In summary, some generalizations about which acid medium to use in a given application can be made. Reaction rates are markedly slower in HzS04 than in the other acidic media, and the equilibrium is least favorable using HzS04. Thus the choice of acids for 12-MPA procedures should be between HClO4 and "03. We have found the detection limits and linearity of reaction-rate measuremerits to be the same for both acids when optimum conditions are employed. Hence either acid may be employed under the conditions of Table I11 with equal success.
LITERATURE CITED (1) E. S.Meehan In "Treatise on A ~ l y t l c eChemistry", l I. M. Kolthoff and P. J. Elvlng, Ed., Part I, Vol. 5, John Wiley and Sons, Inc., New York, N.Y.,
1964,pp 2753-2803.
(2)S.R. Crouch and H. V. Malrnstadt, Anal. Chem., 39, 1090 (1967). (3)A. c. Javler, s. R. Crouch, and H. V. Melrnstadt, Anal. Chem., 41, 239 (1969). (4)s.R. Crouch and H. v, Malrnstadt, AM/. Chem., 39, io84 (1967). (5) H. D. Goldman and L. 0. brgls, Anal. Chem., 41, 490 (1969). (6) A. C. Javler, S.R. Crouch, and H. V. Melrnstadt, Anal. Chem., 40, 1922 (1968). (7)J. L. Dye and V. A. Nicely, J. Chem. Educ., 48,443 (1971). (8) J . L. Dye, Mlchlgen State UnlVerSlty, 1974,personal communication. (9)P. Souchay, Pure Appl. Chem., 6, 61 (1963). (IO) L. Krurnenacker and c. H e k , BU~I,SOC. chlm. Fr., 365 (1971). (1 1) S.R. Crouch, Unhrersity of Illlnolr, 1966,unpublished observatlons. (12)G. C. Dehne and M. 0. Mellon, Anal. Chem., 35, 1382 (1963). (13)J. C. Guyon and L. J. Cline, Anal. Chem., 37, 1778 (1965).
RECEIVEDfor review March 4, 1974. Resubmitted January Accepted June 27, 1975' This work was 201 in part by National Science Foundation Grant GP 18123
Development of an Assay Rate Multiplier for Automatic Analyzer Systems L. F. Cullen, Arthur Schlelfer, M. P. Brindle, and G. J. Paparlello Analytical and Physical Chemistry Section, Wyeth Laboratories, Inc., P.O. Box 8299, Philadelphia, Pa. 19 10 1
The Inherent assay rate llmltatlons In the conventlanal automatlc analyzer system are related to the dual functlon which Is served by the solid sampler's homogenlzatlon vessel: vlr., sample homogenization and sample asplratlon operatlons. To overcome these restrlctlons, a module described as an assay rate multlpller (ARM) has been designed whlch can be Interfaced Into any exlstlng automatlc analyzer system. With thls unit, the sample asplratlon time Is reduced to an extremely rapld 6-sec portlon of the sampler's programmed cycle perlod, completely Independent of the sample assay rate. In exempllfylng the analytlcal utility of thls concept, the unit was evaluated In established ultraviolet spectrophotometric, colorimetric, and fiuorometrlc automated procedures used In the pharmaceutlcal Industry. The ARM produced a twofold Increase In hourly sample rate In all systems examined wlth no loss In method sensltlvlty, accuracy, preclslon, or flow system characterlstlcs. For ex, ample, lorarepam standard solutlons were analyzed at a 60 sample per hour assay rate wlth a method preclslon of f0.4% at the 2-mg lorazepam level. The recording curves demonstrate sample peaks at greater than 99% of steadystate condltlons with a sample-to-sample lnteractlon of less than 0.1%.
The widespread use of automatic analyzer systems in the modern analytical pharmaceutical laboratory testifies to the accuracy, precision, and versatility of this instrumentation (1-5). Automatic preparation and introduction of solid materials directly into the automatic analyzer system is essential, however, if maximum utilization of automation is to be achieved. Holl and Walton (6) first described the development of such a device compatible with continuousflow analysis techniques. Subsequently, the Technicon Instruments Corporation introduced a commercial unit described as a solid sampler (7) which has been modified and is now available as a SOLIDprep Sampler I1 (8). Th'is sampling device has been accepted almost universally with its application extended to encompass a wide spectrum of current analytical techniques. 1938
*
The inherent rate limitations in the conventional automatic analyzer system are related to the dual function which is served by SOLIDprep unit's homogenization vessel: viz., sample homogenization and sample aspiration operations. The sample aspiration operation refers to the presentation of an aliquot of the homogenized sample solution or suspension to the automated manifold. During the homogenization stage of the operating cycle, the vessel performs ita primary function of sample preparation where complete dissolution of the active ingredient is achieved. Throughout this period, however, the analytical system is essentially in a standby state since the system receives only reagent blank stream which yields no analytical information. During the sample aspiration period, the homogenization vessel is further employed as the sample reservoir for the analytical system and is not able to perform its primary function of sample preparation. Multiplexing two SOLIDprep units into a single analytical system was considered and would offer certain advantages in specific applications. However, as a general laboratory technique, the two-sampler concept would be both expensive and complex from a standpoint of manifold flow control and electronics required for instrument synchronization. Clearly, a need existed for the development of a versatile device to separate the sample homogenization and sample aspiration functions of the solid sampler. By this means, it is possible to operate both the sampler and the analytical system at maximum efficiency. A unit described as an assay rate multiplier (ARM) has been designed to overcome these restrictions by reducing the time-consuming sample aspiration stage in the solid sampler cycle period to only 6 sec at any assay rate. This module utilizes programmed micro-solenoid valve control to rapidly withdraw sample from the homogenizer vessel into a storage tank, permitting the sampler to concurrently disrupt the next sample while the previously prepared sample is released a t a desired rate from the storage tank to the analytical system. The ARM has been interfaced into established ultraviolet spectrophotometric, colorimetric, and fluorometric automatic analyzer techniques and has pro-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
duced at least a twofold increase in assay rate in all cases. In the fabrication of this automatic analyzer system module, design considerations related to continuous-flow analysis fluid mechanics, viz., trace characteristics of sample curve, sample-to-sample interaction, and percentage of steady-state condition reached, were considered and optimized. EXPERIMENTAL Assay R a t e Multiplier (ARM) Construction. A detailed schematic diagram of the storage tank system of the ARM, designed to control the flow of homogenized sample into and out of the storage tanks, is illustrated in Figure 1. The unit was fabricated with 10 small internal volume 12-V dc Teflon body solenoid valves, S-1 t o S-10, (Angar Scientific Corp., No. 190-12-vacuum) and two glassblown 50-ml capacity sample storage tanks prepared to order (A. A. Pesce Co.) fitted with 1.5-mm i.d. Teflon tubing. Teflon tubing (2.4-mm i.d. X 3.2-mm 0.d.) was employed from the homogenization vessel to fitting T-1,and to inlet ports and from drain ports on Tanks A and B. The small internal volume, chemically inert Teflon Tee fittings (Angar Scientific Corp., No. 603), T-1 to T-3, and Kel-F ,Tee fittings, T-4 to T-7, (Altex Scientific Inc., No. 200-23) and solenoid valves are interconnected with 1.5-mm i.d. Teflon tubing. All Teflon line to glass connections on storage tanks were sleeved with 2.8-mm i.d. silicone tubing (Technicon Instruments Corp., No. 116-0497-20). Apparatus. A standard automatic analyzer system (Technicon Corp., AutoAnalyzer) was employed consisting of the following modules: (a) SOLIDprep Sampler 11; (b) proportioning pump, Model 111; (c) continuous filter; (d) fluorometer, Model 11; and (e) 25.4-cm (10-inch) linear-log potentiometric strip-chart recorder. Colorimetric and ultraviolet spectrophotometric measurements were made with a spectrophotometer (Beckman Instruments, Inc., Model 25) equipped with a 1.0-cm micro-aperture flow cell (Beckman, No. 886723-X). Interfacing t h e Assay R a t e Multiplier into Analytical System. A diagram demonstrating the interfacing of the ARM into the analytical system is presented in Figure 2. The unit is simply inserted into any conventional automatic analyzer system between the SOLIDprep unit and proportioning pump by connecting Teflon Tee, T-1, to the homogenization vessel with transmission tubing. The operation of the automated system depends upon precise synchronization of the ARM and the SOLIDprep unit with the flowing sample stream. T o eliminate the possibility of synchronization-drift problems from the use of separate timing mechanisms, the required synchronization was accomplished through electrical interconnection of the multiplier to the programming mechanism of the SOLIDprep unit. As illustrated in Figure 2, a 28-V dc auxiliary output terminal, TC-3, on the solid sampler is utilized to sequence the assay rate multiplier controller. This controller automatically coordinates the actions of the 10 solenoid valves on the ARM, S-1 to S-10 (Figure 11, during the 12-step operation cycle of the unit. A schematic wiring diagram of the controlling device is presented in Figure 3. The 12-contact rotary switch is driven by a stepper motor, SMS-1, whose activation is programmed into the operation on the auxiliary-2 track of the SOLIDprep unit's seven-track photo-sensor progranimer system. The 12 contacts on the rotary switch correspond to 12 open-closed configurations for each of the 10 solenoid valves. T3 open a solenoid valve a t a particular position in the operation cycle, the corresponding circuit is completed with the appropriate switch. Figure 3 demonstrates the circuitry for one solenoid valve, S-1. The wiring harnesses for the remaining 9 solenoid valves, S-2 to S-10, are identical and in parallel with the solenoid valve, S-1, a t the connector, C-1. Light emitting diodes have been introduced to provide a convenient means of monitoring individual solenoid operation in each position of the 12-step cycle. As is the case with the conventional analytical system, reagent blank stream is introduced into the automated manifold with the SOLIDprep unit wash distribution solenoid valve, S-11 (Figure 2). In the conventional system, this valve satisfies the hydraulic requirements of the ccintinuous-flow system by releasing reagent blank stream to the manifold during the homogenization period. With the insertion of the ARM, modifications in the operation of the wash distribution valve were required. The 28-V dc signal available a t the auxiliary-1 output terminal, TC-2, on the SOLIDprep unit is employed to activate a 26.5-V dc relay switch, RS-1 (Allied Control Co., [nc., No. BOHRO-6D) which energizes the
i'
i\+
DISCARD
t
IPW SOLlOprw
WASH
DIPIRIBUIION
h NPORT
SOLLMiD V A L Y l
TO AN&LITICAL SVSTiM
Figure 1.
Storage tank system of the ARM
(1) 2.4-mm. i.d. Teflon transmission lines; (2) 1.5-mm, 1.d. Teflon transmlsslon lines
wash solenoid valve, S-11. In the non-sampling mode, the relay switch is inactivated and wash diluent is aspirated into the system a t T-7. The operation is controlled by the auxiliary-1 track on the programmer disc. In reducing the SOLIDprep unit wash cycle to a completely adequate 12-sec period, a cup control device was fabricated to prevent the sample cup from dumping during sampler tray advance and draining of the homogenizer vessel. As shown in Figure 2, this device is activated by the 28-V dc signal available a t the tray advance output terminal, TC-1, on the SOLIDprep unit and is controlled by the tray advance track on the programmer disc. The SOLIDprep Sampler I1 programmer discs for the 30- and 60-sample per hour assay rates considered in this investigation are illustrated in Figure 4. The SOLIDprep unit is commercially supplied with a maximum programmer disc processing rate of 1.5-min per disc. T o effect a 60-sample pet hour assay rate, two complete sample operations are programmed on one disc which is scanned a t a rate of 2-min per disc. Assay R a t e Multiplier Operation. T h e operational sequence of the ARM is based on 12 fixed-time configurations as schematically represented in Figure 5. As previously mentioned, this unit permits the sampler to disrupt the next sample while the preceding sample is released from one of two storage tanks t o the analytical system. Operationally, Position 1 is programmed to coincide with the SOLIDprep unit 12-sec wash cycle and is used to perform two rinsings of Tank B. Tank A contains homogenized sample material obtained subsequent (Positions 10 to 12) to the Position 1tank wash operation. In Position 1, reagent blank stream is introduced into the automated manifold with the SOLIDprep unit wash distribution solenoid valve, S-11. Reagent blank stream is also directed through solenoid valves S-3 and S-8 and Kel-F Tee fittings, T-4 to T-7, to rinse any previous sample remaining and prevent any sample-to-sample carry-over. In Positions 2 and 3, Tank B is dried, then completely evacuated for purposes of removing residual wash solution and to provide an evacuated chamber as the means of rapidly aspirating homogenized sample material a t Position 4. During this homogenization period, Tank A is concurrently releasing sample to the automated manifold for analysis and continues to do such through Position 5. Following homogenization (Position 4) solenoid valve S-9 is opened permitting Tank B to rapidly withdraw sample from the homogenizer. T h e ARM is held in Position 4 for 1-2 sec with the drain solenoid valve, S-6, open to ensure complete discharge of residual wash solution if not removed a t Positions 2 and 3. With the closing of valve S-6 and opening of valve S-7 (Position 5 ) , Tank B begins to fill and valve S-7 is flushed with sample to prevent any sample-to-sample carry-over. At Position 6, Tank B completes the aspiration process with approximately 30-ml of homogenized sample placed in storage for use by the analytical system.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12. OCTOBER 1975
1937
*CUP DUMP CONTROL DNlCf
I
I
I
,I
/’--
i --
1
IO ANALYIICAL SYSTEM CONTINUOUS FILTER UNIT
Figure 2. interfacing of the ARM into an analytical system
(1, 4) 0.056-inch i.d. Silicone manifold tubing: (2)0.1 10-inch i.d. Silicone manifold tubing, sample: (3) 0.045-inch i.d., Tygon manifold tubing, air: (5)0.110-inch i.d. Tygon manifold tubing, air: TC-1, 28-V dc tray advance output terminal: TC-2, 28-V dc auxiliary-1 output terminal: TC-3, 28-V dc auxiliary-2 output terminal: RS-1, 26.5-V dc relay switch: S-11, SOLlDprep unit wash distribution solenoid valve; S-12, solenoid-284 ac. 400-ohm coil (Guardian Electronics Co.). A-1 and D-5, Technicon Corp. glass fittings
The filling operation or sample aspiration cycle (Positions 4 to 6) occurs in a 6-sec period. The contents of the homogenizing vessel, not used in the filling of Tank B, are drawn by vacuum to waste in the conventional manner. Homogenized sample in Tank A, not used by the analytical system, is also drawn by vacuum to waste during Position 6. Reagent blank stream is introduced into the manifold at Positions 6 and 7 to obtain system wash-out properties which demonstrated the absence of any measureable carry-over between samples. The constant air segmentation (Proportioning Tube 3) of the sample line a t h-connector, D-5, further assures sample integrity and optimum wash characteristics. Air injection (Proportioning Tube-5) a t T-connector, A-1, prevents a premature discharge of sample from the homogenizer into the sample line during the nonsampling mode. As is apparent from Positions 7 to 12 (Figure 5), Tank B is releasing sample to the analytical system while Tank A is being prepared to receive the sample undergoing homogenization. Thus, the fixed time stages in the operational sequence a t Positions 7 through 12 are equivalent to the respective configurations in the processing operation of the opposite storage tanks from Positions 1 through 6. The program for the 10 solenoid valves on the ARM, S-1 to S-10, and the SOLIDprep unit wash distribution solenoid valve, S-11,is presented in Figure 6. At any given assay rate, the times allotted to sample homogenization, homogenizer vessel wash and drain, and the various operations of the ARM may be modified to meet the individual requirements of a particular analysis procedure. These alterations can be simply performed by modifying the programmer disc (Figure 4).It has been determined, however, that tank evacuation periods (Positions 3 and 9) of a t least 8 sec are needed to draw sufficient homogenized sample into the storage tanks for subsequent analytical evaluation. Automated Methodologies. A detailed description of the norgestrel (9),penicillins (IO), digitoxin and digoxin (11) automatic analyzer procedures examined in this investigation are presented 1938
ANALYTICAL CHEMISTRY, VOL. 47,
NO. 12,
in the literature. Aside from the insertion of the ARM into the system and the exclusive use of the SOLIDprep Sampler 11, the respective experimental conditions were duplicated exactly as described. The flow diagram of the UV spectrophotometric system used in the analysis of lorazepam and oxazepam solid dosage formulations is shown in Figure 7 . In the UV procedure, samples are introduced into the SOLIDprep unit and dispersed in 60-ml of 85% SD No. 30 alcohol (ethanol-methanol, 101) in water (v/v). In performing the analyses, standards are placed on the sampler tray followed by samples of intact tablets or capsules. At the end of a series of 20 samples, additional standards are introduced into the system. Calculations are made using corresponding instrumental responses of standards and solid dosage formulation samples. Reagents. Reagent grade chemicals were used throughout this study. Working solutions were prepared as described by the respective methodologies examined.
RESULTS AND DISCUSSION Assay Rate Multiplier Design Considerations, In conventional analyzer systems, the available assay rate or minimum operating cycle duration is defined by the time assigned to the sample homogenization, sample aspiration, and homogenization vessel wash operations. This is programmed into the SOLIDprep sampler to accommodate individual assay demands. With the SOLIDprep Sampler 11’s highly efficient blender assembly, any increase in assay rapidity would center around reducing the times normally allotted to the sample aspiration and homogenizer wash periods. One is more restricted in varying the homogenization time because of the need to effect complete dissolution of the drug being analyzed. The SOLIDprep Sampler I1 is commercially available with a maximum sample preparation rate of 40 analyses
OCTOBER 1975
Flguro 3. Schematlc wlring dlagram of ARM controller
SMS-1, 12-posltlon stepper motor awltch (Potter and Brumfleld, Inc. No. GMlN-12A): PS-1, 12-V power supply (Power/Mate Corp. No. EM-l2/16C); R-1 to R-13, reslstora, 2200-ohm, %-watt: L-1 to L-13, 2.84 llght emlttlng diodes (Drake Manufacturlng Co. No. 6039-005-304); SW-1 to SW-12, switches (Alco Ebctronic Products, Inc. No. MTA-206N): D-1 to D-12, dlodes 200-mA (Motorola No. HEP-170); RS-1, 28-V dc relay awltch (Guardlen Electronlcs Co., No. Q-63947); S-1, 1 2 4 dc Teflon solenoid valve: C-1, contact polnt for 9 ldentlcal and parallel clrcults for solenold valves S-2 to S-10; TC-3, 2 8 4 dc auxlllary-2 output termlnel
TRACK NO, (OUTSIDE TO INSIDE) 1) HOMOGENIZATION 2) SAMPLE ASPlRATl 3) AUX 1 (WASH DISTRIBUTION VALVE CONTROL)
51 SOLIDPREP DlLUE
6) VESSEL DRAIN
Flguro 4. SOLiDprep Sampler Ii programmer discs for the 30/hr and 60/hr sample assay rates
per hour. At this assay rate, the homogenizer wash cycle is a fixed time function of 26 sec. Whereas 26 sec of total SOLIDprep cycle time can be dedicated to the non-productive sampler wash operation at assay rates of 40 samples per hour or less, this function represents an unacceptable 43% of the programmed cycle period at the 60 samples per hour assay rate. As indicated previously, a completely adequate 12-sec wash cycle was effected with the insertion of a cup dump control device (Figure 2) and the appropriate programmed reductions in the instrument's wash solution injectiondrain operations (Figure 4). The cup dump control device mechanically retains the sample cup in an upright position
during the rapid sequence of processing functions which result from the significantly reduced blender rinse period. It is apparent that modifications in the sampler's operation to substantially increase the hourly sample rate would center around the sample aspiration function. A simple reduction in the time allotted to this function would, of course, introduce the problem of not having sufficient sample processing time to satisfy the chemistry of the methodology and/or essential flow dynamics of the flow cell. The Technicon Instruments Corporation approached this problem with the introduction of a rapid sampling technique (8) which has been reported to satisfactorily increase assay rapidity (22). Utilizing solenoid valve control,
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
1039
Position 1
\/.::
Posltlon 2
. -
Position 3
\I=
HOMOGENIZATION Cl,r
n
v RESERVOIR
Position 4
Posltlon 7
Position 5
Position 6
Position 9
Position 8
\L
H v
H v Position 10
Position 11
Figure 5. The 12-fixed time configurationsof the ARM
\I==! -
Position 12
DISCARD
4
Flgure 7. Automated UV spectrophotometric procedure flow diagram
this technique rapidly withdraws sample from the homogenizer vessel, following homogenization, into a glass-holding coil. With the sample rapidly withdrawn from the homogenization vessel, the SOLIDprep unit can proceed to the rinsing phase, and subsequently to the preparation of the next sample while the remainder of the analytical system continues to withdraw the previous sample at a normal rate from the holding coil for analysis. This technique was thoroughly examined in our laboratory and found to have a number of major limitations: a) under optimum manifold conditions, the sample aspiration period in the sampler cycle can be reduced to only 25% of the sample processing time required by a given method to produce satisfactory peak characteristics; b) to prevent sample-to-sample interaction, one must experimentally establish the definite and critical pumping rate and volume capacity proportions of the sample storage coil and associated transmission lines and proportioning tubes needed to satisfy individual system hydraulics; c) inert solid dosage formulation components, which are not solubilized during sample homogenization, build up in the storage coil after a series of samples and eventually mechanically obstruct its operation; and d) the conceptual requirements of the rapid sampling device prohibit its use as a modular unit with manifold-to-manifold flexibility. The ARM has been developed (Figure 1) utilizing the concept of the rapid sampling technique but overcoming many of its limitations. With the multiplier, the homogenized sample is aspirated into the analytical system from a storage tank with sample stream air segmentation and reagent blank stream introduction between samples (Figure 2) in a manner analogous to the processing operations of the SOLIDprep unit. Thus, the problems of sample-tosample interaction and mechanical obstructions from inert materials, which are related to the use of a coil storage system, are eliminated. Further, in demonstrating the modular aspect of the ARM, the unit was inserted into various automated methodologies, as described in the Experimental section, without modification. With this unit, the sample aspiration time is reduced to an extremely rapid 6-sec portion of the sampler's programmed cycle period, completely independent of the sample assay rate. The system's samp1e:wash ratio may be set to virtually any value as described by adjusting the ARM and wash distribution solenoid valve program (Figure 4). As described previously, the maximum sample per hour assay rate of a conventional automatic analyzer procedure with a SOLIDprep unit is established by determining the minimum time required to perform each of its three opera-
(6) 0.045-inch i.d., Tygon manifold tubing, air: (7) 0.056-inch i.d. Silicone manifold tubing, debubbler: (8, 10) 0.081-inch i.d. Silicone manifold tublng, 85% SD No. 30 Alcohol in water (v/v) diiuent; (9) 0.110-inch i.d. Silicone manifold tubing, sample; (MC) mixing coil, 7-turn, 2.4-mm. i.d. bore; (PE) 0.034-Inch i.d. polyethylene transmission tubing
tions; viz., sample homogenization, sample aspiration, and homogenizer vessel rinse, on a given sample. With the incorporation of the ARM and reduced vessel rinse period, these sampler operations are occurring completely independent of the chemistry requirements of the analytical system. That is, the analytical system requires sufficient time to aspirate homogenized sample from the storage tank and sufficient reagent blank stream introduction via the wash distribution solenoid valve, S-11 (Figure 2), to maintain an acceptable samp1e:wash ratio. This increased sampling rate can be expressed by the equation:
where AR,,, is the new sample per hour assay rate with the incorporation of the assay rate multiplier; H is existing homogenization time; A is the existing sampler aspiration time; R is the existing homogenizer rinse time; and AR,, is existing sample per hour assay rate. S represents the required sampling time with W the needed wash time to satisfy the analytical system samp1e:wash ratio. With a given conventional automatic analyzer system, the required manifold sampling time, S, would be fulfilled by the SOLIDprep unit and equal to the sampler aspiration time, A . Thus, an assay rate increase would be effected in any analytical system where the wash time, W, is less than the total time required for the sample homogenization, H , and homogenizer rinse, R , cycles. Since the complete dissolution of the active ingredient in solid dosage formulations usually requires protracted homogenization ( 1 3 ) ,the ARM would substantially increase the analytical rate of most automated systems. In the quantitation of lorazepam in tablet formulations, for example, it was necessary to disperse the tablets for a period of 33-36 sec to effect complete dissolution of the drug. For the desired level of system resolution, the UV spectrophotometric methodology (Figure 7) requires 45 sec of sampling time and a minimum of 15 sec of reagent blank stream introduction between samples or a 3:l samp1e:wash ratio. With a 96-sec cycle requirement and the selectable sample rates on the SOLIDprep Sampler 11, the maximum assay rate with the conventional system is 30 samples per hour. By using Equation 1, an increased assay rate of 60 samples per hour is inferred and obtained with the incorporation of the ARM in the manifold. Analytical Properties of the Assay Rate Multiplier; Typical recording curves of lorazepam standard solutions
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
1041
Table I. Comparison of Conventional and ARM Automatic Analyzer Systems in Analyses of Pharmaceutical Products Product
Lorazepam tablets Lorazepam tablets Oxazepam tablets Oxazepam capsules
Labuled amount, mgldorc
1.0 2.0 15 10
Digitoxin tablets
0.10
Digitoxin tablets
0.15
Digitoxin tablets
0.20
Norgestrel tablets
0.075
Sodium nafcillin (monohydrate) capsules Ampicillin capsules
250
Automated ryrtcma
Asna) rate/hr*
Conventional Modified Conventional Modified Conventional Modified Conventional Modified Conventional Modified Conventional Modified Conventional Modified Conventional Modified Conventional Modified
30 60 30 60
30 60
30 60
15 30 15 30 15 30 15 30 15 30
250
*' of claimc
99
100 100 100 101 100 101 102 100 100 99
101 100 100
101 101 104 105
Re1 std dev, %
* 1.2 * 1.0 * 1.3 * 1.2 * 0.9
* 1.1
io.7 i0.6 1.4 1.3 i 1.5 .1: 1.3
*
* 0.9
Method Reference
(UV) (UV)
(UV) (UV) (11)
(11)
(11)
i 1.1
* 1.2 * 1.1
* 0.8 * 0.7 * 0.7
(91 (10)
Conventional 15 103 (10) Mod if ied 30 103 i 0.7 Ampicillin capsules 500 Conventional 15 104 r 0.9 (10) Modified 30 104 i 1.1 Potassium peni250 Conventional 15 103 * 0.8 ( I 0) cillin G tablets Modified 30 103 i 0.7 Potassium peni250 Conventional 15 102 i 0.7 (10) cillin V tablets Modified 30 101 k0.5 a SOLIDprep Sampler I1 used as sample preparation unit in all methodologies examined with the ARM incorporated in modified system. Reported assay rates are maximum obtainable within the experimental conditions of the method. c Average assay value from the unit dose analysis of 30 tablets or capsules.
,
0.m
Flours 8. Reproduction of recording curves of lorarepam standard solutions at the
2-rng level
( A ) 30.sample/hr assay rate with conventlonal system; (8)60-sample/hr assay rate wlth the ARM in the syetem
generated by the automated UV spectrophotometric procedure (Figure 7) both with and without the ARM in the manifold are reproduced in Figure 8. The fine definition of the sample peak tracings and steady-state condition defines the excellent flow characteristics of modified automated system (Curve B)a t this extremely rapid assay rate. Of further significance, no losses in the analytical properties of the manifold were encountered with the twofold increase in assay rate. The recording curves (Figure 8) demonstrate sample peaks a t greater than 99% of steady-state conditions with a sample-to-sample interaction of less than 0.1% and precision of f0.4% for both the conventional and modified methods. In lorazepam and oxazepam tablet and capsule formulation analyses, comparative data between conventional and modified procedures agreed within 1% (Table I). In exemplifying the analytical utility of this concept, the unit was evaluated in established colorimetric and fluorometric automated procedures used in the pharmaceutical industry. As presented in Table I, the equipment was em1042
ployed to analyze successfully commercial digitoxin, norgestrel, and penicillin capsule and tablet formulations. From both mechanical and chemical considerations, these methods are of much greater complexity than the UV technique (Figure 7). They were selected to demonstrate the versatility of the ARM. The methodologies include the hydroxamic acid colorimetric procedure for penicillins, the sulfuric acid-induced fluorescence procedure for norgestrel, and the hydrogen peroxide in HC1-induced fluorescence procedure for digitoxin. In examining the comparative data (Table I), it is evident that the multiplier produced a twofold increase in hourly sample rate without loss in method accuracy or precision. With regard to the sample trace characteristics produced with these methodologies, no losses in system resolution were encountered with the increased assay rate. Specifically, sample peaks a t greater than 95% of steady-state conditions and sample-to-sample interactions at less than 0.6% were obtained both with and without the ARM in the manifold. ACKNOWLEDGMENT The authors acknowledge assistance of A. F. Dewey and J. J. Peterson in the fabrication of the assay rate multiplier. LITERATURE CITED (1) N. R. Kuzel, H. E. Roudebush, and C. E. Stevenson, J. Pharm. Scl., 58, 381 (1969). (2) F. M. Russo-AM, Ann. N.Y. Acad. Scl., 163, 511 (1966). (3) G. J. Paparleilo and L. F. Cullen. "Advances In Automated Analysis, Technicon lnternatlonal Congress 1970", Vol. 2, Medlad, Inc., Whlte Plalns, N.Y., 1971, p 199. (4) M. K. Schwartz, Anal. Chern., 45, 739 (1973). (5) J. T. vanGemert, Talanta, 20, 1045 (1973). ( 6 ) W. W. Holl and R. W. Walton, Ann. N.Y. Acad. Scl., 130, 504 (1965). (7) A. Ferrari, E. Catanzaro, and F. M. Russo-Alesi, Ann. N.Y. Acad. Sci., 130, 602 (1965).
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
( 8 ) P. Grafstein and R. Goldberg, “Advances in Automated Analysis, Technicon International Congress 1972”, Vol. 9, Mediad, Inc.. Tarrytown, N.Y., 1973, p 53. (9) L. F. Cullen, J. G. Rutgers, P. A. Lucchesi, and 0. J. Papariello, J. Pharm. Scb, 57, 1857 (1968). (10) C. E. Stevenson, L. D. Bechtel, and L. J. Coursen, “Advances in Automated Analysis, Technicon International Congress 1969”, Vol. 2, Mediad, inc., White Plains, N.Y., 1970, p 251.
(11) L. F. Cullen, D. L. Packman, and G. J. Papariello. J. Pharm. Sci., 59, 697 (1970). (12) C. W. Gehrke, L. L. Wall, and J. S . Absheer. J. Assoc. Off. Anal. Chem.. 56, 1096 (1973). (13) T. Urbanyi, W. T. Brunsklll, and M. Lin, J. Assoc. Off. Anal. Chem.. 56, 1069 (1973).
RECEIVED’for review May 15,1975. Accepted July 8,1975.
Automated Determinations of Dissolved Organic Carbon in Lake Water P. D. Goulden and Peter Brooksbank Canada Centre for lnland Waters, Burlington, Ontario, Canada L7R 4A6
Automated methods are descrlbed for the determlnatlon of dlssolved organlc carbon In water from the Great Lakes. The lnorganlc carbonate Is removed in a heated packed column, the organlc carbon Is oxldlred and the resultlng carbon dloxlde measured by an Infrared analyzer. Two alternatlve methods of oxldatlon are used, ultraviolet Irradlatlon and silver-catalyzed peroxydlsulfate at 95 O C . When ultravlolet lrradlatlon Is used, the relatlve standard devlatlons obtalned at carbon levels of 50 pg/l., 1 mg/l. and 5 mg/l. are 4.0%, 1.2%, and 0.8% respectively, wlth sllver-catalyzed peroxydlsulfate the relatlve standard devlatlons are 3.2%, l.O%, and 0.7%, respectively. The silver-catalyzed peroxydlsulfate is the more convenlent and precise method but does not completely oxldlre all materlals In water; In a large number of natural water samples analyzed, the oxldatlon completeness averaged 97 %. The llmlt of detectlon for carbon Is 10 pg/l., the analysis rate Is 20 samples per hour.
The measurement of dissolved organic carbon (DOC) is an important part of many water quality studies. The dissolved organic material may represent the degradation products of plant or animal life that lives or has lived in the water, or, alternatively, it may represent pollution by sewage or industrial effluent. The methods commonly used for DOC determination involve oxidation of the organic material with subsequent measurement of CO2. Oxidation can be carried out in the gas phase by passing the sample over a catalyst a t high temperature (1) or by wet oxidation. In its commonly-used form, where the sample is injected in a gas flow with a syringe, the gas phase oxidation technique is convenient and rapid but the necessarily small sample size limits the sensitivity obtained. Wet oxidation, which uses larger sample sizes, gives higher sensitivity. In the method of Menzell and Vaccaro (21, oxidation is carried out by treating the sample with peroxydisulfate in a sealed tube in an autoclave; however, this operation can be quite time consuming and Baldwin and McAtee (3) have used oxidation a t room temperature with silver-catalyzed peroxydisulfate. Oxidation can be carried out using irradiation with ultraviolet light and this has the advantage that it lends itself to an automated process such as that described by Erhardt (4). In all these techniques, a distinction must be made between the inorganic and the organic carbon present in the sample. The distinction can be made by measuring both the carbon dioxide liberated by acidification and also making a “total carbon” measurement. The organic carbon
then is represented by the difference between the two measurements. Alternatively, the acidified sample can be stripped of the “inorganic” carbon dioxide before the oxidation step. The validity of the organic carbon measurement then depends on the completeness of the stripping operation and is also subject to the possibility of volatile organic materials being lost. In the present work, the different techniques have been examined in the development of automated methods for the analysis. An automated system is used in which the inorganic carbonate is removed by stripping the carbon dioxide from an acidified sample in a heated packed column. This enables the stripping to be carried out without any problems of sample contamination and also gives opportunity to examine the stripped gas for volatile organics. Automated methods using both gas phase oxidation and wet oxidation have been developed. T o obtain high sensitivity, wet oxidation is used. In the systems described here, with the large sample flow (-6 ml/min) to the oxidation system, a limit of detection of 10 fig/l. C is obtained. The oxidation process of choice is ultraviolet irradiation, in the work of Erhardt ( 4 ) , Soier and Semenov (5), and, in the present work, this has been shown to oxidize all the organic materials of interest. However, an alternative system that uses silver-catalyzed peroxydisulfate a t 9P°C for the oxidation has some advantages. Although this treatment did not completely oxidize all organic materials studied, e.g., EDTA, it is a convenient and precise method and, in a study of waters from the Great Lakes, it oxidized practically all (97%) of the naturally occurring organic material. A non-dispersive Infrared Analyzer is used to measure the C02 resulting from the oxidation. This was chosen because of its relative freedom from interferences and because the laboratories for which this method was developed already have this equipment as part of a commercially available Total Carbon Analyzer. By making small piping changes, it is possible to utilize this existing equipment either in the original mode or coupled to the automated equipment described here.
EXPERIMENTAL Apparatus. The manifold used is shown in Figure 1. The sampler is an Industrial Sampler (Technicon Corp.) which uses 25-mm X 100-mm borosilicate glass tubes to contain the samples. The sampler is modified by the addition on the sampler arm control shaft of a cam controlling a three-way solenoid valve. In the wash cycle, the wash water after being de-bubbled, passes into a T at the top of the sampler arm. As the sampler arm starts to move to the sample tube, the three-way valve is switched so that the wash water flows directly t o the wash receptacle. The cam is
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
1943
