rotating arm engages the flat edge of the card. The potentiometer arni is geared to the final digit of the print wheel by a worm gear whose ratio corresponds to the range covered by the potentiometer --e.g., if the resistance of the potentiometer covers 1.2 decades in 3603 of rotation, the reduction between the final digit of the printwheel and the niper arm is 120 to 1. Suitable potentiometers wound to these specifications are nom available through E.3I.I. Electronics, Ltd. (Salisbury), South Australia. To avoid ambiguity in the position of the final digit a n indexing mechanism has also been incorporated on the main shaft. This consists of a 10-toothed star wheel, about 7 / ~ inch in diameter, pinned to the main shaft. 4 pawl attached to a fairly heavy solenoid (1- to 2-pound pull), engages the star ahpel when the solenoid is energized, pulling it around into a position corresponding t o an unambiguous, in-line digital read-out. This solenoid “locks” the whole main shaft while the printing movement takes place. The Servobalancing Amplifier. The circuit for this unit is shown in Figure
4. The transmitting and receiving POtentiometers attached to the photometer scale drum and the print wheel servomotor, respectively, form two arms of a Wheatstone bridge, across which is plnccd a small alternating current voltage (6.3 volts from transformer 2‘). The out-of-balance voltage IS amplified, and after a phase inversion,
is fed to the print wheel servomotor t’o drive it to a position of balance. OPERATION AND CALIBRATION OF PRINT-OUT MECHANISM
The sequence of events involved in making a reading is as follows: The photometer servomotor drives the optical Yedge, scale driini, and 10,000-ohm linear potentionieter to a position of balance. The print R heel servomotor drives the print wheel and exponential POtentiometer to a position of balance with the 10,000-ohm linear transmitting potentiometer. The indexing solenoid is energized, locking the main shaft. The printing solenoid is energized and released, pressing the print wheel onto the ribbon and paper. The indexing solenoid is released, freeing the main shaft for the next reading. The tabulator solenoid is energized, operating the tabulator mechanism and moving the carriage t o the next column. Once in every cycle the carriage return motor is switched on to reset the carriage. This sequence of events is controlled by a sequence timer of the type described by Simmonds (3) and Sininionds and Ro\>-lands (4). Such a sequence timer is normally required for the operation of the equipment associated with the print-out mechanism, and so the extra contacts for the above switching operations can readily be incorporated.
Three print-out mechanisms of the type described have been constructcd and tm-o have been used successfully for over a year. The first utilized the switched exponential resistance bank, while the second two used exponentially wound potentiometers. Each was calibrated by rotating the scale drum of the photometer manually and noting the absorbance readings on thc drum and on the print wheel. respectively. Excellent agreement vias achieved in both caqeq. ACKNOWLEDGMENT
It is a pleasure to acknoidedgc tlic assistance of K. I. TTood, who designed and constructed the print wheel servoamplifier, and W.J. Sutherland and E. h’. Paton, Paton Industries Pty., Ltd., Adelaide, South Australia, who constructed the print wheel assembly and carried out the modifications required on the typewriter. The assistance of J. C. Cockram, E.RI.1. Electronics (Sslisbury), Ltd., in the development of the exponential potentiometer is also gratefully acknon-ledged. LITERATURE CITED
Evans, D. S., Brit. Communications and Electronics 4, 334 (1957). (2) Hibbard, L. U., Piddington, J. H., J. Sci. Instr. 24, 92 (1947). (3) Simmonds, D. H., ANAL. CHEW30, 1043 (1958). (4) Simmonds, D. H., Rowlands, R. J., IbLd., 32, 259 (1960). RECEIVED for review April 2, 1959. Accepted October 14, 1959. (1)
Automatic Equipment for Simultaneous Determination of Amino Acids Separated on Several Ion Exchange Resin Columns D. H. SIMMONDS and R. J. ROWLANDS Department o f Agricultural Chemistry, Waite Agricultural Research Institute, University o f Adelaide, South Australia, and Division o f Protein Chemistry, Wool Research laboratories, Commonwealth Scientific and Industrial Research Organization, Parkville N. 2, Victoria, Australia
b Construction of automatic equipment to monitor the effluent of eight ion exchange chromatography columns simultaneously was undertaken to speed amino acid determinations b y the methods of Moore, Spackman, and Stein. A unit fractionates the effluent from each column, treats each fraction with ninhydrin reagent, dilutes, and estimates the resulting color. The measured absorbances are recorded as a printed column of figures on paper tape. This record requires only integration to complete the analysis.
Six analyses for the common amino acids can b e completed in 48 hours. Different samples can b e analyzed simultaneously on different columns, allowing closer and more accurate intercomparison of results. Use of the equipment to analyze four insulin hydrolyzates is described.
A
capable of automatically determining amino acids in mixtures or in protein hydrolyzates has been described (IO). Subsequent MACHINE
publications ( 2 , 8) describe its performance and compare the results with analyses carried out manually by the techniques of Rioore and Stein (5, 6) and Moore, Spackman, and Stein (4). This paper describes a machine which automatically estimates the amino acid composition of up to eight different samples simultaneously. The equipment controls the operation of eight ion exchange chromatographic columns, and presents the results from each as a printed record. This is easier to VOL. 32, NO. 2, FEBRUARY 1960
259
interpret and use as a basis for subsequent calculations than the recorder trace previously obtained. As well as an increased output, this equipment has greatly increased precision because of closer control of analytical variables. DESCRIPTION
OF
EQUIPMENT
The equipment works on the same basis as the single-column machine (10). The processes carried out automatically are : Pumping of buffer solution through the chromatographic columns. Collection of uniform-sized fractions from the column effluent. Addition of ninhydrin reagent to each effluent fraction. Heating to develop the characteristic purple color. Dilution with ethyl alcohol-n ater (1 to 1 v ./v. ). Photometric estimation of the diluted color. Production of a printed, digitized record of the absorbance of each effluent fraction from each column. Because each operation is carried out on up to eight columns simultaneously, the apparatus is more complex than the single-column machine previously described. Pumping Equipment for Buffer Solutions. The glass pumps were modified from t h e design of Edman, Funk, and Sjoquist (I), because certain other commercially available types were unsuitable. Spackman, Moore, and Stein ( I S ) indicate that the Milton Roy Minipump may be made sufficiently accurate for the present aplication by suitable mechanical adjustment. Each pump consists of two carefully ground pear-shaped valves with a small hypodermic syringe (Alga microburet syringe. Burrough. Wellcome and Co., London, England, or 1-cc. tuberculin syringe. Becton, Dickinson & Co., Rutherford, N. J.) located between them. -4 two-position tap enables changes to be rapidly made in the composition of the inflowing buffer solutions. The two inlets below the tap are connected by plastic tubing to separate manifolds and thence to conveniently located 2-liter aspirator bottles which serve as buffer storage containers. Connection to the top of the columns is made through polythene (4 mm. in internal diameter) or Teflon tubing (2 mm. in internal diameter) and a B14 standard-taper joint carrying side lugs for springs. The syringe plunger is operated by a mechanical assembly shown in Figure 1. A chain and sprocket drive from the timing motor rotates the pump camshaft a t approximately 2 r.p.m. Each cam consists of a body and rotor, the latter bearing against the end plate of the pump push rod once each rerolution. Each push rod carries a universal socket and joint, permitting firm but flexible connection to the hypodermic syringe plunger. Return travel is effected by a strong spring located 260
ANALYTICAL CHEMISTRY
around the push rod between the shaft bearings and the end plate. A micrometer stop limits the return travel as the cam rotor moves away from the end plate. The pump units (one for each column in use) are mounted on a base which is bolted to the machine framework (Figures 9 and 10). All parts are chrome-plated, with the exception of the
B CD
Figure 1 . Pumping buffer solutions A. B.
C. D.
E. F.
G. H. 1. J. K.
equipment
for
Micrometer stop adjustmeni Universal joint assembly 1 -cc. hypodermic syringe Clamps for locating syringe Pear-shaped glass valves 2-way tap Rotating cam End-plate of pushrod Return spring Base Push rod
universal coupling between the pushrod and hypodermic plunger, which is aluminum, and the camshaft and push rods, uhich are stainless steel. The push rod bearings are canvas insertion Bakelite. The camshaft runs through four accurately aligned ball bearing races. Chromatography Columns and Ion Exchange Resins. The glass-jacketed chromatography columns (11) are closed a t the bottom with a replaceable sintered disk and length of fine poly(1-inyl chloride) or Teflon tubing (1 to 2 mni. in internal diameter) (Figure 2). This allows the minimum volume for miving below the sintered plate, and pcrmits the columns to be arranged on a rack a short distance array from the machine itself. A volume of up to 1.25 nil. entrained by the tubing does not significantly affect the resolution of the amino acid bands. Dowex 50-X8 and Aniberlite CG-120 ion exchange resins have been used. They \\-ere hydraulically classified before use by the method of Hamilton ( 3 ) using a &liter separating funnel. Particles 35 to 70 mw in diameter were collected at flow rates of 300 to 800 ml. per minute a t a water temperature of 15' C. The buffer solutions and other operating conditions TI ere those described bv Moore, Spackman, and Stein ( 4 ) . Effluent Fraction Dispensers. T h e outlet tube from each chiomatography column is clamped above a fract,ion dispenser. These collector tubes (Figure 2) are a modified form of that described previously (10) I
1 INCH
Figure 2.
Replaceable sintered plate and coupling assembly for bottom
of chromatography columns and effluent fraction dispenser A.
B.
C. D. E. F.
G. H. 1. 1. K.
0.9 X 150 or 0.9 X 2 0 cm. Dawex 50-X8 or similar ion exchange column ground level a t bottom Water-circulating jacket Inlet for constant temperature circulating water Plastic (PVC) tubing sleeve Sintered glass plate 1 cm. in diameter X 2 to 3 mm. thick; porosity 1 Rubber or plastic stopper Hypodermic syringe needle to suit tubing H PVC or Teflon tubing 1 to 2 mm. in internal diameter Fraction dispenser cup Valve-lifting solenoid Metal in glass valve
The glass-enclosed metal valve is lifted by the solenoid coil surrounding it at a predetermined time interval, allowing the collected contents to flow into the appropriate heating vessel just below it. The buffer is pumped through the columns at a constant rate of 2 nil. in 10 minutes adjusted by alteration of the micrometer Retting on the end plate stop of the buffer pumps. Each fraction dispenser carries a graduation mark a t the 2-ml, volume t o allow accurate adjustment of fraction size.
Reagent, Diluent, and Wash Liquor Dispensers. Lack of mounting space a n d difficulties with individual adjustment of delivery volume led t o t h e adoption of a single cam-operated pump for each solution rather t h a n eight reagent a n d diluent dispensers of the type originally described (IO). The outlets from the reagent and diluent pumps are connected via a rotating eight-way t a p (Figure 3) to
each of the eight heating vessels in turn. The outlet from the wash liquor pump leads directly to a nozzle mounted above the cuvette, so that the wash liquid is directed down the walls to rinse the cuvette thoroughly after each reading. Two pump designs have been tried. I n the first the cams are driven by a separate electric motor suitably reduced in speed, which can be snitched on and off as required by the fast sequence timer. I n the second design the camshaft operating the three puinps is driven by chain and sprocket from the main timing motor a t the same speed as the fast sequence timer. The latter design is more foolproof but less flexible in operation, because by suitable wiring, the electric niotor in the first design can be switched off if one or more columns are not in use. The eight-i\ay tap consists of two circular plates 2 inches in diaiiieter Teflon faced joined by a h a f t inch in diameter mounted on tu,o self-aligning ball races. Spring-loaded against the rotating Teflon plates are t n o ground a i d polished qtainless steel plates carrying a. central inlet and eight peripherally arranged outlet tubes. The Teflon plate has a groom cut in its face, so that as it rotates the central tube is joined in turn to each of the peripheral tubes.
H
N
SIDE VIEW Figure 3. Reagent, diluent, and wash liquor dispenser with associated eight-way rotating tap A. Stainless steel plate carrying one inlet and eight outlet tubes for reagent solution 6. Springs for holding stainless steel against Teflon plate Rotating Teflon plate Cams to operate syringe plungers Axle between two eight-way taps Bearing housing G. Eight-toothed step advance g e a r H. Connecting link between cam and syringe 1. 2-mi. hypodermic syringe for reagent J. 20-mi. hypodermic syringe for wash liquor K. All-glass pump valve units 1. 1 0-mi. hypodermic syringe for diluent M . Return spring to operate syringe plunger N. Stainless steel outlet tubes on diluent eight-way t a p connected to corresponding-heating vessel 0. Stainless steel inlet tube connected to outlet of diluent pump P. Main drive shaft C.
D. E. F.
Two driving arrangements of the eight-way t a p unit have been tried. I n the first, this was driven continuously a t the same speed as the slow sequence timer-Le., 1 revolution in 10 minutes. A simpler and more compact arrangement is shown in Figure 3, where the eight-way t a p is located above the cam shaft of the dispenser pump and is advanced stepwise to the next position each time this revolves b> a singletoothed gear nhich engages an eighttoothed gear on the eight-way tap shaft, This ensures that the holes are accurately aligned during each pumping stroke. Both arrangements allow liquid from the reagent and diluent dispenser pumps to be distributed to each heating vessel in turn. The bottom of each dispenser is connected with plastic tubing to the appropriatp storage container-a Miter aspirator in the case of the reagent dispenser, and a 20liter aspirator for the diluent and wash dispensers. Heating Vessels and Supporting Framework. The heating vessels (Figure 4) are arranged in a circle belon t h e fraction-collecting tubes. They are modified from t h e design previously described (10). The outlet of each heating vessel is extended and ground off as illustrated, so that the required number may be arranged compactly around the funnel-shaped opening of the cuvette. Heating vessels are accurately 10VOL. 32, NO. 2, FEBRUARY 1960
261
cated between the fraction-measuring dispensers and the cuvette by clipping them to a framework as illustrated in Figure 4, bvith suitable spring clips (Terry, size 81/3, Terry Spring Manufacturing Co., Birmingham, England). This framework also houses the steam supply manifolds, and provides a clamping bracket for the wash liquor nozzle. Photometer Cuvette. This has been designed t o eliminate t h e necessity for providing a separate diluting vessel, and t o permit aeration and agitation to be carried out 1Tith the minimum incorporation of fine air bubbles into t h e solution. The location of the valve beloJy instead of within the cuvette has enabled the air inlet and light path to be located a t the bottom of the tube, so that small air bubbles are more rapidly eliminated from thia area. This has allon ed the actual taking of readings to be greatly speeded up. The construction of the cuvette has also been simplified by having a circular instead of a square cross section where the light beam passes through. The solenoid coil, controlling the cuvette valve, operates against a light phosphorbronze coil spring which ensures accurate seating of the valve. Self Balancing Photometer. An automatically photometcr is used for measuring t h e absorbance of each effluent fraction. T h e instrument employed is a S i g h t Photoiiieter (Type UP2LD, manufactured by Sigrist a n d Keiss, Ltd., Falkenstrasse 23, Zurich 1/8, Snitzerland). The working principle and modifications required to the standard instrument have been described (10). The 1000-ohm linear potentiometer attached to the rotating shaft carrying the scale drum and optical wedge is replaced by one of 10,000-ohm resistance. Because the drum rotates through only 240' while the wedge moves from open (100%. transmittance) to closed (0% transmittance) , the potentiometer is aligned so that one stop coincides
-
-
Figure 4. A.
6. C.
D. E. F. G.
Arrangement of heating vessels
Fraction dispenser and solenoid coil Solenoid coil to operate heating vessel valve Cooling jacket of heating vessel Diluent inlet tube Reagent inlet tube Steam heating jacket Tube in which color reaction takes place
h'. 1. J.
Valve closing heating vessel Wash liquor nozzle Funnel top of cuvette K. Overflow tube 1. Heating vessel supporting framework M. Iron core
MOTOR SHAFT
38
stow I REV/IOMIN.
I R E V / I%MIN INTERMEDIATE SHAFT
Figure 5, Arrangement of sprocket wheels required to drive buffer pumps, fast, and slow sequence timers Chain size, 8-mm. pitch. Figures refer to number of teeth on each sprocket wheel required to produce desired speed ratios
262
ANALYTICAL CHEMISTRY
-
Figure 6. Cam and switch arm arrangement in eight-way slow sequence timer A. One member of switch arm pair 6. One member of cam pair C. One member of switch contact pair
D.
E.
Spring loading Framework
with the 100% transmittance end of the scale. A second connection is then made at a position corresponding to 5.5% transmittance. This limits the absorbance range which can be recorded to 0.0 to 1.23, a range which should not, in a n y case, be exceeded for accurate analysis.
speed of 9 r.p.m. is suitable. The arrangement of sprocket wheels and gears required to drive the buffer pumps and the fast and slow sequence timers a t the correct speeds for a n eight-column machine is shon-n diagrammatically in Figure 5,
Automatic Print-Out Mechanism. T h e output from t h e linear potentiometer coupled t o the scale drum shaft of t h e photometer could be recorded by a multipoint 10-mv. recording potentiometer ( I O ) . However, in the present system, processing the large number of tracings becomes very time-consuming, especially if the recorder is not provided with an csponential potentiometer. To overcome these disadvantages, the digitizing print-out mechanism described by Simmonds and Roivlands (22) was devised. This permits each photometer reading to be automatically converted into absorbance units and printed onto a roll of paper. Thus a column of figures is produced for each chromatography column being monitored, each figure being the absorbance of one 2-ml. effluent fraction. Sequence Timers and Timing Schedule. T h e sequence of manipulations is controiled by t h e sequence timer, made of two parts: a fast cam-operated program switch similar to that previously described ( I O ) (this controls units required to operate eight tinies in each complete sequence cj-cle) and a slow cam-operated snitch controlling units to be opcmted once only in each complete c>.clc. The shaft speeds of the fast and slon sequence timers are in the ratio 8 to 1. Both switches are driven through chains and sprockets by the motor unit which also operates the pumps A geared motor having a n output shaft
The main shaft of each sequence tinier is extended through the front panel of the apparatus, and carries a hand to indicate its position. The slow clock hand moves around a face graduated to show n-hich column is being switched a t any particular time. The fast clock face may be graduated in seconds, or mith the switching operation being performed at any instant. The exact speed a t n hich the sequence timers rotate depends upon the number of columns being treated by the machine and the time cycle being used. The reagents of Moore and Stein (7) and Spacknian, hloore, and Stein ( I S ) have both been tested with a time cycle of 10 minutes. This allows the sample to be heated with the reagent for 91/2minutes. If the columns are loaded about noon on the first day, with a 10-minute cycle, the buffer change occurs a t 9 ,4.x the following morning, just after the emergence of alanine. A 12-minute cycle allons a n analysis for acidic and neutral amino acids through to 8-alanine t o be completed in 44 hours and may be more convenient for timing the buffer change. Color production n i t h the stannous chloride-ninhydrin reagent of Spackman, Moore, and Stein ( I S ) n-as incomplete even after 60-minute heating under these aerobic conditions. Furthermore, the color produced after long heating n-ith an amino acid solution was a brownish purple instead of the ckar purple obtained n i t h the hydrindantin-ninhydrin reagent of
i iNcn L
I
Figure 7. Alternative selector switch for slow sequence timer A. B. C.
D. E.
Connector strip Rotating contact Bakelite mounting board Brass sector Rotating selector arm
F. G. H. 1.
J.
Central brass ring Engraved dial on outside front panel of equipment Driving shaft Spacing bolts and collars Driving sprocket and chain
Moore and Stein (7). The use of the stannous chloride-ninhydrin reagent, which in a n y case is recommended only for color development under anaerobic conditions, was therefore abandoned. If the reagent described by Rosen (9) is used, a 10-minute cycle should also be satisfactory, but this has not been fully tested. If eight columns are being treated simultaneously, the fast clock rotates eight times per cycle of 10 minutes; if the apparatus is built to handle four columns simultaneously, it rotates four times per cycle, and so on, The slow sequence timer rotates once per cycle. FASTSEQUENCE TIMER.This unit is similar t o t h a t described by Simmonds ( I O ) . A total of 24 cams and arms are required to carry out the snitching operations in each sequence. S L O W SEQUEXCE TIhCER. T T Y O types of slow sequence timer have been used. I n the first, eight pairs of contacts, or the same number of pairs as there are columns being treated, are arranged around each cam pair as shown in Figure 6. Two pairs of cams and two sets of eight pairs of contacts are required, one to operate the fraction diqpensers and one the heating vessels. As the cam pairs rotate they activate each fraction dispenser (1 to 8) arid each heating vessel (1 to 8) in turn. A third pair of cams lifting on1:- one pair of contacts operates the printer csrringe returnmotor ( l a ) . The advantage of this type of snitch is that individual toggle switches can be wired across each set of contacts to allow manual operation of each fraction dispenser and heating vessel valve as required. This is convenient for washing out the glassn-are a t the end of each analytical run. The second type of slon sequence timer used successfully is a multiple selector sn-itch (Figure 7 ) .
It consists of two l,’&icli Bakelite boards mounted rigidly one above the other and spaced about 1’ inches apart, Into each board is recessed a central bearing or hole, a brass ring surrounding this and a number of brass segments, one segment for each column being controlled. A central shaft. rotating once in every 10-minute cycle, carries two Bakelite arnis which. as they rotate, connect the central brass ring with each outer segment in turn. The segments (1 to 8) in one selector bank are connected via a toggle switch to the corresponding fraction dispenser solenoids (1 to S), those in the second bank to the corresponding heating vessel solenoid coils. As the central shaft and selector arms rotate, each segment and, hence, if the appropriate toggle switch is on, each column train is in turn connected to the neutral line of the alternating current supply. The actual witching VOL. 32, NO. 2, FEBRUARY 1960
0
263
Table I.
Cam Kumber 3 and 4 7 and 8 7 and 8 1 and 2 9 and 10 9 and 10 11 and 12 23 and 24 15 and 16 15 and 16 23 and 24 19 and 20 21 and 22 21 and 22 19 and 20 13 and 14 11 and 12 5 and 6 13 and 14 17 and 18 17 and 18
Timing Schedule for Fast and Slow Sequence Timers
Fast ( F ) or Slow (S) Timer F F and F and F F and F and F F F F F F F F F F F
Time from Zero, Sec.
Operation Diluent dispenser operates Heating vessel valve lifts Heating vessel valve closes Reagent dispenser operates Fraction dispenser valve lifts Fraction dispenser valve closes Air supply motor switches off Ultraphotometer servomotor switches on Tabulator solenoid switches on Tabulator solenoid switches off Ultraphotometer servomotor switches off Print wheel indexing solenoid switches on Printing solenoid switches on Printing solenoid switches off Print wheel indexing solenoid switches off Cuvette valve opens Air supply motor switches on Wash dispenser operates Cuvette valve closes Carriage return motor sxitches on0 Carriage return motor switches offa
0 0
S S
7 8 8
S S
12 14 14 40 43 54
55
56 57 58 60 60 62 72 10 17
F F 8
S
This occurs once every revolution of the s l o ~sequence timer-Le., revolutions of the fast sequence timer.
once every eight
0
ACTIVE
112
3e4
5e6
I
I
gal0
-
lis12
- -
of the solenoid coils is then controlled by the cam-operated snap action switches of the fast sequence timer connected into the active line. The addition of a third selector bank connected to the neutral of the reagent dispenser motor allows reagent and diluent to be saved if any columns are not being operated. A disadvantage of the selector switch is that maiiual control of each heating vessel valve and fraction dispenser solenoid is complicated. Double-pole, double-throw snitches must be wired across the coils to supply both active and neutral; otherwise, the coil to which the selector switch is pointing is also activated when the toggle snitch is operated manually. The timing schedule and setting of the cams on these sequence timers are summarized in Table I. Electrical Wiring. The wiring diagram is s h o m in Figure 8. Slight modificatioiis t o the circuit may be required, depending on which slow sequrnce timer is used, and n-hether the reagent and diluent dispenser is electrically or niechanically operated.
CONTACTS ON FAST SEQUENCE Tlh'ER
13e14
-
15e.16 -
17~18 a
- -
19~20
( 1 REVOLUTION 21822
- -
/ Ik MINS)
- -
23924
2 4
9-2
Y
ULTRA PHOTOMETER
".
MOTOR SOLENOID
SOLENOID
u\ SLOW SEQUENCE TIMER
1
1
1
1
REAGENT DILUENT AND WASH LIQUOR DISPENSER MOTOR
3 - B A N K SLOW SEQUENCE Figure 8.
TIMER
1 REVOLUTION/
10 MINS.
Wiring diagram for complete eight-column automatic amino acid analysis equipment Motor-driven reagent, diluent and wash dispenser, and eight-position neutral selector switch
264
a
ANALYTICAL CHEMISTRY
after circulating in parallel through the heating jackets, is condensed and returned to the boiler. Alternatively a small thermosiphon unit (10) may be used to circulate boiling water through the heating vessel jackets. Recent experiments have s h o m t h a t adequate and reliable heating may be more conveniently obtained by winding small elements from resistance wire (28 s.w.g. Nichrome, 30 turns, resistance of each element 16 ohms) onto a n asbestos tube 3/4 inch in outside diameter, having a hole 5/8 inch in diameter through the center. These elements are then slipped over the inner tube of each heating vessel in place of the steam heating jacket (P, Figure 4). By connecting these in series to a 25-watt wire wound resistor and 110-volt
alternating current supply, the heat input to each heating vessel may be accurately controlled. COMPRESSED AIR SUPPLY. A small quantity of compressed air a t a low pressure is required for mixing the ninhydrin reagent and diluent solutions, and to oxidize residual hydrindantin. It is most conveniently supplied by a small aquarium aerating pumpe.g., Hyflo aquarium pump from Exotic Aquarium Supplies Pty.>Ltd., 302 High St., Northcote, Victoria, Australia. The flow of air is controlled by switching the pump motor on and off as required. If the flow of air through the cuvette is too strong, a bypass should be introduced between the pump and the cuvette. OPERATION A N D PERFORMANCE
Table II. Color Factors for Common Amino Acids Obtained by Use of Automatic Equipment
[These figures correspond to the reciprocal
of the “color yields” quoted by Moore and coworkers ( 4 , 6, 7 ) ]
Amino Acid Alanine Ammonia Arginine Aspartic Glutamic Glycine Histidine
Color Factor 1.00 1.08 0.95 1.00 0.97 1.00 0.98
Amino Acid Leucine Lysine Phenylalanine Serine Taurine Threonine Tyrosine“
Color Factor 1.oo 0.89 0 94 0 98 1 38 1 00
(y :3)
Isoleucine 1 08 Valine 1.oo a Because a color yield of 0.93 has given consistently low recoveries of tyrosine from insulin, this has been modified in subsequent work to 1.00, the value recorded by Moore and Stein (‘7).
Table 111.
,4 10-ml. ethyl alcohol-water wash after each solution has drained from the cuvette completely eliminates interference between highly colored aliquots coming from one column train and base line samples from the next column in sequence even under the extreme conditions encountered just after the loading of the short Dowes columns used for analysis of the basic amino acids. Linearity, Accuracy, and Stability of Optical and Recording Section. Standard solutions containing known concentrations of leucine were r u n through t h e machine. Figure 11 s h o m t h a t the relationship between concentration and color developed is linear u p t o a n absorbance of a t least 1.00, and t h a t satisfactory calibration is achieved b e t w e n the printed record and the dial reading of the pho-
tonieter. Using the stepping switch potentiometer described by Simmonds and Rowlands ( I d ) , a n even closer calibration was obtained. Base line stability is also very satisfactory during an analytical run, variations usually being limited t o ~ t 0 . 0 0 3 absorbance unit about the mean reading in the 80 to 100% transmittance range. Quantitative Recovery Experiments on Single Amino Acids and Amino Acid Mixtures Run through Columns. Solutions of purified amino acids (H. hl. Chemical Co., Ltd., Santa Monica, Calif.) either singly or in mixtures, were loaded onto t h e ion exchange columns a t different concentration levels. The total absorbance of each resultant peak, after subtraction of the base line contribution, was plotted against the concentra-
I
2
I
3
4
LEUCINE CONCN., y/P M L .
Figure 1 1, Relation of concentration to print wheel reading Leucine Concn.,
y/2 MI. 0 1 2 3
4
Photometer Reading
Print Wheel Reading
0.165 0.361 0.567 0.745 0.923
0.148 0.337 0.538 0.726 0.919
Summary of Analytical Results Obtained on Four Insulin Hydrolyzates
Results expressed as amino acid riitrogen as a percentage of the total nitrogen 20-Hour 45-Hour Hydrolyzate Hydrolyzate Hydrolyzate Xa Mean S.E.* Mean S.E.b Mean S.E.b 10-Hour
io-Hour
Theoretical NO. of ResiNitro- dues in 1I.W. 5937 gen, % Exptl. Theory
Hydrolyzate _ _ ~ Over-all Mean Amino .hid Mean S.E.b (m, Value Alaninec 3 4 77 0 26 5 24 0 33 4 98 0 07 4 9 3 0 10 4 98(miC 4 6 2 3.~ 2 3 Amide 6 7 67 0 26 8 . 1 6 0 14 8 58 0 07 8 28 0 42 8 . 5 8 ( 4 5 hr.) 9.23 5 . 6 ilrginine 6 5 39 0 36 6 08 0 I G 5 47 0 18 6 12 0 23 6 . 1 0 ( m 20-45 hr.) 6.15 1 . 0 Aspartic acid 3 4 69 0 14 4.68 0 17 4.64 ( m ) 4 8 1 0 14 4 53 0 09 4.62 3 . 0 Glutamic acid 3 10 20 0 25 10.58 0 20 0 i 9 0 49 0 42 0 10 10.50 ( m 10.77 6 . 8 Glycine0 1 0 1 0 22 ... 3 10.77 0 17 10.10 1 33 1 50 0 40 6.15 . . .b Histidine 6 9 11 0 40 9.41 9 50 0 37 9.62 0 44 9.23 2 . 0 9 40 0 :35 Isoleucine 1.52 3 0 . 7 7 0 04 1 55 0 16 1 . 2 6 0 05 1.54 0 . 9 9 1 48 0 06 Leucine 8 77 0 09 9.11 9 62 0 47 3 9 . 0 5 0 14 8 99 0 14 9.23 5 . 9 6 Lysine 3.20 3 26 0 0 2 3 03 0 08 3 32 0 07 3 18 0 14 3.08 1.0 Phenylalanine 3 4.38 4 30 0 05 4 . 3 5 0 14 4 63 0 22 4 22 0 18 4.62 2 . 9 4.23 (m 10-20 hr.) 4 24 0 14 4.62 2 . 8 Serine 4.22 0 04 3 3 06 0 10 3 83 0 22 1.52 (20 hr.) Threonine 1 26 0 06 3 1 38 0 03 1 49 0 10 1 , 5 3 0 07 1 . 5 4 0.99 Tyrosined 5 . 8 9 (m 10,20. 45 hr.) 6 . 1 5 3 . 8 3 5 29 0 18 4 5.74 0 16 5 95 0 09 5 98 0 33 7 56 0 23 7.68 (m 45-70 hr.) Valine 6 . 2 1 0 05 7.69 5 . 0 7.28 0 19 3 7 81 0 36 5 a Number of observations on each hydrolyzate. Standard error of mean. Incomplete separation of cystine from alanine and glycine caused these results to be variable and high. Subsequent analytical runs on 20- and 70-hour insulin hydrolyzates in which cystine was completely separated from glycine and alanine, gave 6.14 f 0.09 and 4.62 i 0.10, respectively, for these two amino acids. These values correspond very closely to theoretical figures. Color factor of 1.00 used, see footnote to Table I.
266
ANALYTICAL CHEMISTRY
ASPARTIC ACID
THREONINE SERINE
tion of a-amino nitrogen in that peak. In every case the resulting graphs nere linear, provided that the highest absorbm c e recorded did not exceed 1.10. Tlw color factors relative to leucine obtiined in this way are sunimariz~d in Table 11. These figures hare been i n all subsequent calculations.
Run on Standard Insulin Four samples (50 mg.) of insulin (Boots Pure Drug Co., batch 2189, 28/11/55, prepared as a standard reference protein), uere 11eighed into hydrolysis tubes, cons t a n t boiling hydrochloric acid (A rid.) was added t o each, and the tubes nere evacuated and sealed. They uere hydrolyzed in an autoclave a t 110" C. for 10, 20, 45, and 70 hours, respectively, after which the contents of the tubes were evaporated to dryne5s and made up to 20 nil. with distilled n atcr. Samples of each hydrolyzate (214 pl.) were loaded in random fashion onto six 150 Dowex 50-XS columns Analytical Preparation.
GLUTAMIC ACID
arranged around the equipment for the determination of acidic and neutral amino acids. The basic amino acids (in 0.5 ml.) were determined simultaneously on two 20-cni. Dowex 50-X8 columns occupying the last two positions of the equipment. One of the analytical records is presented in Figure 12, with a manual plot of the printed absorbance readings. The results of the analyses are summarized and compared n i t h the theoretical composition in Table 111. This table shows that the calculated results agree very closely with the theoretical composition except in the case of ammonia and glycine. It is possible that the amide content of this sample had been reduced during the purification steps. Contamination of the glycine peak by cystine was observed in several of the effluent graphs, but quantitative recoveries of the former were obtained in subsequent analytical runs on the
GLYCINE
ALANINE
20- and 70-hour hydrolyzates in which complete separations were observed. CONCLUSlON
The equipment described, although complex, consists of a logical assembly of several smaller and simpler units. I t s construction should therefore not be too difficult for the average instrument workshop. For most analytical requirements, a four-column machine should have sufficient output, although both machines constructed by the authors have had eight columns, being designed to run continuously with a spare set of eight columns being regenerated while the first set is in use. By enabling multiple determinations to be carried out simultaneously under identical operating conditions, a much closer control of experimental variables is possible, and accuracy is greater. The average standard error has been reduced to about two VOL. 32,
NO. 2, FEBRUARY 1960
267
thirds of that obtained using the manual method. Finally, although designed primarily for amino acid determinations by ion exchange chromatography, this type of equipment with but few niodifications could be adapted to carry out many time-consuming analytical determinations a t present requiring a considerable amount of manual labor. All that is required is a technique for introducing the analytical samples in sequence into the system, which then carries out the steps involved in the colorimetric or turbidimetric assay and records the results.
land in thc construction of this equipment, and by W. B. Hall, Division of Mathematical Statistics, C.S.I.R.O., Kith the experimental design and calculations involved in the standardizing procedure. Thanks are also due to Monica McShane, P. R. Ravenscroft and Steven Gardonyi for technical assistance. The authors particularly thank E. N. Paton, Paton Industries Pty., Ltd., Adelaide, South Australia, for his considerable help in the engineering development and construction of this and subsequent machines. LITERATURE CITED
(1) Edman, Pehr, Funk, Hans, Sjoquist, ACKNOWLEDGMENT
I t is a pleasure to acknorledge the assistance rendered by W, J. Suther-
John, Kgl. Fysiogruj. Sallskap. Lund
Forh. 26, KO.12, 121-4 (1956). (2) Gillespie, M. J., Simmonds, D. H.,
to be submitted to iiustraliun J. Bzol.
Sa.
(3) Hamilton, P. B., ANAL. C H m . 30, 914 (1958). (4) Moore, S., Spackman, D. H., Stein, W. H., Ibid., 30, 1185 (1958). (5) Moore, S., Stein, W. H., J . Bid. Chem. 192, 663 (1951). (6) Ibid., 211, 893, (1954). ( 7 ) Ibid., p. 9Oi. (8) Rogers, G. E., Simmonds, D. H., Nature 182, 186 (1958). (9) Rosen, H., Arch. Biochem Biophys. 67, 10 (1957). (10) Simmonds, D. H., ~ A L CHEAI. . 30, 1043 (1958). (11) Simmonds, D. H., dust>alian J . Bid. Sci. 7, 96 (1951). (12) Simmonds, D. H., Rowlands, R. J., ANAL. CHEN.32, 256 (1960). (13) Spackman, D. H., Moore, S.,Stein, W. H., Ibid., 30, 1190 (1958).
RECEIVEDfor review ilpril 2, 1959. Accepted October 14, 1959.
Photometer for Continuous Determination of Uranium in Radioactive Process Streams F.
A. SCOTT and R. D. DIERKS
Hanford labpratories Operation, General Electric Co., Richland, Wash.
b Uranyl nitrate concentrations in process streams can be monitored colorimetrically. However, commercial process stream photometers cannot easily b e applied to highly radioactive streams. The instrument described is a modified single-beam filter photometer. Because of a unique standardizing system, it is very stable, having a short-term variation of less than 1% and a calibration stability of 3%. The instrument has a remotely located sensing unit with the ruggedness and simplicity required for production plant applications. Its use in process stream monitoring can yield important process data and increase plant operating efficiency.
T
success of a n automated chemical process control system depends, in part, upon its ability to obtain a flow of up-to-the-minute information from key points in the chemical process. Because the most practical method of providing such information is by using continuous process stream analyzers (Z), instrument manufacturers have been adapting more and more laboratory equipment for continuous field service (4, 5 ) , giving the process control engineer a n ever-n idening field of instruments from which to build a control system. Hon-ever, in plants processing irHE
268
ANALYTICAL CHEMISTRY
radiated uranium, the usefulness of many of the early continuous analyzers was sometimes compromised by the hazards associated with radioactive solutions. Such things as large sample holdup, proximity of the sample to operating or maintenance personnel, or general complexity, all posed personnel radiation exposure problems that eliminated their use. As a result, a program to design and test an instrument for the determination of uranium in radioactive streams mas initiated a t the Hanford Laboratories. This paper rerorts the results of this program, which has paralleled similar programs a t other laboratories (3, 6). The Hanford Laboratories instrument is a filter-type photometer, similar to the conventional single-beam photonieter in that it employs a single light path and a single light intensity detector. However, by the alternate positioning of t n o filters in the transmitted beam, many of the desirable features of the more complex dual-beam photometers are obtained. I t s operation is simple. Periodically, the intensity of the light source is automatically adjusted so that the apparent incident light on a sample solution is held constant, this being indicated by the intensity of the transmitted light beam a t 535 mp. The uranium concentration of the sample solution is then indicated directly by
the intensity of the transmitted light beam a t 420 mp. The problems of radiation exposure of operating and maintenance personnel have been minimized by three design features. First, the electronic equipment is separated from the sensing unit and is located outside radiation zones, facilitating routine maintenance. Second, the sensing unit is miniaturized, reducing the sample holdup, shielding requirements, and radiation levels in general. Finally, the sensing unit is kept simple, with a minimum number of easily replaceable parts, minimizing maintenance time in radiation zones. DESCRIPTION
OF
INSTRUMENT
The sensing unit (Figure 1) consists of a light source. a sample cell. t n o light filters, a phototube, and a filterpositioning air cylinder. Because the photometer monitors a continuously flowing sample stream and does not require periodic introduction of standardizing solutions, it is installed directly in the sample strenni n-ithout valving. A gasketed cover (not shown) protects the optics from dust and moisture and inadvertent dousings during n-ashdon-ns or decontamination operations. A bayonet-type, pilot light socket (Dialight Corp. of America, Yo. 91410931) housed in a slightly modified ( l l / l c inch 27NS-2B threads added) cable connector (American Phenolic Corp. No. AF3106.4-168) forms the light
