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 For the analysis of small ions (for example, sodium, potassium, chloride) the magnitude of the EOF is usually not greater than the solute mobilities. In addition, modification of capillary wall charge can decrease EOF while leaving solute mobility unaffected. In these circumstances, anions and cations can migrate in opposite directions. 2.3.2.1 EOF control While the EOF is usually beneficial, it often needs to be controlled. At high pH, for example, the EOF may be too rapid, resulting in elution of solute before separation has occurred. Conversely, at low or moderate pH, the negatively charged wall can cause adsorption of cationic solutes through coulombic interactions. This latter phenomenon has been especially problematic for basic protein separations. In addition, electrophoretic separation modes such as isoelectric focusing, isotachophoresis, and capillary gel electrophoresis often require reduction of EOF. Fundamentally, control of EOF requires alteration of the capillary surface charge or buffer viscosity. There are several methods to accomplish this, as detailed in table 1 and in the following paragraphs. Note that conditions that affect the surface charge of the wall often affect the solute (such as buffer pH). Successful separations are usually obtained when the conditions optimize both EOF and solute mobility properties. The rate of EOF can most easily be decreased by lowering the electric field, as described by equation (7). This action, however, has numerous disadvantages with regard to analysis time, efficiency, and resolution. From a practical point of view, the most dramatic changes in EOF can be made simply by altering the pH of the buffer, as described above (figure 6). Adjusting the pH, however, can also affect the solute charge and mobility. Low pH buffers will proto- 24
Variable Result Comment Principles Electric field Proportional change • Efficiency and resolution in EOF may decrease when lowered • Joule heating may result when increased Buffer pH EOF decreased at low pH ● Most convenient and useful and increased at high pH method to change EOF ● May change charge or structure of solute Ionic strength Decreases zeta potential ● High ionic strength generates or buffer and EOF when increased high current and possible concentration Joule heating ● Low ionic strength problematic for sample adsorption ● May distort peak shape if conductivity different from sample conductivtiy ● Limits sample stacking if reduced Temperature Changes viscosity ● Often useful since tempera- 2 – 3% per°C ture is controlled instrumentally Organic Changes zeta potential ● Complex changes, effect modifier and viscosity (usually most easily determined decreases EOF) experimentally ● May alter selectivity Surfactant Adsorbs to capillary ● Anionic surfactants can wall via hydrophobic and/ increase EOF or ionic interactions ● Cationic surfactant can Neutral Adsorbs to capillary ● Decreases EOF by shielding hydrophilic wall via hydrophobic surface charge and polymer interactions increasing viscosity Table 1 Methods to control electroosmotic flow Covalent Chemical bonding to ● Many modifications possible coating capillary wall (hydrophilicity or charge) ● Stability often problematic nate both the capillary surface and the solute, while high pH buffers deprotonate both. Knowledge of solute pI is often useful in selecting the appropriate pH range of the running buffer. 25
- 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 18 and 19: Principles that ion. The mobility i
- Page 20 and 21: Principles the exact pI of fused si
- Page 22 and 23: Principles µ EOF × 10 -4 (cm 2 /
- 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
- Page 68 and 69: Modes Amplitude 2 a) with a migrati
- Page 70 and 71: Modes CGE t = 0 t > 0 Polymer matri
- Page 72 and 73: Modes Crosslinked polyacrylamide, a
Variable Result Comment<br />
Principles<br />
Electric field Proportional change • Efficiency and resolution<br />
in EOF<br />
may decrease when lowered<br />
• Joule heating may result<br />
when increased<br />
Buffer pH EOF decreased at low pH ● Most convenient and useful<br />
and increased at high pH method to change EOF<br />
● May change charge or<br />
structure of solute<br />
Ionic strength Decreases zeta potential ● <strong>High</strong> ionic strength generates<br />
or buffer and EOF when increased high current and possible<br />
concentration<br />
Joule heating<br />
● Low ionic strength problematic<br />
for sample adsorption<br />
● May distort peak shape if<br />
conductivity different from<br />
sample conductivtiy<br />
● Limits sample stacking if<br />
reduced<br />
Temperature Changes viscosity ● Often useful since tempera-<br />
2 – 3% per°C ture is controlled instrumentally<br />
Organic Changes zeta potential ● Complex changes, effect<br />
modifier and viscosity (usually most easily determined<br />
decreases EOF)<br />
experimentally<br />
● May alter selectivity<br />
Surfactant Adsorbs to <strong>capillary</strong> ● Anionic surfactants can<br />
wall via hydrophobic and/ increase EOF<br />
or ionic interactions<br />
● Cationic surfactant can<br />
Neutral Adsorbs to <strong>capillary</strong> ● Decreases EOF by shielding<br />
hydrophilic wall via hydrophobic surface charge and<br />
polymer interactions increasing viscosity<br />
Table 1<br />
Methods to control electroosmotic flow<br />
Covalent Chemical bonding to ● Many modifications possible<br />
coating <strong>capillary</strong> wall (hydrophilicity or charge)<br />
● Stability often problematic<br />
nate both the <strong>capillary</strong> surface and the solute, while high pH<br />
buffers deprotonate both. Knowledge of solute pI is often<br />
useful in selecting the appropriate pH range of the running<br />
buffer.<br />
25
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