EP0094969A1

EP0094969A1 - Method for eluting samples through a chromatographic column

Info

Publication number: EP0094969A1
Application number: EP19830900255
Authority: EP
Inventors: Donald E. Stevens; Robert J. Ehret
Original assignee: Beckman Instruments Inc
Current assignee: Beckman Coulter Inc
Priority date: 1981-12-07
Filing date: 1982-12-03
Publication date: 1983-11-30
Also published as: WO1983002160A1

Abstract

Le procédé permet l'élution automatique d'échantillons en utilisant un programme parmi une pluralité de programmes différents. La commande automatique de l'analyseur permet de sélectionner avant l'élution les conditions dans lesquelles un échantillon est élué sans qu'il ne soit nécessaire d'utiliser un programme séparé pour réaliser chaque changement au cours du procédé d'élution. La colonne est équilibrée dans des conditions de départ pour un procédé d'élution particulier et l'échantillon est élué au travers de la colonne suivant ce procédé. La colonne est ensuite régénérée et le contrôleur effectue des recherches dans la mémoire de l'analyseur pour détecter un changement dans le procédé. Si un changement dans le procédé d'élution est détecté, la colonne est alors équilibrée sur les conditions de départ pour ce procédé différent et le ou les échantillon(s) est/sont alors élué(s).The method allows automatic elution of samples using one program from a plurality of different programs. The automatic control of the analyzer allows the conditions under which a sample is eluted to be selected before elution without the need to use a separate program to make each change during the elution process. The column is equilibrated under initial conditions for a particular elution process and the sample is eluted through the column according to this process. The column is then regenerated and the controller searches the memory of the analyzer to detect a change in the process. If a change in the elution process is detected, the column is then balanced on the starting conditions for this different process and the sample (s) is / are then eluted.

Description

METHOD FOR ELUTING SAMPLES

THROUGH A CHROMATOGRAPHIC COLUMN

Background of the Invention Field of the Invention The invention relates to the field of liquid chromatography. In still greater particularity, the invention relates to the field of amino acid analysis by eluting a sample through a chromatographic column. By way of further characterization, but not by way of limitation thereto, the invention is a method for automatically changing chro atographic column param¬ eters in order to allow the elution of one or more samples under different operating conditions. Description of the Prior Art In chromatographic analysis, the division of the original sample into its constituent amino acids is a matter of column dynamics in which the ion exchange rate between the stationary phase and moving.phase of the chromatographic process is different for each of the amino acids. Hence, for a given set of conditions (eluting buffer, column temperature, and eluant flow rates), the individual amino acids will move down the column at rates characteristic to each of them, causing them to be eluted from the column in sequential fash- ion. . The exchange rates for the individual amino acids are dependent upon the chemical characteristics of the eluting buffers and the temperature of the column. Therefore, the art of developing useful chromatograms in which all of the amino acids are eluted from the column in a reasonable period of time, separate from each other, involves the manipulation of buffer charac¬ teristics and column temperature.

An elution procedure is comprised of a number of operating conditions such as column bed packing, eluting buffers, column temperature and a flow rate. A program is defined as the control instructions given to the analyzer to execute a specific elution pro¬ cedure. Sophisticated elution procedures involve changing both the eluting buffers and the column temperature during the elution of a sample while flow rates are left constant. The basic chromatographic system can be automated to comprise an analyzer in which samples are automatically injected onto the column in a sequential manner and in which the operat- ing conditions of these analyses are repeated with close precision. For most elution procedures, several of the operating conditions at the end of the run (buffer, temperature) will not be the same as those used at the outset of the analysis. Furthermore, the column must be stripped of any remaining material that may remain bound to the resin at the end of the ana¬ lysis. Usually a base such as NaOH is used. This then necessitates that the column be eluted with the first buffer until it is equilibrated to starting conditions before the next sample is injected.

The control of analyzers to cause them to do such analyses repetitively, including regenerating and equilibrating the column, has been a matter of practice for several years. The control instructions are held in a memory which is easily accessed by the operator. The control instructions, however, have been limited to automating a single elution procedure at one time; i.e., all samples applied to a column are eluted under the same operating conditions. Changing the elution procedure requires programming a new set of control instructions into the control memory. A problem arises, however, in the sequential analyses of samples requiring differing elution procedures. The ion ex¬ change column, having been regenerated following one elution procedure, must now be equilibrated to the starting conditions of the next elution procedure before the sample is injected. The series of instruc¬ tions that the operator must enter into the analyzer control system to effect these changes can become lengthy and involved. Specifically, in order to change from one elution procedure to another, existing ana¬ lyzers require a separate program to instruct the analyzer to make each change. Thus, with four elution procedures, seven programs would be required, thereby making the programming more complicated.

Summary of the Invention The invention is a method for automatically eluting samples through a chromatographic column em¬ ploying varying elution procedures. Automatic analyzer control allows the operating conditions under which a sample is eluted to be selected prior to the elution without the necessity of employing a separate program to make each change of elution procedure.

Using the present method, the column is egui- librated to starting conditions for a particular elu¬ tion procedure and the sample is eluted through the column under that procedure. The column is then re¬ generated and the controller searches the analyzer memory to sense a change in procedure. If a change in elution procedure is detected, then the column is equilibrated to the starting conditions for that different procedure and the sample or samples are then eluted.

Brief Description of the Drawings Fig. 1 is a schematic of the flow system in a chromatographic analyzer;

Fig. 2 illustrates the sequential timing and control in an elution procedure employing two buffers and two temperatures; and Fig. 3 illustrates the automatic timing and control in an analyzer employing more than one elution procedure.

Description of the Preferred Embodiment Referring to Fig. 1, a buffer pump 11 and a reagent pump 12 are positive displacement reciprocating pumps which operate at constant flow rates during the analysis. The displacement of the reagent pump is scaled to meter the reagent at a fixed proportion of the buffer flow. Samples to be loaded onto. the column are injected into the buffer stream at an injector 13. The sample is then eluted through a chromatographic column 14. The column effluent is blended with reagent at a mixing tee 15 before passing into a reactor 16. Color development takes place in reactor 16. The color is detected as the chromatographic stream flows through a photometer 17. The optical absorbance measured by the photometer is recorded on a strip chart recorder 18. In addition, the absorbance data may be operated upon by a computing integrator 19. The effluent stream then passes into a waste collector 20.

Two operating conditions which are varied during the sample elution are the eluting buffer and the column temperature. In the system shown, the eluting buffer may be selected from one of six reser¬ voirs (21 through 26) by a servo valve 27. The system illustrated features control of the column eluant by discrete buffer selections. It is also possible to vary the buffer characteristics as a function of time by the use of a buffer gradient generator. The column temperature is varied by selecting one of three set points ( _χ, ₂ or ₃) for a column thermostat 28. The buffer servo valve and the column thermostat are under control of an automatic control system 29, the control instructions being entered by the operator through a control panel 30. Table One below is a listing of the

OMP typical operating instructions to be entered by the operator to program the automatic sequential operation of an analyzer. With the exception of Link Time, Integration File, and Flow Rate, these operating in- structions are entered in combination with a value of time. The time value defines when, in the elution sequence, each particular operating condition is established.

TABLE ONE - CONTROL INSTRUCTIONS Eluting Buffers

^Bl

^B2

^B3

^B4 B₅

Regeneration Buffer

^B6 • Column Temperatures

^Tl T₂

^T3 Reagent Valve

Reagent Solvent Readout

Recycle Sample Number Link Time Integrator File Flow Rate

Control Instructions B.. through B_β select which buffer is being pumped by the buffer pump. Likewise, instructions T, through T„ define the tem¬ perature setting of the column thermostat. "Reagent" and "Solvent" are the control instructions which control the servo valve that selects either reagent or solvent to be blended with the column eluant at the mixing tee. "Readout" is the instruction which calls for printout, by the computing integrator, of the data pertinent to the analysis just completed. "Recycle" is the instruction which terminates the analysis, com¬ manding the process to be repeated if further analyses are to be done. "Sample Number" is the number of samples to be analyzed in a given set of analyses. "Link Time" is the length of time, when changing from one analytical program to another, for the analyzer to equilibrate to a new set of operating conditions. "Integrator File" controls an output line which is capable of selecting the appropriate set of parameters for data enhancement, which may be stored in an acces- sory computing integrator. "Flow Rate" establishes the flow rate of each eluting buffer. The manner in which these control parameters are utilized to effect auto¬ matic control of an analyzer is shown in Fig. 2. The controller is designed such that the analyzer will not function unless values for "Recycle" and "Sample

Number" are entered for at least one elution procedure. This prevents programming errors which could result in a waste of machine time and materials.

Mode of Operation Fig. 2 illustrates the sequential timing of changing operating conditions in a chromatographic separation that uses two different buffers and two temperatures in the elution procedure. First, the system is equilibrated to starting conditions including Buffer One and Temperature One. The sample is then injected. The first change is to the second buffer, B- , following elution of peaks A, B and C. Next, the temperature is changed to ₂- The next three peaks (D, E, F) are then eluted. Since peak F is the last peak of interest in this procedure, a readout of the data system is requested. The column is then regenerated by

OMP B₆, a basic buffer which scrubs all remaining materials from the column (peak G). The column must then be equilibrated to initial conditions B-. and T-. before the process can be repeated. Therefore, instructions B₁ and T-. are entered following the regeneration step. B., and T-, are the last control instructions selected. After a period of time suitable for column equi¬ libration, the recycle instruction is given which causes the analysis to be repeated for the next sample. If the analyzer is capable of repeating only one routine, the sequence of Fig. 2 will be adequate. However, if the repetitive cycle of Fig. 2 is to be interrupted and the subsequent analysis requires ini¬ tial conditions other than B, and T-. , some added con- trol instructions will be required to recognize the change and establish the correct initial conditions.

In the present method, upon entry into a new elution procedure, the controller will scan all program data, select the last buffer and temperature entries for that elution procedure, and operate the analyzer at those starting conditions. It is the value for "Sample Number" which determines the selection of the elution procedure. That is, upon completion of one elution procedure for a particular sample, the controller will repeat that procedure on another sample as long as the "Sample Number" has not been reduced to zero. If the "Sample Number" is reduced to zero, then the controller will move to the next sequential elution procedure with a "Sample Number" greater than zero. Thus, upon se- lection of an elution procedure, the operator must also select a "Sample Number" for that elution procedure.

Upon moving to a new elution procedure, the analyzer will equilibrate under the new starting condi¬ tions for a period of time selected by the operator before the first sample to be analyzed under the new procedure is injected onto the column. The operator

-f TRE-T

OMPI selects the amount of time by entering a value for the instruction "Link Time." If no reequilibration is required between procedures, "Link Time" is set at zero. Any value other than zero for "Link Time" will cause the search for the new initial conditions. The "Link Time" instruction will be ignored on subsequent repetitions of the same procedure, since the program automatically establishes the correct starting condi¬ tions for repetitive analyses, as shown in Fig. 2. Fig. 3 illustrates the sequential timing of controlled parameters in an analyzer which features storage for four separate elution procedures, to be run sequentially, with several samples being analyzed under each procedure. The program entries which pertain to each method are shown in the four boxes located above the four repetitive procedures in the timing diagram. The timing diagram of Fig. 3 is essentially that of Fig. 2, repeated in succession with the segment of "Link Time" inserted between the repetitive cycles. In this example, it is assumed that the first procedure starts with B.. and T., for the starting conditions. Any of the buffers and temperatures can be used to start or finish, the only restriction being that one of the buffer selections (here taken- as B_β) must be utilized for column regeneration. Each of the four programs illustrates the selection of one buffer change and one temperature change, column regeneration, data readout, plus equilibration to starting conditions, and recycle to repeat the run. In addition, the "Link Time" is specified, plus three parameters that are not time dependent, • the "Integrator File" to be used (I.F.), the number of samples to be analyzed under that procedure (S.N. ), and the "Flow Rate".

It should be understood that it is the last operating conditions which are used to equilibrate the analyzer for the next elution. The reason that the

OMPI last operating conditions are used is due to the format of the program for each procedure. Table Two below illustrates an elution procedure in which the buffer (B) and temperature (T) are changed during the elution procedure.

TABLE TWO Elution Procedure 1 Elution Procedure 2

Time (Minutes) Condition Time (Minutes) Condition

T= 0 (Start) B_Λ T= 0 (Start) ^B3^T1 T T==1100 B ^B2y.T^T2y_ T=10 B₄T₂

T=20 ^B6^T2 T=20 ^B6^T2

T=25 ^BΛ T=25 ^B3^T1

T=35 Recycle T=35 Recycle

At the start of the first procedure (T=0), ^Bι^τ _τ i^s used for 10 minutes (T=0 to T=10), whereupon a. switch to B₂T₂ for 10 minutes (T=10 to T=20) is made. At T=20, B, is used to regenerate the column for 5 minutes. At T=25, B_. is again eluted through the column to equilibrate it. At T=35, the recycle, the recycle command is activated. It is at this point that the controller will look to the sample number to de¬ termine whether or not to repeat the elution procedure. Referring to Table 2, in moving from elution procedure 1 to elution procedure 2, the analyzer will be required to equilibrate to the new starting con¬ ditions (B_T_). However, because any one condition appears only once in any one program, prior art methods required a separate program to change from elution procedure 1 to elution procedure 2. That is, any one condition will occur only once in any program because, whatever the reaction to occur in response to that condition, it will take place the first time that condition occurs. Thus, it is redundant for any condition to appear more than once in any one program. Thus, prior art required the separate program of starting conditions between elution procedures.

In the present method, the controller scans elution procedure 2 for the last set of operating conditions (B._T_ at T=25) which are always the same as the starting conditions. Thus, the analyzer is operated at the B,T₃ conditions for a time period determined by "Link Time". By using this method, the transition from one elution procedure to another may be made without any extra programs. Thus, for four elution procedures, only 4 programs are required in¬ stead of 7.

Referring to Fig. 3, a "Link Time" is used to equilibrate the analyzer prior to the outset of the first program. Equilibration permits fresh reagent and buffers to be installed, with a period of time per¬ mitted for the analyzer to reach equilibration before the first sample is injected. Equilibration could be done manually prior to the first elution procedure in which case "Link Time" for the first program could be zero, and the initial delay (L.T. ) would be omitted. After the first sample is injected, the analyzer will proceed through the analytical routine described earlier for Fig. 2. The first operation upon beginning elution procedure two .is to determine if "Link Time" 2 (L.T.2) is greater than zero. If it is (as shown in Fig. 3), the controller will search to find the equilibrating buffer and temperature (shown for reference as B.E.₂ and T.E.„), . and establish these conditions in the analyzer. The analyzer will then equilibrate for the time designated as L.T.2 before the first sample of the second procedure is injected. Procedure two will then be repeated (ignoring L.T.2) until the number of sam- pies (S.N.) designated to be run under elution pro¬ cedure two have been analyzed. When the last sample for elution procedure two has been analyzed, then, at the Recycle command, the controller will call up pro¬ gram three. The process will then be repeated, the equilibrating buffer and temperature for program three will be set into the controller, and the analyzer will equilibrate at these new conditions for the period of time designated as (L.T.3). The number of samples designated to be run under elution procedure three will be analyzed and in like fashion the equilibrating conditions will be selected for elution procedure four. When the last sample of elution procedure four has been analyzed, the analyzer will go into an automatic sequence which flushes the reagent out of the reactor and turns_ the pumps off. Fig. 3 illustrates the use of the full capac¬ ity of an analyzer having four stored programs. Of course, the controller is so designed as to operate using only one or as many of the programs as desired, skipping over any unused programs. Skipping •a program is done by entering a zero as "Sample Number" for that elution procedure. Additionally, while four programs are used in the preferred embodiment, it is possible to employ any number of programs.

An advantage of the disclosed method is its predictive capability to equilibrate the analyzer to oncoming conditions without requiring the operator to program an involved set of parameter changes between the analytical programs. All the operator need be concerned with is the individual methodologies; the analyzer will automatically proceed from one program to the next. Prior art has accommodated program control for only one elution procedure such that, if more than one elution procedure is used, a separate program is required to make the change. This system features the capability of storing several programs plus the capa¬ bility of automatically moving from one into the next.

OMΓI In the preferred embodiment, the method utilizes microprocessor based technology and is applied to an amino acid analyzer. The method could be applied using other forms of logical elements and memories, such as CMOS circuitry.

Claims

What is claimed is:

1. A method for automatically eluting one or more samples through a chromatographic column by em¬ ploying more than one elution procedure, said method comprising the steps of: a. equilibrating said column to a first starting condition corresponding to a first elution procedure; b. eluting a sample through said column utilizing said first elution procedure; c. regenerating said column; d. sensing a second starting condition for a second elution procedure, said second starting condition being the final operating condition from said second elution procedure; e. equilibrating said column to said second starting condition; and f. eluting said sample through said column employing said second elution procedure.

2. The method of claim 1 wherein step "a" in¬ cludes sensing a final operating condition from said first elution procedure.

3. The method of claim 1 further including, after said step of regenerating, the step of selecting the next elution procedure.

4. The method of claim 3 wherein said step of selecting includes scanning each successive elution procedure for a positive "Sample Number" value.

5. The method of claim 1 wherein said- conditions include a buffer substance and a temperature value.

6. The method of claim 1 further including the step of terminating said elution if a "Recycle" and "Sample Number" instruction have not been entered for at least one said elution procedure.