US3373872A - Apparatus for carrying out a rapid chromatography of amino acid and similar mixtures - Google Patents

Description

March 19, 1968 .1. HRDINA' 3,373,872

APPARATUS FOR CARRYING OUT A RAPID CHROMATOGRAPHY OF AMINO ACID AND SIMILAR MIXTURES Filed March 51, 1965 5 Sheets-Sheet 1 March 19, 1968 J. HRDINA 3,373,872

APPARATUS FOR CARRYING OUT A RAPID CHROMATOGRAPHY OF AMINO ACID AND SIMILAR MIXTURES Filed March 31, 1965 s Sheets-Sheet FIG. 3

3,373,872 APPARATUS FOR CARRYING OUT A RAPID CHROMATOGRAPHY OF AMINO J. HRDINA ACID AND SIMILARMIXTURES 5 Sheets-Sheet 5 8 w 6 w 9 1 1 5 9 h 1 m a .h M m d a m 1 INVENTOR.

United States Patent ABSTRACT OF THE DISCLOSURE A chromatographic apparatus particularly for amino acids and similar mixtures in which a pump feeds eluents from their containers and samples to be analyzed from their respective containers into one or two columns.

The present invention relates to an apparatus for ac complishing a rapid chromatography of complex mixtures such as amino acid mixtures.

Heretofore known semi-automatic and automatic apparatus for the analysis of amino acid and similar complex mixtures use two or three columns which are connected, as a rule, to two or more pumps in order to ob tain a throughflow of elution buffers and of a reagent solution through the column in addition to a pump for a ninhydrin reagent. An apparatus has already been constructed which, in the case of a single column and for practising gradient elution, uses only one pump in addition to that used for ninhydrin. This apparatus requires a rather intricate mixing chamber for varying the quality of the buffer and a special safety device for accurately terminating the period during which liquid is drawn from the mixer in order to prevent any possible intrusion of air into the pump of the column.

The disadvantages of these types of apparatus are obviated by the present invention.

According to the invention there is provided an apparatus for carrying out the separation of complex mixtures having one or more chromatography columns, wherein when the apparatus comprises at least two columns, the inlet of each column is connected through at least one two-way valve to a single pump, the intake side of which is connected to a multiple-way valve, at least three inlets of which are respectively connected directly to supply conduits from at least two elution buffer solution containers and to a supply conduit from a regenerating solution container and at least two sample reservoirs connected either to the intake side of the pump or between the pump and the columns, and wherein when the apparatus comprises a single column, the inlet of said column is connected directly to the pump.

The sample reservoirs may be arranged between the columns and the two-way valve or, alternatively, between the pump and a two-Way valve. Preferably incorporated is a multiple-way automatic valve, the individual branches or ports of which are connected to the sample reservoirs, while its center is connected to the two-way valve which in turn is connected to the column. Further, the sample reservoirs may be connected to an automatic multiple-way valve having an outlet connected to an input of said multiple-way valve.

An advantage of the apparatus according to the present invention consists in the fact that a minimum of mechani cal means is required, whether the work is performed on a single column or on two or more columns. Moreover, it is possible to use even simple pumps for subordinate or mechanical means. The apparatus can be used to advantage primarily with an automatic charging of samples from the reservoirs holding previously prepared samples, this being an important feature in practice in order that the times for performance of complete analyses may be shortened. The apparatus can however, be used to advantage even for a manual dosage to the columns, the entire apparatus being thereby simplified to a point where it can be put together from the above standard parts.

Embodiments of the present invention will now be described, by way of example, with refernce to the accompanying drawings, wherein:

FIG. 1 features schematically an apparatus intended for a pair of columns;

FIG. 2 illustrates schematically the type of connection when pressure reservoirs or dosers are used;

FIG. 3 illustrates an apparatus with a pair of two-way automatic valves;

FIG. 4 shows one alternative of construction of pressure reservoirs or dosers;

FIGS. 5 and 6 illustrate two alternative ways in which one multiple-way valve is used instead of two;

FIG. 7 illustrates the connections in the case when a single column is used.

The apparatus shown in FIG. 1 consists of twochromatography columns 1 and 2 which are connected through their bottom capillary outlets to a four-way distributor valve 3. This distributor valve can connect the outlet of the respective column either to an evaluating device (not shown) by way of a duct 4 or to an outlet by way of aduct 5. The capillary inlets to thecolumns 1 and 2 are connected by way of a two-way valve 6 to the outlet branch of asingle pump 7, the intake branch of which is connected to amulti-way valve 8. Thevalve 8 has six equally spaced inlet branches and a central outlet branch which is connected to thepump 7 as shown. One of the inlet branches to thevalve 8 is connected to the outlet branch of an automaticmulti-way valve 10, the inlet branches of which are connected with a plurality ofsample reservoirs 9. The inlet branches of thevalve 8 located on each side of the inlet branch connected to thevalve 10 are joined together by a common connection which is connected to acontainer 11 holding an elution buffer of a particular pH value. Twoother reservoirs 12, 13 hold elution buflFers of different pH values and are connected to respective inlets of thevalve 8. The sixth inlet of thevalve 8 is connected to acontainer 14 holding a regenerating solution. By rotating thevalve 8 in a clockwise direction the complete separation of a sample may be achieved in one revolution of the valve, i.e., starting from the top inlet of thevalve 8, first the elution buffer fromcontainer 11 is drawn through thepump 7 and pumped into either of thecolumns 1 or 2 depending on how thevalve 6 is arranged, then a sample from one of thereservoirs 9 is pumped into the particular column, then the elution buffer fromcontainer 11 again which is followed successively by elution bulfers fromcontainers 12, 13 and finally at the completion of the separation the regenerating solution fromcontainer 14 is pumped through the respective column.

Thesample reservoirs 9 are unpressurized i.e. arranged on the input side of thepump 7 and thereby the samples in the reservoirs are under ambient pressure. It is an essential feature of this embodiment of the invention that all the passages, including those ofvalves 6, 8 and 10, thepump 7 and thedistributor valve 3, through which a sam ple from any one of thereservoirs 9 flows, are of a capillary type and have a minimum of dead spaces thereby ensuring the maximum amount of the particular sample may be delivered to the respective column.

FIG. 2 to be considered in view of FIG. 1 shows schematically an arrangement for use when pressure sample reservoirs ordosers 15 are used, these dosers being symbolized in the drawing by squares outlined in broken lines. Dosers are reservoirs which have their inlets connected to a source of above ambient pressure which in the present embodiment is the pressure side of thepump 7. The arrangement of the apparatus is such that the pressure reservoirs ordosers 15 are connected between therespective chromatography columns 1, 2 and the outlet branches of the two-way valve 6, the connection between the respective reservoirs ordosers 15 and thecolumns 1, 2 being of capillary type. The inlet branch of thevalve 6 is connected by way of thepump 7 to the center outlet branch of themulti-way valve 8 which may be an automatic six-way valve. This embodiment enables the possibility for a step-type gradient elution to be carried out as will be described in the following. When only three elution buffers, with standard pH values of 3.24, 4.25 and 5.28 are used, their containers are connected toinlet branches 16, 17 and 18 of thevalve 8, theregenerating solution container 14 is connected toinlet branch 19 which leaves two further inlet branches for connection to reservoirs containing a finer, step-type gradient elution.

If it is desired to only use three elution buffer containers and one regenerating solution container thevalve 8 can have four Ways only, or, alternatively it may be substituted by two automatic two-way valves 20 as shown in FIG. 3.

FIG. 4 shows one of the possible arrangements of thepressure reservoir 15 connected for instance, to a six-way capillaryautomatic valve 10, a central outlet of which leads to a capillary two-wayautomatic valve 6 which enables an application of a multiple-way valve 10 to bothcolumns 1 and 2 alternately; this arrangement differs from the embodiment illustrated in FIG. 2, where the two-way valve 6 is connected to the input side of pressure reservoirs ordosers 15 each column having its own pressure reservoir. An advantage of the embodiment illustrated in FIG. 4 consists in a shorter and less complex capillary connection with any one of theclosers 15 to therespective columns 1 and 2. Instead of two automatic multiple-way valves arranged one after another it is pos sible according to FIG. 4 to use only one multiple-way valve 8 as shown in FIGS. and 6. Thisvalve 8 has to be a multiple-way valve so as to permit a successive interconnection of the central outlet to the intake branches for supply from the respective buffer containers and fromsample storage reservoirs 9.

In the embodiment shown in FIG. 5 thevalve 8 is a multiple-way valve having for example six inlet branches. The first, third and fifth of the inlet branches, counting in a clockwise direction are joined together by a commonly connectingsupply branch 21 for individual elution buffers and a regenerating solution is connected to said common connection. Saidsupply branch 21 may be connected to the outlet of a multiple-way valve (not shown) to the inlets of which are connected to the respective containers of said elution buffers and said regenerating solution. The second, fourth and sixth of the said inlet branches of thevalve 8 are separately connectedtosample reservoirs 9. The central outlet from thevalve 8 may be connected either to the intake branch of apump 7 in case thesample reservoirs 9 are of pressureless type or to a chromatography column or columns in case thesample reservoirs 9 are of pressure type. By connecting the outlet of thevalve 8 to the supply branch inlets and to the respective sample inlets for appropriate periods of time, complete separations of the respective samples can be carried out in turn by rotating thevalve 8 in a clockwise direction (FIG. 5). For example, starting with the outlet branch connected to the first inlet, a basic buffer is supplied to the column by way of thesupply branch 21, then the valve is turned so that the outlet is connected to the second inlet through which a sample is conveyed to the column, the valve is then turned so that the outlet is connected to the third inlet through which one or more elution buffers are supplied to the column by way ofsupply branch 21 until the separation of the sample is complete after which a regenerating solution followed by the basic buffer is fed into the column by way ofsupply branch 21, the cycle is then repeated by turning thevalve 8 to the fourth inlet to admit the next sample to the column.

The embodiment shown in FIG. 6 is a modification of that shown in FIG. 5. The main difference between the two embodiments is that instead of thecommon supply branch 21 in FIG. 5 thevalve 8 in FIG. 6 is a bigger valve than that in FIG. 5 and has many more inlet branches e.g., ten shown in FIG. 6. Counting in a clockwise direction, the first, third, sixth and eighth of the inlet branches are joined together by a common connection which may for example, be connected to a first elution buffer container; the fourth and ninth of the said inlet branches are joined together by a common connection which may for example, be connected to a second elution buffer container; the fifth and tenth of the said inlet branches are joined together by a common connection which may for example, be connected to a regenerating solution container, and the second and seventh of the said inlet branches are separately connected torespective sample reservoirs 9. The central outlet branch of thevalve 8 may be connected in either of the ways described with respect to FIG. 5. By successive connection of the inlets of thevalve 8 to the outlet for appropriate periods of time, complete separations of samples can be carried out in turn by rotating thevalve 8 in a clockwise direction (FIG. 6).

The entire connection however, is considerably simplified by the embodiment illustrated in FIG. 7. In this figure a single chromatography column 1, having a capillary outlet 4, is connected by way ofpump 7 to the outlet branch of'a multiple-way valve 8. One inlet branch of thevalve 8 is connected to an outlet branch of an automatic multiple-way valve 10 havingsample reservoirs 9 connected separately to the inlet branches thereof. The inlet branches of thevalve 8 arranged on each side of the inlet branch connected to thevalve 10 are joined together by a common connection which is connected to acontainer 11 of a basic elution buffer. The remaining inlet branches may, for example, be connected to containers holding other elution buffers and a regenerating solution. The connections between thevalve 10 and the column are all of capillary type.

While specific embodiments of my invention have been shown and described in detail to illustrate the application of the principles of my invention, it will be understood that the same may be otherwise embodied without departing from such principles.

What I claim as my invention is:

1. In an apparatus for the chromatography of amino acids and similar mixtures the improvement comprising in combination a plurality of reservoirs for the samples to be analyzed; a first hydraulic multi-way valve distributor having a plurality of intakes each associated with one of said reservoirs and a common first outlet; a plurality of containers for the eluent; a second multi-way valve distributor having a plurality of intakes each associated with one of said containers and a common second outlet leading into said first distributor; not more than two columns arranged in parallel; pumping means drawing liquid from said first outlet and delivering it selectively to one of said columns; third valve means effecting such selective delivery; an outlet line leading from each column; and fourth valve means selectively connecting said outlet lines to an evaluating device.

2. An apparatus for the chromatography of amino acids and similar mixtures according to claim 1 comprising a pressure doser in front of each column.

3. An apparatus for the chromatography of amino acids and similar mixtures according to claim 1 comprising a pressure dosing device between the pumping means and the third valve means.

(References on following page) 5 6 References Cited OTHER REFERENCES UNITED STATES PATENTS Separation of Bases and Amino Acids by Displacement Chromatography on Ion-Exchange Columns, S. M. 13

5g Partridge, Faraday Soc. Disc., No. 7, 1949, pp. 296-305ova 5 2 011 1 1959 coggeshall 55 197 X REUBEN FRIEDMAN, Primary Examiner.

3,097,163 7/ 1963 Riddick 210- J, DE CESARE, Assistant Examiner.

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