High performance capillary electrophoresis - T.E.A.M.
High performance capillary electrophoresis - T.E.A.M. High performance capillary electrophoresis - T.E.A.M.
Principles that ion. The mobility is determined by the electric force that the molecule experiences, balanced by its frictional drag through the medium. That is Electric force (F µ e a = E ) (2) Frictional force (F F ) The electric force can be given by F E = q E (3) and the frictional force (for a spherical ion) by F F = -6 p h r v (4) where q = ion charge h = solution viscosity r = ion radius v = ion velocity. During electrophoresis a steady state, defined by the balance of these forces, is attained. At this point the forces are equal but opposite and q E = 6 p h r v (5) Solving for velocity and substituting equation (5) into equation (1) yields an equation that describes the mobility in terms of physical parameters q m e = (6) 6 p h r From this equation it is evident that small, highly charged species have high mobilities whereas large, minimally charged species have low mobilities. 18
0 µ Full charge Zero charge α = 0 2 pK pK a1 a2 12 pH Figure 3 Mobility of two weak acids as a function of pH Capillary wall = 1/2 α = 1 Diffuse layer Stern layer Figure 4 Representation of the double-layer at the capillary wall 1 The electrophoretic mobility usually found in standard tables is a physical constant, determined at the point of full solute charge and extrapolated to infinite dilution. This usually differs from that determined experimentally. The latter is called the effective mobility and is often highly dependent on pH (that is, solute pK a ) and composition of the running buffer. The differences between the absolute and effective mobilities are demonstrated in figure 3. Here, two hypothetical solutes are shown to possess the same electrophoretic mobility at full charge. From a mobility table, these solutes would appear to be inseparable since there would be no differential migration. However, these species have different pK a values and different mobilities depending on their pH-controlled charge. 2.3.2 Electro-osmotic flow (EOF) A fundamental constituent of CE operation is electroosmotic, or electroendosmotic flow (EOF). EOF is the bulk flow of liquid in the capillary and is a consequence of the surface charge on the interior capillary wall. The EOF results from the effect of the applied electric field on the solution double-layer at the wall (figure 4). The EOF controls the amount of time solutes remain in the capillary by superposition of flow on to solute mobility. This can have the effect of altering the required capillary length, but does not affect selectivity. Under aqueous conditions most solid surfaces possess an excess of negative charges. This can result from ionization of the surface (that is, acid-base equilibria) and/or from adsorption of ionic species at the surface. For fused silica both processes probably occur, although the EOF is most strongly controlled by the numerous silanol groups (SiOH) that can exist in anionic form (SiO - ) (figure 5a). Although Principles 19
- Page 1: An introduction High performance ca
- Page 4 and 5: Copyright © 2000 Agilent Technolog
- Page 6 and 7: Foreword Capillary electrophoresis
- Page 8 and 9: Table of content Foreword .........
- Page 10 and 11: Scope The purpose of this book is t
- Page 12 and 13: Introduction 1.1 High performance c
- Page 14 and 15: Introduction sis, methods for on-ca
- Page 16 and 17: Principles 2.1 Historical backgroun
- Page 20 and 21: Principles the exact pI of fused si
- Page 22 and 23: Principles µ EOF × 10 -4 (cm 2 /
- Page 24 and 25: Principles For the analysis of smal
- Page 26 and 27: Principles µ EOF ( × 10 -4 cm 2 /
- Page 28 and 29: Principles Total length Effective l
- Page 30 and 31: Principles Note that equation (15)
- Page 32 and 33: Principles determined by the capill
- Page 34 and 35: Principles Current (uA) 300 250 200
- Page 36 and 37: Principles The contribution of inje
- Page 38 and 39: Principles k' H N H, µm 0.001 0.58
- Page 40 and 41: Principles Figure 19 Electrodispers
- Page 42 and 43: Principles rapidly eluting ions, th
- Page 44 and 45: Principles 44
- Page 46 and 47: Modes Mode Capillary zone electroph
- Page 48 and 49: Modes 3.1.1 Selectivity and the use
- Page 50 and 51: Modes Name pK a Phosphate 2.12 (pK
- Page 52 and 53: Modes EOF No flow Figure 22 Elimina
- Page 54 and 55: Modes Absorbance 214 nm 0.05 0.04 0
- Page 56 and 57: Modes Type Comment Silylation coupl
- Page 58 and 59: Modes Type Result Comment Extremes
- Page 60 and 61: Modes Figure 29 CZE of reversed pha
- Page 62 and 63: Modes Figure 33 Ion analysis of fer
- Page 64 and 65: Modes The separation mechanism of n
- Page 66 and 67: Modes the stationary phase in LC. S
0<br />
µ<br />
Full charge<br />
Zero<br />
charge<br />
α = 0<br />
2<br />
pK pK<br />
a1 a2<br />
12<br />
pH<br />
Figure 3<br />
Mobility of two weak acids as a function<br />
of pH<br />
Capillary wall<br />
= 1/2<br />
α = 1<br />
Diffuse layer<br />
Stern layer<br />
Figure 4<br />
Representation of the double-layer at the<br />
<strong>capillary</strong> wall 1<br />
The electrophoretic mobility usually found in standard<br />
tables is a physical constant, determined at the point of full<br />
solute charge and extrapolated to infinite dilution. This<br />
usually differs from that determined experimentally. The<br />
latter is called the effective mobility and is often highly<br />
dependent on pH (that is, solute pK a<br />
) and composition of<br />
the running buffer.<br />
The differences between the absolute and effective mobilities<br />
are demonstrated in figure 3. Here, two hypothetical<br />
solutes are shown to possess the same electrophoretic<br />
mobility at full charge. From a mobility table, these solutes<br />
would appear to be inseparable since there would be no<br />
differential migration. However, these species have different<br />
pK a<br />
values and different mobilities depending on their<br />
pH-controlled charge.<br />
2.3.2 Electro-osmotic flow (EOF)<br />
A fundamental constituent of CE operation is electroosmotic,<br />
or electroendosmotic flow (EOF). EOF is the bulk<br />
flow of liquid in the <strong>capillary</strong> and is a consequence of the<br />
surface charge on the interior <strong>capillary</strong> wall. The EOF<br />
results from the effect of the applied electric field on the<br />
solution double-layer at the wall (figure 4). The EOF<br />
controls the amount of time solutes remain in the <strong>capillary</strong><br />
by superposition of flow on to solute mobility. This can<br />
have the effect of altering the required <strong>capillary</strong> length, but<br />
does not affect selectivity.<br />
Under aqueous conditions most solid surfaces possess an<br />
excess of negative charges. This can result from ionization<br />
of the surface (that is, acid-base equilibria) and/or from<br />
adsorption of ionic species at the surface. For fused silica<br />
both processes probably occur, although the EOF is most<br />
strongly controlled by the numerous silanol groups (SiOH)<br />
that can exist in anionic form (SiO - ) (figure 5a). Although<br />
Principles<br />
19
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