Abstract
The retention data of the components of the peptide mixture obtained by the enzymatic digestion of cytochrome-c were measured under different gradient conditions, using different organic modifiers (acetonitrile, methanol, and tetrahydrofuran) in a 0.1% (v/v) aqueous solution of trifluoroacetic acid. The influences of the nature of the organic modifier, of the gradient steepness, and of the column length on the retention and the resolution of these peptides were studied. Peak capacity and retention factor calculations for gradient separations were helpful in determining a desired gradient for this separation. All three organic modifiers are shown to cause reversal orders of some peaks in the cytochrome-c digest separation.
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Rajan A, Mullen J, Bhatanagar N, Dubey A, Niemz A, Chakravarti B, Chakravarti DN (2004) J Assoc Lab Automation 9:312–318
Mant CT, Hodges RS (1991) High-performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis, and Conformation, CRC Press, Boca Raton, FL
Stone KL, Williams KR (2002) Reverse-phase HPLC separation of enzymatic digests of proteins. In: Walker JM (ed) The protein protocols handbook, Humana Press, Totawa, NJ, pp. 533–542
Larive CK, Lunte SM, Zhong M, Perkins MD, Wilson GS, Gokulrangan G, Williams T, Afroz K, Schoneich C, Derrick TS, Middaugh CR, Bogdanowich-Knipp S (1999) Anal Chem 71:389R–423R
Sereda TJ, Mant CT, Hodges RS (1997) J Chromatogr A 776:153–165
Marchetti N, Guiochon G (2005) Anal Chem 77:3425–3430
Mao Y, Zhang X (2003) Electrophoresis 24:3289–3295
Diaz RS, Regueiro P, Monreal J, Tandler CJ (1991) J Neurosci Research 29:114–120
Glajch JL, Quarry MA, Vasta JF, Snyder LR (1986) Anal Chem 58:280–285
Kunitani M, Johnson D, Snyder LR (1986) J Chromatogr 371:313–333
Dolan JW, Lommen DC, Snyder LR (1989) J Chromatogr 485:91–112
Chloupek RC, Hancock WS, Snyder LR (1992) J Chromatogr 594:65–73
Chloupek RC, Hancock WS, Marchylo BA, Kirkland JJ, Boyes BE, Snyder LR (1994) J Chromatogr A 686:45–59
Snyder LR, Dolan JW (1998) The linear-solvent-strength model of gradient elution. In: Brown PR, Grushka E (eds.) Advances in chromatography. Marcel Dekker, New York, pp. 115–187
Dolan JW, Snyder LR, Djordjevic NM, Hill DW, Heukelem LV, Waeghe TJ (1997) J Chromatogr A 857:1–20
McCalley DV (2004) J Chromatogr A 1038:77–84
Neue UD (2005) J Chromatogr A 1079:153–161
Snyder LR, Kirkland JJ, Glajch JL (1997) Practical HPLC Method Development, Wiley, New York
McCalley DV (2005) LC-GC (North America) 23:162–180
Neue UD, Mazzeo JR (2001) J Sep Sci 24:921–929
Gillar M, Daly AE, Kele M, Neue UD, Gebler JC (2004) J Chromatogr A 1061:183–192
Shen Y, Zhang R, Moore RJ, Kim J, Metz TO, Hixson KK, Zhao R, Livesay ER, Udseth HR, Smith RD (2005) Anal Chem 77:3090–3100
Davis JM, Giddings JC (1983) Anal Chem 55:418–424
Giddings JC (1991) Unified separation science, John Wiley and Sons, New York
Horvath CG, Lipsky SR (1967) Anal Chem 39:1893
Gritti F, Guiochon G (2003) J Chromatogr A 1021:25–53
Qian MG, Lubman DM (1995) Anal Chem 67:2870–2877
Snyder LR, Saunders DL (1969) J Chromatogr Sci 7:195–208
Jandera P, Churacek J (1985) Gradient elution in column liquid chromatography, Elsevier, Amsterdam
Snyder LR, Stadalius MA (1986) High-performance liquid chromatography: advances and perspectives, Academic Press, New York
Dolan JW, Snyder LR, Quarry MA (1987) Chromatographia 24:261–276
Jupille TH, Dolan JW, Snyder LR (1988) American Laboratory 20:20–24
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Riddle, L.A., Guiochon, G. Influence of Mobile Phase Gradients on the Retention and Separation of Peptides from a Cytochrome-c Digest by Reversed-Phase Liquid Chromatography. Chroma 64, 1–7 (2006). https://doi.org/10.1365/s10337-006-0839-1
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DOI: https://doi.org/10.1365/s10337-006-0839-1