0
PREFACE TO THE FIRST EDITION
Capillary electrophoresis (CE) or high-performance CE (HPCE) is making the transition
from a ...
XIV Preface
development evolves from this treatment. This is the goal of my course, and hope-
fully, I have translated thi...
PREFACE TO THE SECOND EDITION
It is hard to believe that seven years have passed since I wrote the first edition of this
b...
MASTER SYMBOL LIST
A Corrected peak area
corr r^
A Raw peak area
raw r
a Fraction ionized
a Molar absorptivity
a Separatio...
XVi Master Symbol List
/ Frictional force (Stoke's law)
g Gravitational constant
Y Field enhancement factor
Y Obstructive ...
Master Symbol List XVll
R Resolution
s
r Ionic radius (Stokes' law)
r Capillary radius
S/N Signal to noise ratio
a Peak va...
CHAPTER 1
Introduction
1.1 Electrophoresis
1.2 Microchromatographic Separation Methods
1.3 Capillary Electrophoresis
1.4 C...
^ Chapter 1 Introduction
capillary wall. The theory and practice of electrophoresis have been the subject
of many textbook...
1.2 Microchromatographic Separation Methods 3
Finally, the gel must be sufficiently viscous to provide physical support. L...
Chapter 1 Introduction
If
FIGURE 1.2 Slab-gel electrophoresis of a 500-mer double-stranded PCR reaction product in a
1.8% ...
1.2 Microchromatographic Separation Methods
i
500
IL 1746
303 ^^
10 15
TIME (min.)
20
FIGURE 1.3 Capillary gel electrophor...
6 Chapter 1 Introduction
Most of work with i-LC employs 250 |im i.d. packed columns, and so the
advantages enjoyed by open...
1.3 Capillary Electrophoresis 7
Dispersive transport, or band broadening, is the sum of processes of the dis-
persing zone...
8 Chapter 1 Introduction
W^
wW u
TIME (MIN.) 11
FIGURE 1.4 Reversed-phase liquid chromatography of barbiturates. Column: E...
1.3 Capillary Electrophoresis
X
TIME (MIN.) 10
FIGURE 1.5 Micellar electrokinetic capillary chromatography of barbiturates...
10 Chapter 1 Introduction
TABLE 1.1 Comparison of Slab-Gel Electrophoresis, p-LC, Conventional LC, and HPCE
Speed
Intrumen...
1.6 Historical Perspective 11
Typically, 50 |im i.d. capillaries are used though larger diameter tubes can be
employed at ...
12 Chapter 1 Introduction
Background Electrolyte
A
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FIGURE 1.6 Layout...
1.6HistoricalPerspective13
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1.7 GENERIC HPCE SYSTEMS
While application specific DNA systems are becoming wildly successful, ...
1.8 Instrumentation 17
10. Since a single HPCE instrument can replace as many as ten liquid chro-
matographs, the size of ...
18 Chapter 1 Introduction
to inadequate grounding and shielding. This problem has been solved in com-
mercial instrumentat...
1.9 Sources of Information on HPCE 1 9
autosamplers. Data systems that are specifically designed for HPCE are found
on mos...
20 Chapter 1 Introduction
Table 1.4 Capillary Electrophoresis Books and Proceedings
Grossman, ED., Colbum,J.C., eds. Capil...
References 21
capillary gel electrophoresis (CGE)^
capillary isotachophoresis (CITP)-^
micellar electrokinetic capillary c...
22 Chapter 1 Introduction
20.Manz, A., Harrison, D. J., Verpoorte, E. M. J., Fettinger, J. C, Ludi, H., Widmer, H. M. Mini...
References 2 3
46. Guttman, A., Paulus, A., Cohen, A. S., Grinberg, N., Karger, B. L. Use of Complexing Agents for
Selecti...
CHAPTER 2
Capillary Zone
Electrophoresis
Basic Concepts
2.1 Electrical Conduction in Fluid Solution
2.2 The Language of El...
26 Chapter 2 Capillary Zone Electrophoresis
solution, the current is carried by cations and anions. The molecular weight o...
2.1 Electrical Conduction in Fluid Solution 27
POWER SUPPLY
CAPILLARY
DETECTOR
OH GENERATED
AT CATHODE
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2 8 Chapter 2 Capillary Zone Electrophoresis
The mobility of ions in fluid solution is governed by their charge to size ra...
2.2TheLanguageofElectrophoresis29
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MOBILITY
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2.3 Electroendoosmosis 31
Expressions for some other fundamental terms are given in the following
equatio...
32 Chapter 2 Capillary Zone Electrophoresis
be removed to create a ultraviolet (UV) transparent optical window for detecti...
2.3 Electroendoosmosis 33
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34 Chapter 2 Capillary Zone Electrophoresis
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2.3 Electroendoosmosis
HIGH pH
35
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36 Chapter 2 Capillary Zone Electrophoresis
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2.3 Electroendoosmosis 37
71
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50 100 150 200
E (V/cm)
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FIGURE 2.9 Effect of buffer concentration a...
3 8 Chapter 2 Capillary Zone Electrophoresis
be carefully controlled. In this regard, wholly aqueous separations are often...
2.4 Efficiency 3 9
2.4 EFFICIENCY
The high efficiency of HPCE is a consequence of several unrelated factors:
1. A stationa...
40 Chapter 2 Capillary Zone Electrophoresis
Jorgenson and Lukacs derived the efficiency of the electrophoretic system
from...
2.5 Resolution 41
Diffusion in liquids that leads to broadening of an initially sharp band is
described by the Einstein eq...
42 Chapter 2 Capillary Zone Electrophoresis
DIFFUSION
RAPID
SLOW
MOBILITY
HIGH
LOW
SMALL
MOLECULES
LARGE
MOLECULES
FIGURE ...
2.6 Joule Heating 43
approach. Another means of improving resolution as predicted by Eq. (2.17)
is to adjust the EOF. Akho...
44 Chapter 2 Capillary Zone Electrophoresis
dT
kV'
(2.19)
The amount of heat that must be removed is proportional to the c...
2.6 Joule Heating 45
The requirement for narrow-bore capillaries comes with a price due to the
short optical path length. ...
46 Chapter 2 Capillary Zone Electrophoresis
Even these electropherograms must be carefully interpreted. In both cases,
the...
2.7 Optimizing the Voltage and Temperature
11
5 9
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50 100 150
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200 250
FIGURE 2.16 Effect o...
48 Chapter 2 Capillary Zone Electrophoresis
Some Ohm's law plots are shown in Figures 2.17 and 2.18 for an air-cooled and
...
2.7 Optimizing the Voltage and Temperature 49
1
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5 0 Chapter 2 Capillary Zone Electrophoresis
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2.8 Capillary Diameter and Buffer Ionic Strength 51
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Practical capillary electrophoresis
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Transcript of "Practical capillary electrophoresis"

  1. 1. PREFACE TO THE FIRST EDITION Capillary electrophoresis (CE) or high-performance CE (HPCE) is making the transition from a laboratory curiosity to a maturing microseparations technique. Now used in almost 1000 laboratories worldwide, CE is employed in an ever-widening scope of applications covering both large and small molecules. The inspiration for this book arose from my popular American Chemical Society short course entitled, as is this text, "Practical Capillary Electrophore- sis." During the first 18 months since its inception, nearly 500 students have enrolled in public and private sessions in the United States and Europe. I have been amazed at the diversity of the scientific backgrounds of my stu- dents. Represented in these courses were molecular biologists, protein chemists, analytical chemists, organic chemists, and analytical biochemists from indus- trial, academic, and government laboratories. Interestingly enough, CE provides the mechanism for members of this multidisciplinary group to actually talk with each other, a rare event in most organizations. But the diverse nature of the group provides teaching challenges as well. Most of the students are well versed in the art and science of liquid chromatography. However, CE is not chromatography (usually). It is electrophoresis, and it is gov- erned by the art and science of electrophoresis. For those skilled in electrophore- sis, CE offers additional separation opportunities that are not available in the slab-gel format. Furthermore, the intellectual process of methods development differs from that in either slab-gel electrophoresis or liquid chromatography. The key to grasping the fundamentals of CE is to develop an understanding of how ions move about in fluid solution under the influence of an applied electric field. With this background, it becomes painless to wander through the elec- trophoretic domain and explain the subdeties and permutations frequently illus- trated on the electropherograms. Accordingly, a logical approach to methods
  2. 2. XIV Preface development evolves from this treatment. This is the goal of my course, and hope- fully, I have translated this message into this text. Since I work independently, without academic or industrial affiliations, the writing of this text would have been impossible without the help of my friends and colleagues. In particular, 1 am grateful to Professor Ira Krull and his grad- uate student, Jeff Mazzeo, from Northeastern University for reviewing the entire manuscript; Dr. Michael Albin from Applied Biosystems, Inc., for providing his company's computerized bibliography on HPCE; and the Perkin-Elmer Corpo- ration including Ralph Conlon, Franco Spoldi, and librarian Debra Kaufman and her staff for invaluable assistance. I am also thankful to my associates throughout the scientific instrumentation industry for providing information, intellectual challenges, hints, electropherograms, comments, etc., many of which are included in this text. Last, I thank my students for helping me con- tinuously reshape this material to provide clear and concise explanations of elec- trophoretic phenomena. Finally, many of the figures in this text were produced by scanning the illus- tration in a journal article with subsequent graphic editing. While all efforts were made to preserve the integrity of the original data, subtle differences may appear in the figures produced in this book. Robert Weinberger Chappaqua, NY August 1992
  3. 3. PREFACE TO THE SECOND EDITION It is hard to believe that seven years have passed since I wrote the first edition of this book. The time is ripe for a second edition. Not only has capillary electrophoresis matured, but my ability to articulate the field has improved as well. I have reorganized this book to better reflect usage in the field. There are now ten chapters instead of twelve. The material on isotachophoresis has been com- bined with the section on stacking, and the special topics chapter has been elimi- nated. With the exception of the introduction and the chapter on basic concepts, all of the other material has been extensively reorganized and rewritten. Empha- sis has been placed on commercially available apparatus and reagents, although gaps in the commercial offerings are discussed as well. Note that micellar electro- kinetic capillary chromatography (MECC) is considered as a variant of capillary zone electrophoresis (CZE) and is included in the chapter on secondary equilib- rium. Cyclodextrins and chiral recognition are covered here as well. Many thanks to Dr. Bruce McCord, Mr. Ira Lurie, and Professor Ira KruU for reviewing some of the chapters in this second edition. The author gratefully acknowledges the support of Hewlett-Packard and in particular Dr. David Heiger. Much has been said about the ability of capillary electrophoresis (HPCE) to replace liquid chromatography (HPLC). Clearly it has not. As the first high- performance condensed phase technique, HPLC quickly replaced gas chro- matography as the method of choice for separating polar molocules. As food for thought, imagine if capillary electrophoresis had a 25-year head start over HPLC. Then perhaps the chromatographers would be fighting the uphill battle of displacing HPCE. As noted in this text, HPCE is clearly superseding the slab gel, at least in the fields of DNA separations. Robert Weinberger Chappaqua, NY June 1,1999
  4. 4. MASTER SYMBOL LIST A Corrected peak area corr r^ A Raw peak area raw r a Fraction ionized a Molar absorptivity a Separation factor h Detector optical pathlength C, c Concentration C Coefficient for resistance to mass transfer in the mobile phase C^ Coefficient for resistance to mass transfer in the stationary phase CLOD Concentration limit of detection CMC Critical micelle concentration %C Percentage of crosslinker in a gel D Capillary diameter D, D Diffusion coefficient m D^^ Solute diffusion in stagnant mobile phase DR Dynamic reserve d Particle diameter, chromatography AH Height differential between capillary inlet and outlet Ap^ Difference in mobility between two solutes AP Pressure drop 6 Debye radius ^ Zeta potential e Charge per unit area E Field strength E Acceptable increase in H E Detector efficiency 8 Dielectric constant 8 Molar absorptivity 8o Permittivity of vacuum
  5. 5. XVi Master Symbol List / Frictional force (Stoke's law) g Gravitational constant Y Field enhancement factor Y Obstructive factor for diffusion, Van Deemter equation H Height equivalent of a theoretical plate dH/dt Rate of heat production I Current If Fluorescence intensity I Excitation source intensity k Conductivity k' Capacity factor k' Capacity factor in MECC K, X Thermal conductivity K Equilibrium constant L Length of capillary L^ Length of capillary to detector I^ Length of the detector window Lf Length of capillary from detector to fraction collector L^ ^^ Length of the unpacked portion of a CEC capillary L ^^^^^ Length of the packed portion of a CEC capillary L^ Total length of capillary l.^. Length of an injection plug X Tortuosity factor, Van Deemter equation m Mass M Actual mass MLOD Mass limit of detection N Number of segments in a polymer chain N Number of theoretical plates n Number of charges r Viscosity P Partition coefficient between water and micelle wm AP Pressure drop O Polymer concentration, size separations O Quantum yield O Overlap threshold Oj Fluorescence quantum yield O* Entanglement threshold, size separations p Density p Resistivity Q Quantity of injected material q Ionic net charge R Resistance R Peak ratio R Displacement ratio
  6. 6. Master Symbol List XVll R Resolution s r Ionic radius (Stokes' law) r Capillary radius S/N Signal to noise ratio a Peak variance a Peak variance due to capillary wall effects cap ^ J a^ Peak variance due to the detector det a, „ Peak variance due to diffusion diff a^^ Peak variance due to electrodispersion ^heat P^ak variance due to Joule heating a Peak variance due to injection mj J a^ Peak variance in units of length o Peak variance from all sources tot Time Absorption time to a stationary phase or wall Desorption time from a stationary phase or wall Lag time Migration time Migration time for a micellar aggregate Migration time for a neutral "unretained" solute Retention time T Temperature TR Transfer ratio %)T Percentage of monomer and crosslinker in a gel 0/ |JL Ionic mobility |i^ Apparent (measured) mobility |Li^^ Electroosmotic mobility |Li^ Electrophoretic mobility V Partial molar volume of micelle V Voltage 1) Ionic velocity 1) Mean linear velocity D^ Electrophoretic velocity 1) Electroosmotic velocity eo J 0)^ ^^ Solute velocity in the unpacked portion of a CEC capillary ) ^^^^^ Solute velocity in the packed portion of a CEC capillary W Power W.^ Width of an injection plug W^ Spatial width of a sample zone W^ Temporal width of a sample zone X. Intital length of an injection plug X^ Zone length after stacking Z Number of valence electrons Z Charge
  7. 7. CHAPTER 1 Introduction 1.1 Electrophoresis 1.2 Microchromatographic Separation Methods 1.3 Capillary Electrophoresis 1.4 Capillary Electrochromatography 1.5 Micromachined Electrophoretic Devices 1.6 Historical Perspective 1.7 Generic HPCE Systems 1.8 Instrumentation 1.9 Sources of Information on HPCE 1.10 Capillary Electrophoresis: A Family of Techniques References 1.1 ELECTROPHORESIS Electrophoresis is a process for separating charged molecules based on their move- ment through a fluid under the influence of an applied electric field. If two solutes have differing electrophoretic mobilities, then separation v^U usually occur. The separation is performed in a medium such as a semisolid slab-gel. Gels provide physical support and mechanical stabiUty for the fluidic buffer system. In some modes of electrophoresis, the gel participates in the mechanism of separation by serving as a molecular sieve. Nongel media such as paper or cellulose acetate are alternative supports. These media are less inert than gels, as they contain charged surface groups that may interact with the sample or the run buffer. A carrier electrolyte is also required for electrophoresis. Otherwise known as the background electrolyte (BGE), the carrier electrolyte, or simply the run buffer, this solution maintains the requisite pH and provides sufficient conduc- tivity to allow the passage of current (ions), necessary for the separation. Fre- quently, additional materials are added to the BGE to adjust the resolution of the separation through the generation of secondary equilibria. Additives can also serve to maintain solubility and prevent the interaction of solutes or excip- ients with the gel matrix or, in the case of capillary electrophoresis, with the
  8. 8. ^ Chapter 1 Introduction capillary wall. The theory and practice of electrophoresis have been the subject of many textbooks and conference proceedings (1-9). Apparatus for conducting electrophoresis, such as that illustrated in Figure 1.1, is remarkably simple and low cost. The gel medium, which is supported on glass plates, is inserted into a Plexiglass chamber. Two buffer reservoirs make contact at each end of the gel. Electrodes immersed in the buffers complete the electrical circuit between the gel and power supply. Many samples can be sepa- rated simultaneously, since it is possible to use a multilane gel. One or two lanes are frequently reserved for standard mixtures to calibrate the electropherogram. Calibration is usually based on molecular size or, in isoelectric focusing, pi. Gels such as polyacrylamide or agarose serve several important functions: 1. they may contribute to the mechanism of separation; 2. they reduce the dispersive effects of diffusion and convection; and 3. they serve to physically stabilize the separation matrix. The gel composition is adjusted to define specific pore sizes, each for a nom- inal range of molecular sizes. This forms the basis for separations of macro- molecules based on size. By proper calibration, extrapolation to molecular weight is straightforward. Reduction of convection and diffusion is an important function of the gel matrix. The production of heat by the applied field induces convective move- ment of the electrolyte. This movement results in band broadening that reduces the efficiency of the separation. The viscous gel media inhibits fluid movement in the electric field. Such a material is termed anticonvective. Since the gel is of high viscosity, molecular diffusion is reduced as well, further enhancing the effi- ciency of the separation. CATHODE ANODE BUFFER SOLUTION GEL BUFFER SOLUTION FIGURE 1.1 Drawing of an apparatus for slab-gel electrophoresis.
  9. 9. 1.2 Microchromatographic Separation Methods 3 Finally, the gel must be sufficiently viscous to provide physical support. Low viscosity solutions or gels would flow if the plate is not held level. Immersion in detection reagents would be impossible, since handling or contact with fluid solutions would destroy the matrix and separation. In the capillary format, the gel is unnecessary since the walls of the capillary provide the mechanical sta- bility for the separation. The basic procedure for performing gel electrophoresis is as follows: 1. prepare, pour and polymerize the gel; 2. apply the sample; 3. run the separation; 4. immerse the gel in a detection reagent;^ 5. destain the gel; 6. preserve the gel; and 7. photograph or scan the gel for a permanent record.^ These steps are extremely labor intensive. High performance capillary elec- trophoresis (HPCE) is the automated and instrumental version of slab-gel elec- trophoresis. In the DNA applications arena, the most important of which include DNA sequencing, human identification, and genetic analysis, HPCE is rapidly replacing the slab-gel as the separation method of choice. The separation of some polymerase chain reaction (PCR) products is shown in Figure 1.2. A restriction digest, used as a sizing standard, appears in the outer lanes. The middle three lanes of the gel show a triplicate run of a 500-mer dou- ble-stranded DNA PCR reaction. Quantitation for such a separation is difficult and often imprecise, but such information can be obtained with the aid of a gel scanner. Recoveries of material from the gel are performed using procedures such as the Southern blot (10). Sufficient material is recoverable for sequenc- ing or other bioassays. Separations of the sizing standard and 500-mer PCR product by HPCE using a size selective polymer network are shown in Figure 1.3. Quantitation is read- ily performed using peak area comparison with the standard. However, fraction collection is difficult relative to the slab-gel, particularly for trace impurities, since only minuscule amounts of material are injected into the capillary. 1.2 MICROCHROMATOGRAPHIC SEPARATION METHODS The evolution of chromatographic methods over the last 40 years has produced a systematic and rational trend toward miniaturization. This is particularly true lOn-line detection is performed on an instrument such as an automated DNA sequencer. ^Automated gel scanners can be used in place of gel archiving or photography.
  10. 10. Chapter 1 Introduction If FIGURE 1.2 Slab-gel electrophoresis of a 500-mer double-stranded PCR reaction product in a 1.8% agarose ethidium bromide gel. Courtesy of Bio-Rad. for gas chromatography, where the advantages of the open tubular capillary dis- placed the use of packed columns for most applications. Chromatographic separations all function via differential partitioning of a solute between a stationary phase and a mobile phase. A packed column offers solutes "a multiplicity of flow paths, some short, the majority of average length, and some long (11)." Solute molecules select various paths through the chro- matographic maze. The detected peak suggests this distribution and is broad- ened. In the open tubular capillary, the choices for solute transport are limited, so that the solute elutes as a narrow band. In order for the open tubular capillary to function properly, its diameter must be quite small. Larger diameter capillaries present a problem, since solutes away from the walls do not sense the stationary phase in a timely fashion. However, a major problem with narrow inner diameter (i.d.) capillaries is loading capac- ity. Injection sizes must be kept small to avoid overloading the system. In gas chromatography (GC) this problem is overcome in part, since sensitive detec- tors such as the flame ionization detector (FID), electron capture detector (ECD), and mass spectrometer are easily interfaced. Improved efficiency is one of several advantages obtained through minia- turization. The most important of those is improved mass limits of detection
  11. 11. 1.2 Microchromatographic Separation Methods i 500 IL 1746 303 ^^ 10 15 TIME (min.) 20 FIGURE 1.3 Capillary gel electrophoresis of a 500-mer (top) double-stranded PCR reaction prod- uct and a low molecular weight sizing standard (bottom). Capillary: 50 cm x 50 [im i.d. Bio-Rad coated capillary; buffer: 100 mM tris-borate, pH 8.3, 2 mM EDTA with linear polymers; injection: electrokinetic, 8 kV, 8 sec; detection UV, 260 nm. Courtesy of Bio-Rad. (MLOD). Since dilution of the solute is minimized in the miniaturized system, better MLODs are obtained than in large scale systems. This is particularly important when the available sample size is small, as sometimes happens in bio- molecule separations. Miniaturization of GC has been exquisitely successful. These triumphs could not be directly transferred to liquid chromatography (LC) for several reasons. The most important is the lack of good detectors. Interface to the FID and ECD is not practical due to the incompatibility of the mobile phase with each detec- tor. Pumping of the mobile phase at the low flow rates required by miniatur- ization is also more complex, particularly when gradient elution is required. Despite these problems, |I-LC systems are useful in sample-limited situations and for mass spectrometry where the reduced liquid flow rate is advantageous. Several books have been devoted to this important field (12-14).
  12. 12. 6 Chapter 1 Introduction Most of work with i-LC employs 250 |im i.d. packed columns, and so the advantages enjoyed by open tubular GC are not realized in |Li-LC. The instru- mental problems of injection and detection posed by open tubular LC have inhibited most people from using this technology 1.3 CAPILLARY ELECTROPHORESIS The arrival of HPCE solved many experimental problems of gels. Use of gels is unnecessary since the capillary walls provide mechanical support for the car- rier electrolyte.3 The daunting task of automation for the slab-gel format is solved with HPCE. Sample introduction (injection) is performed in a repeatable manner. Detection is on-line, and the instrumental output resembles a chro- matogram. The use of narrow diameter capillaries allows efficient heat dissipa- tion. This permits the use of high voltage to drive the separation. Since the speed of electrophoresis is directly proportional to the field strength, separations by HPCE are faster than those in slab-gels. On the other hand, the relative speed of the slab-gel is enhanced, since multiple samples can be separated at once. HPCE is a serial technique; one sample is followed by another. This limitation has been overcome through the use of the capillary array for high throughput applications such as DNA sequencing (15,16) and serum protein analysis (17). Commercial instruments are now available for these applications. HPCE represents a merging of technologies derived from traditional elec- trophoresis and high performance liquid chromatography (HPLC). Both HPCE and HPLC employ on-line detection. Developments in on-column micro-LC detec- tion have directly transferred over to capillary electrophoresis. One of the modes of HPCE, micellar electrokinetic capillary chromatography (Chapter 4), can be considered a chromatographic technique. Electrically driven separations through packed columns (Chapter 7) have been reported from many laboratories. While there is much in common between chromatography and electrophoresis, the fun- damentals of HPCE are based on electrophoresis, not chromatography. Professor Richard Hartwick, formerly from the State University of New York at Binghamton, started many of his lectures on capillary electrophoresis with a discussion of transport processes in separations. While performing a separation, there are two major transport processes occurring: Separative transport arises from the free energy differences experienced by molecules with their physicochemical environment. The separation mechanism may be based on phase equilibria such as adsorption, extraction, or ion exchange. Alternatively, kinetic processes such as electrophoresis or dialysis provide the mechanism for separation. Whatever the mechanism for separation, each indi- vidual solute must have unique transport properties for a separation to occur. ^Gels are occasionally used in HPCE for running size separations. Pumpable polymer networks are preferred, since they can be changed for each run.
  13. 13. 1.3 Capillary Electrophoresis 7 Dispersive transport, or band broadening, is the sum of processes of the dis- persing zones about their center of gravities. Examples of dispersion processes are diffusion, convection, and restricted mass transfer. Even under conditions of excellent separative transport, dispersive transport, unless properly con- trolled, can merge peaks together. According to the late Professor Calvin Giddings as paraphrased by Hartwick, "separation is the art and science of maximizing separative transport relative to dispersive transport." In this regard, capillary electrophoresis is perhaps the finest example of optimizing both transport mechanisms to yield highly effi- cient separations. Figures 1.4 and 1.5 illustrate this concept, using a series of barbiturate sep- arations to compare HPCE and HPLC. The mode of electrophoresis used in Fig- ure 1.5 is micellar electrokinetic capillary chromatography (MECC), an electrophoretic technique that resembles reversed-phase LC. In the LC separa- tion amobarbital and pentabarbital coelute, but they are resolved by HPCE. With some optimization work, amobarbital and pentabarbital can be sepa- rated by HPLC. But with HPCE, methods development often progresses rapidly because of the enormous peak capacity of the technique. Peak capacity simply describes the number of peaks can be separated per unit time. With a couple of hundred thousand theoretical plates,"^ many separations occur without exten- sive optimization efforts. In addition, peak symmetry is excellent using HPCE unless wall effects (Section 3.3) occur. With the absence of a stationary phase, many factors that contribute to peak broadening and tailing are minimized. It would be misleading to state that all separations are superior by HPCE or that methods development will always be straightforward. It is realistic, how- ever, based on the experiences of many separation scientists skilled in the art of both techniques, to predict that HPCE will provide the requisite speed and res- olution in the shortest possible run time with the least amount of methods development, under most circumstances. These same two figures illustrate an important limitation of HPCE, the con- centration limit of detection (CLOD). In Figure 1.4, the LC separation requires a 1.25 |Lig/mL solution to give full scale peaks with 1-2% noise (the postcolumn reagent merely alkalized the mobile phase, permitting sensitive detection at 240 nm). The CLOD is approximately 30-fold better by HPLC. The MECC separa- tion shown in Figure 1.5 required a solute concentration of 100 |ag/mL for a similar response, although the noise was lower (0.5%).^ On the other hand, the MLOD by capillary electrophoresis exceeds HPLC by a factor of 100. The ideal detector for HPCE will be mass sensitive and not depend on the narrow optical pathlength defined by the capillary itself. Descriptions, advantages, and limita- tions of many HPCE detectors can be found in Chapter 9. ^The theoretical plate (N) is a measure of the efficiency of a chromatographic of electrophoretic peak; N = 5.5'(t^/Wiy, where t^ is the migration time and W is the peak width at half height. 5The CLOD can easily be improved through the use of stacking and/or extended pathlength flowcells.
  14. 14. 8 Chapter 1 Introduction W^ wW u TIME (MIN.) 11 FIGURE 1.4 Reversed-phase liquid chromatography of barbiturates. Column: Econosphere Cis, 25 cm X 4.6 mm i.d.; mobile phase: acetonitrile : water, 55/45 (v/v); injection size: 20 jxL; flow rate: 1.2 mL/min; postcolumn reagent: borate buffer, pH 10, 0.2 mL/min; detection: UV, 240 nm; solutes: (1) barbital, (2) butethel, (3) amobarbital and pentabarbital, (4) secobarbital; amount injected: 25 ng of each barbiturate from a 1.25 |Llg/mL solution. The preceding comparison is significant since a |Li-separation technique is compared with conventional HPLC using a 4.6 mm i.d. column. Would it be bet- ter to compare HPCE with |i-LC? Perhaps so from an academic standpoint, but this would not reflect the current usage and thinking in the real world. Chemists are contemplating using HPCE to replace or augment conventional HPLC as well as |i-LC. Table 1.1 provides a comparison of slab-gel electrophoresis, |I-LC, HPLC, and HPCE. Two disadvantages of HPCE compared to conventional HPLC are sensitivity of detection and precision of analysis. These have prevented the most widespread use of HPCE. On the other hand, HPCE is replacing the slab- gel for most high-throughput DNA applications. In this case, the ease of automa- tion, precision and ruggedness of HPCE supercede the slab-gel.
  15. 15. 1.3 Capillary Electrophoresis X TIME (MIN.) 10 FIGURE 1.5 Micellar electrokinetic capillary chromatography of barbiturates. Capillary: 50 cm (length to detector) X 50 |lm i.d.; buffer: 110 mM SDS, 50 mM borate, pH 9.5; injection: 1 sec vac- uum (5 nL); detection: UV, 240 nm; solutes: (1) phenobarbital, (2) butethel, (3) barbital, (4) amo- barbital, (5) pentobarbital, (6) secobarbital; amount injected: 500 pg of each barbiturate from a 100 |lg/mL solution. HPCE is a novel and alternative format for both liquid chromatography and electrophoresis. The unique properties of this technique include the use of: 1. capillary tubing in the range of 25-100 jim; 2. high electric field strength; 3. on-line detection in real time; 4. only nanoliters of sample; 5. limited quantities of mostly aqueous reagents; and 6. inexpensive capillaries relative to HPLC columns. The molecular weight range of analytes separable by HPCE is enormous. A search of the literature reveals applications covering small ions, small molecules,
  16. 16. 10 Chapter 1 Introduction TABLE 1.1 Comparison of Slab-Gel Electrophoresis, p-LC, Conventional LC, and HPCE Speed Intrumentation cost Sensitivity CLOD MLOD Efficiency Automation Precision Quantitation Selectivity Methods development Reagent consumption Preparative mode Ruggedness Separations DNA Proteins Small molecules Slab-Gel slow low poor poor moderate Htde poor difficult moderate slow low good good excellent excellent poor p-LC moderate high poor good moderate yes good easy moderate moderate low fair good fair good excellent HPLC moderate moderate excellent poor moderate yes excellent easy moderate moderate high excellent excellent fair good excellent HPCE fast moderate poor excellent high yes good easy high rapid minimal poor good excellent excellent excellent peptides, proteins, DNA, viruses, bacteria, blood cells, and colloidal particles. The molecular weight range of HPCE is easily from 3 for a lithium ion to 100,000,000 for a virus or particle. 1.4 CAPILLARY ELECTROCHROMATOGRAPHY A hybrid of chromatography and electrophoresis, capillary electrochromatog- raphy (CEC) employs the electrically driven electroosmotic flow (EOF) to pump a mobile phase through a packed capillary. The use of the EOF to gener- ate flow solves some of the instrumental problems of pumping at nL flow rates. Capillary electrochromatography employs small diameter capillaries filled with a stationary phase. Reversed-phase packings are most often used, although an application with a cation-exchange material has been reported (18). An amazing efficiency 8 million plates per meter was reported in that paper, though the mechanism and reproducibility of the effect are still unclear.
  17. 17. 1.6 Historical Perspective 11 Typically, 50 |im i.d. capillaries are used though larger diameter tubes can be employed at the expense of efficiency. Particle diameters of 3-5 |im porus mate- rial are most common, though it is possible to employ 1.5 |Lim pellicular pack- ing. Since there is no pressure drop with an electrically pumped system, relatively long capillaries can be employed to generate hundred of thousands of theoretical plates. The reduction of eddy diffusion also contributes to the enhanced efficiency (19). The mobile phase is pumped using the EOF generated by both the wall of the capillary and the chromatographic packing. Formulation of the mobile phase is similar to conventional reversed-phase chromatography, except that a dilute buffer—for example, 1-10 mM tris, borate, or phosphate—is added to ensure sufficient electrical conductivity The capillary is usually pressurized to a few atmospheres to suppress bubble formation. The least mature of the electrically driven techniques, CEC capillaries and second generation instruments are now available. One promise for this tech- nique is the ability to employ the vast existing chromatographic database to speed methods development. 1.5 MICROMACHINED ELECTROPHORETIC DEVICES Employing technology used in the fabrication of integrated circuits, it is now possible to create an electrophoretic apparatus on a chip (20-28). Designed for dedicated applications such as clinical analysis, genetic analysis, or DNA sequencing, chips can be manufactured at low cost in commercial quantities. These devices can form the basis of an automated laboratory, where the dispos- able chip serves as the separations device. A diagram of a simple micromachined HPCE chip is shown in Figure 1.6. The technological advantage of this device compared with a conventional capillary is its ability to perform extremely small injections (29). As a result, a shorter separation channel is required, again compared with the conventional capillary. Detection problems resulting from the small injection are solved through the use of laser-induced fluorescence (LIE). Micromachined electrophoretic devices are expected to have a huge impact in the DNA applications area. 1.6 HISTORICAL PERSPECTIVE A century of development in electrophoresis and instrumentation has provided the foundation for HPCE. Reviews describing the history of electrophoresis were published by Vesterberg (30) and Compton and Brownlee (31). The highlights in the development of HPCE are given in Table 1.2.
  18. 18. 12 Chapter 1 Introduction Background Electrolyte A B oil o- Sample o- Separation Channel Detector Window FIGURE 1.6 Layout of the channels in a planar glass substrate. Channels are referred to by num- ber and inlet points (reservoirs) as letters. Each channel is labeled with its content or its function. Overall dimensions are 14.8 cm x 3.9 cm x 1 cm thick. The location of one pair of platinum elec- trodes is shown; for clarity, the others are not. (A) BGE reservoir; (B) sample reservoir; (C) outlet reservoir. (1) BGE inlet; (2) sample inlet; (3) separation channel; (4) sample outlet. Injection is made where 4 crosses 3. Redrawn with permission from Anal. Chem., 64, 1926 (1992), copyright © Am. Chem. Soc. A direct forerunner of modem CZE was developed by Hjerten in 1967 (32). To reduce the detrimental effects of convection caused by heat production, the 3 mm i.d. capillaries were rotated. While heat dissipation was unchanged, the rotating action caused mixing to occur within the capillary, smoothing out the convective gradients. In the 1970s, techniques using smaller i.d. capillaries were successfully developed (34). Superior heat dissipation permitted the use of higher field strength without the need for capillary rotation. In 1981, Jorgenson and Lukacs (35) solved the perplexing problems of injection and detection with 75 |Lim i.d. capillaries. Their advances clearly defined the start of the era of HPCE. Fluorescence detection was required at that time to record the electropherogram. The 1980s proved ripe for invention. Adaptation of gel electrophoresis (36) and isoelectric focusing (38) to the capillary format was successful. In 1984, Terabe et al. (37) described a new form of electrophoresis called micellar elec- trokinetic capillary chromatography (MECC). Chromatographic separations of small molecules, whether charged or neutral, were obtained by employing the micelle as a "pseudo-stationary" phase. Great advances in detection occurred during the 1980s to overcome, in part, the serious limitation of the short pathlength defined by narrow i.d. capillaries
  19. 19. 1.6HistoricalPerspective13 u Pu PC o u u a ^ PI o u ^ 1 IBU C/5 r2 'PH c« CJ o U 2 o c_o (U Pi 15 b CC p. u O <^ Zfi uu o CIS D2 JOi :3 c^ o •rH 1ro _g W N UU W5 c/5 c« 'tob ^ so o in ci o(N •S wN U f^ m U w u D. c« u O c:3 f^ TJ so o fN .swN U ^•^ CD <U ^^ 7^ PH CC u T3 in h- ,P W N U vo ro W O U m U U P-) S II II ^in W) CT" UO < § ^JS ;=i U «,a •^ •.-!^->s vp (N ^-^a U TS <u fl ^o o _o < P <Tl ^-? SSJ II X ^h- ^ ^_^ ^in 1ON <! P (J J^ ON r-H 00 o m00 ON T^ 00 ON in 00 ON ^X ON J-~ X ON 00 X ON ON X ON O ON ON (N ON ON in ON ON X ON ON
  20. 20. 1 4 Chapter 1 Introduction (57, 58). One ofJorgenson's first papers in the field employed fluorescence (59). Gassmann et al. (39) employed LIF, improving detectability to the attomole range. Olivares et al (42) interfaced CZE to the mass spectrometer via the elec- trospray. The use of on-line mass spectrometry is significant because of the dif- ficulty of carrying out fraction collection. Wallingford and Ewing (43) developed electrochemical detection, sensitive enough to measure catecholamines in a sin- gle snail neuron. Kuhr and Yeung (44) employed indirect detection to measure solutes that neither absorbed nor fluoresced. More exotic detection techniques include electrochemical detection (43, 60) nuclear magnetic resonance (56), Raman (45), chemiluminescence (55) and radioactivity (61). The problem of protein adherence to the capillary wall was addressed from sev- eral fronts. The use of treated capillaries was described by Hjerten (40) in 1985. Around the same time, Lauer and McManigill (41) employed alkaline buffers above the pi of the protein to effect solute repulsion from the anionic capillary wall. Based on these and related developments, wall effects have been substantially reduced. The relative instability of crosslinked polyacrylamide gel-filled capillaries for protein and DNA separations was addressed by the first reports of polymer net- works (47, 48). This led to the commercial introduction of kits for separations of proteins, oligonucleotides, and DNA. DNA sequencing can now be performed using various low viscosity polymer solutions (62, 63) The first commercial instrument was introduced in 1988 by the late Bob Brownlee's company, Microphoretics. The following year, new instruments from Applied Biosystems, Beckman, and Bio-Rad were introduced. Later, Spectra- Physics, Isco, Europhor, Dionex, Waters Associates, Hewlett-Packard, and Uni- cam entered the fray. Modular systems from Lauer Labs, Groton Technologies, Jasco, and Europhor became available over the next few years. In the mid 1990s, the slow development of the HPCE generic marketplace caused an industry shakeout as a number of instruments were withdrawn from the marketplace. In 1990, the first report employing a multiple capil- lary system was published (54). The mid to late 1990s provided the first application specific instruments for performing serum protein analysis (Beck- man) and DNA sequencing (Beckman, PE Biosystems, Molecular Dynamics). Instruments from the latter two companies are sold with 96 capillary arrays. These instruments are designed for high throughput DNA sequencing as required by the Human Genome Project. It is expected that human identifi- cation, another area that requires high throughput, will be implemented on these instruments. In 1998, Covergant Bioscience Limited of Ontario, Canada, reported on a new dedicated instrument for capillary isoelectric focusing. The entire capillary is imaged using a charged coupled device camera. The advantage of whole cap- illary imaging is the elimination of the mobilization step. Electrochromatogra- phy has attracted intense interest in the late 1990s. The present state of the commercial offerings is given in Table 1.3.
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  22. 22. 16 Chapter 1 Introduction 1.7 GENERIC HPCE SYSTEMS While application specific DNA systems are becoming wildly successful, generic HPCE systems have not provided the returns expected by the scientific instru- ment manufacturers. The generic HPCE system is designed for the user to develop his or her own methods. In HPLC, this type of system forms the largest segment of this multibillion dollar market. There are numerous reasons, beyond the scientific, that this has not occurred with HPCE. 1. Liquid chromatography (HPLC) is the greatest analytical instrumenta- tion success story in history. With a 23 year head start over HPCE, most problems have been worked out. Methods development is straightfor- ward, chemists are trained, and troubleshooting is usually simple. 2. HPLC scales up for preparative work and scales down to the capillary format with relative ease. It is possible to have a single method for ana- lytical, preparative, and commercial scale separations. 3. Many chromatographers consider HPCE to be the separations technique of last resort. 4. It is far more difficult for an instrument company sales force to sell HPCE. With quotas high and bonuses tied to performance, the sales- person goes where the money is. Setting up a demo instrument in a users lab is prone to failure, since the chemist is probably not trained in cap- illary electrophoresis. Postsales customer support is also quite high. 5. Capillary electrophoresis is electrophoresis, not chromatography. Chro- matographers must first master the principles of electrophoresis in order to effectively develop and troubleshoot methods. The training require- ments are not trivial. Methods development can seem overwhelmingly complex to the new user. 6. Private industry is so downsized that scientists have no time to learn new techniques. Many purchased instruments sit idle because of initial fail- ures of methods development. Instrument companies are downsized as well and have cut back on customer applications efforts. When faced with a problem chemists retreat to the familiar, and that is frequently HPLC. 7. Capillary electrophoresis is not as rugged as HPLC. Changes in the cap- illary surface chemistry lead to variable electroosmotic flow This in turn causes changes in the solute migration time. 8. The sensitivity of HPCE is lower than HPLC. This has become less of an issue as stacking techniques coupled with extended pathlength capillar- ies come into play. The training issues prevail, as many chemists are unaware of the variety of stacking techniques that exist. 9. There are few official methods of analysis employing HPCE. However, much is now in the pipeline. A number of pharmaceutical companies have submitted new drug applications to the Food and Drug Adminis- tration citing HPCE methodology.
  23. 23. 1.8 Instrumentation 17 10. Since a single HPCE instrument can replace as many as ten liquid chro- matographs, the size of the market may become self-limiting. The prospects for HPCE are not so bleak since once the learning curve is scaled. Successful methods development and routine implementation has been accomplished in many organizations. 1.8 INSTRUMENTATION The instrumental configuration for HPCE is relatively simple. Before 1988, all work was done on simple homemade systems of a design similar to Jorgenson and Lukacs's original work (35, 59, 64). A schematic of a homemade system is shown in Figure 1.7. The system con- sists of a high voltage power supply, buffer reservoirs, an HPLC ultraviolet detec- tor, a capillary, and a Plexiglas cabinet. A safety interlock can be employed to prevent activation of high voltage when the cabinet is open. The capillary can be filled with buffer by a vacuum, generated using a syringe or handpump. Sam- ples are injected either by siphoning (elevating the capillary for a defined time at a specified height) or by electrokinetic injection. While these simple systems provide good separations, precision may be poor due to the lack of temperature control and system automation. Another common problem in homemade systems is excessive detector noise. The capillary is threaded through the detector and generally passes close to sensitive elec- tronics, where the high electric field frequently causes electrical disturbances due POWER SUPPLY CAPILLARY DETECTOR CATHOLYTE ELECTRODES ^ ^ Q ^ ^ T E (OUTLET) (INLET) FIGURE 1.7 Basic schematic of an HPCE Instrument.
  24. 24. 18 Chapter 1 Introduction to inadequate grounding and shielding. This problem has been solved in com- mercial instrumentation. The advantage of homemade systems is primarily in the area of detection. It is easy to interface HPCE to fluorescence detection and in particular laser- induced fluorescence. With the introduction of commercial modular systems, the advantages of homebuilt systems have all but disappeared excepting cost. The arrival of commercial instruments has facilitated substantial growth in the field. An illustration of the now obsolete Applied Biosystems 270A is shown in Figure 1.8. This instrument provides the following basic features: a high volt- age power supply that can provide up to 30 kV, an autosampler, electrodes, a separation capillary, an air-cooled capillary temperature controller, a UV detec- tor, a capillary filling apparatus, and microprocessor control. Newer instruments have random access where any vial can be designated as the inlet or outlet. Most of the newer instruments contain the capillary within a cartridge for efficient cooling with either air or fluids. Pressure is used rather than vacuum for filling the capillary; this is an advantage when using viscous polymers or interfacing to the mass spectrometer. Many instruments can also perform voltage programming and fraction collection, have alternative detec- tors such as fluorescence, photodiode array, or conductivity, and possess cooled T Microprocessor Reporting Integrator Regulated Vacuum Reservoirs Vent Solenoid Valves csfe zDetector 1 Thermostated Compartment Sample Buffer Reservoir Sample Vials Vacuum Buffer Reservoir Auto Sampler Carousel FIGURE 1.8 Schematic of the Apphed Biosystems 270A. Courtesy of Apphed Biosystems.
  25. 25. 1.9 Sources of Information on HPCE 1 9 autosamplers. Data systems that are specifically designed for HPCE are found on most units. Computers now provide for system control on all fully auto- mated units. 1.9 SOURCES OF INFORMATION ON HPCE Keeping up with the hterature in HPCE is no small task. Through 1998, about 7000 English language papers have appeared in the literature. The growth of the literature in the field is illustrated in Figure 1.9. Note the large increase that began in 1988, the year of commercial introduction of HPCE instrumentation. The conference proceedings of the International Symposia on High Perfor- mance Capillary Electrophoresis that have appeared in the Journal of Chro- matography, Vols. 480, 516,559,608, 652,680, 717, 744, 745, 781,817, and 853, contain an impressive concentration of state-of-the-art results. Other journals containing numerous papers on HPCE are Analytical Chemistry,Journal ofMicro- column Separations, Chromatographia,Journal ofHigh Resolution Chromatography, Electrophoresis, Journal of Liquid Chromatography and Related Techniques, and Journal of Capillary Electrophoresis. Many dedicated issues from some of these journals covering HPCE, notably Electrophoresis and Journal of Liquid Chro- matography, have been published as well. For a comprehensive review of the literature, the biannual editions of Analytical Chemistry entitled "Fundamental Reviews" should be consulted. For general information on the theory of electromigration techniques, see (65) for an excellent review. Two recent editions of Electrophoresis, Vol. 18 (1997) No. 12-13 and Vol. 19 (1998) No. 16-17, contain outstanding 1400 r -^ ^ - ^ ~ - - - ~- - ^ -^.-.--.- -; 123^ laooh 1000 h 800 j-- 600 400 200 1" 0 9 • i 3 HumbBf of P 1 6 4 apers il ^^ ^m 80 81 82 83 84 86 86 87 BB 89 90 91 92 93 94 95 96 97 FIGURE 1.9 The growth of the hterature of HPCE.
  26. 26. 20 Chapter 1 Introduction Table 1.4 Capillary Electrophoresis Books and Proceedings Grossman, ED., Colbum,J.C., eds. Capillary Electrophoresis: Theory and Practice. 1992, Acadenic Press. Vindevogel, J., Sandra, P Introduction to Micellar Electrokinetic Chromatography. 1992, Huthig. Guzman, N., ed. Capillary Electrophoresis Technology. 1993, Marcel Dekker. Weinberger, R. Practical Capillary Electrophoresis. 1993, Academic Press. Camilleri, P., ed. Capillary Electrophoresis: Theory and Practice. 1993, CRC Press. Foret, E, Krivankova, L., Bocek, P Capillary Zone Electrophoresis. 1993, VCH. Jandik, P, Bonn, G. Capillary Electrophoresis of Small Molecules and Ions. 1993, VCH. Baker, D. Capillary Electrophoresis. 1995, Wiley. Righetti, P G., ed. Capillary Electrophoresis in Analytical Biotechnology. 1995, CRC. Engelhardt, H., Beck, W, Schmitt, T. Capillary Electrophoresis: Methods and Potentials. 1996, Vieweg. Cohen, A. S., Terabe, S., Deyl, Z., eds. Capillary Electrophoretic Separation of Drugs. 1996, Elsevier. Altria, K.D., ed. Capillary Electrophoresis Guidebook: Principles, Operation, and Applications. 1996, Humana Press. Jackim, E., ed. Capillary Electrophoresis Procedures Manual. 1996, Elsevier. Lunte, S. M., Radzik, D. M., eds. Pharmaceutical and Biomedical Applications of Capillary Elec- trophoresis. 1996, Pergamon. Coleman, D., ed. Directory ofCapillary Electrophoresis: New Completely Revised Edition, 1996, Elsevier Chankvetadze, B. Capillary Electrophoresis in Chiral Analysis. 1997, John Wiley 62: Sons. Parvez, H., Caudy, P, Parvez, S., Roland-Gosselin, P, eds. Capillary Electrophoresis in Biotechnol- ogy and Environmental Analysis. 1997, VSP Shintani, H., Polonsky J., ed. Handbook of Capillary Electrophoresis Applications. 1997, Blackie. Weston, A., Brown, PR., HPLC and CE: Principle and Practice. 1997, Academic Press. Heller, C, ed. Analysis of Nucleic Acids by Capillary Electro phoresis. 1997, Vieweg. Khaledi, M.G., ed. High Performance Capillary Electrophoresis. Theory, Techniques, and Appli- cations. 1998, Wiley reviews of most aspects of HPCE. There have been numerous textbooks and conference proceedings in this field; a compilation is given in Table 1.4. 1.10 CAPILLARY ELECTROPHORESIS: A FAMILY OF TECHNIQUES Capillary electrophoresis comprises a family of related techniques with differ- ing mechanisms of separation. These techniques, which are covered in the fol- lowing chapters of this book, are: capillary zone electrophoresis (CZE) capillary isoelectric focusing (CIEF)
  27. 27. References 21 capillary gel electrophoresis (CGE)^ capillary isotachophoresis (CITP)-^ micellar electrokinetic capillary chromatography (MECC)^ capillary electroosmotic chromatography (CEC). ^CGE is now performed using replaceable polymer network reagents. 7CITP is considered here only for trace enrichment or sample stacking. ^MECC is the most significant application employing secondary equilibrium with CZE. REFERENCES 1. Westheimer, R., Electrophoresis in Practice: A Guide to Methods and Applications ofDNA and Pro- tein Separations, 2nd Ed. 1997, Wiley. l.Mosher, R. A., Saville, D. A., Thormann, W, The Dynamics of Electrophoresis. 1992, VCH. 3.Rickwood, D., Hames, B. D., Gel Electrophoresis of Nucleic Acids: A Practical Approach, 2nd Ed. 1990, IRL Press. 4. Rickwood, D., Hames, B. D., Gel Electrophoresis of Proteins: A Practical Approach, 2nd Ed. 1990, IRL Press. 5. Andrews, A. T., Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications. 1981, Clarendon Press. 6. Righetti, P. G., Isoelectric Focusing: Theory, Methodology and Applications. Laboratory Techniques in Biochemistry and Molecular Biology, ed. T. S. Work and R. H. Burdon. 1983, Elsevier Bio- medical Press. 7. Chrambach, A., The Practice of Quantitative Gel Electrophoresis. 1985, VCH. S.Dunn, M. J., ed. Gel Electrophoresis of Proteins. 1986, Wright. 9.Jorgenson, J. W, Phillips, M., eds. New Directions in Electrophoretic Methods ACS Symposium Series 335. 1987. American Chemical Society. 10.Southern, E. M.J. Mol. Biol, 1975; 98:503. 1 I.Jennings, W, Analytical Gas Chromatography. 1987, Academic Press, p.5. 12.Novotny, M., Ishii, D., eds. Microcolumn Separations. 1985, Elsevier. 13.1shii, D., ed. Introduction to Microscale High Performance Liquid Chromatography. 1988, VCH. 14. Yang, E J., ed. Microbore Column Chromatography: A Unified Approach to Chromatography. 1989, Marcel Dekker. 15.Carrilho, E., Miller, A. W, Ruiz-Martinez, M. C, Kotler, L., Kesilman, J., Karger, B. L. Factors to Be Considered for Robust High-Throughput Automated DNA Sequencing Using a Multiple- Capillary Array Instrument. Proc. SPIE-Int. Soc. Opt. Eng., 1997; 2985 (Ultrasensitive Biochem- ical Diagnostics II):4. 16.Huang, X. C, Quesada, M. A., Mathies, R. A. Capillary Array Electrophoresis Using Laser- Excited Confocal Fluorescence Detection. Anal. Chem., 1992; 64:967. 17.Bienvenu, J., Graziani, M. S., Rpin, E A., Bernon, H., Blessum, C, Marchetti, C, Righetti, G., Somenzini, M., Verga, G., Aguzzi, E Multicenter Evaluation of the Paragon CZE 2000 Capillary Zone Electrophoresis for Serum Protein Electrophoresis and Monoclonal Component Typing. Clin. Chem., 1998; 44:599. 18. Smith, N. W, Evans, M. B. The Efficient Analysis of Neutral and Highly Polar Pharmaceutical Compounds Using Reversed-Phase and Ion-Exchange Electrochromatography. Chro- matographifl, 1995; 41:197. 19.Dittman, M. M., Wienand, K., Bek, E, Rozing, G. P. Theory and Practice of Capillary Elec- trochromatography LC-GC, 1995; 13:800.
  28. 28. 22 Chapter 1 Introduction 20.Manz, A., Harrison, D. J., Verpoorte, E. M. J., Fettinger, J. C, Ludi, H., Widmer, H. M. Minia- turization of Chemical Analysis Systems—A Look into Next Century's Technology or Just a Fashionable Craze. Chimia, 1991; 45:103. 21.Manz, A., Harrison, D. J., Verpoorte, E. M. J., Fettinger, J. C, Paulus, A., Ludi, H., Widmer, H. M. Planar Chips Technology for Miniaturization and Integration of Separation Techniques into Monitoring Systems. Capillary Electrophoresis on a Chip. J. Chromatogr., 1992; 593:253. 22.Woolley, A. T, Mathies, R. A. Ultra-High-Speed DNA Sequencing Using Capillary Elec- trophoresis Chips. Anal. Chem., 1995; 67:3676. 23. Chiem, N. H., Harrison, D. J. Microchip Systems for Immunoassay: An Integrated Immunoreactor with Electrophoretic Separation for Serum Theophylline Determination. Clin. Chem., 1998; 44:591. 24. Colyer, C. L., Tang, T, Chiem, N., Harrison, D.J. Clinical Potential of Microchip Capillary Elec- trophoresis. Electrophoresis, 1997; 18:1733. 25.Effenhausen, C. S., Manz, A. Miniaturizing a Whole Analytical Laboratory Down to Chip Size. Am.Lah., 1994; 26:15. 26.Harrison, D. J., Manz, A., Fan, Z., Ludi, H., Widmar, H. M. Capillary Electrophoresis and Sam- ple Injection Systems Integrated on a Planar Glass Chip. Anal. Chem., 1992; 64:1926. 27.Jacobson, S. C, Hergenroder, R., Koutny, L. B., Ramsey, M. J. High Speed Separations on a Microchip. Anal. Chem., 1994; 66:1114. 28.Jacobson, S. C, Hergenroder, R., Koutny, L. B., Ramsey, M.J. Open Channel Electrochro- matography on a Microchip. Anal. Chem., 1994; 66:2369. 29.Jacobson, S. C, Hergenroder, R., Koutny L. B., Warmack, R. J., Ramsey M. J. Effects of Injec- tion Schemes and Column Geometry on the Performance of Microchip Electrophoresis Devices. Anal. Chem., 1994; 66:1107. 30.Vesterberg, O. History of Electrophoretic Methods. J. Chromatogr, 1989; 480:3. 31. Compton, S. W, Brownlee, R. G. Capillary Electrophoresis. BioTechniques, 1988; 6:432. 32.Hjerten, S. Free Zone Electrophoresis. Chromatogr Rev., 1967; 9:122. 33.Pretorius, V, Hopkins, B. J., Schieke, J. D. A New Concept of High-Speed Liquid Chromatogra- phy J. Chromatogr, 1974; 99:23. 34.Mikkers, F E. R, Everaerts, F M., Verheggen, T P. E. M. High Performance Zone Electrophore- sis. J. Chromatogr, 1979; 169:11. 35.Jorgenson, J. W, Lukacs, K. D. Zone Electrophoresis in Open Tubular Glass Capillaries. Anal. Chem., 1981; 53:1298. 36.Hjerten, S. High-Performance Electrophoresis: The Electrophoretic Counterpart of High Per- formance Liquid Chromatography. J. Chromatogr, 1983; 270:1. 37.Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A., Ando, T. Electrokinetic Separations with Micellar Solutions and Open-Tubular Capillaries. Anal. Chem., 1984; 56:111. 38.Hjerten, S., Zhu, M.-D. Adaptation of the Equipment for High-Performance Electrophoresis to Isoelectric Focusing. J. Chromatogr, 1985; 346:265. 39.Gassmann, E., Kuo, J. E., Zare, R. N. Electrokinetic Separation of Chiral Compounds. Science, 1985; 230:813. 40.Hjerten, S. High-Performance Electrophoresis: Elimination of Electroendosmosis and Solute Adsorption. J. Chromatogr, 1985; 347:191. 41.Lauer, H. H., McManigill, D. Capillary Zone Electrophoresis of Proteins in Untreated Fused Sil- ica Tubing. Anal. Chem., 1986; 58:166. 42.01ivares, J. A., Nguyen, N. T, Yonker, C. R., Smith, R. D. On-Line Mass Spectrometric Detec- tion for Capillary Zone Electrophoresis. Anal. Chem., 1987; 59:1230. 43. Wallingford, R. A., Ewing, A. G. Capillary Zone Electrophoresis with Electrochemical Detec- tion. Anal. Chem., 1987; 59:1762. 44.Kuhr, W G., Yeung, E. S. Indirect Fluorescence Detection of Native Amino Acids in Capillary Zone Electrophoresis. Anal. Chem., 1988; 60:1832. 45. Chen, C. Y., Morris, M. D. Raman Spectroscopic Detection System for Capillary Zone Elec- trophoresis. Appl. Spectrosc, 1988; 42:515.
  29. 29. References 2 3 46. Guttman, A., Paulus, A., Cohen, A. S., Grinberg, N., Karger, B. L. Use of Complexing Agents for Selective Separation in High-Performance Capillary Electrophoresis: Chiral Resolution via Cyclodextrins Incorporated Within Polyacrylamide Gel Columns. J. Chromatogr., 1988; 448:41. 47.Hjerten, S., Valtcheva, L., Elenbring, K., Eaker, D. High-Performance Electrophoresis of Acidic and Basic Low-Molecular Weight Compounds and of Proteins in the Presence of Polymers and Neutral Surfactants. J. Liq. Chromatogr., 1989; 12:2471. 48.Zhu, M., Hansen, D. L., Burd, S., Gannon, F. Factors Affecting Free Zone Electrophoresis and Isoelectric Focusing in Capillary Electrophoresis. J. Chromatogr., 1989; 480:311. 49. Cohen, A. S., Najarian, D. R., Karger, B. L. Separation and Analysis of DNA Sequence Reaction Products by Capillary Gel Electrophoresis. J. Chromatogr, 1990; 516:49. 50.Drossman, H., Luckey J. A., Kostichka, A. J., D'Cunha, J., Smith, L. M. High-Speed Separations of DNA Sequencing Reactions by Capillary Electrophoresis. Anal. Chem., 1990; 62:900. 51.Luckey, J. A., Drossman, H., Kostichka, A. J., Mead, D. A., D'Cunha, J., Norris, T. B., Smith, L. M. High Speed DNA Sequencing by Capillary Electrophoresis. Nud. Acids Res., 1990; 18:4417. 52.Swerdlow, H., Gesteland, R. Capillary Gel Electrophoresis for Rapid, High Resolution DNA Sequencing. Nud. Adds Res., 1990; 18:1415. 53. Swerdlow, H., Wu, S., Harke, H., Dovichi, N. J. Capillary Gel Electrophoresis for DNA Sequencing: Laser-Induced Fluorescence Detection with the Sheath Flow Cuvette.J. Chromatogr, 1990; 516:61. 54.Zagursky, R. J., McCormick, R. M. DNA Sequencing Separations in Capillary Gels on a Modi- fied Commercial DNA Sequencing Instrument. BioTechniques, 1990; 9:74. 55.Dadoo, R., Colon, L. A., Zare, R. N. Chemiluminescence Detection in Capillary Electrophore- sis. HRC & CC, 1992; 15:133. 56. Wu, N., Peck, T. L., Webb, A. G., Magin, R. L., Sweedler, J. V Nanoliter Volume Sample Cells for ^H NMR: Application to Online Detection in Capillary Electrophoresis. J. Am. Chem. Soc, 1994; 116:7929. 57.Walbroehl, Y., Jorgenson, J. W On-Column UV Absorbance Detector for Open Tubular Capil- lary Zone Electrophoresis. J. Chromatogr, 1984; 315:135. 58. Green, J. S., Jorgenson, J. W Design of a Variable Wavelength UV Absorption Detector for On- Column Detection in Capillary Electrophoresis and Comparison of Its Performance to a Fixed Wavelength UV Absorption Detector. J. Liq. Chromatogr, 1989; 12:2527. 59.Jorgenson,J. W, Lukacs, K. D. Free-Zone Electrophoresis in Glass Capillaries. Clin. Chem., 1981; 27:1551. 60. Wallingford, R. A., Ewing, A. G. Capillary Zone Electrophoresis with Electrochemical Detec- tion in 12.7|Lim Diameter Columns. Anal. Chem., 1988; 60:1972. 61.Pentoney, S. L., Zare, R. N., Quint, J. F. On-Line Radioisotope Detection for Capillary Elec- trophoresis. Anal. Chem., 1989; 61:1642. 62.Salas-Solano, O., Carrilho, E., Kolter, L., Miller, A. W, Goetzinger, W, Sosic, Z., Karger, B. L. Routine DNA Sequencing of 1000 Bases in Less than One Hour by Capillary Electrophoresis with Replaceable Linear Polyacrylamide Solutions. Anal. Chem., 1998; 70:3996. 63. Kim, Y., Yeung, E. S. Separation of DNA Sequencing Fragments up to 1000 Bases by Using PolyCethylene Oxide)-Filled Capillaries. J. Chromatogr, A, 1997; 781:315. 64.Jorgenson, J. W, Lukacs, K. D. Zone Electrophoresis in Open-Tubular Glass Capillaries: Pre- liminary Data on Performance. HRC & CC, 1981; 4:230. 65.Kleparnik, K., Bocek, P Theoretical Background for Clinical and Biomedical Applications of Electromigration Techniques. J. Chromatogr, 1991; 569:3.
  30. 30. CHAPTER 2 Capillary Zone Electrophoresis Basic Concepts 2.1 Electrical Conduction in Fluid Solution 2.2 The Language of Electrophoresis 2.3 Electroendoosmosis 2.4 Efficiency 2.5 Resolution 2.6 Joule Heating 2.7 Optimizing the Voltage and Temperature 2.8 Capillary Diameter and Buffer Ionic Strength 2.9 Optimizing the Capillary Length 2.10 Buffers 2.11 Temperature Effects 2.12 Buffer Additives 2.13 Capillaries 2.14 Sources of Band Broadening References 2.1 ELECTRICAL CONDUCTION IN FLUID SOLUTION Several simple concepts are important for understanding the physical processes that occur upon passage of an electrical current through an ionic solution. ^ These processes are far more complex than the passage of current through a metal. In metals, uniform and v^eightless electrons carry all the current. In fluid ^See any basic text on physical chemistry for a thorough description of electrical conduction in fluid solution. 25
  31. 31. 26 Chapter 2 Capillary Zone Electrophoresis solution, the current is carried by cations and anions. The molecular weight of these charge bearing ions ranges from a simple proton to tens of thousands for large complex ions such as proteins and polynucleotides. Conduction in fluid solution is still described by Ohm's law, E = IR, (2.1) where E is the voltage or applied field, I is the current that passes through the solution, and R is the resistance of the fluid medium. The reciprocal of resistance is conductivity. Kohlrausch found that the con- ductivity of a solution resulted from the independent migration of ions. As illustrated in Figure 2.1, when a current passes through an ionic solution, anions migrate toward the anode (positive electrode) while cations migrate toward the cathode (negative electrode) in equal quantities. Despite the passage of current, electroneutrality of the solution is always maintained because of electrolysis at each electrode. This is important because electrolysis produces protons at the anode and hydroxide at the cathode (Figure 2.2). The resultant pH changes are due to the process known as buffer depletion (1-3). Since pH is the single most important experimental parameter in capillary electrophoresis, this effect must be mini- mized by 1. Using the appropriate buffers 2. Having sufficiently large buffer reservoirs 3. Replacing buffers frequently The introduction of a sample into the capillary changes the situation dra- matically (Figure 2.3). The Ohm's law equation changes as well to that for a series circuit: E = IR^ + IRj . (2.2) This process and the equation have important implications in HPCE. When low- conductivity samples (relative to the BGE) are injected, a process known as stacking (Section 8.6) occurs. This permits the use of large-volume injections CATHODE ANODE FIGURE 2.1 The independent migration of ions.
  32. 32. 2.1 Electrical Conduction in Fluid Solution 27 POWER SUPPLY CAPILLARY DETECTOR OH GENERATED AT CATHODE H GENERATED AT ANODE CATHOLYTE (OUTLET) ELECTRODES FIGURE 2.2 Buffer depletion. ANOLYTE (INLET) to be employed without excessive band broadening because zone compression occurs. On the other hand, if high-conductivity samples are injected relative to the BGE, antistacking or zone broadening will occur. The conductivity of a solution is determined by two factors: 1. The concentration of the ionic species. 2. The speed of movement or mobility of the ionic species in an electric field. In other words, highly mobile species are also highly conductive, and vice versa. IR IR, ©, FIGURE 2.3 Impact of the sample injection on the IR drops in a capillary.
  33. 33. 2 8 Chapter 2 Capillary Zone Electrophoresis The mobility of ions in fluid solution is governed by their charge to size ratio. The size of the molecule is based on the molecular weight, the three-dimensional structure, and the degree of solvation (usually hydration). Data given in Table 2.1 (4) for alkali metals illustrate several of these important points: 1. The orders for the mobilities of the metal ions are the reverse of what is expected based on the metal or crystal radii data. These smaller ions are more hydrated than their larger counterparts. 2. The current generated by 100 mM solutions of various acetate salts is proportional to the ionic mobility of the cation. This feature becomes important when selecting the appropriate counterion for preparing buffer solutions. The forces governing this behavior are expressed by Stoke's law, / = 67rrirv , (2.3) where 7] = viscosity, r = ionic radius, and v = ionic velocity. The competing forces of mobility (velocity) and viscosity are illustrated in Figure 2.4 for an ion of radius r. Ionic size modifies mobility because of a solute's exposure to fric- tional drag as it migrates through the supporting electrolyte. The frictional drag is directly proportional to viscosity, size, and electrophoretic velocity. An expres- sion for mobility that contains these terms is M—)=-5^^^^^ = ^ - , (2.4) Vs E(V/cm) 6nrir where q = the net charge and E = the electric field strength. Thus, mobility is considered a charge-to-size ratio. Since the units for velocity are centimeters per second and the field strength is expressed as volts per centimeter, the units of mobility are cm^A^s. 2.2 THE LANGUAGE OF ELECTROPHORESIS There are several distinguishing differences between the terminology of chro- matography and that of capillary electrophoresis. For example, a fundamental para- meter in chromatography is the retention time. In electrophoresis nothing should ever be retained (except for CEC), so a more descriptive term is migration time: the time it takes a solute to travel from the beginning of the capillary to the detec- tor window.
  34. 34. 2.2TheLanguageofElectrophoresis29 < I—I 2 o 1^ T1 <SJ t<U to 4:5 v^> »-l 3 0u fio 2»- OS,^ B X 0 TJ !U P^nd OH <;>> o< "rn 3 Tl rr! X^ II UPi Sf2 hs.in^in ^ONOrH ON ON O rn 00 d —1.-I(N su u =^0 ^r^ON 5o^j <-.
  35. 35. 30 Chapter 2 Capillary Zone Electrophoresis MOBILITY FRICTIONAL FORCES FIGURE 2.4 The competing forces of electrophoretic mobility and viscous drag. The use of a detection window in HPCE (on-capillary detection) as opposed to postcolumn detection must also be considered. In HPLC, the length of the chromatographic column must be included in all methods. Figure 2.5 is a draw- ing of a capillary. Both the total length of the capillary (L^ or L) and the length to the detector (L^ or I) must be described. The segment of capillary that occurs after the detector window is necessary to make electrical contact with the out- let or detector-side electrolyte reservoir. Ideally, L^ - L^ should be as short as practical. Otherwise, some system voltage (V) is wasted on maintaining field strength (E) over part of the capillary that lies beyond the detector window and hence does not participate in the separation. Lt DETECTION WINDOW FIGURE 2.5 Illustration of a capillary defining the total length (Lj) and the length to the detec- tor (Ld).
  36. 36. "ep Mep E .jjtrn 2.3 Electroendoosmosis 31 Expressions for some other fundamental terms are given in the following equations: (2.5) (2.6) The preceding include the electrophoretic mobility (^ep^ cm^A^s), the elec- trophoretic velocity (Vgp, cm/s), and the field strength (E, V/cm). These equa- tions define some fundamental features of HPCE: 1. Velocities are measured experimentally (Eq. 2.5). They are determined by dividing the length of capillary, from the injection side to the detec- tor window (Ld), by the migration time t^. 2. MobiUties are calculated by dividing the electrophoretic velocity v^p by the field strength (Eq. 2.6). The field strength is simply the voltage divided by the total capillary length (L^). The field strength is the important parameter governing electrophoretic migration. Field strength is changed when either the voltage or the capillary length is altered. Mobility is the fundamental parameter of capillary electrophoresis. This term is independent of voltage and capillary length. Equations (2.6) and (2.7) define only the relative mobility. To calculate the true mobility, a correction for a phenomenon known as electroendoosmotic flow (Section 2.3) must first be made. 2.3 ELECTROENDOOSMOSIS A. THE CAPILLARY SURFACE One of the fundamental processes that accompanies electrophoresis is electroos- mosis. One of the "pumping" mechanisms of HPCE, electroosmosis occurs because of the surface charge, known as the zeta potential, on the wall of the cap- illary. Fused silica is the most common material used to produce capillaries for HPCE. Technology developed for manufacturing capillary columns for GC read- ily transferred to HPCE. Fused silica is a highly crosslinked polymer of silicon dioxide with tremendous tensile strength (5), although it is quite brittle. With its polyimide coating, fused silica is quite durable, although some polyimide must
  37. 37. 32 Chapter 2 Capillary Zone Electrophoresis be removed to create a ultraviolet (UV) transparent optical window for detection. Other materials such as Teflon and quartz have been used (6), but performance and cost are less favorable. Before use, capillaries are usually conditioned with 1 N sodium hydroxide. The base ionizes free silanol groups and may cleave some silica epoxide linkages as well. An anionic charge on the capillary surface results in the formation of an electrical double layer. The resulting ionic distribution is shown in Figure 2.6 (7). Anions are repelled from the negatively charged wall region, whereas cations are attracted as counterions. Ions closest to the wall are tightly bound and immobile, even under the influence of an electric field. Further from the wall is a compact and mobile region with substantial cationic character. At a greater distance from the wall, the solution becomes electrically neutral as the zeta potential of the wall is no longer sensed. Expressions describing this phenomenon were derived by Gouy and Chapman in 1910 and 1913, respectively This diffuse outer region is known as the Gouy-Chapman layer. The rigid inner layer is called the Stem layer. When a voltage is applied, the mobile positive charges migrate in the direc- tion of the cathode or negative electrode. Since ions are solvated by water, the fluid in the buffer is mobilized as well and dragged along by the migrating charge. Although the double layer is perhaps 100 A thick, the electroendoos- motic flow (EOF) is transmitted throughout the diameter of the capillary, pre- sumably through hydrogen bonding of water molecules or van der Waals interactions between buffer constituents. The electroosmotic flow as defined by Smoluchowski in 1903 is given by Veo = ^ E , (2.8) where £ is the dielectric constant, 77 is the viscosity of the buffer, and f is the zeta potential of the liquid-solid interface. The equation is only valid for capil- laries sufficiently large that the double layers on opposite walls do not overlap each other (8). Practical use of this equation is not forthcoming, as the zeta potential is rarely measured and data for the dielectric constants of mixtures are not readily available. Like electrophoretic mobility, the EOF is inversely pro- portional to the viscosity of the BGE. B. MEASURING THE ELECTROOSMOTIC FLOW Since the migration time of a solute is influenced by the EOF, calculation of the actual mobility requires measurement of the EOF: Here jH^^ = the actual mobility, jU^pp = apparent (observed) mobility, and jLl^o = electroosmotic mobility. The use of mobility as the "migration parameter"
  38. 38. 2.3 Electroendoosmosis 33 inttrfaisa c . ^ g—: SI ®, '"^lA J Z*^^ /'""x x*i*' I • X - adsorbed compact diffuse layer layer layer B « 4N# 0 fit i interf8€0 1 i s 1 1 u r L <*• 1 1 ^ l ^ compact. dlffuM***" layer 1 ''yr ' ^ • HMliiiiiiliilnpi dislanai from lh# eolynin wail FIGURE 2.6 Representation of the electrical double layer versus distance from the capillary wall. Reprinted with permission from J. Chromatogr., 559, 69 (1991), copyright © 1991 Elsevier Science Publishers. will frequently yield greater precision compared to the use of migration time, since the impact of the EOF is factored out of the calculation (Section 10.6). Routine measurement of the EOF is also necessary to ensure the integrity of the separation. If the EOF is not reproducible, it is likely that the capillary wall is being affected by some component in the sample or an experimental para- meter is not being properly controlled (see Section 2.3F).
  39. 39. 34 Chapter 2 Capillary Zone Electrophoresis The simplest method for measuring the EOF is to inject a dilute solution con- taining a neutral solute and measure the time it takes to transit the detector (9-11). Since the capillary length is known, the velocity in centimeters per second is eas- ily calculated. Dividing that value by the field strength yields the electroosmotic mobility in units of cmWs. Neutral solutes such as methanol, acetone, benzyl alcohol, and mesityl oxide are frequently employed. When MECC is the mode of separation (Chapter 4), a further requirement that the marker solute not partition into the micelle is imposed. When the EOF is slow, the migration time can be quite long. To reduce the experimental time, it is useful to use the short end of the capillary to make the measurement. The short end is the section of capillary normally found between the detector window and capillary outlet. The injection can be made at the out- let side and the system operated using reversed polarity Now the EOF is mea- sured using a short capillary length of 6-10 cm depending on the brand of instrument. As will be shown later, the short end of the capillary can be very useful when performing screening runs during methods development. When the EOF is very slow, as in the case with certain coated capillaries, spe- cial techniques must be employed (12). It is seldom necessary to measure very weak EOF, since it does not notably affect mobility or experimental precision. C. EFFECT OF BUFFER P H The impact of pH on the EOF and the mobility is illustrated in Figure 2.7. At high pH the silanol groups are fully ionized, generating a strong zeta potential and dense electrical double layer. As a result, the EOF increases as the buffer pH is elevated (9, 13). A robust flow, typically around 2 mm/s at pH 9 in 20 mM borate buffer at 30 kV, 30°C is realized. For a 50 |Lim capillary, this translates to 235 nL/min. Since the total volume of a 50 cm x 50 jim i.d. capillary is only 980 nL, a neutral compound would reach the detector in 4.2 min. At pH 3, the EOF is much lower, about 30 nL/min. The EOF must be controlled or even suppressed to run certain modes of HPCE. On the other hand, the EOF makes possible the simultaneous separation of cations, anions, and neutral species in a single run. For example, a zwitterion like a peptide will be negatively charged at a pH above its pi. The solute will elec- tromigrate toward the positive electrode. However, the EOF is sufficiently strong that the solute's net migration is toward the negative electrode (Figure 2.7, top). At low pH, the zwitterion has a positive charge and will migrate as well toward the negative electrode (Figure 2.7, bottom). In untreated fused-silica capillar- ies, most solutes migrate toward the negative electrode unless buffer additives or capillary treatments are used to reduce or reverse the EOF (Section 3.3). The EOF is exquisitely sensitive to pH (9,14,15). Hysteresis effects have been reported (15) wherein the direction of approach to a particular pH value produces a different pH (Figure 2.8). When approaching from the acid side, the measured
  40. 40. 2.3 Electroendoosmosis HIGH pH 35 ++++•'•+•'•+++++++++++++++++++++++++"'•+•*'+''" M,0 / * ep 4 . 4 . ^ 4 . 4 , 4 » 4.4> 4. 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 * 4 . + + + + + 4* + 4- + + +^+ + -f + -I" + -f + FIGURE 2.7 Behavior of electroendoosmotic flow and electrophoretic migration of a zwitterion (pi = 7) at high and low pH. EOF is always lower, and vice versa. This means there is a kinetic parameter with regard to the estabUshment of a stable charge on the capillary wall. Longer equili- bration times would reduce hysteresis at the expense of increased total run time. Since the EOF will affect migration time precision, it is important to design experiments with these features in mind. The problems with EOF repro- ducibility are often most severe in the pH range 4-6 (15). D. EFFECT OF BUFFER CONCENTRATION The expression for the zeta potential is (16) c = 47r5e (2.10) where £ = the buffer's dielectric constant, e = total excess charge in solution per unit area, and 5 is the double-layer thickness or Debye ionic radius. The Debye radius is 5 = (3 x 10'')(Z)(Ci/^), where Z = number of valence electrons and C = the buffer concentration. As the ionic strength increases, the zeta potential and, similarly, the EOF decreases in proportion to the square root of the buffer concentration. This was
  41. 41. 36 Chapter 2 Capillary Zone Electrophoresis 10 9 + 8+ E o % 6 X u. o 5 4+ 3+ H 1 1 1 1—I 1 1 1 1 2 3 4 5 6 7 8 9 10 11 PH FIGURE 2.8 Effect of experimental design on the EOF. Key: • , high pH titrated to low pH; •, low pH titrated to high pH. Data from reference (15). confirmed experimentally (17) for a series of buffers where the EOF was found hnear to the natural logarithm^ of the buffer concentration. It was reported that equivalent EOF is found for different buffer types as long as the ionic strength is kept constant (17). The effect of buffer concentration and field strength is shown in Figure 2.9 (18). The electroosmotic mobility is plotted against field strength for phosphate buffer at three different concentrations using a 50-|Lim-i.d. capillary. As expected, the higher buffer concentrations showed lower EOF at all field strengths. Since ^The linear relationship of EOF with the buffer concentration is a square root relationship as indicated by Eq. (2.10).
  42. 42. 2.3 Electroendoosmosis 37 71 u. ^^ u z o Sliini UJ 50 100 150 200 E (V/cm) 250 FIGURE 2.9 Effect of buffer concentration and field strength (E, V/cm) on the electroosmotic flow in a 50-|xm-i.d. capillary. Buffer: phosphate at a concentration of (a) 10 mM; (b) 20 mM; (c) 50 mM. Redrawn with permission from J. Chromatogr., 516, 223 (1990), copyright © 1990 Elsevier Science Publishers. mobility was plotted, all three lines should be flat. Slight positive slopes were reported for all three concentrations, presumably due to heating effects (Sec- tion 2.6). The same data produced using a lOO-jiim-i.d. capillary will be exam- ined in that section. E. EFFECT OF ORGANIC SOLVENTS Organic solvents can modify the EOF because of their impact on buffer viscos- ity (17) and zeta potential (19). Linear alcohols such as methanol, ethanol, or isopropanol usually decrease the EOF because they increase the viscosity of the electrolyte. Acetonitrile either does not affect or may slightly increase the EOF (20). Organic solvents are often employed in HPCE to help solubilize the sam- ple. Selectivity can be affected as well in both CZE (20) and MECC (21). Because of the sensitivity of organic solvent concentration on selectivity, evaporation must
  43. 43. 3 8 Chapter 2 Capillary Zone Electrophoresis be carefully controlled. In this regard, wholly aqueous separations are often advantageous. F. CONTROLLING THE ELECTROOSMOTIC FLOW The EOF is a double-edged sword. It allows the separation of cations, anions, and neutral solutes in a single run. It is also the single most important contrib- utor to migration time variability on a run-to-run, day-to-day, and capillary-to- capillary basis. The EOF is affected by many parameters, including Buffer pH Buffer concentration Temperature Viscosity Capillary surface Field strength Organic modifiers Cellulose polymers Surfactants In this list, the only factor not under direct experimental control is the cap- illary surface. This single factor is often implicated as the cause for migration time variation in HPCE. It is important to ensure that the capillary surface is properly reconditioned after each run to maintain a reproducible surface. Coated capillaries that suppress the EOF are useful here, as long as the coating is sta- ble. Some new reagents^ that form a dynamic surface coating show great promise for stabilizing the capillary surface (Section 3.3). For some modes of HPCE, it is advantageous to suppress the FOE Capillary isoelectric focusing (CIEF) and capillary isotachophoresis (CITP) separations are usually performed under conditions of very low or carefully controlled EOF Additives such as 0.5% hydroxypropylmethyl cellulose are effective in sup- pressing the EOF, particularly in conjunction with a coated capillary (22). Cationic surfactants such as cetyltrimethylammonium bromide can actually reverse the direction of electroosmotic flow (14). This can be employed to pre- vent proteins from sticking to the capillary wall (23, 24). While complete sup- pression of the EOF is unnecessary for most applications, control is critical to obtain reproducible migration times and resolution. ^CElixir, Scientific Resources, Inc., Eatontown, NJ.
  44. 44. 2.4 Efficiency 3 9 2.4 EFFICIENCY The high efficiency of HPCE is a consequence of several unrelated factors: 1. A stationary phase is not required for HPCE. The primary cause of band broadening in LC is resistance to mass transfer between the stationary and mobile phases. This mass transfer problem is illustrated in Figure 2.10. When a solute is in the mobile phase, its linear velocity is determined by the linear velocity of the mobile phase. When attached to the stationary phase, the linear velocity becomes zero. The solute is not of a single velocity as it moves down the chro- matographic tube. Whenever differing velocities occur during a separation, band broadening will occur. Minimizing the particle size of the packing improves but does not eliminate this problem. Thus, the parameter that results in separation also causes band broadening. The greater the retention, the greater the prob- lem—as evidenced by broadened peaks as retention time increases. For most modes of HPCE (except CEC), this dispersion mechanism does not operate. Similarly, other HPLC dispersion mechanisms such as eddy diffusion and stag- nant mobile phase are unimportant in HPCE. 2. In pressure-driven systems such as LC, the frictional forces of the mobile phase interacting at the walls of the tubing result in radial velocity gradients throughout the tube. As a result, the fluid velocity is greatest at the middle of the tube and approaches zero near the walls (Figure 2.11). This is known as laminar or parabolic flow. These frictional forces, together with the chromato- graphic packing, result in a substantial pressure drop across the column. In electrically driven systems, the EOF is generated uniformly down the entire length of the capillary. There is no pressure drop in HPCE, and the radial flow profile is uniform across the capillary except very close to the walls, where the flow rate approaches zero (Figure 2.11). FIGURE 2.10 The mass transport problem in HPLC.
  45. 45. 40 Chapter 2 Capillary Zone Electrophoresis Jorgenson and Lukacs derived the efficiency of the electrophoretic system from basic principles (25-27) using the assumption that diffusion is the only source of band broadening. Other sources of dispersion—including Joule heat- ing (Section 2.6), capillary wall binding (Section 3.5), injection (Section 9.1), detection (Section 9.5), and electromigration dispersion (Section 2.13)—lead to fewer theoretical plates than the simple theory predicts. The migration velocity for a solute is V = luE = HV (2.11) where // = the mobility, E = field strength, V = voltage, and L = capillary length. The time t for a solute to migrate the length L of the capillary is V fiV (2.12) ELECTROOSMOTIC FLOW HYDRODYNAMIC FLOW FIGURE 2.11 Capillary flow profiles resulting from electroosmotic and hydrodynamic flow.
  46. 46. 2.5 Resolution 41 Diffusion in liquids that leads to broadening of an initially sharp band is described by the Einstein equation (yl = 2Dt = =^^, (2.13) where D = the diffusion coefficient of the individual solute. The number of the- oretical plates N is given by N = — . (2.14) Substituting Eq. (2.11) into Eq. (2.12) gives an expression for the number of theoretical plates: N = ^ . (2.15) 2D Some important generalizations can be made from this expression: 1. The use of high voltage gives the greatest number of theoretical plates, since the separation proceeds rapidly, minimizing the effect of diffusion. This holds true up to the point where heat dissipation is inadequate (Section 2.6). 2. Highly mobile solutes produce high plate counts, because their rapid velocity through the capillary minimizes the time for diffusion. 3. Solutes with low diffusion coefficients give high efficiency due to slow diffusional band broadening. Points 2 and 3 appear contradictory. This is clarified by Figure 2.12 and sup- plemented with some calculations in Table 2.2. Because of the indirect but inverse relationship between mobility and diffusion, high-efficiency separations occur across a wide range of molecular weights. HPCE can yield high-efficiency separations for both large and small mole- cules. The greatest number of theoretical plates is found in capillary gel elec- trophoresis (CGE). The use of an anticonvective gel matrix furthers the advantages of HPCE. The combination of HPCE in the gel or polymer network format (Chapter 6) can yield millions of theoretical plates. 2.5 RESOLUTION While high efficiency is important, resolution is the key for all forms of sepa- ration. In a high-efficiency system, inadequate resolution may result in a single very sharp peak.
  47. 47. 42 Chapter 2 Capillary Zone Electrophoresis DIFFUSION RAPID SLOW MOBILITY HIGH LOW SMALL MOLECULES LARGE MOLECULES FIGURE 2.12 Diffusion and mobihty of small and large molecules. The resolution (R) between two solutes is defined as R. = 1 AAI^PVN 4 Mep + Meo (2.16) where A^ is the difference in mobility between two solutes, /i^^ is the average mobility of the two solutes, and N is the number of theoretical plates. Substi- tuting the plate count equation (Eq. (2.15) and V = EL) yields (25) R, = O.UlAfl^ EL (/^ep + I^J^n (2.17) This expression suggests that increasing the voltage is not very effective in improving resolution, since that parameter falls inside of the square root of the resolution equation. A doubling of voltage results in only a 41% improvement in resolution. The production of heat quickly limits this Table 2.2 Calculated Theoretical Plates for a Small and Large Molecule Solute Horse heart myoglobin Quinine sulfate MW 13,900 747 Mobility (10-^ cmW-s) 0.65 4 Diffusion Coefficient (10-^cmVs) 1 7 N 975,000 857,000
  48. 48. 2.6 Joule Heating 43 approach. Another means of improving resolution as predicted by Eq. (2.17) is to adjust the EOF. Akhough this also falls under the square root sign of the resolution equation, this technique can be quite effective. There are three cat- egories in this regard: 1. Both electrophoresis and electroosmosis are in the same direction. This normally occurs when cations are being separated. In this case, decreas- ing the EOF will enhance resolution at the expense of run time. Dou- bling the run time produces a 41% improvement in resolution. 2. Electrophoresis and electroosmosis are in opposite directions. This occurs on bare silica capillaries when anions are separated. Decreasing the EOF will enhance run time at the expense of resolution, and vice versa. 3. Electrophoresis and electroosmosis are equal but in opposite directions. Here the resolution is infinite, but so is the separation time. However, this concept was used to generate ultrahigh theoretical plate numbers (28). It is clear that improvements in resolution are best addressed by adjustments to AjUgp, the difference in mobility between the two most closely eluting solutes in a separation. Since A/i^p falls outside of the square root sign of the resolution equation, the improvement in resolution is directly proportional to the change in mobilities. This subject forms the basis for many of the chapters in this book. 2.6 JOULE HEATING The conduction of electric current through an electrolytic solution generates heat via frictional collisions between migrating ions and buffer molecules. Since high field strengths are employed in HPCE, ohmic orJoule heating can be sub- stantial. There are two problems that can result from Joule heating: 1. Temperature changes due to ineffective heat dissipation 2. Development of thermal gradients across the capillary If heat is not dissipated at a rate equal to its production, the temperature inside the capillary will rise and eventually the buffer solution will outgas. Even a small bubble inside of the capillary disrupts the electrical circuit. At moder- ate field strengths, outgassing is not usually a problem, even for capillaries that are passively cooled. The rate of heat production inside the capillary can be estimated by ^ = - ^ , (2.18) dT LA where L = capillary length and A = the cross-sectional area. Rearranging this equation using J = V/R, where the resistance R = L/kA and k = the conductivity.
  49. 49. 44 Chapter 2 Capillary Zone Electrophoresis dT kV' (2.19) The amount of heat that must be removed is proportional to the conductivity of the buffer, as well as the square of the field strength. Lacking catastrophic failure (bubble formation), the problem of thermal gra- dients across the capillary can result in substantial band broadening (29-31). This problem is illustrated in Figure 2.13. The second law of thermodynamics states heat flows from warmer to cooler bodies. In HPCE, the center of the cap- illary is hotter than the periphery. Since the viscosity of most fluids decreases with increasing temperature, Eq. (2.4) and (2.8) predict that both mobility and EOF increase as the temperature rises. This situation becomes similar to laminar flow where the electrophoretic or electroosmotic velocity at the center of the capillary is greater than the velocity near the walls of the capillary. The temperature differential of the buffer between the middle and the wall of the capillary can be estimated from AT = 0.24 4K (2.20) where W = power, r = capillary radius, and K = thermal conductivity of the buffer, capillary wall, and polyimide cladding. A 2-mm-i.d. capillary filled with 20 mM CAPS buffer draws 18 rtiA of current at 30 ky giving a AT of 75°C. A 50-|lm-i.d. capillary filled with the same buffer draws only 12 |LIA of current, yielding a AT of 50 m°C. Since the thermal gradient is proportional to the square of the capillary radius, the use of narrow capillaries facilitates high resolution. On the other hand, the use of dilute buffers or isoelectric buffers (32) permits the use of wider bore capillaries, but the loading capacity of the separation is reduced. ep rAT FIGURE 2.13 Impact of the radial temperature gradient on electrophoretic and electroosmotic flow.
  50. 50. 2.6 Joule Heating 45 The requirement for narrow-bore capillaries comes with a price due to the short optical path length. If a solution is injected equivalent to 1% of the capil- lary volume of a 50 cm x 50 |im i.d. capillary, the injection size is 9.8 nL. This small-volume injection coupled to a 50-|im optical path length provides for con- centration limits of detection (CLOD) that are about 50 times poorer than by LC. Fortunately, through the use of stacking procedures (33) and extended path length capillaries (34), this gap has been narrowed considerably The compromise between sensitivity and resolution is illustrated in Figures 2.14 and 2.15. Note in particular the cluster of peaks centered at a migration time of 31 min (26 min in Figure 2.15) in Figure 2.14. With the 50-|im-i.d. cap- illary, none of these peaks are baseline-resolved, but there is virtually no noise in the electropherogram. Separation of the same sample in a 25-|im-i.d. capil- lary (Figure 2.15) presents a different picture. The peaks are nearly baseline- resolved, but there is substantial noise in the output. This presents one of several compromises that must be made in HPCE. In this case, sensitivity and resolu- tion are competing analytical goals. 0.300-! 0.262H 0.225H 0. I87H 0. 150H 0. uaH 0.075H 0.037H 0.00OH MJ WAAAJ L io —J— 15 20 —~r-—1—- 25 30 MINUTES 3S 40 " "1— 45 •'"••'" ! " " • " 50 '" 1 55 FIGURE 2.14 Separation of heroin impurities by MECC on a 50-|lm-i.d. capillary. Buffer: 85 mM SDS, 8.5 mM borate, 8.5 mM phosphate, 15% acetonitrile, pH 8.5; capillary: 50 cm (length to detec- tor) X 50 (Xm i.d.; voltage: 30 kV; temperature: 50°C; detection: UV, 210 nm. Reprinted with per- mission from Anal. Chem., 63, 823 (1991), copyright © 1991 Am. Chem. Soc.
  51. 51. 46 Chapter 2 Capillary Zone Electrophoresis Even these electropherograms must be carefully interpreted. In both cases, the injection time was kept constant at 1 s. This means that the amount of mate- rial injected in the 25-|Ltm-i.d. capillary was a factor of four lower relative to the 50-|Llm-i.d. tube (Section 8.1). This contributes to the decreased signal-to-noise ratio observed when using the 25-|lm-i.d. capillary. The problem of Joule heating depends on the capillary diameter, the field strength, and the buffer concentration. Recalling Figure 2.9 (50-|Lim-i.d. capillary), there was a slight increase in |Lieo as the field strength was increased. Figure 2.16 contains data from the same experiments, except a 100-|Lim-i.d. capillary is used. A marked departure from linearity is found at the higher buffer concentrations. Higher concentration buffers are more conductive, draw higher currents, and produce more heat than more dilute solutions. In the 100-|im-i.d. capillary, this heat is not properly dissipated. As a result, the internal temperature rises, reduc- ing the viscosity of the buffer. Since Eq. (2.8), the basic expression for elec- troosmotic velocity, contains a viscosity parameter in the denominator, Vgo increases with decreasing buffer viscosity. Because the buffer viscosity depends on temperature, the capillary heat removal system plays an important role in deciding the maximum field strength, buffer concentration, and capillary diam- 0.080-1 0.07CH 0.060H 0. 050H 0. 040-H 0.030H 0. 020H 0. oiCH 0. GOOH N'wWW ^IKJMM ] 12 I 16 1 1 20 24 MINUTES 1 28 1 32 1 36 1" 40 FIGURE 2.15 Separation of heroin impurities by MECC on a 25-fxm-i.d. Conditions as per Fig- ure 2.14 except for capillary diameter. Reprinted with permission from Anal. Chem., 63, 823 (1991), copyright © 1991 Am. Chem. Soc.
  52. 52. 2.7 Optimizing the Voltage and Temperature 11 5 9 u. ^ o E7 to ' o j>5 47 50 100 150 E (V/cm) 200 250 FIGURE 2.16 Effect of buffer concentration and field strength (E, V/cm) on the electroosmotic flow in a 100-|lm-i.d. capillary. Buffer: phosphate at a concentration of (a) 10 mM; (b) 20 mM; (c) 50 mM. Redrawn with permission from J. Chromatogr., 516, 223 (1990), copyright © 1990 Elsevier Science Publishers. eter that can be successfully employed. Insufficient heat removal begins a vicious cycle leading to viscosity reduction, greater current draw, and higher temperature, further reducing the viscosity. 2.7 OPTIMIZING THE VOLTAGE AND TEMPERATURE A. OHM'S LAW PLOTS A means of optimizing the voltage and/or the temperature despite the buffer concentration and capillary cooling system is very desirable. An Ohm's law plot provides this tool with very little experimental work (35, 36). Simply fill the capillary with buffer, set the temperature, vary the voltage, record the current, and plot the results.
  53. 53. 48 Chapter 2 Capillary Zone Electrophoresis Some Ohm's law plots are shown in Figures 2.17 and 2.18 for an air-cooled and water-cooled temperature control system, respectively. Whenever the graph shows a positive deviation from linearity, the heat removal capacity of the system is being exceeded. Operating on the linear portion of the curve will generally yield the high- est number of theoretical plates. As a rule of thumb, it is best to keep operating currents below 100 joA. Often, separations are run in the nonlinear section to opti- mize speed at the expense of plates, but it is not wise to push things too far. Lowering the temperature below ambient can be used to extend the linear range of the Ohm's law plot. This is useful when high-ionic-strength buffers are < UJ O 100 80 60 40 20 A ^ •BV • • • " • .c •V *>' ' XT ^ • ^ 0 V • ^ D X T . ^ D •^ 0 • = - • ^ ^ D V ^- u" ¥ ^ D I ^^o° 1 ^ ^t°^¥ ^0° ¥^^D l±lll 1 H _— 1- 10 20 VOLTAGE (kV) 30 FIGURE 2.17 Ohm's law plots for capillary temperature control by air circulation. (A) no con- trol; (B) 25°C; (C) 10°C; (D) 4°C. Redrawn with permission from J. High Res. Chromatogr., 14, 200 (1991), copyright © 1991 Dr. Alfred Heuthig Publishers.
  54. 54. 2.7 Optimizing the Voltage and Temperature 49 1 LU OL O 100 80 60 40 20 A ' 1 "V • • B • V • • • V • • ^ C A 0 ^ • ^ 0 • ^ 0 -^ A „ « • A ° X -6. a • -<^ 0 • ^ 0 • ^ 0 • ^ 0 ,^^t°• ^ a * i ^ a «ELf 1 1 I — 10 20 VOLTAGE (kV) 30 FIGURE 2.18 Ohm's law plots for capillary temperature control by water circulation. (A) no con- trol; (B) 25°C; (C) 10°C; (D) 4°C. Redrawn with permission from J. High Res. Chromatogr., 14, 200 (1991), copyright © 1991 Dr. Alfred Heuthig Publishers. necessary. These concentrated buffers are particularly useful in microprepara- tive CE (Section 9.10), increasing the linear dynamic range (Section 10.4) and suppressing wall effects (Section 3.3). Increasing the temperature can also be employed to speed the separation, since both v, due to the decreased viscosity of the buffer medium For various instruments the Ohm's law plot is an effective means of evaluat- ing the efficiency of their capillary cooling systems. Fluid-cooled systems are and Vep increase about 2%/K
  55. 55. 5 0 Chapter 2 Capillary Zone Electrophoresis generally more effective than air-cooled systems, since the heat capacity of most fluids exceeds that of air. B. CONSTANT VOLTAGE OR CONSTANT CURRENT? Power can be applied to the system in one of two ways. The voltage can be fixed, allowing the current to float based on the resistance of the buffer. Alternatively the current can be fixed. Most published work in HPCE is in the constant-voltage mode. There has been one report that found constant current more reproducible than constant voltage (37). Until this is better understood, both modes should be studied during methods development for CZE, MECC, and CGE separations. CITP is typically performed in the constant-current mode, or else separation time becomes long. CIEF may also benefit from the constant-current mode as well, although there is no evidence published to that effect. 2.8 CAPILLARY DIAMETER AND BUFFER IONIC STRENGTH Some very subtle effects due to Joule heating can occur when comparing sepa- rations run on capillaries with different inner diameters, or even the same cap- illaries run on various instruments with different capillary cooling systems. Some of these issues are illustrated in Figures 2.19 and 2.20. The ionic strength of the buffer influences not only the EOF and jil^^, but indirectly the viscosity of the medium. More concentrated buffers have greater conductivity and generate more heat when the voltage is applied. The viscos- ity depends on the temperature, and so there is also a dependence on the cap- illary diameter. This is shown for a series of runs in 50- and 75-|Llm-i.d. capillaries (38). With the 50-|lm capillary, the migration times lengthen as the buffer con- centration is increased. Ions in solution are always surrounded by a double layer of ions of the opposite charge. The migration of these counterions is in a direction opposite to that of the solute (Figure 2.21); hence, increasing the concentration of the buffer reduces the mobility of the solute, due to increased drag caused by countermigration of the more densely packed counterions. With the 75-|Lim capillary, the solute migration times first increase as expected, but then they decrease. This decrease is a consequence of the sig- nificant effects of Joule heating at higher buffer concentrations. Note as well the impact of buffer concentration on peak width. Sharper peak widths at the higher buffer concentrations are due to stacking (Section 8.6). The
  56. 56. 2.8 Capillary Diameter and Buffer Ionic Strength 51 .32 50nm Capillary 0200M ^'ULlJlJLJIjLii^ liujLi 0.075M 0.050M O.O^M 8 10 12 14 16 TIME Crnin,) FIGURE 2.19 Effect of buffer ionic strength on peptide separations in a 50-|im-i.d. capillary. Buffers: 0.025-0.200 M phosphate, pH 2.44; voltage: 30 kV; capillary: 50 cm to detector x 50 |Lim i.d.; key: (1) bradykinin; (2) angiotensin 11; (3) TRH; (4) LRHR; (5) bombesin; (6) leucine enkephalin; (7) methio- nine enkephalin; (8) oxytocin; (9) dynorphin. Reprinted with permission from Techniques in Protein Chemistry 11, 1991, Academic Press, 3-19, copyright © 1991 Academic Press. Stacking effect is more evident when the 75-|im-i.d. capillary is used. Injec- tion time was held constant for this comparison; thus, a larger injection was made on the latter capillary. Stacking is not very noticeable when small injections are made.
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