Focusing Review

Chromatography, Vol.26 No.1 (2005)

Focusing Review

Development of Capillary Liquid Chromatography
Toyohide Takeuchi
Department of Chemistry, Faculty of Engineering, Gifu University, 1−1 Yanagido, Gifu 501−1193, Japan
Received for review November 22, 2004. Accepted January 18, 2005.

Keywords: liquid chromatography, capillary columns, instrumentation[2] and packed microcapillary columns [3] were then appeared and1. Introduction
Ishii’s group at Nagoya University started microcolumn liquid

expected to provide higher resolution than densely packed columnschromatography (µLC) more than 30 years ago [1]. His ideas haveowing to their higher permeability, these separation methods still

been realized in user−friendly µLC systems which are now com-have not been popularized, yet. One of the plausible reasons lies inmercially available from a number of manufacturers. In the mean-

the development of electrically−driven separation methods such as

time, injection valves with low dead volume, pumping systems

capillary zone electrophoresis [5], electrokinetic chromatography

working at low flow rates, high−resolution columns, and novel de-[6] and capillary electrochromatography, which have a potential totection systems have been developed by manufacturers and re-produce much higher theoretical plates than pressure−driven sepa-search institutes. The present review will focus on methods andration methods. Monolithic silica capillary columns, which haveideas for improving µLC systems proposed in the author’s labora-

intermediate permeability between open−tubular columns andtory.

densely packed columns, are expected to produce higher resolution
in the LC mode [9]. It should be noted that all these capillary−based separation methods have expanded owing to the develop-

2. Development of related techniques

ment of fused silica capillaries [4].

Table 1 shows development history of capillary−based separation methods and their related techniques [1−9]. In the early stageof the development of µLC, the researchers competed for achieving

3. Features of capillary LC

higher resolution. However, the pressure drop across the separation

LC can be classified based on the column diameter. Columns

columns was a restring parameter as long as densely packed col-with the diameter smaller than 0.075 mm are used in nano−LC andumns were employed. Although open−tubular capillary columns

its flow rate is less than 1 µL/min, whereas the column diameter inµLC is 0.2−0.8 mm and its flow rate is 1−20 µL/min. In this article

Table 1. Development history of capillary−based separation methods and their related techniques
Year1974
1978
1978
1979
1981
1985
1987
1998

Developed capillary separation
methods and their related technique

ReferencesµLC
Open−tubular capillary LC
Packed microcapillary LC
Fused−silica capillary
Capillary zone electrophoresis
Electrokinetic chromatography
Capillary electrochromatography
Monolithic silica capillary columns

1234567,89capillary LC is defined as the one involving nano−LC and µLC.
The features of capillary LC are attributed to the use ofTable 2. Classification of LC

Correspondence:
E−mail: takeuchi@apchem.gifu−u.ac.jp―７―

PurposeClassification

ID
mm

AnalyticalNano−LCµLC
Semi−µLC
Conventional LC〜0.0750.2~0.8
1.0~2.1
4.0~6.0

Preparative

Preparative LC10~

Chromatography, Vol.26 No.1 (2005)

Fig. 1. Features of capillary LCsmaller−diameter columns and lower eluent flow rate, as shown in
Fig. 1 [10]. The lower flow rate saves solvents, reagents and packing materials, compared with conventional LC using 4−6 mm ID

Fig. 2. Comparison of chromatograms obtained in the absence orpresence of the stationary phase.

columns. This situation is environmentally friendly. Improved
mass sensitivity can be expected owing to small−volume detection

Indirect photometric detection

in capillary LC. This is especially of benefit when the sample
amounts available are restricted as in the case of biological sam-

When appropriate detection methods are not available, indi-ples. Low flow rate allows us to use exotic mobile phases or mo-rect detection can be a candidate in ion chromatography, reversed−

bile phase additives, leading to achievement of novel separation se-phase LC and ion−pair chromatography. Indirect detection is clas-

lectivity or detection systems. Low heat capacity of capillary col-sified according to the type of interaction between the analytes andumns facilitates the control of column temperature. This feature al-

the visualization agents. Analytes are usually visualized in indirectlows us to carry out temperature programming more easily not only

detection via ion exchange or an interaction with a mobile−phasein capillary LC but also in supercritical fluid chromatography andadditive in the column. The following equation was derived for thegas chromatography (GC). The most significant feature of capillaryindirect detection of non−electrolytes in reversed−phase LC [12].

columns lies in direct coupling of LC and mass spectrometry (MS),The peak area (Sp) of the analyte can be correlated with operatingwhich is an ultimate detector for chromatography.

conditions and the parameters.
Sp ∝ CadM Wan0fkad (kan + 1)/(kan − kad)4. Novel detection systems based on capillary columns
On−column fluorimetric detection(1)

where CadM is the concentration of the additive in the eluent, Wan0On−column detection is sometimes indispensable for narrow−is the amounts of the analyte injected, f is the coefficient account-bore columns in order to avoid additional extra−column banding for the signal direction and intensity, kad and kan are the reten-broadening. This is not convenient to handle, and it leads to dete-tion factor of the additive and the analyte, respectively. Eq. (1) in-rioration of the sensitivity. However, in some cases the sensitivity

dicates that the analyte signal is higher when the analyte elutes

can be improved by detecting analytes in the presence of the sta-close to the additive system peak and that the signal direction

tionary phase. It can be expected that the signal intensity does notchanges between before and after the system peak.

depend on the retention time in on−column detection because boththe analytes existing in the stationary and mobile phase are sub-Direct coupling with MS

jected to the detection in on−column detection. Fig. 2 compares theLow flow rate is advantageous for direct coupling LC with MS.

chromatograms obtained in the absence or presence of the station-

We have developed an interface for capillary LC and fast atom

ary phase.

bombardment (FAB) MS [13]. The interface was composed of cap-

As for fluorimetric detection of dansyl amino acids, the signal

illary tubing, the top of which was attached with a metal frit. Thewas increased with increasing retention time, owing to the fact thateluent containing a matrix was eluted on the surface of the frit,the fluorescence was enhanced by the hydrophobic stationary phase

where xenon beam was irradiated and ionized. The stainless steel[11]. The on−column fluorimetric detection can improve the detec-frit (1/16”) employed for this purpose is illustrated in Fig. 3. Thetion limits of the analyte eluting late.

Frit FAB LC−MS interface is now commercially available.

―８―Chromatography, Vol.26 No.1 (2005)

Toyohide TakeuchiFig. 3. Stainless steel frit for Frit FAB LC−MS
tration of ethanol in the breath was determined to be 0.29 mg L−1.

5. Use of accessories for capillary columns
Unions, 3−way connectors and 6−way switching valves for

The time course of the breath sample could be monitored. As a re-capillary tubing or columns are now commercially available. Thesesult, the concentrations in the breath of the person in 5, 10, 30 andaccessories possess low dead volumes as low as a few tens of nL,

60 min after drinking 350 mL beer were 0.044, 0.021, 0.015 andand they therefore allow us to conduct pre−column enrichment

0.007 mg L−1, respectively.

[14], recycle separation [15], split and bypass flow system [16] andAn alternate−pumping recycle system was developed for cap-post−column reaction [17] with tolerable band broadening.

illary LC with an aid of a commercially available low dead−vol-An on−line sample enrichment system was designed using

ume 6−way switching valve [15]. The recycle system utilized twomonolithic pre−columns in capillary LC [14]. The monolithic ODS

separation monolithic columns, and the dead volume of the recy-

capillary columns were prepared via in−situ sol−gel processes. The

cling lines was kept to a minimum by avoiding passing the sample

limits of detection were improved by more than 2000−fold and thethrough the pump chamber and sample injector. It was observed

system was applied to the determination of phthalates contained inthat the theoretical plate number (N ) increased linearly with in-laboratory distilled water and tap water samples.

creasing number of cycle, and the N per unit time increased with

The pre−column enrichment system was applied to the deter-

increasing inlet pressure.

mination of ethanol in breath [18]. Fig. 4 demonstrates the detec-

Split flow and bypass flow systems were assembled using

tion of ethanol in breath of a volunteer who drank 350 mL beer.

Nano Y Connectors with low dead volume commercially available

The breath sample was taken right after the drinking. The concen-for capillary LC [16]. The split ratio could be controlled by changing the dimension of restriction tubing and applied back pressure tothe restriction tubing. The bypass flow system uses two Nano YConnectors, where the eluent split at the first Nano Y Connector,
which is located in the inlet of the separation column, is merged
again into the effluent from the column at the second Nano Y Connector. The bypass flow system could avoid on−column detection
and allowed us to use flow cells, leading to improved sensitivity.
6. Future prospects of capillary LC
Development of capillary columns with much higher efficiency than conventional LC columns will shift users from conventional to capillary LC, as seen in the development of capillary GC[10]. Columns with higher column efficiency must have higherFig. 4. Determination of ethanol in breath.
Column: Develosil C 30−UG−5, 100 × 0.32 mm I.D. Eluent:aqueous solution of 0.5% propionic acid. Flow rate: 4.2 µL min−1.
Enrichment column: quartz wool, 1.7 × 0.25 mm ID. Sample:
Sample: 2 mL of breath of a person who drank 350 mL beer.
Wavelength of UV detection: 210 nm.

permeability in comparison with common densely packed columns.
Open tubular capillary columns and monolithic columns are expected to achieve higher column efficiency because of their higherpermeability. At this moment, monolithic silica columns can beone of the most hopeful candidates for such a highly permeable―９―Chromatography, Vol.26 No.1 (2005)

143.

separation system. On the other hand, when the pressure limit can[9]be increased up to much higher levels, densely packed columns

Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.,
Tanaka, N. J. High Resolut. Chromatogr. 1998, 21, 477−

will come back to the candidate.

479.

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nique, downsizing of a whole capillary LC system, etc. are to be[11] Takeuchi, T.; Miwa, T. J. Chromatogr. A 1995, 696, 185−192.

considered in the near future.

[12] Takeuchi, T.; Watanabe, S.; Murase, K.; Ishii, D. Chromatographia 1988, 25, 107−110.

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lished result.

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