RPLC-HILIC

HILIC RPLC UV, MS Protein mix, cell lysates 8-port Murphy (2001)... 

We use the second-dimension separation from Fig. 6.6 with a 25 pL injection volume and 2.5 min sampling time the separation is an RPLC method that uses a monolithic column. Thus, 10 pL/min is the maximum flow rate in the first-dimension. Fig. 6.7 shows the development of the first-dimension column that utilizes a hydrophilic interaction (or HILIC) column for the separation of proteins at decreasing flow rates. The same proteins were separated in Fig. 6.6 (RPLC) and 6.7 (HILIC) and have a reversed elution order, which is known from the basics of HILIC (Alpert, 1990). It is believed that HILIC and RPLC separations are a good pair for 2DLC analysis of proteins as they appear to have dissimilar retention mechanisms, much like those of NPLC and RPLC it has been suggested that HILIC is similar in retention to NPLC (Alpert, 1990). Because the HILIC column used in Fig. 6.7 gave good resolution at 0.1 mL/min and no smaller diameter column was available, the flow was split 10-fold to match the second-dimension requirement... 

HILIC is a variant of normal-phase chromatography that employs polar stationary phases and RPLC-type mobile phases. Because HILIC separations occur by a normal-phase mechanism, the organic component of the mobile phase... 

Additional modes of HPTC include normal phase, where the stationary phase is relatively polar and the mobile phase is relatively nonpolar. Silica, diol, cyano, or amino bonded phases are typically used as the stationary phase and hexane (weak solvent) in combination with ethyl acetate, propanol, or butanol (strong solvent) as the mobile phase. The retention and separation of solutes are achieved through adsorp-tion/desorption. Normal phase systems usually show better selectivity for positional isomers and can provide orthogonal selectivity compared with classical RPLC. Hydrophilic interaction chromatography (HILIC), first reported by Alpert in 1990, is potentially another viable approach for developing separations that are orthogonal to RPLC. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal phase retention behavior. Typical stationary phases include silica, diol, or amino phases. Diluted acid or a buffer usually is needed in the mobile phase to control the pH and ensure the reproducibility of retention times. The use of HILIC is currently limited to the separation of very polar small molecules. Examples of applications... 

Figure 2 Comparison of HPLC (top panels) and chrom-NMR (bottom panels) for a series of homologous aromatic molecules, recorded under RPLC (left panels ODS solid phase and water/acetonitrile mobile phase) and HILIC conditions (right panels bare silica solid phase and water/acetonitrile mobile phase). From Ref. 49. Copyright 2006 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim.

M, the compounds being separated do not sufficiently interact with the stationary phase. The problem occurs most commonly in analyses of urine containing sodium chloride at a concentration exceeding 100 mM. Salinity of analyzed solutions is also problematic in RPLC (because of solvation of the investigated compounds) and in HILIC (as a result of competitive interactions with the stationary phase). These unfavorable phenomena often require salt removal. The procedure can be performed by SPE, among other methods [165]. 

Quantitative speciation analyses have been dominated by the coupled HPLC/GC ICP MS techniques on account of the good separation, sensitivity, and isotopic specificity of detector response. The same stams in qualitative analysis is held by ESI MS coupled with RPLC and, increasingly, with HILIC. Sometimes, it becomes an alternative system in quantitative analysis for assays involving larger quantities of investigated compounds. 

Reversed-phase LC (RPLC) is applied most often in LC-MS, although attention is also paid to the use of hydrophilic interaction chromatography (HILIC, Ch. 11.7.3) and the need for chiral separations (see Ch. 11.7.4). 

The bioanalysis of LOR and DCL was also reported with hydrophilic-interaction LC (HILIC, Ch. 1.4.5), i.e., utilizing a 50x3-mm-ID silica column (5 pm) and an aqueous acetonitrile mobile phase (90% acetonitrile containing 0.1% TFA) [66]. In HILIC, the retention order is reversed relative to RPLC, i.e., highly polar analytes are more strongly retained. The analytes were extracted from alkalized human plasma by LLE with hexane. The extract was evaporated to dryness and reconstituted in 0.1% aqueous TFA. The flow-rate was 0.5 ml/min the run time 2... 

HILIC (Ch. 1.4.5) was promoted for apphcation in bioanalytical LC-MS by Naidong et al. [66-67, 131-132]. In initial studies [131], 5-8-fold and 20-fold gain in sensitivity was achieved in HlLlC for basic and acidic compounds, respectively, compared to reversed-phase LC. The use of HlLlC in the analysis of LOR and DCL was discussed in Ch. 11.4.5 [66]. Different from RPLC, an organic solvent is a weak solvent in HlLlC. This opens the possibility to direct injection of organic extracts from LLE, as demonstrated with MTBE extracts after 96-well plate LLE of LOR and DCL [67]. The potential of HlLlC in bioanalysis with LC-MS was reviewed by Naidong [132]. 

Fig. 2 Comparison of RPLC and HILIC/CEX elution profiles of cyclic peptides. RPC Zorbax 300XDB-Cg column linear gradient (0.5% acetonitrile/min) from 0.05% aqueous trifluoro-acetic acid (TFA) to 0.05% TFA in acetonitrile at 1 mL/min at 70°C and detection at 210 nm. HILIC/CEX Polysulfoethyl A column 5 min isocratic elution with buffer A (20 mM aqueous triethylammonium phosphate with 90% acetonitrile), followed by linear gradient (2.5 mM sodium perchlorate/min) from buffer A to buffer B (buffer A containing 400 mM sodium perchlorate with 80% acetonitrile) at 30°C and detection at 210 nm. [Reprinted from C. T. Mant, L. H. Kondejewski, and R. S. Hodges, /. Chromatogr. 816 (1998), p. 79, with permission of Elsevier Science. ...

To better distinguish the contributions of polar interactions to retention, the LEER model was transformed into the so-called hydrophobic subtraction model (HSM) for RPLC, where the hydrophobic contribution to retention is compensated for by relating the solute retention to a standard nonpolar reference compound. This approach was applied to characterize more than 300 stationary phases for RPLC, including silica gel supports with bonded alkyl-, cyanopropyl-, phenylalkyl-, and fluoro-substituted stationary phases and columns with embedded or end-capping polar groups. The QSRR models can be used to characterize and compare the suitabihty of columns not only for reversed-phase, but also for NP and HILIC systems. 

Figure 7.18 Separation of acryiic acid and its oligomers by RPLC on Acciaim OA (a) and by HILIC on Acciaim HILIC-10 (b). Chromatographic conditions for Acciaim OA coiumn dimensions 250mmx4mm i.d., Spm column temperature 30°C eluent (A) MeCN and (B) 2.5 mmol/L methanesulfonic acid gradient 5% A for 1 min isocratically and then linearly to 90% A in 19 min flow rate 0.75 mU/min ...

Because gabapentin lacks chromophores, UV detection is difficult. Therefore, analysis was usually carried out by precolumn derivatization followed by RPLC with UV or fluorescence detection. In contrast, the method of Jia et al. [156] does not require any derivatization. In their study, the performance of CAD and ELSD were compared on four different HILIC columns. It was found that the sensitivity achieved by ELSD was comparable to that with CAD and both superior to UV detection in HlLlC mode. However, employing a conventional CIS column, CAD was about 25-times more sensitive than ELSD. Jia et al. also introduced HPLC-CAD analysis of gabapentin in rat serum and mine, which is the first report of a successful application of CAD in such matrices [157],... 

Next to MALDI-MS, the use of ESt-MS is important in oligosaccharide and glycan characterization, especially because it can be combined with on-line LC separation of complex mixtures. The most important LC methods in glycan and oligosaccharide separation are HILIC [184], HPAEC, and, after derivatization by reductive amination with 2-aminobenzamide or 2-aminoacridone, RPLC on either Cjg or porous graphitized carbon materials [185]. 

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