to dry between applications. Typically 5µl of the solution was applied to each target
For the DCTB experiments, the DCTB solution and sample were mixed and applied together l of this solution was used per target spot.
Figure 5.7: Structures of 2.5 (above left) and 2.6 (above right)
A typical sample preparation method involved dissolving the sample in toluene at a 1 mg/ml
DCTB was prepared in a 10 mg/ml solution, which was added to the sample as required,
6B and 8B Staedtler branded Mars Lumograph pencils were used throughout and were ed individually on the target spots. A Grafix branded HB pencil was also tested. A pressure equivalent to writing was used to transfer pencil lead to the target plate, except in the experiments where the pressure used to apply was harder or lighter, in an attempt to increase and decrease the amount of pencil lead on the target plate. The entire slide was held in an air stream to remove any loose pencil lead prior to sample application.
l Barky µltipipette and l of the solution was applied to each target
Results
Pencil as a matrix
As an initial step, test experiments were performed to evaluate the viability of pencil as a matrix for derivatised fullerenes. Experiments were performed in both ion modes and in comparison with direct LDI and DCTB-MALDI. The methano-bridged [60]fullerenes: isopropyl mono, mono, bis and tris (all displayed in the introduction to this chapter) were chosen as the test compounds, as their behaviour under LDI and MALDI conditions has been described in detail earlier4.
Isopropyl Mono
Isopropyl mono was chosen as an ideal representation of a fullerene derivative with which to test the use of pencil as a matrix, as it severely dissociates under direct LDI conditions, but delivers a molecular ion almost free of decomposition when protected under true MALDI conditions. Comparing standard DCTB-MALDI and LDI spectra of isopropyl mono, below, shows how important a matrix is in order to achieve a molecular ion peak:
It was noted that the laser power had to be markedly enhanced for LDI compared to MALDI, in order to obtain a molecular ion peak, however, this also results in extensive fragmentation. By comparing actual intensity values it is obvious how few ions are produced with the LDI method, there is also a smaller signal-to-noise ratio when compared to DCTB- MALDI.
Initial positive-ion mode spectra of isopropyl mono on 6B and 8B pencil are below. The fragmentation peaks at m/z 720 (C60) and 733 (C61H) are immediately obvious. Also, there is
not an obvious peak at m/z 906, but peaks are present at m/z 929 and 945, which indicate addition of sodium and potassium.
Figure 5.9: Spectra of isopropyl mono on 6B and 8B pencil
Varying the application of the pencil
The matrix-to-analyte ratio is of crucial influence to the outcome of a MALDI experiment. Therefore, it was decided to test how different applications of the pencil might affect its ability to behave as a matrix. For the majority of experiments, the pencil was applied at a normal writing pressure. For the present experiments the pencil was either lightly scraped over or heavily pressed across the surface of the target plate, leaving less or more pencil lead attached to the target plate, respectively, the excess material was blown from the target