Reasons to be optimisticReport on the 27th International Symposium on Capillary Chromatography, held in

Riva del Garda, Italy, 31 May–4 June 2004

Steve Weber

1. Introduction

The setting of the 27th International Symposium onCapillary Chromatography, chaired by Professor PatSandra, was marvelous. The meeting brought togetherinnovators, users and sellers of capillary separationstechniques. Oral sessions were augmented by posters.The papers from the meeting will be published in aforthcoming issue of J. Chromatogr. A. The overall tone ofthe meeting was very optimistic. Two-dimensionalcomprehensive separations, electromigration techniquesand fundamental approaches to optimization werecommon themes. What follows is a selective reporting onsome of the oral presentations.

2. M.J.E. Golay Award

The meeting opened with the presentation of theM.J.E. Golay Award (sponsored by Perkin–Elmer) toWilfried Koenig (Hamburg) and Volker Schurig(Tuebingen) for their pioneering work in the develop-ment of chiral separation techniques. Dr. Palladini (P-E)and Professor Novotny introduced the awardees.

Koenig was, during his graduate studies, inspired byhis mentor, Professor Bayer, to use a ‘‘Golay’’ (capillary)column to separate components of rum aroma by gaschromatography (GC). The power of the techniqueinspired him to continue to work on separations, and,in particular, chiral GC stationary phases based oncyclodextrins. He reviewed the history of contributionsstarting with Koscielski and Sybilska’s contribution ofintroducing b-cyclodextrin (b-CD) as a chiral selector inGC. He noted that major progress in developing newphases stopped in 1992. Despite this, this class ofselectors has impressive properties. The thermal stabilityof peralkyl b-CDs at up to nearly 400�C allows forseparations of compounds containing 1–28 carboncompounds. Even simple non-functional hydrocarbonenantiomers are separable; 3-methyl hexane, heptane,octane, but not nonane, are separated by a c-CD col-

umn. Capillary columns provide high sensitivity. Forexample 99.98–99.99% enantiomeric excess (ee) ismeasurable for ibuprofen. The recent trend to use lowertemperature with short columns saves time with almostno sacrifice in resolution. CD columns can give kineticinformation, too. A cyclophane with a C8 bridge isstable; with a C9 bridge, it is metastable, while a C10

bridge is labile.How can enantioselectivity be enhanced? Koenig hy-

pothesized that monofunctional substitution of a singleglucose in CD might provide novel and powerful selec-tors. Selectivity depended on the added functional groupand the position. 2,3-Methyl-30-acetyl derivativesseemed to be the best. Are there other selectors thatmight be better? 3,6-anhydro and b-cycloaltrin are notbetter. Cyclofructin, with a crown-ether structure, sep-arates trifluoroacetyl amides of amino acid methyl esters.CDs are still most versatile chiral selectors for GC.

3. Electromigration

Professor Volker Schurig shared the spotlight. Hedescribed various modes of chiral separations byelectromigration. He recounted his earliest work inwhich he first separated chiral olefins on a metallo-organic stationary phase. He saw a strong temperaturedependence of selectivity. Retention time differences,initially of one sign, become smaller, pass through zeroand then become of the opposite sign as T is changed.Metal complexes also separate N chiral centers.

Professor Schurig described his understanding ofseparations of metastable species. One can use thetemperature dependence of coalescence to give theconversion barrier. The US Food and Drug Administra-tion (FDA) has a policy on drugs that interconvert.Enantiopure compounds are not required if the enanti-omers interconvert rapidly. Schurig uses his chromato-graphic method to evaluate this.

Another really interesting direction began withcreating a covalently bound CD to make something likethe successful phase created from permethyl CDs indimethylsiloxane (Chirasil-Dex). Once this phase was inhand, and open tubes were coated with Chirasil b-Dex, itbecame possible to use a single column for four separa-

Department of Chemistry, Chevron Science Center, University of Pittsburgh,

Pittsburgh, PA115260, USA

E-mail: sweber@pitt.edu

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tion techniques! (GC, SFC, LC, CEC). Why does it work?There is balance of k0 and D. Because the k0 in GC is about100, the solute spends a lot of time diffusing in thestationary phase. In LC, the k0 is 0.1. Thus the optimumvelocity for GC and LC are not as far apart as might beexpected. What is in the future? High-throughputscreening of selectors for ee, ultrafast ee analysis, 1part in 105 analysis. Finally, we must understandmechanisms.

4. Single biomolecules

In a plenary address, Professor Ed Yeung (Iowa StateUniversity/Ames Lab) spoke on chromatography ofsingle biomolecules. The apparatus is based on themicroscopic observation of the region of solution near aprism supporting an evanescent wave from a laser.Labeled DNA molecules are visualized in the evanescentfield. To study the effect of surface chemistry on thepropensity of DNA to adsorb, Yeung modified the prismsurface with Au and an alkanethiol. He also preparedmonolayers terminated in –NH2 or –COOH. Patterningof the surface modifications aided in discriminating whatwas adsorbing where. DNA preferentially adsorbs on Crvs. Au surfaces. The use of a square capillary allowed forthe visualization of DNA molecules in a more realisticseparations situation. Interestingly, DNA becomesfocused in the center of the capillary when a balancinghydrodynamic flow is imposed on the driving electro-migration. The focusing effect is size dependent, and canbe used for separating molecules (5 vs. 48 kbase pairs).This simple separation approach facilitated separatinglabeled antigens from their antibody-bound analogs forhomogeneous immunoassay.

5. Peptide and protein separations

Peptide and protein separations occupied a considerablefraction of the meeting. C.G. Huber (Saarland) demon-strated impressive performance in HPLC with ESI MSbased on PS-DVB (polystyrene/divinylbenzene) mono-liths. Additives for peptides aid their separation, but alsoinfluence the mass spectrometry. Common additivesHFBA (heptafluorobutyric acid), TFA (trifluoroaceticacid), and FA (formic acid) all functioned. The S=N wasbest for TFA. In fact, when comparing HPLC-UVchromatograms and HPLC-MS chromatograms (PSDVBmonolith 60�0.2 mm), one can see that peptide-to-peptide differences are far larger than acid-to-aciddifferences. Thus, generalizing about what is the bestadditive is pointless. This separation system can identifyisoforms of membrane proteins.

James Jorgenson (UNC-Chapel Hill) is continuing hisdevelopment of ultra-high-pressure LC MS of pep-tides and proteins. Initial experiments with alkylsilane-

modified 1.0-lm solid silica spheres (Stoeber synthesis)showed a very strong dependence of van Deemtercurves on capillary diameter (smaller capillaries arebetter). The A term is most influenced by capillarydiameter. This system is capable of a peak capacity of300 in 6 h with a 40-cm column, gradient elution.However, a BSA tryptic digest, 120-fmol injection on a50-lm ID, 40-cm long column shows peak broadeningfrom overload, so porous particles are needed.

Waters prepared some 1.5-lm porous particles withfairly good size distribution. They are nearly 100 timesbetter in loading capacity. Surprisingly, despite thesmall pore diameter (150 �A), they worked very well forproteins (cytosolic E. coli proteins). Is pressure playing arole? Jorgenson hypothesized that complete denatur-ation is achieved at high pressure. He obtained 5-lmparticles of the same bridged chemistry. Solutes wereRNAase A, BSA, myoglobin, and ovalbumin. Significantghosting occurred on the 5-lm column (P � 2:3 kpsi).The magnitude of the problem correlated with k0. On1.5-lm particles (23 kpsi), there was no ghosting. In acontrol experiment at low pressure runs on 1.5-lmparticles, ghosting does occur, but only for the well-retained ovalbumin. When the pressure is brought upto 23 kpsi, the ghosts appear without another injection.Thus, the slowly reversed adsorption leading to ghost-ing does not exist at high pressure. Interestingly, whenthe gradient is slowed down, the high-pressure systemresolves the broad BSA peak into several peaks. MSshows that these are different molecules.

Milos Novotny (Indiana University) once againdemonstrated powerful separation/detection combina-tions for bioanalysis. For many years, Novotny haschampioned the importance of the various carbo-hydrate oligomers and their conjugates with proteins.In a more global approach to the problem, he demon-strated the use of affinity columns to trap glycosylatedpeptides. He makes the point strongly that the genomedoes not tell all. Proteins are modified post-transla-tionally. Also, protein synthesis is not the same in allorganelles. Using a combination of CEC and capillaryLC, eluted compounds were combined with matrix forMALDI–TOF–TOF. The distributions of protein peaks forvarious cellular fractions are different.

6. GC

GC also occupied a large portion of the meeting. RichardSacks (U. Michigan) showed his progress towards acomplete chip-based GC–GC. The engineering is alreadybeyond the design stage. Particularly important has beenlowering the thermal mass of the column to allow forrapid temperature control. Data on various generationsof instruments demonstrated impressive performance.The column is 3 m long, 240 lm high, 150 lm wide

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made by reactive ion etching in Si. The rectangularcross-section columns are coated at Restek. Filmsare 1–2 lm thick. Air is the carrier gas, pulled in byvacuum. This works surprisingly well. There is noobvious oxidation problem for temperatures below200�C. Theoretical plate counts of 8000 at the optimalvelocity (compare N ¼ 6000 for H2 carrier at uopt. Thereis not such a great advantage to the low MW carrierbecause the films are thick). Native and oxidized Sisurfaces both work. but the native surface shows 25%more theoretical plates (N). A test mixture of low MWpollutants showed 20 peaks in 600 s.

Now to the tricky part. There are two columns inseries with a valve between them. The first column isnon-polar (PDMS), while the second is polar (trifluoro-propyl methyl silicone). When the order of elution of twopeaks is opposite on the two columns, the second columnwould undo the separation from the first. The valve canbe used to park a peak in the first column while its nearlyco-eluting partner gets a head start in the second.Resolution can be achieved in this way. The advantage isthat you get whatever selectivity you want but with onlyone detector. The instrument also has a preconcentratormade of a stack of four adsorbents. A sample is broughtin from right to left which is from low to high polarityadsorbents, then pushed out left to right. More adsorb-able compounds stick to weak adsorbents, so desorptionis complete for compounds from C4 to C13. The injectedpeak width is controlled with splitter and temperaturegradient in the precolumn.

Milton Lee (Brigham Young University) spoke onorthogonal acceleration TOF-MS. There are half-a-million GCs in the world, with detection limits in the ngto pg range. We can do better. TOF-MS is great – it canrun 10,000 spectra per second. The weak link is the EIefficiency. This is only 0.1%. Lee has created an ioniza-tion source from a quadrupole. The ions are trapped, butelectrons are not. A magnetic field applied in the rightway in the quadrupole traps electrons and the quadru-pole traps product ions. Detection limits are 10–2 fg foralkyl benzenes, a real improvement. Further improve-ments are realized by using a bigger and permanentmagnet (500 G vs. 300 G) and from creating electrodearrays in the quadrupole to trap ions. The detection limitdrops another order of magnitude with the bettermagnet, and with the trapping.

J. Luong (Dow Chemical) addressed the topic of lowthermal mass GC. The problem with the classicalapproach is oven speed and cool down. Microwaveallows higher speed gradients, but special coatings andequipment are required. Resistive heating works, but thepower requirement is high. Thus, low thermal mass GCis the best option.

The column coil includes heaters and sensors. Thesystem uses 5 W for 40–180�C, whereas a normal ovenuses 850 W. Cooling is faster too. A 5 m column takes

8 min in a normal GC to cool from 220�C to 40�C,whereas the LTM GC takes only 30 s. The device isattached to the door of a standard GC. One can move itfrom one GC to another to use various injectors/detec-tors. Applications show remarkable improvements inspeed of analysis.

For stacked injection, use two modules. Hydrocarbonspass through the first column and separate on anon-polar column while a more polar column separatesoxygenated species. Negative temperature programmingis now possible because of the low thermal mass system.

F. Munari (Thermo Electron) spoke on variousmethods of injection, particularly of large volumes, inGC. Depending on the situation, type of solvent andsolutes, there are different methods that essentiallyperform a batch separation of solvent from solutes. Thesolvent may be vented, or the large change in volumefrom its volatilization may be used as added carrier gas toaid the separation. Large-volume injection GC allows fordramatic on-column preconcentration. With sufficientlygood understanding of boiling points, cosolvents can beused to ‘manage’ solutes and solvent behavior.

7. Advances in LC

Advances in LC featured in several talks. J. Finch(Waters) described how two new systems, one a proto-type and one just introduced, had demonstratedimpressive performance for direct flow at nL/min flowrates.

The prototype system creates a gradient at lowpressure by mixing two syringe outflows in a column,then pushing it out at high pressure with a bellows. Thesystem is highly reproducible. At 1 lL/min for 100injections with a 60 min gradient, the standarddeviation of the retention times (about 27 min) was0.041 min. The prototype system is best for 100-lmcolumns or smaller. The new, commercially availablesystem is called nanoACQUITY. This system is alsohighly reproducible. It works as well at 250 nL/min with75-lm columns as it does at higher flow rates for300-lm columns. With a new packing based on 1.7-lmhybrid RP materials, a 30-cm column needs 9000 psi.Typical applications are to peptide separations. The peakwidth at half height is only 3 s. For a 13.5-cm column,the baseline width is 5.3 s.

P. Dugo presented work on comprehensive techniquesfor the analysis of natural matrices. Two impressiveapplications in the food business demonstrated thepower of 2-D comprehensive LC separations. In one,reversed phase chromatography on a 4.6-mm-diametercolumn operating at high flow rate follows a normalphase microbore column. A monolith, with its highpermeability, works well for the second column too. A10-port valve for storing and injecting two different

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fractions assures smooth integration of the two systems.The solvent compatibility problem is avoided by using asmall volume loop for the second separation (20 lL) andhigh flow rate to dilute the problem away. However, toget focusing, the initial solvent strength should be low inthe second dimension, which has a 1-min cycle time.Application to oils of citrus fruits proved the worth of thesystem. In another example, a Ag column in the firstdimension and a non-aqueous reversed phase column inthe second, triacylglycerols were cleanly separated. Inthe first dimension, again a small column works best(1 mm�150 mm, 13 lL/min). The second is a monolith(100 mm�4.6 mm, 4 mL/min). The Ag dimensionseparations are based on double bonds (oleoyl vs.palmitoyl). The RP dimension separates based on size,palmitoyl vs. stearoyl.

Peter Schoenemakers (University of Amsterdam) in apaper of broad scope first reminded the audience that 2-Dseparations require that the two dimensions beorthogonal. He then described what he called a Poppe plotbased on a 1997 paper of Hans Poppe. It is time per platevs. N (log/log). It embodies the van Deemter (or other)dependence of plate height on velocity, and suggests per-formance specifications given the limitations of particularcolumns and pumps. Importantly, for polymers,where thecoupling term in the H vs. v relationship is dominant athigh v, the Poppe plot takes a completely different char-acter. This makes fast SEC practical.

Speaking of monoliths, Frank Svec (Berkeley)presented his latest work on monolith preparationchemistry and technology. The research is focused oncapillaries. Isco and LC Packings have products. Theadvantages of monoliths are that permeability is high,and there is no packing process. After reviewing thermaland redox methods for initiation of acrylate polymeri-zation, he turned to the newest work – photochemicalinitiation. It is fast – only 10 min required. It enablespatterning (a disadvantage being the need for UVtransparent containers). He demonstrated applicationsto proteins. Two materials were demonstrated(0.66- and 2.2-lm pore size (controlled by porogen)).Good separation of a standard protein mixture occurredin only 45 s.

G. Bonn (Innsbruck) discussed a variety of phasesbased on PS-DVB. His tag-affinity chromatographyworks well in coated capillaries. Monolithic discs 3 mmlong with 1 cm ID function well. Interestingly, mixing insilica with PS-DVB materials leads to a fritless system.The media are useful in CEC as well as HPLC.

8. Activity in electrokinetic phenomena

Finally, a significant amount of activity in electrokineticphenomena shared the stage with chromatography.Robert Kennedy (U. Michigan) described his approaches

to microfluidic physiological monitoring. He studiespancreatic Islets of Langerhans, specifically, insulinrelease. The best current technology is to capturefractions and run ELISA on each. This is very hard. TheKennedy group has developed a CE-based immunoassay.The assay is rapid, 10 s, with quantitation in the nMconcentration range. Based on this assay, the group hasdeveloped a microfabricated Islet Work Station in glass.Electroosmotic flow (EOF) adds immunoassay reagents tothe samples from the Islet, or adds glucose to the Islet.The system is heated to 37�C. This temperature is notonly good for the Islets, but it improves forward reactionrate of the antibody and the insulin. Control of evapo-ration from the various vials is necessary for reproduc-ibility. The system is remarkably stable. Bound/free (B/F)ratios have relative standard deviations of a few percentin an hour. For times much longer than an hour, bufferelectrolysis contributes to signal variability. The Islet isperfused at high flow rate (1 lL/min) by pressure, butsampled by EOF at 1 nL/min. Secretion spikes of insulinare clearly seen. The maximum release rate of insulin isabout 1000 pg/min and each spikes is about 100 s wide.Current work is using the same device to sample frommicrodialysis tubing directly.

Norm Dovichi (U. Washington) spoke on 2D capillaryelectrophoresis for proteomic analysis of singlemammalian cells. Why? (1) Samples are small, (2)cancer diagnosis is better (hypothesis: protein distribu-tion changes as cell becomes cancerous), and (3) tocharacterize heterogeneous systems. The methodinvolves picking a cell, injecting it into capillary, lysingthe cell (SDS), labeling the proteins (FQ for e amino oflysine) and laser-induced fluorescence. The problem ofhomogeneous labeling is solved by labeling in the pres-ence of 5 mM SDS. Separation with a sieving matrixreveals peaks. What are they? The cells were fractionatedto determine the origin. In samples of neonatal ratneurons, the sieving separation is very different from cellto cell. For 2D, sieving is followed by fast MEKC. Transferis efficient. It involves placing the capillary ends neareach other in buffer. The second dimension separates200 fractions from the first dimension (300 s each) foran 8 h total run time. Analysis of data shows that a plotof log (number of spots) vs. log (signal intensity) has aslope of )1. Better detection limits will reveal moreinformation. Recent changes have improved the analysistime to 3 h. Current single-cell work is on precursor cells(which become osteoblasts).

Shigeru Terabe (Hyogo) spoke on the challenges ofinterfacing MS with chip CE. A lot of the trick is ap-propriate control of voltages. Using labeled species, heshowed that MS peaks are broadened some by theinterface. An interface of 50-lm ID tubing is pretty bad.A 20-lm interface is better. If one increases the columnlength for better separation, the peaks get a bit wider.The MS effect is then relatively lower for a longer

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separations channel. The effect of organic solvent isconsiderable. When analytes are peptides, injection canbe a problem. At low pH, EOF goes down. At high pH,mobility of peptides is low. He therefore used a suc-cessive multi-ionic layer coating [1] to keep EOF con-sistent. Quantitation of creatine and creatinine ispossible. Linearity is not always good probably becauseinjection is irreproducible.

Chris Culbertson (Kansas State) spoke on a very nicenew material for microfluidics. Sylgard 184 containssignificant silica. Culbertson wanted a low EOF, andthought that it was probably the silica causing it, so theresearch group created their own PDMS with vinylterminated oligos, but with no silica. EOFs in channelsformed in the pure material are lower, but still signifi-cant. NaOH treatment leads to the same EOF ascommercial Sylgard (with silica), so they decided to addback silica, but not as particulate matter, but bymaking sol/gel material. They make chips in thenormal way, then immerse the chip in TEOS, then inbase. The TEOS dissolves in the polymerized chip. Thebase creates the gel material in the silicon matrix. Thechip remains transparent in visible light. The estimatedparticle size (TEM) is 10 nm. The formation of the gelyields a 3.1% weight increase. The contact anglechanges from 108� (Sylgard) to 90�. The best part of all– there is no detectable dissolution of rhodamine dyeinto the matrix. This is a welcome change from Sylgard.The surfaces are stable under dry storage conditions.Separations of labeled amino acids are better than withPDMS, especially with a rhodamine-based label. Thesurface can be modified with normal silane chemistry.A microfabricated system generated 40,000 theoreticalplates in 8 s for proteins with non-ionic surfactantadditives.

B. Ga�s (Charles Univ. Prague) spoke about hiscomputer program for optimization of backgroundelectrolytes in electrophoresis. He uses an exact solutionof the transport equations. There is also a simpleprogram in which the equations are linearized (velocityindependent of concentration of analyte). He used these

to gain insight into electropherograms of underivatizedamino acids in two different electrolytes of same pH.Acetate was much better than HCl. The important lessonis that background electrolyte of N constituents hasalways N eigenzones (system peaks). When systemeigenmobility is close to an analyte there is a‘resonance’. This resonance causes analyte peaks to bemisshapen. Using the program, a scientist can modifybuffer conditions to move system peaks away fromanalytes.

K.D. Patel (Sandia) discussed progress on EK pumpsfor UHPLC. They packed beds with 0.2–1-lm non-porous silica. The pressure developed by enclosing thecathode chamber. When flow is zero (capillary plugged),EK flow is same is pressure flow. A parameter, DP=DU,results. For example, a 100 lm�7 cm column packedwith 0.5-lm particles gives 10 psi/volt. On a glass chip,1–200 nL/min at 100 bar is achievable. In capillaries,10–1500 nL/m at 700 bar is achievable. The theory thatsays that DP=DU is inversely proportional to particlediameter works. Though columns with 0.2-lm particlescan be packed, 0.4-lm particles seem to provide the bestoverall performance. Sandia has developed a set ofconnectors, tees, and so on, for facilitating the use ofthese pumps. The fittings compress directly onto thesilica tubing.

C.F. Ivory (Washington State) presented work ondynamic electrofocusing at mg scales. The apparatus isan annular boron nitride inside with coolant runningthrough it. Outside that, and defining an annular space,is a Lucite tube. Rotation of the inner boron nitride partprevents natural convection from occurring. Using aseries of electrodes in the boron nitride core, a voltagegradient of any shape can be established. Ions can bepropelled with the flow, against the flow, or held to aconstant position with zero net velocity.

Reference

[1] H. Katayama, Y. Ishihama, Y. Asakawa, Anal. Chem. 70 (1998)

5272.

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