Reversed peptide separation

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

The application of polymer monoliths in 2D separations, however, is very attractive in that polymer-based packing materials can provide a high performance, chemically stable stationary phase, and better recovery of biological molecules, namely proteins and peptides, even in comparison with C18 phases on silica particles with wide mesopores (Tanaka et al., 1990). Microchip fabrication for 2D HPLC has been disclosed in a recent patent, based on polymer monoliths (Corso et al., 2003). This separation system consists of stacked separation blocks, namely, the first block for ion exchange (strong cation exchange) and the second block for reversed-phase separation. This layered separation chip device also contains an electrospray interface microfabricated on chip (a polymer monolith/... 

Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled peptides from a (strept)avidin affinity column. The use of 14C isotopes instead of deuterium labels permits precise reverse phase separations prior to mass spec that show no elution peak time differences between isotope-labeled and normal atom-labeled peptides.

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

The ability to prepare monoliths within a mold of any shape was used by Lee et al. [128] who prepared monolithic ST-DVB microbeads within pulled fused silica needles and used them for the reversed-phase separation and on-line electrospray ionization mass spectrometry (ESI-MS) detection of proteins and peptides. As illustrated by Fig. 18, these monolithic microcolumns separated proteins far better than capillaries packed with commercial C18 silica or polymeric beads. 

D. J. Throckmorton, T.J. Sheppodd and A.K. Singh, Electrochromatography in microchips Reversed-phase separation of peptides and amino acids using photopattemed rigid polymer monoliths. Anal. Chem. 74 (2002) 784—789. 

In addition to classical reverse phase separation of peptides on octadecyl derivatized silica monoliths, sugars and peptides as well as proteins and nucleosides have been analyzed on a 20-cm-long silica-based poly(acrylic acid) column (ID. 200 pm), employing HILIC and weak cation-exchange chromatography, respectively [194]. Furthermore, HILIC fractionation of polysaccharides delivered remarkable and promising results [84,194]. 

The Nature of the In k versus [S]m Dependencies and Their Impact on Peptide Separation in Reversed-Phase Chromatography... 

COI.ITMNST7F. Analytical reversed phase columns for proteins and peptides range from 50 to 250 mm in length having 4 to 5mm i.d. Columns with 2mm i.d. have also been employed for peptide separations in microsequencing applications. The use of microbore columns with less than 2 mm i.d. is severely hampered by instrumental constraints due to small extra-column dead volumes and difficulties in obtaining accurate... 

These capillary columns possessing positively charged surface functionalities were used for the reversed-phase separations of basic and acidic peptides. Figure 6.17 shows the separation of three angiotensins and insulin with plate numbers as high as... 

Bizanek R, Manes JD, Fujinari E, Chemiluminescent nitrogen detection as a new technique for purity assessment of synthetic peptides separated by reversed-phase HPLC, Peptide. Res., 9(l) 40 44, 1996. 

Figure 9.22 Separation of hydrolysis products of the action of tadpole collagenase on DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Argby reversed-phase HPLC. Upper tracing shows the gradient employed. The reaction was initiated at zero time by the addition of 4 /xg of enzyme. At the indicated times, a 4.0 /xL aliquot erf the reaction mixture was injected onto the HPLC column and the peptides separated. (From Gray and Saneii, 1982.)...

An inherent drawback of this methodology is that quantitative information is only obtained from those peptides which are subjected to MS/MS analysis. This fact stresses the necessity for efficient peptide separation via multidimensional chromatography (e.g., SCX/reversed-phase HPLC) prior to tandem MS analysis. Moreover, ion trap instruments cannot be employed in quantitative iTRAQ experiments since their peptide fragmentation spectra do not contain information... 

Shibue, M., Mant, C.T., and Hodges, R.S. Effect of anionic ion-pairing reagent hydro-phobicity on selectivity of peptide separations by reversed-phase liquid chromatography. J. Chromatogr. A. 2005, 1080, 68-75. 

Studies with larger peptides, e.g., polypeptide hormones, have indicated that the resolving power of reversed-phase separations may decrease as the peptide chain length increases. These results stress the importance of using elution conditions which generate adequate relative... 

---