EO = +1.52 volts (1) 7H20 Cr20,2- 14H’ EO = +1.33 volts (2) 2e == Mn2+ 2Hz0 MnOp 4H’ Eo = +123 volts (3) I+(Cl-) e == %I2 C1- Eo = +1.19 volts@) (4) Fe3+ e e Fez+ Eo +0.77 volts (5) Thus, there is no problem of any interference from Mn2+
+
+ 6e
+
== Zr3+ +
+
+
+
+
+
5
or C r s which are in general present in rocks in small amounts (Mn from 0.05 to 0.2%, Cr from 1to 4000 ppm). The presence of an appreciable amount of “acid decomposable sulfide” invalidates the ferrous iron determination. Pyrite is not appreciably attacked by mixture of HF and HC1 but other sulfides, such as pyrrhotite, are more extensively decomposed liberating hydrogen sulfide which
will result in higher values of ferrous iron. Organic matter other than graphite will completely invalidate the meth-
od. The relative 70 deviation has been calculated on the amounts present.
CONCLUSIONS Ferrous iron can be determined by the iodine monochloride method without any possible aerial oxidation and without any interference from manganese or chromium. Ferrous iron in carbonate and other acid decomposable rocks (which are attacked by HCl or HCl and HF) can also be determined. Acid decomposable sulfides and organic matter other than graphite invalidate the method. Received for review October 6, 1973. Accepted January 7, 1974.
Automated Loading of Discrete, Microliter Volumes of Liquids into a Miniature Fast Analyzer C . A. Burtis, W. F. Johnson, and J. B. Overton Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
A miniature Fast Analyzer is under development at the
Oak Ridge National Laboratory (1-4). The analyzer combines the inherent advantages of the Anderson fast analyzer concept (5) with those of miniaturization. One of the advantages of the latter is a further decrease in sample and reagent volume requirements. For example, for each assay, the miniature analyzer requires only from 2 to 10 ~1 of sample, which is analyzed in a total reaction volume of only 120 to 130 rl. Consequently, to fully realize the advantages of the analyzer, it is necessary to have the capability of precisely and accurately introducing volumes of this magnitude into the system. Samples and reagents are introduced into the miniature Fast Analyzer via a 17-cuvet rotor into which aliquots of sample(s) and reagent(s) are loaded, transferred, and mixed (within their respective cuvets). The ensuing reactions are then photometrically monitored. One of the two modes in which the aliquots of sample(s) and reagent(s) can be introduced into the rotor is a discrete mode ( 3 ) , in which a dispensing device is used to obtain aliquots of the liquids and dispense them into their respective sample and reagent cavities in the rotor. Using two commercially available, automatic pipets and a unique carousel-tumtable assembly, a sample-reagent loader which automatically performs this discrete loading operation has been designed and fabricated.
EXPERIMENTAL Instrument Description. The sample-reagent loader (Figure 1) sequentially and automatically obtains and dispenses aliquots of reagent(s) and sample(s) into the corresponding cavities in the (1) N. G. Anderson. C. A. Burtis. J. C. Mailen. C . D. Scott, and D. D. Willis, Anal. Lett. 5, 153 (1972). (2) C . A . Burtis, J . C. Mailen, W . F. Johnson, C. D. Scott, T . 0. Tiffany, and N. G.Anderson, Clin. Chem., 18,753 (1972). (3)C D . Scott and C . A . Burtis, Anal. Chem. 45,327A (1973). (4)C . A. Burtis, W . F. Johnson, J . C. Mailen, J . 8.Overton, T. 0.Tiffany, and M. B. Watsky, Clin. Chem. 19,895 (1973). (5) N. G.Anderson, Amer. J . Clin. Pathol., 53, 778 (1970).
786
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 6, M A Y 1974
rotor. Primary components of the loader are two Micromedic automatic pipets (Model 25004, Micromedic Systems, Inc., Philadelphia, Pa.), which obtain and deliver aliquots of reagent and sample respectively. Both of these pipets are operated in a sample-diluting mode, which entails aspirating an accurately measured volume of sample or reagent into a small-diameter, flexible delivery tube and then dispensing and following it with a preset quantity of diluent (usually distilled water). As shown in the flow schematic in Figure 2, the loader utilizes one of the pipets to obtain aliquots of sample and reagent and the other to dispense and dilute the sample and reagent aliquots into their respective cavities in the rotor. This unique arrangement of pipets, pumps, and delivery lines is an important factor in minimizing the carryover of the loading system and will be discussed later in greater detail. The volumes aspirated and dispensed with these pipets are set by adjusting index counters on the control panel of the pipets. This adjustment allows the volumes to be varied from 5 to 100% of full pump capacity. To give a selectable volume range within that required by the miniature Fast Analyzer, 20-4 sample and reagent pumps and 50-pl diluent pumps have been utilized. Typically, the pumps of the pipets are set to obtain and deliver from 2 to 10 p1 of sample followed by 50 p1 of diluent, and 20 pl of concentrated reagent followed by 50 pl of diluent. To automate the sample- and reagent-loading process, a unique turntable and carousel assembly was developed for use with the Micromedic pipets. In an earlier evaluation, it was found that, during work with microliter volumes, maximum performance was obtained from the Micromedic pipet when its probe was kept stationary. Consequently, during routine operation of the automated sample-reagent loader, the sample probe remains stationary and the reagent probe moves only slightly, while the sample and reagent cups and the respective receiving cavities in the rotors are sequentially brought to the probes. Thus in a typical loading operation, (a) the sample and reagent cups are positioned and aligned below the sample and reagent probes; (b) the turntable is raised, the probes enter the liquids in the cups, and aliquots of sample and reagent are aspirated into the probes; (c) the turntable is lowered, thereby removing the probes, and then moved laterally to position and align the sample and reagent cavities of the rotor under the probes; (d) the turntable is raised, and the probes enter the rotor cavities, where the aliquots are dispensed and diluted with a preset quantity of diluent; (e) the turntable is lowered, thereby removing the probes, and then moved laterally to position and align the two cups of the wash station under the
...
Figure 2. Flow Schematic of t h e pipets and delivery lines of the
automated sample-reagent loader
Figure 1. Automated sample-reagent loader developed for use with the miniature Fast Analyzer probes; and (0 the turntable is again raised, the pmhes enter the wash cups where liquids adhering to the external surface of the pmhes are rinsed into the wash cups, the diluting pipet is activated, and a second aliquot of diluent is dispensed through each of the probes into the wash cups (Note: This second cleansing of the probes with diluent is an important step in a Loading operation since it minimizes carryover due to material adhering to the internal walls of the probes). The turntable is then lowered, which necessitates removal of the probes, and rotated and indexed one position. Subsequently, it is moved laterally to position and align the next sample and reagent cups under the probes. Finally, the process is repeated until the rotor is completely loaded, which requires 5 min for a 17-cuvet rotor. The turntable and carousel assembly of the automated samplereagent loader was also designed far operational flexibility and versatility. This was accomplished hy designing the turntable to accept either or both of two concentric carousels that contain compartments for individual cups of samples or reagents. These carousels rotate with the turntable, allowing single aliquots fmm the cups to be obtained and dispensed as the turntable is mtated through each of its loading positions. In addition, either or both of the carousels may be replaced with a cup holder that remains stationary as the turntable rotates through each of its loading positions. Thus, a series of repetitive aliquats of samples and/or reagent can he loaded. Consequently, by selecting any pair of the carousels or stationary cup holders, the loader can be operated in one of the following four loading modes: (a) test, (h) multiplesamples:single-reagent, (c) single-sample:multiple-reagents, or (d) multiple-ssmp1es:multiple-reagents.
Table I. Gravimetric Evalution of the Micromedic A u t o m a t i c Pipets Used in the A u t o m a t e d S a m p l e R e a g e n t Loader
Evaluation Procedure. To determine the precision and accuracy of the automated sample-reagent loader, both gravimetric and photometric techniques were used. In the gravimetric studies, ten individual aliquots of distilled water were dispensed at each of the selected volume settings. These aliquots were dispensed into preweighed vials and weighed. Assuming a density of 1.0 gram/ml, the measured weights of the individual aliquots are also equal to their volumes. A Mettler Microbalance (Type M5,Mettler Instrument Corporation, Hightatom, N.J.) having a specified precision of zk1 pg was used for these measurements. In the photometric experiments, various volumes of a concentrated solution (7.4 mg/ml) of Blue Dextran (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) were diluted to a final volume of 0.130 ml. The absorbances of these diluted solutions were measured at 620 nm using the computer-interfaced miniature Fast Analyzer. The operation of this analytical system has been previously described ( 4 ) . To evaluate the loader's overall performance in loading reagents and samples for routine clinical analyses, a series of clinical analyses was performed. The clinical procedures and their analysis with the mi niature analytical system have been previously described (4).
RESUI,TS AND DISCUSSION Precision and Accuracy. .. ... As shown .. by. the. data . . .sum, . marized in Table I, m e Mlcromealc aucomacic pipers 01 the automated sample-reagent loader were gravimetrically evaluated at the volume settings at which the instmment would he routinely operated. In regard to accuracy, the volumetric delivery of the pipets was found to be within 1%of the indicated pump setting. The precision of the delivery procedure was found to be between *0.02 and 0.10%, depending on the volume dispensed. In a series of repetitive experiments, various volumes of Blue Dextran solution were each diluted to a final volume of 0.130 ml and their absorbances measured at 620 nm. Results of these experiments indicated that the delivered volumes of the sample and reagent pumps were linear 1Pable 11. Precision of the A u t o m a t e d Sample. Reagent Loader at Various Sample Volumes
volume aelffted, "1-
ReSUlW DWi*tiW fmm
Diluting pipet Samp7ingpipet
"OlUme,
~
ReaSample
gent
0 10 0 0
0 0 0 20
ReaSample gent diluent diluent
50 50 0 0
.
0 0 50 50
M
e
d volume, g1
=peaed
Mean*
RSD; %
"olumep % '
49.51 59.53 49.63 69.56
0.02 0.02 0.08 0.10
-1.0 -0.8 -0.7 -0.6
Sample = 20 d;reagent = 20 A sample diluent = 50 pl; "^
&>-.--
.I I "
e Den
a
d
n
1 2 5 10 15 20
I.6
Six-
I..fi I.6 I.6 lti 16 ~
Marbanma
0.0324
sigma
0 ,0027
n ~nfiafi
n ,0024
0.1611 0.3282 -
0 .0018 0 ,0025 u ,0016 0.0023
u .41(4Y
0 .I3433
-
RSD,
9%"
3.48 2.67 0.50 0.58 0.41 0.25
replicate diquats of a concentrated Blue Dextran solution (7.4
m -
(standard deviation)/(mean) X 100. d Deviation, % = (measured volume)/ (sum of selected volume) x 100. sample volume 59.53 - 49.51 = 10.02 ,,I. b m m t = 69.56 - 49.63 = 19.93 ,,I. Total reaction volume = 59.53 69.56 = 129.1 "1.
+
Sample
to a &la1 volume of 0.130 mL The abso?bancea of the diluted solutions were then m e a d at 620 m using the miniature Fsst Analyzer. Mean absorbance of 16 measuremente. RSD % = relative standard deviation = (standard deviation)l(m-) X 100.
'
ANALYTiCAL CHEMISTRY, VOL. 46. NO. 6. MAY 1974 * 787
0.7
I
I
Sample Pump
I
I
I
I
R e a g e n t Pump
I
I
Table 111. Analytical Results Obtained from the Miniature Fast Analyzer System in Which the Automated Sample-Reagent Loader Was Used To Load the Rotors Sample volume,
rl
&Y
Enzymes, I.U. 1.-1 min-1, 30 "C ALP 10 ALT 10 AST 10
9 4 O0 23 I
I
/
--0
i 4
8
12
IS
20
Volume ( p l )
Volumetric linearity of the sample and reagent pumps used in the automated sample-reagent loader Figure 3.
over the 1- to 20-pl voumetric range of the pumps (Figure 3) and that precision ranged from 3Z3.5% at 1 pl to 0.25% at 20 p1 (Table 11). It should be noted that these data reflect the precision and accuracy not only of the automated sample-reagent loader but of the entire system, and compare favorably with previously published data (6-8) for the larger Fast Analyzer. Carryover. In discrete sampling systems, such as the automated sample-reagent loader, intercompartmental carryover is a potential problem. To determine the extent of carryover in the automated sample-reagent loader, the sample and reagent pumps were set to obtain 10- and 2 0 4 aliquots, respectively. Both of the diluent pumps were set to dispense and dilute these sample and reagent volumes with 50 p1 of diluent. In one experiment, the turntable was set in the multiple-samp1e:single-chemistry mode and distilled water was placed in the cup of the stationary reagent holder. Cups containing concentrated dye solution were placed in compartments 2, 7, and 12 of the sample carousel. Cups containing distilled water were placed in compartments 1, 3-6, 8-11, and 13-17 of the sample carousel. A rotor was loaded, placed in the analyzer, and its contents were transferred and mixed; then the absorbance of each of the resulting solutions was measured at 620 nm. Carryover would be observed by an increase in absorbance, primarily in cuvets 3, 8, and 13. Under the conditions used in this experiment, carryover was found to be less than 0.1%. In a second experiment, the turntable was set for the single-samp1e:multiplechemistry loading mode, and the reverse of the first experiment was performed. The reagent carryover was approximately 0.2% under these conditions. Performance under Analytical Conditions. To test the automated sample-reagent loader under conditions associated with an analytical environment, 16 replicate aliquots of a single serum sample were assayed for seven blood constituents by previously reported ( 4 ) procedures. The results from these analyses are summarized in Table 111. Excellent precision was obtained. The relative standard deviation ranged from only 0.4% for the CK assay to 3.2% for the AST assay. The data from the glucose and triglyceride analyses were quite impressive when one considers that the volume of sample used in each assay was only 2 (6)
c . A.
Burtis. W. F . Johnson, J. E. P.ttril1, C . D. Scott, N. Cho, and N. G . Anderson, Clin. Chern. 17,686 (1971) (7) C. A. Burtis, W. F. Johnson, J. C Mailen, and J . E. Attrill, Clin. Chern., 18, 433 (1972). (8) T. 0 Tiffany, G. F. Johnson, and M . E Chilcote, Clin. Chern., 17,
715 (1971). 788
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, MAY 1974
CK LD-L Substrates, mg/dl Glucose Triglyceride
Analytical results
RSD, Meanb
10 10
44.6 12.3 17.1 87.7 66.7
1.8 2.9 3.2 0.4 1.6
2 2
86.7 141.7
0.9 1.7
a Abbreviations used: ALP, alkaline phosphatase; ALT, serum glutamic pyruvic transaminase; AST, serum glutamic oxaloacetic transaminase; CK, creatinine phosphokinase; LD-L, lactic dehydrogenase-lactate substrate. = relative standard deviation = Mean activity of 16 observations. RSD, (standard deviation)/(mean) X 100.
pl. In comparison to previously published data (7, 8), these results are equal to or better than those reported for the larger Fast Analyzers. In order to evaluate the reproducibility of the analytical system on a run-to-run basis, a serum sample was repetitively assayed for its alkaline phosphatase and serum glutamic oxaloacetic transaminase (AST) activity. Consequently, seven rotors of samples were assayed for each of the two enzymes, resulting in 112 analyses for each. As shown in Table IV, the analytical precision of the system was quite good. The within-run variation for the alkaline phosphatase analyses averaged *0.75% and increased to only 3~1.15%for the run-to-run variation. At a lower enzyme activity, as indicated by the AST data, the withinrun and run-to-run variations were h5.30 and *6.88%, respectively. However, even at this enzyme activity, where the optical noise of the system becomes the limiting precision factor, the observed precision is adequate; in fact, it compares quite favorably with results obtained with the larger Fast Analyzers. CONCLUSIONS The automated sample-reagent loader has been shown to fulfill six design criteria: (a) by using 20- and 50-pl sampling and diluent pumps, its range of dispensed volumes is within the volumetric requirements of the miniature Fast Analyzer; (b) it has been demonstrated to autoTable IV. Analytical Precision of the Miniature Fast Analyzer System in Which the Automated Sample-Reagent Loader Is Used to Load the Rotors Average withinrun variationa Enzyme
Alkaline phosphatased ASTe
Run-to-run variationb
Mean activityc
RSD
Mean activityC
RSD
43.43
0.75
43.43
1.51
10.93
5.30
10.93
6.88
a Sixteen replicate aliquota were assayed during a single run, and the resulting data were statistically analyzed. One hundred twelve replicate aliquota were assayed over a 8even-run period, and the resulting data were statistically analyzed. CEnzyme activity is expressed as I.U. I.-' rnin- 'at 30 "C. Reaction conditions: wavelength, 400 nm; sample volume, 10 sl; total volume, 130 total run time, 200 sec. e Reaction conditions: wavelength, 340 nm; sample volume, 10 pl; total volume, 130 d ; total run time, 400 sec.
*
matically load microliter aliquots of sample and reagent precisely and accurately; (c) both internal and external probe carryover has been minimized in its operation; (d) since only 5 min is required to completely load a 17-cuvet rotor, the loading time per rotor is not the rate-limiting factor in determining the analytical throughput of the system; (e) the loader is quite versatile and can be used in either of four loading modes; and (f) the loader has only a few control switches, thus is very simple to operate. An additional advantage of the loader is that it allows for rapid changeover when proceeding from one type of clinical analysis to another. Since both the sample and the reagent are loaded by operating the pipets of the loader in a sample-diluting mode, the aliquots of sample and reagent enter only a short section of the terminal tips of their probes. Thus, the pump chambers and most of the delivery lines contain only diluent (i.e., usually distilled water) during operation; hence, it is not necessary to flush and reprime them before loading new solutions. To introduce new solutions (either reagent or sample, depending on which loading mode has been chosen), one merely changes cups in the carousel or stationary cup holder. The results obtained in the initial evaluation of the automated sample-reagent loader were quite encouraging since they demonstrate that small, microliter volumes of samples and reagent can be quickly and automatically loaded into a rotor. The precision and accuracy of the loading operation are better than or equal to data pub-
lished for a loading system used in the larger Fast Analyzers (6). A question that remains to be answered concerns the operational and mechanical reliability of the automated sample-reagent loader under routine operating conditions. This information will soon be available as analytical systems, each of which contains an automated samplereagent loader, are currently being evaluated in the clinical laboratories of the Health Division of the Oak Ridge National Laboratory, Oak Ridge, Tennessee, and a t the NASA Johnson Space Center, Houston, Texas.
ACKNOWLEDGMENT The indispensable assistance of R. A. Mathis, W. A. Walker, and C. M. Benge in designing, fabricating, and assembling the system is greatly appreciated. The technical advice and consultation provided by c. D. Scott, J. c. Mailen, and T. 0. Tiffany are also gratefully acknowledged. Received for review September 27, 1973. Accepted January 11, 1974. This work is a part of the Molecular Anatomy (MAN) Program supported by the National Institute of General Medical Sciences, the National Aeronautics and Space Administration, and the U S . Atomic Energy Commission. Oak Ridge National Laboratory is operated by Union Carbide Corporation, Nuclear Division, for the U S . Atomic Energy Commission.
Spectrophotometric Determination of Picein and p-Hydroxyacetophenone in Needles of Picea abies with 2,4=Dinitrophenylhydrazine Hermann Esterbauer, Dieter Grill, and Gerhard Beck lnstitut fur Biochem~eund lnstitut fur Anatomie and Physiologie der Pflanzen, Universitzet Graz, A-8070 Granz, Austria
Picein (p-hydroxyacetophenone D-glucoside) is the major component of the phenolic compounds in the needles of spruce (Picea abies (L.) Karsten, Pinaceae). Picein and/or p-hydroxyacetophenone is also found in Salix spp. (Salicaceae), Amelanchier oualis (Rosaceae), and in Fabiana inbricata (Solanaceae) ( I , 2 ) . Only a few reports are available on the biosynthesis, metabolism, and function of picein in plants (3, 4 ) . We have reported previously that SOz-containing emissions affect phenolic compounds in spruce needles, most probably' by alteration of the ratio of picein to p-hydroxyacetophenone ( 5 ) . This finding led us to investigate in detail the factors influencing acetophenones in spruce needles and to work out a method for their determination. The methods described in the literature for the picein determination are time consuming and have the disadvantage of low specificity and sensitivity. Usually picein is isolated by paper chromatography and estimated enzymaKarrer, "Konstitution und Vorkommen der organischen Pflanzenstoffe,"Birkhauser Verlag, Basel, 1958, p 177. J. B. Harborne. "Biochemistry of Phenolic Compounds," Academic Press, London-New Y o r k , 1964, p 328. P. Dittrich, Thesis, University of Munchen, 1970. P. Dittrich and 0. Kandler. Ber. Deut. Bot. Ges., 84, 465 (1971). D. Grill. Int. J Environ. A n a / . Chern.. 1, 293 (1972).
(1) W. (2) (3) (4) (5)
tically with /?-glucosidase (3, 6) or colorimetrically with Millon's reagent (7,8). In the method described here, picein and p-hydroxyacetophenone are converted to the 2,4-dinitrophenylhydrazones, isolated by thin-layer chromatography, and determined spectrophotometrically in solution. The proposed procedure allows a specific, accurate, and rapid determination of picein and p-hydroxyacetophenone in plant materials.
EXPERIMENTAL Apparatus Absorbance measurements were made with a Zeiss PMQ I1 Spectrophotometer using 1-cm quartz cells. Homogenization of plant material was done with an Ultra Turrax (Janke u. Kunkel KG). Reagents. 2,4-Dinitrophenylhydrazine (DNPH) reagent A: 1 gram DNPH (Fluka) was dissolved in 7.5 ml concentrated sulfuric acid; to this solution 75 ml ethanol-methanol 95:5 ( u : u ) was added slowly under cooling. 2.4-Dinitrophenylhydrazine reagent B: 1.2 grams DNPH was dissolved in 50 ml30% HC104. Materials. Picein (p-hydroxyacetophenone 6-D-glucopyranoside) and p-hydroxyacetophenone were obtained from the Koch(6) E. Eourquelot, C. R. Acad. So., 171, 423 (1920) (7) H. Thieme. Pharrnazie, 19, 535 (1964). (8) H. Thieme, Pharrnazie, 19, 471 (1964). A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 6, M A Y 1974
789
