2466
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 & 0.41
4.54 i 0.36 10.14 i 0.81
4.35 ?r 0.49
10.04 jl 0.71
determination limitC
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 6.03
(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.;
6.54
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, Japan 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.
EXPERIMENTAL SECTION Apparatus. Figure 1 shows the overall view of the title apparatus, 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
'3
C
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
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
2407
v e r t i c a l section, side view
v e r t i c a l section, f r o n t view
vertical sec:im
stage
-Lm
transverse section
'p Flgure 2. Cross section of the top cover: ( a )flow line, ( b )Teflon tube. Other symbols are the same as those given in Figure 1. All scales are in mlllimeters.
stage
-/7m.3
Flgure 4. Diagrammatic expresslon of each stage of precolumn labeling.
tw surface I
0
I
10
I
20
I
30
I
40
E l u t i o n t i m e (inin)
Figure 5. High-performance liquid chromatographic analysis of w,w'-diamino-n-alkanes by precolumn labeling with fluorescamine by using the title apparatus. Sample scale was 12.5 pmol each. Key: (1) putrescine, (2) cadaverine, (3) 1,6-diamino-n-hexane, (4) 1,7-diamino-n -heptane. vertical s e c t i m
I
1 I
Flgure 3. l o p surface and vertical section of the rotatable body: @) rotating shaft, (i) flow line, ( I ) leaf springs, ( n ) safety nail, ( 0 ) ball bearings. Other symbols are the same as those given in Figures 1 and 2. All scales are In millimeters.
of the outer block. Ball bearings (0)set under the leaf springs effect smooth rotation of the shaft.
Precolumn Labeling of w,w'-Diamino-n-alkanes with Fluorescamine. A 1:l (by volume) mixture of 0.3% fluorescamine in acetone and 0.1 M borate buffer (pH 10.0) was used as a reagent solution. An aqueous solution of a diamine or a mixture of diamines was added to the reagent solution and the mixed solution was allowed to stand for 5 min at room temperature. The fluorescent products were separated on a column of LiChrosorb RP-18 (4 mm i.d., 15 cm) at room temperature with a 1:l (by volume) mixture of methanol and 0.05 M borate buffer (pH 10.0) as eluant at a flow rate of 1.0 mL/min and monitored at 390 (excitation)/475 (emission) nm.
RESULTS AND DISCUSSION Figure 4 shows the flow diagram at each stage of precolumn derivatization. At stage 1 a reagent solution is led into the cuvette from one of the flow lines 2 and 4 (side view) by using a small injector. Excess solution is drained from the other line. At this stage an eluant is pumped to the column via the flow lines 1-3 (front view). At the subsequent stage (stage 2) the stream of the reagent solution is stopped, and a sample is loaded into the cuvette via the Teflon tube by using a microsyringe. The eluant is allowed to continue flowing. At stage 3 the Diflon block is turned to the right by 4 5 O . The cuvette is naturally closed tight, and the eluant stream is also stopped. By allowing the mixture to stand in the cuvette for an appropriate period in this state, derivatization is completed. Finally a t stage 4 the block is turned to the right by another 45'. The eluant flows into the cuvette, and washes out the whole reaction mixture into the column. We present a typical example of application of this device. Although the shape and the capacity of the derivatization cuvette may be varied, a 7.5-pL crescent was used in this experiment. The efficiency of sample-reagent mixing was excellent, giving reproducible results, provided that sample volume was less than 1pL. Larger volumes resulted in loss
2468
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
all diamines. The internal standard method taking 1,7-diamino-n-heptane as the standard gave a slightly higher upper limit (500 pmol). The precision data in Table I indicate that reproducibility of this determination was reasonable for practical analysis of biological samples, though coefficient of variation was rather high at the lowest limit of the linearity range in the absolute method. The internal standard method showed higher reproducibility than the absolute method a t all sample levels examined. Application of the established method to the analysis of urinary diamines indicated that a sample scale as small as 6 MLwas sufficient. Large peaks of putrescine (1,4-diamino-n-butane) and cadaverine (1,5-diamino-n-pentane) were observed in the chromatogram of a urine sample from a patient with gastric cancer, whereas no peaks of diamines were detected for a sample from a normal subject.
Table I. Reproducibility of the Determination of w ,w '-Diamino-n-alkanes
coefficient of variation (%)" 10 pmol
50 pmol
250 pmol
Absolute Method putrescine (n = 4) 8.8 cadaverine ( n = 5) 7.2 1,6-diaminohexane (n = 6) 7.5 1,7-diaminoheptane ( n = 7) 6.9
6.7 6.4 6.2 5.9
5.8 4.7 5.4 4.8
Internal Standard Method putrescine ( n = 4) 6.4 cadaverine ( n = 5) 6.2 1,6-diaminohexane ( n = 6 ) 5.8
5.2 4.7 5.1
4.1 4.0 3.9
The number of determinations was 10 in all cases. 1,7-Diaminoheptane was used as the internal standard.
a
ACKNOWLEDGMENT
of sample due to overflow. Samples of w,w'-diamino-n-alkanes were reacted with fluorescamine and the fluorescent derivatives were separated by high-performance liquid chromatography, under the conditions obtained by optimization studies. Under these conditions picomole amounts of the C4-C7 diamines could be analyzed in ca. 40 min, including precolumn labeling process, as seen from Figure 5. The lower limit of detection was ca. 1 pmol for a signal to noise ratio of 2. Calibration curves obtained by the absolute method gave good linearity for sample amounts ranging from 5 to 250 pmol for
We thank K. Hishikawa of Gasukuro Kogyo for his skillful assistance in preparing the precolumn apparatus. Registry No. Putrescine, 110-60-1; cadaverine, 462-94-2; 1,6-diamino-n-hexane,124-09-4;1,7-diamino-n-heptane,646-19-5; fluorescamine, 38183-12-9.
LITERATURE CITED (1) Gfeller, J. C.; Huen, J. M.; Thevenin, J. P. J . Chrometogr. 1078, 766, 133-140.
RECEIVED for review June 27,1983. 1983.
CORRECTIONS Liquid Chromatography/Proton Nuclear Magnetic Resonance Spectrometry Average Composition Analysis of Fuels James F. Haw, T. E. Glass, and H. C. Dorn (Anal. Chem. 1983, 55, 22-29). There are unfortunate errors in eq 24 and 26, appearing on p 27. The correct equations should read
Na = HaCH3 - 3HaCH - [Fq~at(~~CH,)l (24)
MWa =
6
+
+
1 5 * C " c ~ ~ 14*caCH2 13*caCH
3
+ 12Fquat(*CaCH3) (26)
Computer-Assisted Optimization for Flow Injection Analysis of Isoprenaline D. Betteridge, Timothy J. Sly, Adrian P. Wade, and John E. W. Tillman (Anal. Chem. 1983,55,1292-1299). There is an unfortunate error in citation of ref 22, under "Literature Cited" appearing on page 1299. The correct citation should read Stieg, s.;Nieman, T. A. Anal. Chem. 1980, 52, 800-804.
Accepted September 6,
