US3714035A

US3714035A - Method and apparatus for thin layer chromatography

Info

Publication number: US3714035A
Application number: US00146577A
Authority: US
Inventors: D Jones
Original assignee: Individual
Current assignee: ANALYTICAL SYSTEMS Inc A CORP OF; TOXI-LAB Inc A CORP OF CA; Marion Laboratories Inc
Priority date: 1971-05-19
Filing date: 1971-05-19
Publication date: 1973-01-30
Anticipated expiration: 1990-01-30
Also published as: FR2138020A1; DE2224411A1; DE2224411C3; FR2138020B1; CA971112A; GB1362463A; DE2224411B2; CH571880A5; SE390216B; JPS5441238B1

Abstract

METHOD AND APPARATUS ARE PROVIDED FOR CARRYING OUT CHROMATOGRAPHS ON A THIN LAYER OF ADSORBENT, WHERE THE THIN LAYER OF ADSOREBET HAS A PLURALITY OF HOLLOWS ADJACENT TO ONE END. INSERTED IN AT LEAST ONE OF THE HOLLOWS IN AN INSERT WHICH IS IMPREGNATED WITH AT LEAST ONE COMPOUND OF KNOWN CHEMICAL COMPOSITION AND INSERTED IN AT LEAST ONE OF THE OTHER HOLLOWS IS AN INSERT IMPREGNATED WITH A SOLUTION HAVING ONE OR MORE UNKNOWN COMPOUNDS. THE DEVELOPMENT OF THE CHROMATOGRAPH IS THEN CARRIED OUT IN A NORMAL MANNER. BY COMPARING THE DISTANCE THE UNKNOWN COMPOUND HAS TRAVELED WITH THE DISTANCE THE KNOWN COMPOUND OR COMPOUNDS HAVE TRAVELED AND THKEIR RESPONSE TO VARIOUS DEVELOPING MATERIALS, SUCH AS DYES, OXIDANTS, ETC., THE UNKNOWN COMPOUND CAN BE DETERMINED, AS WELL AS A QUALITATIVE ESTIMATE OF THE AMOUNT.

Description

Jan. 30, 1973 D. w. JONES 3,714,035

METHOD AND APPARATUS FOR THIN LAYER CHROMATOGRAPHY Filed May 19, 1971 |s /'O I 1 Fl G 2 FIG .3 I DONALD W S R J ES ATTORNEYS US. Cl. 210-31 C 22 Claims ABSTRACT OF THE DISCLOSURE Method and apparatus are provided for carrying out chromatographs on a thin layer of adsorbent, where the thin layer of adsorbent has a plurality of hollows adjacent to one end. Inserted in at least one of the hollows is an insert which is impregnated with at least one compound of known chemical composition and inserted in at least one of the other hollows is an insert impregnated with a solution having one or more unknown compounds. The development of the chromatograph is then carried out in a normal manner. By comparing the distance the unknown compound has traveled with the distance the known compound or compounds have traveled and their response to various developing materials, such as dyes, oxidants, etc., the unknown compound can be determined, as Well as a qualitative estimate of the amount.

BACKGROUND OF THE INVENTION Field of the invention Thin layer chromatography (TLC) is an extremely versatile and economical method for separation analysis of unknown compositions. Extremely simple equipment is employed, and relatively simple manipulation and handling is required. Thin layer chromatography has found widespread use in the biomedical field for analyzing for drugs, e.g. narcotics, tranquilizers, barbiturates, amphetamines, etc., naturally occurring biologically active materials, e.g. hormones, prostaglandins, steroids, amino acids and polypeptides, as well as other materials, such as pesticides, organic toxins, etc. Thin layer chromatography requires extremely small amounts of sample, usually in the order of a fraction or a few micrograms, so that it is ideally suited for situations where extremely low concentrations of materials are encountered.

Because of the convenience of thin layer chromatography, an extensive literature has developed detailing the use of solvents, adsorbents and developing agents in the analysis of a wide variety of compounds. The R values have been reported for a wide range of compounds under diverse conditions. (R, is the ratio of the distance the compound travels to the distance the solvent boundary travels.) Nevertheless, this value is sensitive to variations in conditions and it is usually desirable to have a standard, so as to provide a direct comparison. While some information about the nature of the unknown material may be determined by the method of isolation, there frequently remains a wide range of possibilities as to the chemical composition of the unknown. Therefore, it has been found necessary in most instances to maintain a relatively large number of standard solutions with which to compare the unknown to the standard.

In addition, in preparing the chromatograph, it is desirable to have a relatively discrete spot of the unknown solution. The larger or more diffuse the spot, which is initially introduced onto the chromatograph, the larger and more diffuse the spot becomes as it moves along the chromatograph. This has required relatively painstaking eiiorts of slowly introducing minute quantities of the un- United States Patent O known solution onto the chromatograph, drying the spot, and then adding another minute quantity, until the total amount of unknown material has been added.

Description of the prior art For a general discussion of thin layer chromatography see Stahl, Thin Layer Chromatography, Springer-Verlag, New York, 1969; and Thin Layer Chromatography Technical Bulletin No. 22 Brinkmann Instruments, Inc., New York 1962.

SUMMARY OF THE INVENTION Thin layer chromatographs are scored, so that by removal of the scored area, hollows are provided into which inserts may be introduced. The scored areas are adjacent one end of the chromatograph. Inserts are provided which are impregnated with one or more standardized compositions and which may be inserted snugly into the scored area. In addition, blank inserts are provided for impregnation with unknown solution which maybe introduced into one or more of the scored areas. The inserts fit snugly into the scored area so as to provide a substantially con tinuous surface. The chromatograph is then developed in the normal way by allowing the solvent system to migrate from one end of the chromatograph toward the other end for a substantial distance. The spots are then developed according to known means. By comparison of the standard with the unknown, the presence of any one of the standard compounds in the unknown may be determined.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of a thin layer chromatop FIG. 2 is a side elevational view of a thin layer chromatograph with a backing; and

FIG. 3 is a perspective view of a disc.

DESCRIPTION OF THE SPEOIFIC EMBODIMENTS A variety of thin layer chromatographs are available which employ different absorbents and ditferent means for stabilizing the adsorbents. Usually, the thickness of the adsorbent layer will be from about 0.2 to about 2 millimeters, more usually from about 0.5 to about 1.5 millimeters. Adsorbents which find widespread use are silica gel, alumina, kieselguhr, carbon black, magnesium oxide, etc. The chromatographs may be stabilized in a variety of ways, by adhering the adsorbent to a backing, such as foil or glass, or by impregnating a web of glass fiber, the latter being preferred for this invention.

The usual thin layer chromatograph will be about 1 to 10 cm. in width, and from about 10 to cm. in length. Of course, any width or length may be used in this invention, depending on the size of the equipment available, the number of materials involved, as well as the availability of such thin layer chromatographs.

In carrying out this invention, a conventional thin layer chromatograph can be employed and at a distance from about 1 to 3 centimeters, more usually about 2 centimeters, from one end, a number of small areas are scored, so as to allow for easy removal of the scored area to provide a hollow area. These scored areas may be removed at the time of fabrication of the thin layer chromatograph or may be supplied as present in the thin layer chromatograph, prior to use by the chromatographer. To the extent a plurality of areas are scored, it will be more convenient to leave the scored areas present, so that only that number which is required may be removed to provide a hollow for insertion of an insert.

Standard inserts are provided which are impregnated with one or more standard compositions at known concentrations. One or more of the inserts impregnated with the same or different standards are pressed firmly into the hollows or openings so as to provide a substantially continuous surface for the chromatograph. One or more inserts are impregnated individually with one or more of the same or different unknowns and similarly inserted into available hollows.

The normal chromatography procedure is then followed, by introducing the chromatograph into a jar having sufiicient solvent, so that the chromatograph is immersed to the extent of a few millimeters. The solvent is allowed to migrate, normally to a fixed distance from the end, at which time the chromatograph is removed from the jar and dried.

Depending on the compounds involved, the compounds may be observed for fluorescence, or developed with dyes, oxidants, etc. Chromatographic adsorbents frequently have materials incorporated which permit direct observation of spots with certain compounds. For example, fluorescing materials may be incorporated and compounds which quench fluorescence would be observable. By comparison of the spot formed by the unknown with the spots from the standards, a determination may be made if one or more of the standard materials was present in the original unknown and qualitatively the amount of the material.

The inserts which are provided may be of any shape which conforms with the scored area on the chromatograph. Most conveniently, the scored area will be circular, but may be rectangular, square or other shape. The size of the scored area will normally have as its largest dimension approximately A; inch to inch, preferably about 3 inch, Blank inserts are provided which are used for the unknown, by dipping the insert into a solution containing the unknown and then drying the insert. The inserts may be made of any appropriate material, such as supported adsorbent (see subsequent discussion), filter paper or other adsorbent material.

The standards are conveniently prepared by spraying a sheet of the adsorbent material, drying the sheet and cutting out the inserts.

The standard inserts may have more than one component, particularly where there are a family of compounds which have similar properties and are separated by the same adsorbent and solvent system. Desirable combinations would be morphine and morphine analogs; barbiturates, certain groups of tranquilizers, e.g. benzdiazacycloheptanes; various groups of steroids; prostaglandins; popular phosphate insecticides; popular carbamate insecticides; halogenated fungicides; halogenated insecticides; amphetamines, etc. Depending upon the ease of detection, as little as one microgram of the unknown compound need be present and almost never more than one milligram, more usually in the range of about 2 micrograms to 500 micrograms and preferably in the range of about 2 micrograms to 50 micrograms. The amount of material impregnated in the insert can be easily determined by the amount of solution of known concentration adsorbed by the insert.

The following are illustrative families of materials which may be impregnated in a single disc:

Morphine and related compounds normally have a tertiary amine and one or more oxy or x0 functionalities. Illustrative morphine and physiomimetic analogs are hydromorphone, oxymorphone, codeine, hydrocodone, pholcodine, dextromethorphan, phenazocine and dionin.

Physiomimetic analogs to morphine include methadone and structural analogs, illustrated by dextromoramide, dipipanone, phenadoxone, propoxyphene (Darvon) and acetylmethadone.

Another class of compounds includes meperidine and its analogs, such as alphaprodine, alvodine and anileridine.

Another class of compounds are the catecholamines, illustrated by cotainine, narceine, noscapine, papaverine, epinephrine, and L-dopa. Analogous compounds of catecholamines are those having unhydroxylated phenyl rings which include amphetamine, paredrine, ephedrine, methamphetamine and norephedrine.

Another class of compounds are the barbiturates which are 5,5-disubstituted barbituric acids or their sodium salts. The substituents at the 5 position are normally hydrocarbon, usually from 1 to 6 carbon atoms. Illustrative barbiturates are veronal, medinal, luminal, prominal, soneryl, nembutal, amytal, dial, phenadorn, seconal, evipan, phenobarbital and pentothal.

Another drug of importance is cocaine and its hydrolytic product ecgonine.

Among the steroidal families of interest are the androgens (male hormones), estrogens (female hormones), gestogens and corticosteroids, including the mineralcorticolds and glucocorticoids.

Another compound of interest is tetrahydrocannibinol which is an active ingredient of marijuana.

Another group of compounds which are of interest are tranquilizers, which include Meprobamate, benzdiazocycloheptanes, such as Librium, Diazepam or Oxazepam, and phenothiazin such as Chlorpromazine, Vesprin, Phenergan, Diparcol, and Pyrrolazote.

Another group of compounds are the sapogenins and saponins, of which digitalis finds widespread use.

Other groups of compounds include the vitamins, which because of their widely different structure, would probably be analyzed only for members having similar chemical structure, such as the D vitamins. Also, various saccharides and polysaccharides may be analyzed. As already indicated, pesticides can be analyzed, normally having such groups as phosphates, carbamates, thiocarbamates, sulfenamides, halogenated polycyclics, etc.

Also, various polypeptides and amino acids may be analyzed, such as tryptophan, alanine, glutamine, aspartic acid, tyrosine, cysteine, angiotensin, luteinizing hormone, insulin, follicle stimulating hormone, etc.

Of course, the above list is intended only to be illustrative, since the subject invention may be employed with any material which can be chromatographed, providing a convenience both in preparing the spot for the chromatograph and the standard for comparison with the unknown.

The method of this invention will now be indicated with a specific example with consideration of the draw ings. First, an unknown sample is obtained, either from such biological fluid as urine, saliva, blood, or other fluid, and treated in an appropriate manner, so as to permit extraction of a class of compounds. For example, if basic compounds such as amines were to be extracted, normally a high pH would be used. If acid compound were to be extracted, normally a low pH would be used. (Of course, the subject invention may be used with other than body fluids, such as scrapings from plants, digested plants, pollutants, or any material which has a component which is to be assayed.)

After treating the biological fluid in an appropriate way, the solution is then extracted with a water immiscible solvent so as to provide an organic and an aqueous layer. The organic layer should have the unknown material to be assayed.

Depending on the particular assay, the organic solution may then be treated in a variety of ways to remove possible interferants, to stabilize the solution, or even to modify the unknown, so as to make it more amenable to chromatography. After the appropriate treatment, the organic solution is then concentrated to the desired concentration, so as to provide suflicient material on the insert.

In accordance with this invention, a large number of inserts 10 are provided, as depicted in FIG. 3, which are most conveniently circular. The chromatograph 12 is usually rectangular having a locating marker 14 at one end. If desired, a boundary line 16 is drawn which indicates the distance to which the boundary of the solvent is to be allowed to go.

A plurality of scored areas 18 are provided which are adjacent to the edge opposite the locating marker 14. Two or more scored areas are provided, which are scored so as to allow for the easy removal of the scored portion to leave a hollow area. The insert is shaped the same as the scored area and fits snugly therein, providing a substantially continuous surface through which the solvent migrates.

Two or more of the scored areas 18 are removed so as to provide places for the introduction of inserts. A blank insert 10 is taken and dipped into the concentrated unknown solution absorbing the unknown solution. The insert is then allowed to dry and inserted into one of the hollow areas formed by removal of the scored portion. Preferably, although not essential, the unknown is placed in the middle. Assuming one is interested in a particular class of compounds, or a number of different classes of compounds which can be chromatographed by the particular absorbent and solvent system employed, one or more standards may be inserted into one or more of the hollow areas which are provided.

The inserts are self-supporting so that means must be provided for their not crumbling under normal handling conditions. This can be achieved in a variety of ways. With chromatographys which have a backing 20 such as glass or a foil, supporting an adsorbent layer 21, the insert may also be made of an adsorbent which is adhered to a backing, Where the backing is glass, it will normally be preferable to provide the necessary stability using glass fiber, such as employed in conventional, commerically available thin layer chromatographs. Other layers may also be provided for stabilizing the insert. It is found, that the insert which is supported by glass fiber can be used as an insert in the other chromatographs, such as the foil and glass backed chromatographs. Alternatively, various grades of filter paper may be employed advantageously.

After inserting the desired number of inserts with the appropriate standards and unknows, the chromatograph 12 is introduced into a jar which has a sufiicient amount of the solvent system to immerse the bottom of the chromatograph a few millimeters. A cover is put over the jar to prevent evaporation, and the solvent is allowed to migrate until it reaches the boundary marker 16. This is usually a factor of fifteen minutes to a few hours. The chromatograph is then removed from the jar, the solvent allowed to evaporate and then, depending upon the particular compounds to be observed, the chromatograph developed in a number of ways.

As already indicated, a number of compounds respond in specific ways to certain dyes. Alternatively, the chromatography may be introduced into a jar of iodine vapor and developed. In addition, certain materials are sensitive to oxidants and either decolorize the colored oxidant, or become colored so as to be identifiable. By comparison of the color of the standard, in relation to the unknown,

a qualitative determination of the amount of unknown may be made.

In FIG. 1, the chromatograph is indicated after developing, where two unknowns were employed, and a single standard having four difierent constituents. The chromatograph would indicate that the second sample does have one of the components present in the spots provided by the standard 24. The other unknown would have a component 26 which is not present in the standard.

In accordance with this invention, a simplified method is provided whereby a large stock pile of standards can be conveniently maintained for use in thin layer chromatography. In this manner, thin layer chromatographic analysis can be greatly simplified by direct comparison of the standard to the unknown on the same chromatograph. In addition, applying the unknown to the chromatograph is greatly simplified providing a simpler more efficient way of insuring localized application of the unknown to the chromatograph.

The subject invention is extremely advantageous in analytical laboratories. The number of even common drugs, which are subject to abuse and over-dosage, is great. To have smaller laboratories obtain a library of pure drugs in the proper form is presumptuous. Even large laboratories with unlimited facilities are deficient in not having enough standard drugs to fully identify recovered spots in unknowns. Pure compounds are generally unavailable through chemical supply houses and frequently must be obtained from manufacturers. When one considers the large number of drug manufactures, some idea is obtained as to the difliculty of forming a drug library. The problem is enhanced since the use of pills or capsules to obtain standard drug material is frequently unsatisfactory. Additionally, because of the abuse potential and associated stringent laws, many of the drugs, e.g. morphine and heroin, are obtained only after great difficulty. The subject invention obviates these problems and provides an efiicient and convenient library of drugs for thin layer chromatographic analysis.

What is claimed is:

1. Method for chromatographing an unknown solution employing a thin layer chromatograph having an adsorbent surface with a small hollow area adjacent one end of said chromatograph which comprises:

immersing in said solution at the desired concentration,

a self supporting insert, which is shaped to fit snugly in said hollow area;

inserting the insert into said hollow; and

chromatographing with a solvent system, wherein the solvent system migrates from the boundary adjacent said hollow toward the opposite end of said thin layer chromatograph.

2. A method according to claim 1, wherein said insert is dried after immersing and prior to inserting into said hollow.

3. A method according to claim 1, wherein said adsorbent surface is comprised of silica gel or alumina and is supported by glass fiber, glass or foil.

4. A method according to claim 1, wherein said insert is circular and is of from A3" to A" in diameter.

5. A method according to claim 1, wherein a plurality of hollow areas are provided which are equidistant from one end of said thin layer chromatograph, and wherein in at least one hollow area, an insert impregnated with at least one compound of known composition is inserted prior to chromatographing.

6. A method according to claim 5, wherein said compound of known composition is a biologically active compound.

7. A method according to claim 1, wherein said insert is impregnated with a plurality of compounds of known composition belonging to the class of narcotics, barbiturates, amphetamines, steroids, catecholamines, or tranquilizers.

8. A thin layer chromatograph having a plurality of scored or hollow areas, the hollow areas obtained by removal of the scored portion;

the scored or hollow area being in a linear arrangement paralleling and adjacent one end of said thin layer chromatograph. v

9. A thin layer chromatograph according to claim 8, wherein the adsorbent of said chromatograph is silica gel or alumina and said adsorbent is supported by glass fiber or foil.

10. Self-sustaining adsorbent inserts usable in a method according to claim 1;

which are of substantially regular shape, having a selfsustaining adsorbent layer; and

their largest dimension in the range of A3" to A".

11. Inserts according to claim 10, wherein said insert is composed of silica gel or alumina supported by glass fiber or foil.

12. Insert according to claim 11, wherein said insert is composed of silica gel supported by glass fiber.

13. Insert according to claim 10, impregnated with at least one member of the class of barbiturates in an amount of about 1 to 500 micrograms.

14. Insert according to claim 10, impregnated with at least one member of the class of narcotics in an amount of about 1 to 500 micrograms.

15. Insert according to claim 10, impregnated with at least one member of the class of tranquilizers in an amount of about 1 to 500 micrograms.

16. Insert according to claim 10, impregnated with at least one member of the class of steroids in an amount of about 1 to 500 micrograms.

17. Insert according to claim 10, impregnated with at least one member of the class of amphetamines in an amount of about 1 to 500 micrograms.

18. Insert according to claim 10. impregnated with at least one member of the class of catecholamines in an amount of about 1 to 500 micrograms.

19. Insert according to claim 10, impregnated with at least one member of the class of polypeptides in an amount of about 1 to 500 micrograms.

20. Insert according to claim 10, wherein said selfsustaining adsorbent layer is filter paper.

21. Insert according to claim 10, impregnated with at least one member of the class of hypnotics in an amount of about 1-500 micrograms.

22. Insert according to claim 10, impregnated with at least one member of the class of central nervous system stimulants, in an amount of about 1-500 micrograms.

References Cited UNITED STATES PATENTS 3,046,779 7/1964 Coleman 21031 C 3,318,451 5/ 1967 Przrbrlowicz 210-31 C 3,449,083 6/1969 Pelick 210-31 C 3,513,092 5/19 Matheane, Jr. 21031 C 3,600,306 8/1971 Tocci 210-31 C JOHN ADEE, Primary Examiner US. Cl. X.R. 210-198 C