Continuous flow measurement of beta radiation using suspended

F. Morley, I. K. O'Neill, M. A. Pringuer, and P. B. Stockwell. Microprocessor-controlled digital integrator for nuclear magnetic resonance measurement...
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Chemical Instrumentation Edited by GALEN W. WING, Seton Hall University, So. Orange, N. J.

07079

T&.w articles are in^ to serve ihe readers of m m JOURNAL by calling a t t e n t h lo n m developmen& i n the themy, & n i p , or availobili& of and ezchemical l a h a t m y instrumenlath, or by prcmting d u l i-h& planations of topics thohat ore of practical imporhnce lo Ihow who we, or L%h the use of, modern instrumenlotiun and inshrmenlol techniques. Tlu edilm in&s cmespondmce from proapeeliw contribulms.

XLIX. Continuous Flow Measurement of Beta Radiation Using Suspended

EUGENE T. McGUlNNESS and MARTIN C. CULLEN, Department of Chemistry, Seton Hall University, South Orange, N. J. 07079 Continuous Bow detection methods moviding mnrlwrrurtive rcndmt and based on themnl, opricd, e l r < t r ~ a or l rrliorhemirnl p r u p ~ r ~ of i e immiturcd ~ cftluents have been variously adapted for use in conjunction with liquid column chromsr tography. Two types of continuous measurement detectors based on optical principles seem t o predominate in this area. Quartz flow cells, mounted in any one of a. number of oommercial spectrophotometers, or flowstream absorbance monitors, are used routinely t o assay nucleosides, nueleotides and proteins in the 240, 260 or 280 nm range of the spectrum. I n contrast, continuous monitoring using B differential refractometer provides an indiscriminate and sensitive detection system which depends on the magnitude of the refractive index difference between a n eluting solvent and the eluant solute. Both types of optical detection systems are adaptable t o single solvent or gradient elution chromatography. The thermal-responding micro-adsorption detector, which can sense less than 5 X 10-S"C variations in stream tempersture, is likewise a n indiscriminate response system, and has been used in the analysis of carbohydrates, proteins, lipids and hydrocarbons in the micro- t o nano-molar coneentmtion range. Another widely applicable method which deserves t o be hetter known is the onst,ream measurement of beta radiation. 1 Specifications contained in this article are based on information supplied by the manufacturer and do not imolv . . endorsement. No attempt is made to evaluate equipment on a eompetitive basis. Exclusion of a product is not to be interpreted as a negative evaluation. While we have attempted to survey the field, it is not a l w a y ~possible to obtain the desired information.

t Circle No. 149 m Readers' Service Card

Dr. McGuinness i.:wwi:ttr 1,rofnior chemistry nt Selon I I d l 1-niverdy. Previous to this nfli1i:ition he 1,ursued a bnsic interest in l~iael~erniatt~y i n the pharmaceutical (Wnllnce & Tieinan. he.) md fermentation (P. Ballantine Q Sons) ~ndustries. Heis n gradunte of St. Peter's College (B.S., 1949). Fordhnm University (M.S., 1954) and llutgers University (Ph.D.. 1961). Current and recent research interests tnd publications have been centered on radiochemienl synthesis of biochemicnlly important compounds, new radiochromntographio asscry methods and charactencation of fungal enzymes. 3f

This method is readily available to anyone with access t o a. liquid scintillation spectrometer. Within the limitations of the method, to be discussed in this article, i t is possible, with a minimum of expense, to adapt a variety of currently discrete sampling and beta activity mensurements to automated nondestructive readout in aqueous systems. Det,eetor response in the piwmolar concentraiion range is possible. I n principle liquid scintillation spectrometry provides s t least four options for the measurement of sample activity in conjunction with the detection of p radiation. Beta radiation can he mensured (it) directly on discrete samples in the presence of dissolved scintillator, or (b) continuously in the presence of suspended scintillators. Alternatively, p activity can be monitored indirectly by measuring the Cerenkov radiation generated by 6 particles of energy greater than 0.265 Mev. This Cerenkov radiation can be measured in t h e absence of scintillator, (a) in individually prepared samples, or (d) in a continuously flowing stream. Current practioe is directed predominantly t o (a), the discontinuous measurement of beta radiation in individual samples discretely constituted with appropriate scintillators, solvents and soluhilisem in a nonaqueous environment. A variation of this method, whereby the liquid scintillation spectrometer is used as a quantum counter to measure the number of photons arising from the bioluminescence generating adenosine triphasphate luciferin-luciferase reaction, has received increasing attention as a n nssay method for A T P i n the nano- to pica-molar range of concentration. Recently, Haberer and Kolle (I), Clausen ( 8 ) and Elrick and Volume

Dr. Cullen mceiied his A R . iiegrre 'ron~Catholic 1-liversits (IO(iU) s n d hi3 KS. ( ~ m nlld ) PIID. (Inno) degrees

'rom Seton IInll University. hr the mriod from 1960-1'367 he taught chemis.ry and mathematics in regular end ipeoial courses in severill of the high :ohools run hy the Christian Brothers. :erving as science department head in one ,f them in 1965. 'Ie has worked as a asenroh soientist ~t Sohering Corpora.ion. His research interests lie in areas of xotein structure and function, drug netnholism and analytical instrumenta.ion. He has done research in the areas ,f protein structure determination using .adioisotopesand flow cell detectors, synhesis of labeled organic compounds, and nfluenee of drug levels on hone structure. 3r. Cullen is ourrently a post-doctoral ellow a t Stanford TJniversity Medical lohool.

(Continued on page AlO)

47, Number I , January 1970

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Chemkal Instrumentation Parker ( 3 ) have drawn attention to the

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aqueous systems, no soluhiliirers are needed. The separation and purification of a labeled product from excess labeled reagent can be oarried out simultaneously with the assay by introducing the reaction mixture onto a gel-permeation or ionexchange chromatography column inserted into the flow system prior to the beta monitor. The read-out is nondestructive. The labeled sample can be directed through other d e t e c t o r ~ a nultraviolet or gamma-ray spectrometer, gas chromatograph, or differential refractometer-when additional read-out is called for, then recovered by directing t,he effluent to 8. fraction collector. Where the results gotten by (a) and (h) are of comparable quality, flow counting is more economical since no counting vials, sohbilisers or solvents are required.

tinoous monitoring of Cerenkov radiation, (d), has been confined to the examination of liquid wastes from reactor sites ( 6 ) . Rapkin has summarized the status of liquid scintillation counting for the period 1957-1963, in a copiously referenced review (6). He noted that commercial equipment for continuous flow measurement was only then becoming wailable. The most recent review of detailed developments in liquid scintillation counting since 1963, dealing with instrumental developments, scintillators and solvents and methods, is that of Parmentier and Ten H a d (7). I t was noted here that little 1. INSTRUMENTATION progress could be reported in connection with the use of suspended scintillators in I n addition to the flow-cell (see Section the interval since the review by Rapkin 2) the minimum equipment necessary in(6). The substance of each of these recludes a detector assembly with apviews deals almost exclusively with (a). propriate photomultiplier tubes, 8. rateThe most extensive reports available on meter and a strip chart recorder. With continuous measurement of beta radiation this arrangement i t is necessary to proover suspended scintillators are those of vide some method of integrilbing the area Rapkin (8) and Schram (9). Our subunder a radioactive peak to quantitate sequent discussion will be confined to this the results. This can be done manually type of 0-measurement. by triangulation or by equipping the reThe uti1it.y of this approach can be corder with one of t,he commercially traced to tbe seminal work of Schram and available integrators. Alternatively, a Crnkaert (lo), Schram and Lomhaert ( i f ) scaler and interconnected high speed and Sleinberg (18, 13, 14). Schram and digital printer can replace the ratemeter coworkers mmitored IzSS]taurinc a t 6% and recorder. This is a, preferred comefficiency using plastic scintillatom in bination since the statistical uncertainty what is probably the first description of a associated wit,h the time oonstmt of the biochemical experiment employing conratemeter is eliminated. I n this arrangetinuous monit,oring over a stationary ment the printer is set to repetitively scintillator (lo). Steinberg, in a eomprint out the rtccumulated count in a. parstive evaluation of several scintilpreset time interval (e.g., 1 mi", 6 min). lators, demonstrated t,he advantages that Where the level of activity in the sample accrue fram using crystalline anthmeene is very high, i t may he necessary to correct as the suspended scintillator (14). The for the count not accumulated by the subsequent literature associated with (b), scaler during the short (-1 sec.) interval which can be traced to the work of Piez of print-out. (16), has been largely confined to csrThe optimum equipment for flow countban-14 labeled compounds where the ing utilizes the coincidence counting scintillator of choice is anthraeene. Howcircuitry of beavy-shielded dual photoever, the same properties of anthracene multiplier tubes found in most liquid that make i t the preferred sointillator scintillation counters. No alteration of (16)-the delocalieed or T electrons of its existing electronics is required to operate polycyclic aromatic ring system-are. them in a flow counting mode. These inthose responsible for its ability to function struments are usually equipped with s n as a Lewis base, to form a complexes elevator-interrupt mechanism or equivawith certain electropositive centers. For lent device to aceomodate the flow-cell. this reason Lewis acids of transition and When this mechanism is inactivatedheavy metals, far example, ohromiurn as i t is during flaw counting-the operator and mercury and their salts, are sequesosmmes ihe ~esponsibilityfor preuating tered by anthracene. The accompanystray light from striking the photomultiplier ing build up of radi0activit.y in the flow tubes when the high voltage of the instvument cell det,ectar gives rise to a marked inis on. Commercially available flow-cells creasein background, renderingsubsequent can be sccommodated interchangeably in measurement useless. I n addition, the different make liquid scintillation counters. tendency of anthracene, glass and plastic to st,rangly adsorb the various phosphate ion species bas limited the use of the 2. FLOW-CELLS method for studies involving the phosphorus-32 nuclide. CommernalFlow-Cells. Flow-cell equipIn mite of these aouarent limitations ment is available from a number of snp.. emt~it.t~.,t~i flow nwi.urernwar uf beta pliers (18). The major factors that must ~ t d i a r i ww e r x q w d r d :cin~illa~c~r is a be considered in constructing a flow-cell, powrrflll 1001 oil?rille, *ever~ldisriwt adincluding fluid volume, the surface/ vantages over static eount,ing. For volume ratio, flow characteristics of the example '%hemica1 quenching" is comchamber, and characteristics of the fiuor pletely eliminated (17). The counting and cell material, have been discussed environment is entirely compatible with (10). Cells of nomind volumw of 0.5,

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Journal of Chemical Education

Figure 1. Nuclear-Chicogo Model 6 7 7 0 Chroma/Ceil Ilucite). Nominal volume, 2 ml.

1.0, 2.0 and 4.0 ml me commonly used. The configuration of the NuclearChicago (DesPlaines, Ill. 60018) Model 6770 Chroma /Cell, 2 ml volume, is shown in Fig. 1. The cell is constructed of

Figwe 2. Nuclear-Chicago glugr Row-cell, Model 6901 (nominal volume, 2 mll. Phofa#mph courtesv o/ Nuclear-Chicapo Co7p.

Lueite. A glass fldw-cell, Model 69006908, available from Nuclear-Chicago, is shown in Fig. 2. The various model numbers pertain to glass cells of nominal volumes 1, 2, and 4 ml, filled with anthracene, plastic or glass scintillators. The cells in all of these models are demountable from the cylindrical metal top referred to as the manipulstor. Details of these configurations are available in NuclearChicago Publication 716620. A flow-cell adapter with an associated Kel-F cell, Model 3040, available from the Packard Instrument Campany (Downers Grove, Ill. 60515) is shown in Fig. 3. The flow-cell adapter (Models 3042 or (Continued on page A1B)

3043) does not include the Bow-cells which are available in a. number of sizes, including a 10 ml flow-cell which can be used

Figure 3. Flow.cell adapter with on orrocioted Kel-F cell, Model 3040, Pockord lnrtrurnent Company. P i o t o ~ r a g hC O Y T ~ C W of Pnckord Indrumen1 Co.

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to monitor the effluent from s. gas chromatograph. Specificstions and extensive details for these flow-cell sdapters are presented in Packard Instrument Company, Manual 2039. Lucite flow-cells cannot be used when organic solvents such as alcohol, benzene or toluene, etc., are employed as eluting agents. Quarts U-tube detectors, based on the suggestions of Piez (16), are available from Picker Nuclear Corp. (White Plains, N. Y. 1060.5) in sizes of 1, 2 and 4 ml capacity (Catalog Nos. 652-146, 147, 148, 149). The design of this @-cellassembly is shown in Fig. 4. I n this arrangement the end or base plate swings aside permitting easy interchange of the different capacity U-tubes. End closure of the relatively large orifices of these tubes is made with silicone rubber septa. The Beckman (Beckman Instruments, Inc., Fullerton, Calif. 92634) flow-cell is illustrated in Fig. 5. This det,eetor utilizes europium doped calcium fluoride as the scintillator and has a packed volume of approximately 1 ml. Intertechnique Instruments, Inc. (Dover, N. J. 07901) offers Flo-Cell adapters which accommodate a seamless, quartz body or a seamless, molded polymethacrylate body for their SL-40, -30, -20 series spectrometers. A quartz cell, suitable for use with liquid or gas streams, is shown mounted in an SL 20 spectrometer (Figs. 6 and 7).

Figure 4. Picker ~ u c l e a rquartz V-tube detec. tor, 4 rnl volume, mounted in o &cell asrembly, Model 652-149. P h o l w r a ~ hC O U T ~ S S I Iof P~cEBI. Nudam.

(Continued on page A14)

Chemical Instrumentcrtion

Figure 5. Bsckmon Row-cell. The detector of nominal 2 ml volume is packed with europiumdoped calcium fluoride. Photoproph courtesy of Bcckmon Indiumenta, Inc.

Figure 6. Intertechnique SL-20 spectrometer. Phoiooinph couilasg alInlertechninue Inaln~ma&

fifodified Flow-Cells. The investigator has the option of modifying available equipment, or fabricating his own flow-cell from readily avdnhle materials to cope with individual counting problems. For example, phosphorus-32 cannot be counted as phosphate in an ant,hmeene packed flow-cell under ordinary conditions bec a u e of the at,tendant, build-up of background radioactivity arising from the interaction of ant,hracene and phosphate. I n view of the high energy of the beta-particle emitted by the *%Pnuclide, Martin (SO) has described s cell configuration in which 7 ft of Teflon tubing (i.d. 0.022 in.) is coiled loosely in a standard glass colmting vial completely filled with scintillation fluid. The tubing ends were passed through holes drilled in a polyet,hylene screw cap attached to the threaded end (Catinued on page A161 ~~

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of a. metal rod which served as a handle. Using this arrangement Martin reported an efficiency of about 65% for orthophosphate with essentially complete washout of activity from the tubing. Solutions containing Hsa2P04,with carrier KHsPOa, a t the relative levels of 0.1, 1.0, 5.0 and 25.0 were counted with relative efficiencies of 54, 52, 60 and 59% respectively. Iodine-131 was counted in t,his cell as N a W with an efficiency of 14%. l b b a , Rancucci and Lambelin (81)employed a commercially available flow-cell (Kel-F, Paekard) modified partially aecording to Martin (SO) to continuously monitor hard beta-cmitt,ing isotopes in small laboratory animals with a. reported efficiency of about 5070. Clifford, Hewett and Popjak (88) have described a scintillation counter suitable for the continuous measurement of radioactivity in solutions irrespective of the nature of the solvent. Beads of lithiumcerium glass itre placed in a shallow flowcell 1 mm deep fabricated of borosilicrtte Counting efficiencies of better than 2 0 7 ~ are reported far oarbon-14 and sulfur-35 with 90% efficiency for phosphorus-32. We have found it useful, when counting beta- and gamma-emitting nuclides, to include a flaw-cell inserted in a. 2-in. well type thallium-activated sodium iodide detector in the counting stream either just prior to or after the B Bow-cell detector. The flow-cell for this detector consists of a coil of '/$> in. i.d. Teflon tubing wound in a spiral about a glass rod and inserted in a. plast,io or glass well scintilla-

Figure 7. an

Quartz U-tube detector mounted in

SL-20 spectrometer.

P h m g r o p h c o u i l e ~ vofinlertechnfuc Inatrumenla.

tion vial. We have used this flow-cell mounted in a Baird-Atomic 810C well detector connected to s. Nuclear-Chicago Model 1820 single channel gamm%ray spectrometer. The output of the spectrometer was monitored on a 10 mv rewrder. The volume difference for the same peak of radioactivity appearing on the digital printer and recorder is readily determined from the diameter and length of the tubing between the two detectors. This arrangement is especially useful when studying nuclide hold-up on anthracene or other scintillators, since only the Teflon of the gamma flow-cell is contacted by the column elfiuent. Column Packing and Connections. For a variety of reasons it becomes necessary to empty and repack the flow-cell. For example, i t is easier and quicker to empty

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and repack a, cell than attempt to remove entrapped air. We frequently have had occasion to deliberately contaminate a cell with phosphorus-32 as phosphate or chromium-51 as chromate when studying cgmpamtive retention characteristics of different suspended scintillators and different surfactatits. Since anthracene is the most widely used scintillator, our comments pertaining to repacking are directly applicable to this material. Flow-cells require about 0.5,l and 2.5 g of anthracene for the 1, 2 and 4 ml cells, respectively. Commercidly available blue-violet fluareseencc grade anthracene crystals that can pass through a 30 mesh screen but not a 100 mesh screen should be used. A 50% excess of the estimated qumtity of material is suspended in water in a small beaker or flask and a few drops of any commercial surfactant is added to wet the crysi,als. A small wet plug of spun glass wool is inserted in the exit side of the water-filled cell which is connected by in. i d . blackened tubing to a 20 ml syringe. I t is essential that. all air be excluded from the system prior to filling. As the entrance limb of the flow-cell is slowly emptied by syringe suction, the suspension of snthracenc in detergent solution is poured into the cell. This action is continued until the cell is filled. A gentle back-and-forth motion on the syringe is sufficient to properly seat the crystals. A netted plug of the glass wool is then inserted into the entrance side of the cell. The cell should be packed to a. degree of tighbness that will prevent motion of the crystals if the flow of effluent is reversed through the cell, and avoid the appearance of empty space under the glass wool after prolonged directional flow. Several fillings sre sufficient for one to develop a. degree of proficiency in the operation so that the entire filling process is completed in less than 5 min. Since it is frequently necessary to connect and disconnect tubing in the flow system to accommodate to different counting situations and a variety of detectors, d l junotions of the '/ax in. i.d. TcflonTeflon flow-through tubing arc made by inserting the tubing into a sleeve of '/a in. id., in. wall polyethylene tubing. The sleeve is then mounted and pressure held in the striotured end of a cuboff 6 mm 0.d. T-shaped connecting tube (Corning No. 9187 or Kimble No. 45020). This arrangement, shown in Fig. 8, permits rapid connections to be made and 1s leak proof when incorporated into a. liquid flow system pumped a t a rate of up to 150 &hr. Calculation of counting efficiency requires a knowledge of the effective fluid volume of the packed cell. I n order to determine this parameter for suspended scintillators, Schram and Lombaert (23) have wed an acid solution of known molarity which is circulated through the cell until it reaches the outlet. Inlets and outlets are then carefully taken off, filled with distilled water and reinstalled. More water is passed through the cell while collecting the effluent until it is neutral. Titration of the acid effluent with alkali permits cdculation of the effective volume of the cell.

Journol o f Chemical Education

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(Continued on page A18)

A variety of suspended fluom have been used in nddition to anthrseene for the flow monitoring of aqueous solutions. For certain counting situations a particular fluor may prove more advantageous than anthracene. The properties of organic scintillators have been described by Sohram (64) and more recently by Horrocks ($6). Mohgissi ($6) reports using calcium fluoride as a.scintillator with favorable results. We have examined cerium activated silicate glass with 2.5% natural lithium content and found it shows less retardation of certain nuclides hut is less efficient than anthracene. The packing of any of these materials is carried out essentially as described for anthracene, care being taken to exclude fines which would tend to clog the flaw system. Low temperature operatmn, below O0C, of the liquid scintillation counter should generally be avoided when flow monitoring an aqueous system.

3. CALCULATIONS Before the flow-cell is placed in onstream operation, i t is necessary to establish the optimum counting conditions for the particular radioactive nuclide to be monitored. To do this, the cell is filled with a solution of the isotope to be counted, placed in the counter, and the voltages and discriminator settings corresponding to the best (efficiency)a/hackground are established in the manner of statio counting. Onoe the counting conditions are established, the cell is incorporated into the column efluent side of the stream using the connections described. When a solution containing a bolus of radioactivity of total activity A(dpm) flows a t a constant rate u(ml/min) through a sow-cell with a fluid volume V(m1) in a counting system of efficiency E(%), the total sample count which will he callected, C,, can be calculated (19) from the equation

Both V and u are largely determined by the characteristics of the chromatographic system used to effect separation of the nuclides of interest. Where one or several well separated peaks of radioactivity are involved, a large cell can he used for maximal sample counts. Small cells are preferred where closely spaced peaks are met. The fluid volume should always be less than the volume between adjacent radioactive peaks. The choice of flow rate is usually governed by the nature and conditions of the experiment. Rate of flow can he maintained constant by gravity feed using a Mariotte bottle or by pumping the column influent. Estimation of Eficiency. The determination of efficiency using a liquid. scintillation counter equipped with a digital printer is carried out as follows: a known amount of radioactivity is placed on the ehramatoera~h column and the .. tirnrt. is set fwr n n i l l I l . ~ ddependent 01. the chuirr of erperi,ner>tul wnditions n t d .," ",. A 18 / Journal of Chemicol Education

.

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Figure 8. Arrangement showing quick-fit connections "red to incorporate a 2 ml (Packordl Lucite detector into a flow syrtem.

the resolution. An interval equal to V/u i., a reasonable initial choice of measurement time. At the end of the interval the accumulated counts are printed out on tape and the scaler resets to collect the counts in the next time segment. The total number of counts in any peak is obtained by summing the counts accumulated for each increment within the peak. The total activity due to sample is obtained by adding the counts in the region of each radioactive peak and subtracting the background, calculated for an equal time interval, from an adjacent region free of sample count. The following data, obtained in the authors' laboratory, illustrate the ealculs, tion of a representative efficiency: one milliliter of a uniformly labeled sample of [WjLleucine, containing 0.1 wCi/ml, was loaded on a. column of Bio-Rex 70(1.2 X 25 cm) and eluted with 0.2 M citrate-phosphate buffer, pH 5.85, through a 2 ml Packard flow-cell packed with anbhracene and mounted in s. NucleerChicago Model 703P liquid scintillation counter. The eluant volume, 59.5 ml, was collected over 249 mi". The fluid volume of the cell was 1.0 ml, total count was 244,618, and the background count was 2,015. Therefore

=

0.262 -v 26%

Porcellati and diJeso (97) report s. similar value, 26.9%, for the efficiency of carbon-14 using [W]leucine. I t should be noted that the efficiency calculated for a particular nuolide will vary with the counting equipment, discriminator and high voltage settings, and, as Steinberg (14) has pointed out, with differenthatches of anthraeene. I t is good practice, therefore, to periodically check the efficiency of a flow-cell bv addine a standard amount of radioactivit,; to a Gmple placed on the chromatography eolumn. The effioienoy (Continued on page A6O)

Chemical Instrumentation for a particular cell should also be redetermined each time i t is cleaned and repacked.

4. A SURVEY OF SOME NUCLIDES COUNTED Rzpkin has presented a. tabular summary (17) of some efficiencies for e,@ and r emitters assayed over anthracene. Carbon-14 and Hvdrogen-S. The hulk of the literature dealing with continuous flow measurement over suspended scintillator has been concerned with amino acids, containing a carbon-14 label. Rapkin has made some interesting comments (.8,) on the extensive work of Piez (16) using 14C and 8H.This investigator employed a quarts cell with fluid volume of 1.1 ml and obtained efficiencies of 38 and 0.9% for "C and aH,respectively with s. background of 18 cpm when counting in integral mode. Using differential counting mode, efficiencies of 27 and 0.7y0 and B background of 7 cpm were obtained. With these conditions grass count rates of twice background would correspond to a tritium activity of 1O8 dpm/ml and a. carbon-14 rtctivity of 26 dpm/ml. In spite of the low efficiency for tritium, flow counting of this nuclide should not he lightly dismissed, since many tritiumlabeled compounds are available a t lower cost and higher specific activities than the corresponding carbon-14 labeled compounds, and since A in Eq. (1) can he readily increased for enhanced sensitivity. There appears to be no problem of holdup of carbon-14 activity on anthracene using labeled amino acids. Flow-cell use is reported questionable with large molecules such as polysaecharides, nucleic acids and certain types of proteins. However, no systematic investigation of the interaction of these macromolecules with anthracene has been reported. Phosphorus-3% Funt and Hetherington have reported (38) the retention of phosphorus-32 as JaP043-in a flow-cell consisting of a spiral of plastic scintillator. Versene (EDTA) added to the eluant removed the residual contamination. Although can be counted over anthracene with efficiencies in excess of 90%, the strong interaction of phosphate with mthracene, noted by several investigators, precludes the use of this fluor to monitor a2P04a-. We have observed (29) that aZP labeled sugar phosphates are less strongly adsorbed on anthcteene than inorganic phosphates. In a preliminary investigation we have found that if the anthracene is precoated with an anionio surfactant (e.g., Tergitol 15-S-3A) and the eluting solvent is made 1% ( u / v ) ,in this wetting agent, the phosphate budd up on anthracene is markedly attenuated hut not completely eliminated, following repetitive input of 0.01 pCi samples of 'TO,'-. Unfortunately these surfactants will be of limited utility since they are not entirely compatible with certain chromatography systems. Mercury-803 and Ch~omiumdl. Mercury-203 as mercuric ion, organic mereuri d s and chromium-51 as chromate appear

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to be entirely incompatible with anthracene since t,hey are very tenrtciously bound by this scintillator. The resulting eontaminetion can be removed only by extensive cleaning and repacking of the cell. A solution to the problem of heavy metal retention by anthracene would constitute an important development in analytical hioohemistry since i t would provide us with a capability to assay funotional groups (e.g. sulfhydryl groups) in proteins simultaneously with the separsr tion and purification of the macromolecule using high pressure liquid ehromatography. The semitivity of the assay, limited only by the specific activity incorporated into a group-specific reagent, would he considerably greater than is inherent in an amperometric (SO) or spectraphotometric (31) method and eomparable to that reported by Leach, et al. (38) using a static radioehemical assay of sulfhydryl groups in proteins. We have had some success, a t least with mercury labeled compounds, by using a detergent-anthracene system in which a cationic surfactant, Hyainine 2389, is added as a wetting agent when packing the cell and to the extent of 1% ( u / v ) to the citrate or citrate buffer used as eluting agent (33). Using gel permeation chromatography with this detector we have been able to separate and assay 'OJHg(II), [103HglCH.HgN08 ($4) and [XWHg] methyl mercuri-labeled proteins a t 60% counting efficiency. The count returns rapidly to background after each radioactive peak if the pH is kept between 2.0-3.0. Chromium-51 as chromate appears to be held more avidly by anthracene than mercury and thus far is intractable on this scintillator.

5. STATISTICS OF FLOW COUNTING As previously mentioned, since both v and V are fixed by the nature of the experiment, it is difficult or impossible to alter the measurement time. The only variables which are subject to the control of the experimenter to increase the sensitivity are the efficiency, E, and the total activity, A . It is not possible, however, using flow monitoring, to compensate for either low efficiency or activity by lengthening the counting time. The influence of background on the standard counting error is given (19) by the equation:

where c is the standard counting error (%), CT is the total count, B is the hackground (cpm), t is the time required for the radioactive peak to completely traverse the cell (min) and C, is the net sample count (CT - Bt). I n spite of the fact that measurement time is not under the contml of the investigator, the quality of a measurement of radioactivity is time dependent. The ramifications of this fact have been discussed by Rapkin (8). He has pointed out that while transient measurements provide an accurate reflection of high (Continued on page A S )

Chemical Instrumentation activity levels, the same is not true far low levels. A useful approximation applicable to low level counting is:

where t is the counting time, K is a eonstant indicative of the oonfidence level, a is the accuracy, N is the net counting rate (cpm), and B is the background (cpm). In static counting the operator is free to select the counting time corresponding

typical continuom-flow system is 20 cpm-and making the not-so-valid assumption that the activity peak 1s perfectly rectangular and flat-topped-the data in the Table can be fitted to various operating patrameters. The numbers approximate the minimum activity which must be present in a peak to make a. measurement having a particular quality. If the investigator can accept these figures, continuous Bow measurement using suspended scintillator should be given serious consideration as an extremely sensitive method of detection.

to a desired accuracy and confidence level from a knowledge of the net count rate and background. In dynamic counting, since the counting time is not a. variable and the background is constant, the investigator must base his decision as to the qudity of his measurements on the net counting rate. The following Table, taken from Rapkin's paper (a), provides us with a basis bv rphich we can ~ u d e ethe a u a l i t ~of our r&ults where thk net count rate is a. limiting factor, provided we are willing to make certain assumptions. If it is assumed that there is no mixing or peak dispersion and that the background of a.

(Continued on page A341

Table 1. Table of Confidence Levels for a Variety of Counting Parameters Applicable to @Flow Monitoring Over Suspended Scintillator. The Numbers Approximate the Minimum Activity Which Must b e Present in o Peak to M a k e a Measurement Having a Particular Quality. These Data, Taken from Rapkin (8),are Reproduced b y Permission and with the Courtesy of Picker Nuclear Corp. Time Confidence level

1%

Accuracy

0.5 min

1 mi"1%-

5%

5%

1%

1%

5%

5%

Effioienoy

(%I

1 2 5 10 20 50 100

2x10' 106 4 X 105 2 X 1 0 106 4 X 10' 2 X 104

5 X 101 2.3 X 10' 2.5 X 10' 1.2 X 10' 101 4 . 7 X 1 2.5X10' 2.3X108 1.3 X 10. 1.2 X 1 0 2 10' 4.7 x 10' 5 X 10-2. X 10'

1

2 5 10 20 50 100

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8.4X10' 4.2 X 10' 1.7 X 10, 0 4 X 101 4.2 X 108 1.7 X 10' 8 . 4 X 10'

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8 X 1 0 ~ 3 . X I 0 18 . 4 X 1 0 ' 101 23x10' 4 X 1 0 U . 6 X 101 4.2 X 10' 5 X 105 1.2 X 10' 4.6 X 103 1.6 X 106 6.4 X 10' 1.7 X 10' 2 X 105 105 2.3 X 103 8 X 1 0 V . 2 X 10' 8.4 X 10" 4 X 108 1.6 X 10' 4.2 X 10' 5 X 10' 1.2 X 10' X 10' 4.6 X 1OL 1.6 X 1 0 0 . 4 X 1 0 V . 7 X 10" 2.3 X 101 8 X 10' 3.2 X 1 0 M . 4 X 10' 10' 2 mi" 7.6 X 1 0 4 2 X 1 0 8 8 . 4 X 10' 2.3 X 1 0 4 2 X 10' 3.8 X 10" 10' 4.2 x 10' 1.2 X 10' 1W 0 ~ . 5 X 1 O ~ X X O ~ . 7 X 1 40. 7' X 1 0 8 4 x 1 0 ' 7.6X10' 2 x 1 0 1 8.4X10' 2.3X103'2XlO' 3.8 X 102 1 0 V . 2 X 10' 1.2 X 10' 10' 1.5 x 10' 4 x 104 1.7 x 1 o q . 7 x 10s 4 x 1 0 76 2 X 10' 8.4 X 10' 2.3 X 10% 2 X 10'

Journal o f Chemical Education

4.4 2.2 8.7 4.4 2.2 8.7 4.4

X X X X X X X

10' 4 X 10' 1.6 X 10" 4.4 X 10' 10X 10' 8 X 1 0 q . 2 X lo4 103 8 X 10' 3.2 X 10' 8.7 X 10" 10" X l O V . 6 X 104 4.4 X 10" 10' 2 X lo1 8 X 1 0 U . 2 X 10' 10% 8 X 10' 3.2 X 1 0 a . 7 X 10% 4 X 10' 1.6 X 10' 4.4 X LO' 1Ua 5 min 4 X 10' 8 X 10Q.6 X 10' 1.1 X 10' X 10X lo' 1.8 X 10' 5.5 X 10" 8X101 1 . 6 X 1 0 V . 1 X 1 0 ~ 2 . 2 X 1 0 ' 4x10' 8 x 1 0 ' 3.6X1OV1.1X1O8 2 x 1 0 ~ 4x10' 1.8X10V5.5X101 80 1.6 10' 7.1 x 10' 2.2 lo* 8 X 1 0 V . 6 X 10' 1.1 X 10' 40

10' 1.3 10' 6 . 5 10m.6 10' 1.3 10' 6.5 10P 2.6 10' 1.3

1.1 X 10' 5.5 x 102.2X10; 1.1X10 5.5~10' v . z x lol 1.1 X l o P

X X X X X X X

x

x

REFERENCES (1) HABERER,K., AND KOLLE,W., Atomprazis, 11, 1 (1965). T., Anal. Bioehem., 22, 70 (2) CLAUSF-N, IlQRR) ~-..-,. (3) ELRICH,R. H., AND PARKER,R. P. (a) Int. J . Appl. Radial. Isotopes, 14, 71 (1963); (b) ibid., 19, 263 (1968). (4) MATTHEWS, H. K., J . Chromatog.,36, 302 (1968). S., in (5) TAKUMI,K., A N D YAMADA, "Nuclear Electronics," Vol. 111, p. 459, I.A.E.A., Vienna. (1962).

RAPKIN,E., Pieker Nuclew Lab. Scintillator, 11 (6L) (1967). Scnnnw., E.., "Flow-Monitorine of Aouwus Solutions Cont&ine Weak @-Emitters," presented at the International Symposium an The Current St,stus of Liquid Scintillation Counting, Cambridge, Msss., April, 1969. To appear in "The Current Status of Liquid Sointillation Counting," E. D. BRANSOME, ed., Grune and S t r a t ton, New Ynrk, N. Y., in press. (10) SCHRAM, E. AND CROKA~RT, R., Proc. Biochm. Soe., 66, 20P (1957).

R., (11) SCHRAM,E., A N D LOMBAERT, Anel. Chim. Ada, 17, 417 (19.57). (12) STEINBEBO,D., Nature, 182, 740 (1958). (13) STEINBEBO,D., Ibid., 183, 1253 (1959). D.. Anal. Biochem., 1.. (14) , , STEINBERG. 23 ( m o j . ' (15) PIEZ,K. A., ibid., 4, 444 (1962). (16) WILLIAMB,R. T., AND BRIDGES, J. W., J . Clin. Path., 17, 371 (1964). (17) RAPKIN,E., Packard Technical Bulletin. No. 11. Fehruarv. 1963. (18) " ~ u i d e to scientific Instruments," Sciace, 162A, No. 3856A, 82 (1968); 165, No. 3900, 83 (1969). (19) Nuclear-Chicago Technical Bullelzn No. 15, 1963. (20) MARTIN,J. K . , A n d . Biochmn., 22$ 238 (1968). (21) ROBA,J., RoNcuccr, R., AND LAMBELIN, G., Anal. I~tlers,2 (6), 325 (1969). K. H.. HEWETT,A. J. W., (22) CLIFFORD, A N D POPSAK, G., J. Chromalog.,40, 377 (1969). E. A N D LOMBAERT, R., Anal. (23) SCHRAM, Bioehem., 3, 68 (1962). E., "Org.gitnic Scintillation (24) SCHRAM, Detectors," Elsevier, Amsterdam, 1963. (25) Honnoc~s, D. L., ed., "Organic Scintillators," Gordon and Breach, New York, N. Y., 1968. (26) Moanrssr, A. A,, private communication. (27) PROCELLATI, G., A N D n d ~ s o ,F., J . Label. Compounds, 3,205 (1967). A., (28) FUNT,B. L.,A N D HETHERINGTON, Science, 129, 1429 (1959). E. T. A N D BCEBI,:, (29) MCGUINNESS, J. L., J Label. Compounds, 3, 419 (1967). (30) ORII, Y., TSUDZUKI, T., A N D OKUNUKI, K., J . Biochem. (Tokyo), 58. 373 11965). , . (31) BENESCH, R. A N D BENESCH, R., Determination of Solfhydryl Groups in Proteins in "Methods of Biochemical Analysis," I). Glick, ed., Vol. 10, p. 43, J. Wiley & Sans, New York, N. Y., 1962. , AND (32) LEACH,S. J., M C S C I I ~ SA., SPRINGALL, P. H., Anal. Biochem., 15, 18 (1966). M. AND MCGUINNESS, E. T. (33) CULLEN, "A Suspended Scintilliltnr il4ethod for the Det.ection of Radioactive Mercurials in Protein Structure Studies," Abstract of Papem, 156t,h National A.C.S. Meeting, Atlant,ic City, N. J., September, 1968. E. T. (34) CULLEN,M. A N D MCGUINNESS, J . Label. Compounds, 5 (2), 152 (1969).

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Coming in the nezt issue: "Pulse Polarography" by Dr. David E. Burge.

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Journal o f Chemical Education