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Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions
Journal of Chromatography A, 1218 (2011) 8021–8026
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions Reinhard I. Boysen a , Yuanzhong Yang a , Jamil Chowdhury a , Maria T. Matyska b , Joseph J. Pesek b , Milton T.W. Hearn a,∗ a b
Australian Research Council Special Research Centre for Green Chemistry, Monash University, Clayton, Victoria 3800, Australia Department of Chemistry, San Jose State University, San Jose, CA 95192, USA
a r t i c l e
i n f o
Article history: Received 10 December 2010 Received in revised form 1 September 2011 Accepted 5 September 2011 Available online 9 September 2011 Keywords: Aqueous normal phase conditions Silica hydride Hydrophobic peptides Hydrophilic peptides High resolution separation Electrospray ionization mass spectrometry
a b s t r a c t The application of a silica hydride modified stationary phase with low organic loading has been investigated as a new type of chromatographic material suitable for the separation and analysis of peptides with electrospray ionization mass spectrometric detection. Retention maps were established to delineate the chromatographic characteristics of a series of peptides with physical properties ranging from strongly hydrophobic to very hydrophilic and encompassing a broad range of pI values (pI 5.5–9.4). The effects of low concentrations of two additives (formic acid and acetic acid) in the mobile phase were also investigated with respect to their contribution to separation selectivity and retention under comparable conditions. Significantly, strong retention of both the hydrophobic and the hydrophilic peptides was observed when high-organic low-aqueous mobile phases were employed, thus providing a new avenue to achieve high resolution peptide separations. For example, simultaneous separation of hydrophobic and hydrophilic peptides was achieved under aqueous normal phase (ANP) chromatographic conditions with linear gradient elution procedures in a single run, whilst further gradient optimization enabled improved peak efficiencies of the more strongly retained hydrophobic and hydrophilic peptides. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In many fields, from clinical diagnosis to pharmaceutical manufacture to process monitoring in the chemical industry, the availability of analytical methods that allow rapid, high-resolution separation and identification of compounds of interest present in complex mixtures, and the characterization of the associated impurities, is essential. For many compound classes, such analytical methods are based on liquid chromatography (LC), a key technology in separation science. A major strength of LC arises from its ability to achieve separation in various application modes by exploiting specific properties of the stationary phase, mobile phase and analytes. Over the past two decades, reversed-phase liquid chromatography (RPLC) has dominated the field of LC analysis, relying largely upon the hydrophobic characteristics of the sorbent and analytes, the organic solvent content and the presence of ion-pair additives, such as trifluoroacetic acid, in the mobile phase, to achieve retention and resolution [1]. Various modes of detection are feasible with RPLC
∗ Corresponding author. Tel.: +61 3 9905 4547; fax: +61 3 9905 8501. E-mail address: [email protected] (M.T.W. Hearn). 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.09.009
methods, but increasingly these procedures are used in conjunction with electrospray ionization mass spectrometry (ESI-MS) or other MS techniques. However, many current analytical tasks deal with molecules that have chemical properties at the hydrophilic extreme of the hydrophobicity/hydrophilicity scale and these molecules fall outside the capability of RPLC methods to resolve. As documented in our previous studies [2], as well as reviewed elsewhere in the scientific literature [3], a major challenge is the lack of suitable chromatographic methods for the analysis of low abundance, very polar molecules present in complex mixtures, and particularly the need to simultaneously resolve analytes that vary significantly over a very wide range of hydrophobicity/hydrophilicity properties. In this context, hydrophilic interaction chromatography (HILIC) [4] has attracted interest for the separation of several classes of polar compounds, e.g. simple mono, di- and oligosaccharides, neurotransmitters or antibiotics [5]. This method can also be interfaced with mass spectrometric (MS) detection methods [6]. However, with complex mixtures of hydrophilic compounds, and particularly with the structurally more complex biological molecules, their resolution has proven to be difficult, and in many cases impossible, to achieve with current RPLC and HILIC techniques. This is because the ‘chromatographic resolution window’ of the current generation
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of these adsorbents is frequently too narrow to permit simultaneous separation of analyte mixtures encompassing a broad range of molecular properties. Overcoming these constraints and limitations is the motivation for the development of the next generation of high performance chromatographic materials capable of mixed-mode separation of hydrophilic compounds. An example of a stationary phase material which meets these requirements has been recently developed, based on silica hydride [7]. This new type of stationary phase has significant differences in terms of chemical structure to the traditional type-A/type-B silicas, which are mainly populated with polar acidic silanol (Si–OH) groups. In contrast, the type-C silicas predominantly have silicon–hydride (Si–H) groups at their surfaces [8]. With such stationary phases, additional regions of the chromatographic separation space can be interrogated, through the modality of aqueous normal phase chromatography (ANPC) [9–12]. ANP retention behavior of solutes is analogous to that found in normal phase chromatography but with the important difference that the mobile phase contains water as part of the eluent [13]. Moreover, by exploiting the principle of normal phase, ANP retention becomes greater for polar solutes, such as acids in their anionic form or protonated bases, when the concentration (or volume fraction) of the least polar solvent component in the mobile phase is increased. For example, if the mobile phase consists of water and acetonitrile, retention will increase as the amount of acetonitrile increases. For most hydrophilic compounds the amount of the less polar (solvent) component in the mobile phase is usually 60% (v/v) or greater, with the extent of retention depending on the physicochemical properties of the solute and the mobile phase composition. However, for all hydrophobic compounds investigated with silica hydride materials to date, retention has been found to increase as the amount of water increases in the mobile phase, a behavior also typified by reversed-phase adsorbents [10,14]. With hydrophilic solutes, enhanced retention is observed as the amount of organic solvent (e.g., acetonitrile) present in the mobile phase is increased for both silica hydride and HILIC stationary phases; retention behavior that is common also with normal phase adsorbents [15]. An interesting possibility with silica hydride materials is thus their potential to concomitantly resolve highly polar and nonpolar compounds with both having reasonable retention using an organic solvent, such as acetonitrile or acetone, in the mobile phase. Previously, ANP retention behavior has been demonstrated with polar compounds, using silica hydride-based stationary phases and neutral or acidic pH eluents [9,16–19]. As a consequence, high pH mobile phases may therefore not be necessary to separate polar compounds using ANPC adsorbents. Moreover, for some ANP applications, the mobile phase can be chosen to be compatible with mass spectrometric analysis, e.g. 0.1–0.5% (v/v) formic or acetic acids, or 5–15 mM ammonium acetate or formate. The focus of this investigation was to examine the performance attributes of a new type of silica hydride separation material of low carbon content in order to provide significantly improved ANP chromatographic capabilities for the separation of mixtures of hydrophobic and hydrophilic peptides. Previous investigations have shown that several different classes of metabolites [16] and nucleotides [17] can be readily analyzed with such types of stationary phase. However, to date these materials have not been investigated for the separation and analysis of complex peptide mixtures. The objective of this current study was not to carry out an exhaustive evaluation of the retention characteristics of all available types of stationary phases with HILIC-like or ANP-like features, but rather to explore the retention capabilities of this silica hydridebased stationary phase to simultaneously retain and resolve both hydrophilic and hydrophobic peptides using mobile phases compatible with on-line mass spectrometric detection.
2. Experimental 2.1. Chemicals and materials The peptides 1 (Gly-Arg-Ala-Asp-Ser-Pro-Lys), 2 (Arg-GlyAsp), 3 (Lys-Gln-Ala-Gly-Asp-Val), 4 (Tyr-Ile-gly-Ser-Arg), 5 (Trp-His-Trp-Leu-Gln-Leu), 6 (Tyr-Gly-Gly-Phe-Leu) and 7 (TyrTyr-Tyr-Tyr-Tyr-Tyr) were purchased from Sigma Aldrich (St Louis, USA); formic acid (FA, 99%, v/v) were obtained from AJAX Chemicals, Australia; acetic acid (AA, 100%, v/v) was purchased from BDH Chemicals Australia Pty. Ltd. HPLC grade acetonitrile (ACN) was obtained from Merck (Darmstadt, Germany). Water was distilled and deionized in a Milli-Q system (Millipore, Bedford, MA, USA). The stationary phase (4 m particle size, 100 A˚ pore size), called Diamond Hydride (DH), contains a small amount of an organic moiety (∼2% bonded alkyl moiety carbon loading as determined by elemental analysis) on a silica hydride surface. This stationary phase was packed into capillary columns (0.3 mm × 150 mm) by MicroSolv Technology (Eatontown, NJ, USA) based on specifications developed by the authors of this study to meet the requirements of industry standards. 2.2. Liquid chromatography and mass spectrometry The chromatographic analysis of peptides was conducted on an Agilent 1100 capillary LC system (Agilent Technologies, Palo Alto, CA, USA) coupled to an ion-trap MS system (Agilent 1100 series LC/MSD-SL). The studies with the modified silica hydride capillaries were performed at room temperature by using a capillary pump. The outlet of the capillary was connected to the diode array detector and the electrospray source of the ion-trap mass spectrometer. The experimental data for the retention maps were acquired using isocratic elution with different acetonitrile–water mixtures containing between 30 and 95% (v/v) acetonitrile and 0.1% (v/v) formic acid or 0.1% (v/v) acetic acid, respectively. The flow rate was 4 L/min. The ANP gradient separations of the peptides were performed using acetonitrile as the weak eluent (A) and water as the strong eluent (B), with both eluents containing either formic acid or acetic acid as mobile phase additive. Further details of the experimental conditions are noted in the respective figure captions. ESI-MS analysis was carried out in the positive ion mode. The electrospray voltage was 3.5 kV and ion trap mass spectrometer was operated in full scan mode in the range of 200–1200 m/z. The nebulizing gas (N2 ) pressure, the drying gas (N2 ) flow rate, and the drying gas temperature were set at 10 psi, 5 L/min, and 300 ◦ C, respectively. The ion accumulation time was automatically adjusted using the Ion Charge Control (ICC) feature of the instrument. The maximal accumulation time was set at 300 ms and the ICC target was set at 30,000. All system control and data acquisition were conducted with Agilent ChemStation and MSD Trap Control software. 3. Results and discussion The analytes employed in this study comprised some very hydrophobic and hydrophilic peptides with diverse isoelectric points (pIs), and thus served as useful probes to explore the potential of the silica hydride phase in this field of bioanalysis. The attribution of ANP modality for the separation of peptides follows from the nature of the mobile phases employed and distinguishes these silica hydride materials from alternative HILIC stationary phases. Depending on the amount of water in the mobile phase, silica hydride phases can operate either under ANP or reversedphase conditions. HILIC materials do not have this capability and thus can only retain polar compounds under such conditions. The
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Table 1 Amino acid sequence, calculated hydrophobicity [21] and isoelectric point (pI) [22] of peptides (1)–(7). Peptide code
Sequence
Calculated hydrophobicity
pI
1 2 3 4 5 6 7
H-Gly-Arg-Ala-Asp-Ser-Pro-Lys-OH H-Arg-Gly-Asp-OH H-Lys-Gln-Ala-Gly-Asp-Val-OH H-Tyr-Ile-Gly-Ser-Arg-OH H-Trp-His-Trp-Leu-Gln-Leu-OH H-Tyr-Gly-Gly-Phe-Leu-OH H-Tyr-Tyr-Tyr-Tyr-Tyr-Tyr-OH
−2.31 −0.84 0.35 4.11 9.65 10.61 11.34
9.4 6.1 5.8 9.1 7.1 5.5 5.5
silica hydride material (DH) used in this study has previously been shown to be a highly effective stationary phase for the retention of polar low molecular weight metabolites [16–19]. In order to explore the range of retention characteristics that this type of silica hydride phase might encompass with hydrophilic peptides, a low molecular mass peptide set ranging in hydrophobicity values from −2.31 (most hydrophilic) to 11.34 (most hydrophobic) and in pI values from 5.5 to 9.1 was investigated, with the individual peptides small enough to not show any secondary structural elements. The structures and physical properties of these peptides are listed in Table 1. Fig. 1 shows a plot of retention time vs. percent composition of organic solvent in the mobile phase for the isocratic elution of the three most hydrophilic peptides (1–3) listed in Table 1. As might be expected for polar compounds with the DH adsorbent, typical ANP behavior was observed, i.e., peptide retention increased as the amount of the less polar component (acetonitrile) of the mobile phase was increased. In addition, at a given mobile phase composition, the most hydrophilic peptide (1) was the most strongly retained, followed by lower retentions of the less polar peptides (3) and (2). Thus, for the hydrophilic species, the behavior of peptides with the DH adsorbent paralleled the behavior of small polar solutes, such as amino acids, organic acids, carbohydrates and nucleotides [16]. A more surprising result was the retention behavior (Fig. 2) of the four hydrophobic peptides (4)–(7) determined under similar ANP conditions, and again using low concentrations of formic acid as an additive. These peptides range in hydrophobicity values from 4.11 to 11.34. As evident from Fig. 2, these hydrophobic peptides are retained under conditions that are normally used for polar compounds, i.e., high-organic low-aqueous mobile phases. The precise mechanism controlling the strong retention of these hydrophobic peptides under these experimental conditions has yet to be fully
Fig. 2. Effects of acetonitrile concentration in the mobile phase on the retention of hydrophobic peptides. Chromatographic conditions as in Fig. 1.
Fig. 1. Effects of acetonitrile concentration in the mobile phase on the retention of hydrophilic peptides. Chromatographic conditions: isocratic elution using a Diamond Hydride capillary column (0.3 mm × 150 mm, 4 m) with water–organic solvent mixtures at different volume fractions of acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 4 L/min. The sample contained peptides (1)–(3) at a concentration of 10 g/mL each in 0.1% formic acid, 50% acetonitrile (v/v); the injection volume was 0.1 L.
Fig. 3. Effects of acetonitrile concentration in the mobile phase on the retention of hydrophobic peptides. Chromatographic conditions: isocratic elution using a Diamond Hydride capillary column (0.3 mm × 150 mm, 4 m) with water–organic solvent mixtures at different volume fractions of acetonitrile containing 0.1% (v/v) acetic acid at a flow rate of 4 L/min. The sample contained peptides (4)–(7) at a concentration of 10 g/mL each in 0.1% acetic acid, 50% acetonitrile (v/v); the injection volume was 0.1 L.
elucidated; however, it can be noted that the interaction between the polar amide backbone moieties of the hydrophobic peptides and the stationary phase through hydrogen bonding effects would account for this behavior. Moreover, as shown in Fig. 2, the elution order of these peptides (4)–(7) at a given mobile phase composition paralleled the results obtained for peptides (1)–(3) (Fig. 1), with the least hydrophobic of the four peptides having the greatest retention whilst the most hydrophobic peptide having the lowest retention, e.g. with 80% (v/v) acetonitrile in the mobile phase, the elution order was 7 < 6 < 5 < 4. The higher retention of peptides 4 and 5 may be attributed to the presence of the ␥-amido- and guanidinyl-amino groups of Gln and Arg, respectively, which may interact with the stationary phase. This is in agreement with earlier work on small molecules containing similar types of amino groups [16]. With 50% (v/v) acetonitrile in the mobile phase, all four peptides co-eluted in the void volume of the packed capillary. Another interesting manifestation of this novel retention behavior was observed for the hydrophobic peptides (4)–(7) when the additive in the mobile phase was changed from formic acid to acetic acid. Fig. 3 shows the retention map for these four hydrophobic peptides (4)–(7) using 0.1% (v/v) acetic acid as the additive in the mobile phase. The elution times for all of the peptides increased in comparison to the data obtained when 0.1% (v/v) formic acid was employed as the additive (Fig. 2). For example, the retention times of peptides (6) and (7) with a mobile phase consisting of 90:10 (v/v) acetonitrile/water with 0.1% (v/v) formic acid were similar to those obtained for a mobile phase consisting of 80:20 (v/v) acetonitrile/water with 0.1% (v/v) acetic acid. Thus, the presence of acetic acid increased the ANP selectivity
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of the silica hydride adsorbent, indicating that weaker hydrogen ion activity leads to stronger retention. A similar effect has been observed for the retention of metabolites with similar silica hydride adsorbents [16]. Further comparison of the data shown in Figs. 2 and 3 reveals that the elution order of peptides (4) and (5) is reversed. Thus, in addition to controlling the elution strength, changes to the composition of the mobile phase additives can also alter selectivity, an important feature that can be used to optimize separations. Finally, it can be noted that whilst all of the hydrophobic peptides eluted near to the void volume when a mobile phase comprising 50:50 (v/v) acetonitrile/water with 0.1% (v/v) formic acid was used, peptides (4) and (5) had considerable retention when the mobile phase was changed to 50:50 (v/v) acetonitrile/water with 0.1% (v/v) acetic acid and this retention increased further as the organic solvent content was increased to higher values. Whilst some ANP materials can exhibit reversed-phase features, such behavior is less pronounced with silica surfaces used in hydrophilic interaction chromatography, as recent investigations indicate [20]. With mobile phases of high organic content, e.g., 50–95% acetonitrile such as those employed in the current study, it is unlikely that any RP-like features will be manifested with the selected peptides, due to contributions from the Si–H groups or the very low level (2%) of bonded n-alkyl groups in the DH silica hydride phase. The significant differences thus noted in Figs. 1 and 2 for the retention behavior of the selected peptides when either 0.1% acetic acid or 0.1% formic acid was used with a mobile phase of the same organic solvent content highlight the important role that secondary modifiers can play with ANP materials. These results thus indicate that significant retention and selectivity effects can occur when either water-rich or waterlean mobile phases are employed, depending on which region of the ANP separation space is employed. While HILIC phases display predominantly normal phase retention, various commercially available ANP materials can also exhibit reversed-phase features. In this context, however, the solvent dependencies of materials, such as this DH silica hydride phase, with peptides significantly differ to that observed [1,2] with traditional RPC adsorbents with mobile phases of high organic solvent content. The origins of this novel attribute of the DH silica hydride phase are undergoing further investigation with structurally more diverse peptide mixtures and these results will be described more fully in a subsequent report. The plots shown in Figs. 1–3 document the broad range of retention properties of this particular type of silica hydride stationary phase. In order to apply these capabilities to samples containing peptides with a wide range of hydrophobicities, gradient elution methods are needed to accomplish such analyses in a reasonable time frame. An example of a gradient separation of the four hydrophobic peptides (4)–(7) with 0.1% (v/v) formic acid as the additive in the mobile phase is shown in Fig. 4A. Almost complete baseline separation was achieved with a 5 min hold period at 90% (v/v) A (weak solvent) followed by a linear gradient to 50% (v/v) A over 20 min. Although further gradient optimization could be undertaken to achieve improved separation or shorter analysis times, by using MS detection in the extracted ion chromatogram (EIC) mode, as is the case in this study, the need for complete baseline separation of all components is removed. A similar strategy was developed using 0.1% (v/v) acetic acid as the additive in the mobile phase. Because of the higher retention of the peptides with acetic acid, the mobile phase utilized an 80:20 (v/v) acetonitrile/water composition and the 20 min gradient went to 30% (v/v) acetonitrile. The resulting base peak chromatogram (BPC represents the intensity of the most intense peak at every time point in the analysis) is shown in Fig. 4B, where an elution order reversal for peptides (4) and (5) in comparison to the results obtained in 0.1% (v/v) formic
Fig. 4. Separation of hydrophobic peptides on a Diamond Hydride capillary column. (A) Eluent A: 0.1% (v/v) formic acid in acetonitrile, eluent B: 0.1% (v/v) formic acid in water; gradient: 0.0–5.0 min 90% (v/v) A, 5.0–25.0 min to 50% (v/v) A. (B) Eluent A: 0.1% (v/v) acetic acid in acetonitrile, eluent B: 0.1% (v/v) acetic acid in water; gradient: 0.0–5.0 min 80% (v/v) A, 5.0–25.0 min to 30% (v/v) A; flow rate: 4 L/min; sample: peptides (4)–(7), 10 g/mL each in 50% (v/v) B; 0.1 L injection.
acid is illustrated. The separations achieved here were similar to those obtained when 0.1% (v/v) formic acid was employed, although the peak widths for peptides (4) and (5) were broader, (cf. Fig. 4A and B). Further enhancement of peak shape can be achieved by making the gradient steeper. In order to show the full range of separation capability of the DH support material, a mixture of all seven peptides was injected onto the capillary. In this case, the concentration of the mobile phase additive, formic acid, was increased to 0.5% (v/v) to reduce the retention of the most hydrophilic species. The base peak chromatogram of this mixture, using the same ANP gradient as shown in Fig. 4A, is illustrated in Fig. 5A. With no gradient optimization, all seven peptides were almost baseline separated. The elution order followed closely the calculated hydrophobicities of the seven compounds, i.e., the most hydrophobic peptide was eluted first and the remainder in close correlation with decreasing hydrophobicity with the most hydrophilic peptide eluting last. As also evident from Fig. 1, only the elution order of peptides (2) and (3) did not match their calculated hydrophobicity. Under these non-optimized conditions, the peak widths of the most hydrophobic peptides (6) and (7) and the most hydrophilic peptide (1) were significantly broader than those for peptides (2)–(5) with intermediate hydrophobicity. The extracted ion chromatogram of peptide 1 is shown in Fig. 5B. Such broad peaks are often the result of slow mass transfer and can be improved by adjusting the gradient to enhance this process. In general, the most efficient way to improve peak shape for the LC separation of polar compounds is to increase the gradient steepness profile, so that over the total elution time the change in
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Fig. 5. (A) Base peak chromatogram (BPC) of peptides (1)–(7) separated on a Diamond Hydride capillary column. (B) Extracted ion chromatogram (EIC) of peptide (1). Conditions: eluent A: 0.5% (v/v) formic acid in acetonitrile, eluent B: 0.5% (v/v) formic acid in water; gradient: 0.0–5.0 min 90% (v/v) A, 5.0–25.0 min to 50% (v/v) A, 25.0–25.1 min to 30% (v/v) A, 25.1–32.0 min hold 30% (v/v) A; flow rate: 4 L/min; sample concentration: 10 g/mL in 50% (v/v) B; 0.1 L injection.
the solvent composition is more rapid. Thus to generate the data shown in Fig. 5 the gradient time was modified according to these general principles with a hold used at a relatively high composition of the weak solvent in the mobile phase in order to have sufficient retention of the earliest eluting solutes. The result of further optimization of the separation is shown in Fig. 6A, where both an acetonitrile and a formic acid gradient, were employed. With this modification, the most hydrophobic compounds had longer retention times but the peaks were much narrower with resolution approximately the same. A more dramatic improvement is seen for peptide (1), the most hydrophilic species. In this case, the peak width was considerably narrower than under the previous gradient conditions as can be seen by the MS-EIC of this compound in Fig. 6B. As evident from the chromatogram shown in Fig. 6A lower resolution between peptides (4) and (2) occurred under these elution conditions compared to the results illustrated in Fig. 5A. As noted above, further optimization of the gradient slope would be anticipated to improve the resolution further, however, since MS detection is now performed with many types of peptide analyses, from a practical perspective such gradient optimization procedures may not be necessary. With most modes of MS detection, since the EICs of each peptide can be used to simultaneously delineate individual peptides in terms of retention and mass, only peptides having the same nominal molecular mass or those differing by, for example, by 1 amu, need to be chromatographically separated. With more advanced tandem MS spectrometers, e.g. triple quadrupole MS/MS instrumentation, having accurate mass capabilities, even this requirement can be substantially circumvented.
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Fig. 6. (A) Base peak chromatogram (BPC) of peptides (1)–(7) separated on a Diamond Hydride capillary column. (B) Extracted ion chromatogram (EIC) of peptide (1). Conditions: eluent A: 0.1% (v/v) formic acid in acetonitrile, eluent B: 0.5% (v/v) formic acid in water; gradient: 0.0–5.0 min 90% (v/v) A, 5.0–10.0 min to 70% (v/v) A, 10.0–20.0 min to 60% (v/v) A, 20.0–20.1 min to 30% (v/v) A, 20.1–30.0 min hold 30% (v/v) A; flow rate: 4 L/min; sample concentration: 10 g/mL in 50% (v/v) B; 0.1 L injection.
4. Conclusions In this investigation the capability of a new class of ANP stationary phase for use in the simultaneous separation of hydrophilic and hydrophobic peptides has been documented. This new separation material was derived from silica hydride with properties that can be modulated in multiple ways, through the appropriate use of isocratic mobile phases of high water content, or in the case of gradient elution procedures, increasing water content and the use of mobile phase additives. The application of silica hydride stationary phases under ANP conditions for the separation of peptides of widely different hydrophilicities/hydrophobicities properties over an extended chromatographic window as demonstrated in this study is anticipated to provide separation scientists and analytical chemists with access to an additional and novel modality of high-resolution separation capabilities that can be readily interfaced with mass spectrometric and other detection methods.
Acknowledgments The financial support of the Australian Research Council, the National Institutes of Health (GM079741-01) and the National Science Foundation (CHE 0724218) is gratefully acknowledged. One of the authors (J.J.P.) would like to acknowledge the support of the Camille and Henry Dreyfus Foundation through a Scholar Award.
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Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions Reinhard I. Boysen a , Yuanzhong Yang a , Jamil Chowdhury a , Maria T. Matyska b , Joseph J. Pesek b , Milton T.W. Hearn a,∗ a b
Australian Research Council Special Research Centre for Green Chemistry, Monash University, Clayton, Victoria 3800, Australia Department of Chemistry, San Jose State University, San Jose, CA 95192, USA
a r t i c l e
i n f o
Article history: Received 10 December 2010 Received in revised form 1 September 2011 Accepted 5 September 2011 Available online 9 September 2011 Keywords: Aqueous normal phase conditions Silica hydride Hydrophobic peptides Hydrophilic peptides High resolution separation Electrospray ionization mass spectrometry
a b s t r a c t The application of a silica hydride modified stationary phase with low organic loading has been investigated as a new type of chromatographic material suitable for the separation and analysis of peptides with electrospray ionization mass spectrometric detection. Retention maps were established to delineate the chromatographic characteristics of a series of peptides with physical properties ranging from strongly hydrophobic to very hydrophilic and encompassing a broad range of pI values (pI 5.5–9.4). The effects of low concentrations of two additives (formic acid and acetic acid) in the mobile phase were also investigated with respect to their contribution to separation selectivity and retention under comparable conditions. Significantly, strong retention of both the hydrophobic and the hydrophilic peptides was observed when high-organic low-aqueous mobile phases were employed, thus providing a new avenue to achieve high resolution peptide separations. For example, simultaneous separation of hydrophobic and hydrophilic peptides was achieved under aqueous normal phase (ANP) chromatographic conditions with linear gradient elution procedures in a single run, whilst further gradient optimization enabled improved peak efficiencies of the more strongly retained hydrophobic and hydrophilic peptides. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In many fields, from clinical diagnosis to pharmaceutical manufacture to process monitoring in the chemical industry, the availability of analytical methods that allow rapid, high-resolution separation and identification of compounds of interest present in complex mixtures, and the characterization of the associated impurities, is essential. For many compound classes, such analytical methods are based on liquid chromatography (LC), a key technology in separation science. A major strength of LC arises from its ability to achieve separation in various application modes by exploiting specific properties of the stationary phase, mobile phase and analytes. Over the past two decades, reversed-phase liquid chromatography (RPLC) has dominated the field of LC analysis, relying largely upon the hydrophobic characteristics of the sorbent and analytes, the organic solvent content and the presence of ion-pair additives, such as trifluoroacetic acid, in the mobile phase, to achieve retention and resolution [1]. Various modes of detection are feasible with RPLC
∗ Corresponding author. Tel.: +61 3 9905 4547; fax: +61 3 9905 8501. E-mail address: [email protected] (M.T.W. Hearn). 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.09.009
methods, but increasingly these procedures are used in conjunction with electrospray ionization mass spectrometry (ESI-MS) or other MS techniques. However, many current analytical tasks deal with molecules that have chemical properties at the hydrophilic extreme of the hydrophobicity/hydrophilicity scale and these molecules fall outside the capability of RPLC methods to resolve. As documented in our previous studies [2], as well as reviewed elsewhere in the scientific literature [3], a major challenge is the lack of suitable chromatographic methods for the analysis of low abundance, very polar molecules present in complex mixtures, and particularly the need to simultaneously resolve analytes that vary significantly over a very wide range of hydrophobicity/hydrophilicity properties. In this context, hydrophilic interaction chromatography (HILIC) [4] has attracted interest for the separation of several classes of polar compounds, e.g. simple mono, di- and oligosaccharides, neurotransmitters or antibiotics [5]. This method can also be interfaced with mass spectrometric (MS) detection methods [6]. However, with complex mixtures of hydrophilic compounds, and particularly with the structurally more complex biological molecules, their resolution has proven to be difficult, and in many cases impossible, to achieve with current RPLC and HILIC techniques. This is because the ‘chromatographic resolution window’ of the current generation
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of these adsorbents is frequently too narrow to permit simultaneous separation of analyte mixtures encompassing a broad range of molecular properties. Overcoming these constraints and limitations is the motivation for the development of the next generation of high performance chromatographic materials capable of mixed-mode separation of hydrophilic compounds. An example of a stationary phase material which meets these requirements has been recently developed, based on silica hydride [7]. This new type of stationary phase has significant differences in terms of chemical structure to the traditional type-A/type-B silicas, which are mainly populated with polar acidic silanol (Si–OH) groups. In contrast, the type-C silicas predominantly have silicon–hydride (Si–H) groups at their surfaces [8]. With such stationary phases, additional regions of the chromatographic separation space can be interrogated, through the modality of aqueous normal phase chromatography (ANPC) [9–12]. ANP retention behavior of solutes is analogous to that found in normal phase chromatography but with the important difference that the mobile phase contains water as part of the eluent [13]. Moreover, by exploiting the principle of normal phase, ANP retention becomes greater for polar solutes, such as acids in their anionic form or protonated bases, when the concentration (or volume fraction) of the least polar solvent component in the mobile phase is increased. For example, if the mobile phase consists of water and acetonitrile, retention will increase as the amount of acetonitrile increases. For most hydrophilic compounds the amount of the less polar (solvent) component in the mobile phase is usually 60% (v/v) or greater, with the extent of retention depending on the physicochemical properties of the solute and the mobile phase composition. However, for all hydrophobic compounds investigated with silica hydride materials to date, retention has been found to increase as the amount of water increases in the mobile phase, a behavior also typified by reversed-phase adsorbents [10,14]. With hydrophilic solutes, enhanced retention is observed as the amount of organic solvent (e.g., acetonitrile) present in the mobile phase is increased for both silica hydride and HILIC stationary phases; retention behavior that is common also with normal phase adsorbents [15]. An interesting possibility with silica hydride materials is thus their potential to concomitantly resolve highly polar and nonpolar compounds with both having reasonable retention using an organic solvent, such as acetonitrile or acetone, in the mobile phase. Previously, ANP retention behavior has been demonstrated with polar compounds, using silica hydride-based stationary phases and neutral or acidic pH eluents [9,16–19]. As a consequence, high pH mobile phases may therefore not be necessary to separate polar compounds using ANPC adsorbents. Moreover, for some ANP applications, the mobile phase can be chosen to be compatible with mass spectrometric analysis, e.g. 0.1–0.5% (v/v) formic or acetic acids, or 5–15 mM ammonium acetate or formate. The focus of this investigation was to examine the performance attributes of a new type of silica hydride separation material of low carbon content in order to provide significantly improved ANP chromatographic capabilities for the separation of mixtures of hydrophobic and hydrophilic peptides. Previous investigations have shown that several different classes of metabolites [16] and nucleotides [17] can be readily analyzed with such types of stationary phase. However, to date these materials have not been investigated for the separation and analysis of complex peptide mixtures. The objective of this current study was not to carry out an exhaustive evaluation of the retention characteristics of all available types of stationary phases with HILIC-like or ANP-like features, but rather to explore the retention capabilities of this silica hydridebased stationary phase to simultaneously retain and resolve both hydrophilic and hydrophobic peptides using mobile phases compatible with on-line mass spectrometric detection.
2. Experimental 2.1. Chemicals and materials The peptides 1 (Gly-Arg-Ala-Asp-Ser-Pro-Lys), 2 (Arg-GlyAsp), 3 (Lys-Gln-Ala-Gly-Asp-Val), 4 (Tyr-Ile-gly-Ser-Arg), 5 (Trp-His-Trp-Leu-Gln-Leu), 6 (Tyr-Gly-Gly-Phe-Leu) and 7 (TyrTyr-Tyr-Tyr-Tyr-Tyr) were purchased from Sigma Aldrich (St Louis, USA); formic acid (FA, 99%, v/v) were obtained from AJAX Chemicals, Australia; acetic acid (AA, 100%, v/v) was purchased from BDH Chemicals Australia Pty. Ltd. HPLC grade acetonitrile (ACN) was obtained from Merck (Darmstadt, Germany). Water was distilled and deionized in a Milli-Q system (Millipore, Bedford, MA, USA). The stationary phase (4 m particle size, 100 A˚ pore size), called Diamond Hydride (DH), contains a small amount of an organic moiety (∼2% bonded alkyl moiety carbon loading as determined by elemental analysis) on a silica hydride surface. This stationary phase was packed into capillary columns (0.3 mm × 150 mm) by MicroSolv Technology (Eatontown, NJ, USA) based on specifications developed by the authors of this study to meet the requirements of industry standards. 2.2. Liquid chromatography and mass spectrometry The chromatographic analysis of peptides was conducted on an Agilent 1100 capillary LC system (Agilent Technologies, Palo Alto, CA, USA) coupled to an ion-trap MS system (Agilent 1100 series LC/MSD-SL). The studies with the modified silica hydride capillaries were performed at room temperature by using a capillary pump. The outlet of the capillary was connected to the diode array detector and the electrospray source of the ion-trap mass spectrometer. The experimental data for the retention maps were acquired using isocratic elution with different acetonitrile–water mixtures containing between 30 and 95% (v/v) acetonitrile and 0.1% (v/v) formic acid or 0.1% (v/v) acetic acid, respectively. The flow rate was 4 L/min. The ANP gradient separations of the peptides were performed using acetonitrile as the weak eluent (A) and water as the strong eluent (B), with both eluents containing either formic acid or acetic acid as mobile phase additive. Further details of the experimental conditions are noted in the respective figure captions. ESI-MS analysis was carried out in the positive ion mode. The electrospray voltage was 3.5 kV and ion trap mass spectrometer was operated in full scan mode in the range of 200–1200 m/z. The nebulizing gas (N2 ) pressure, the drying gas (N2 ) flow rate, and the drying gas temperature were set at 10 psi, 5 L/min, and 300 ◦ C, respectively. The ion accumulation time was automatically adjusted using the Ion Charge Control (ICC) feature of the instrument. The maximal accumulation time was set at 300 ms and the ICC target was set at 30,000. All system control and data acquisition were conducted with Agilent ChemStation and MSD Trap Control software. 3. Results and discussion The analytes employed in this study comprised some very hydrophobic and hydrophilic peptides with diverse isoelectric points (pIs), and thus served as useful probes to explore the potential of the silica hydride phase in this field of bioanalysis. The attribution of ANP modality for the separation of peptides follows from the nature of the mobile phases employed and distinguishes these silica hydride materials from alternative HILIC stationary phases. Depending on the amount of water in the mobile phase, silica hydride phases can operate either under ANP or reversedphase conditions. HILIC materials do not have this capability and thus can only retain polar compounds under such conditions. The
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Table 1 Amino acid sequence, calculated hydrophobicity [21] and isoelectric point (pI) [22] of peptides (1)–(7). Peptide code
Sequence
Calculated hydrophobicity
pI
1 2 3 4 5 6 7
H-Gly-Arg-Ala-Asp-Ser-Pro-Lys-OH H-Arg-Gly-Asp-OH H-Lys-Gln-Ala-Gly-Asp-Val-OH H-Tyr-Ile-Gly-Ser-Arg-OH H-Trp-His-Trp-Leu-Gln-Leu-OH H-Tyr-Gly-Gly-Phe-Leu-OH H-Tyr-Tyr-Tyr-Tyr-Tyr-Tyr-OH
−2.31 −0.84 0.35 4.11 9.65 10.61 11.34
9.4 6.1 5.8 9.1 7.1 5.5 5.5
silica hydride material (DH) used in this study has previously been shown to be a highly effective stationary phase for the retention of polar low molecular weight metabolites [16–19]. In order to explore the range of retention characteristics that this type of silica hydride phase might encompass with hydrophilic peptides, a low molecular mass peptide set ranging in hydrophobicity values from −2.31 (most hydrophilic) to 11.34 (most hydrophobic) and in pI values from 5.5 to 9.1 was investigated, with the individual peptides small enough to not show any secondary structural elements. The structures and physical properties of these peptides are listed in Table 1. Fig. 1 shows a plot of retention time vs. percent composition of organic solvent in the mobile phase for the isocratic elution of the three most hydrophilic peptides (1–3) listed in Table 1. As might be expected for polar compounds with the DH adsorbent, typical ANP behavior was observed, i.e., peptide retention increased as the amount of the less polar component (acetonitrile) of the mobile phase was increased. In addition, at a given mobile phase composition, the most hydrophilic peptide (1) was the most strongly retained, followed by lower retentions of the less polar peptides (3) and (2). Thus, for the hydrophilic species, the behavior of peptides with the DH adsorbent paralleled the behavior of small polar solutes, such as amino acids, organic acids, carbohydrates and nucleotides [16]. A more surprising result was the retention behavior (Fig. 2) of the four hydrophobic peptides (4)–(7) determined under similar ANP conditions, and again using low concentrations of formic acid as an additive. These peptides range in hydrophobicity values from 4.11 to 11.34. As evident from Fig. 2, these hydrophobic peptides are retained under conditions that are normally used for polar compounds, i.e., high-organic low-aqueous mobile phases. The precise mechanism controlling the strong retention of these hydrophobic peptides under these experimental conditions has yet to be fully
Fig. 2. Effects of acetonitrile concentration in the mobile phase on the retention of hydrophobic peptides. Chromatographic conditions as in Fig. 1.
Fig. 1. Effects of acetonitrile concentration in the mobile phase on the retention of hydrophilic peptides. Chromatographic conditions: isocratic elution using a Diamond Hydride capillary column (0.3 mm × 150 mm, 4 m) with water–organic solvent mixtures at different volume fractions of acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 4 L/min. The sample contained peptides (1)–(3) at a concentration of 10 g/mL each in 0.1% formic acid, 50% acetonitrile (v/v); the injection volume was 0.1 L.
Fig. 3. Effects of acetonitrile concentration in the mobile phase on the retention of hydrophobic peptides. Chromatographic conditions: isocratic elution using a Diamond Hydride capillary column (0.3 mm × 150 mm, 4 m) with water–organic solvent mixtures at different volume fractions of acetonitrile containing 0.1% (v/v) acetic acid at a flow rate of 4 L/min. The sample contained peptides (4)–(7) at a concentration of 10 g/mL each in 0.1% acetic acid, 50% acetonitrile (v/v); the injection volume was 0.1 L.
elucidated; however, it can be noted that the interaction between the polar amide backbone moieties of the hydrophobic peptides and the stationary phase through hydrogen bonding effects would account for this behavior. Moreover, as shown in Fig. 2, the elution order of these peptides (4)–(7) at a given mobile phase composition paralleled the results obtained for peptides (1)–(3) (Fig. 1), with the least hydrophobic of the four peptides having the greatest retention whilst the most hydrophobic peptide having the lowest retention, e.g. with 80% (v/v) acetonitrile in the mobile phase, the elution order was 7 < 6 < 5 < 4. The higher retention of peptides 4 and 5 may be attributed to the presence of the ␥-amido- and guanidinyl-amino groups of Gln and Arg, respectively, which may interact with the stationary phase. This is in agreement with earlier work on small molecules containing similar types of amino groups [16]. With 50% (v/v) acetonitrile in the mobile phase, all four peptides co-eluted in the void volume of the packed capillary. Another interesting manifestation of this novel retention behavior was observed for the hydrophobic peptides (4)–(7) when the additive in the mobile phase was changed from formic acid to acetic acid. Fig. 3 shows the retention map for these four hydrophobic peptides (4)–(7) using 0.1% (v/v) acetic acid as the additive in the mobile phase. The elution times for all of the peptides increased in comparison to the data obtained when 0.1% (v/v) formic acid was employed as the additive (Fig. 2). For example, the retention times of peptides (6) and (7) with a mobile phase consisting of 90:10 (v/v) acetonitrile/water with 0.1% (v/v) formic acid were similar to those obtained for a mobile phase consisting of 80:20 (v/v) acetonitrile/water with 0.1% (v/v) acetic acid. Thus, the presence of acetic acid increased the ANP selectivity
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of the silica hydride adsorbent, indicating that weaker hydrogen ion activity leads to stronger retention. A similar effect has been observed for the retention of metabolites with similar silica hydride adsorbents [16]. Further comparison of the data shown in Figs. 2 and 3 reveals that the elution order of peptides (4) and (5) is reversed. Thus, in addition to controlling the elution strength, changes to the composition of the mobile phase additives can also alter selectivity, an important feature that can be used to optimize separations. Finally, it can be noted that whilst all of the hydrophobic peptides eluted near to the void volume when a mobile phase comprising 50:50 (v/v) acetonitrile/water with 0.1% (v/v) formic acid was used, peptides (4) and (5) had considerable retention when the mobile phase was changed to 50:50 (v/v) acetonitrile/water with 0.1% (v/v) acetic acid and this retention increased further as the organic solvent content was increased to higher values. Whilst some ANP materials can exhibit reversed-phase features, such behavior is less pronounced with silica surfaces used in hydrophilic interaction chromatography, as recent investigations indicate [20]. With mobile phases of high organic content, e.g., 50–95% acetonitrile such as those employed in the current study, it is unlikely that any RP-like features will be manifested with the selected peptides, due to contributions from the Si–H groups or the very low level (2%) of bonded n-alkyl groups in the DH silica hydride phase. The significant differences thus noted in Figs. 1 and 2 for the retention behavior of the selected peptides when either 0.1% acetic acid or 0.1% formic acid was used with a mobile phase of the same organic solvent content highlight the important role that secondary modifiers can play with ANP materials. These results thus indicate that significant retention and selectivity effects can occur when either water-rich or waterlean mobile phases are employed, depending on which region of the ANP separation space is employed. While HILIC phases display predominantly normal phase retention, various commercially available ANP materials can also exhibit reversed-phase features. In this context, however, the solvent dependencies of materials, such as this DH silica hydride phase, with peptides significantly differ to that observed [1,2] with traditional RPC adsorbents with mobile phases of high organic solvent content. The origins of this novel attribute of the DH silica hydride phase are undergoing further investigation with structurally more diverse peptide mixtures and these results will be described more fully in a subsequent report. The plots shown in Figs. 1–3 document the broad range of retention properties of this particular type of silica hydride stationary phase. In order to apply these capabilities to samples containing peptides with a wide range of hydrophobicities, gradient elution methods are needed to accomplish such analyses in a reasonable time frame. An example of a gradient separation of the four hydrophobic peptides (4)–(7) with 0.1% (v/v) formic acid as the additive in the mobile phase is shown in Fig. 4A. Almost complete baseline separation was achieved with a 5 min hold period at 90% (v/v) A (weak solvent) followed by a linear gradient to 50% (v/v) A over 20 min. Although further gradient optimization could be undertaken to achieve improved separation or shorter analysis times, by using MS detection in the extracted ion chromatogram (EIC) mode, as is the case in this study, the need for complete baseline separation of all components is removed. A similar strategy was developed using 0.1% (v/v) acetic acid as the additive in the mobile phase. Because of the higher retention of the peptides with acetic acid, the mobile phase utilized an 80:20 (v/v) acetonitrile/water composition and the 20 min gradient went to 30% (v/v) acetonitrile. The resulting base peak chromatogram (BPC represents the intensity of the most intense peak at every time point in the analysis) is shown in Fig. 4B, where an elution order reversal for peptides (4) and (5) in comparison to the results obtained in 0.1% (v/v) formic
Fig. 4. Separation of hydrophobic peptides on a Diamond Hydride capillary column. (A) Eluent A: 0.1% (v/v) formic acid in acetonitrile, eluent B: 0.1% (v/v) formic acid in water; gradient: 0.0–5.0 min 90% (v/v) A, 5.0–25.0 min to 50% (v/v) A. (B) Eluent A: 0.1% (v/v) acetic acid in acetonitrile, eluent B: 0.1% (v/v) acetic acid in water; gradient: 0.0–5.0 min 80% (v/v) A, 5.0–25.0 min to 30% (v/v) A; flow rate: 4 L/min; sample: peptides (4)–(7), 10 g/mL each in 50% (v/v) B; 0.1 L injection.
acid is illustrated. The separations achieved here were similar to those obtained when 0.1% (v/v) formic acid was employed, although the peak widths for peptides (4) and (5) were broader, (cf. Fig. 4A and B). Further enhancement of peak shape can be achieved by making the gradient steeper. In order to show the full range of separation capability of the DH support material, a mixture of all seven peptides was injected onto the capillary. In this case, the concentration of the mobile phase additive, formic acid, was increased to 0.5% (v/v) to reduce the retention of the most hydrophilic species. The base peak chromatogram of this mixture, using the same ANP gradient as shown in Fig. 4A, is illustrated in Fig. 5A. With no gradient optimization, all seven peptides were almost baseline separated. The elution order followed closely the calculated hydrophobicities of the seven compounds, i.e., the most hydrophobic peptide was eluted first and the remainder in close correlation with decreasing hydrophobicity with the most hydrophilic peptide eluting last. As also evident from Fig. 1, only the elution order of peptides (2) and (3) did not match their calculated hydrophobicity. Under these non-optimized conditions, the peak widths of the most hydrophobic peptides (6) and (7) and the most hydrophilic peptide (1) were significantly broader than those for peptides (2)–(5) with intermediate hydrophobicity. The extracted ion chromatogram of peptide 1 is shown in Fig. 5B. Such broad peaks are often the result of slow mass transfer and can be improved by adjusting the gradient to enhance this process. In general, the most efficient way to improve peak shape for the LC separation of polar compounds is to increase the gradient steepness profile, so that over the total elution time the change in
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Fig. 5. (A) Base peak chromatogram (BPC) of peptides (1)–(7) separated on a Diamond Hydride capillary column. (B) Extracted ion chromatogram (EIC) of peptide (1). Conditions: eluent A: 0.5% (v/v) formic acid in acetonitrile, eluent B: 0.5% (v/v) formic acid in water; gradient: 0.0–5.0 min 90% (v/v) A, 5.0–25.0 min to 50% (v/v) A, 25.0–25.1 min to 30% (v/v) A, 25.1–32.0 min hold 30% (v/v) A; flow rate: 4 L/min; sample concentration: 10 g/mL in 50% (v/v) B; 0.1 L injection.
the solvent composition is more rapid. Thus to generate the data shown in Fig. 5 the gradient time was modified according to these general principles with a hold used at a relatively high composition of the weak solvent in the mobile phase in order to have sufficient retention of the earliest eluting solutes. The result of further optimization of the separation is shown in Fig. 6A, where both an acetonitrile and a formic acid gradient, were employed. With this modification, the most hydrophobic compounds had longer retention times but the peaks were much narrower with resolution approximately the same. A more dramatic improvement is seen for peptide (1), the most hydrophilic species. In this case, the peak width was considerably narrower than under the previous gradient conditions as can be seen by the MS-EIC of this compound in Fig. 6B. As evident from the chromatogram shown in Fig. 6A lower resolution between peptides (4) and (2) occurred under these elution conditions compared to the results illustrated in Fig. 5A. As noted above, further optimization of the gradient slope would be anticipated to improve the resolution further, however, since MS detection is now performed with many types of peptide analyses, from a practical perspective such gradient optimization procedures may not be necessary. With most modes of MS detection, since the EICs of each peptide can be used to simultaneously delineate individual peptides in terms of retention and mass, only peptides having the same nominal molecular mass or those differing by, for example, by 1 amu, need to be chromatographically separated. With more advanced tandem MS spectrometers, e.g. triple quadrupole MS/MS instrumentation, having accurate mass capabilities, even this requirement can be substantially circumvented.
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Fig. 6. (A) Base peak chromatogram (BPC) of peptides (1)–(7) separated on a Diamond Hydride capillary column. (B) Extracted ion chromatogram (EIC) of peptide (1). Conditions: eluent A: 0.1% (v/v) formic acid in acetonitrile, eluent B: 0.5% (v/v) formic acid in water; gradient: 0.0–5.0 min 90% (v/v) A, 5.0–10.0 min to 70% (v/v) A, 10.0–20.0 min to 60% (v/v) A, 20.0–20.1 min to 30% (v/v) A, 20.1–30.0 min hold 30% (v/v) A; flow rate: 4 L/min; sample concentration: 10 g/mL in 50% (v/v) B; 0.1 L injection.
4. Conclusions In this investigation the capability of a new class of ANP stationary phase for use in the simultaneous separation of hydrophilic and hydrophobic peptides has been documented. This new separation material was derived from silica hydride with properties that can be modulated in multiple ways, through the appropriate use of isocratic mobile phases of high water content, or in the case of gradient elution procedures, increasing water content and the use of mobile phase additives. The application of silica hydride stationary phases under ANP conditions for the separation of peptides of widely different hydrophilicities/hydrophobicities properties over an extended chromatographic window as demonstrated in this study is anticipated to provide separation scientists and analytical chemists with access to an additional and novel modality of high-resolution separation capabilities that can be readily interfaced with mass spectrometric and other detection methods.
Acknowledgments The financial support of the Australian Research Council, the National Institutes of Health (GM079741-01) and the National Science Foundation (CHE 0724218) is gratefully acknowledged. One of the authors (J.J.P.) would like to acknowledge the support of the Camille and Henry Dreyfus Foundation through a Scholar Award.
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