CONCLUSIONS
In the past, computerized chemical analysis systems have not been widely accepted in the laboratory because an easy, conversationql programming language has not been available. Although LABTRAN is not as flexible as other higher level languages, e.g., BASIC, its simplicity and interactive editing features greatly speed the programming of chemical analysis procedures. The sequential testing principle, although described in a clinical laboratory setting, has applicability in other testing areas where one must first discern a malfunction and then pin-point its cause through analytical procedures. A new configuration using a 24-port valve with the elimination of the holding coil is presently being incorporated into the system. The possibility of carryover will therefore be greatly minimized. The addition of a computer-operated, spectrophotometer wavelength drive together with other improvements will provide for the simultaneous automation of chemical analysis experiments requiring a wide variety of reagents and wavelengths. Other readout devices, e.g., electrodes, thermal conductivity detectors, etc., can be easily added to the system.
Finally, the adaptation of the external control interfaces from the ELLA system (4) permits even a small computer like the p-LINC to be used in a time-sharing mode to control and monitor data from several sets of instruments simultaneously. Therefore, the completely automated analytical laboratory becomes a definite possibility. ACKNOWLEDGMENT
The authors are indebted to Walt Pankonin for technical assistance in the construction of equipment and to Arthur A. Eggert for constructive criticism in the preparation of this paper. RECEIVED for review July 6, 1971. Accepted September 22, 1971. This work was supported by grants from the National Institutes of Health, G M 10978, the National Science Foundation, G P 26505, the University of Wisconsin Medical School, and an institutional grant from the American Cancer Society. One of us, R.N.C., gratefully acknowledges an NDEA Title IV Traineeship administered by Duke University.
A New Machine for Automated Solid Phase Peptide Synthesis Victor J. Hruby, Leon E. Barstow, and The0 Linhart Department of Chemistry, University of Arizona, Tucson, Ariz. 85721 A versatile, fail-safe, and simple machine for automated solid phase peptide synthesis was designed and constructed. A dual programming scheme was used. The variable operations such as kind and quantity of solvent or reagent, reaction or wash times, and other variables, are controlled by a Drum Programmer. The repetitive operations needed to get the solvent or reagent into and out of the reaction vessel are controlled by a Card Programmer. A number of failsafe devices were built into the machine to shut it off if electrical or mechanical failures occur. The operation is also monitored with a strip chart recorder which gives a permanent record of a synthesis. The machine can be operated in both automatic and manual modes.
IN 1963, R . B. Merrifield (1) introduced a new approach to polypeptide synthesis called solid phase peptide synthesis (SPPS). The large body of literature on this method (2, 3) attests to its utility and potential. For example, Gutte and Merrifield have used the method for the total synthesis of enzymatically active ribonuclease A (4,5). The same principle has also been applied to other synthetic and degradative problems, and this aspect is receiving increasing attention and investigation (2). The solid phase method can readily be automated (6) so that much of the routine work of solid phase polypeptide and protein syntheses can be done by a machine. (1) R. B. Merrifield, J. Amer. Chem. Soc., 85, 2149 (1963). (2) R. B. Merrifield, Aduan. Enzymology, 32,221 (1969). (3) J. M. Stewart and J. D. Young, “Solid Phase Peptide Synthesis,” W. H. Freeman and Co., San Francisco, Calif., 1969. (4) B. Gutte and R. B. Merrifield, J . Amer. Chem. SOC.,91, 501 (1969). (5) B. Gutte and R. B. Merrifield, J. B i d . Chem., 246, 1922 (1971). (6) R. B. Merrifield, J. M. Stewart, and N. Jernberg, ANAL.CHEM., 38, 1905 (1966).
An examination of the needs of solid phase chemistry and of existing machines (6-11) uncovered no automated instrument with all the requirements we felt to be important. Hence we designed and constructed a machine for automated solid phase synthesis which includes many new desirable features (12). GENERAL DESIGN OF THE INSTRUMENT
Simplicity, versatility, fail-safe operation, and easy maintenance were primary requirements in our design considerations. Standard and easily repaired circuitry was used so that the machine can readily be maintained and modified to suit particular needs. Solid phase peptide synthesis usually consists of the following kinds of methodology. An amino acid is attached to an insoluble, unreactive polymer support and the reactions needed to form a peptide bond are performed on this resin. After each reaction, a series of solvent washes are used to remove unreacted reagent(s) and side product(s) of the re(7) A. B. Robinson, Dissertation, University of California, San Diego, Calif., 1967. (8) A. Loffet and J. Close, in “Peptides, 1968,” E. Bricas, Ed., North-Holland Publishing Co., Amsterdam, Holland, 1968, p 189. (9) K. Brunfeldt, J. Halstrsm, and P. Poepstorff, Acta Chem. Scand., 23, 2830 (1969). (10) G. W. H. A. Mansveld, H. Hindriks, and H. C. Beyerman, in “Peptides, 1968,” E. Bricas, Ed., North-Holland Publishing Company, Amsterdam, Holland, 1968, p 197. (11) A. M. Tometsko, J. Garden, 11, and J. Tischio, Reu. Sci. Instrum., 42, 331 (1971). (12) V. J. Hruby, L. E. Barstow, and T. Linhart, Abstracts, 60th
National Meeting of the American Chemical Society, Chicago, Ill., Sept. 1970, No. 114 (Org.).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
343
PWR INTER. 8 MALE IND
IND LIGHT
!EFlLL
t !EFILL
PROGRAM ADV BUS W
a K W
V-17
~~
Figure 1. Block diagram of electronic portion of machine
action. The resin-reactant is then prepared for the next reaction by another series of washes, and the process is continued until the synthesis is completed. The product is then removed from the resin and purified. In general there are two different kinds of operations: (1) variable operations such as the kind and quantity of reagent or wash solvent, the reaction or wash times, and the number of times each such operation is consecutively repeated, and (2), repetitiue operations which involve transfer of a measured quantity of reagent or solvent into and out of the reaction vessel. A programming scheme separating these two different kinds of operations was desirable and hence the machine was designed such that the aariable operations are controlled by one programming scheme (the Drum Programmer) and the repetitioe operations by a second (the Card Programmer). This design greatly increases the flexibility of the machine. The design can also readily accommodate new or improved chemical methodologies as they are developed. Figure 1 is a block diagram of the electronic components of the machine. The machine can be operated in both automatic and manual modes. In the automatic mode, the Card Programmer System activates the MEASURE, CLEAR, TRANSFER, VENT, MIX, AND EMPTY functions (all repetitive operations for the transfer of solvent or reagent into and out of the reaction vessel). (A diagram of the mechanical portion of the machine is shown in Figure 2). In the Manual mode, choice of solvent or reagent is made by simultaneous use of a switch to activate the solenoid to the solvent or reagent of choice 344
and a switch t o activate the MEASURE and CLEAR functions. The other functions such as TRANSFER, VENT, etc. are activated by their individual switches. A timer (Timer 6, Figure 1) for use in the Manual mode has been added to control operation during the MIX function and the machine can thus be left unattended during this period. A pressure-vacuum system is used to transfer the solvents and reagents from their storage bottles to the measuring vessel. Valves are used to control the pressure or vacuum supply to the bottles, but the valves d o not come in contact with the solvents and reagents. This prevents cross contamination and eliminates corrosion. In general, a measured quantity of solvent or reagent is to be transferred to the reaction vessel. Since different solvents and reagents have different densities, surface tensions, and other properties that result in different flow rates, it is necessary to make a volumetric measurement of the various solvents and reagents. The measuring vessel has level sensors which measure the quantity of liquid to be used. Each solvent and reagent has its own Teflon line to the measuring vessel to minimize cross contamination. The instrument was designed with a VENT function that purges the vapors from the measuring vessel to minimize contamination. A cold finger trap is provided just before the vacuum pump to condense all of the vapors that might otherwise escape into the atmosphere. Argon or purified nitrogen is used as the pressure source to prevent contamination of the solvents and reagents by water vapor and oxygen.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
The Manual mode option allows routine servicing of the machine with little or no machine shutdown time. The servicing is done during the MIX function of a coupling step which is usually 2 hr or greater (3). During this time it is possible to empty the waste flask (Figure 2) and change the program on the Drum Programmer without disturbing the coupling reaction. The bottles can also be refilled at this time by use of the manual controlled fill-refill switch (Figure 1). In the refill position, the valve corresponding to the switch used is opened and a vacuum is placed on the corresponding solvent or reagent bottle. The line leading to the metering flask is replaced by a line leading t o a bottle or reservoir of the solvent or reagent and the fluid in the reservoir is transferred to the bottle. Suitable precautions to protect sensitive reagents are readily incorporated. Operation of the machine is monitored and a number of fail-safe devices are used which shut off the machine should any electrical or mechanical failures occur. The various functions of MEASURE, CLEAR, TRANSFER, VENT, MIX, and EMPTY utilized to transfer accurately measured quantities of solvents and reagents into and out of the reaction vessel are monitored electronically and recorded on a strip chart recorder for a permanent record of the synthesis. Indicator lights corresponding to the solvent or reagent bottle in use, to the Card Programmer function activated, and to the number of times (or cycles) a particular solvent or reagent has been consecutively repeated are constantly on as these functions are performed so that the exact status of a program can be determined by reference to the Drum Programmer position and the illuminated indicator lights. In case of power interruption, the machine is shut down to ensure that no important steps in the programmed scheme are skipped. During the MEASURE function the machine will shut off if the desired amount of solvent or reagent is not obtained due to mechanical or electronic failure. Any mechanical or electrical failure resulting in the programmed amount of solvent or reagent not being transferred into and out of the reaction could cause an error in the synthesis. A postmetering vessel between the reaction vessel and the waste flask is provided to safeguard against such failures. The contents of this vessel are emptied into waste each time material is transferred to it, but nor during the EMPTY function. In this way we can “look at” the effluent from the reaction vessel after it is emptied into the postmetering vessel. A level sensor detects if the proper amount of effluent is not obtained in the postmetering vessel and the machine is shut off. DESCRIPTION OF APPARATUS
The apparatus consists of three major parts: The mechanical portion (Figure 2) which consists of the reaction vessel, various solvent and reagent storage vessels, metering flasks, pressure-vacuum system, waste flask, and other components needed to transfer the reagents and solvents into and out of the reaction vessel; the Card Programmer System, which consists of the timers, relays, program cards, valves, fail-safe circuitry, and other electronic circuitry necessary to control the transfer of solvents and reagents throughout the mechanical portion of the machine; and the Drum Programmer, which selects the solvent or reagent used, the number of times the solvent or reagent is used, the volume of solvent or reagent used, the washing or reaction time, and other specific functions required by the particular synthetic scheme that is programmed. The major parts for the instrument are listed below.
T O AIR
c
--
I I
-J1 v-20
v-22
TO
LS 4 4
Figure 2.
Diagram of mechanical portion of machine
V-1-V-9 = Asco 2-way valves with ethylene-propylene seats V-10-V-17 = Asco 2-way valves with ethylene-propylene seats V-18, V-21, V-22, V-26, V-30 = Asco 3-way solenoid valves with ethylene-propylene seats V-19, V-20, V-25, V-27 = Asco 2-way solenoid valves with ethylenepropylene seats V-23, V-24 = 3-Way pneumatically activated valves of Kel-F V-31 = 1-way check valve (to air only) B-1-B-9 = Solvent and reagent bottles (5 I., 1 I., and 250 ml) B-10-B-17 = Amino acid bottles (15, 35 or 50 ml graduated tubes) MV = Measuring vessel (see text) RV = Reaction vessel (see text) PMV = Postmetering vessel (see text) Tr 1, Tr 2 = Vapor traps; 30-ml capacity LS 2, LS 3 = Level sensors (measuring) (see text) LS 1, LS 4 = Level Sensors (fail-safe) (see text) PG = Pressure gauge VG = Vacuum gauge T = Imperial-Eastman Brass T’s, lia-in.size T’ = Kel-F T(Chromatronix,Inc.) L = Elbow, l/An. size WF = Waste flask (5 I.) VT = Vent trap (1 1.) B = Ballast tank, 12-in. X 24411. (A. C. Tank Co., Burlington, Wis.) Teflon Tubing = Chemfluor TFE Spaghetti Tubing, Size AWG 12; standard wall Electronic Parts (see Figure 1). PROGRAMMER. Stepping Drum Programmer, Model 200B-31-EZ-100-120U, Tenor Company, Milwaukee, Wisconsin. TIMERS. Timers 1, 2, 3, 4, and 6; Tenor Multirange Reset Timers, Model 650-0-B, 5 ranges. Timer 5; Tenor Multirange Reset Timer, Model 650-1-B. SHAKER. Motor driven to turn reaction vessel through a 180O arc (see text). VALVES V-1 to V-17, V-19, V-20, V-25, V-27: Asco Solenoid Valves; Model 8262C6: 2-way. Valves V-18, V-21, V-22, V-26, V-30: Asco Solenoid Valves; Model 8320A49; 3-way. Automatic Switch Company, Florham Park, N. J. Valves V-23, V-24: Chromatronix 3-way, CAV Valves; 0.090-in. bore; Kel-F; with Pneumatic actuation. For control of penumatic actuation use Chromatronix Solenoid Air Valves, Model No. SOL-3-24VDC. Chromatronix, Inc., Berkeley, California. TIMERST-7, T-8, T-9, T-10, T-11: Amperite Thermal Time Delay Relays. STRIPCHARTRECORDER.Simpson Model 2750 Miniature Strip Chart Recorder. ELAPSED TIMEINDICATOR. Haydon Model ER 3022 Elapsed Time Indicator. ROTARY SOLENOID.Guardian Rotomite DC Stepping Relay; Model 12P24D.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
345
Mechanical Parts (see Figure 2 for most parts). VACUUM SOURCE.Dyna Pump (Van Water and Rogers No. 54904006), o r Water Aspirator. All of the parts of the instrument that come in contact with solvents or reagents are made of glass, Teflon, or Kel-F. Glass t o Teflon connections are made using Chromatronix connectors G2-C or G3-C, and Teflon t o Teflon connections are made using Chromatronix Tube End Fittings (No. TEF 136) and Coupling (No. 107A3) pieces. PRESSURE-VACUUM SYSTEM. A pressure-vacuum system is utilized to transfer solvents and reagents t o the reaction vessel and from there to waste. The direction of flow is controlled by 2-way and 3-way solenoid valves. The 2-way valves connected to bottles containing strong acids such as trifluoroacetic acid or HCl are protected from vapors by use of a small anhydrous K O H trap. All 2-way valves are activated either by the Drum Programmer System (solvent and reagent valves V-1 , , , .V-17) or by the Card Programmer System (vacuum and pressure valves V-19, V-20, V-25, V-27). The 3-way valves directly controlling the pressure-vacuum system (V-18, V-30, V-21, V-22, V-26) are shown in their unenergized mode in Figure 2 and are controlled by the Card Programmer System. The 3-way valves V-23 and V-24 are special all Kel-F valves which control the flow of reagent and solvents into and out of the reaction vessel, and are also used for the VENT function (oide infra). The pressure in the system is provided from a n argon source, and the vacuum by a pump or water aspirator system. A large ballast is used t o minimize fluctuations in vacuum, and a vacuum trap is used to collect solvent vapors. The normal operating pressure has been about 1.15 atmospheres. The normal operating vacuum has been about 0.80 atmosphere. A single rectangular glass manifold of 7 mm glass tubing (27411. X 4-in.) is used to connect the solvent and reagent bottles (B-1 . . . B-17) t o the pressure and vacuum system. The manifold is mounted to the back of the center panel of three 6-in. X 45-in. X '/*-in. aluminum panels mounted on the back of a 24-in. X 45-in. X 1-in. plywood surface that holds the solvent and reagent bottles. Solvents used in large SOLVENT AND REAGENT RESERVOIRS. quantities such as methylene chloride, ethanol, dimethylformamide, chloroform, etc. are stored in 5-liter bottles with 28/ 45 S T female joints. The tops of the bottles are 28/45 ST male joints fitted on top with two Chromatronix glass connectors, and a Teflon stopcock. One Teflon line is connected t o the appropriate valve. The other Teflon line runs directly to the measuring vessel, Steel springs attached t o the glass hooks hold the top piece firmly on the bottle. Solvents and reagents used in lesser quantities or requiring frequent fresh preparation such as diisopropylethylamine/ chloroform, dicyclohexylcarbodiimideimethylene chloride, trifluoroacetic acidimethylene chloride, etc., are stored in either I-liter or 250-ml bottles. The bottles are constructed with tops similar to those of the larger bottles. Solutions of the appropriate protected amino acids or amino acid active esters are stored in 15-ml, 35-1111, or 50-ml calibrated, conical shaped screw cap vials with Teflon caps. The Teflon lines t o the appropriate valve and t o the measuring vessel are inserted through slightly undersized holes in the caps. MEASURING VESSEL. The measuring vessel consists of a conical shaped glass vessel with ST joint and two side arms (for attachment of the level sensors), a small outlet at the bottom of the vessel (for transfer of liquid t o the reaction vessel), and a machined Teflon cap containing 18 carefully 346
machined slightly undersized holes for inserting the Teflon tubing from bottles B-1 through B-17 and from valve 19. (Three short steel pins are pressfit at the periphery of the cap, and steel springs securely hold the cap t o the vessel.) The vessel can be used t o measure volumes of 2.5-70 ml to * 3 % accuracy, and is calibrated at the side arms. The volume is detected and measured by a level sensor attached in the desired position on the side arm. REACTION VESSEL. The two-piece reaction vessel described by Merrifield (6) is used with a few modifications. The bottom outlet of the vessel is a Chromatronix glass connector (No. G3-C). The vessel is made with several indentations just above the coarse frit to prevent caking of the resin. The stopper for the top end of the vessel consists of a IO-mm EC fritted disk (Kontes) inserted into the end of the ST 14/20 male joint. The joint is reground to fit flush with the ST 14/20 female joint on the top of the reaction vessel. In this way the frit does not extend into the reaction vessel where it can provide a cavity for accumulation of the resin material. The added surface area of the frit also greatly facilitates the flow of liquid into and out of the reaction vessel. POSTMETERING VESSEL. The postmetering vessel is very similar in construction to the metering vessel except that only one side arm for attachment of a level sensor device is used and the top of the vessel terminates in a 24/40 ST joint. The bottom of the vessel terminates in an inverted 7-mm glass U-tube. The inverted U-tube rises just above the height of the upper end of the side arm and the exit is terminated by a Chromatronix glass connector. This design prevents solvent entering the postmetering vessel from flowing into the waste flask during the emptying of the reaction vessel. A 24/40 ST Teflon stopper with a 'i2-in. lip is used for the top of the postmetering vessel. Two slightly undersized holes are drilled in the stopper for insertion of the Teflon lines leading to the top of the vessel. WASTEFLASKAND VAPORTRAP. The waste flask and vapor trap can be of any suitable design for the collection of solvents and vapors. For the waste flask, we have used a 5-liter round bottom flask with ST joint and have added a spigot with stopcock at the lower quarter of the vessel for easy emptying of the flask contents. For the vapor trap we have used a I-liter round bottom flask having a 15 X 5-cm neck (such that the bulb end of the trap will fit down into a large Dewar containing a suitable coolant). SHAKER,The design is virtually the same as that used by Robinson (7), and moves the reaction vessel through a 180" arc during the various shake periods. The duration of shaking is controlled by the use of adjustable synchronous motor timers. Provisions are made in the timer circuitry to ensure that the reaction vessel is stopped in the upright position by use of a micro magnetic reed interlock switch (No. MSRR2-185, Hamlin Tnc., Lake Mills, Wis.). LEVELSENSORS.All sensor devices used either as fill level sensors or as sensors for fail-safe devices are designed to detect the presence or absence of fluid in 7-mm 0.d. borosilicate glass tubing. The fluid-level sensors utilize the following technique. A pre-focused light source is radially directed through a section of glass tubing into a LASCR (Light Activated Silicon Controlled Rectifier, G E L8F). Since the LASCR is essentially a digital device, when it is subjected to light levels below a specific threshold, it remains in a nonconducting state. However, when the light level exceeds the threshold point, the LASCR instantaneously switches to a fully conducting or on state. Its power handling capabilities are sufficient to allow direct control of relays and other devices
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
1
a
3
4
5
6
7 X
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
X
X
X
X
X X X
x X
X
X X
X
X
X X X
X
X X
x
X
X X X
X X X
X
X
X
X
X X
X X
X X
X X
X X
X X X
X X X
X X X
X X X X
X X X X
X X X
X X X
X X
X X X X
X
X
X X X X X X X X X X
X
X
X
X
X X X
X X X X X X X X
X
20 21 22 23 24
2
X X X
X X
X X
X X X
An X indicates the position of a nylon plug on the programmer drum. DIEA = diisopropylethylamine; TFA = trifluoroacetic acid.
Figure 3. Drum program for addition of 2 amino acids. in this equipment. When the glass tube fills with liquid, the liquid acts as a lens and increases the light intensity at the LASCR, causing it to turn on and apply power to the appropriate circuitry for stopping the liquid flow or performing other control functions. The light source and detector are mounted in two separate sections of a simple aluminum mounting (overall dimensions, 2.20 in. X 1.00 in. X 0.625 in.) with a light shaft (0.41 in.) between the source and detector. A 0.281-in. diameter hole in the center of the light shaft between the bulb and detector permits the two compartments to fit onto the appropriate side arm tubing. PROGRAMMER
A dual interconnected programming scheme is utilized to control the proper sequential operation of the various components of the machine. Drum Programmer. The central programming for the instrument in the automated mode is provided by a Tenor Stepping Drum Programmer. In this machine the following functions are controlled by the Drum Programmer: 1) choice of solvent, reagent, or amino acid valve (switches 1-17); 2) the number of consecutive times (cycles) a particular solvent or reagent is to be used on that particular step (switches 18-21 and H); 3) the various liquid level sensors which control the amount of material measured by the measuring vessel (switches 22-23); 4) the various timers which control the length of time the reaction vessel and its contents are to be mixed (switches 25-29); and 5) control to skip the VENT or MIX and EMPTY functions of the Card Programmer (switches 30 and 31). A typical drum program for the addi-
tion of one amino acid to a polypeptide-resin by the dicyclohexylcarbodiimide method, followed by a second addition of one amino acid to the chain by the active ester method is given in Figure 3. Since lengthening the peptide chain by one amino acid residue requires only 12 steps of the programmer drum for this scheme, the addition of up to eight amino acids to a polypeptide-resin chain is possible with each complete revolution of the programmer drum. If a greater number of solvents, reagents, or steps is needed for the addition of one amino acid (3, 13), this can readily be programmed. The instrument has a greater flexibility in the number of possible cycles that can be programmed than indicated in Figure 3, since it is actually possible to have as many as 12 cycles by simple internal wiring changes. Also since switch 24 of the Drum Programmer is currently not used, an additional fill level sensor, or an additional timer, or some other equipment can be added to the machine. A program change from the dicyclohexylcarbodiimide method to the active ester method can be made by the simple removal of a few plugs and their replacement by a few different plugs (compare steps number 8, 10, and 1 1 with steps number 20, 22, and 23). A drum step in a particular program can be readily skipped by inserting a pin the in “H” position on the Tenor drum. Card Programmer System. The valve configurations necessary to effect the repetitive functions of MEASURE, CLEAR, TRANSFER, VENT, MIX, and EMPTY are con(13) W. S. Hancock, D. J. Prescott, W. L. Nulty, J. Weintraub, P. R. Vagelos, and G. R. Marshall, J . Ameu. Clzem. SOC.,93, 1799 (1971).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
347
+24V
32 P O L E GROUNDING R E L A Y
VALVES
Figure 4. Wiring for a program card trolled by means of program cards. As shown in Figure 4, all of the pressure-vacuum valves are permanently connected to the +24 volt dc bus and can be actuated by simply grounding the return lead. If for example it is desirable to have V-22 actuated, it is necessary merely to jumper pins 6 and 6’ on the program card. The card receptacle-grounding relay wiring is duplicated in each of the six positions corresponding to the MEASURE, CLEAR, TRANSFER, VENT, MIX, and EMPTY functions. In this way a different valve configuration corresponding to each of these functions can be readily programmed. The solvent and reagent valves are actuated in a similar manner except that only one of the valves (as determined by the drum program) is energized when pins 1 and 1 ’ are jumpered and the grounding relay is closed. A typical programming scheme for the Card Programmer System is shown in Figure 5 . The automatic sequencing of the MEASURE, CLEAR, TRANSFER, VENT, MIX, and EMPTY functions is controlled by a solenoid activated rotary stepping relay (Figure 1). This relay is stepped by the application of a 24-V dc signal. In order to minimize the power required of this “advance signal” and thus extend the life to the thermal time delay relays, an electronic relay driver is used eliminating noise induced stepping and providing a positive and reliable signal to the stepping relay. Operation of Card Programmer System. When the stepping relay is in position 1, 24-V is applied t o the measure relay that activates the various valves as specified by the program card in order to accomplish the MEASURE function (Figure 5). Simultaneously, power is applied to the selected level sensor, the thermal fail-safe timer and the overfill sensor. When the liquid level in the metering vessel reaches the correct height, the level sensor is activated and a 24-V signal is applied to the program advance bus. This advances the stepping relay to position No. 2. In the event of a failure of the level sensor or if the proper amount of fluid is not transferred in a specified time, fail-safe devices are provided to shut down the instrument. When the stepping relay is in position No. 2, the clear relay is energized and the fluid line is purged of fluid. The duration of this function is determined by a thermal timer. In position No. 3 of the stepping relay, the liquid in the measuring vessel is transferred to the reaction vessel and the 348
Program Card Connections Valve No. 1 . . . . . . . . . . . . . . . . . 1-17 (common to programming drum) 18 19 4 . . . . . . . . . . . . . . . . . 20 5~ . . . . . . . . . . . . . . . . 21 6 . . . . . . . . . . . . . . . . . 22 7 . . . . . . . . . . . . . . . . . 23A 8 . . . . . . . . . . . . . . . . . 23B 9 . . . . . . . . . . . . . . . . . 24A 10 . . . . . . . . . . . . . . . . . 24B 11 . . . . . . . . . . . . . . . . . 25 12 . . . . . . . . . . . . . . . . . 26 13 . . . . . . . . . . . . . . . . . 21 14 . . . . . . . . . . . . . . . . . 30 15 . . . . . . . . . . . . . . . . . EFC Light MEASURE Card MIX Card 1 Common 8 23B 2 V-18E 9 24A 3 V-19E 5 V-21E 7 V-23A EMPTY Card 10 V-24B 1 Common 4 V-20E CLEAR Card 6 V-22E 1 Common 1 V-23A 3 V-19E 10 V-24B 11 V-25E TRANSFER Card 12 V-26E V-30E 4 V-20E 15 Empty Failure Cir7 V-23A cuitry Light 9 V-24A
VENT Card 3 V-19E 8 V-23B 9 V-24A 15 V-27E Figure 5. Card programmer system liquid in the postmetering vessel is transferred to waste. Again, after the prescribed time, an “advance” signal from a thermal relay timer is applied to the program advance bus and the stepping relay is advanced to position No. 4. If the VENT function is desired, pin position No. 31 of the Tenor drum is left blank and the VENT function occurs exactly the same as the TRANSFER and CLEAR functions described above. If no VENT is required, a pin is placed in position No. 31 on the program drum. Under these circumstances the Tenor drum switch is closed and thus the stepping relay immediately advances to position No. 5. The MIX function occurs exactly as did the CLEAR and TRANSFER functions except that the clock timer, as selected by the program drum, performs the timing function. The MIX and EMPTY functions can both be skipped by insertion of a pin into hole No. 30 in the Tenor Drum. After the programmed time has elapsed, the shaker interlock switch closes as soon as the reaction vessel is in an upright position advancing the stepping relay to position No. 6. The EMPTY function is similar to the MEASURE function. However in this case, closure of the thermal time delay relay applies a signal simultaneously to a level sensor on the postmetering flask, and to an electronic time delay circuit. With a proper transfer to liquid into the postmetering vessel, the level sensor detects the correct fluid level and applies a signal moving the stepping relay to position No. 7. If, however, the fluid transfer has not been properly effected, the level sensor will detect no fluid, and consequently will nor generate an “advance” signal. After approximately 3 sec, the elec-
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
tronic time delay circuit applies a signal to the malfunction bus and the machine is shut down. In position 7 of the program stepping relay, a signal is applied t o the program repeater circuitry. The program repeater stepping relay is advanced one step. The panel indicator lamp indicating the completion of one cycle is illuminated, and this information is compared with the program drum instructions. If the required number of cycles has not been completed, a signal is sent to the program advance bus, the stepping relay is advanced to position No. 1 and the entire sequence described above is repeated. When a comparison of the number of cycles completed matches the number of cycles required, a signal is sent to the drum step mechanism, and the drum advances one step. At the same time, the repeater stepping relay is reset to zero, and a signal is sent to the drum step mechanism, and the drum advances one step. At the same time, the repeater stepping relay is reset to zero, and a signal is applied to the program advance bus which resets the stepping relay to position No. 1. Fail-safe Devices. POWERINTERRUPTION. A safety switch is incorporated into the machine so that the machine shuts down in case of power interruption. The time and position in the program when shut-down occurred can readily be determined by reference to the total elapsed time indicator, to the Tenor drum's position, to the strip chart recorder, and to the front panel indicator lights. Manual or automated adjustments can then be made if necessary and the program continued. DURING THE MEASURE FUNCTION. During the MEASURE function, a 2-min thermal relay timer is utilized with the measure card. If solvent or reagent is not transferred to the metering flask during this 2-min period, the machine is shut down. A second fail-safe device is used during the MEASURE function. This consists of a photocell device positioned at a level greater than any of the programmed fill indicators so that if an excess of solvent or reagent is added to the metering flask, the over-fill sensor will shut down the instrument. DURING THE EMPTYFUNCTION. T o monitor the effluent from the reaction vessel, we have incorporated a postmetering vessel, a level sensor, and the necessary circuitry to shut off the machine if the desired volume of reagent or solvent is not transferred from the reaction vessel to the postmetering vessel during the EMPTY function. CARDPROGRAMMER MONITOR. The various functions of MEASURE, CLEAR, TRANSFER, VENT, MIX, and EMPTY are monitored electronically and recorded on a strip chart recorder. The pattern of a particular programming scheme is reproducible, and changes from the usual will be easily observed as an aid to troubleshooting and also as a record that the proper sequence of reactions and washes of the solid phase methodology used has been utilized. Operation of the Machine. The general methodology for regular as well as automated solid phase synthesis has been adequately discussed in detail elsewhere (2, 3, 6, 7), and no attempt will be made here t o review the various precautions and procedures necessary to undertake successful solid phase polypeptide synthesis. However, because of the various improvements and changes in this machine, a few factors which permit maximum utilization of our machine should be discussed. Before beginning, the level sensors are carefully set at the proper volume levels and are then firmly anchored to the side arms of the measuring vessel. We have found the following timers to be of greatest utility; 1) CLEAR function, a 3-sec timer; 2) TRANSFER function, for volumes of 10-25 ml, a
45-sec timer; for volumes of 2 5 4 0 ml, a 60-sec timer; 3) VENT function, a 60-90 sec timer is found to clear the metering vessel and lines of noxious vapors; 4) EMPTY fu,nction, for volumes of 10-25 ml, a 45- or 60-sec timer has been used; for volumes of 25-40 ml, a 60- or 90-sec timer. The variability of timers used here is a function of the reaction vessel used, the volume of solvent, the number of grams of resin material, and the concentration of peptide material on the resin. Before beginning operation, it is also necessary t o charge the Dewar flask containing the VENT trap and the small traps, T r 1 and Tr 2, with dry ice. Tr 1 is also charged with a few grams of sodium carbonate so that any acidic vapors condensed in the trap will be neutralized. The operation of the machine in the automated mode is very simple. The Tenor drum is programmed as shown in Figure 3 and the program is extended to all 100 positions on the Tenor drum as desired. The Card Programmer is programmed as shown in Figure 5. The reagent and solvent bottles are filled, the reaction vessel is charged with the desired amino acid resin, the various level sensors and fail-safe devices are adjusted, and the other appropriate preparations mentioned in the preceding section are made. The automatic mode switch is turned on and the instrument will proceed as programmed. In the manual mode the functions of MEASURE, CLEAR, TRANSFER, VENT, MIX, and EMPTY are operated by manually turning on the desired switch. During the MEASURE and CLEAR functions the desired solvent or reagent must also be chosen by turning on the appropriate switch. Timing of the MIX function can be done with Timer 6 or an outside Timer. At the end of the polypeptide synthesis, the polypeptide resin is usually washed several times with methylene chloride, dimethylformamide, and ethanol to remove all material not covalently bound t o the resin. The resin is removed from the vessel and is then dried in L'UCUO. Finally the polypeptide is cleaved from the resin material by some suitable procedure (2,3,)and the polypeptide obtained is purified. Reliability and Maintenance of Machine. The automated machine described above has been used for over 2000-hr operating time and has shown a high degree of reliability. From our experience, certain kinds of maintenance should be regularly performed. For example, it is important to check the valve seats from time t o time to ensure that they have not been worn. It is also desirable to change the bulbs in the level sensors about every 800 hours of machine operating time. Under these conditions we have experienced no failures of these devices. In the machine it is possible to isolate the reaction vessel from the rest of the system. During the coupling step in an automated program, reaction times of 2-12 hr are usually required, and this time is excellent for filling the bottles and rinsing out the metering flask. The metering flask can be readily rinsed using the vent cycle. Approximately every 200 hr of operating time, it is desirable to clean out the vacuum trap and the small vapor traps. This can also be done during the SHAKE function, though it is necessary t o discontinue briefly the shaking of the reaction vessel while the line to Tr 1 is disconnected from the trap and closed off. However it is possible to connect a second 3-way Chromatronix valve on V-23 such that the valve is between the reaction vessel and Tr 1. In this way the reaction vessel is shut off from Tr 1 during the shake function and hence Tr 1 can be readily emptied during this function without any shutdown time. Applications. We have used the automated apparatus described above for the successful synthesis of a number of
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
349
polypeptides. Among the polypeptides we have synthesized are the following; the protected tetrapeptide to oxytocin, Cbz-S-Bzl-Cys-Pro-Leu-Gly(NH,) (14), the C-terminal hexapeptide to lysozyme, Ile-Arg-Gly-Cys(CM)-Arg-Leu (12), and several other peptides related to lysozyme including Trp-TrpCys(CM)-Asn-Asp-Gly-Arg (12), Gly-Asp-Gly-Met-Asn-Ala, Gly-Asn-Gly-Met-Asn-Ala, and Gly-Ser-Thr-Asp-Tyr-GlyIle-Leu-Gln-Ile-Asn-Ser-Arg-Gly. For these syntheses the usual methodologies of SPPS were followed (2, 3, 5 ) and purification was accomplished by general procedures. The purity and homogeniety of these various polypeptides were determined by quantitative amino acid analysis, use of TLC and electrophoresis, and in several cases by direct comparison with authentic samples. (14) V. J. Hruby and L. E. Barstow, Macromol. Syi., in press (1972).
The flexibility, reliability, simplicity, and fail-safe features of this instrument make it an extremely useful tool for solid phase synthesis of polypeptides and for other solid phase methodologies. ACKNOWLEDGMENT
We wish to thank John Rupley for his help and encouragement, and A. B. Robinson and John Sharp for their advice and the drawings for the shaker. RECEIVED for review August 2 , 1971. Accepted September 22, 1971. The work was supported in part by a Fredrick Gardner Cottrell Grant from the Research Corporation and in part by Grant AM-13411 from the U. S. Public Health Service.
Errors in Theoretical Correction Systems in Quantitative Electron Probe Microanalysis-A Synopsis Kurt F. J. Heinrich Institute for Materials Research, National Bureau of Standards, Washington, D.C. 20234 Accurate electron probe microanalysis with simple reference materials requires the use of the theoretical models describing the emission of direct and indirect characteristic X-radiation, and of an iterative calculation procedure. The presently accepted theoretical equations for the calculation of intensities are presented, with references to the main sources of error. An efficient iteration procedure which uses a hyperbolic approximation to the analytical calibration curve is discussed in detail.
THE ACCURACY of electron probe microanalysis is limited by errors in the measurement of X-ray intensity ratios between specimen and standards, by inaccuracies in the correction calculation procedures (models), and by uncertainties in physical constants and parameters used in the correction calculation. The accuracy of test procedures for the quality of the correction is further restricted by the scarcity of adequate test specimens of well known composition. In the past, limitations in computational facilities have forced the analyst to drastically simplify the correction calculations. It has recently been pointed out that such simplifications may introduce substantial errors ( I ) . It is difficult to study the effects of individual sources of errors, since the effects of many sources of error combine in the final result. However, progress is achieved through the study of propagation of errors (2), the use of reliable standard reference materials (3, 4), and the availability of computer programs in which the use of sjmplifying assumptions is minimized. A very detailed program named COR, which considers (1) K. F. J. Heinrich and H. Yakowitz, Mikrochim. Acta, 1970, 123. (2) K. F. J. Heinrich and H. Yakowitz, “Proc. Fifth International Congress on X-ray Optics and Microanalysis,”Springer-Verlag. Berlin, 1969, p 151. (3) K. F. J. Heinrich, Nat. Bur. Stand. (US.), Tech. Nore, 521, 11 (1970). (4) NBS Certificates of Analysis, Standard Reference Materials 480 (1968); 481,482 (1969). 350
rigorously all correction factors known at present, was developed at NBS by HCnoc, Heinrich, and Myklebust (5). The full expressions for generated X-ray intensities, which will be discussed in this paper, are used in the program COR, without simplifying shortcuts. The combination of the aforementioned factors enables us to perform a critical study of the correction procedures and can be expected to significantly reduce the uncertainty of quantitative electron probe microanalysis. We will now briefly outline the principles involved in the correction procedure, with some remarks concerning the potential sources of error. GENERAL PROCEDURE
The ratio of characteristic emitted X-rays (after background correction), k , is expressed by the equation k = I,* Ip
+ XI,* + IC*
+ BIf + I C
(1)
In this equation, I denotes emitted X-ray intensity (counts/ sec), the superscript * denotes the specimen, while the corresponding intensities foI the standard are not superscripted, and the subscripts p , f ; c, denote, respectively, primary radiation, fluorescence due to characteristic lines, and fluorescence due to the continuum. The summation indicates that more than one line may excite the line under observation. If the standard is an element, ZZf = 0. The calculations for fluorescent contribution due to both characteristic lines and continuum take into account the loss of radiation upon emergence. For the primary radiation, it is customary to calculate separately the intensity of radiation generated within the sample, I,’, and the attenuation during emergence, f, [usually denoted by J ( x ) ] . Neglecting the aperture angle of the spectrometer, which is a common multiplication factor for all emergent intensities, we can write: I p = Ip’j,. In ~
(5) J. Hhoc. K. F. J. Heinrich, and R. Myklebust, Nat. Bur. Stand. (US.), Tech. Note, in press (1971).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
