AND MASS SPECTROMETRY 5
11. MONOLITHIC LAYERS FOR THIN-LAYER CHROMATOGRAPHY e MASS SPECTROMETRYSPECTROMETRY
11.2 TWO-DIMENSIONAL THIN-LAYER CHROMATOGRAPHY SEPARATION AND MASS SPECTROMETRIC DETECTION OF BIOMOLECULESMASS SPECTROMETRIC DETECTION OF BIOMOLECULES
identity of the labeled proteins. The fourth spot with anm/z¼34841.579, which had the smallestRf, was assigned to a myoglobin dimer, which is a known impurity in the commercial protein. Although the MS signal of this peak was weak because of the low concentration of the dimer in the sample, it contained two fluorescamine labels that enhanced the fluorescence and enabled visualization of its spot. The repeatability ofRfwas again excellent with an RSD of less than 2.4%.
An additional benefit of the chemistry of these thin layers lies in their ability to get hyper- crosslinked. The FriedeleCrafts alkylation reaction catalyzed by ferric chloride is known to form a large number of mesopores and leads to a significant increase in the surface area. Hypercrosslinking of the monoliths containing chloromethylstyrene units was introduced for monolithic capillary columns only recently (Maya and Svec, 2014; Skerı´kova´ and Urban, 2013; Urban et al., 2010a,b;
Urban and Skerı´kova´, 2014) and so has not been used previously for thin layers. In contrast to the original plate, a hypercrosslinked layer was able to separate even small molecules such as dyes (Lv et al., 2013).
11.2 TWO-DIMENSIONAL THIN-LAYER CHROMATOGRAPHY SEPARATION AND
Photografting was used for creation of the ion exchange channel. To do so, the pores of the entire superhydrophobic layer were filled with a mixture of 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-hydroxyethyl methacrylate, and benzophenone dissolved intert-butanolewater. UV-initiated pho- tografting was then carried out through a simple home-made mask to produce a 0.6 mm wide channel across one side of the plate.Fig. 5.16Ashows the optical microscopy image of the cross section of the patterned hydrophilic virtual channel filled with an aqueous solution of red dye. The aqueous phase is retained within the three dimensional channel by surface tension at the interface, which prevents it from entering the adjacent superhydrophobic areas of the monolithic thin layer.
The prepared 2D plates were then used for the separation of fluorescamine-labeled peptides:
leucine enkephalin, bradykinin, angiotensin II, and valetyreval. An aqueous solution of the mixture of UV-labeled peptides was spotted close to the beginning of the grafted hydrophilic channel. The separation in the first dimension was then carried out using a mobile phase consisting of 30 vol%
acetonitrile in 0.2 mol/L aqueous ammonium acetate at pH 7. To avoid the mobile phase running over the plate instead of through the channel, a paper tissue wick saturated with the mobile phase was placed at the sampling end of the ion exchange channel. The hydrophilic channel then pulled the aqueous mobile phase from the wick through the channel. For the separation in the second dimension orthogonal to the development in the first dimension, a standard chamber containing 0.1 vol%
FIGURE 5.16
Optical microscopic image of cross section of the superhydrophilic channel filled with aqueous solution of red dye (A) and schematic illustration of desorption electrospray ionization scanning of surface of poly(butyl acrylate-co- ethylene dimethacrylate) monolithic layer to visualize the 2D separation (B).
trifluoroacetic acid in 40 vol% aqueous acetonitrile was used. After completion of the 2D separation, the plate was air-dried and peptide spots were detected by illumination with UV light as shown in Fig. 5.17. Interestingly, valetyreval separated in two spots. Mass spectral analysis of the commercial tripeptide revealed that this tripeptide contained molecules with the expected molecular mass of 380 and a dimer with a mass of 760, thus explaining the origin of the two spots.
The unidirectional scanning described in the literature (Kertesz and Van Berkel, 2008) was then used for MS detection of nonlabeled peptides. Each selected lane on the surface of the plate was scanned in the same direction. The plate was placed on an insulated sample holder mounted on ax/y/z stage.Fig. 5.16Bshows that the first lane was scanned by moving the plate in a direction parallel to the x-axis, which represents the direction of the separation in the first dimension, i.e., in the grafted channel, from low to highRf. At the end of the first lane, the surface was moved back to the beginning of the lane. Then the surface was moved parallel to they-axis and scanning of the next lane was carried out. The y-axis represents the direction of the separation in second dimension. This process was repeated until the entire plate was scanned.
Fig. 5.18 presents results of the separation of nonlabeled peptides in the first dimension. Their positions along the virtual channel were constructed from extracted ion profiles using DESI-MS. The separation profile also indicated a certain overlap of spots of the separated peptides and the mass spectra of the individual spots were not very clean.
FIGURE 5.17
Two-dimensional Thin-layer chromatograohy separation of a mixture of labeled peptides including leucine enkephalin, bradykinin, angiotensin II, and valetyreval on monolithic polymer layer with dual chemistry detected using UV detection.
FIGURE 5.18
Desorption electrospray ionization (DESI) mass spectrometry (MS) scan of the first dimension separation of peptides leucine enkephalin, bradykinin, angiotensin II, and valetyreval achieved in the 30 mm long virtual channel grafted with the 2-acrylamido-2-methyl-1-propanesulfonic acide2-hydroxyethyl methacrylate mixture (top spectrum) and DESI-MS spectra of individual peptides.
Fig. 5.19then shows mass spectra corresponding to individual peptides found during the scanning of the plate after 2D separation. The spectra are significantly cleaner, thus demonstrating that the resolution improved after separation in the second dimension. Also, the order of the monitored pep- tides changed to leucine enkephalin, valetyreval, angiotensin II, and bradykinin, and it was different from that observed in the first dimension.
Although in the previous study photografting was used to create the virtual channel, this technique can also be used for the preparation of plates with a gradient of hydrophobicity designed for 2D separations. The parent monolithic porous layer supported at a glass plate was prepared from glycidyl methacrylate and ethylene dimethacrylate and then hydrolyzed using dilute sulfuric acid to produce a poly(glycerol methacrylate-co-ethylene dimethacrylate) thin layer. Although this reaction increased the hydrophilicity of the layer, it remained insufficient to completely avoid adsorption of some more hydrophobic compounds. Therefore, all the pores were first photografted with poly(ethylene glycol) methacrylate, thus producing a surface that exhibited a water contact angle close to 0 degrees, rendering the entire layer superhydrophilic. This hydrophilized monolithic layer was then wetted with a solution of benzophenone in lauryl methacrylateeethanol mixture. The wetted layer was covered with the quartz plate, and the top of this plate with a mask nontransparent to the UV light. Creation of the diagonal gradient was very simple. The mask was attached with a string to a syringe pump set to a constant speed and moved diagonally across the mold over the course of 5 min. The ideal situation is modeled inFig. 5.20A. The actual gradient was confirmed by measuring the water contact angle at different surface locations. The bottom left corner was the most hydrophilic part, with a contact angle of 0 degrees, whereas the most hydrophobic part at the top right corner exhibited a contact angle of 135 degrees. The gradient profile was also visualized in UV light after labeling the plate with 1-anilinonaphthalene-8-sulfonic acid solution (Fig. 5.20B).
The positive effect of TLC with the gradient of lauryl methacrylate is demonstrated with separa- tions shown inFig. 5.21using four different plates. Clearly, the initial poly(glycidyl methacrylate-co- ethylene dimethacrylate) layer did not produce any good 2D separation (Fig. 5.21A). All peptides are retained close to the sampling corner and their spots after 2D development were smeared. Similarly, a plate with all epoxide groups hydrolyzed to reduce interactions with peptides did not provide a good result. The peptides migrated quickly through the plate without being adequately separated, and the spot shapes were poor (Fig. 5.21B). A slightly better separation was achieved using a monolithic TLC plate with a homogeneously grafted layer of poly(lauryl methacrylate).Fig. 5.21C shows that the peptides are separated with leucine enkephaline and oxytocin producing the smallest and the largest retardation factor,Rf. However, valetyreval and glyetyr coeluted and their spot was located between the other two peptides.
Fig. 5.21Dthen represents the best separation using a plate with diagonally grafted hydrophobicity.
The peptide mixture was spotted at the most hydrophilic part of the plate and the first dimension separation was carried out using acidic aqueous acetonitrile. This separation was very fast and accomplished in less than 1 min. At that time, the front of the mobile phase almost reached the end of the layer. This separation afforded three distinct spots of leucine enkephalin, coeluted valetyreval with glyetyr, and oxytocin. This result was almost identical with that achieved with the homoge- neously grafted hydrophobic plate. However, after turning the plate perpendicularly and developing it in the second dimension with acidic aqueous methanol, valetyreval and glyetyr were clearly separated from each other with valetyreval having the largerRfvalue.
FIGURE 5.19
Desorption electrospray ionization (DESI) mass spectrometry (MS) spectra of leucine enkephalin, bradykinin, angiotensin II, and valetyreval observed during scan of the entire plate after two dimensional separation using monolithic polymer layer with dual chemistry.
The reason for the better separations on the diagonally grafted layer was considered to be the following. On the plate grafted in the “linear” manner, the peptides come into contact with the same hydrophobicity in both the first and the second dimensions. Therefore, all the spots are lined up along the diagonal. In contrast, the diagonally grafted layer provides a gradient of hydrophobicity during development in both dimensions and enables fine tuning of the separation. The oxytocin separated into two spots. The mass spectral analysis of the commercial nonapeptide indicated that it contained molecules with the expected molecular mass and that of a dimer, thus explaining the origin of the two spots. It is worth noting that the separation in the second dimension was completed in less than 3 min.
The feasibility of mass spectrometric detection was then demonstrated with leucine enkephalin and oxytocin. These two unlabeled larger peptides were mixed in equivalent quantities, and their sepa- ration carried out using conditions applied to the labeled peptides. After the separation in both di- mensions was completed, the monolith was sprayed with the matrix solution and dried.Fig. 5.22shows the mass spectra of leucine enkephalin and oxytocin, after the separation in the first and second di- mensions, respectively. The signal-to-noise ratio is good even after separation in the first dimension, with no signal of the other peptide observed. However, the spectra of both peptides obtained after the 2D separation are cleaner because impurities typically contained in man-made peptides are separated away from the major spot. This “cleanup” advantage of the 2D TLC separations is similar to that observed above and presented inFigs. 5.18 and 5.19.
FIGURE 5.20
Artistic rendition of the gradient of hydrophobicity at a monolithic thin-layer chromatography plate increasing in the direction of thearrowtogether with suggested directions of the separations in first (1st D) and second dimension (2nd D) (A), and visualization of the gradient of hydrophobicity using fluorescent labeling with 1- anilinonaphthalene-8-sulfonic acid (B). The bright area at the left down corner represents the most hydrophilic part and the dark at top right most hydrophobic part.