The perchlorate anion is more effective than the trifluoroacetate anion as an ion-pairing reagent for reversed-phase chromatography of peptides

Shibue, Mitsukuni; Mant, Colin T.; Hodges, Robert S.

doi:10.1016/j.chroma.2005.02.063

Cited by 54 publications

(43 citation statements)

References 31 publications

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“…This is in agreement with many highperformance liquid chromatography (HPLC) studies, which show that trifluoroacetic acid (besides other perfluorinated acids), especially, is often used to improve resolution and peak shape of peptides [13][14][15]. Formic acid, instead of trifluoroacetic acid, is also often used in column chromatography, despite its worse ion-pairing capability.…”

Section: Discussionsupporting

confidence: 87%

Retention and separation efficiency of some synthetic oligopeptides in reversed-phase thin-layer chromatography

Gwarda¹,

Tomczyszyn²,

Misicka³

et al. 2015

Acta Chromatographica

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Summary.Influence of the following variables such as adsorbent type, type and concentration of organic modifier in mobile phase, type and concentration of ion-pairing reagent, or pH of the mobile phase buffer on retention of some synthetic peptides in reversed-phase high-performance thin-layer chromatography systems has been investigated. The investigations have been also focused on influence of the variables mentioned on solute zone shape regarding optimization of their separation. Remarks about solute retention mechanism have been also provided.

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Section: Discussionsupporting

confidence: 87%

Retention and separation efficiency of some synthetic oligopeptides in reversed-phase thin-layer chromatography

Gwarda¹,

Tomczyszyn²,

Misicka³

et al. 2015

Acta Chromatographica

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“…1 and 2), the effect of increasing counter-ion concentration, at least over the 10-30 mM range, appears not so clear cut, where the response of all three groups of peptides to increasing concentrations of H 3 PO 4 , TFA, PFPA and HFBA were similar despite the increase in counterion hydrophobicity of phosphate < TFA − < PFPA − < HFBA − (Table 2). This phenomenon is investigated in greater detail in a companion study [25] which investigates the effect on peptide retention behaviour of anionic ion-pairing reagent concentration over a much wider concentration range (1-60 mM) compared to the present study. Table 3 reports the effect of increasing concentration (10-30 mM) of the four ion-pairing reagents on the resolution of representative peptide pairs 1a/1b and 5h/5j, i.e., resolution of peptides with the same net positive charge (+1 and +5, respectively).…”

Section: Effect Of Concentration Of Ion-pairing Reagent On Peptide Elmentioning

confidence: 99%

Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography

Shibue

Mant

Hodges

2005

Journal of Chromatography A

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Despite the continuing dominance of trifluoroacetic acid (TFA) as the anionic ion-pairing reagent of choice for peptide separations by reversed-phase high-performance liquid chromatography (RP-HPLC), we believe that a step-by-step approach to re-examining the relative efficacy of TFA compared to other ion-pairing reagents is worthwhile, particularly for the design of separation protocols for complex peptide mixtures, e.g., in proteomics applications. Thus, we applied RP-HPLC in the presence of different concentrations of anionic ion-pairing reagents -phosphoric acid, TFA, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA) -to a mixture of three groups of four 10-residue peptides, these groups containing peptides of +1, +3 or +5 net charge. Overall separation of the 12-peptide mixture improved with increasing reagent hydrophobicity (phosphate − < TFA − < PFPA − < HFBA − ) and/or concentration of the anion, with reagent hydrophobicity having a considerably more pronounced effect than reagent concentration. HFBA, in particular, achieved an excellent separation at a concentration of just 10 mM, whereby the peptides were separated by charged groups (+1 < +3 < +5) and hydrophobicity within these groups. There was an essentially equal effect of reagent hydrophobicity and concentration on each positive charge of the peptides, a useful observation for prediction of the effect of varying counterion concentration hydrophobicity and/or concentration during optimization of peptide purification protocols. Peak widths were greater for the more highly charged peptides, although these could be decreased significantly by raising the acid concentration; concomitantly, peptide resolution increased with increasing concentration of ion-pairing reagent.

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“…In addition, 3 mmol/L sodium perchlorate as an anionic ion pair reagent in the eluent was effective for excellent separation of idebenone from other fluorescent substances in blood. 7,32) On the other hand, 2-cyanoacetamide was soluble and stable in the eluent; therefore, this post-column derivatization HPLC system was simplified because only two pumps were used to deliver the eluent containing 2-cyanoacetamide and the alkaline solution. Figure 3A shows a chromatogram of the standard solution of idebenone.…”

Section: Resultsmentioning

confidence: 99%

Determination of Idebenone in Plasma by HPLC with Post-Column Fluorescence Derivatization Using 2-Cyanoacetamide

Nohara

Suzuki

Yamazaki

et al. 2012

CHEMICAL & PHARMACEUTICAL BULLETIN

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Idebenone is a benzoquinone analog that is used in the treatment of several neurological disorders including Friedreich's ataxia. It was found that the reaction of idebenone with 2-cyanoacetamide under alkaline conditions generates fluorescent products, and the reaction was considered to proceed via Craven's reaction. The reaction mixture from idebenone gave fluorescence with excitation and emission maximum wavelengths at 358 nm and 409 nm, respectively. It was adopted for HPLC with post-column fluorescence derivatization of idebenone. Idebenone in the plasma showed a linear response in the range of 0.5-32 ng (25-1600 ng/mL), and the quantitation limit (S/N 10) was 12.5 ng/mL. The detection limit (S/N 3) of the standard solution of idebenone was 0.1 ng. The HPLC system was applied to the human blood plasma obtained by finger prick. The plasma sample obtained by finger prick gave a similar chromatogram to that of venous blood obtained by venipuncture.Key words idebenone; finger prick; 2-cyanoacetamide; fluorescence derivatization; HPLC Idebenone is a synthetic analog of ubiquinone, 1) which is an essential component of the mitochondrial respiratory chain responsible for oxidative phosphorylation, and may prevent oxidative damage in the human body as an antioxidant. 2,3)Idebenone also has potent antioxidant activity and facilitates the flux of electrons along the mitochondrial electron transport chain, increasing the production of ATP, and has already been clinically investigated for the treatment of several neurological disorders, which include Friedreich's ataxia and Leber's hereditary optic neuropathy. 4,5) The pharmacokinetic properties of idebenone have been investigated by HPLC. Several procedures have been developed to analyze idebenone in biological samples by HPLC coupled with UV, electrochemical, or mass spectrometry detection. [6][7][8][9] HPLC methods employing mass spectrometry and electrochemical detectors have higher selectivity and sensitivity than those with a UV detector; however, these methods are not available in many laboratories because of a lack of specialized equipment. HPLC coupled with a fluorophotometric detector has been widely used in the determination of various substances in biological samples because of its high sensitivity and selectivity. However, idebenone needs fluorescence derivatization for the development of fluorimetric detection because it is not a fluorescent substance itself. 2-Cyanoacetamide has been used as a post-column derivatization reagent of fluorimetric HPLC for catecholamines, 3,4-dihydroxyphenylalanine, unsaturated disaccharides, and benzoquinones. [10][11][12][13][14][15] In the present study, 2-cyanoacetamide was tested for the fluorophotometric reagent of idebenone because idebenone is a benzoquinone compound. It was found that idebenone reacted with 2-cyanoacetamide under alkaline conditions with heating to form fluorescent products. This reaction was applied to HPLC with post-column fluorescence derivatization of idebenone.The salting out effect is useful a...

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The perchlorate anion is more effective than the trifluoroacetate anion as an ion-pairing reagent for reversed-phase chromatography of peptides

Cited by 54 publications

References 31 publications

Retention and separation efficiency of some synthetic oligopeptides in reversed-phase thin-layer chromatography

Retention and separation efficiency of some synthetic oligopeptides in reversed-phase thin-layer chromatography

Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography

Determination of Idebenone in Plasma by HPLC with Post-Column Fluorescence Derivatization Using 2-Cyanoacetamide

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