High Resolution Chromatography and Sensitive Retention: Optimization of the Experimental Conditions for Proteins Extraction by Preparative HPLC

Loai AlJerf, Nuha AlMasri

Abstract


High performance liquid chromatography (HPLC) is employed with its various elution systems for the fractionation (by isocratic or gradient elution) of peptides and proteins. Lichrosorb Diol (pore size 100 Ǻ) was chosen for normal partition chromatography of proteins. More details about these separations are illustrated in the current research. The chromatographic capacity and its resolution are investigated and the guidelines are widely defined. Reverse-phase (RP) (more suitable with Lichrosorb RP-8) sorbent was partially dissolved during elution with n-propanol (˂ 40 vol. /vol.) and lyophilized during fractionation. An outstanding resolution of these compounds was seen both at pH 4.0 and 7.5 under room temperature and low flow rate at linear gradient of n-propanol. Selective adsorption had been initiated at pH < 4 and peak broadening was observed when salts eliminated from the eluents. It is suggested by the results of this paper, the use of normal phase with Lichrosorb Diol for the isolation of the soluble heterogeneous proteins in extremely elevated concentrations of organic solvents. Consequently, an exceptional resolution, high capacity, and diminutive elution times were verified for peptides and proteins separations.

Keywords


High performance liquid chromatography; Gradient elution; Proteins; Peak broadening; Adsorption.

Full Text:

PDF

References


Martin, D.G., Peltonen, R.E., Nielsen, J.W. (1986). Preparative resolution of an actinomycin complex by countercurrent chromatography in the Ito coil planet centrifuge. J. Antibiot., 39(5), 721-723. https://doi.org/10.7164/antibiotics.39.721

Minshull, T.C., Cole, J., Dockrell, D.H., Read, R.C., Dickman, M.J. (2016). Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase×reverse phase chromatography in conjunction with porous graphitic carbon stationary phase. J. Chromatogr. A., 1453, 43-53. https://doi.org/10.1016/j.chroma.2016.05.025

Potts, A.R., Psurek, T., Jones, C., Parris, L., Wise, A. (2012). Validation of a quantitative HPLC method for bacitracin and bacitracin zinc using EDTA as a mobile-phase modifier. J. Pharm. Biomed. Anal., 70, 619-623. https://doi.org/10.1016/j.jpba.2012.06.016

Alwera, S., Bhushan, R. (2017). RP-HPLC enantioseparation of β-adrenolytics using micellar mobile phase without organic solvents. Biomed. Chromatogr., 31(11), e3983. https://doi.org/10.1002/bmc.3983

Yphantis, D.A. (2006). Rapid determination of molecular weights of peptides and proteins. Ann. N. Y. Acad. Sci., 88(3), 586-601. https://doi.org/10.1111/j.1749-6632.1960.tb20055.x

Aljerf, L., Choukaife, A.E. (2017). A novel method to chromatographically resolution of sulphonamides by vapour-programmed Thin-Layer Chromatography. MOJ Bioorg. Org. Chem., 1(4), 00024. https://doi.org/10.15406/mojboc.2017.01.00024

Meyerson, L.R., Abraham, K.I. (1986). High performance liquid chromatographic properties of peptides and proteins on a dihydroxyalkyl bonded silica stationary phase. Peptides., 7(3), 481-489. https://doi.org/10.1016/0196-9781(86)90018-5

Aljerf, L., Alhaffar, I. (2017). Salivary distinctiveness and modifications in males with Diabetes and Behçet's disease. Biochem. Res. Int., 2017, 1-12. https://doi.org/10.1155/2017/9596202

Anspach, F.B. (1994). Silica-based metal chelate affinity sorbents II. Adsorption and elution behavior of proteins on iminodiacetic acid affinity sorbents prepared via different immobilization techniques. J. Chromatogr. A., 676(2), 249-266. https://doi.org/10.1016/0021-9673(94)80425-7

Říčný, J., Tuček, S., Vinš, I. (1992). Sensitive method for HPLC determination of acetylcholine, choline and their analogues using fluorometric detection. J. Neurosci. Methods., 41(1), 11-17. https://doi.org/10.1016/0165-0270(92)90119-x

Johnsen, A.A. (1991). Nondestructive amino acid analysis at the picomole level of proline-containing peptides using aminopeptidase M and prolidase: Application to peptides containing tyrosine sulfate. Anal. Biochem., 197(1), 182-186. https://doi.org/10.1016/0003-2697(91)90376-5

Kondo, N., Imai, K., Isobe, M., Goto, T. (1984). A low picomole fluorometric detection system for amino acid analysis. Agric. Biol. Chem., 48(6), 1595-1601. https://doi.org/10.1271/bbb1961.48.1595

Sasaki, Y., Coy, D.H. (1987). Solid phase synthesis of peptides containing the CH2NH peptide bond isostere. Peptides., 8(1), 119-121. https://doi.org/10.1016/0196-9781(87)90174-4

Prokopov, S.V., Kurbatova, S.V., Davankov, V.A., Il’in, M.A. (2012). Chromatographic retention of adamantylamidrazones and triazoles by octadecyl silica gel and hypercrosslinked polystyrenes from water-acetonitrile solutions. Russ. J. Phys. Chem. A., 86(5), 852-859. https://doi.org/10.1134/s0036024412050299

Kalchenko, O.I., Cherenok, S.O., Savonik, L.I., Solovyov, A.V., Gorbachuk, V.V., Kalchenko, V.I. (2014). Investigation of sorption of calix[4]arene and calix[4]resorcinarene tetraalkyl derivatives with the LiChrosorb RP 18 surface by RP HPLC and molecular modelling methods. Ž. org. farm. hìm., 12(3), 17-22. https://doi.org/10.24959/ophcj.14.799

Naito, N., Takahashi, A., Nakai, Y., Kawauchi, H. (1984). Immunocytochemical identification of the proopiocortin-producing cells in the chum salmon pituitary with aantisera to endorphin and NH2-terminal peptide of salmon proopiocortin. Gen. Comp. Endocrinol., 56(2), 185-192. https://doi.org/10.1016/0016-6480(84)90029-7


Refbacks

  • There are currently no refbacks.


Copyright © 2018 Journal of Progressive Research in Modern Physics and ChemistryAll rights reserved.

ISSN: 2456-6438

For any query/support contact us at jprmpceditor@scischolars.com, ssroscischolars@gmail.com,

editorial@scischolars.com