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306	Santana Rodrı´guez

limits is limited owing to the increase in background noise when using a micellar medium.

When the influence was studied of different surfactants on the CL intensity of the reaction of lucigenin with isoprenaline, it was found that while cationic surfactants such as HTAH and HTAB and anionic surfactants such as SDS decrease the CL signal, the presence of Brij-35 increases the signal by a factor of 2.1 compared to that obtained in an aqueous medium [61]. As a result, a quite sensitive analytical method has been established for determination of isoprenaline, using Brij-35 as a CL enhancer. Application of the method has been satisfactorily verified with the determination of isoprenaline in pharmaceutical preparations.

3.1.1.4 Zwitterionic Surfactants

One of the few zwitterionic surfactant used in CL reactions is N-dodecyl-N,N- dimethyl-ammonium-3-propane-1-sulfonate (SB-12). Particularly, SB-12 has been assayed in the study of the CL reaction of lucigenin with various biological reductants [10]. The results show that SB-12 enhances CL intensity of the luci- genin-glucose and lucigenin-fructose systems by factors of 3.0 and 1.5, respectively, compared to the intensity obtained in aqueous medium. In these conditions detection limits were found for-both analytes of 0.7 10 4 and 2.5 10 5 M, respectively.

3.1.2Reversed Micelles

As mentioned earlier, reversed micelles have different properties from normal micelles. These properties have the potential to favorably affect the sensitivity and other analytical aspects of CL reactions. Thus, reversed micelles have been used to prolong the duration of the observed CL of various oxalate ester (or acid)–hydrogen peroxide–sensitizer reaction systems for application as chemical light sources [62].

The CL system luminol–hydrogen peroxide was characterized by Hoshino and Hinze in HTAC reversed micelles, formed in a 6:5 (v/v) chloroform-cyclo- hexane mixture [63]. The results indicate that such a CL system can be used from an analytical point of view in a pH interval of 7.8–9.0 without the need to add a catalyst or a co-oxidant. In these conditions an analytical method was established for determination of hydrogen peroxide that, apart from supplying much milder conditions compared to the usual situation in an aqueous medium, is also acceptably precise and reproducible.

Studies have also been made on the coupling of the CL luminol–hydrogen peroxide in HTAB reversed micellar medium detection system with enzyme reactions [64]. The use of HTAB reversed micellar medium permits the simultaneous performance of both reactions, enzyme and CL detection, at a mild pH in the

Organized Media in Chemiluminescence

307

Figure 13 Schematic representation of the coupled substrate-enzymatic-luminol CL reaction system in a reversed micellar medium. (From Ref. 64 with permission.)

absence of a co-oxidant or catalyst. Figure 13 shows a scheme of the coupled substrate-enzymatic-luminol CL detection reaction system in a reversed micellar medium.

Based on these results, a simple and unique determination of 14 L-amino acids and glucose as substrates was developed. Thus, the calibration graph for a representative amino acid, L-phenylalanine was linear in the concentration range 1.0 10 6–2 10 8 M with a relative standard deviation of 5.78% and a correlation coefficient of 0.9974. The detection limit obtained was 1.05 10 8 M. In the case of glucose the calibration graph was linear in the concentration range 2.7 10 6–2.7 10 8 M with a relative standard deviation of 4.27% and a correlation coefficient of 0.9980. The detection limit was 2.7 10 8 M. The method was successfully applied to the determination of glucose in human blood serum.

3.2 Cyclodextrins

Grayeski and Woolf have studied the influence of the presence of cyclodextrins in chemiluminescent systems [65, 66]. They have found that aqueous solutions of α-, β-, and γ-cyclodextrins enhance the CL intensity of the reaction of luci-

308	Santana Rodrı´guez

genin with hydrogen peroxide [65]. The greatest enhancement was observed for a solution 1 10 2 M of β-cyclodextrin, presumably due to restrictions of size in accommodating the corresponding species. Different experiments show that the CL enhancement can be attributed to an increase in the excitation efficiency and in the rate of the reaction through the inclusion of a reaction intermediate in the cyclodextrin cavity.

The same authors studied the CL of 4,4′-[oxalylbis(trifluoromethylsulfo- nyl)imino]bis[4-methylmorphilinium trifluoromethane sulfonate] (METQ) with hydrogen peroxide and a fluorophor in the presence of α, β, γ, and heptakis 2,6- di-O-methyl β-cyclodextrin [66]. The fluorophors studied were rhodamine B (RH B), 8-aniline-1-naphthalene sulfonic acid (ANS), potassium 2-p-toluidinylnaph- thalene-6-sulfonate (TNS), and fluorescein. It was found that TNS, ANS, and fluorescein show CL intensity enhancement in all cyclodextrins, while the CL of rhodamine B is enhanced in α- and γ-cyclodextrin and reduced in β-cyclodextrin medium. The enhancement factors were found in the range of 1.4 for rhodamine B in α-cyclodextrin and 300 for TNS in heptakis 2,6-di-O-methyl β-cyclodextrin. The authors conclude that this enhancement could be attributed to increases in reaction rate, excitation efficiency, and fluorescence efficiency of the emitting species. Inclusion of a reaction intermediate and fluorophore in the cyclodextrin cavity is proposed as one possible mechanism for the observed enhancement.

4. CONCLUSIONS

In accordance with the above, it is clear that the organized media may play an important role in the development of CL reactions. This role may be shown in the improvement of the sensitivity, precision, and selectivity of many CL reactions, due principally to the change of the microenvironment of the CL system. Organized media can alter the microviscosity, local pH, polarity, reaction pathway or rate, etc. This situation allows application of these organized media to determination of organic and inorganic analytes in different kinds of matrices using CL reactions. A summary of these applications is shown in Table 5.

Moreover, the organized systems can be used to smooth the required experimental conditions of CL reactions, above all pH conditions. In this way, it is possible to carry out CL reactions more easily, as well as to develop simultaneously these reactions with other kinds of reactions, such as enzymatic ones, that are of great biochemical interest.

Finally, it is possible to use these media to solubilize some products of CL reactions, which due to their low solubility in aqueous medium can produce interferences in the development of CL reaction as well as in the detection of CL signal. Obviously, this leads to an improvement in the precision of the CL reaction.

Table 5	Analytical Applications of CL Systems in the Presence of Organized Media				a
			Organized
Analyte		CL system	medium		Analytical parameters
Ascorbic acid		Lucigenin–ascorbic acid	HTAC	LDR		b	:	0.5		10		5		–0.2
		Alkaline medium		10		3		mol.L		1
				Slope: 1.447;								c		: 0.9996
										CC
						d	:	1.0		10		6		mol.L	1
				LOD
Ascorbic acid		Lucigenin–ascorbic acid	SDS	LDR:			8.5 10				5		–2.0		10	3
		Alkaline medium		mol.L					1
				Slope: 0.951; CC: 0.9961
				LOD:			5.0			10	5			1
														mol.L
Glucose		Lucigenin-glucose	HTAC	LDR:			1.3 10				4		–4.5		10	3
		Alkaline medium		mol.L					1
				Slope: 0.664; CC: 0.9933
				LOD:			0.8			10	4			1
														mol.L
Glucose		Lucigenin-glucose	SDS	LDR:			1.3 10				4		–4.5		10	3
		Alkaline medium		mol.L					1
				Slope: 0.788; CC: 0.9944
				LOD:			0.9			10	4			1
														mol.L
Glucose		Lucigenin-glucose	SB-12	LDR:			0.6 10				4		–2.6		10	3
		Alkaline medium		mol.L					1
				Slope: 0.783; CC: 0.9959
				LOD:			0.7			10	4			1
														mol.L
Uric acid		Lucigenin–uric acid	HTAC	LDR:			0.6 10				5		–5.0		10	4
		Alkaline medium		mol.L					1
				Slope: 0.639; CC: 0.9958
				LOD:			2.9			10	6			1
														mol.L

Applications Ref.

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Chemiluminescence in Media Organized

309

Table 5	Continued
			Organized
Analyte		CL system	medium
Glucuronic acid		Lucigenin–glucuronic acid	SDS
		Alkaline medium
Fructose		Lucigenin-fructose	HTAC
		Alkaline medium
Fructose		Lucigenin-fructose	SB-12
		Alkaline medium
Fructose		Lucigenin-fructose	SDS
		Alkaline medium
Hydrogen peroxide		Lucigenin–hydrogen peroxide	SB-12
		Alkaline medium
Hydrogen peroxide		Lucigenin–hydrogen peroxide	HTAC
		Alkaline medium

Analytical parameters
LDR: 3.5 10			4	–1.0	10	2
mol.L	1
Slope: 0.887; CC: 0.9981
LOD: 8.4		10	5	1
				mol.L
LDR: 1.2 10			4	–1.3	10	3
mol.L	1
Slope: 1.100; CC: 0.9961
LOD: 4.0		10	5	1
				mol.L
LDR: 1.2 10			4	–1.3	10	3
mol.L	1
Slope: 0.934; CC: 0.9955
LOD: 2.5		10	5	1
				mol.L
LDR: 0.6 10			4	–1.0	10	3
mol.L	1
Slope: 1.052; CC: 0.9998
LOD: 2.0		10	5	1
				mol.L
LDR: 1.0 10			6	–1.0	10	3
mol.L	1
Slope: 0.9998, Intercep: 6.18
CC: 0.9998
LOD: 7.5		10	7	1
				mol.L
LDR: 3.0 10			6	–2.0	10	3
mol.L	1
Slope: 0.98; Intercep: 5.66
CC: 0.9999
LOD: 9.5		10	7	1
				mol.L

Applications Ref.

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Hydrogen peroxide

Hydrogen peroxide

Lucigenin

Lucigenin

Lucigenin

Fructose

Hydrogen peroxide

Lucigenin–hydrogen peroxide
Alkaline medium
Lucigenin–hydrogen peroxide
Alkaline medium
Lucigenin–hydrogen peroxide
Alkaline medium
Lucigenin–hydrogen peroxide
Alkaline medium
Lucigenin–hydrogen peroxide
Alkaline medium
Lucigenin-fructose
Fluorescein-H	O	2
2
pH: 7.0

Brij-35

β-CD

HTAC

Brij-35

β-CD

HTAH

HTAC

LDR:	2.0 10	6	–2.0	10	3
mol.L	1
	;
Slope: 0.99; Intercep: 5.86
CC: 0.9999

LOD: 7.4
LDR: 2.0
mol.L	1
	;
Slope: 0.99;
CC: 0.9989

10	7	1
		mol.L
10	6	–6.0 10	4
Intercep: 6.62

LOD: 8.0
LDR: 5.0
mol.L	1
	;
Slope: 0.71;
CC: 0.9998

10	6	1
		mol.L
10	7	–5.0 10	3
Intercep: 5.14

LDR:	5.0 10	7	–5.0	10	4
mol.L	1
	;
Slope: 0.92; Intercep: 6.58
CC: 0.9984

LOD: 2.9
LDR: 5.0
mol.L	1
Slope: 0.82;
CC: 0.9984

10	7	1
		mol.L
10	7	–3.0 10	4
Intercep: 6.33

LOD:	1.2 10	7		1
			mol.L
LDR: 6.5–84 mg.L				1
Slope: 12.57; Intercep: 361.5
			e	1.6%
CC: 0.9997; RSD :
LOD:		1
	2.3 mg.L
LDR:	5.0 10	9	–1.0		10	6
mol.L	1
Slope: 0.63

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Chemiluminescence in Media Organized

311

Table 5	Continued
Analyte
Dansyl chloride

Dansyl chloride

Rhodamine B (RH B)

Rhodamine B (RH B)

8-anilino-1- naphthalenesulfonic acid (ANS)

		CL system
f			O			–dansyl chloride;
METQ - H
	2			2
pH: 7.0
METQ- H		O			–dansyl chloride;
	2			2
pH: 7.0
METQ- H		O			–RH B pH: 7.0
	2			2
METQ-H	O			–RH B pH 7.0
2			2
METQ-H O				-ANS pH: 7.0
2			2

Organized

medium

Brij-35

HTAC

Brij-35

HTAC

Brij-35

Analytical parameters

LDR: 0.6 10

6

–3.0

10

6

mol.L

1

Slope: 0.15

g

:

3.43–15.2%

RSD

LOD

h

3

1

4.0 10

mol.L

LDR: 3.0 10

6

–10.0

10

6

mol.L

1

Slope: 2.31

g

:

2.24–5.05%

RSD

LOD

h

:

2.0

10

3

mol.L

1

LDR: 5.5 10

8

–1.1

10

4

mol.L

1

Slope: 4.11

g

:

6.4–11.5%

RSD

LOD

h

:

2.0

10

3

mol.L

1

LDR:5.5 10

8

–5.5 10

5

mol.L

1

Slope: 1.13

g

:

4.75–14.5%

RSD

LOD

h

:

4.0

10

3

mol.L

1

8

–5.5 10

5

LDR:5.5 10

mol.L

1

Slope: 9.94;

f

RSD

:0.46–6.10%

LOD

h

:

3.0

10

3

mol.L

1

Applications Ref.

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8-anilino-1-			METQ-H	O	-ANS
			2	2
naphthalenesulfonic acid			pH: 7.0
(ANS)
Albumin–dansyl chloride			METQ-H	O	-albumin–dansyl
			2	2
			chloride
			pH: 7.0
Albumin–dansyl chloride			METQ-H	O	-albumin–dansyl
			2	2
			chloride
			pH: 7.0
Avidin–RH B			METQ-H	O	-avidin-RHB;
			2	2
			pH: 7.0
Avidin–RH B			METQ-H	O	-avidin-RHB;
			2	2
			pH: 7.0
H O		-ANS	METQ-H O		-ANS;
2	2		2	2
			pH: 7.0

HTAC

Brij-35

HTAC

Brij-35

HTAC

Brij-35

LDR:

5.5 10

8

5.5

10

5

mol.L

1

Slope: 3.74

g

: 1.74–2.26%

RSD

h

: 2.0 10

3

1

LOD

mol.L

7

–8.7 10

7

LDR:0.7 10

mol.L

1

Slope: 3.80

g

: 5.86–6.84%

RSD

h

: 6.0 10

3

1

LOD

mol.L

7

–8.7 10

7

LDR:0.7 10

mol.L

1

Slope: 14.2

g

: 1.87–2.05%

RSD

h

: 2.0 10

3

1

LOD

mol.L

7

–3.6 10

5

LDR:3.6 10

mol.L

1

Slope: 0.62

g

: 0.20–17.4%

RSD

h

: 4.0 10

3

1

LOD

mol.L

7

–3.6 10

5

LDR:3.6 10

mol.L

1

Slope: 0.36

g

: 3.30–5.93%

RSD

h

: 2.0 10

3

1

LOD

mol.L

LDR:

1.1 10

3

–5.5 10

2

mol.L

1

Slope: 4.42

g

: 2.54–2.75%

RSD

h

: 3.0 10

3

1

LOD

mol.L

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313

Table 5			Continued
Analyte
H O		-ANS
2	2

H O		–RH B
2	2

H O		–RH B
2	2

Dopamine

Dopamine

			Organized
	CL system		medium
METQ-H O		-ANS;	HTAC
2	2
pH: 7.0
METQ-H	O	-RH B;	Brij-35
2	2
pH: 7.0
METQ-H O		-RH B;	HTAC
2	2
pH: 7.0
Lucigenin-dopamine			Brij-35
Lucigenin-dopamine			HTAH

Analytical parameters
LDR:5.5				5			–5.5				10	2
				10
mol.L			1
Slope: 1.80;
g	: 1.32–6.46%
RSD
h		: 4.0 10				3					1
LOD									mol.L
LDR:		5.5 10			5			–5.5		10			2
mol.L			1
Slope: 2.27
g	: 2.35–4.06%
RSD
h		: 2.0 10				3					1
LOD									mol.L
LDR:5.5				5			–5.5				10	2
				10
mol.L			1
Slope: 5.56
g	: 3.42–5.22%
RSD
h		: 2.0 10				3					1
LOD									mol.L
LDR:5.0				7			–1.0				10	3
				10
mol.L			1
Slope: 0.88
RSD: 2.5%
LOD:		5.0 10			7				mol.L		1
LDR:3.0				7			–1.0				10	3
				10
mol.L			1
Slope: 0.60
RSD: 2.5%
LOD:		3.0 10			7				mol.L		1

Applications Ref.

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Norepinephrine	Lucigenin-norepinephrine	Brij-35
Norepinephrine	Lucigenin-norepinephrine	HTAH
Epinephrine	Lucigenin-epinephrine	Brij-35
Epinephrine	Lucigenin-epinephrine	HTAH
Co(II)	2,6,7-trihydroxy-9-(4-	HTAB
	chlorophenyl)-3-fluorone–H O -
	2	2
	Co(II)
	Alkaline medium
Pyrogallol	N-bromosuccini-mide(NBS)-	HTAB
	pyrogallol
	Akaline medium and hydrox-
	ylammonium chloride
Glucose	Luminol-glucose–glucose oxi-	HTAB
	dase
	pH: 7.25

		7		–1.0				10	3
LDR:5.0 10
mol.L	1
Slope: 0.84; RSD: 2.5%
LOD:	5.0	10	7			mol.L		1
		7		–1.0				10	3
LDR:5.0 10
mol.L	1
Slope: 0.68
RSD: 2.5%
LOD:	5.0	10	7			mol.L		1
		8		–1.0				10	4
LDR:5.0 10
mol.L	1
Slope: 1.40
RSD: 2.5%
LOD:	5.0	10	8			mol.L		1
		8		–1.0				10	4
LDR:5.0 10
mol.L	1
Slope: 1.40
RSD: 2.5%
LOD:	5.0	10	8			mol.L		1
LDR:0.5–6.0 ng.mL							1
Slope: 13.05
RSD: 2.1%
LOD: 0.07 ng.mL						1
		7		–3.0				10	5
LDR:5.0 10
mol.L	1
f	2.1–1.6%
RSD :
LOD:	2.0	10	7			mol.L		1
LDR:	3.0	10	7		–3.0 10					4
mol.L	1
RSD: 3.8%
LOD:	1.0	10	7			mol.L		1

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Water (river,	43
lake)

—44

Blood serum

45

Chemiluminescence in Media Organized

315