Anal. Chem. 1903, 55,2464-2466
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aspiration system deserves merit for those applications requiring a minimum amount of sample usage.
LITERATURE CITED (1) (2)
Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1983, 745, 1-15. Riley, C.; Asslett, L. H.; Rocks, B. F.; Sherwood, R. A,; Watson, J. D.;
Morgon, J. Clin. Chem. (Winston-Salem, N . C . ) 1983, 29, 332-335. (3) Rocks, B.; Riley, C. Clln. Chem. (Winston-Salem, N . C . ) 1982, 28, 409-421,
(4) Ramsing, A. U.; Janata, J.; 1980, 118, 45-52.
Ruzicka, J. J.; Levy, M. Anal. Chim. Acta
RECEIVED for review July 5, 1983. Accepted September 6, 1983. The financial support of NIGMS Grant No. 22952 and the donation of items by Reedex Corp. and Critikon, Inc., are gratefully acknowledged. Jeff Harrow was supported by the NHLBI Training Grant No. 5T32HL0752002.
Aluminum-Coated Cell for Fluorescence Signal Enhancement A. J. M. Vermorken,* C. M. A. A. Goos, and M. W. A. C. Hukkelhoven Research Unit for Cellular Differentiation and Transformation, University of Nijmegen, Geert Grooteplein Noord 21, 6525 EZ Nijmegen, T h e Netherlands
M. Coelen Technical Workshop of the University of Nijmegen, F.C.Donderslaan 2, 6525 GJ Nijmegen, T h e Netherlands Fluorescence spectrometry is increasingly being used in chemistry. The reason is that it allows determination of chemical parameters with a higher sensitivity than the formerly used absorbance spectrometry ( I , 2). The tendency, for example in biochemistry, to measure parameters in smaller and smaller samples requires the use of very sensitive techniques (3). An improvement in the setup that would lead to a higher sensitivity would be very helpful for the development of techniques for, for example, population studies that require the use of human biopsy tissue (4-6). In this report a relatively simple design is described that results in a marked improvement in fluorescence spectrometry. An example is given of an application where the increased sensitivity enables one to work with a smaller, acceptable amount of tissue specimens.
EXPERIMENTAL SECTION Materials. Quinine hydrobromide (quinine-HBr)was obtained from Sigma Chemical Co. (St. Louis, MO). For fluorimetric measurements two spectrofluorometerswere used: a Perkin-Elmer 3000 and a Perkin-Elmer 650-40.Fluorescent cells were purchased from Hellma GmbH & Co. (Miillheim-Baden, GDR). The cell holder device with two concave mirrors was obtained from Perkin-Elmer and was especially developed for the 650-40 Model. The coating of a fluorescept cell with aluminum was carried out as follows: After the fluorescent cell was thoroughly cleaned with Alconox and rinsed with alcohol and redistilled water, the cell was placed inside the coating chamber of an Edwards high vacuum coating unit, Model 12E6/579M (Crawley, U.K.).Two sides of the cell were covered to prevent disposition of aluminum onto these sides. The coating chamber was put under vacuum and aluminum of the highest purity was evaporated from a tungsten boat onto the rotating work holder containing the fluorescent cell. To prevent scratching of the aluminum coated cell a protective lacquer film was applied onto the cell and hardened in an oven. Methods. A. Determination of SignallNoise Ratio (Using the Raman Band of Distilled Water). The following controls on the spectrofluorometer were selected as indicated: excitation wavelength, 350 nm; emission wavelength, 397 nm; excitation slit, 10 nm; emission slit, 10 nm; scan speed, 60 nm/min. The emission spectrum of distilled water was recorded with the excitation wavelength set at 350 nm. The signal height of the Raman peak was then measured from the base line along a vertical to the peak (the maximal signal was reached at a wavelength of 397 nm). The spectrofluorometer was then prepared for time base measurements, and the noise level at the peak height of the Raman band (at 397 nm) was recorded. B. Sensitivity of the Fluorescent Signal. A standard solution of quinine-HBr was used with a concentration of 2 ng/mL in 0.1 N H,S04. The fluorescent signal was measured under the following conditions: excitation wavelength, 350 nm; emission wavelength, 450 nm; excitation slit, 10 nm; emission slit, 10 nm.
Table I. Determination of SignallNoise Ratio by Use of the Raman Band of Distilled Watera fluorescence spectrophotometer
Ib
SIN ratio I1
IIId
A. Perkin-Elmer 3000 26.0 i 1.4e 36.1 i 1.9 B. Perkin-Elmer 650-40 87.6 c 7.1 113 109 i 1 0 + 9 a Excitation wavelength, 350 nm; emission wavelength, 397 nm; slit widths, 10 nm. Normal cell. Normal Aluminumcell (cell holder with concave mirrors). coated cell. e Results are expressed as mean c standard deviation ( n = 5).
C. Sensitivity of the Aryl Hydrocarbon Hydroxylase Assay. To test the usefulness of the cell device in a practical test, the enzyme aryl hydrocarbon hydroxylase was measured in human hair follicles. Collection of hair follicles and determination of the enzyme activity, based on the formation of phenolic benzo[a]pyrene metabolites, were performed as described earlier. (6).
RESULTS AND DISCUSSION The intensity of the fluorescent signal depends on several fadors such as the intensity of the exciting radiation, the molar absorption of the sample a t the excitation wavelength, the sample path length along the axis of irradiation, the concentration of the fluorescing material, and the geometry, depending on the effective angle viewed by the detector (7). Figure 1gives a schematic representation of the different cells used for improvement of the effective angle, that would result in a higher yield of fluorescent signal. Figure 1A represents the traditionally used cell for fluorescence measurements. In Figure 1B the cell is placed in a holder containing two concave mirrors, designed for the Perkin-Elmer 650-40 fluorometer. The mirror directly opposite the incoming excitation beam refocuses transmitted excitation light back onto the sample, thereby increasing the intensity of the exciting radiation. Light is emitted by the sample in all directions. The mirror directly opposite the emission slit reflects the emitted light, back to the emission collection optics, resulting in the increase of the fluorescent signal. In Figure 1C the alternative device is demonstrated: two sides of a conventional cell are coated with an aluminum layer that reflects the light, thereby resulting in a higher yield of fluorescent signal. The choice of aluminum as coating metal was based on the following considerations (8,9):the metals that are mostly used for the preparation of mirrors are aluminum and silver, since they give the highest reflection. In Figure 2 the reflection
0003-2700/83/0355-2464$01.50/0 0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
2465
Table 11. Sensitivity of Fluorescent Signal* Ufl(max)
fluorescence spectrophotometer
Ib
A. Perkin-Elmer 3000 B. Perkin-Elmer 650-40
52.1 ?: 3.7 86.7 ?r 4.5
I1
ratio IIId
II/I
140.9 f 4.1 222.7 i 9.2
2.6
III/I
III/II
2.7 2.6 1.0 As a standard a solution of quinine-HBr was used with a concentration of 2 ng/mL in 0.1 N H,SO,: excitation wavelength, 350 nm; emission wavelength, 450 nm; slit widths, 1 0 nm. The values given in the table were obtained after extracting the blanks (mean i: standard deviation (n = 5)). Normal cell. Normal cell (cell holder with concave mirrors). Aluminum-coated cell. A
223.4 * 13.7
1
DETECTOR
A 2 I FLUORESCENT1
A1 IEXCITINGI
,pi
A' ITRAA$=
SAMPLE
A21 FLUORESCENT)
AZIFLUORESCENT)
B
~
I
DETECTOR
300
1
A Z I FLUORESCENT)
\I
U
/ l \
A2 (FLUORESCENTI
M C
L
I
DETECTOR
A21 FLUORESCENT1
'LE
~
/
I('
I
X21FLUORESCENTI
I
i
4
XZIFLUORESCENTI
Figure 1. (A) Normal cell. (B) Normal cell, placed In a cell holder contalnlng two concave mlrrors (opposlte the excitation light source and opposite the detector of the emitted Ilght). (C) Aluminum-coated cell. The two sides of the cell that are coated with an aluminum layer are the sides opposite the excitation light source and opposite the detector of the emitted light (heavy striped line). The dotted lines Indicate the light that is reflected by the aluminum coating.
Characteristicsof both metals over a wide range of wavelengths are compared. It can be seen that silver has the highest reflection in the visible range. However, silver mirrors rapidly deteriorate on exposure in sulfur-contaminated atmospheres. Moreover, at a wavelength under 400 nm the reflection of silver falls abruptly, so that aluminum has to be used when UV light is employed. From Figure 2 it can be concluded that aluminum has very good reflection characteristics from short-wave ultraviolet to the infrared region. Furthermore, aluminum is resistant to corrosion due to the very thin oxide layer that is formed immediately when aluminum comes into contact with air. This layer is very hard and resistant to chemicals, so that aluminum mirrors retain their high re-
400
500
600
700
800
900 h(nrn)
Flgure 2. Comparlson of the reflection characteristics of aluminum and silver (adapted from ref 9).
flectivity over long periods of time. However, the thickness of this oxide layer is not sufficient to protect the underlying aluminum from mechanical damage. Therefore, a protective lacquer was applied. This protected the aluminum layer from scratches during handling, even after prolonged use. The signal to noise ratio of the cells in the two fluorometers was determined by using the Raman band of distilled water. The results are shown in Table I. A marked improvement in the signal to noise ratio is obtained with the use of the aluminum-coated cell and the cell with the mirror device as compared to the normal cell (note: the cell holder with the two concave mirrors was especially designed for the 650-40 Model and could not be used in the other spectrofluorometer). The effect of the different cells on the sensitivity of the fluorescent signal was determined by using quinine-HBr as a standard. The results of these measurements are summarized in Table 11. The fluoregcent signal was improved by a factor 2.6-2.7 with both the aluminum-coated cell and the cell with the mirror device (as compared to the normal cell). To test the usefulness of the aluminum-coated cell in a practical system, the activity of the enzyme aryl hydrocarbon hydroxylase was determined in human hair follicles. The enzyme activity in this human biopsy tissue might be related to individual cancer susceptibility in humans (10). For the determination of aryl hydrocarbon hydroxylase activity we used 30 hair follicles. The results are summarized in Table 111. When we define the determination limit (XD)as a function of the standard deviation of the blank (uo)according to the formula XD = X O (k21/2uo/n1/2) (where Xois the mean value of the blank and n is the number of blanks (II)), the enzyme could not be adequately measured in a normal cell. However, with the mirror construction and the aluminumcoated cell, enzyme activity was above the determination limit. This example shows the usefulness of the both cells in population studies where high sensitivity with little biopsy material is required. Although both the cell with the mirror construction and the cell with aluminum coating resulted in an equal increase in sensitivity of the fluorescent signal {Table I1 and Table 111), the cell with the mirror device has the disadvantage that it
+
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Anal. Chem. 1983, 55,2466-2468
ACKNOWLEDGMENT
Table 111. Aryl Hydrocarbon Hydroxylase Assaya hair blank follicles ( n = 10) (n = 5 ) normal cell normal cell (cell holder with concave mirrors) aluminum-coated cell
2.44 ?r 0.50 4.19 &
determination limitC
6.03
0.41
4.54 i 0.36 10.14 i 0.81
4.35 ?r 0.49
10.04 jl 0.71
6.54
The authors wish to thank E. W. M. Vromans for her skillful technical assistance. Registry No. Al, 7429-90-5;aryl hydrocarbon hydroxylase, 9037-52-9.
LITERATURE CITED
4.68
(1) Brehe, J. E.; Burch, H. B. Anal. Biochem. 1978, 74, 189, (2) Singh, J.; Wiebel, F. J. Anal. Blochem. 1979, 98, 394.
(3) Vermorken, A. J. M.; Van Bennekom, C. A.; De Bruyn, C. H. M. M.;
a Measurements were performed with a Perkin-Elmer 650-40 fluorescence spectrophotometer. The values given in the table are expressed in units of fluorescence (mean i standard deviation). The deterenation limit was calculated by using the formula XD = X, t (k21'20,/ nl") (with k = 10).
is especially developed for one type of fluorometer and cannot be used in other fluorometers. The cell with the aluminum coating is a universal design, that can be used in all types of fluorometers and is therefore much simpler to introduce in fluorescence spectrometry. Reflectorized fluorometer cells have been commercially available for some time (Perkin-Elmer, Oak Brook, IL).
Oei, T. L.; Frohlich, J. Bf. J. Dermafol. 1980, 703,101. (4) Hukkelhoven, M. W. A. C.; Vromans, E.; Markslag, A. M. G.; Vermorken, A. J. M. AnficancerRes. 1981, 7, 341. (5) Hukkelhoven, M. W. A. C.; Vromans, E. W. M.; Vermorken, A. J. M.; Bloemendal, H. FEBS Lett. 1982, 144, 104. (6)Hukkelhoven, M. W. A. C.; Vromans, E. W. M.; Van Diepen, C. A.; Vermorken, A. J. M.; Bloemendal, H. Anal. Biochem. 1982, 725,370. (7) Parker, C. A.; Rees, W. T. Analyst (London) 1980, 85,587. (8) Holland, L. "Vacuum Deposltlon of Thin Films", 6th ed.; Chapman and Hall Ltd.: London, 1970;Chapter 11. (9) AndBrs, H. "Dunne Schichten Fur die Optik"; Wissenschaftliche Verlaggesellschaft mbH: Stuttgart, 1965;Chapter 2. (IO) Vermorken, A. J. M.; Bloemendal, H. I n "Tumour Markers, Impact and Prospects"; Boelsma, E., Rumke, Ph., Eds.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1979;p 305. (11) Kateman, G.;Pljpers, F. W. "Quality Control in Analytical Chemistry"; Wlley: New York, 1981; Chapter 3.
RECEIVED for review March 14, 1983. Resubmitted August 15,1983. Accepted August 29,1983. Acknowledgment is made to the Netherlands Cancer Society (Koningin Wilhelmina Fonds) for financial support.
Precolumn Labeling Device for Liquid Chromatography Susumu Honda* and Hiroko Kuwada Faculty of Pharmaceutical Sciences, Kinki University, Kowakae, Higashi-Osaka, J a p a n Photometric as well as fluorimetric labeling of samples has played an important role in liquid chromatography, because there are no universal methods for detection, such as those based on thermal conductivity and flame ionization in gas chromatography. Of the two types of labeling, postcolumn labeling is more frequently used, because it is more easily automated. Although automated precolumn labeling was attempted by conducting reactions in flow analysis mode ( I ) , this method analyzes only small portions of derivatized products and hence requires large amounts of samples. The present paper describes a convenient device for in situ precolumn labeling, which allows direct application of samples and introduction of the whole derivatization mixture onto columns.
'3
C
EXPERIMENTAL SECTION Apparatus. Figure 1 shows the overall view of the title ap-
paratus, which is composed of a top cover (A), a rotatable body (B), and a fixing plate (C). The top cover is a thick, round-shaped plate, made of stainless steel, and contains four symmetrically arranged flow lines ( a ) , as shown in Figure 2. One end of each flow line is opened on the side surface and the other end on the under surface. The top cover also has a small hollow and a narrow-bore Teflon tube (b) plugged in it, through which samples are loaded along the bisector of two adjacent flow lines, a little off the plate center. A plastic syringe guide (c) is attached to the top surface to effect reproducible sample loading, and the under surface is made flat by metal vaporization. The side ends of flow lines can be connected to stainless steel or Teflon tubes for eluant and reagent delivery by ferrules and setscrews in the ordinary fashion. Figure 3 illustrates the structure of the rotatable body. The inner part is composed of a Diflon cylindrical block ( e ) and a 0003-2700/83/0355-2466$0 1.50/0
Flgure 1. Overall view of the device for in situ precolumn labeling: (A) top cover; (B)rotatable body: (C) fixing plate, (c) syringe guide, ( d ) screw bolt, (e) Diflon block, ( f ) block holder, ( h )derivatlzation cuvette, (i) inlet and outlet of a flow line, (k) outer block. ( m ) handle.
stainless steel holder (f, having a slightly larger diameter, the latter being connected to a rotating shaft (g). The Diflon block has a small cuvette (h)for derivatization, carved on the top surface, and two small holes (i) leading to an inside flow line 0'). The Diflon block with its holder is covered by an outer stainless steel block ( k ) and is pressed upward by leaf springs ( I ) to make the Diflon block touch closely with the under surface of the top cover. The shaft may be rotated by a handle (m),but rotation angle is restricted to 90° by a safety nail ( n )attached' to the inner wall
D 1983 American Chemical Society
