New directions in isotachophoresis

New directions in isotachophoresis

Citation for published version (APA):

Everaerts, F. M., Verheggen, T. P. E. M., & Reijenga, J. C. (1983). New directions in isotachophoresis. TRAC,

Trends in Analytical Chemistry, 2(9), 188-192. https://doi.org/10.1016/0165-9936(83)85040-7

DOI:

10.1016/0165-9936(83)85040-7

Document status and date:

Published: 01/01/1983

Document Version:

Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers)

Please check the document version of this publication:

• A submitted manuscript is the version of the article upon submission and before peer-review. There can be

important differences between the submitted version and the official published version of record. People

interested in the research are advised to contact the author for the final version of the publication, or visit the

DOI to the publisher's website.

• The final author version and the galley proof are versions of the publication after peer review.

• The final published version features the final layout of the paper including the volume, issue and page

numbers.

Link to publication

General rights

Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain

• You may freely distribute the URL identifying the publication in the public portal.

If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement:

www.tue.nl/taverne

Take down policy

If you believe that this document breaches copyright please contact us at:

openaccess@tue.nl

providing details and we will investigate your claim.

trends in analytical chemistry, vol. 2, no. 6, 1983

Electrophoresis,

isotachophoresis,

isoelectric

focusing

This issue of

TrAC

highlights electrophoresis, in many areas of biological and biomedical research. isotachophoresis and isoelectric focusing, all of which Isoelectric focusing now has not only remarkable separate charged species under the influence of an resolving power, but also a greatly increased protein electric field. Like chromatographic systems they can loading capacity on the preparative scale. Advances in be used for the separation of both high and low isotachophoresis equipment and detection systems, molecular weight compounds. They do, however, combined with microprocessors for instrument handl- possess a number of advantages; particularly signifi- ing and data processing have contributed to the great cant is their requirement for only minimal sample flexibility, reproducibility, accuracy and extremely low

pretreatment. running costs of this technique.

Recent years have seen several important advances, and these are covered in this issue of

TrAC.

High-resolution Z-dimensional electrophoresis has made the analysis of complex protein mixtures a practical possibility, rendering it a potent research tool

In the following articles the growing versatility of electrophoresis, isotachophoresis and isoelectric focus- ing will be amply demonstrated.

Peter T. Shepherd

New directions

in isotachophoresis

The great flexibility, reproducibility, accuracy and extremely

low running costs of isotachophoresis make it an attractive

alternative to HPLC for a number of applications. Recent

developments in isotachophoresis equipment, detection

systems and the advent of the microprocessor have

enhanced the technique’s capabilities.

F. M. Everaerts, Th. P. E. M. Verheggen and

J. C. Reijenga

Eindhoven, The Netherlands

The development of analytical isotachophoresis (ITP) in narrow-bore tubes began in 1964 at the Eindhoven

University of Technology with the work of A. J. P. Martin and F. M. Everaerts. Both anions and cations

(as well as amphoteric species) can be separated by ITP, and the system chosen depends on the nature of the sample constituents to be separated (separands). A schematic diagram of an ITP apparatus is shown in Fig. 1. For the separation of anions, the separation compartment and the anode compartment are filled with the ‘leading electrolyte’ (L). This electrolyte consists of an anion with a high effective mobility

(velocity per unit of fieldstrength) and a counter ion with low effective mobility, and buffering capacity. The cathode compartment is filled with the ‘terminating electrolyte’ (T), which contains an anion with a low effective mobility. The counter ion is of minor importance since it will not enter the separation compartment. The sample is introduced into the injection compartment at the boundary between the leading electrolyte and the terminating electrolyte (Fig. 2). If the sample load is properly chosen with respect to the separation capacity of the separation compartment, all separands Xi fitting into the mobility frame men, L> merr, Xi> men; T will finally migrate with constant

hem& in anaIytica1 chemistty, ~01. 2, no. 9, 1983 189 a b Terminating compartment

I

H 2 I h&

L

t injection block Separation compartment

IF-8

I ‘-f 1 Leading compartment

a

111

‘1,

isotachophomsis

Fig. 1. (le)) Schematit diagram of an ITP apparatus. a = Ptzlcctrode, b = krminating electrolyk, c = drain, d = sepwn, c = UV-a?kckr, f = conductimekr~ g = sefitwn, h = semi-pcnncablc membrane, i = Pt&ctro&, p

and q are lea& to a currmt-stabil&dpower srgply. The separation compartment is a PTFE capillary (I.D. = 0.2 mm, O.D. = 0.35 mm, length is about 2OCt?l).

fig. 2. (above) Principle of isotucha@resis. A&r &rod&on of the separandr A and B between the leading clcctrolyk L and the terminating electrolyk T (Fig. 2 I), the concentrations are a&skd to the conditions of the leading cldrolyrc during the moving boundary phase (Fig. 2 II). Afi reaching the sktiy-stak (Fig. 2 III), the zones can be dckckd isotachophorettially.

I

$7

H

I

b

I

Fig. 3. Isotacho~horetic annlyti of 50 nl of a French wine (MuscaaYet dc Sivrc et Maine) at pH 3.0 with 0.01 M ch.lor&#-akmine as leading electrolyte (L) and acetate as kn&ator (T). (a) Trace of conductivity &kctor. (b) The com/mkrzonverkd bt~~hopherogram which has the p+rties of a chromatog~am, and is keakd as such. I = sul’hak, 2 = sul’hik, 3 = phos@tak, 4 = malonak, 5 = tartrak, 6 ,= citrate, 7 = malak, 8 = la&k, 9 = gluconak, 10 = sac&ate and I1 = &hydroascorbak.

190

velocity during the steady-state period. The concentra- tions of the separand zones are adapted to the conditions of the leading electrolyte according to the Kohlrausch Law’. Therefore, the length of the zones provides quantitative information, while the electrical conductivity provides qualitative information. Other zone characteristics are temperature, electric-field strength, pH, UV-light absorption, fluorescence and radioactivity. The strength of this separation method, compared with its chromatographic equivalent (dis- placement chromatography), is its limited dif- fusion2-4, due to the adaptation of all concentrations of the zones in the separation compartment. Injecting a sample thus causes a concentration or dilution step

(Fig. 2).

A characteristic isotachopherogram is shown in Fig. 3a. This isotachopherogram shows the steady state in the analysis of a wine sample. It was analysed at low pH for organic and inorganic acids and an a.c.

conductivity detector2 was used. In standard

a

R

I

x_

’

2 3 2

b

5 4 Al I

T

10% ,3 5 1 2

C

A2j

T

T

F ’ PA I1

Fig. 4. Selective fluorescence detection of VI Itc;

d

\ t

lmin B constituents, analysed as cations at pH 5.0 with 0.01 Mpotassiumlacetate as leading electrolyte and H’

as terminator. (a) Conductivity trace, (b) W-trace at 254 nm, (c) W-trace at 340 nm, the wavelength of excitation, and (d)$uorescence emission above 350 nm. Approximately 1 nM each of thefollowing separands were injected: I

= thiamine Bl, 2 = pyridoxamine B6,3 = pyridoxine B6,4 = pyridoxal B6 and 5 = nicotinic acid amide.

trends in analytical chemistry, vol. 2, no. 9,’ 1983

isotachophoretic equipment minimal detectable

amounts are of the order of 100 pmol. Specially adapted equipment and/or sample pretreatment can lower the minimal detectable concentration to c. 1 pM.

On-line and off-line combinations of ITP-MS,

ITP-HPLC and ITP-HPLC-MS are currently under

investigation by groups in Vienna5, Bratislava5 and Eindhoven’. On-line ITP-HPLC looks particularly promising and the results obtained are comparable with the experiments on disc-electrophoresis, per- formed in 1964 by Ornstein’ and Davis*.

Further lowering ofthe minimum detectable amount has been achieved by decreasing the inner diameter of the separation compartment to 0.1 mm (Verheggen’), by using volume-coupling (Verheggen”, Shimadzu) and by two-dimensional column-coupling (Verheg- gen”, Kaniansky12, Eriksson13). These developments have enabled the minimum detectable amount to be decreased by a factor of c. 100, compared with commercially available standard isotachophoretic equipment.

Detection systems

Although accurate, the thermal detector provides insufficient resolution and almost all information collected with a thermometric detector can be obtained using the high-resolution conductometric (potential gradient) detector. (A comprehensive comparison of possible ITP detectors can be found in Refs 2 and 3.)

With the conductivity (potential gradient) detector, the adjoining zones can be resolved in zone volumes as small as 3 nl, which under standard operational conditions is equivalent to 30 pmols of analyte. The nature ofthe universal detector signal in ITP. however, makes signal processing by commercially available

equipment (chromatographic peak integrators)

impossible. The amplitude of the signal provides only qualitative information, whereas the time axis contains both qualitative (sequence of zones) and quantitative (length of zones) information. The differential of the signal is widely used for measuring zone-lengths manually and attempts at automation have not thus far been successful. The only signal processor for ITP currently available (type I-ElB Shimadzu) is, in fact, a modified integrator for chromatography and makes use of the differential of the isotachopherogram for the detection of the zone transitions. Failure to detect a zone transition obscures the quantitative results of other zones, whereas the qualitative accuracy is determined by the stability of the universal detector.

Reijenga14 has introduced a signal processing method for ITP which converts the linear trace of the isotachopherogram to a signal with chromatographic properties (Fig. 3b) which is then treated as such. The amplitude ofthe converted signal provides quantitative information. Thus, a great deal of software and hardware developed for chromatography can be used for ITP.

A computer programme for the conversion of ITP signals, written in BASIC, can be used on any

trmak in adytical chemistry, 001. 2, no. 9, 1983

191

Fia. 5. (abow and right) Schematic diagram of an ITP abbaratus in which column

microprocessor with an 8 bit ADC and c. 10 kbyte of

RAM. With this programme it is possible to resolve

zones, e.g. in trace analysis, which approach the

theoretical minimum detectable volume in the detector

probe used. Quantitative accuracy in ITP, with a

well-defined leading electrolyte transport number, is

determined only by the stability of the driving current

and the accuracy with which the zonelengths are

measured. The method described takes both these

effects into account, as the microprocessor also

measures the driving current with an absolute accuracy

of 0.1%. It has been found sufficient to measure the

qualitative information with a resolution of 0.5%. 200

stepheight intervals are available with the micro-

processor, which means that, in principle, one can

qualitatively identify 199 separands between the

leading electrolyte and the terminating electrolyte.

The use of specific detectors, such as UV-absorption

or fluorescence detectors, has provided useful addi-

tional information in isotachophoresis, especially since

at the steady-state the separand zone is mixed only

with the counter ion (Fig. 2 III). The concentration is

adjusted to the concentration of the leading electrolyte,

which makes it necessary to use detector cell volumes

less than 10 nl. The introduction of dual-wavelength

detection, making use of such a measuring cellI with

computerized signal processing, has recently been

introduced by the Eindhoven laboratory”. Multiple

wavelength detection is possible, especially if optical

fibers are used. Scanning detectors are not yet available

for ITP because the scan must be completed within

0.1 s to allow the resolution of short zones. Moreover,

such a detector would need a more complicated

data-system such as that used for GC-MS. At the

present time it is possible to choose two wavelengths

from 206, 254, 280 and 340 nm with the plasma

lamp/filter combinations currently available. Making

‘ION BLOCK

s

I

PRE-SEPARATION TUBE (0.8 mm) BIFURCATION BLOCK / --‘TELL-TALE-DETECTOR SEPARATION TUBE (0.2 mm) CONDUCTIVITY

I

DETECTOR --- LECTRODE COMPARTMENT

use of the UV-absorption (or absorbance) ratios, the

method has been extremely useful for identification and

quantification of steady-state zones, even where these

were short.

The detection unit developed for dual-wavelength

UV-absorption detection has made it possible to apply

fluorescence detection (see Fig. 4). An even more

specific detection method uses radioactivity,

as

introduced by Kaniansky12.

Recent developments in instrumentation

Column coupling (Fig. 5), nowadays equipped with

a microprocessor

for handling the system, for

controlling various operations and for stabilizing the

electric driving current, enhances the versatility of

isotachophoresis without requiring more complex

equipment. Column coupling makes use of two

PTFE-tubes with different internal diameters. In the

pre-separation tube, which has the larger internal

diameter, a high pre-separation current is permitted.

At a well defined distance from a conductivity detector

- a ‘tell-tale detector’ - the final separation

compartment is coupled to the pre-separation capillary

192

trmds in analytical chmirtty, vol. 2, no. 9, 1983

in the bifurcation block. The zones of interest can easily

be selected from the sample train, migrating

isotachophoretically in the pre-separation compart-

ment via the tell-tale detector. The smaller internal

diameter ofthe final separation compartment permits a

higher current density during detection by means ofthe

high resolution detectors described earlier. This system

possesses several advantages

over conventional

isotachophoretic equipment:

*A higher sample load can be handled in the same

analysis time.

@Higher concentration

ratios of separands are

permitted.

l

Different operational systems can be applied in the

two separation

compartments

(multidimensional

isotachophoresis).

l

Various electrophoretic separation principles can be

combined, e.g. isotachophoresis followed by zone-

electrophoresis.

ITP LITERATURE CA vol. 90-97 (1979-1982) ~232 APPLICATIONS ISOTACHOPHORESIS CA vol. 90-97 (1979-1982)

Fig, 6. A survey of recent literature on ITP, divided into papers on instrumentation (I), theory (T), patents (P), applications (A) and reviews (R). The field of ITP-applications is broad and shows an overlap with HPLC. CA = Chemical Abstracts.

Conclusions

Capillary isotachophoresis makes it possible to

analyse both low and high molecular-weight charged

substances with a minimum of sample pretreatment. A

survey of recent ITP literature (Fig. 6) indicates that

there is a considerable overlap in applications with

HPLC. Modern developments in isotachophoretic

equipment and detection systems, combined with the

use of microprocessors for equipment handling and

signal processing make this analytical separation

technique attractive because of its flexibility, repro-

ducibility, accuracy and its extremely low running

costs.

References

1

2

Kohlrausch, F. (1897) Ann. Phys. (Leipzig) 62, 209

Everaerts, F. M., Beckers, J. L. and Verheggen, Th. P. E. M. (1976) J. Chromatogr. Lib. Vol. 6, Isokuhophoresis, Theory, Instrumentation and Applications, Elsevier Scientific Publishing Co., Amsterdam.

Deyl, Z. (ed.) (1979) J. Chromatogr. Lib. Vol. 18, Electropharesis Part A, Techniques, Elsevier Scientific Publishing Co., Amsterdam 3 4 5 6 7 8 9 10 11 12 13 14

Deyl, Z. (ed.) (1983) J. Chromatogr. Lib. Vol. 18Electrophoresi.s Part B, Applications, Elsevier Scientific Publishing Co., Amsterdam Kenndler, E. and Kaniansky, D. (1981) J. Chromatogr. 209, 306 Schoots, A. C. and Everaerts, F. M., J. Chromatogr. Biomed. Appl.

(in press)

Ornstein, L. (1964) Ann. N.Y. Acad. Sci. 121, 321 Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404

Verheggen, Th. P. E. M., Mikkers, F. E. P. and Everaerts, F. M. (1977) J. Chromatogr. 132, 205

Verheggen, Th. P. E. M. and Everaerts, F. M. (1982) J. Chromatogr. 249, 22 1

Verheggen, Th., Mikkers, F. E. P., Kroonenberg, D. M. J. and Everaerts, F. M. (1980) inBiochemicalandBiologica1 Applications of Isotachophoresis (Adam, A. and Schots, C., eds), Elsevier

Scientific Publishing Co., Amsterdam

Kaniansky, D. (1982) Thesis, Comenius University ofBratislava (CSSR)

Eriksson, G. (1983) Thesis, University of Lund, Sweden Reijenga, J. C.et al. Isotachophoresis 1982 ‘Goslar’(FRG), Analytical Symposia Series, Elsevier Scientific Publishing Co., Amsterdam

(in press)

15 Verheggen, Th. et al. Isotachophoresis 1982 %oslar’ (FRG), Ar&ytical Symposia Series, Elsevier Scientific Publishing Co., Amsterdam (in press)

16 Reijenga, J. C., Verheggen, Th. P. E. M. and Everaerts, F. M.

J. Chromatogr. (in press)

Frans M. Everaerts graduated in analytical chemistry in 1965,finished his Ph.D. thesis on Displacement Electrophoresis in I968 and in 19W was nominated professor in analytical separation methods in the Laboratory of Instrumental Analysis at the Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.

Theo P. E. M. Verheggen joined the Research Group on Electrophoresis of the Laboratory of Instrumental Analysis in 1967. His major contribution consists of instrumental development of tiotachophoresis.

Jetse C. Reijenga graduated in analytical chemistry in 1978 from the Eindhoven University of Technology, worked on HPLC for 2 years and in 1980 started researchfor his Ph.D. thesis on theoretical andpractical aspects of isotachophoresis (especially detection systems and signal processing) in Eindhoven.