US3649203A

US3649203A - Automatic analyzer

Info

Publication number: US3649203A
Application number: US778048A
Authority: US
Inventors: Eugene Louis Schneider
Original assignee: Ralston Purina Co
Current assignee: Nestle Purina PetCare Co
Priority date: 1968-11-22
Filing date: 1968-11-22
Publication date: 1972-03-14
Anticipated expiration: 1989-03-14

Abstract

An amino acid analyzer having an ion exchange column through which buffer reagents are passed is provided with circuitry means for automatically effecting the various steps of the analysis. The circuitry means being effective to allow continuous automatic supply of samples to the ion exchange column and also to automatically control the elution process, permits uninterrupted continuous operation of the analyzer without the necessity of operator attention. A central timer mechanism effects actuation of a sample supply system to place the sample on the ion exchange column and thereafter the central timer mechanism sequentially activates other timers to automatically control the eluting process. During the eluting process the analysis is carried out in the conventional manner, i.e., using a ninhydrin system and a colorimeter. The colorimeter detects the concentration of amino acids present in the column effluent and sends electrical signals to a recorder mechanism to record or plot the quantity of specific amino acids present in the sample.

Description

United States Patent Schneider Mar. 14, 1972 [54] AUTOMATIC ANALYZER Primary Examiner-Morris O. Wolk [72] Inventor. Eugene Louis SchneIder, St. Louis, Mo. Assistant Examinelgk' E Serwin [73] Assignee: Ralston Purina Company, St. Louis, Mo. Attorney-R Bl'ukaldt and Hurst [22] Filed: NOV. 22, 1968 57 ABSTRACT [21] App]. No.: 778,048 An amino acid analyzer having an ion exchange column through which buffer reagents are passed is provided with cir- 52 US. (:1. ..23 2s3 R, 23/230 A, 23/253 A, witty means P effeFting theyarious Steps of 210/31, 210/198, 210/200, 73/61 1 C the analysis. The circuitry means being effectiveto allow con- [51 im. Cl ..G01n 31/04, GOln 31/08 autommlc supply Samples exchange 531 Field of Search ..23 253, 253 A, 259, 230 A, aummatically OM01 F Pmcess, 23/230; 73 /61 1 C; 2l0/31 198 200 permits uninterrupted continuous operation of the analyzer without the necessity of operator attention. A central timer [56] References Cited mechanism effects actuation of a sample supply system to place the sample on the ion exchange column and thereafter UNITED STATES PATENTS the central timer mechanism sequentially activates other timers to automatically control the eluting process. During the 3,230,048 H1966 Skeggsm ..23/253 eluting process the analysis is carried out in the conventional 3,266,322 8/1966 Negersmnh et X manner, i.e., using a ninhydrin system and a colorimeter; The 3'334969 8/1967 Catravas "23/253 X calorimeter detects the concentration of amino acids present 3,341,299 9/1967 Catravas "23/253 X in the column effluent and sends electrical signals to a gase at recorder mechanism to record or plot the quantity of specific r ma l 3373 872 3/1968 Hrdina ..23/253 UX ammo aclds m the Sam 6 3,518,874 7/ 1970 Hrdina ..23/253 R 12 Claims, 4 Drawing Figures PATENTEDMAR 14 1972 3. e49 ,203

SHEET 1 0F 3 9 8 Illllllllllllll Q -2 12e 1 2d 12c 12b 6 t1 t1 (:1 b 1 INVENTOR EUGENE LOUIS SCHNEIDER BY ATTORNEY FIG. 3

AUTOMATIC ANALYZER BACKGROUND OF THE INVENTION Amino acid analyzers which utilize the principle of ion exchange have been fairly common in the instrumentation field. These analyzers make use of the principle of depositing a sample to be analyzed on the resin of an ion exchange column and thereafter pass various buffer reagents successively through the column, a ninhydrin reaction and a colorimeter to obtain data which reflects the concentration of the various amino acid constituents present in the sample being analyzed.

In the prior art, it has been necessary for an operator to present each sample which is to be analyzed to the machine by manually inserting this sample on top of the ion exchange column. Not only was this undesirable due to the necessity of an operator manually placing each sample to be analyzed into the machine but also it was necessary that the seals on the ion exchange column be opened to permit the insertion of such samples. The necessity of continuously opening these seals resulted in the attendant problems of seal leakage due to the high pressures of the fluids used in the ion exchange columns.

Other prior art attempts to automate the amino acid analysis system have utilized an automatic method of inserting a cartridge containing the sample onto the end of the ion exchange column. Although this method eliminates some of the necessary operator attention, it still requires the periodic opening of the seal on the column to receive samples thereby permitting leakage of the fluids around the seal. In addition, the samples were mounted in such a manner that the heat generated by the analyzer in its operation was effective to destroy some of the amino acid constituents, such as glutamine and methionine, in the sample prior to its analysis.

In an effort to overcome these aforementioned undesirable features, applicant has developed a system which utilizes time controlled circuitry means for actuating the various components of the analyzer enabling the operation of such an analyzer to be completely automatic without the necessity of operator attention.

Another object of the present invention is to provide an analyzer which permits samples to be maintained separate from the column portion of the analyzer. These samples may even be maintained under refrigerated conditions prior to their analysis, thereby minimizing the possible deterioration of the various amino acid constituents.

Another object of the present invention is to provide circuitry means effective for actuating a sample feed mechanism which delivers the sample to a control valve and in response to other electrical impulses from said circuitry means said control valve is effective to supply the sample to the ion exchange column and control the eluting process.

Another object of the present invention is to provide an automatic amino acid analyzer which permits sequential feeding of samples to be analyzed to the apparatus without the necessity of opening the high-pressure seals on an ion exchange column.

Another object of the present invention is to provide an amino acid analyzer which is provided with a central timer mechanism to sequentially effect the analysis of a sample and said timer mechanism being responsive to the completion of the analysis to automatically supply another sample to be analyzed.

Another object of the present invention is to provide circuitry means on the amino acid analyzer which permits automatic operation and which permits the columns to be run in overlap condition. It should be understood that overlap condition means that when two columns are used for the analysis, the eluting process may be initiated on the long column prior to the completion of the eluting process on the short column. The long column may be started prior to completion of the short column due to the existence of an initial period of operation on the long column when there are no amino acids of interest which are eluted.

Still another object of the present invention is to provide automatic circuitry means which permits the short column eluting process to begin as the long column is being stripped and prepared for another sample.

These and other objects and advantages of the present invention will become more apparent in the description hereinafter disclosed.

Briefly, the present invention comprises an automatic amino acid analyzer having an ion exchange column, a control valve for controlling the supply of samples to the column and for controlling the eluting process, sample supply means for supplying a predetermined amount of sample to the control valve, and a central timer means for controlling the analysis cycle which includes energizing said sample supply means to supply the sample to said control valve, said central timer means activating said control valve to initially supply the sample to the ion exchange column and thereafter controlling the eluting process, and said central timer means being responsive to the completion of the eluting process to begin the analysis cycle for another sample.

DESCRIPTION OF THE DRAWINGS FIG. 1 is a view of the amino acid analyzer embodying the present invention,

FIG 2 is a diagrammatic view of the fluid pressure system of the amino acid analyzer of FIG 1,

FIG 3 is an enlarged diagrammatic view of the flow path through one of the control valves of the amino acid analyzer, and

FIG 4 is a schematic wiring diagram of a typical electrical circuit for the present amino acid analyzer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring now to FIG 1, an amino acid analyzer or chromotography apparatus 1 is shown as having a cabinet 2 which is provided with a refrigeration portion 3. The refrigeration unit 3 houses or contains a turntable 4 adapted to receive samples 5, for subsequent analysis by the apparatus 1. Ion exchange resin columns 6 and 7 are provided on the cabinet 2 with the resin length of column 6 being approximately 7 cm. while the resin length of column 7 is approximately 51 cm. Solenoid operated control valve means 8 and 9 are provided on the cabinet 2 for controlling the flow of samples and buffer reagents to the columns 6 and 7, respectively. Sample feed mechanism or dipper 10 is provided in the refrigeration unit 3 for selectively supplying samples 5 to the

control valves

8 and 9, and the samples 5 are subsequently deposited or absorbed on the resin of columns 6 and 7. A central time control mechanism 11 is affixed to the cabinet 2 for automatically effecting movement of the various components of the amino acid analyzer 1 to automatically complete an analysis of a sample in a predetermined time sequence, as will be described in detail hereinafter. A series of

individual timers

12a, 12b, 12c, 12d, 12c and 12f are provided for controlling specific portions of the amino acid analysis as it is carried out in an automatic manner by the apparatus 1. A recorder 13 is provided for recording curves or peaks which reflect the concentrations of the various amino acids present in the samples as they are separated or eluted from the resin columns in response to the passage of buffers therethrough. It should be understood that as an alternative, a computer or an integrator, such as available from Infotronics Company model CRS-l in, may be utilized as a digital readout system or that both a computer and recorder 13 may be utilized to compile the results of the analy- SIS.

Referring now to FIG 2, a diagrammatic view of the fluid pressure system of the analyzer is shown with the flow lines in the

control valves

8 and 9 illustrating their activated positions to receive a portion of a sample 5 from the turntable 4. The sample feed mechanism or dipper 10 (shown here in its activated position) is controlled by way of a motor 14 and gears 15 to effect its movement between an upright or inactivated position and an activated position wherein the dipper is in the on the turntable 4. The dipper 10 is connected by means of teflon tubing 16 to the inlet port 17 of the eight-port solenoid operated control valve 9.

Bypass ports

18 and 19 of the control valve 9 are connected to each other by means of teflon tubing 20 and the

sample ports

21, 22 of said valve are connected by the teflon tubing 23. The tubing 23 has therein a series of sample retention loops 24 arranged so that a sample of convenient size, such as 1 millileter, may be held in such loops. The buffer inlet port 25 is connected by means of tubing 26, a pump 27 and tubing 28 to a solenoid operated selection valve 29. The selection valve 29 is connected by means of

tubing

30a, 30b, 30c and 30d to the

buffer reagent supplies

31, 32, 33 and 34.

The

buffer reagents

31, 32 are selected to carry out the complete eluting process on the column 7. The

buffers

33 and 34 are selected to regenerate the resin of said column with the buffer 33 serving to strip the residue of the sample from the column and the buffer 34 reconditioning the resin in said column. In this particular apparatus, it has been found desirable to utilize the buffer 31 as a solution having a pH of approximately 3.25, and the buffer reagent 32 as a solution having a pH of approximately 4.1 l. The buffer reagent 33 is NaOH and the buffer 34 is equilibration, which are selected to completely strip the sample from the resin of column 7 and prepare it for receiving another sample. The selection of these buffers may depend on the operator's preference.

The outlet port 35 of the control valve 9 is connected by teflon tubing 36 to the sample inlet port 37 of the eight-port solenoid operated control valve 8, Another outlet port 38 of the control valve 9 is connected by tubing 39 to the inlet port of the long ion exchange column 7. The outlet port of the long column 7 is connected by teflon tubing 40 to a solenoid operated valve 41 which may be operated to divert the fluid supplied thereto to the the drain line 42 or to the teflon tubing 43 which is connected to a mixing valve 44.

Bypass ports

45 and 46 of the control valve 8 are connected to each other by means of teflon tubing 47 and the

sample ports

48, 49 of said valve are connected by the teflon tubing 50. The tubing 50 has therein a series of sample retention loops 51 arranged so that a sample of convenient size, such as l millileter, may be held in such loops. The buffer inlet port 52 is connected by means of teflon tubing 53, a pump 54 and tubing 55 to the valve 56. The valve 56 is connected by means of tubing 57 to buffer supply 58. The buffer is a solution having a pH of approximately 5.18, however, this may be varied depending upon the operator's preference. The outlet port 59 of the control valve 8 is connected by tubing 60 to the sample supply pump 61 and said pump is connected to a drain 62. Another outlet port 63 of the control valve 8 is connected by tubing 64 to the inlet port of the short ion exchange column 6. The outlet port of the short column 6 is connected by tubing 65 to a valve 66 which may be operated to divert the fluid supplied thereto to the drain line 67 or to the tubing 68 which is connected to the mixing valve 44. A ninhydrin system 69, which is well known in the art, is connected by means of tubing 70 to the mixing valve 44 and tubing 71 connects the mixing valve 44 with a standard reaction bath 72. Tubing 73 is connected to the reaction bath 72 and passes through a colorimeter 74 and therefrom to a drain.

Referring now to FIG 3, the flow diagram for the control valve 9 is shown. Since the control valve 9 and control valve 8 are identical only the flow pattern through the control valve 9 will be discussed in detail, it being understood that the flow through the control valve 8 will be identical. The control valve 9 is a commercially available eight-port valve having a plunger 9a therein which control the pressure fluid flow through the valve. The plunger 9a is spring biased to its normal or deactivated position establishing pressure fluid flow through the valve 9 as shown by the solid lines of FIG 3 whereby the sample may be deposited on the ion exchange resin column 7 and whereby the buffers supplied through the tubing 26 may be passed through said column. The plunger 9a is movable in response to fluid pressure from a source (not shown). The fluid pressure is applied to one end of the plunger 90 by a solenoid valve to move said plunger to a position establishing pressure fluid flow through the valve as shown by the dotted lines whereby the sample may be supplied from the tubing 16 to the sample retention loop 24. More particularly, when solenoid controlling the control valve 9 is deactivated the plunger 90 is in its normal or deactivated position, and the bufi'er supplied to the column 7 will pass through tubing 26, sample loop 24 and therefrom through the port 38, tubing 39 and into the column 7. When the solenoid is activated, the plunger mechanism 9a is activated or moved from its eluting position. The fluid flow between the

ports

25, 22 and

ports

21, 38 is interrupted and pressure fluid communication is established between the

ports

17, 35 through the loop 24 and the

ports

25, 38 through the bypass tubing 20. This enables the sample to be delivered from the tubing 16 to the sample retention loop 24.

Referring now to FIG 4, a simplified schematic drawing of the typical electrical circuits to be used with the apparatus of FIG 1 is shown. The circuits primarily consist ofa long column timing circuit indicated generally at 75, a short column timing circuit indicated generally at 76 and the remaining portion of the circuitry controlled by the central timer mechanism 11. Central timer mechanism 11 consists of a standard industrial sequential l0 terminal timer which is commercially available from a supplier such as Mallory & Company. A manual start switch 77 is provided between a power source (not shown) and terminal 780 of the central timer 1 l.

The terminal 78b is connected to another terminal 79b on the central timer l1 and to the motor 80 of said timer. It should be noted that the

switches

78, 79 connecting the terminals 78a, 78b and 79a, 79b are so provided that one or the other of them is closed at anytime. Therefore, when the terminals 78a, 78b are connected, the terminals 79a, 79b are open and when 78a, 78b are open, the terminals 79a, 79b are closed, The switch 81 on the timer 11 is connected to the power source and to the motor 14, said motor being effective through means of the gears 15 to raise and lower the dipper mechanism 10.

Another switch 82 on the timer 11 is connected between the power source and the motor 83 which is effective in rotating the turntable 4 to present a new sample 5 to the dipper mechanism 10. The

switches

84, 85 and 86 on the timer 1 1 are connected between the power source and the motor of the sample supply pump 61, and the

solenoids

87, 88 respectively. The

solenoids

87, 88 are effective in controlling the actuation of the plungers ofthe

control valves

8 and 9, respectively.

The short column timer circuit 76 has a standard commercially available timer 12a therein, available as model 3053 from such suppliers as ATC. The timer 12a is connected to a power source. Also, the timer 12a is provided with the normal means of manually setting the time period which will elapse before the contacts therein are closed and the timer times out. The timer 12a is connected to switch 90 in the timer l1 and a manual push button switch 91 may also be provided between the timers 1 l and 12a. The terminal 92 of the timer 12a is connected to the motor of the buffer pump 54, the solenoid 93 of the valve 41 and to the

terminals

94, 95 which serve as the power source for the pump of the ninhydrin system 69 and the recording mechanism 13. Another terminal 96 of the timer 12a is connected to the terminal 790 of the timer l1 and a switch 97 is provided intermediate the two timers.

The long column timer circuit 75 is provided with a series of

individual timers

12b, 12c, 12d, 12c and 12f. All of these timers are provided with the normal means of manually setting the time period which will elapse before the contacts therein are closed and the timer times out. Each of the

timers

12b, 12c, 12d and 12e are effective in controlling the length of time that the

buffers

31, 32, 33 and 34 are permitted to flow through the long column 7. The timer 12b is connected to a power source and to the switch 98 on the central timer 11. A manual pushbutton switch 99 may be provided between the timers 11 an 12b, The terminal 100 on the timer 12b is connected to the motor of the buffer supply pump 27 and to the timer 12f. Terminal 101 of timer 12b is connected to the solenoid 102 of the selection valve 29 and to the timer 120. Also, terminal 103 on timer 12b is connected to terminal of timer 12c.

The terminal 120 is connected to a solenoid 105 of the selection valve 29 and to the timer 12d. Timer 12d is connected to the solenoid 106 of the selection valve 29 and the timer 12e. It should be noted that the

timer

12e and 12f are connected to a power source, Also, it should be noted that terminal 107 of the timer 12e is connected to the switch 97 which in turn is connected to the terminal 79a.

The terminal 108 of the timer 12f is connected to the solenoid 109 of the valve 66 and to the

terminals

110, 111 which serve as the power source for the pump of the ninhydrin system 69 and the recording mechanism 13. The terminal 112 of the timer 12f is connected to the terminal 780 of the central timer 11 and a switch 113 is provided intermediate to these two timers.

The switch 113 is maintained in a closed position when a sample 5 is presented beneath the dipper mechanism 10 by means of a spring arrangement 114 and an actuation arm or sample detector 115 which is provided in a collar 116 on the machine 1. When the actuation arm or sample detector 115 is in contact with the sample 5 in the turntable 4 the switch 113 is closed, however, if a sample 5 is not present beneath the dipper 10 the spring 114 will force the sample detector rightwardly to open the switch 113.

Samples 5 for the amino acid analyzer are prepared by the standard hydrolysized process, e.g., a 125 ml. sample is mixed with six normal HCl, purged with nitrogen, sealed and heated at 110 C. for approximately 22 hours, An internal standard may be added which is a natural amino acid and which will be eluted as a separate peak. To complete the sample 5, an aliquot sample is taken, dried, and thereafter a buffer having a pH of 2.2 is added to obtain a 10 ml. sample. The samples are then placed in cups on the rotary turntable 4. Since the turntable 4 is located in the refrigeration portion 3, a large number of samples 5 may be placed therein without concern that some of the amino acid constituents of the samples will be destroyed.

In the operation of the amino acid analyzer 1 the

control valves

8 and 9 are normally in a position as shown in FIG 3, that is, permitting fluid flow from the buffer system to the ion exchange columns 6 and 7, through the

sample retention loops

24, 51. Also, the heating unit of the reaction bath 72 and the colorimeter 74 are maintained energized; the turning on of the recorder 13 being effective to receive the signals from the colorimeter. In response to the closing of the manual switch 77 current from the power source flows through the switch 78 to energize the timer motor 80. After the elapse of a predetermined amount of time, the

switches

84, 85 and 86 are closed thereby serving to energize the pump 61 and activate the

control valves

8 and 9. The

control valves

8 and 9 are then in a position to establish pressure fluid communication therethrough as shown by the lines in FIG. 2. After the elapse of another predetermined amount of time, the switch 82 is closed serving to activate the motor 83 and rotate the turntable 4. The switch 82 is opened after a predetermined time has elapsed which is sufficient to permit the turntable 4 to rotate an amount to properly locate a sample 5 below the dipper mechanism 10. It should be understood that since the sample supply pump 61 has been activated while the dipper 10 is not in the sample 5, air is passed through the supply line and

sample loops

24, 51 to serve as a flushing or cleansing action prior to insertion of the sample into said loops.

Thereafter, the timer 1] closes the switch 90 thereby serving to energize the circuit 76 and activate the short column timer 120. This serves to connect the power source of the timer 12a to the terminal 92. When the power source of timer 12a is connected to terminal 92 the buffer pump 54 is activated serving to force the buffer through the short column 6 to develop the desired fluid pressure in said column prior to injection of the sample. The timer 12a is also effective in energizing the solenoid 93 to activate the control valve 41 to establish pressure fluid communication between the

conduits

40 and 43. Simultaneously, the power to the

terminals

94, 95 serves to activate the pump of the ninhydrin system 69 and to activate the recorder 13. Once the timer 12a has been activated the switch 90 in the central timer may then be opened and the timer 12a will operate for the period of time set on said timer. The timer mechanism 11 then closes the switch 81 which serves to insert the dipper 10 into the sample 5 and since the sample supply pump 61 is already activated the sample is drawn into the

sample loops

24 and 51 of the

control valves

9 and 8, respectively. After a period of time which is sufficient for the sample 5 to be pumped into the

control sample loops

24 and 51, as previously described, the timer 11 opens the switch 84 which serves to turn off the sample supply pump 61.

Thereafter the switch 86 is opened to deactivate the solenoid 88 which serves to move the plunger of the control valve 8 to its normal or deactivated position establishing pressure fluid flow through said control valve as shown by the solid lines of FIG 3. Also, the switch 78 is opened while the switch 79 is closed which serves to interrupt the power source to the motor 80, thereby turning off the timer mechanism 11. The manual switch 77 may then be opened and thereafter the analyzer 1 will operate in a completely automatic manner as described hereinafter. The total elapsed time from the start until the manual switch may be opened is approximately 8 minutes.

The buffer 58 is pumped through the sample loop 51 and into the column 6 which permits the sample contained in said loop to be absorbed on the resin of said column. Thereafter as the buffer 58 passes through the column 6 the eluting process is performed wherein the effluent from said column passes through the mixing valve 44 to be admixed with the ninhydrin and therefrom through the reaction bath 72 where the effluent is heated and therefrom through the colorimeter 74. The colorimeter 74 is effective to transmit electrical signals, in response to the concentration of amino acids present, to the recorder 13 to form peaks or curves, in the conventional manner. The eluting operation on the short column 6 is effective to produce peaks which typically are l) a total of the amino acids other than the bases, (2) lysine, (3) histidine, (4) ammonia, and (5) arginine. This eluting process on the short column 6 takes approximately minutes, however, it should be understood that the timer 12a may be set for any period of time desired. Upon completion of the time cycle of the timer 12a, the power source is disconnected from terminal 92 and connected with terminal 96. This connection will be maintained until another impulse is received by the timer 12a through the switch or pushbutton 91. This serves to turn off the pump 54 and to deactivate the solenoid 93 which permits the valve 41 to connect the tubing 40 with the drain line 42. In addition, the power supply to the

terminals

94 and 95 is interrupted to turn off the pump of the ninhydrin system 69 and the recorder 13. It should be understood that since the buffer 58 is effective to remove all of the sample from the resin of column 6 it is not necessary to pass further reagents through the column before another sample is presented thereto.

The connection of the power source to the terminal 96 of the timer 12a is effective through the switch 79 to transmit an impulse from the power source to the motor 80 and again energize the timer mechanism 11. Thereafter, the switch 98 is closed which transmits an impulse to the timer 12b and actuates the long column timer circuit 75, as will be discussed in detail hereinafter.

It should be understood that the power source of timer 122 is normally connected with terminal 104 and therefore with terminal 103 on timer 12b. Thus, when the timer 12b is turned on, the terminal 103 is connected with terminal 100 which serves to turn on the motor of the buffer pump 27. The activation of the buffer pump 27 serves to force the buffer 31 through the long column 7 to develop the desired fluid pressure in said column prior to injection of the sample. The energizing of the timer 12b is also effective to transmit an electrical impulse to the timer 12f which turns on the timer 12f and connects its power source with the terminal 108. The timer 12f is effective in energizing the solenoid 109 to activate the control valve 66 to establish pressure fluid communication between the

tubing

65 and 68. Simultaneously, the power to the terminals 1 10 and 11 1 serves as the power source to activate the pump of the ninhydrin system 69 and to activate the recorder 13 for receiving signals from the colorimeter 74. Once the timer 12b has been activated the switch 98 in the central timer may be opened. Thereafter, the switch 85 is opened to deactivate the solenoid 87 which serves to move the plunger of the control valve 9 to its normal position establishing pressure fluid flow through the sample loop 24 of said control valve as shown by the solid lines of FIG 3. The timer 11 then opens the switch 79 while the switch 78 is closed which serves to interrupt the power source to the motor 80, thereby turning off the timer mechanism 11.

With the control valve 9 moved to its normal position, the buffer 31 is pumped through the sample loop and into the column 7 which permits the sample contained in said sample retention loop to be absorbed on the resin of said column. Thereafter, the eluting process is carried out by the buffer 31 for the period of time set on the timer 12b. The buffer 31 passes through the column 7 and then follows the normal course through the mixing valve 44 where ninhydrin is added and therefrom through the reaction bath 72 and colorimeter 74. When the period of time set on the timer 12b has elapsed the eluting process to be carried out by the buffer 31 is completed. in a typical operation of the analyzer 1, the timer 12b is set for approximately 95 minutes, but this may be varied depending on the operators preference. The timer 12b then connects the terminal 103 with the terminal 101 while maintaining the connection of terminal 103 with the terminal 100. The connection of terminal 101 with the power source serves to supply an electrical impulse to the timer 12c thereby beginning the timing cycle controlled by this timer. The impulse which activates the timer 120 also activates the solenoid 102 which moves the selection valve 29 to a position establishing pressure fluid flow of the buffer 32 through the ion exchange column 7 and the remainder of the analysis system. Upon completion of the final time set on the timer 120, amino acids which were not separately eluted during the short column run will have been recovered. In a typical operation of the analyzer l, the timer 12c is set for approximately 60 minutes, however, this may be varied by the operator. Therefore,

timer

12b and 12c control the complete eluting cycle to recover the amino acids not previously separately recovered on the short column 6. In addition, by including an internal standard in the sample as previously discussed, the curve produced in the recovery of this standard may be compared with a known curve so that the true ratio of recovery from the sample 5 will be known.

Upon completion of the timing cycle of timer 12c, an impulse is sent to the timer 12d and to the solenoid 105. The solenoid 105 being effective to move the selection valve 29 to a position permitting the buffer reagent 33 to be passed through the ion exchange column 7.

Since the buffer 33 is NaOH the passage of this buffer through the column 7 serves to completely strip the remaining sample present on the resin of said column. it should be understood that the material remaining on the resin column which is stripped by the buffer 33 is the same amino acid constituents which were previously eluted from the column 6. Upon completion of the timing cycle of the timer 12d the ion exchange column is completely stripped of all of the sample 5 deposited thereon. An impulse is sent from the timer 12a to the timer l2e and also to the solenoid 106 which moves the selection valve to a position permitting pressure fluid flow of the buffer 34 through the column 7. The passage of the buffer 34 through the column 7 serves to recondition the resin in said column so it will be receptive and have a high degree of affinity for the introduction of another sample to said column. Thus, the

timers

12c and 12d control the complete regeneration operation on column 7. In a typical operation each of the

timers

12c and 12d are set for 35 minutes but this may be varied. The eluting operation and the regeneration operation are both controlled by the long column timing circuit 75. Upon completion of the timing cycle [22, the power source to the terminal 104 and the timer 12b is interrupted, serving to effect a deenergization of the buffer pump 27. This also permits each of the

timers

12b, 12c, 12d and 12e to return to their original positions and time settings.

In addition, it should be understood that the timer 12f may be set to time out or elapse simultaneously with the timing out of the timer 12e. The timing out of the timer 12f serves to interrupt the power supply to the terminal 108 thereby deactivating the equipment connected thereto and also serves to connect the terminal 112 to the power source of said timer. With the terminal 112 connected to the power source an impulse is sent to the switch 78 of the timer mechanism 11 and thereafter another analysis cycle as previously described is begun. The power source will be maintained in contact with the terminal 112 until another impulse is received by the timer 12f from the terminal 100. It should be noted that with the sample detection switch 113 in the line connecting the terminal 112 to the switch 78 the absence of a sample 5 on the turntable 4 permitting the switch 113 to open will prevent a further power supply to the motor and shut down the analyzer 1. However, if another sample 5 is present, the analyzer 1 will continue to operate in the analysis cycle as previously described.

Further, it should be understood that by adjusting the timer 12f such that it expires upon completion of the complete eluting operation or simultaneously with the completion of the eluting operation controlled by the timer 12c, it is possible to begin another analysis cycle while the timers 12d and 122 carry out the regeneration operation of stripping and reconditioning on the long column 7. This is achieved due to the ability of the control valve 9 to permit the

buffer reagents

33 and 34 to be cycled into the column 7 through the bypass 20, while a sample is simultaneously being pumped into the loop 24 as previously described. Thus upon completion of the eluting operation of timer 120, the timer 12f would time out deactivating the equipment connected to timer 12f and connecting terminal 112 with terminal 78 as described heretofore. Since pump 27 is connected to timer 12b, it will not be deenergized until the timer 12c has timed out. In this manner, it is possible to supply another sample to the

control valves

8, 9 and begin analysis of the sample on the short column 6 while the regeneration steps are carried out of the column 7.

It should also be understood that when the switch 97 is in the position shown in FIG 4 the apparatus is in a position to operate in its normal manner. By this is meant that upon completion of the short column analysis a signal is sent through the switch 97 to the timer 1] and thereafter the long column analysis is begun. By moving the switch 97 to its lower position to connect the terminal 107 of timer 12 with the timer 79a, the analyzer 1 may be operated in the condition known as the overlap. Operation of the analyzer 1 in overlap condition means that the long column 7 eluting operation is begun before the eluting operation is completed on the short column 6. This may be accomplished due to the fact that an initial period of time elapses during the eluting operation on the column 7 before any of the amino acids of interest are recovered. In this manner, upon completion of the timing cycle 122, an impulse is sent from terminal 107 through the switch 97 to begin the operation of the long column analysis rather than relying on an impulse to be sent from the short column timer 12a. in this manner it is possible for the long column analysis to begin while the short column analysis is still being carried out thereby providing optimum operating effciency. It should be understood that the timing for the eluting step on the short column and the long column are so arranged that the long column cycle may be begun prior to the termination of the short column without loss of any of the amino acids of interest being eluted.

From the foregoing, it is now apparent that a novel automatic amino acid analyzer meeting the objects and advantages set out hereinbefore, as well as other objects and advantages apparent in the disclosure, is provided and that changes or modifications as to the precise configurations, shapes and details of the construction set forth in the disclosure and drawings by way of illustration may be made by those skilled in the art without departing from the spirit of the invention, as defined by the claims which follow.

lclaim:

1. Apparatus for determining the quantity of amino acids present in a hydrolized sample which is deposited on and eluted from an ion exchange column comprising a frame, an ion exchange column on said frame, a solenoid operated control valve for controlling the supply of sample and buffer reagents to said column, said control valve having sample retention means connected thereto of a predetermined size for retaining a predetermined quantity of sample, said control valve being movable between a deactivated position permitting pressure fluid flow of the buffer reagents through said sample retention means to the ion exchange column for effecting the eluting of the sample and an activated position permitting pressure fluid flow of a sample into said sample retention means to obtain the predetermine quantity of said sample while said control valve maintains pressure fluid flow of the buffer reagents directly with the ion exchange column, means on said frame for determining and recording the quantity of amino acids present in the sample as eluted from said column by the buffer reagents and automatic circuitry means on said frame for controlling said solenoid operated control valve and said determining and recording means to perform the steps of the analysis cycle at predetermined times, said automatic circuitry means including a portion responsive to the completion of an analysis cycle to move said solenoid operated valve from the deactivated to the activated position and to supply another sample to said sample retention means on said control valve and start another analysis cycle.

2. The apparatus according to claim 1 wherein the sample supply means comprises a turntable adapted to receive a plurality of samples, a dipper mechanism insertable into successive ones of the samples, said dipper mechanism being in fluid flow connection with said sample retention means for supplying sample thereto, said automatic circuitry means being ef fective in timed sequence to perform the analysis cycle including the steps of energizing said turntable to present a sample to said dipper mechanism, activating said dipper mechanism to supply a sample to said control valve, activating said control valve to supply the sample to the sample retention means, and deactivating said control valve permitting the eluting of the sample to be completed 3. Apparatus for determining the quantity of amino acids present in a hydrolized sample comprising a frame, a short ion exchange column on said frame, a long ion exchange column on said frame, a pair of solenoid operated control valves having sample retention means of a predetermine size connected thereto for retaining a predetermined quantity of a sample, said control valves controlling the supply of sample and buffer reagents to said long and short columns, respectively, said control valves being movable between a deactivated position permitting pressure fluid flow of the buffer reagents through said sample retention means to the ion exchange columns for effecting the sorbing of the sample onto the ion exchange column and the eluting of the sample therefrom and an activated position permitting pressure fluid flow of a sample into said sample retention means while establishing pressure fluid flow of the buffer reagents directly with the ion exchange columns, transmitting means on said frame responsive to the effluent of the eluting operation from said long and short columns for transmitting signals to indicate the quantity of amino acids present in the sample, automatic circuitry means on said frame for controlling the analysis cycle, said automatic circuitry means being effective to control the eluting operation on said long and short columns and including a portion thereof for supplying an impulse to urge said solenoid valves from the deactivated to the activated positions, said automatic circuitry means being effective to deactivate the short column solenoid operated control valve for sorbing and eluting the sample on said short column, upon completion of the short column eluting operation said automatic circuitry means being effective to deactivate the long column solenoid operated control valve for sorbing and eluting the sample on said long column and another portion of said automatic circuitry means being responsive to the completion of the analysis cycle to start another analysis cycle.

4. The apparatus according to claim 3 including sample detection means in said automatic circuitry means for turning off said automatic circuitry mean in the absence of a sample.

5. The apparatus according to claim 3 wherein the sample supply means comprises a turntable adapted to receive a plurality of samples, a dipper mechanism insertable into successive ones of the samples, said dipper mechanism being in fluid flow connection with said long and short column control valves for supplying sample thereto, said turntable being movable in response to an impulse from said automatic cir cuitry means to present a sample beneath said dipper mechanism, said dipper mechanism being inserted into a sample for a predetermined time in response to a signal from said sample retention means of the automatic circuitry means to supply a sample to said long and short column control valves, and sample detection means engageable with the sample to be presented to the dipper mechanism, said sample detection means in the absence of a sample being effective to turn off said automatic circuitry means.

6. The apparatus according to claim 5 including a refrigeration unit connected to said frame, the turntable of samples being maintained in said refrigeration unit to permit the storage of a plurality of samples substantially without deteri oration of any of the amino acids present in the samples.

7. The apparatus according to claim 5 including a pump means connected to said dipper mechanism for effecting fluid flow of the samples to said long and short column control valves, said pump means being responsive to an impulse from said automatic circuitry means to initially flush the sample supply system by passing air therethrough and thereafter said dipper mechanism is inserted into the sample to supply the sample to said long and short column control valves.

8. The apparatus according to claim 3 wherein said automatic circuitry means includes a central timer adapted to control the analysis of a sample in a predetermined timed sequence, a short column timer connected to said central timer for controlling the amino acid analysis to be carried out on the short column, and a plurality of other timers connected to said central timer for controlling the amino acid analyses to be carried out on the long column, said short column timer and said other timers being energized in response to impulses from said central timer to effect the complete analysis of a sample, and one of said other timers controlling the long column analysis being effective to transmit an impulse to said central timer upon completion of the analysis cycle to begin an analysis cycle on another sample.

9. The apparatus according to claim 8 wherein said one timer is effective to time the complete eluting operation on said long column thereby transmitting an impulse to said central timer as the regeneration of said long column is completed.

10. The apparatus according to claim 8 wherein upon completion of the analysis on said short column the short column timer transmits an impulse to the central timer to energize said other timers.

11. The apparatus according to claim 10 including a switch therein, said switch being movable between a first position connecting said short column timer with said central timer and a second position connecting a second of said other timers with said central timer, the second of said other timers being effective to time the complete eluting and regeneration operation on said long column, when said switch is in the second position the analysis will be operated in overlap condition and upon completion of the complete eluting and regeneration operation on said long column an impulse from the second of said other timers will be transmitted to said central timer to begin an analysis cycle on another sample.

12. Apparatus for determining the quantity of amino acids present in a hydrolyzed sample comprising a frame, short and long ion exchange columns on said frame, a short column buffer reagent supply, a long column buffer reagent supply, a control valve connected to said long column and controlling the long column buffer reagents supplied thereto, another control valve connected to said short column and controlling the short column buffer supplied thereto, sample supply means connected to said long and short column control valves, said long and short column control valves each including a sample retention means of a predetermined size for retaining a predetermined quantity of sample and controlling the supply of sample to said long and short columns, said control valves being movable between a deactivated position permitting pressure fluid flow of the buffer reagents through said sample retention means to the respective ion exchange column for effecting the eluting of the sample and an activated position permitting pressure fluid flow of a sample into said sample retention means to obtain the predetermine quantity of said sample while said control valve maintains pressure fluid flow of the buffer reagents directly with the respective ion exchange column, said long and short columns being connected with the remainder of the eluting system including a ninhydrin system and colorimeter, said colorimeter being effective to determine the concentration of amino acids present in the effluent from the eluting operation of said long and short columns to send signals to a recorder mechanism and record same, automatic circuitry means for sequentially effecting the complete analysis cycle and including timer means for the short column and timer means for the long columns, said automatic circuitry means activating said sample supply means to supply a sample to the sample retention means of said long and short column control valves, said automatic circuitry means thereafter permitting said short column control valve to connect the sample in the sample retention means and said short column buffer reagent with said short column, while substantially simultaneously energizing the short column timer means to control the eluting system for said short column, upon completion of the eluting operation and deenergization of the short column timer means the automatic circuitry means permitting said long column control valve to connect the sample retention means and said long column buffer reagents with said long column while substantially simultaneously energizing the long column timer means to control the eluting system for the long column, and upon completion of the eluting operation and deenergizing of said long column timer means an impulse is sent to said automatic circuitry means serving to present another sample to said sample supply means and begin another analysis cycle

Claims

2. The apparatus according to claim 1 wherein the sample supply means comprises a turntable adapted to receive a plurality of samples, a dipper mechanism insertable into successive ones of the samples, said dipper mechanism being in fluid flow connection with said sample retention means for supplying sample thereto, said automatic circuitry means being effective in timed sequence to perform the analysis cycle including the steps of energizing said turntable to present a sample to said dipper mechanism, activating said dipper mechanism to supply a sample to said control valve, activating said control valve to supply the sample to the sample retention means, and deactivating said control valve permitting the eluting of the sample to be completed.
3. Apparatus for determining the quantity of amino acids present in a hydrolized sample comprising a frame, a short ion exchange column on said frame, a long ion exchange column on said frame, a pair of solenoid operated control valves having sample retention means of a predetermine size connected thereto for retaining a predetermined quantity of a sample, said control valves controlling the supply of sample and buffer reagents to said long and short columns, respectively, said control valves being mOvable between a deactivated position permitting pressure fluid flow of the buffer reagents through said sample retention means to the ion exchange columns for effecting the sorbing of the sample onto the ion exchange column and the eluting of the sample therefrom and an activated position permitting pressure fluid flow of a sample into said sample retention means while establishing pressure fluid flow of the buffer reagents directly with the ion exchange columns, transmitting means on said frame responsive to the effluent of the eluting operation from said long and short columns for transmitting signals to indicate the quantity of amino acids present in the sample, automatic circuitry means on said frame for controlling the analysis cycle, said automatic circuitry means being effective to control the eluting operation on said long and short columns and including a portion thereof for supplying an impulse to urge said solenoid valves from the deactivated to the activated positions, said automatic circuitry means being effective to deactivate the short column solenoid operated control valve for sorbing and eluting the sample on said short column, upon completion of the short column eluting operation said automatic circuitry means being effective to deactivate the long column solenoid operated control valve for sorbing and eluting the sample on said long column and another portion of said automatic circuitry means being responsive to the completion of the analysis cycle to start another analysis cycle.
4. The apparatus according to claim 3 including sample detection means in said automatic circuitry means for turning off said automatic circuitry means in the absence of a sample.
5. The apparatus according to claim 3 wherein the sample supply means comprises a turntable adapted to receive a plurality of samples, a dipper mechanism insertable into successive ones of the samples, said dipper mechanism being in fluid flow connection with said long and short column control valves for supplying sample thereto, said turntable being movable in response to an impulse from said automatic circuitry means to present a sample beneath said dipper mechanism, said dipper mechanism being inserted into a sample for a predetermined time in response to a signal from said sample retention means of the automatic circuitry means to supply a sample to said long and short column control valves, and sample detection means engageable with the sample to be presented to the dipper mechanism, said sample detection means in the absence of a sample being effective to turn off said automatic circuitry means.
6. The apparatus according to claim 5 including a refrigeration unit connected to said frame, the turntable of samples being maintained in said refrigeration unit to permit the storage of a plurality of samples substantially without deterioration of any of the amino acids present in the samples.
7. The apparatus according to claim 5 including a pump means connected to said dipper mechanism for effecting fluid flow of the samples to said long and short column control valves, said pump means being responsive to an impulse from said automatic circuitry means to initially flush the sample supply system by passing air therethrough and thereafter said dipper mechanism is inserted into the sample to supply the sample to said long and short column control valves.
8. The apparatus according to claim 3 wherein said automatic circuitry means includes a central timer adapted to control the analysis of a sample in a predetermined timed sequence, a short column timer connected to said central timer for controlling the amino acid analysis to be carried out on the short column, and a plurality of other timers connected to said central timer for controlling the amino acid analyses to be carried out on the long column, said short column timer and said other timers being energized in response to impulses from said central timer to effect the complete analysis of a sample, and one of said other timers cOntrolling the long column analysis being effective to transmit an impulse to said central timer upon completion of the analysis cycle to begin an analysis cycle on another sample.
9. The apparatus according to claim 8 wherein said one timer is effective to time the complete eluting operation on said long column thereby transmitting an impulse to said central timer as the regeneration of said long column is completed.
10. The apparatus according to claim 8 wherein upon completion of the analysis on said short column the short column timer transmits an impulse to the central timer to energize said other timers.
11. The apparatus according to claim 10 including a switch therein, said switch being movable between a first position connecting said short column timer with said central timer and a second position connecting a second of said other timers with said central timer, the second of said other timers being effective to time the complete eluting and regeneration operation on said long column, when said switch is in the second position the analysis will be operated in overlap condition and upon completion of the complete eluting and regeneration operation on said long column an impulse from the second of said other timers will be transmitted to said central timer to begin an analysis cycle on another sample.
12. Apparatus for determining the quantity of amino acids present in a hydrolyzed sample comprising a frame, short and long ion exchange columns on said frame, a short column buffer reagent supply, a long column buffer reagent supply, a control valve connected to said long column and controlling the long column buffer reagents supplied thereto, another control valve connected to said short column and controlling the short column buffer supplied thereto, sample supply means connected to said long and short column control valves, said long and short column control valves each including a sample retention means of a predetermined size for retaining a predetermined quantity of sample and controlling the supply of sample to said long and short columns, said control valves being movable between a deactivated position permitting pressure fluid flow of the buffer reagents through said sample retention means to the respective ion exchange column for effecting the eluting of the sample and an activated position permitting pressure fluid flow of a sample into said sample retention means to obtain the predetermine quantity of said sample while said control valve maintains pressure fluid flow of the buffer reagents directly with the respective ion exchange column, said long and short columns being connected with the remainder of the eluting system including a ninhydrin system and colorimeter, said colorimeter being effective to determine the concentration of amino acids present in the effluent from the eluting operation of said long and short columns to send signals to a recorder mechanism and record same, automatic circuitry means for sequentially effecting the complete analysis cycle and including timer means for the short column and timer means for the long columns, said automatic circuitry means activating said sample supply means to supply a sample to the sample retention means of said long and short column control valves, said automatic circuitry means thereafter permitting said short column control valve to connect the sample in the sample retention means and said short column buffer reagent with said short column, while substantially simultaneously energizing the short column timer means to control the eluting system for said short column, upon completion of the eluting operation and deenergization of the short column timer means the automatic circuitry means permitting said long column control valve to connect the sample retention means and said long column buffer reagents with said long column while substantially simultaneously energizing the long column timer means to control the eluting system for the long column, and upon completion of the eluting operation aNd deenergizing of said long column timer means an impulse is sent to said automatic circuitry means serving to present another sample to said sample supply means and begin another analysis cycle.