RP-columns

Standardisation of RP columns is an issue of debate. RPLC stationary phases were reviewed and compared and the ideal properties were defined [543]. For the development of RP packing materials, see Dolan [544]. 

In a sense each monolithic column is unique, or produced as a product of a separate batch, because the columns are prepared one by one by a process including monolith formation, column fabrication, and chemical modification. Reproducibility of Chro-molith columns has been examined, and found to be similar to particle-packed-silica-based columns of different batches (Kele and Guiochon, 2002). Surface coverage of a Chromolith reversed-phase (RP) column appears to be nearly maximum, but greater silanol effects were found for basic compounds and ionized amines in buffered and nonbuffered mobile phases than advanced particle-packed columns prepared from high purity silica (McCalley, 2002). Small differences were observed between monolithic silica columns derived from TMOS and those from silane mixtures for planarity in solute structure as well as polar interactions (Kobayashi et al., 2004). 

NaCl or KC1 (Peng et al., 2003 Ballif et al., 2004 Beausoleil et al., 2004 Wilmarth et al., 2004 DeSouza et al., 2005 Vitali et al., 2005) may be used for the SCX fractionation, in spite of the incompatibility of these salts with mass spectrometers. When using KC1, for example, the sample must be desalted off-line (Ballif et al., 2004 Beausoleil et al., 2004), on the RP column before MS/MS acquisition (DeSouza et al., 2005 Vitali et al., 2005), with a vented column (Peng et al., 2003), or with a RP-trap (Vollmer et al., 2004 Wilmarth et al., 2004). The configuration with a RP-trap is shown in Fig. 11.1, and in this case, a flow splitter is used to reduce the flow rate from hundreds of microliters per minute to hundreds of nanoliters per minute. However, HPLC pumps of lower flow rate are now available and could eliminate the need for a flow splitter. 

Fig. 11.1. In addition, one area that has yet to be characterized is the use of sequential RP columns with large dead volumes. It seems unlikely that peptides eluting from the RP trap would reconcentrate on the RP column directly in front of the mass spectrometer diminishing the value of the second RP column. This needs further analytical investigation.

FIGURE 13.1 Schematic of a comprehensive 2DLC (IEX/RP) configuration with alternating second-dimension analytical RP column sampling of first-dimension eluent. A salt diversion valve is present to divert salts from the IEX dimension to waste, and prevent contamination of downstream collected fractions or mass analyzer. 

FIGURE 13.3 Raw and deconvoluted mass spectra of a yeast ribosomal protein (L16) from a 2DLC(SCX/RP)/MS experiment were obtained, where mass spectral adducts were observed because of insufficient washing of the second-dimension RP column (Panel a, 7 column volumes of wash). Panel b shows mass spectra for the same protein from an experiment with sufficient second-dimension wash volumes (Panel b, 14 column volumes of wash). 

The separations of OTC have been carried out, in most instances, with reversed phase (RP) and polymeric ODS column. One difficulty in the analysis of OTC using RP-HPLC is the interaction of OTC with metal ions to form chelate complexes and the adsorption of OTC on RP columns, which consequently leads to severe peak... 

Polymer Labs. PLRP-S Gradient A 0.001 M oxalic acid, 0.5% formic acid + 3% THF in water, B = THF Positive ESF MS/MS OTC, CTC, TC, DC, and its 4-epimer in pig tissues Extraction with sodium succinate solution, protein removal with TCA, SPE cleanup on polymeric RP column DL = 0.5-4.2 ng/g [57]... 

This multidimensional protein identification technology (MudPIT) specifically incorporates a strong cationic exchange (SCX) column in tandem with an RP column to achieve maximal resolution and exquisite sensitivity. MudPIT is effective for studying complex proteomes such as mammalian cellular samples. It has been applied to large-scale protein characterization with identification of up to 1484 proteins from yeast in a single experiment.12... 

NPEOs and OPEOs (rcEo = 3-10) as industrial blends or standard compound (Triton X-100), respectively, were separated together with linear alkylbenzene sulfonates (LASs) on a Ci-RP column [10]. The intensive ions that could be observed in the spectra were mono-, di- and tri-sodium adduct ions [M + Na]+ (m/z 581), [M + 2Na]+ (m/z 604) and [M + 3Na]+ (m/z 626) of the EO7 homologue. The intensity of the molecular [M + H]+-ion, however, was small compared with the sodium adduct ions. The compounds had been concentrated prior to separation on Cis and SAX SPE cartridges. Samples from river water were handled in the same way. 

In one study, however, atmospheric pressure chemical ionisation (APCI)-MS was applied for the simultaneous determination of LAS and octylphenol ethoxylates (OPEO) in surface waters after preconcentration by solid-phase extraction (SPE) on Cis cartridges [1]. In the chromatogram from a Ci-reversed phase (RP) column, peaks arising from both the anionic LAS and the non-ionic OPEO were detected after positive ionisation, while in negative ionisation mode, OPEO were discriminated and only the anionic surfactant was observed. Surprisingly, the relative sensitivity for detection of LAS was approximately five times higher in positive ion mode, which led the authors to the conclusion that this ionisation mode was desirable for quantitative work. 

For sufficient retention of these very polar sulfonated carboxylates on RP columns, the addition of an ion-pairing (IP) agent such as tetraethylammonium acetate (TEAA) to the LC buffer was compulsory [13]. To maintain the compatibility of the eluent with the MS interface, the use of such an involatile cationic additive entailed a cation exchanger to be placed between the column and the interface [13]. Alternatively, equimolar amounts (5 mM) of acetic acid and triethyl-amine, which form the volatile IP agent triethylammonium, were added to the mobile phase in order to improve the retardation of very polar SPC [14]. While the first approach with TEAA was effective in retaining even the very short-chain C3- and C4-SPC (Fig. 2.10.4), the weaker IP agent triethylammonium notably increased the retention of C5-SPC and higher, whereas C4-SPC elutes almost with the dead volume of the LC (Fig. 2.10.5). Addition of commonly used ammonium acetate as buffer component led to the co-elution of the short-chain SPC ([Pg.322]

A C30 RP column has been applied for the separation of carotenoids present in various matrices such as human serum, raw and thermally processed carrots, a Duniella algae-derived preparation and a poultry feed. Samples were mixed with deionized water and CaC03, homogenized and extracted with acetone-hexane (1 1, v/v). 

Figure 6. scheme for on-line orthogonal HILIC-RP HPLC system and one detector step (A) sample injection into HILIC column (for polar analytes) and RP column (for non-polar analytes), step (B) analysis of HILIC retained analytes, step (C) analysis of RP phase retained analytes. 

Liquid chromatographic clean up [441,443,450] has been used either in normal phase flow using alumina, silica, or florisil [22,189,403,481,484] or with reverse-phase (RP) columns [409,452,480]. In most cases these techniques are well established and are used in an off-line mode, primarily to remove the bulk of co-extracted materials prior to a more refined clean-up prior to the final determination. These columns may be prepared in the laboratory [22,403 -405] or commercial solid phase extraction (SPE) cartridges can be used [409,452, 463,470,485,486]. In both cases, the normal phase cartridges and column materials are disposable since many of the polar co-extractants bind firmly to the substrate surface and are difficult to remove. This has been overcome to some... 

HPLC system consisting of a model LC-20 AT pump unit SPD-20A uv-vis detector, 7,725 20 iL sample injection, a computer and a 10 gm alphabond Cj (300 x 3.9 mm) RP column was used. Elution was obtained by using the following gradient steps of acetonitrile and 0.03 M pH 3 phosphate buffer. Acetonitrile Buffer (15 85)... 

RP Columns with Shorter Alkyl Chains or Other... 

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