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GH MULTIMEDIA
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Selectiv ity Petr oc Bøek
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Radim Vespalec
Academy of Sciences of the Czech Republic
Roger W. Giese Northeastern University
Definitions, concepts, and, most impor clear guidelines for optimizing selec
f you add up the pluses and minuses of capillary electrophoresis (CE), selectivity emerges as one of the most powerful features. Selectivity in CE is the ability to give different molecules varying velocities and, thereby, to separate them rapidly (1–3). In this article, we present an overview of CE selectivity that includes some basic concepts, guidelines, terminology, and an organizational scheme. To put this discussion in perspective, we compare selectivity for CE with that for HPLC. We hope that our presentation will deepen the reader’s appreciation and knowledge of this subject, serve as a roadmap for those in the laboratory, and stimulate further understanding. Although selectivity is an outstanding feature of CE,
other aspects of the technique are less impressive. CE has low capacity (a tiny separation pathway in the capillary) and often moderate sensitivity (typically a small injection volume). Its performance is easily disturbed by real samples, primarily because the matrix of real samples affects two delicate aspects of CE: injection and electroosmotic flow (EOF). Consequently, CE tends to deliver low precision for real samples. As Georges Guiochon has pointed out, standard deviations in CE for quantitative analytical results are typically 10-fold worse than for HPLC (4). Given these problems, CE may never catch up to HPLC, its major competitor in the commercial market, unless major applications for CE emerge, such as clinical
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diagnostics on disposable chips. For example, the separation of dideoxy DNA sequencing ladders already is a great commercial success for CE. It is also encouraging that the number of articles reporting the analysis of real samples is growing. We believe that HPLC will continue to be the first choice for most liquid-phase separations, and setting up an alternative or better selectivity will continue to be a major reason for using CE.
Some ground rules
When a voltage is applied across the ends of a capillary tube filled with an electrolyte solution, the ions move with respect to the surrounding solvent molecules (electrophoretic movement or electromigration) and the bulk solvent moves with respect to any fixed charged surface, such as an ionized silica capillary wall (electroosmotic movement). Mobility is simply the analyte’s velocity normalized for the electric field strength, E. Thus, mobility equals velocity divided by E. E, in turn, is given by the total length of the capillary, LC , and by the ap– plied voltage, V, where E = V/LC. (Note that we Power ordinarily ignore the dissupply tance from the ends of the capillary to the electrodes because of the negligible D voltage drop there.) A schematic of a typical CE system is shown in Figure 1. What we usually detect and measure is the movement (migration) of an E analyte from the injection point to the detector, which is the result of electromigration and EOF. Hence, the apparent (total) velocity, v, is
In this article, CE is de+ fined broadly. It includes capillary zone electrophoresis, whether in solution (including one containing an entangled polymer) or in a gel; micellar electrokiC netic chromatography (MEKC, 5); and other related techniques. However, capillary electrochromatography (CEC, 6) is not included, because it is E a combination of CE and chromatography with a major contribution from the latter. (CE and CEC Sample share a fuzzy boundary, for example, CE with an ionized gel.) Excluded as Inlet Outlet well are electrophoretic v = LD /tm (1) techniques in which the separation of species ocin which LD is the discurs in a steady-state, such tance (capillary length) D as isoelectric focusing (stafrom injection (capillary tionary steady-state) or inlet) to the detector (deisotachophoresis (steadytection window), and tm state of bands migrating is the migration time. with a constant velocity). Unless otherwise desigThese techniques require nated, migration reflects their own concepts of Figure 1. Schematic of electrophoretic equipment. the combined effects of selectivity. electromigration and elecE, electrodes; C, separation capillary; D, detector. (Adapted with permission from Ref. 7.) Another definition is troosmosis, and is considered to be the apparent that analytes are the chemmobility, mapp (7). ical substances to be measured, whereas everything else in the solution is additives, mapp = LCLD/tmV (2) ingredients, or components of the background electrolyte. The related terms “velocity”, “mobility”, and “migration” In other words, the mapp of an analyte inside a fused describe the movement of the analyte due to electrophoresilica capillary, or any other capillary that is superficially sis and electroosmosis. Thus, any hydrodynamic flow of charged by contact with a solution, typically consists of the liquid inside the capillary—usually a rare and unweltwo contributions. The first is the electrophoretic movecome event in CE except when gravity, pressure, or vacument of the analyte with respect to the electrolyte, which um is used for hydrodynamic injection—is factored out.
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is characterized quantitatively by the effective mobility, meff ; and the second is the EOF of the liquid with respect to immobile, charged surfaces, which is characterized by the electroosmotic coefficient, meof . These contributions lead to v = (meff
⫹
meof)E
(3)
Keep in mind that meff and meof are signed quantities (7). Cathodic electromigration of an analyte, that is, electromigration toward the cathode, is designated as positive (⫹) along with the mobility (⫹). Anodic electromigration and mobility are negative (⫺). The mobility of cations therefore is (⫹) and of anions is (⫺). Cathodic and anodic electroosmosis are designated (⫹) and (⫺), respectively. If an analyte is neutral and is driven at the speed of the liquid due to electroosmosis, then its effective mobility meff is equal to zero and mapp = meof . Such an analyte can be used as an EOF marker. Separation selectivity, S, in electrophoresis is defined as the relative velocity difference, if it exists, between two migrating analytes (8).
CE, unlike HPLC, migrates differentially with respect to the liquid phase, creating a more intense interaction. (Imagine walking against, rather than with, a crowd.) Yet, even when this mechanism yields only a small selectivity, the impact
A very small selec tivity is frequently useful in routine CE but
S = ∆v/vav
(4)
in which ∆v is the velocity difference between the two analytes and vav is the velocity average for the two analytes. (Note that the velocity difference refers to the peak apexes; peak widths come into the picture when the discussion turns to resolution.) Thus, for CE of the types considered here, and where selectivity is fully controlled by meff , selectivity is expressed as S = ∆meff /(meff, av + meof)
(5)
in which meff, av is the average of the effective mobilities. For closely migrating analytes, the average of the effective mobilities may be replaced by the effective mobility of one of them.
CE versus HPLC Although CE and HPLC both involve separations in liquids and offer significant selectivity, they differ in how they commonly provide this property. CE ordinarily relies entirely on solution-based effects, including subtle ones, to achieve selectivity. (MEKC is not an exception, because micelles are dissolved species.) This can yield significant selectivity because the analyte in
on the separation can still be important. This finding relates to the peaks in CE, which tend to be much sharper than those in HPLC due to fundamental factors such as mass transfer. It is well known that high separation efficiencies reduce the demands on selectivity. A very small selectivity, therefore, is frequently useful in routine CE but rarely for HPLC. Complex additives that enhance selectivity—such as surfactants, micelles, cyclodextrins, polymers, metal ions, and other affinity agents—tend to broaden peaks and contaminate columns more often in HPLC than in CE. Moreover, some complex additives are not very pure or have poor batch-to-batch reproducibility. However, when they do work, the cost of obtaining very pure, and therefore expensive, additives can be more readily borne by CE because of its negligible volume. HPLC can take advantage of these complex additives by attaching them to the stationary phase, and this approach can be effective and convenient. However, changing to a different additive means changing columns, and these columns, particularly the specialty ones, are expensive. On the other hand, attaching additives to the wall in CE compromises performance, because the contact between analytes and the wall in CE is inefficient at ordinary internal diameters, causing peak broadening. Unfortunately, the remedy of reducing the capillary diameter tends to increase clogging and lower sensitivity. Thus, CE and HPLC have contrasting advantages for selectivity, making them a good team for liquid-phase separtions. CEC combines the advantages but also the disadvantages of both techniques and thus faces the challenge of being more complex. What are the origins of the solution-based events that ordinarily determine selectivity in CE? Separation selectivity in CE basically depends on the effective electro-
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phoretic mobilities of the analytes of interest. (SomeExamples of CE selectivity times the separation is modified by an additional efCE’s selectivity is powerful across a wide range of molefect, such as the interactions of analytes with the capilcules—small to large, neutral or charged. Impressive lary wall.) The effective electrophoretic mobility (total examples of small molecule separations include normal mobility minus electroosmosis) of an analyte depends and a-trideuterated acetophenone (11); dansyl derivaon its charge, size (strictly speaking, the charge-to-size tives of normal and trideuterated methylamine (12); ratio), shape, number and relative abundance of any of ortho, meta, and para as well as cis- and trans-isomers its alternate forms, and the volume of the comigrating of cationic drugs (13); the 12-, 7-, 6-, and 5-methyl solvation layer of each form. (The solvation of the anaisomers of benz[a]anthracene isomers (11); and 12 hylyte actually exchanges, droxy polyaromatic hybut it is modeled as drocarbons (14). Note 3 bound solvent.) that some of these sepaCharge can be a comrations are of neutral plex concept, because it compounds. So far, how4 involves more than just ever, very small neutrals, the net charge. For exsuch as methanol and 5 ample, the mobility of ethanol, have not been the common alkali metals separated by CE. 2 in water increases in the With large molecules, 0.005 AU 1 order Li+< Na+ < K+. some spectacular separations have been achieved, This increase is commonsuch as the separation of ly attributed to a correDNA sequencing ladders sponding decrease in the >1000 bases (15). Modsolvation volume of these ern DNA sequencing reions, making the solvated lies heavily on CE with lithium ion the effectively 96-capillary instruments, larger species. 30 40 50 which are responsible for Other “anomalies” Time (min) deciphering the human exist. Trichloroacetate is and other genomes (16). larger than acetate and Figure 2. CE separation of ovalbumin. the ions have a similar The analysis of protein Purified ovalbumin (500 µg/mL) separated in an uncoated 87-cm fused-silica capillary containing 100 mM borate and 1 mM putrescine at pH 8.5. (Adapted with permisshape, yet trichloroacmicroheterogeneity by etate migrates faster. This sion from Ref. 17.) CE also is very impressive. speed is attributed to a For example, Figure 2 lower dielectric friction shows the separation of (transient solvation) for trichloroacetate (9). In some ovalbumin by CE, revealing 5 major peaks and 10–15 cases, the preferred orientation of the migrating ions minor peaks, largely due to variations in carbohydrate within the applied electric field can also significantly instructure on this glycoprotein (17 ). For this separation, fluence mobility. Such a mechanism has been used to borate buffer was selected because of this species’ ability explain differences in the migra-tion of closely related to form anionic, borate–sugar diol complexes. 1,4-Dialkylpyridines (10). aminobutane was added to create a long migration time For an analyte A present in its zone migrating through by reducing the EOF (via electrostatic masking of the the capillary in various subspecies A1, A2, A3, . . . An, negative charge at the wall). Last but not least, a big sucwhich have mobilities m1, m2, m3, . . . mn and molar cess story for CE is its selectivity in chiral separations (18). fractions x1, x2, . . . xn, the effective mobility is given by
Selectivity menu—POINT by point m eff = ∑ xn mn
(6)
Equation 6 covers MEKC, providing that a given species incorporated into a micelle is considered as a subspecies. Obviously, any change to the background electrolyte that changes the mobility of any of the subspecies, and/or shifts the above equilibrium, changes meff.
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We have set up a “selectivity menu”—categories of agents that can influence separation selectivity—for CE in Table 1 and have arranged the entries to spell the acronym “POINT”. We have also listed the usual, primary effects for each type of selectivity agent. Admittedly, these listings are an oversimplification, because variations in selectivity agents typically have secondary
UV absorbance (AU)
effects on a CE separation, such as changes in electroos- conditions was required to achieve the remarkable semosis (variations in ionic strength and viscosity of the lectivity, although the analysis time is long and the plate liquid, and the capillary wall charge), temperature (due count is low. Phosphoric acid was selected over hydroto Joule heating), analyte solubility and adsorptivity, and chloric acid, because the latter has a higher conductance the selectivity agent’s interactions with the background and carries a greater current. A C14 detergent, rather electrolyte components. Also, the sample’s matrix may than a C12 one, interacted less strongly with the PAHs interact with the components of the background elecand thereby provided less selectivity, whereas the C16 detrolyte. Nevertheless, Table 1 helps to organize the subtergent had too low a solubility. Initially, some of these ject and emphasize the primary events. compounds were separated in 100% methanol, but this Generally, the most step necessitated a low powerful selectivity agent current to minimize eluin CE is pH, and thereant evaporation in the elecfore it requires special trode reservoirs. Adding consideration and conwater dealt with this prob3 trol. Often, adequate selem and also boosted the 6 1 2 9 4 7 lectivity can be obtained selectivity, perhaps because 5 8 by simply adjusting the it strengthened the solvopH, and then the other phobic interaction of the separation conditions can detergent with the PAHs. be optimized for fur ther Various interaction purposes such as separaagents have been used to tion speed. pH can affect improve selectivity in CE, selectivity even when varranging from small ions 6 8 10 12 14 16 18 20 22 24 26 28 30 ied in a range that is far and molecules to polymers. Retention time (min) from the analyte’s pKa, This category, as we have defined it, is very broad but near the buffer’s and includes agents whose pKa (19). Figure 3. Separation of nine PAHs in methanol:water (75:25 v/v). interactions with the anaAn organic solvent like Electrolyte is 10 mM H PO with 70 mM sodium n-tetradecyl sulfate. 3 4 1, benzo[a]perylene; 2, perylene; 3, benzo[a]anthracene; 4, pyrene; 5, 9-methylanlyte range from weak to acetonitrile or methanol thracene; 6, anthracene; 7 , fluorene; 8, napthalene; 9, benzophenone. (Adapted with strong. Generally, the often is added to the CE permission from Ref. 25.) weakest interaction agent buffer, typically in the is a buffer, which in this range of 5–30% (20). context can be varied in composition or concentration The usual purpose is to increase the solubility of the ana(pH changes are a separate category). Changing the conlyte, but selectivity can also be affected. This tendency is most likely if an interaction agent (which is discussed centration of a small ion (i.e., ionic strength) is more later) is present, especially a complex one. The organic likely to modify selectivity when separating ions that solvent could then modify selectivity by influencing this differ in the number of their charges, or when another interaction. This has been observed with cyclodextrins interaction agent is present. However, the choice of the that complex anionic enantiomers (21) and with sulfon- monovalent ion of a buffer rarely influences the selecated ß-cyclodextrin used to resolve racemic terbutaline tivity at a given pH and concentration. One interesting (22). The use of “exotic” organic solvents (also discussed exception is the elution order of some ß-blockers, which later) in concentrations
