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 Current (uA) 300 250 200 150 100 50 Liquid thermostating 10 m/s air thermostating No thermostating 0 5 10 15 20 25 30 Voltage (kV) Figure 14 Ohm’s law plots to monitor Joule heating voltage is increased. Another indication is the disproportionate increase in EOF or solute mobility with increasing voltage. Similarly, a disproportionate increase in current with voltage (Ohm’s law) indicates a temperature increase (figure 14). A variety of methods to limit Joule heating are described in table 5. Equation (17) indicates that temperature gradients will be reduced linearly with a reduction in power. This can be accomplished by lowering either the applied voltage or decreasing the buffer conductivity by lowering the ionic strength or decreasing buffer ion mobility. The latter methods may be useful but have practical limitations. Reduced buffer concentration may decrease buffering capacity and also may lead to increased solute-wall interactions (see below). Variable Effect Decrease electric field ● Proportional decrease in heat generated ● Reduces efficiency and resolution Reduce capillary inner ● Dramatic decrease in current (i a r 2 ) diameter ● Decreases sensitivity ● May cause increased sample adsorption Table 5 Methods to control Joule heating and temperature gradients Decrease buffer ionic strength or concentration Active temperature control ● Proportional decrease in current ● May cause increased sample adsorption ● Thermostats and removes heat from capillary An alternative is the use of low mobility buffers which contain large, minimally charged ions, such as tris, borate, histidine, and CAPS. Additional comments regarding buffer selection can be found in section 3.1.1.1. A dramatic decrease in temperature differences can also be realized by reducing the capillary internal diameter due to the squared dependence of area (and thus electrical cur- 34
ent) with radius. Capillaries with internal diameters as small as 5 to 10 µm have been used. However, such small internal diameters are not practical for routine use due primarily to detection, sample loading, and capillary clogging difficulties. Internal diameters of 25 to 50 mm are more practical. Principles Removal of heat from the outer capillary wall is extremely important. This can most easily be accomplished by use of a fan to blow ambient temperature air around the capillary. Dramatic improvements can be made by use of a cooling system which thermostats the capillary with high velocity air or liquid. While liquid cooling is theoretically more efficient (k Liquid > k air ), under typical conditions of less than 5 to 7 W/m power generation, high-velocity air cooling is sufficient. This is illustrated in the Ohm’s law plots of figure 14. Resolution 12 9 6 3 0 0 0.2 0.4 0.6 0.8 1.0 Injection width [cm] Figure 15 Effect of injection plug length on resolution 6 Calculation using: Upper curve = 20 kV, lower curve = 10 kV, m e1 = 3.0 × 10 -4 cm 2 /Vs, m e2 = 3.15 cm 2 /Vs, m EOF = 3.0 × 10 -4 cm 2 /Vs, diffusion coefficients = 7 × 10 -5 cm 2 /s Active temperature control is not only important for heat dissipation but also for maintaining constant capillary temperature. Even in the absence of Joule heating phenomena, variations in migration time can result from changes in ambient temperature and the resulting 2 to 3 % per °C changes in viscosity. Further, since sample loading is also dependent on the temperature, small variations can have profound effects on the quantity injected. Further details regarding temperature control and reproducibility can be found in the chapter 4. 2.3.4.3 Injection plug length During injection it is important that the sample plug length be minimized. If the length is longer than the dispersion caused by diffusion, efficiency and resolution will be sacrificed. This is illustrated in figure 15. 35
- 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 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 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
- Page 74 and 75: Modes a) ds 500 base pairs This sam
- Page 76 and 77: Modes and resolution with respect t
- Page 78 and 79: Modes 3.5 Capillary isotachophoresi
- Page 80 and 81: Modes 80
- Page 82 and 83: Instrumentation/Operation Diode-arr
ent) with radius. Capillaries with internal diameters as<br />
small as 5 to 10 µm have been used. However, such small<br />
internal diameters are not practical for routine use due<br />
primarily to detection, sample loading, and <strong>capillary</strong><br />
clogging difficulties. Internal diameters of 25 to 50 mm are<br />
more practical.<br />
Principles<br />
Removal of heat from the outer <strong>capillary</strong> wall is extremely<br />
important. This can most easily be accomplished by use of<br />
a fan to blow ambient temperature air around the <strong>capillary</strong>.<br />
Dramatic improvements can be made by use of a cooling<br />
system which thermostats the <strong>capillary</strong> with high velocity<br />
air or liquid. While liquid cooling is theoretically more<br />
efficient (k Liquid<br />
> k air<br />
), under typical conditions of less than<br />
5 to 7 W/m power generation, high-velocity air cooling is<br />
sufficient. This is illustrated in the Ohm’s law plots of<br />
figure 14.<br />
Resolution<br />
12<br />
9<br />
6<br />
3<br />
0<br />
0 0.2 0.4 0.6 0.8 1.0<br />
Injection width [cm]<br />
Figure 15<br />
Effect of injection plug length on<br />
resolution 6<br />
Calculation using: Upper curve = 20 kV,<br />
lower curve = 10 kV, m e1<br />
= 3.0 × 10 -4 cm 2 /Vs,<br />
m e2<br />
= 3.15 cm 2 /Vs, m EOF<br />
= 3.0 × 10 -4 cm 2 /Vs,<br />
diffusion coefficients = 7 × 10 -5 cm 2 /s<br />
Active temperature control is not only important for heat<br />
dissipation but also for maintaining constant <strong>capillary</strong><br />
temperature. Even in the absence of Joule heating phenomena,<br />
variations in migration time can result from changes in<br />
ambient temperature and the resulting 2 to 3 % per °C<br />
changes in viscosity. Further, since sample loading is also<br />
dependent on the temperature, small variations can have<br />
profound effects on the quantity injected. Further details<br />
regarding temperature control and reproducibility can be<br />
found in the chapter 4.<br />
2.3.4.3 Injection plug length<br />
During injection it is important that the sample plug length<br />
be minimized. If the length is longer than the dispersion<br />
caused by diffusion, efficiency and resolution will be<br />
sacrificed. This is illustrated in figure 15.<br />
35
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