Dried-droplet method, preparation

The solid-sample preparation is usually achieved by the deposition on a metallic surface of the solution of matrix and analyte with a concentration suitable to obtain the desired analyte/matrix ratio. The solution is left to dry under different conditions (simply at atmospheric pressure, reduced pressure, or under a nitrogen stream). This method is usually called the Dried Droplet Method. In all cases, what is observed is the formation of an inhomogeneous solid sample, due to the different crystallization rate of the matrix and analyte. Consequently, the 10 molar ratio is only a theoretical datum In the solid sample, different ratios will be found in different positions and the only way to overcome this is to average a high number of spectra corresponding to laser irradiation of different points. 

The dried droplet method is the method originally introduced by Hillenkamp and Karas (Fig. 4.7). A saturated matrix solution, 5-10 g depending on the solubility of the matrix, is prepared in water, water-acetonitrile, or water-alcohol mixtures. In a second vessel, the sample is diluted to about 100 mg L Mn a solvent that is miscible with the matrix solution. The matrix and sample solutions are then mixed such that the final molar ratio is 10,000 1 with a final volume of a few tiL. 

For sample preparation, the lignin water solution samples were dissolved in acetone (4 mg/ml, 50/50 vol%) and mixed with an acetone solution (10 mg/ml in acetone) of the matrix. As the matrix 2,5-dihydroxy benzoic acid was used. For the enhancement of ion formation, NaCl was added to the matrix (10 mg/ml in water). The solutions of the sample and the matrix were mixed in the proportion 3 parts matrix solution -I- 3 parts sample solution -I-1 part NaCl solution, and 0.5 to 1 j1 of the resulting solution mix were placed on the MALDI target. After evaporation of the solvent the MALDI target was introduced into the spectrometer. The dry droplet sample preparation method was used. 

Electrospray sample deposition (ESDEP) is a sample preparation method where matrix and analyte solutions are sprayed on the target surface under the influence of a high-voltage electric field [44,45]. ESDEP is reported to yield much better shot-to-shot and spot-to-spot reproducibility than the dried-droplet method. The improved results are ascribed to the small and evenly sized crystals that are formed, and as a consequence, improved homogeneity of the MALDI sample surface. Hanton et al. [45] analyzed PEG1450, a narrowly distributed poly(ethylene oxide) sample, using an uncommon... 

This does not mean that dried-droplet preparations cannot be imaged. Indeed, clearly dependent on the chemical structure, certain compounds do remain in the tissue (e.g., the (prenol) lipid ion at 610.444). For specific analyte molecules such as these latter compounds, the dried-droplet method can thus be used as a specific sample preparation method to wash away unwanted compounds. In this respect, it is interesting to note that the corpora cardiaca are the main synthesis sites of the insect juvenile hormone (JH), also a prenol lipid of the class of terpenoids. JH as such is not secreted in the adult stage of the insect imaged here, but the presence of other terpenoids in this tissue is expected. 

FIGURE 9.9 Water solutions of P-amyloid (42-1), bovine insulin, and amixture of the two small proteins were prepared and 50 pmol applied to the glass slide using the dried droplet method and 2,5-DHB as matrix. Heat (ca. 225 °C) was applied to the copper transfer tube with the ion source set at 150 °C. Mass spectra of (A.l) P-amyloid (42-1), (B.l) hovine insulin, and (C.l) of the mixture. The DriftScope tj v,s. miz representations are displayed in (A.2) to (C.2) showing clean separation of the two peptides in (C.2). 

Figure 6 Segregation of a poly(butylene oxide) 1000 (PBO, in MeOH) in a CCA matrix sample spot prepared by the dried droplet method (left) [PBGJaNa ion at m/z732.2g moL" (right) [PBGJuNa ion at m/z1164.5g mol". Image spot size 100 pm. Reprinted from Weidner, S. M. Falkenhagen, J. Rapid Commun. Mass Spectrom. 2009, 23 (5), 653-660, with permission of John Wiley and Sons.

Since one of the issues raised in this paper is whether the objects seen in the TEM images are a proper representation of the structures present in solution, we will describe briefly sample preparation strategies. For solutions of micelles in hexane, a very volatile solvent, samples for TEM studies could be obtained by aspirating a dilute solution directly onto a carbon-coated copper grid. Most of the solvent likely evaporated as the sample was deposited on the substrate. Alternatively, the TEM substrate could be dipped briefly into a dilute solution of the micelles and allowed to dry. This method also worked for less volatile solvents like decane. For decane, we could also place a small drop (a few pi) of solution on the grid and then touch the edge of the droplet with a Kimwipe to remove excess solvent. For several samples these methods were compared, and we observed the same morphology. 

A number of different sample preparation methods have been described in the literature [37,38], A collection of these protocols is accessible on the Internet [39,40], The original method that is always the most widely used has been called dried-droplet. This method consists of mixing some saturated matrix solution (5-10 pi) with a smaller volume (1-2 pi) of an analyte solution. Then, a droplet (0.5-2 pi) of the resulting mixture is placed on the MALDI probe, which usually consists of a metal plate with a regular array of sites for sample application. The droplet is dried at room temperature and when the liquid has completely evaporated to form crystals, the sample may be loaded into the mass spectrometer. 

There are two common matrix preparation methods, thin-layer and dried droplet preparation. For thin-layer preparations the matrix is tqiplied to the MALDl target plate in a volatile solvent, such as acetone. The solvent spreads and evaporates... 

The most common method for preparing a MALDI target, known as the dried droplet technique, is to mix the sample and matrix, usually in a ratio of about 1 5000 in lpl of solvent, and allow the mixture to dry on the target. Mixing can occur either before addition to the target or, more commonly, solutions of the sample and matrix are added to the target independently and allowed to mix. Most... 

For the MALDI-MS analysis of intact proteins, FI CCA (alpha-cyano-4-hydroxycinnamic acid), SA (sinapinic acid) or DHB (2,5-dihydroxybenzoic acid) matrices and the dried-droplet deposition method for sample preparation are typically used [13, 14] (Table 3.1). Depending on the properties of the protein, it is often necessary to test a series of solvents and matrices to optimize the outcome of the MALDI-MS experiment. Peptides and small proteins below molecular weight 20000 Da are often amenable to analysis using HCCA matrix and reflector TOF-MS mode, whereas larger proteins may produce better results with SA or DHB matrix in the linear TOF-MS mode. Hydrophobic proteins can be analyzed using the HCCA matrix dissolved in high concentrations of formic acid (up to 30%) [15]. When using cirmamic acid matrices, SA and HCCA, and the... 

This approach resulted in bacteria being evenly distributed across the deposited sample (Caution the spray method is not suitable for clinical samples ). Subsequent MALDI-MS analyses of these homogenous sample/matrix preparations yielded highly reproducible mass spectra, regardless of the spatial coordinates of the laser shot on the sample. When compared to the manuaFpipette dried-droplet... 

Similar materials could be obtained by an emulsification method [253]. Nematic liquid crystal is emulsified into an aqueous dispersion of a water-insoluble polymer colloid (i.e., latex paint). An emulsion is formed which contains a droplet with a diameter of a few microns. This paint emulsion is then coated onto a conductive substrate and allowed to dry. The polymer film forms around the nematic droplets. To prepare an electrooptical cell a second electrode is laminated to the PDLC film [253]. In the phase separation and solvent-casting methods the chloroform solutions of liquid crystal and polymer are also used [254, 255]. The solution is mixed with the glass spheres of the required diameter to maintain the desired gap thickness and pipetted onto a hot (140 °C) ITO-coated glass substrate [255]. After the chloroform has completely evaporated another ITO-coated glass cover is pressed onto the mixture and then it is cooled down. Structural characteristics of the PDLC films are controlled by the type of liquid crystal and polymer used, the concentration of solution, the casting solvent, the rate of solvent evaporation, perparation temperature, etc. [254]. 

The most common sample-preparation method is the dried-droplet technique in which a droplet of the matrix containing the analyte is deposited on the metal plate and then dried. Typically, the ratio of analyte to matrix is 1 10 to 1 10 Analyte concentrations arc usually in the micromolar range. 

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