Skip to Main Content
 

Global Search Box

 
 
 

ETD Abstract Container

Abstract Header

OPTIMIZATION OF GRADIENT CHROMATOFOCUSING FOR THE PROTEIN SEPARATION AND LC-MS/MS DETERMINATION OF NGP1-01 IN MOUSE SERUM, BRAIN, AND RETINA

Jogiraju, Harini
http://rave.ohiolink.edu/etdc/view?acc_num=csu1389704171

Abstract Details

2013, Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, College of Sciences and Health Professions.
Gradient chromatofocusing (GCF) is a chromatographic technique developed by our laboratory that generates linear pH gradients using weak anion-exchange HPLC with inexpensive low-molecular buffer components. This method shows significant advantages over conventional chromatofocusing and salt gradient ion-exchange chromatography in protein separation on DEAE column. In GCF an elution buffer (consisting of multiple acidic buffers with evenly spaced pKa values) is mixed in successively greater proportions with application buffer (composed of multiple basic buffers with evenly spaced pkas), to generate a smooth linear pH gradient. GCF has the advantage of giving an estimate of the protein pI and being able to be directly interfaced to a mass spectrometer when volatile buffers are used. Although GCF yields considerable success in protein separation, extensive study has not been done yet. Further characterization and method optimization studies in GCF are done in the present work. Chapter 1 describes the characteristics of the conventional ion-exchange chromatography, chromatofocusing, and gradient chromatofocusing for protein separation. In the second chapter, a new method with smooth linear pH- gradients on self packed DEAE anion-exchange column was developed by introducing “bridging” buffers between application and elution buffers. This was employed for the chromatographic separation of a mixture of conalbumin, rat albumin, and ß-lactoglobulin which resulted with good resolutions. In the third chapter, the effect of the number of buffer components on protein separation in high-performance gradient chromatofocusing ion-exchange using linear pH-gradients was studied. The results showed that the number of buffer components has significant effect on protein separation. Results showed the increased resolutions and decreased peak widths at half height were obtained with increased number of buffer components. This optimized GCF technique was applied to the separation of prolactin isoforms by a PEI weak anion exchange column. In chapter four, the development and validation according to FDA guidelines of a sensitive quantitative LC-MS/MS method for the determination of a neuroprotective compound, NGP1-01, in mouse serum is described. The developed method was applied to preliminary pharmacokinetic studies to quantify NGP1-01 in limited number of dosed mouse serum samples to show the suitability for undertaking full NGP1-01 pharmacokinetic studies.
David J. Anderson, PhD (Committee Chair)
Amin Zhou, PhD (Committee Member)
Xue-Long Sun, PhD (Committee Member)
Xiang Zhou, PhD (Committee Member)
Robert Mensforth, PhD (Committee Member)
Chemistry

Recommended Citations

Citations

  • Jogiraju, H. (2013). OPTIMIZATION OF GRADIENT CHROMATOFOCUSING FOR THE PROTEIN SEPARATION AND LC-MS/MS DETERMINATION OF NGP1-01 IN MOUSE SERUM, BRAIN, AND RETINA [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1389704171

    APA Style (7th edition)

  • Jogiraju, Harini. OPTIMIZATION OF GRADIENT CHROMATOFOCUSING FOR THE PROTEIN SEPARATION AND LC-MS/MS DETERMINATION OF NGP1-01 IN MOUSE SERUM, BRAIN, AND RETINA . 2013. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1389704171.

    MLA Style (8th edition)

  • Jogiraju, Harini. "OPTIMIZATION OF GRADIENT CHROMATOFOCUSING FOR THE PROTEIN SEPARATION AND LC-MS/MS DETERMINATION OF NGP1-01 IN MOUSE SERUM, BRAIN, AND RETINA ." Doctoral dissertation, Cleveland State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=csu1389704171

    Chicago Manual of Style (17th edition)

csu1389704171
1,307
© 2013, all rights reserved.
This open access ETD is published by Cleveland State University and OhioLINK.
Release 3.2.12