Whole-Column-Imaging Detection for Capillary Isoelectric ...files.pharmtech.com/alfresco_images/pharma/2014/08/...capillary isoelectric focusing: Figure 2 illustrates the basic concept

526 LCGC VOLUME 19 NUMBER 5 MAY 2001 www.chromatographyonline.com

Despite the significant advantages of cap-illary isoelectric focusing over gel isoelectricfocusing, widespread acceptance of the tech-nique as a replacement for gel isoelectricfocusing has not occurred. Several factorscontribute to capillary isoelectric focusing’sslower-than-expected adoption; perhaps themost important are the performance andprocedural difficulties introduced when cap-illary isoelectric focusing is performed onconventional CE instruments. A conven-tional CE instrument is equipped with a single-point, on-column, UV–vis absorptiondetector that is located at one end of the cap-illary. Thus, most capillary isoelectric focus-ing is performed in a two-step operation(4–9). In the first step, samples such as pro-teins are focused (separated and concen-trated) to stationary sharp zones accordingto their respective pI values in a coated cap-illary, in which electroosmotic flow is elimi-nated by the coating. Then as a second step,the focused protein zones are moved by elec-trophoretic or hydrodynamic mobilizationso they can pass the detector point to bemeasured and recorded. The second mobi-lization step introduces several problems tocapillary isoelectric focusing, such as unevenseparation resolution, poor reproducibility,and increased analysis times. An alternative

In this article, the authors review recent developments in the research ofwhole-column-imaging detection for capillary electrophoresis (CE). Whole-column-imaging detection was developed for capillary isoelectric focusing,for which it proved to be an ideal detector. Several whole-column-imagingdetectors — including refractive index gradient imaging, UV-absorptionimaging, and fluorescence imaging detectors — have been studied. Thecapillary isoelectric focusing UV-absorption imaging technique even hasbeen commercialized. The development of whole-column-imagingdetection itself facilitates CE studies in many directions such as inelectrophoretic dynamics within narrow channels, new separation modes,and two-dimensional separations. Whole-column-imaging detection alsofinds application in capillary zone electrophoresis.

Whole-Column-Imaging Detectionfor Capillary Isoelectric Focusingand Capillary Electrophoresis

Xing-Zheng Wu, Jiaqi Wu*,and Janusz PawliszynDepartment of Chemistry,University of Waterloo, 200 University Avenue West,Waterloo, Ontario N2L 3G1,Canada

* Convergent Bioscience Ltd., 27 Coronet Road, Unit 18,Toronto, Ontario 8MZ 2L8,Canada

Address correspondence to J. Pawliszyn.

soelectric focusing is the highest reso-lution technique used in biomoleculeanalysis (1,2). It is used routinely tocharacterize biological fluids and

extracts, monitor protein purification, evalu-ate the stability or microheterogeneity ofproteins, and determine isoelectric points(pI). Isoelectric focusing is an electropho-retic method traditionally performed in slabgels. As an analytical technique, gel isoelec-tric focusing is slow, labor intensive, andgenerally not quantitative (3). With theintroduction of capillary electrophoresis(CE) and its high speed and quantitativeproperties, researchers recognized that if iso-electric focusing could be performed in acapillary format, it could greatly decreasetime requirements, improve quantitation,facilitate automation, and enhance separa-tion resolution.

In 1985, Hjertén and Zhu (4) firstreported high-resolution isoelectric focusingperformed in a gel-free capillary column.Since then, other researchers have reportedmany studies of capillary isoelectric focusing(5–9), and CE manufacturers began devel-oping and eventually commercializing kitsand accessories that provided capillary iso-electric focusing analysis capabilities forexisting CE instruments.

I


tion window of a UV absorbance (14) or flu-orescence detector (15). Although the prob-lems of distorted pH gradient and unevenresolution in the focused-zones-moved cap-illary isoelectric focusing have improved, themechanical movement of the capillaryincreases dynamic noise and analysis time.Also, it is difficult to apply to monitoring afast capillary isoelectric focusing process(16).

An ideal approach to capillary isoelec-tric focusing is real-time, whole-column-imaging detection without moving any partin the system. Pawliszyn and Wu (17) havedemonstrated this approach. They and theircolleagues have developed several types ofreal-time, whole-column-imaging detectorsfor capillary isoelectric focusing (18–24).Their approach combines gel-like isoelectricfocusing separation and detection with the

approach is to perform capillary isoelectricfocusing in uncoated capillaries by usingmethyl cellulose as a buffer additive toreduce electroosmotic flow and allow attain-ment of steady-state conditions at whichprotein samples are focused (10–13). Thereduced electroosmotic flow will continuemobilizing the focused protein zones pastthe detection point. Although this approachis a one-step procedure, it still has limita-tions associated with uneven mobilizationspeeds, long mobilization times for acidicproteins, and incomplete pattern detectionat column locations near the capillary end.

To overcome the drawbacks of single-point detection, some have proposed opticalwhole-column detection methods based onmoving the separation capillary to the opti-cal detection point; for example, by movingthe separation capillary through the detec-

automation, speed, and quantitation of acolumn-based separation technique. Morerecently, real-time, whole-column-imagedcapillary isoelectric focusing technology hasbeen commercialized and applied in a widevariety of fields.

In this article, we will discuss the princi-ples of isoelectric focusing and, specifically,whole-column-imaged capillary isoelectricfocusing. We will describe real-time, whole-column-imaging detectors for capillary iso-electric focusing and their applications.Finally, we also will discuss the current statusof whole-column-imaged capillary isoelec-tric focusing and future trends.

Principle of Real-Time, Whole-Column-Imaged Capillary Isoelectric FocusingPrinciple of capillary isoelectric focusing:Figure 1 illustrates the concepts of standardand capillary isoelectric focusing. For anamphoteric (zwitterionic) compound suchas a protein in a buffer with a certain pH, ifthe pH is lower or higher than its isoelectricpoint, the protein molecule will be positivelyor negatively charged. When an electric fieldis applied, the positively charged proteinmolecule will electromigrate toward thecathode, and the negative one toward theanode. If a pH gradient exists between theanode and cathode, the protein will electro-migrate to a point at which the pH is equalto its pI. At the isoelectric point, the netcharge of the protein is zero, thus the elec-tromigration is stopped. The protein mole-cules distributed in the whole pH gradientwill be focused or concentrated at the iso-electric point to form a sharp narrow zone. Itusually is difficult to fix a pH gradient in abulk solution, however, because of convec-tion. On the other hand, convection can beeliminated in a gel, which is why most earlyisoelectric focusing experiments were per-formed in gels. A stable pH gradient also canform in a coated capillary filled with carrierampholyte, in which electroosmotic flow iseliminated or reduced nearly to zero, in thepresence of an electric field (25). Therefore,if protein samples are mixed with the carrierampholyte, the proteins will be focused —concentrated and separated — at differentpoints along the capillary according to theirpI values in the presence of an electric field(Figure 1b).

Resolution of capillary isoelectric focus-ing: Separation resolution is the most signif-icant concern for a separation method. Maoand Pawliszyn (16) addressed a theoreticalconsideration of the separation resolution ofcapillary isoelectric focusing. For a sample

Figure 1: Illustration of the concepts of (a) isoelectric focusing and (b) capillary isoelectric focus-ing.

Focused sample zones

Distance

(b)

(a)

(�)(�)

H� OH�

NH3� NH3

�

COO�

COOHCOOH

COOH

Net charge:

pH:

pH

pH � pI pH � pI pH � pI

(�2)

Anode (�)

Capillary (�10�100 �m)

Cathode (�)

(0) (�3)

NH3� NH3

�

COOH

COO�

COO�

NH2 NH2

COO�

COO�


zone focused in a capillary by the isoelectricfocusing process, concentration has aGaussian distribution with a variance ()defined as

[1]

[2]

where C0 is the maximum concentration, pis the mobility slope (�d�/dx), E is the fieldstrength, D is the diffusion coefficient, andx is the position along the capillary (1,2).Using the criterion of three times the vari-ance for resolved adjacent proteins, theresolving power or resolution (pI) of iso-electric focusing in terms of can beexpressed as (1):

[3]

Equation 3 shows that good resolution isfavored by a high field strength, a low diffu-sion coefficient, a high mobility slope(d�/d [pH]), and a narrow pH gradient. Ofthe variables, the diffusion coefficient andthe mobility slope are intrinsic properties ofthe analytes, so only pH gradient and thefield strength can be varied experimentally.

Equation 3 also shows that the resolutionhas no direct relation to the length of sepa-ration capillary. A short column can be usedin capillary isoelectric focusing without sac-rificing any separation resolution. Using ashort column has several advantages: First,

pI �DE

d (pH)�d�

d (pH)dx

3

�DE

dx�d�

�

C �C 0

(�pEx2)

2D

the separation process over the whole col-umn can be studied with a charge-coupleddevice camera imaging detector. Second,the capillary isoelectric focusing process willbe very fast in a short column. Third, ashort column requires a smaller amount ofthe sample than a long column, because thesample mixed with carrier ampholytes usu-ally fills the whole column. This usage isimportant for expensive and scarce biologi-cal samples. It also simplifies instrumenta-tion, because a relatively low direct current(dc) power supply can provide high electricfield for a short column. Conventional cap-illary isoelectric focusing with a long capil-lary (usually �10–100 cm) requires a highdc power supply (usually �10 kV) to pro-vide the high electric field (usually 300–600V/cm) across the capillary. Using a high dcpower supply requires additional safety con-siderations in operation and instrumenta-tion.

Based on the above theoretical considera-tions, we propose the concept of short-column capillary isoelectric focusing withwhole-column-imaging detection.

Whole-column-imaging detection ofcapillary isoelectric focusing: Figure 2illustrates the basic concept of real-time,whole-column-imaging detection for capil-lary isoelectric focusing. As stated above,protein samples in the capillary are focused— concentrated and separated — into nar-row sharp zones at different pI points dur-ing capillary isoelectric focusing. High con-centration gradients are created at theboundaries of the separated zones inside thecapillary. The concentration gradients willinduce refractive index gradients, which inturn cause the deflection of light passed

through the gradients. The deflection willchange the intensity distribution of a trans-mitted light from the capillary, which ishomogeneously illuminated along the capil-lary axis. Therefore, the focused samplezones can be imaged by measuring thechange in intensity distribution of the trans-mitted light with a charge-coupled device.

If samples have optical absorption for thelight, the focused sample zones can be imag-ined simply by measuring the opticalabsorption. Users can measure a decrease inthe intensity of the transmitted light. If thesamples are fluorescent, fluorescence will beproduced by illuminating light with a suit-able wavelength. Then, the focused samplezones also are imagable by measuring thefluorescence distribution along the capillary.Therefore, three types of whole columnimaging detectors based on concentrationgradient, optical absorption, and fluores-cence are possible. Analysts should assemblea suitable optical arrangement, including aset of lenses and filters, between the capil-lary and charge-coupled device to achievethe maximum detection sensitivity. Basedupon these considerations, we have devel-oped real-time, whole-column refractiveindex gradient, absorption, and fluores-cence imaging detectors for capillary iso-electric focusing.

Development of Real-Time,Whole-Column-Imaging Detectorsfor Capillary Isoelectric FocusingRefractive index gradient imaging detec-tor: As stated above in the “Whole-column-imaging detection of capillary isoelectricfocusing” section, the focused zones can bedetected through refractive index gradient(concentration gradient) imaging detection.A typical instrument setup can be con-structed on the basis of either the schlierenshadow graph method (18) or a dark-fieldToepler schlieren system (19). Comparedwith the schlieren shadow graph method,the dark-field Toepler schlieren system ismore reliable (20). Figure 3 shows the lattersystem. Because the concentration changegenerates a refractive index change for allcompounds, the refractive index gradientimaging detector is universal and providesresults in real time. We obtained a detectionlimit of 10�6 M for all proteins tested underoptimal conditions.

Fluorescence imaging detector: Fluores-cence detection is one of the most sensitivemethods for CE detection (26). A high-sensitivity charge-coupled device camera isused in the whole-column fluorescenceimaging detector for capillary isoelectricfocusing (22,27). Filters are placed in front

Figure 3: Illustration of a dark-field Toeplerschlieren refractive index imaging systems forcapillary isoelectric focusing.

Charge-coupled device sensor

Laser beam

Cylindricallens

Cylindricallens

Optical stopElectrolytereservoir

Electrode

Capillary

Figure 2: Illustration of the basic concept ofthe whole-column-imaging detection for capil-lary isoelectric focusing.

Reservoir

Separationcapillary

Transmittedlight or

fluorescencelight

Homogeneouslyilluminating light

H� OH�

Anode(�)

Charge-coupleddevice camera

Optical components(lens, filters)

Cathode(�)


of the charge-coupled device to remove scat-tered light. Researchers have examined threemethods to introduce the excitation light toilluminate the whole column homoge-neously with an Ar� laser light (22,27).

The first was a fiber array along the capil-lary. Whole-column fluorescence imagedcapillary isoelectric focusing of the fluores-cent protein �-phycoerythrin at a concen-tration of 10�7 M has been demonstrated(17). This excitation light introductionmethod makes the imaging system verysimple, but the sensitivity is not the highestbecause of the inefficient coupling of theexcitation light from the fiber array to thecapillary.

The second introduction method was tofocus the central part of an expanded laserbeam into the capillary using a cylindricallens (22). This method provides an efficientand homogeneous illumination of thewhole capillary, thus it has high sensitivity

for fluorescent and fluorescently labeledproteins. Three fluorescein isothiocyanate–labeled proteins — albumin, insulin, andcasein — were successfully separated in a4.5-cm capillary by the method. Further-more, heterogeneity in the fluorescein isoth-iocyanate labeling reaction of proteins canbe understood easily from the broader cap-illary isoelectric focusing peaks in compari-son with nonlabeled proteins. Their detec-tion limits are approximately 10�11 M orattomoles (10�18 mol) (22).

The third method involves axially illumi-nating the whole column by introducingthe excitation light from one end of the cap-illary (Figure 4a) (27). When the refractiveindex of the liquid medium inside the cap-illary is larger than that of the capillary wall,total internal reflection of the excitationlight occurs. For a fused-silica capillary, totalinternal reflection occurs when it is filledwith an organic solvent such as dimethyl

sulfoxide (28,29). On the other hand, totalinternal reflection occurs easily in a polyte-trafluoroethylene (PTFE) capillary filledwith carrier ampholyte used in capillary iso-electric focusing by adding 20% of glycerol(27). Figure 4b shows the dynamic focusingprocess of the two naturally fluorescent pro-teins R-phycoerythrin and green fluores-cence protein monitored by axially illumi-nated whole-column-imaging detection.Because the intensity of the excitation lightin the capillary is very strong for this intro-duction method, fluorescence detectionsensitivity is the highest of the three meth-ods. Detection of 10�13 M or subattomole-level proteins was accomplished easily, evenwithout optimizing the optical detectionsystem (27). Because of its extremely highsensitivity, we expect this whole-column flu-orescence imaging detector to be the mostpowerful tool for capillary isoelectric focus-ing of trace biomolecules in a single cell.

Absorption imaging detector: Wu andPawliszyn (23) also developed a UV–visabsorption imaging detection system.Although absorption imaging is less sensi-tive than the fluorescence, it is more sensi-tive than refractive index imaging. It also isquantitative and can be used for most pro-teins. Therefore, absorption-imaged capil-lary isoelectric focusing has become themost practical mode, and a commercialinstrument, which we will describe later, hasbeen developed.

Capillary cartridge: In the early develop-ment stages of the whole-column-imagedcapillary isoelectric focusing technique, thecartridge was very simple: a 4–5 cm capil-lary connected to two electrolyte reservoirsat its ends. However, this cartridge wasinconvenient for sample injection. A newcartridge, shown in Figure 5, has beendeveloped (16,22). The separation capillaryis internally coated, and its outer polyimidecoating is removed. Two pieces of hollow-fiber membrane are glued to the ends of theseparation column. Two connection capil-laries for sample introduction are glued tothe other ends of hollow-fiber membranes.Two glass plates hold the capillary, and oneslit is used to cut off stray light. The mem-branes in the electrolyte reservoirs allowsmall ions such as H� and OH� to passfreely and, thus, allow capillary isoelectricfocusing to occur normally. By using thistype of configuration, analysts can easilyintroduce sample solutions mixed with car-rier ampholytes without disturbing the elec-trolytes. Workers can also use multiple cap-illaries for increased throughput.

Figure 4: Illustration of (a) an axially illuminated, whole-column fluorescence imaged capillaryisoelectric focusing setup and (b) a separation example of two fluorescent proteins. Sample: R-phycoerythrin (1.7 10�10 M) and green fluorescence protein (1.8 10�8 M).

3.0

2.0

1.0

0.0

Fluo

resc

ence

inte

nsit

y (

10�

4 )

Distance (cm)

8 min

6 min

4 min

2 min

0 min

Green fluorescence protein

Excitationlight

Electrolyte reservoir

Fluorescenceemission


UV lens

Filter

Optical fiber

Sample injection capillary

Separation capillaryHollow fiber

Electrode

R-Phycoerythrin(b)

(a)

0.1 1.1 2.1 3.1 4.1 5.1 6.1


A commercial imaged capillary isoelec-tric focusing instrument: The technique ofimaged capillary isoelectric focusing wascommercialized in 1998 (3). Figure 6 showsa block diagram of the first imaged capillaryisoelectric focusing instrument (modeliCE280m, Convergent Bioscience Ltd.,Toronto, Ontario, Canada). It is based oncapillary isoelectric focusing, whole-column, UV-absorption imaging detection.

A 280-nm light beam from a xenon lamp isfocused on the separation column by a bun-dle of optical fibers and a set of lenses. Theinstrument uses a cartridge, as described inthe “Capillary cartridge” section above. Thetwo electrolyte tanks are filled with anolyte(usually 100 mM phosphoric acid) andcatholyte (usually 40 mM sodium hydrox-ide). The inlet capillary in the cartridge isconnected with a finger-tightened nut to a

two-position, eight-port polyetheretherke-tone (PEEK) switch valve. Sample is intro-duced either manually or automaticallythrough the switch valve. In the automaticmode, the sample throughput can be ashigh as 7 samples/h.

The instrument applies a 3-kV dc voltagefor the isoelectric focusing. The focusingprocess usually lasts 5–7 min and can bemonitored by taking the whole-columnabsorption image every 30 s. The UV-absorption image then can be analyzed withquantitation software.

Applications of Whole-Column-Imaged Capillary Isoelectric FocusingIn this section, we will discuss several appli-cations of whole-column-imaged capillaryisoelectric focusing.

pI determination: Imaged capillary iso-electric focusing provides fast and precise pI determination. Unlike the conventionalcapillary isoelectric focusing method,imaged capillary isoelectric focusing deter-mines the pI values of proteins and peptidesdirectly from their peak positions along theseparation column. Conventional capillaryisoelectric focusing must use a difficultmethod based upon retention time to estab-lish pI values.

The imaged capillary isoelectric focusingmeasurement of a protein’s pI value is per-formed by running it with two pI markersthat are mixed in the sample. Ideally, thetwo pI markers’ peaks bracket the samplepeaks. Usually, low molecular weight syn-thetic pI markers (such as BioMarkers, Bio-Rad Laboratories, Hercules, California) areused in the measurement. Figure 7 showsthe experimental relationship between pIand the peak positions along the 5-cm-longseparation column when using Pharmalytecarrier ampholytes (pH 3–10) (SigmaCanada, Oakville, Ontario, Canada). Theresult is a linear pH gradient throughout thewhole pH range.

If we assume that the pH gradientbetween two pI markers is linear, we candetermine the pI value of an unknown sam-ple by bracketing the sample peaks with thetwo pI marker peaks. Figure 8 shows anexample. The monoclonal antibody sampleis focused with two pI markers, and the pIvalues of its six peaks can be calculated auto-matically by the capillary isoelectric focus-ing instrument’s software.

This method uses different pH-range car-rier ampholytes. Figure 8 shows the sameprotein separated in pH 3–10 and pH 4–7carrier ampholytes. Under those conditions,

Figure 5: Illustrations of a capillary isoelectric focusing cartridge. Shown are (a) side and (b) topviews.

(b)

(a) Electrolyte reservoirs

Separation capillary

Separation capillary

Slit (50 �m)

50 mmGlue

25 mm

Hollow-fibermembrane

Connectioncapillary

Glass plate (75 mm 25 mm 10 mm)

Figure 6: Block diagram of the iCE280m capillary isoelectric focusing instrument.

Injectionport

Outletcapillary

Inlet capillary

Two-position,eight-port

switchvalve

Separationcolumn

Xenonlamp

WasteHollow-fibermembrane

From pumpFinger-tightenednut

Fiber-opticsbundle

Charge-coupleddevicecamera

Highvoltage

Electrode (�)

Catholyte (OH�)

Column cartridge

Anolyte (H�) � �


the resolution is much better with the pH4–7 carrier ampholytes. We achieved base-line resolution for two peaks that had a pIdifference of 0.05 pH units. The relativestandard deviation (RSD) of pI measure-ment for this sample is less than 0.5%.Analysis times for the pI determination rou-tinely are less than 6 min.

Protein identification: Most proteinsexhibit intrinsic microheterogeneity. Iso-electric focusing often is the separationtechnique of choice for characterizingmicroheterogeneity. The imaged capillaryisoelectric focusing’s high precision in pIdetermination makes it a valuable tool forquality-control identification of proteinproducts. The method for protein peakidentification is the same as that for pI measurement.

In isoelectric focusing, the salt concentra-tion in the sample’s matrix affects thefocused pattern. The salt in the samplecompresses the pH gradient created by thecarrier ampholytes. This effect is illustrated

in Figure 9, which shows the focused pat-terns of the same protein in different saltconcentrations. The focused pattern of theprotein becomes narrower with the increaseof the salt concentration. This factor makesit difficult to identify peaks for proteins indifferent matrices.

By using the two pI markers for pI deter-mination, we could compensate for thematrix effect of the sample. Figures 9 and10 show an example. Figure 10 shows thesame electropherograms as those depicted inFigure 9, but they are aligned with the twopI marker peaks. The precision in the peakidentification using this method is 0.01 pHunits.

Quantitation: Imaged capillary isoelectricfocusing is a quantitative analysis methodbecause the imaging system is based on UVabsorption of the protein samples. Thedetector’s linear range is as high as 160 (3).The RSD we obtained in the quantitationfor major peaks (�20% of the total) was5% for sample concentrations in the 0.1–1mg/mL range.

During quality-control analysis of pro-teins with multiple peaks in isoelectricfocusing (such as the protein in Figure 8),we sometimes use a parameter called averagepI to identify the proteins. The average pI ofa protein is calculated by averaging the pIvalues of all its peaks weighed by each peak’srelative concentration. Thus, the relativeconcentration of each species is very usefulin characterizing the protein. The determi-nation of the relative concentration of eachspecies can be performed automatically

using the capillary isoelectric focusinginstrument’s software. Using the capillaryisoelectric focusing instrument for determi-nation, we obtained RSDs of 2% for majorspecies (�30% of the total species) and15% for minor species (approximately 3%of the total species).

Analysis of acidic proteins and pep-tides: In the conventional capillary isoelec-tric focusing method, the direction of themobilization typically is from the anodicend of the capillary column toward thecathodic end. Users can perform the mobi-lization three ways: by adding salts to thecatholyte to create a pH shift toward thecathodic end of the column; by using theelectroosmotic flow, the direction of whichis from anodic end of the column towardthe cathodic end; and by applying pressureto one end of the capillary column. Com-bining the three methods can optimize themobilization process.

The first method usually provides goodresolution for proteins close to the cathodicend of the column, which include high pIand basic proteins, because mobilizationspeed is even and slow at the beginning ofthe mobilization. However, the speedbecomes faster and uneven during themobilization process. Its resolution for pro-teins focused at the anodic end of the col-umn, which include low pI and acidic pro-teins, is inferior to that of the basic proteinsthat are focused close to the cathodic end ofthe column. Mobilization may take as longas 40 min for proteins with pI 3–4 valuesusing this method.

Figure 7: Capillary isoelectric focusing sepa-ration of pI markers. Shown are (a) electro-pherograms and (b) a plot of the relationshipbetween peak position and pI. Sample: methylred (2 �g/mL) and pI markers (BioMarkers, Bio-Rad) (2 �g/mL).

10

9

8

7

6

5

4

3

pI v

alue

Distance from anodic end (mm)

(b)

(a)

Methylred 3.8

pI 5.3

6.4

7.4

8.5

0 10 20 30 40 50

Abs

orba

nce


0 20 40

Figure 8: Capillary isoelectric focusing separation of monoclonal human anti-�-chorionicgonadotropin (0.2 mg/mL) in 4 M urea. (a) pH 3–10 carrier ampholytes, (b) pH 4–7 carrierampholytes.


(b)

(a)pI 5.3

pI 5.3

6.6

7.4

6.015.96

10 20 30 40


Because the second method uses electro-osmotic flow to mobilize the focused pro-teins within the column, the electroosmoticflow of the column cannot be eliminated orreduced substantially. Analysts mix addi-tives with the samples to regulate the elec-troosmotic flow. In this way, the concentra-tion of the additives must be optimized toachieve the optimal mobilization speed fordifferent samples. Because the electro-osmotic flow is dependent upon pH values,

the mobilization speed is uneven along thecolumn. Usually it is slower for acidic pro-teins, which makes mobilization a longprocess. It is difficult to mobilize acidic pro-teins using this method alone.

Pressure mobilization provides an evenspeed, and the speed is easy to control. How-ever, the pressure creates a parabolic flowprofile within the column, which distortsthe focused pattern. It has the lowest resolu-tion of the three mobilization methods (30).

The above problems caused by mobiliza-tion do not exist in imaged capillary isoelec-tric focusing, because it detects all focusedprotein zones within the column simultane-ously. Figure 11 shows an example of thecapillary isoelectric focusing of an acidicprotein with an approximate pI value of 3.6.The analysis time is only 5 min, which ismuch shorter than the time required withconventional capillary isoelectric focusing.

Imaged capillary isoelectric focusing alsocan be used to analyze peptides. Figure 12shows two analytical runs of an acidic pep-tide. Its pI value is 3.8. Peptides usually aredifficult to analyze by gel isoelectric focus-ing because they are washed away duringthe staining process.

Gel isoelectric focusing uses a poroussupport media such as polyacrylamide. Thegel’s pore size limits the molecular weight ofproteins that gel isoelectric focusing cananalyze. Figure 13 shows electropherogramsof a recombinant vaccine, which has a mol-ecular weight of approximately 6 million,that was analyzed by imaged capillary iso-electric focusing. Its pI value is approxi-mately 2.5. Because of its low pI value andhigh molecular weight, it is difficult to ana-lyze by either conventional capillary isoelec-tric focusing or gel isoelectric focusing.

Comparing imaged and conventionalcapillary isoelectric focusing: Imaged cap-illary isoelectric focusing’s elimination ofthe mobilization process in conventionalcapillary isoelectric focusing yields three sig-nificant advantages: reduced analysis andmethod development time, extended pIanalysis range, and improved reproducibil-ity of the focused pattern.

In conventional capillary isoelectricfocusing, optimizing the mobilizationprocess takes most of the method develop-ment time. The mobilization process itselfoften takes more than 15 min, and it maytake as long as 40 min for some acidic pro-teins (low pI proteins). This long mobiliza-tion process prolongs method development.It may take weeks to develop a capillary iso-electric focusing method for a new sample.On the other hand, the throughput of theimaged capillary isoelectric focusing is thesame for all protein samples regardless oftheir pI values, and this throughput can beas high as 7 samples/h. For this reason,method development for a sample usingimaged capillary isoelectric focusing oftenrequires only a day or two instead of theweeks necessary for conventional capillaryisoelectric focusing.

The focusing voltage in conventionalcapillary isoelectric focusing still is applied

Figure 9: Separation of 0.2-mg/mL monoclonal human anti-�-chorionic gonadotropin in 4 Murea using pH 4–7 ampholytes with (a) 7.5 mM, (b) 15 mM, and (c) 25 mM phosphate-bufferedsaline.


(b)

(a)

(c)

5.3

5.3

5.3

6.6

6.6

6.6

10 20 30 40

Figure 10: Compensation of salt effect in the sample matrix by aligning the pH 5.3 and pH 6.6marker peaks. (a) 7.5 mM phosphate-buffered saline, (b) 15 mM phosphate-buffered saline, (c) 25mM phosphate-buffered saline. Sample: same as in Figure 9.

0.32

0.24

0.16

0.08

0.00

Abs

orba

nce

(AU

)

pI

(a)

(b)

5.3

5.89

5.96 6.01

6.116.21

6.6

(c)

4.0 4.5 5.0 5.5 6.0 6.5 7.0


during the mobilization process, after focus-ing. Thus, the focusing time for a proteinzone equals the zone’s retention time. Pro-teins with different pI values experience dif-ferent focusing times. The focusing timecan be 40 min for some acidic proteins withlow pI values. Confining proteins at the iso-electric point (zero net charge status) for along time increases the chance of sampleprecipitation. In imaged capillary isoelectricfocusing, however, all protein samples haveequivalent focusing time, which also is theanalysis time. The short analysis time of theimaged capillary isoelectric focusing reducesthe chance for sample precipitation duringfocusing. This reduction in sample precipi-tation extends the application of the isoelec-

tric focusing method to proteins that aredifficult to analyze using conventional cap-illary isoelectric focusing because of its longmobilization time. Various additives, suchas detergents and urea, can be used inimaged capillary isoelectric focusing analy-sis. All of these advantages significantlyincrease the likelihood that a protein can besuccessfully analyzed by the imaged capil-lary isoelectric focusing method.

Current Status and Future Trends of Whole-Column-Imaging DetectionIsoelectric focusing of proteins in amicrochip: Recently, scientists have shownan increasing tendency toward miniaturiza-

tion or microfabrications of a chemical oranalytical system on a glass chip (31,32).For example, many separations based onliquid chromatography and electrophoresishave been performed on chips several cen-timeters in size (33–36). Schmalzing andcolleagues (37) reported deoxyribonucleicacid (DNA) sequencing on a microfabri-cated chip. So far, most experiments per-formed on a chip use a single-point fluores-cence detection method. In principle, thereal-time, whole-column-imaging detectorsdescribed above also are ideal detectiontools for the chip format, because they canobtain the real-time dynamic informationof a chemical or analytical process.

This ability has been demonstrated byusing a whole-column-imaging absorptiondetector to monitor an isoelectric focusingprocess of proteins performed in a micro-channel fabricated in a quartz chip (38).The microchannel, fabricated by photolith-ography and a chemical etching process,was 40-mm long, 100-�m wide, and 10-�m deep. Its inner surface was coated withlinear polyacrylamide to reduce electro-osmotic flow. Mao and Pawliszyn (38)mixed the protein myoglobin and a pImarker with 4% carrier ampholyte solutionand introduced the mixture into themicrochannel. After they applied a 3-kVelectric voltage across the microchannel, theauthors obtained well-focused protein andpI marker in approximately 10 min. Figure14 shows a separation example of two pImarkers obtained from chip isoelectricfocusing with absorption imaging detec-tion. The detection limit was approximately0.3 �g/mL or 24 pg for the pI marker and30 �g/mL or 2.4 ng for myoglobin. Weexpect that whole-column-imaging detec-tion will find wide application in the field ofmicrochips.

On-line sample preparation and two-dimensional separation of high perfor-mance liquid chromatography–capillaryisoelectric focusing: Biological samplessuch as protein samples usually containlarge amounts of low molecular weight con-taminants such as salts, simple sugars, orother metabolites. Using a high electric fieldis restricted in a capillary that contains ahigh concentration of these salts, because alarge electric current, and thus a largeamount of Joule heat, will be produced.Restricted use of a high electric field meanslow separation resolution for capillary iso-electric focusing or CE. Therefore, desaltingis necessary for real sample analysis. How-ever, conventional off-line desalting meth-ods result in sample loss and are time con-suming. An on-line sample preparation

Figure 11: Capillary isoelectric focusing of an acidic protein with a pI of approximately 3.6.Sample: amyloglucosidase (0.2 mg/mL); focusing time: 5 min.

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Figure 12: Two runs from a capillary isoelectric focusing analysis of acidic peptide. Sample: Arg-Asp-Tyr[SO3H]-Thr-Gly-Trp-Nle-Asp-Phe-NH2 (5 �g/mL); focusing time: 5 min.

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542 LCGC VOLUME 19 NUMBER 5 MAY 2001www.chromatographyonline.com

Although high performance liquid chro-matography (HPLC) and capillary isoelec-tric focusing are the two best separationmethods with the highest resolution pow-ers, the separation efficiency still is insuffi-

cient for complex biochemical samples. It isdesirable to combine these techniques, thatis, to develop a two-dimensional (2-D)HPLC–capillary isoelectric focusing separa-tion. Recently, Tragas and Pawliszyn (40)demonstrated the first step toward 2-D sep-aration — the on-line coupling of HPLCand capillary isoelectric focusing withwhole-column-imaging detection.

Figure 15 illustrates the on-line couplingof HPLC with capillary isoelectric focusing.In this instrument, an HPLC system wasconnected to the imaged capillary isoelectricfocusing system with an on-line desaltingdevice. Protein samples of albumin frombovine serum and myoglobin from horseskeletal muscle were first separated byHPLC, based upon size difference using agel filtration chromatography column, anddetected with a UV-absorbance detector.Each eluted protein was sampled anddirected to a microdialysis hollow-fibermembrane device, in which simultaneousdesalting and carrier ampholyte mixingoccurred. The sample then was driven tothe separation column in an on-line fash-ion, and capillary isoelectric focusingoccurred and was monitored by the whole-column-imaging detector.

Figure 16 shows that HPLC separation of protein myoglobin resolves one peak,whereas capillary isoelectric focusing dis-tinctly shows the presence and separation ofboth isoforms at their expected pI values of6.8 and 7.2.

Fast capillary isoelectric focusing ofproteins without carrier ampholytes: Asstated above, carrier ampholytes commonly

method for capillary isoelectric focusing isdesirable. Recently, Wu and Pawliszyn (39)reported an on-line desalting and carrierampholyte mixing device for capillary iso-electric focusing.

Figure 13: Capillary isoelectric focusing analysis of 4-�g/mL recombinant vaccine. Focusingtime: 4 min.

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Figure 14: Separation of pI markers on amicrochannel with a whole-column-imagingabsorption detector. Sample concentration: 1 mg/mL each; focusing time: 11 min.

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Figure 15: Schematic of an HPLC system coupled on-line with a capillary isoelectric focusing system.

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MAY 2001 LCGC VOLUME 19 NUMBER 5 543www.chromatographyonline.com

are used in gel or capillary isoelectric focus-ing. In protein purification with an isoelec-tric focusing process, the purified proteinsmust be further separated from the carrierampholytes. Also, carrier ampholytes mayinteract with some protein samples, reduc-ing the sensitivity of UV detection, andcomplicate the matrix or backgrounds whenusing mass spectroscopy for characteriza-tion. Accordingly, it is ideal to perform iso-electric focusing or capillary isoelectricfocusing without carrier ampholytes.

chrome c proteins without carrier ampho-lytes. The dynamic process of capillary iso-electric focusing without carrier ampholytewas monitored with the axially illuminatedfluorescence and absorption imaging detec-tion for the fluorescent and nonfluorescentproteins, respectively.

Figure 17 shows one example of thedynamic process of capillary isoelectricfocusing without carrier ampholytes. Exper-imental results show that capillary isoelec-tric focusing without carrier ampholyte is very fast. Usually, it takes less than 30 s,but a capillary isoelectric focusing with car-rier ampholytes requires several minutes or more. Huang and co-workers (41)explained that the capillary isoelectric focus-ing without carrier ampholytes is related toelectrolysis of water in the anode and cath-ode. This phenomenon provides an expla-nation of the mechanism for velocity-difference induced focusing in CE (42).

An alternative method to perform capil-lary isoelectric focusing without carrierampholytes is to create a temperature gradi-ent along the capillary, because somebuffers’ pH depend upon temperature (43).Pawliszyn and Wu (44) demonstrated thisalternative using a 3–4 cm long tapered cap-illary filled with Tris–HCl buffer that con-tained methemoglobin (pI 7.2) and oxyhe-moglobin (pI 7.0). When they applied anelectric voltage across two ends of thetapered capillary, the system generated agradient of electric current density along thecapillary because the cross-section area wasdifferent at different points. This gradientof electric-current density induced a tem-perature gradient related to Joule heat andin turn a pH gradient. The two proteinswere separated at approximately 30 min,after applying approximately 1-kV dc volt-age. Furthermore, Fang and colleagues (45)developed a mathematical model for calcu-lating the temperature gradient in thetapered capillary and capillary isoelectricfocusing with a vertically held tapered capil-lary.

Theoretical study of capillary isoelectricfocusing dynamics: An alternative methodto study capillary isoelectric focusing isdynamic computer simulation that consid-ers the principles of electroneutrality andconservation of mass and charge during theprocess (46–48). So far, the simulation hasbeen successful for predicting separationdynamics, focusing behavior of amphotericsample components, and pH-gradient for-mation and stability in the presence of asmany as 15 amphoteric carrier componentsand 3 proteins. However, capillary isoelec-

Recently, Huang and co-workers (41)demonstrated a simple method for capillaryisoelectric focusing without carrier ampho-lytes using whole-column-imaging detec-tion (41). Using either a 6 cm 200 �mPTFE separation capillary coated withhydroxypropylmethylcellulose or a 5 cm 100 �m fused-silica capillary coated withfluorocarbon, the authors focused and sepa-rated fluorescent R-phycoerythrin andgreen fluorescence proteins and nonfluores-cent human hemoglobin control and cyto-

Figure 16: Separation of bovine serum albumin and horse myoglobin using the coupledHPLC–capillary isoelectric focusing system.

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Figure 17: An example of capillary isoelectric focusing without carrier ampholyte using whole-column-imaging absorption detection. Sample: hemoglobin control (3.1 10�6 M) andcytochrome c (6.1 10�4 M).

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kinetically injected a protein sample ofcytochrome c and myoglobin into theanode end of a 4.5 cm 250 �m capillarythat was filled with 0.5% agarose gel as anti-convection medium. As Figure 18 shows,the two proteins were completely separatedin approximately 1 min. Therefore, rapidseparation easily could be accomplished inwhole-column-imaging CE.

Furthermore, Palm and colleagues (50)demonstrated that interaction between bio-logically active substances such as proteins,detergents, enzymes, and substrate can bestudied by the technique. Monitoringresults of CE behavior of negative hemo-globin A1c before and after interacting with positive hexadecyltrimethylammo-nium bromide micelles that they havestrong interaction. A complex with a posi-tive charge between hemoglobin A1c andhexadecyltrimethylammonium bromidemicelles apparently formed. They alsodemonstrated that whole-column-imagingdetection could be used for multiple-column or capillary-array CE with a 2-Dcharge-coupled device imaging sensor.

In addition to the whole-column detec-tion described above, researchers have usedreal-time, part-column fluorescence imag-ing for studying the dynamic process of iso-tachophoresis of rhodamine B (51) and CEseparation of DNA fragments (52). In theseexperiments, the researchers imaged fluores-cence from the middle 7 cm of a 18-cm-long capillary. More recently, the samegroup used a 10-cm-wide fiber array to cou-ple fluorescence from the middle part of a28-cm-long capillary, in which they per-

formed CE enantiomer separations of dan-sylated amino acids using cyclodextrins(53).

Alternatively, Razee and co-workers (54)used a charge-coupled device video camerato image fluorescence from a capillary. Theyimaged approximately 10 cm of a 59-cm-long capillary, in which electrophoreticachiral separation of dansyl-DL-amino acidswas performed, using a charge-coupleddevice video camera. This video imagingsystem also has been used for direct imagingof the step-wise elution process in HPLC(55).

Researchers also have reported part-column absorption imaging detection witheither a photodiode (56) or charge-coupleddevice (57) array detector for CE. Spectralinformation also can be obtained with a 2-D charge-coupled device sensor and a grating (23,57). Although these part-column imaging detections cannot providethe separation dynamics available from thewhole column, they provide much moreuseful dynamics than those obtainable byconventional single-point detection.

ConclusionWhole-column-imaging capillary isoelectricfocusing and CE allow rapid separation andvisual on-line direct observation of the sep-aration process and molecular interactioninvolving proteins. In addition, analysts canobtain information involving protein struc-ture by coupling whole-column-imagingcapillary isoelectric focusing with mass spec-troscopy. Therefore, we expect that thistechnique will become a powerful tool inprotein separation and determination.

AcknowledgmentThe authors gratefully acknowledge editor-ial assistance by Heather Lord of the Uni-versity of Waterloo (Waterloo, Ontario,Canada).

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Figure 18: Whole-column-imaging CE separation of myoglobin and cytochrome c. Electro-kinetic injection voltage: 500 V; time: 5 s; CE voltage: 500 V; buffer: 0.05 M phosphate (pH 2.5).Peaks: 1 � myoglobin (1 mg/mL), 2 � cytochrome c (1 mg/mL).

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Whole-Column-Imaging Detection for Capillary Isoelectric ...files.pharmtech.com/alfresco_images/pharma/2014/08/...capillary isoelectric focusing: Figure 2 illustrates the basic concept