The "Steady-Migration" Mechanism Of The Two Dipeptides In Reverse Phase Liquid Chromatography

Polypeptides have been attracting scientist attention in many fields,such as biotechnology,biomedical sciences,peptide feed,cosmetology,etc.The separation and purification of peptides have became the key to the development of polypeptides.Although there is no a strict boundary between polypeptides and proteins,the molecular mass of most the polypeptides,especially oligopeptides(dipeptides in this thesis)are far less than proteins.Peptide chain is short,and the changes in molecular conformation in environment is commonly considered very small.However,peptide is quite different from the usual small molecules,but similar to proteins due to the connection among amino acid residues of both peptide and proteins is peptide bonding.Thus,peptides have polar,apolar,and electrostatic groups,performing different characters as the changes in environments.This function of proteins,or peptides involve not only number and kind of amino acid residues,but also its connecting manner of peptide bonding,either C-terminal,or N-terminal.When amino acids link together via peptide bonding to form polypeptides/proteins,the amine group of one amino acid interacts with the carboxyl group of another.An end with an amine group on one end of the protein and a carboxyl group on the other is named the end with the amine on it the"N terminus" because the chemical formula for amine is NH2,while the end with the carboxyl group on it is named the "C terminal" because the chemical formula of a carboxyl is COOH.The difference of connection between the two peptide bonding should be characterized by liquid chromatography(LC).Reverse phase liquid chromatography(RPLC)due to a high resolution has been widely used in the purification of peptides.So the investigation of retention behavior of peptides in LC plays an important role in the purification of peptides and the adsorption theory in liquid-solid interface.The thesis mainly studies on the difference of the retention behavior of dipeptides,Phenylalanine-Isoleucine(Phe-Ile)with N-terminal and Isoleucine-Phenylalanine(Ile-Phe)with C-terminal in RPLC which contains four chapters as follows:Chapter 1.Introduction.The one of the most effective methods for the separation of peptides is liquid chromatography(LC),Many kinds of LC,such as ion-exchang chromatography,hydrophobic interaction chromatography,reverse phase chromatography,and mixed mode chromatography are widely employed in scientific research and applications in industry.Many retention models,such as Synder’ s empirical formula,solubility parameter model,solvatochromic models,solvophobic models,and stoichiometric displacement models were briefly introduced.Chapter 2.The separation of two dipeptides in RPLC.Use various RPLC column lengths for the separation of the two dipeptides of Phe-Ile and Ile-Phe in the system of RPLC.This chapter studies the behavior of the two dipeptides by column length,flow rate of mobile phase,gradient elution mode,and temperature.It was found that different chromatographic conditions lead to the changes in the retention time(Δt),peak width(w),and resolution(Rs)of the dipeptides:(1)The two dipeptides can be completely separated under either gradient,or isocratic elution,and Phe-Ile was found to be firstly eluted out.(2)Under isocratic elution,TR,Δt,w and R were found totally to increase with column length,which are identical to conventional small solutes.However,under gradient elution,when column length increased from 5 cm to 25 cm,the At almost remained invariance,but peak width became narrow.(3)When flow rate increased from 0.50ml/min to 2.0 mL/min,the w decreased,but At increased.(4)When column temperature increased from 10℃-50℃,At decreased,but w kept invariance.(5)When linear gradient elution time increased from 15 min to 40 min,both of Δt and w increased.Both of At and w are the main factors influencing the resolution,so compared to other factors,the flow rate is the most.Chapter 3.The migration mechanism of the dipeptides in RPLC.The "steady migration" mechanism of the two dipeptides is firstly found in RPLC.The relationship of retention time,sampling time and the elution concentration between the two dipeptides during gradients elution in RPLC were investigated and found to compose of the"steady region"(SRN),in which the dipeptides did not move,followed by the "migration region"(MRN),in which the dipeptides moved normally.With non-synchronous sampling,the span of both the SRN and MRN could be quantitatively measured by experiment.The boundary between the two "regions" is called as critical migration time point(TCMP)..Chapter 4.Thermodynamics of the two dipeptides in RPLC.The TCMP and the changes in the migration free energy(ΔG)of dipeptides under the different temperatures were measured and found that temperature effects on the the TCMP.Both of log I and Z values which from stoichiometric displacement theory of the dipeptides were also measured under different temperatures.The mechanism of the "steady migration phenomenon" from thermodynamic was explained from the changes in enthalpy,entropy and the enthalpy-entropy compensation of the two dipeptides.