Chemiluminescence detection coupled to capillary electrophoresis

Trends in Analytical Chemistry, Vol. 28, No. 8, 2009

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Chemiluminescence detection coupled to capillary electrophoresis Ana M. Garcı´a-Campan˜a, Francisco J. Lara, Laura Ga´miz-Gracia, Jose´ F. Huertas-Pe´rez In recent years, chemiluminescence (CL)-based detection coupled to capillary electrophoresis (CE) as separation technique has attracted much interest due to new advances in home-made configurations, sample-treatment techniques for application to real matrixes, development of a commercial instrument and use of miniaturization techniques to obtain micro total analysis systems incorporating CE separation and CL detection in microchips. We present some developments, key strategies and selected analytical applications of CE-CL since the year 2000 in diverse fields (e.g., clinical and pharmaceutical, environmental or food analysis). ª 2009 Elsevier Ltd. All rights reserved. Abbreviations: ABEI, N-(4-aminobutyl)-N-ethylisoluminol; BMP, Bone morphogenic protein-2; CE, Capillary electrophoresis; CEC, Capillary electrochromatography; CEIA, Capillary electrophoresis immunoassay; CL, Chemiluminescence; CMC, Carboxylmethylcellulose; CPE, Carbonpaste electrode; CTAB, Cetyltrimethyl ammonium bromide; DNPO, bis-(2,4-dinitrophenyl) oxalate; ECL, Electrogenerated chemiluminescence; FA, Folic acid; FITC, Fluorescein isothiocyanate; FR, Fluorescamine; HSA, Human serum albumin; HP-b-CD, Hydroxypropyl-b-cyclodextrin; HRP, Horseradish peroxidase; HV, High voltage; IAP, Immunosuppressive acidic protein; ILITC, Isoluminol isothiocyanate; ITO, Indium/tin oxide; LIF, Laser-induced fluorescence; MWCNTPE, multi-walled carbon-nanotube paste electrode; MEKC, Micellar electrokinetic chromatography; NSAID, Non-steroidal anti-inflammatory drug; PDMS, Poly(dimethylsiloxane); PDMSAO, Poly(dimethoxysilane)-Al2O3; PMP, 1,2,2,6,6-pentamethylpiperidine; PO, Peroxyoxalate; lTAS, Micro total analysis system; TCPO, bis-(2,4,6-trichlorophenyl)oxalate; TDPO, bis[4-nitro-2-(3,6, 9-trioxadecyloxycarbonyl)phenyl] oxalate; S-b-CD, Sulfate-b-cyclodextrin; SDS, Sodium dodecyl sulfate Keywords: Capillary electrophoresis (CE); Chemiluminescence (CL); Clinical analysis; Environmental analysis; Food analysis; Microchip; Micro total analysis systems (lTAS); Pharmaceutical analysis

1. Introduction Ana M. Garcı´a-Campan˜a*, Francisco J. Lara, Laura Ga´miz-Gracia, Jose´ F. Huertas-Pe´rez Department of Analytical Chemistry, Faculty of Sciences, Campus Fuentenueva, University of Granada, E-18071 Granada, Spain

*

Corresponding author. Tel.: +34 958 242385; Fax: +34 958 249510; E-mail: [email protected] This article is dedicated to Professor Willy R.G. Baeyens

In the past two decades, chemiluminescence (CL) has provided a well-established spectrometric branch of analytical chemistry [1,2]. This technique is based on the production of electromagnetic radiation observed when a chemical reaction yields an electronically-excited intermediate or product, which either luminesces (direct CL) or donates its energy to another molecule that then luminesces (indirect or sensitized CL). CL measurements are strongly affected by experimental factors, including temperature, pH, ionic strength, and solvent and solution composition. Because of the dependence of the reaction rate on the concentration, CL techniques may be satisfactorily used for quantitative analysis of a variety of species that can participate in the CL process [CL precursors, reagents, species that affect the rate or the efficiency of CL reaction (e.g., activators, catalysts or

0165-9936/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2009.05.003

inhibitors), species that are not directly involved in the reaction but that can react with other reagents to generate a product that participates, or species that can be derivatized with some CL precursors or fluorophores]. Several limitations apply to CL analysis, such as:  the control of factors that affect the CL emission;  the lack of selectivity because a CL reagent is not limited to just one analyte; and, finally,  like other mass-flow-detection approaches, since CL emission is not constant but varies with time (light flash composed of a signal increase after reagent mixing, passing through a maximum, then declining to the baseline), and this emission versus time profile can widely vary in different CL systems, care must be taken so as to detect the signal in the flowing stream at strictly defined periods. 973

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Several reviews have been published about CL [3–8]. Due to its simplicity, low cost and high sensitivity, CLbased detection has become a useful tool in flow-injection analysis (FIA), liquid chromatography (LC) and capillary electrophoresis (CE), making this technique an interesting research field in clinical [9,10], biomedical [11], environmental and food analysis [12]. The main advantages of CE are low reagent and sample consumption, high separation efficiency and reduced analysis time. However, due to the ultra-small sample volumes used in the system and the requirement for small internal diameter (i.d.), limits of detection (LODs) are poor, limiting its usefulness. Due to the advantages of CL detection and its potential when combined with the high separation ability offered by CE, research in this area has significantly increased since the 1990s. In the past decade, there have been some reviews and book chapters related to the coupling CE-CL [13–18]. In spite of its great potential, this coupling is not widespread, mainly

because there are no commercially available instruments, except in the case of CE coupled to electrogenerated CL (ECL). Basically, a CE-CL instrument is a common CE instrument with some modifications to incorporate a channel with the CL reagents in order to carry out the CL reaction once the analytes have been separated in the electrophoretic capillary. Different interfaces have been used to introduce this channel and to comply with CL requirements (Fig. 1), on-column, off-column and endcolumn interfaces, depending on the way the analytes and the CL reagents are mixed [13]. Various CL reagents have been used in CE-CL, mainly ruthenium complexes (Table 1), luminol and some derivatives [e.g., isoluminol isothiocyanate (ILITC) or N(4-aminobutyl)-N-ethylisoluminol (ABEI) (Table 2) and peroxyoxalates (POs) (Table 3)]. Also, although less common in CE-CL, CL emissions have been explored, based on the interaction of analytes  with strong oxidants [e.g., MnO 4 , ClO , Ce(IV), H2O2,

Figure 1. Devices using capillary electrophoresis with chemiluminescence (CE-CL) with different interfaces. CP, Separation capillary; HV, Highvoltage supply; G, Ground; B, Buffer reservoir, R, CL reagent; D, Detector; M, Semi-permeable membrane; T, Four-way connector; W, Waste; CF, Coaxial flow connector; GE, Grounding electrode; V, Reaction and grounding vial; PMT: Photomultiplier tube (adapted from [18]).

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Trends in Analytical Chemistry, Vol. 28, No. 8, 2009

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Table 1. Some applications of capillary electrophoresis with chemiluminescence (CE-CL) using the tris-(2,2 0 -bipyridyl)ruthenium (II) system Analyte

CL system/CE buffer RuðbpyÞ3þ 3

Detection mode

LOD

Applications

Ref.

Peptides

ECL Borate buffer (pH 9.0)

End-column ECL

2.5 pmol gly-phe-ala 80 fmol val-pro-leu

Compositional analysis of peptides and proteins

[26]

Tramadol, lidocaine

ECL RuðbpyÞ3þ 3 Phosphate buffer (pH 9.0)

End-column ECL

6.0 · 108 M, 4.5 · 108 M

Human urine

[27]

Polyamines

ECL RuðbpyÞ3þ 3

End-column ECL

1.9 · 109 M putrescine and cadaverine 7.6 · 109 M spermidine and spermine

Human urine

[28]

Phosphate buffer (pH 2.0)

Benzhexol hydrochloride

ECL RuðbpyÞ3þ 3 Phosphate buffer (pH 8.0)

End-column ECL

6.7 · 109 M

Pharmaceutical formulations

[29]

Tripropilamine, lidocaine

ECL RuðbpyÞ3þ 3 Phosphate buffer (9.5)

End-column ECL

5.0 · 1011 M, 2.0 · 108 M

Human urine

[30]

Proline

ECL RuðbpyÞ3þ 3 Phosphate buffer (pH 7.6)

End-column ECL commercial instr.

12.2 fmol

Prolidase activity in serum of diabetic patients

[31]

Proline

ECL RuðbpyÞ3þ 3 Phosphate buffer (pH 7.5)

End-column ECL commercial instr.

5.0 · 106 M

Kinetic study of NSAIDs on prolidase activity

[32]

Tripropilamine, proline

ECL RuðbpyÞ3þ 3 Phosphate buffer (pH 7.0)

Solid-state endcolumn ECL commercial instr.

0.002 lM, 2 lM

Instrumental development

[33]

Tripropylamine, lidocaine, proline

ECL RuðbpyÞ3þ 3 Phosphate buffer (pH 9.0)

Solid-state endcolumn ECL RuðbpyÞ2þ 3 immobilized in Zirconia/Nafion modified electrode

5.0 · 109 M, 1.0 · 108 M, 5.0 · 106 M

Instrumental development

[34]

Trimethylamine, triethylamine, tripropylamine, tributylamine proline, hydroxyproline

ECL RuðbpyÞ3þ 3 /ITO electrode Phosphate buffer (pH 9.5)

End-column ECL

5-5 lM

Instrumental development

[35]

Ethambutol, methoxyphenamine

ECL RuðbpyÞ3þ 3 /ITO electrode Phosphate buffer (pH 10.0)

End-column ECL

1.0 ng/mL, 0.9 ng/ mL

Human plasma

[36]

Tripropylamine, proline, oxalate

ECL RuðbpyÞ3þ 3

1.5 · 108 M, 3.1 · 108 M, 4.4 · 108 M

Instrumental development

[37]

Phosphate buffer (pH 9.0)

Miniaturized chiptype cell suitable for FI and CE End-column ECL

Proline

ECL RuðbpyÞ3þ 3 /ITO electrodeBorate buffer (pH 9.2)

Microchip CE End-column ECL

1.2 lM

Instrumental development

[38]

Tertiary amines, lidocaine

ECL RuðbpyÞ3þ 3

Flow cell (PDMSAO)-ITO electrode End-column ECL

20–32 nM

Human urine

[39]

Borate buffer (pH 8.0)

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Table 1. (continued ) Analyte

CL system/CE buffer

Detection mode

LOD

Applications

Ref.

Triethylamine, tri-npropylamine

ECL RuðbpyÞ3þ 3 /electrically heated carbon- paste electrode Phosphate buffer (pH 8.0)

End-column ECL RuðbpyÞ2þ 3 immobilized into a CPE

0.003 lM, 0.002 lM

Instrumental development

[41]

Tri-n-propylamine, acephate, dimethoate

ECL RuðbpyÞ3þ 3 /electrically heated multi-wall-carbonnanotube-paste-electrode Borate buffer (pH 6.5)

End-column ECL

5.9 · 109 M, 2.0 · 107 M, 3.5 · 107 M

Instrumental development

[42]

Disopyramide (chiral separation)

ECL RuðbpyÞ3þ 3 Phosphate buffer (6.5/4.5 separation/detection buffer pH) s-b-CD in the separation buffer (capillary inlet)

End-column ECL

8.0 · 108 M and 1.0 · 107 M for the enantiomers

Plasma

[77]

Alanine, glutamate, aspartate

ECL RuðbpyÞ3þ 3

Porous etched joint off-column ECL

37.3 fmol glutamate

Aspartate aminotransferase and alanine aminotransferase activities in biofluids

[78]

Phosphate buffer (pH 2.1)

RuðbpyÞ2þ 3 immobilized into a MWCNTPE

a-Ketocarboxylic acids

Ce(IV) (for chemical generation of RuðbpyÞ3þ 3 Phosphate buffer (pH 9.5)/0.7 mM CTAB (reverse EOF)

Off-column coaxial flow

1.3 · 109 M3.7 · 108 M

Honey

[79]

Trimethylamine

ECL RuðbpyÞ3þ 3 Borate buffer (pH 9.2)

End-column ECL

8.0 · 109 M

Fish

[80]

Enrofloxacin, Ciprofloxacin

ECL RuðbpyÞ3þ 3 Phosphate buffer (pH 8.5)

End-column ECL

10 ng/mL, 15 ng/mL

Milk

[81]

CPE, Carbon-paste electrode; CTAB, Cetyltrimethyl ammonium bromide; s-b-CD, Sulfate-b-cyclodextrin; ITO, Indium/tin oxide; MWCNTPE, Multi-wall carbon-nanotube paste electrode; NSAID, Non-steroidal anti-inflammatory drug; PDMSAO, Poly(dimethoxysilane)-Al2O3.

IO 4 , Br2, N-bromosuccinimide] or reductants, under different chemical conditions.

2. Tris(2,2 0 -bipyridine) ruthenium (ii) reaction ECL is based on the production of CL emission directly or indirectly as a result of electrochemical reactions. Reactive species formed electrochemically diffuse from the electrode and react, either with each other or with chemicals to produce light from a CL reaction in the vicinity of the electrode. (II) complex Tris-(2,2 0 -bipyridyl)ruthenium 2þ [RuðbpyÞ3 ] is the inorganic compound most exploited 2þ in ECL [19–23]. RuðbpyÞ3 is a stable species in solution, 3þ and the reactive species, RuðbpyÞ3 , can be generated 2þ from RuðbpyÞ3 on the electrode surface by oxidation. 3þ RuðbpyÞ3 can react with analytes containing tertiary, secondary and primary alkyl amine groups to form ex976

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2þ cited state [RuðbpyÞ3 ]*, which decays to the ground state, emitting orange light at 610 nm. In this CL system, for which derivatization is not required for many classes of compounds, the reagent is regenerated and can be recycled. ECL employing RuðbpyÞ2þ 3 offers an attractive detection scheme for CE because of the solubility and the stability of the reagents in aqueous media and the high efficiency over a wide pH range, making it compatible with most buffer systems used. Table 1 summarizes some applications. Most of the contributions in this area that appeared before 2000 suffered from poor separation efficiency, low sensitivity and complicated instrumentation [24,25]. In previous designs, an electric-field decoupler was used to eliminate the influence of the high-voltage (HV) field on the ECL detector [26]. However, its use brought peak broadening and involved experimental skill for its manufacture, while the capillary was easily blocked and also made it more difficult to align capillary to electrode.

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Table 2. Some applications of capillary electrophoresis with chemiluminescence (CE-CL) using the luminol reaction Analyte

CL system/CE buffer

Detection mode

LOD

Applications

11

Luminol, glycine, glycylglycine, and glycylglycylglycine

HRP-luminol- H2O2 Phosphate buffer (pH 10.8)

End-column

1.5 · 10

Luminol

Luminol- H2O2- K3[Fe(CN)6] Phosphate buffer (pH 10.8)

On-column coaxial flow

Rutin, chlorogenic acid

Luminol-K3[Fe(CN)6] Borate buffer (pH 10.0)

Rutin, chlorogenic acid

9

–2.0 · 10

M

Ref.

Instrumental development

[44]

2.0 · 108 M

Instrumental development

[45]

On-column coaxial flow

1.3 · 105 M, 2.2 · 105 M

Cigarettes

[46]

Luminol-K3[Fe(CN)6] Borate buffer (pH 8.5)

Off-column coaxial flow

0.22 lg/mL, 0.50 lg/mL

Pharmaceutical formulations and cigarettes

[47]

Phenothiazines

Luminol-K3[Fe(CN)6] Borate buffer (pH 8.5)

On-column coaxial flow

Injection by gravity: 80 ng/ mL promazine 334 ng/mL promethazine Electrokinetic injection: 1 ng/mL promazine

Pharmaceutical formulation and human urine

[48]

Co(II), Cu(II)

Luminol - K3[Fe(CN)6] Acetate buffer (pH 4.8)

On-column coaxial flow

7.5 · 1011 M, 7.5 · 109 M

Electroplating wastewater

[49]

Polyphenols

Luminol- K3[Fe(CN)6] Phosphate buffer (pH 7.8)

Off-column coaxial flow

1.0 · 1010 M catechol 1.0 · 109 M hydroquinone 3.0 · 1011 M pyrogallol

River water

[50]

Biogenic amines

Analytes labeled with ABEI- H2O2- K3[Fe(CN)6] Borate buffer (pH 9.3)

Off-column coaxial flow

3.5 · 108 M diaminopropane 3.5 · 108 M putrescine 3.9 · 108 M cadaverine 1.2 · 107 M diaminohexane

Lake water

[51]

Hemoglobin

Luminol-H2O2 Phosphate buffer (pH 10.0)

Off-column coaxial flow

1.0 · 109 M

Lysate of human red blood cell

[54]

Cr(III), Cr(VI)

Luminol- H2O2 Acetate buffer (pH 4.7)

On-column coaxial flow

6 · 1013 M, 8 · 1012 M

Water

[55]

Cr(III), Cr(VI)

Luminol- H2O2 Acetate buffer (pH 4.7)

On-column coaxial flow

6 · 1013 M, 8 · 1012 M

Water

[56]

Bone morphogenic protein-2

CEIA, HRP-p-iodophenol-luminolH2O2 Phosphate buffer (pH 6.5)

Off-column coaxial flow

6.2 pM

Rat vascular smooth muscle

[58]

Amino acids

Luminol-BrO Borate buffer (pH 9.2)

On-column coaxial flow

0.35 · 106 - 7.2 · 106 M

Pharmaceutical formulation

[59]

Amino acids

Luminol – BrO Borate buffer (pH 9.4)

Off-column coaxial flow

1 · 107 M glutamic acid 1.3 · 107 M aspartic acid

Rat brain tissue and monkey plasma

[60]

Antioxidants

Cu(II)-Luminol-H2O2 Carbonate buffer (pH 10.8)

Microchip

0.1 mM catechin 0.1 mM nitroblue tetrazolium 0.05 mM superoxide dismutase

Catechin in commercial green tea beverages

[62]

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Table 2. (continued) Analyte

CL system/CE buffer

Detection mode

LOD 11

Co(II)

Luminol- H2O2 Acetate buffer (pH 4.5)

Microchip

5.0 · 10

Human serum albumin and immunosuppressive acidic protein

ILITC-H2O2-microperoxidase Phosphate buffer (pH 7.3) 4 lM microperoxidase

Microchip

Hemoglobin

Luminol-H2O2 Phosphate buffer (pH 10.0)

Agmatine

M

Applications

Ref.

Instrumental development

[63]

1.0 · 107 M HAS 1.0 · 107 M IAP

Human serum

[64]

On-column coaxial flow

0.17 lg/mL

Single human red blood cells

[82]

Luminol – BrO Borate buffer (pH 9.3)

Off-column coaxial flow

4.3 · 106 M

Rat brain, and rat and monkey stomachs

[83]

Medroxy-progesterone acetate

HRP-Luminol-H2O2 Phosphate buffer (pH 6.5)

On-column

0.9 nM

Pork tissues

[84]

Folic acid

Luminol-BrO Borate buffer (pH 9.4)

Off-column coaxial flow

2.0 · 108 M

Pharmaceutical tablets, apple juices, human urine

[61]

ABEI, N-(4-aminobutyl)-N-ethylisoluminol, CEIA, Capillary electrophoresis-immunoassay; HAS, Human serum albumin; HRP, Horseradish peroxidase; IAP, Immunosuppressive acidic protein; ILITC, Isoluminol isothiocyanate.

More recently, important contributions improved the design of the ECL end-column, detection cell and applied CE-ECL to the analysis of biochemicals and pharmaceuticals in real samples. WangÕs group proposed the use of a narrow capillary and a low-conductivity buffer to eliminate the influence of the HV field on the ECL detector [27]. Using this approach, drugs containing tertiary amine groups were monitored in biological fluids and pharmaceutical formulations [27–29]. Although employment of a lowconductivity CE buffer and a narrow capillary effectively decreased the influence of the CE voltage on the ECL detection, it also limited application of this technique. Consequently, the same group explored the use of a 75-lm i.d. capillary and high-conductivity CE buffers to investigate the influence of the HV field without using an electric-field decoupler [30]. The hydrodynamic cyclic voltammogram and corresponding 2þ ECL of RuðbpyÞ3 under the HV field showed that the electrophoretic current did not annihilate the ECL of 2þ RuðbpyÞ3 , but made the ECL-potential shift more positive and demonstrated that the capillary-to-electrode distance was crucial to achieving high sensitivity. Based on that, the authors proposed a new end-column ECL detector without the need for the electric-field decoupler. This device was used to monitor lidocaine in urine [30]. Wang et al. have recently developed a commercial computer-controlled CE–ECL system, integrating CE HV power together with the electrochemical and ECL system, and including data acquisition and data treat978

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ment [19]. Relevant applications in clinical analysis (see Table 1) have been developed using this device [31,32]. Also, a CE-solid-ECL-detection system, using a large i.d. capillary without decoupler, was proposed [33]. Although the solid-ECL system gives high LODs, it is cost effective and regenerative. It is prepared by immobilizing in poly-(p-styrene-sulfonate)-silica-poly(vinyl RuðbpyÞ2þ 3 alcohol) grafted onto 4-vinylpyridine copolymer films. Fabrication of this solid-ECL detector consumed only 0.1 2þ lL of 10 mM RuðbpyÞ3 , which was less than the hun2þ dreds of lL of RuðbpyÞ3 in general solution CE–ECL systems. Sol-gel-derived ZrO2–Nafion composite films have also proved to be an effective matrix for immobi2þ lizing RuðbpyÞ3 at an electrode surface [34]. Indium/tin oxide (ITO) was also used as an electrode to construct a cost-effective, end-column ECL detector for CE [35,36]. The ITO electrode simplified alignment between the separation capillary and the working electrode. However, the ITO-electrode system is limited by a need for machine work to fabricate the working electrode, large void volumes at the detection cell and inconvenience in the cell assembly. As miniaturization is an important trend in analytical chemistry, some instrumental developments have addressed the coupling of CL detection with CE separation on a chip. In this way, WangÕs group proposed an instrument with miniaturized ECL detection in flow systems with a detection cell as part of the instrument [37]. The most important features of this ECL-detection cell were its suitability for CE measurements and the continuous flow of RuðbpyÞ2þ 3 solution through the capillary-

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Table 3. Some applications of capillary electrophoresis with chemiluminescence (CE-CL) using peroxyoxalates (POs) Analyte

CL system/CE buffer

Detection mode

LOD

Applications

Ref.

Amino-PAHs and compounds tagged with chemilumophores

TCPO-H2O2 catalyzed by PMP and triazole Non-aqueous CE

On-column

0.939–2060 nM

Checking the catalysis by PMP for ultrafast POCL reaction

[67]

3-Amino-fluoranthene, 1-aminopyrene 1-aminoanthracene, dansylhydrazine

TCPO-H2O2 CEC (90:10 ACN:H2O, l mM tris, 10 mM PMP, 5 mM H2O2, 20 mM triazole)

End-column

53.7–863 nM

Instrumental development

[68]

Bovine serum albumin

TDPO or TDPO-H2O2derivatized with FR Phosphate buffer (pH 3.0)

On-column flow-type detector

6 · 107 M lysozyme (TCPO) 6 · 108 M lysozyme (TDPO)

Instrumental development

[70]

a-Amino acids, proteins, and phenolic compounds

TDPO- H2O2-dansyl chloride/ FITC Tris–borate buffer (pH 7.0) Tris–borate buffer (pH 8.4)/0.1% CMC/1 mM EDTA Phosphate buffer (pH 8.0))/50% ACN/2 mM SDS

Multi-capillary device for simultaneous operation End-column detection cell

—————

Instrumental development

[71]

Phenolic compounds

TDPO- H2O2 Derivatization with dansyl chloride. Phosphate buffer (pH 8.0)/50% ACN/ 2 mM SDS

Comparison of 2 batchtype CL detection cells End-column

1 · 107–5 · 107 M

Surface and reused waters

[72]

Dansyl-amino acids

TDPO- H2O2 CE: Tris-Borate buffer (pH7.0) MECK: Borate buffer (pH 8.3)/ 150 mM SDS

Batch-type and flowtype detection cells

0.43 fmol (batch-type) 1.3 fmol (flow-type) for dansyl-Trp

Instrumental development

[73]

Dansyl-lysine, dansylglycine

TDPO- H2O2 Tris-borate buffer (pH 7.0)

Microchip

1 · 105 M dansyl-lysine

Instrumental development

[75]

Dansyl amino acids

TCPO- H2O2 HP-b-CD Chiral recognition

Microchip

—————

Instrumental development

[76]

DNA and proteins (lysozyme, cytochrome C, RNase A)

TCPO-H2O2 Derivatization with fluorescein Tris-glycine buffer (pH 8.4)/0.5% CMC/5 mM EDTA/2.5% dextrane sulfate for DNA Tris-borate buffer (pH 8.4)/0.5% CMC/5 mM EDTA for proteins

Batch-type and flowtype detection cells

6 · 107 M lysozyme

Instrumental development

[74]

Human serum albumin

TCPO - H2O2 – dyestuff (Eosin Y-containing liposome) Carbonate buffer (pH 9.0)

On-column coaxial flow

1 · 106 M

Human serum

[85]

lysozyme, cytochrome C

CEC, Capillary electrochromatography; CMC, Carboxylmethylcellulose; FITC, Fluorescein isothiocyanate; FR, Fluorescamine; HP-b-CD, Hydroxypropyl-b-cyclodextrin; PMP, 1,2,2,6,6-pentamethylpiperidine; SDS, Sodium dodecyl sulfate; TCPO, bis-(2,4,6-trichlorophenyl)oxalate); TDPO, bis [(2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate.

electrode interface as a result of hydrostatic pressure without the use of a syringe pump, thus simplifying the ECL-instrument design (Fig. 2). The CE effluent came directly in contact with the surface of the working elec-

trode in CE-measurement mode, so the dead volume of 3þ the cell was greatly decreased. RuðbpyÞ3 was generated in situ at the surface of the working electrode by electrochemical oxidation; however, the flow of effluent from http://www.elsevier.com/locate/trac

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Figure 2. Process for constructing the chip-type electrochemiluminescence (ECL) flow cell and schematic view of capillary electrophoresis (CE) and flow-injection (FI) measurement modes. (A) Design pattern of flow channel and accommodating grooves, (B) etched glass plates, (C) accommodating pipette tips and guides, (D) CE, and (E) FI measurement modes: (1) cover plate, (2) bottom plate, (3) pattern designs of flow channel, (4) working electrode, (5) reference electrode, (6) capillary accommodating grooves, (7) flow channel, (8) accommodating grooves of working electrode, (9) capillary, (10) reference electrode, (11) RuðbpyÞ2þ 3 solution reservoir, (12) waste reservoir, (13) working electrode guide, (14) capillary guide, (15) rubber septum [accommodated at the bottom of (10)], (16) connecting capillary, (17) working electrode, (18) separation capillary, (19) silver-wire quasi-reference electrode, (20) inlet capillary, (21) outlet tubing, and (22) rubber septa seal (from [37]).

the electrophoresis capillary over the electrode could re3þ duce the concentration of RuðbpyÞ3 , thereby reducing the efficiency of the light-producing reaction. The dis980

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tance between the separation capillary outlet and the working electrode therefore needed to be optimized to obtain higher separation efficiency and ECL signals.

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The ITO electrode can be also integrated into an ECL detector to give a microchip CE device [38]. The system (Fig. 3) used an ITO-coated glass slide as the chip substrate with a photolithographically-fabricated ITO electrode located at the end of the separation channel. Separation and injection channels were contained in a poly(dimethylsiloxane) (PDMS) layer, which was reversibly bound to the ITO-electrode plate. With this construction, the photon-capturing efficiency was enhanced. To improve sensitivity, an approach was developed based on a pH junction and field amplification for determining tertiary amines by CE–ECL [39]. To simplify the connection between the separation capillary and the ECL detector and to increase light-collection efficiency, a flow cell of PDMS-Al2O3 (PDMSAO) was fabricated and used.

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In the microchip area, Ding et al. proposed a simpler, universal wall-jet configuration combined with an endcolumn ECL-detection system, which applied both detection modes (i.e. pre-column and post-column) [40]. A glassy-carbon-disc electrode used as a working electrode and aligned with the outlet of the separation 2þ channel electrochemically oxidized the RuðbpyÞ3 to the 3þ active RuðbpyÞ3 form, which then reacted with analytes at the cathodic cell and produced light. The device was evaluated using local anesthetic-containing tertiary amines. In some of the above-mentioned contributions, 2þ RuðbpyÞ3 was usually immobilized on various working electrodes. These conventional, modified electrodes worked at room temperature and had to be ground to get fresh surfaces for reproducibility. By contrast, using an

Figure 3. Microchip device for capillary electrophoresis with electrochemiluminescence (CE-ECL). (A) Top view of the PDMS layer and electrode plate. Separation channel dimensions: 15 lm deep · 48 lm wide at the bottom and 60 lm wide at the top · 45 mm from the intersection to the detection reservoir, and 5 mm from the intersection to the sample, buffer, and unused reservoirs. (B) An enlargement of the detection region. Distance between the separation channel outlet and the indium/tin-oxide (ITO) electrode, 30 lm. (C) The entire system (from [38]).

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electrically-heated carbon-paste electrode (CPE) [41] had several advantages: (i) with moderate heating, the peak width of analyte was decreased and the shape of the peak was obviously improved, and this was favorable for the separation in CE; (ii) the surface-fouling effects could be minimized by moderate heating, which led to satisfactory reproducibility; and, (iii) the application of a heated CPE in CE-ECL provided a higher sensitivity, wider linear range and lower LOD. 2þ Recently, an electrically-heated RuðbpyÞ3 /multi2þ walled carbon-nanotube paste electrode [RuðbpyÞ3 / 2þ MWCNTPE] was developed [42],] with the RuðbpyÞ3 embedded in the MWCNTPE. The analytes would react with the electrochemically-generated RuðbpyÞ3þ and 3 emit light when they were exiting the capillary. The RuðbpyÞ2þ 3 /MWCNTPE can be renewed easily by polishing the surface of the electrode.

3. Luminol and derivatives In the 1990s, applications of luminol (5-amino-2,3dihydro-1,4-phthalazinedione) and luminol-derivative reaction dealt mostly with the determination of metal ions, considering their catalytic effect. Nowadays, the applications have evolved towards the clinical field. Most of them deal with the determination of small molecules and macromolecules in complex matrices, such as biological fluids. Although the 6-amino isomer of luminol (isoluminol) is around 100 times less chemiluminescent than luminol, it has been widely used in CL reactions because its amino group is less sterically hindered than luminol. It has therefore been derivatized for CL labeling far more often than luminol. Luminol-type CL-derivatization reagents used mainly in LC and CE have been reviewed [43]. For coupling the luminol reaction in CE, basically two interfaces have been used (Fig. 1): end-column (or batchtype) and on-column (or flow-type). End-column detection simply immerses the outlet end of a separation capillary in an outlet reservoir containing certain CL reagents and electrolyte buffer. This mode has been applied for the detection of luminol or compounds labeled with isoluminol isothiocyanate [44]. Its advantages are simplicity and sensitivity but its reproducibility and separation efficiency can be problematic. After several injections the outlet vial containing the CL reagent can be diluted so the electrophoretic buffer needs to be renewed. Also, contamination in the outlet reservoir could occur. On-column detection comprises an electrophoretic capillary, a reagent tube and a reaction capillary 982

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connected by a three-way or four-way joint [45]. This design has proved to be effective in a high number of applications. However, it is more complex than end-column detection because reagent flow affects sensitivity and the possible mismatch between electroosmotic flow (EOF) and reagent flow can result in turbulent mixing decreasing the peak efficiency, so it needs to be optimized. Table 2 summarizes selected applications. In the luminol reaction, the CL emission is produced by oxidation of luminol or that of a derivative with an oxidant in alkaline medium and, in some cases, in the presence of a catalyst. Oxidants (e.g., permanganate, hypochlorite, iodine or hydrogen peroxide) can be used, the reaction being catalyzed by metal ions, ferricyanide, some metallocomplexes (hemin, hemoglobin) and peroxidases. Although H2O2 is the preferred reagent for macromolecules, K3[Fe(CN)6] has some advantages for its utilization in CE: (i) it comprises both oxidant and catalyst, so the CECL configuration is simpler; (ii) there are no problems with disruption of current due to bubble formation, which occurs with H2O2; (iii) it is soluble in alkaline media; and, therefore, (iv) it is suitable for the luminol reaction (pH 10–11). The reaction of luminol with K3[Fe(CN)6] produces an intense CL emission and the analytes can enhance or inhibit the emission. Some analytes (e.g., rutin or chlorogenic acid) present both enhancement and inhibition, depending on experimental conditions. Jiang et al. developed a method to determine these analytes in cigarettes by indirect CL [46], but sensitivity was better using the direct method [47]. Phenothiazines have also been determined using the direct CL approach, allowing separation of promethazine and promazine hydrochloride in less than 4 min [48]. The method was characterized using two different injection modes: (i) gravity, to determine promethazine as an impurity in thiazinamium methyl-sulfate ampoules; and, (ii) electrokinetic injection to determine promazine in human urine. No interferences comigrating with analytes were found (Fig. 4). K3[Fe(CN)6] is the preferred oxidant for environmental applications and it has been used to determine Co(II) and Cu(II) in wastewater from electroplating [49]. It is also known that polyphenols (important due to their toxicity and widespread use in numerous commercial products, including pesticides, wood preservatives, dyes and synthetic intermediates), can enhance the CL emission of luminol with K3[Fe(CN)6] as well, so they can be determined by CE-CL with LODs 1–5 orders lower than those from other methods [50]. Also, micellar electrokinetic chromatography (MEKC) has been used to determine biogenic amines with this system in lake-water samples with

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Figure 4. Electropherograms of a blank and a urine sample spiked with 10 ng/mL of prometazine. Electrokinetic injection: 5 kV for 30 s; running buffer: pH 8.5 using 20 mM borate containing 1.25 mM luminol; separation voltage: 15 kV; chemiluminescence reagents reservoir at 30 cm of height containing 0.27 mM ferricyanide and 0.01 N NaOH (from [48]).

previous labeling of the analytes with N-(4-aminobutyl)N-ethylisoluminol [51]. H2O2 is still used as oxidant, in presence of a catalyst [e.g., metal ion (mainly Co(II) or Cu(II)) or horseradish peroxidase (HRP)]. One of the problems of using a metal ion is the incompatibility with the alkaline pH conditions of the CL emission. At basic pH, the metal ions precipitate as hydroxides, avoiding the catalytic effect and producing other problems (e.g., clogging the capillary). Different strategies must be used to avoid it (e.g., utilization of an acidic electrophoretic buffer containing the metal ion and luminol). In this case, the alkaline CL reagent determines the pH of the reaction zone, as its flow is much faster than the flow of the electrophoretic capillary [52]. A different approach that avoids precipitation is use of a complexing agent. This strategy was adopted to determine biomolecules (e.g., alpha amino adipates (aAAs), peptides, and proteins) [53]. Potassium sodium tartrate was used as masking agent to avoid the formation of Cu(OH)2 and allowed the interaction of Cu(II) with biomolecules because it was found that Cu(II) was more catalytically active when it interacted with biomolecules to form Cu(II)-biomolecule complexes. In some cases, a metal ion contained in a macromolecule is used to determine the macromolecule [e.g., in the determination of hemoglobin, which reacts with H2O2 and leads to fluorescent products and Fe(III)]. Zhou et al. applied CE-CL to monitor way that hemoglobin reacted with H2O2. They found an intermediate that greatly enhanced the CL emission of luminol-H2O2 [54]. Using H2O2 as oxidant, luminol-based systems in CE can be a powerful tool to check the contamination of

water by metal ions due to the enhanced effect. In this way, Cr(III) and Cr(VI) (CrO2 4 ) were determined in tapwater, surface-water, well-water, river-water and wastewater samples (i.e. allowing speciation) [55,56]. This method was based on in-capillary reduction [i.e. Cr(VI) could be reduced by acidic sodium hydrogen sulfite to form Cr(III), while the sample was flowing through the capillary]. Other metal ions were masked with EDTA, taking advantage of the slow kinetics of the formation of the Cr(III)-EDTA complex, so Cr(III) could be determined selectively. HRP has been widely used as catalyst in the luminol reaction. However, because the catalytic activity is quite weak, it is conventional to find an appropriate enhancer, which strongly increases the sensitivity, such as p-iodophenol or p-iodophenylboronic acid. This strategy was used in CE-immunoassay (CEIA) with CL detection, which was characterized by high efficiency, fewer samples, short analysis time, ease of automation and high sensitivity [57]. For example, Wang et al. [58] detected up to zeptomoles (zmol) of bone morphogenic protein-2 (BMP-2) in rat vascular smooth-muscle cell using HRP linked to BMP-2 in non-competitive format by CEIA, based on its catalytic effect on the luminol/ H2O2/p-iodophenol reaction. This method allowed the baseline separation of HRP complexes from free HRP in less than 3 min. It was applied successfully to arteriosclerosis-pathology research. BrO has also been used as an oxidant, mainly in clinical applications. For example, Yang et al. [59] determined seven underivatized AAs based on their inhibitory effect in the CL reaction between luminol and BrO and the method was satisfactory applied to AA injections, achieving recoveries higher than 94%. http://www.elsevier.com/locate/trac

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Also, Zhao et al. [60] developed a method to determine excitatory AAs (glutamic acid and aspartic acid) in ratbrain tissue and monkey plasma. In this case, amino acids enhanced the CL emission of luminol and BrO. Lower LODs were achieved with the direct approach; the sensitivity was comparable with laser-induced fluorescence (LIF) detection. Folic acid (FA) was also determined in apple juice, pharmaceutical tablets and human urine [61] by its enhancement of the CL reaction between luminol and BrO in alkaline aqueous solution. No solid-phase extraction was needed due to the high selectivity. Recently, the suppression of CL from luminol in the presence of antioxidants was, for the first time, introduced into microchip CE-CL as the principle for antioxidant detection [62]. Luminol-H2O2 in presence of Cu(II) was used in this system, which generates active oxygen species (e.g., superoxide radical anions). Nitroblue tetrazolium, superoxide dismutase and catechin were analyzed as model analytes of antioxidants. Negative peaks from baseline formed by the CL reaction were observed based on the reaction between active oxygen (superoxide radical anion) and antioxidants (analytes). The analytes were separated and detected within 2 min. Mixing the reagent flow and the electrophoretic buffer using the luminol system was simpler in a microchip since the different channels could be easily crossed [63]. TsukagoshiÕs group developed a micro total analysis system (lTAS), in which the CL reaction of isoluminol

isothiocyanate (ILITC)-microperoxidase–H2O2 was used to determine human serum albumin or immunosuppressive acidic protein as a cancer marker in human serum [64]. The three processes (immune reaction, electrophoresis and CL) were compactly integrated onto the microchip to give the lTAS.

4. Peroxyoxalate reaction The PO reaction involves H2O2 oxidation of an aryloxalate ester in the presence of a fluorophore, following a chemically-initiated electron-exchange luminescence mechanism via a high-energy intermediate, 1,2-dioxetanedione [65]. The high sensitivity, the versatility and the relative absence of interferences make this system very useful [66]. Its main disadvantages are the insolubility of the POs in water and their instability towards hydrolysis, which requires the use of organic solvents that are insufficient for CE separation. Also, the high separation voltage can affect the stability of the POs. The most widely used POs are bis-(2,4,6-trichlorophenyl)oxalate (TCPO), bis-(2,4-dinitrophenyl) oxalate (DNPO) or bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO). Although impressive LODs and satisfactory selectivity were obtained in the first contributions, applications to real samples were not reported until recently (see Table 3). The efficiency of POs in CE-CL is limited by the relatively slow kinetics of the reaction, so the use of a

Figure 5. Detection system for capillary electrophoresis with chemiluminescence (CE-CL) using a polyacrilamide-coated capillary (from [70]).

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catalyst (e.g., imidazole) is considered. Kuyper et al. [67] presented a method for coupling an ultrafast PO-CL reaction to CE based on the use of ancillary base 1,2,2,6,6- pentamethylpiperidine (PMP) to assist nucleophilic reagent 1,2,4-triazole (triazole). Under certain conditions, this PO-CL reaction exhibits a half life of less than 2 s with light yields that are twice as great as those of the imidazole-catalyzed PO-CL reaction. Post-separation electrokinetic delivery of TCPO was accomplished using a micro-tee. Electrokinetic addition of TCPO allowed precise control of the ratio of TCPO to CL reagents (PMP and triazole), spiked into the running buffer. This method for CL-reagent delivery avoided the problems and the costs associated with using pressure or mechanical pumps and proved its usefulness for environmental analysis of amino-polyaromatic hydrocarbons (amino-PAHs) and compounds tagged with dansylhydrazine. To extend the range of analytes, the same group reported a new detector cell for use with an ultra-fast co-catalyzed PO-CL reaction coupled to capillary electrochromatography (CEC) [68]. The method avoids problems associated with tedious reactor configurations and expensive syringe pumps. Compared with their previous paper [67], there was a significant improvement in separation efficiency employing electrokinetic reagent delivery using this CL configuration. Using separation buffers containing small percentages of water yielded excellent CEC separations, while CL reagent degradation was minimized. Previous CE-CL devices using the PO system, applying untreated fused-silica capillaries [69], were improved by introducing a polyacrylamide-coated capillary and attaching a flow line for an aqueous buffer solution and a mixing filter in order to provide the conductivity required for CE (Fig. 5) [70]. The stable, constant electric current in the system allowed separation and detection of a mixture of proteins labeled with fluorescamine (FR). Although more expensive, TDPO was used in this case, providing better CL performance than TCPO. Considering that CE-CL detection, mainly carried out end-column, possesses a useful ‘‘micro-space area’’ for reaction or detection at the tip of the capillary outlet, Tsukagoshi et al. [71] proposed to take advantage of this area for simultaneously operating multiple separation modes in the CE-CL detection system using multiple capillaries and different migration buffers. Analysis of aAAs, proteins, and phenolic compounds were simultaneously performed using three capillaries, including usual, polymer-containing and SDS-containing migration buffers for separation, previously described in individual papers for each of the three types of compounds, respectively [72–74]. The samples eluted from the capillaries, which were inserted into the CL-detection cell, were mixed with CL reagent at the tips of the capillaries to generate visible light.

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Also, an on-line quartz-microchip CE-CL device was used to apply the PO reaction for the detection of dansylAAs, using TDPO as CL reagent [75], and, by using microchip CE-CL, chiral recognition was demonstrated, using hydroxypropyl-b-cyclodextrin as an effective enantiomer selector for chiral dansyl-AAs [76]. 5. Conclusions As a sensitive detection system, CL offers interesting perspectives by coupling to CE in the analysis of a great variety of molecules, increasing the advantages presented by both methodologies. Most of the efforts to improve CE-CL instruments focus on the design of CE-CL interfaces. Important developments in this aspect have considerably increased the applications of CE-CL to real matrixes in relevant fields (e.g., biomedical, environmental and food analysis). Also, construction of miniaturized devices has provided chip-type instruments with low LODs, short analysis times, high efficiency and low consumption of sample and reagents. This CE-CL combination can be considered as an alternative to LC and other powerful modes of detection. Acknowledgements The Spanish Ministry of Science and Innovation (Projects Ref. DEP2006-56207-C03-02 and AGL200764313/ALI) and EU funds (FEDER) supported this work. References [1] A.M. Garcı´a-Campan˜a, W.R.G. Baeyens (Editors), Chemiluminescence in Analytical Chemistry, Marcel Dekker, New York, USA, 2001. [2] Y. Su, H. Chen, Z. Wang, Y. Lv, Appl. Spectrosc. Rev. 42 (2007) 139. [3] F. Li, C. Zhang, X. Guo, W. Feng, Biomed. Chromatogr. 17 (2003) 96. [4] Y. Ohba, N. Kuroda, K. Nakashima, Anal. Chim. Acta 465 (2002) 101. [5] A.M. Garcı´a-Campan˜a, W.R.G. Baeyens, Analusis 28 (2001) 686. [6] A.M. Garcı´a-Campan˜a, W.R.G. Baeyens, L. Cuadros-Rodrı´guez, F. Ale´s-Barrero, J.M. Bosque-Sendra, L. Ga´miz-Gracia, Curr. Org. Chem. 6 (2002) 2001. [7] N.W. Barnett, P.S. Francis, Chemiluminescence: liquid-phase chemiluminescence, in: P.J. Worsfold, A. Townshend, C.F. Poole (Editors), Encyclopedia of Analytical Science, 2nd Edition., Elsevier, Oxford, UK, 2005, pp. 511–520. [8] A.M. Garcı´a-Campan˜a, F.J. Lara, Anal. Bioanal. Chem. 387 (2007) 165. [9] C. Dodeigne, L. Thunus, R. Lejeune, Talanta 51 (2000) 415. [10] L.J. Kricka, Anal Chim Acta 500 (2003) 279. [11] A. Roda, M. Guardigli, P. Pasini, M. Mirasoli, Anal. Bioanal. Chem. 377 (2003) 826. [12] L. Ga´miz-Gracia, A.M. Garcı´a-Campan˜a, J.J. Soto-Chinchilla, J.F. Huertas Pe´rez, A. Gonza´lez Casado, Trends Anal. Chem. 24 (2005) 927. [13] X.J. Huang, Z.L. Fang, Anal. Chim. Acta 414 (2000) 1. [14] A.M. Garcı´a-Campan˜a, W.R.G. Baeyens, N.A. Guzman, in: A.M. Garcı´a-Campan˜a, W.R.G. Baeyens (Editors), Chemiluminescence

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