V O L U M E 26, NO. 10, O C T O B E R 1 9 5 4 from each other hut difficult when no separation occurred. Upon ignition of the paper strip containing the two metals, the recovery of iron and molybdmum was not found to be quantitative. However, when the deterinination was made by the colorimetric methods of Fortune and Nellon (5’) and Evans, Purvis, and Bear ( 2 ) the quantitative determination of iron and molybdenum, in the presence of each other, was possible with good precision. ACKNOWLEDGMENT
The authors are indebted to E. F. Reese for the many helpful suggestions and encouragements tendered, and to E. F. Stevenson for the spectrophotometric determinations.
1549 LITERATURE CITED (1)
Codell, 11.. AIikula, J. J., and Norwitz, G., ANAL.CHEM.,25, 1441 (1953).
(2) Evans, H. J . , Purvis, E. R., and Rear, F. E., Ibid., 22, 1568
.
,
(1950).
Fortune. TV. R., and hlellon, 11. G., IND. ENG.CHEM.,ANAL. ED., 10, GO (1938). (4) Godar, E. 31.. and Alexander, 0. Ii., Ibid., 18, 681 (1946). ( 5 ) Knanishu, ,J., and Rice, T., I b z d . . 17. 4 4 4 (1945). (6) Lederer, E., and Lederer, AI., “Chromatography,” pp. 67, 317, London, Elsevier Publishing Co., 1053. (7) Smith, 0. C., “Inorganic Chromatography,” p. 57, New York, D. I‘an Nostrand Co., 1953. (3)
for review March 26, 1954. RECEIVED
Accepted August 12, 1951
Indicator Chromatographic Analysis of Organic Mixtures H. S. KNIGHT
and
SIGURD GROENNINGS
Shell Development Co,, Emeryville, Calif.
A n indicator-chromatographic methocl has been developed for the analysis of mixtures of polar organic liquids such as alcohols, ketones, and amines. The completeness of displacement of a sample by an eluent was found to depend on the ideality of the solution as well as on the relative adsorbabilities of the materials; in nonideal systems the sample is abnormally strongly adsorbed and may be bypassed by the eluent. Techniques for utilizing or avoiding bypassing by careful selection of eluent are described and examples of displacement development and frontal analysis by indicator adsorption methods are given. The equipment and mechanical procedure are adapted from the fluorescent-indicator chromatographic method of Criddle and LeTourneau for the deterniination of hydrocarbon types, but daylight dyes are used. The analysis requires 1 to 2 hours of elapsed time and about 15 niinutes of operator time. The accuracy varies from 1 0 . 1 to *4%, depending on the sisteni.
I
S THE: chromatographic analysis of colorless materials i t is often helpful to employ colored substances as indicators, whose locations on the column relative to the colorless materials are known. I n this way the course of the separation may be followed visually and often zones can be measured whose lengths are relntcd to the sample composition ( 2 ) . Criddle and LeTour~ieau(3) have described a fluorescent-indicator method for the deterniination of hydrocarbon types in gasoline and other petroleum fractions, in which a small sample containing traces of fluorescent dyes is forced through a long, narrow column of d i c a gel with alcohol as the displacing agent The sample components ai e aligned in the column on the basis of adsorbability, with saturate= hrst, followed by olefins, aromatics, and alcohol last. The bounciai ies between the zones are made visible in ultraviolet light by the fluorescent dyes, and the composition of the sample is determined by measuring the zones, the lengths of which are proportional to the concentrations of the types in the sample. Reclentlv L:llis and LeTourneau ( 4 ) have employed a mixturc of daylight .iud fluorescent dyes as indicators for the determination of hydi ocw-bon type and total ovygenated solvent content in lacquer thinners. I n the present work indicator adsorption methods were developed for the determination of individual compounds in oxygenated solvent and related systems of known qualitative coniposition. The eluotropic series or order of adsorbability of the components of each system was determined, and suitable oilsoluble dyes were selected to serve as indicators. The displace-
ment development technique, outlined above for hydrocarbons could usually be employed. However, in many nonideal systems the sample components failed to align themselves according to adsorbability; yet,, in most such instances the separation could be improved by using special eluents or by resorting to special techniques. A few systems were analyzed by frontal analysis where a larger sample is forced through the column without the use of developer, and at the liquid front the most weakly adsorbed component forms a zone, the length of which was indicated by the appropriate dj-e. The technique of indicator analysis is very simple; the theory, however, is complex and incompletely understood. I n this paper a qualitative theory is presented to explain the observed phenomena and assist analytical chcmists in establishing similar methods. APPARITUS AND PROCEDURE
The apparatus and procedure devised for hydrocarbon analysis ( 3 ) are suitable with certain modifications for other organic mixtures. The design of the analytical column employed in this work is shown in Figure 1. Specifications for the column truebore tubing are given in ASTM standards (1). The column is packed with about 7 ml. of Davison’s Gradc 923 (100- to 200-mesh) silica gel, and a few cubic millimeters of dyed gel-made by slurrying 0.1 gram of dye with 2 ml. of gel in a solvent and evaporating off the solvent-is inserted during the packing so that it appears somewhere in the separator section, below the surface of the adsorbent. For displacement development, the sample, usually 0.5 ml., is added to the surface of the adsorbent from a pipet, or a 1.00-ml. syringe for greater accuracy. Air pressure of 0.5 to 1 pound per square inch is applied until the sample is taken up by the adsorbent; then a 1- or 2-cm. layer of silica gel is added slowly to absorb any sample on thr walls of the column and prevent the eluent and sample from mixing. The eluent i3 added and pressure is again :tpplicd until the sample reaches the lower part of the analyzer wction. The sample component zones are niade visihle by the indicator dyes a t their boundaries. The zone Icngths a x measured and the sample composition in per cent by volume is calculated from the ratio of each zone length to the total sainple length, provided all the sample components are determined directly; otherwise the calculation is made from the equation Component, % v. =
zone length, mni. ~sample volume, ml.
where F is determined for the particular column design by displacing a known volume of a hydrocarbon such as iso-octane (2,2,4-trimethyl pentane) or benzene with isopropyl alcohol, and
ANALYTICAL CHEMISTRY
1350 calculating F from the resulting data. (The value of F is about 0.11 for the column shown in Figure 1.1 For frontal analysis the column is prepared as usual except that the gel quantity is critical and must be constant within 1%. More than enough sample is added to fill the packed section of the column and no eluent is employed. The zone length and the sample composition are correlated empirically, as discussed below. The analysis requires l or 2 hours of elapsed time, and about 15 minutes of operator time. When it is completed, the column may be cleaned by flushing with water from a long hypodermic. tube.
Table I.
Table 11. Elution in Nonideal Systems (Expected aone length of 0.5-ml. sample, assuming complete separation, 440 mm.) .lctirity Coeficientn for Dilute Zone, C’ Sample in Eluent Sample hlm. Bypassed Eluent Water 243 44 5 Acetone Water Acetone 155 A4 7 Methanol n-Heptane 0 100 20 Ethyl alcohol n-Heptane 440 0 13 Water Ethvl ether 0 100 (100) dcetone Ethyl ether 420 3 2 Ethyl alcohol Ethyl ether 400 10 2 5 0 Activity coefficient at infinite dilution with pure liquid as reference state. zC
Dyes Used as Indicators
Trade Name Oil Scarlet ZBL
Manufacturer Calco Chemical Division, American Cyanamid Co., Bound Brook, N. J.
Nile Blue B B Ind Plasto Orange RS
National Aniline Division, Allied Chemical and Dye Corp., New York, S . T.
FIA dye
Patent Cheniicals Inc., Patterson, N. J.
Anthraquinone Violet Base Capracyl Red B Luxol Fast Red B
Organic Chemicals Department, E. I . du Pont de Semours 8- Co., Wilmington, Del.
Air Pressure
DETERMINING THE ELUOTROPIC SERIES
The eluotropic series or order of adsorbability was determined by frontal analysis of solvent pairs a t 1 to 1 (by volume) composition. An excess of each blend (more than enough to fill the packed section of the column) was forced through the column and the first few drops of effluent were analyzed by refractive index. A representative eluotropic series on silica gel, in decreasing order of adsorbability, is as follows: Pyruvic acid Methanol Acetone Water Ethyl alcohol sec-Butyl alcohol Ethyl acetate
Ethyl ether Diisopropyl ether Isopropyl chloride Benzene Diisohutylene Heptane
Long Taper
True Bore Capillary Tubing
1. 6 0 - 1 . 6 5
i in
The adsorbability depends on the functional groups and on the molecular weight. Additional series for specific solvent systems were determined as needed or estimated by analogy with the above series. CLASSIFYING THE INDICATOR DYES
The oil-soluble dyes employed as indicators were classified in groups according to adsorbability by placing a small amount of each dye a t the top of a 10 X 100 mm. silica column and adding successively stronger eluents until the dye was displaced. Dyes for specific systems were selected from w i t h the appropriate groups in the same manner, using the system components as eluents. Usually the analyt,ical column (Figure 1) was employed for the final selection. The dye? used are listed in Table I. INDICATOR ANALYSIS AND ACTIVITY
In attempting to displace a sample with an eluent, using a dye to indicate the extent of displacement, it is often observed that the sample volume ahead of the dye becomes progressively smaller as the material proceeds t,hrough the column of adsorbent. If the column is sufficiently long, the sample is entirely “bypassed” by the eluent. This may occur even though the sample is much less strongly adsorbed than the eluent, according to the eluotropic series, if the sample and eluent form a sufficiently nonideal solution. -2 theoretical treatment of batchwise adsorption, including the effect, of nonideality, is given by Kipling and Tester ( 5 ) . Consider the sample-eluent boundary in the column, where a concentration gradient has developed. On the eluent side a dilute solution of sample in eluent exists in the interstices between
.
Dimensions in millimeters
1220
Analyzer
w
I
30
Tip
Figure 1. .4dsorption Column
the gel particles, and if the solution is nonideal the sample molecules have abnormally high activity and tend to escape from the solution by being adsorbed. This is analogous to a vapor-liquid equilibrium system where the sample or solute molecules escape into the vapor in abnormally high concentration. Qualitatively, then, the completeness of displacement may be said to depend on two factors: the relative adsorbability as represented by the eluotropic series, and the ideality of the solution. Although silica gel was used exclusively in this investigation, it is reasonable to assume that the same factors may help explain adsorption on many if not all adsorbents. I n Table I1 some examples of elution in nonideal binary systems are given, together Kith the pertinent activity coefficients in round numbers. It is shown that acetone and water tend to bypass each other to an extent that is a direct function of the activity coefficient for dilute sample in eluent. Methanol fails to displace n-heptane, whereas ethyl alcohol displaces this hydrocarbon completely. Methanol-hydrocarbon solutions are sufficiently more nonideal than ethyl alcohol-hydrocarbon solutions to overcome the effect of relative adsorbability, for according to
V O L U M E 26, NO. 10, O C T O B E R 1 9 5 4 the eluotropic series methanol would have been deemed the better eluent. Acetone is a better eluent for ether than ethyl alcohol, because acetone is more strongly adsorbed and forms a more nearly ideal solution with ether than does alcohol. Water bypasses ether completely. although it precedes ether in the eluotropic series. 4SORBOTROPES
The net result of lionideality ie to cause a component which is present in low concentration to be strongly adsorbed. This gives rise to the familiar S-shaped “adsorption isotherm” ( 5 ) , which represents data obtained by analyzing a series of blends of components -4and B that have been allowed to come to equilibrium with a given quantitl- of adsorbent. .4t lorn- values of -4, A is adsorbed preferentially, while at high values of A, B is more strongly adsorbed. .it some intermediate concentrat,ion the adsorbed material will have the same composition as the solution. This composition, which does not change on addition of the adsorbent, has been called an adsorption azeotrope, hut the authors believe that the term “asorbotrope” is more descriptive. The composition of the “asorbotrope” can also he determined by frontal analysis of mixtures of A and 13. If .i is above B in the eluotropic series, and a 1 to 1 (volume) mixture is passed through a column, niore .i than B will be adsorbed and B will appear a t the liquid front. This is the way the eluotropic series given above was determined At lower concentrations of B, B will be adsorbed and -4will appear a t the liquid front, and a t the asorbotrope composition the misture ivill pass through the column unchanged. If a dye is available which is displaced by A but not by B, the frontal analysis may be carried out with the aid of the indicator. The asorhotrope composition is the maximum concentration of I3 a t which no B appears ahead of the dye. Aiseither A or B m:+y iippear :it the front, depending on the composition, it follows that the asorhotrope is more strongly adsorbed than either A or B. Thus, for acetone-water blends a dye (Lusol Fast Red B, Table I ) is available which is not displaced by either acetone or water, though soluble in both, but is displaced by a mixture of thr two. This dye appears a t the liquid front only if the asorhotrope itself is being forced through the column. Some esaniples of asorbotrope compositions on silica gel are given b(!lo\v, with the more strongly adsorbed member on the left. .I*oi.hotrope Coiii~iosition.Voluine Acetone 98 0 Ether Methanol 75 .hetone Acetone 03 Water Methanol 81 Iso-octane
2 0 25
37 19
The variat)les sff(v;ting the composition of the asorbotrope have not been studied. Because the asorbotrope is more strongly adsorbed than either of its memhcrs, and an excess of either member tends to be separated during frontal analysis, it appeared t,hat the asorbotrope should he a perfect eluent for either of its members. Unfortutiately, experiments showed that this was often incorrect, probably because the adsorbability difference between the asorbotrope and the menihrr is often not very great. Thus the acetone-ethcr asorhotrope which contains only 2y0 ether could not be much more strongly adsorbed than acetone itself. For this reason a n asorbotrope is generally unsuitable as an eluent for its more strongly adporbed member, although it is usualll- a satisfactory eluent for its mor? weakly adsorbed member. SELECTIIVC, ELUENTS FOR INDICATOR ANALYSIS
In selecting eluents for given solvent systems, certain rules may be set forth as guides. In this discussion a “normal” system is defined as one in which any component will displace any other component which is lower in the eluotropic series. rln example of a normal system is methanol-butyl alcohol-butyl ether. Such mixtures can be readily separated if a normal eluent is available, or the most strongly adsorbed member may be used as the eluent and its.concentration in the sample determined by difference.
1551
If the sample components are not normal, an eluent which is normal with component A and abnormal with the others may displace A while bypassing the other components. If the mixture is partially normal, an eluent which is normal with respect to A would elute -4and any sample components below A in the eluotropic series with which -4is normal. hn example of the first case is the system water-ethvl alcohol-ethyl ether, in which acetone displaces only the ether. A butyl ether solution in butyl alcohol containing a little water falls in the second cat’egory; here methanol displaces the alcohol and ether, and the water is bypassed. These systems are discussed further below. Khet,her a given binary system will be normal may be decided from relative adsorbability and ideality considerations. The relative adsorbabilit,y may be estimated by analogy with known compounds of similar structure, or det,erniined as outlined in connection with the eluotropic series. Compounds having a given functional group but differing by at least 2 in carbon number have often been found to be normal, as would be expected, since they differ in adsorbability and form relatively ideal solutions. Conversely, compounds of the same carbon number but with different functional groups are usually abnormal unless one functional group confers much greater adsorbability than the other, as such solutions are usually nonideal. Water is generally abnormal with organic materials. An asorbotrope containing as its more weakly adsorbed member the component to be displaced is often a useful eluent,. Ultimately the behavior of any system must be verified by experiment. Two indicator chromatographic procedures niay be employed to determine whether a binary system is normal. The eluent E, the more strongly adsorbed member, may be uJed to displace a known volume of the other memher, A, and the zone occupied by A in the analyzer may be compared with the calculated length using factor F . A more sensitive method involves testing to see whether an asorbotrope is formed, using the method described earlier. If an asorbotrope containing less than 1% of A is formed, the displacement of A by E will still be complete within the limits of accuracy of the method (except for special cases where lox concentrations of A are to he determined). There must be an adequate adsorbabilit,y difference b e h e e n E and .4or the displacement will not be complete even if the solution is ideal. The presence of a third component B may affect the separation markedly. SYSTElIS ANALYZED
Displacement Development Analysis. Although most solvent blends are sufficiently nonideal to affect chromatographic separations on silica gel, some systems are normal as defined above and niay be completely analyzed by the displacement development technique. I n Table I11 some esamples of this type of analysis are given. The simulated product of alkaline hydrolysis of isopropyl alcohol, diisopropyl ether, unreacted chloride, and diisobutylene (subst,ituted for the propylene) was separated using isopropyl alcohol as the eluent. This mixture was analyzed completely, the alcohol being determined by difference. However, in the isopropyl alcohol-diisopropyl ethw-water system, the ether alone was det,ermined and the water was bypassed. In the next example, ethyl alcohol was separated quantitatively from a mixture of ethyl acetate and acetic acid, which were determined t.0gether. The butyl alcohol-butyl ether blend was analyzed completely with methanol as the displacing agent. A 5.0-nil. sample of o.5yOtert-hutyl chloride in tert-butyl alcohol was analyzed by a two-column technique which has been found generally applicable for determining low concentrations of completely separable, less st,rongly adsorbed solutes. The chloride in the sample was concentrated into a 0.5-nil. portion by passing the sample through a column holding about 20 grams of adsorbent. The first 0.5 ml. (approximately) of effluent, containing all of the chloride. was analyzed in the usual manner and the chloride zone length was converted to per cent hy volume of the original sample.
1552
ANALYTICAL CHEMISTRY
Small amounts of readily separable, more strongly adsorbed solutes may be determined by employing a larger sample and ultimately measuring the zone occupied by the solute. As an example of the determination of one component in a mixture by employing an eluent which displaces that component while bypassing the others, the analysis of ether-alcohol-water blends may be considered. Acetone displaced the ether nearly quantitatively while bypassing the alcohol and water. In order to obtain complete bypassing, a special column having 70-cm. separator and 70-cm. analyzer sections and holding 17 inl. of gel was employed. The use of asorbotropes as eluents is illustrated by the etheralcohol-water system. Acetone alone is a fair eluent for ether, but the acetoneether asorbotrope containing 2.0% ether elutes ether quantitatively. Thus, samples containing 0.5, 1.5, and 4.0 volume % ether in alcohol and water gave 0.7, 1.5, and 3.6 volume % ether by displacenleiit development with the acetone ether asorbotrope as eluents. In attempting to determine water in isopropyl alcohol, using the acetonewater asorbotrope as the eluent, it was found that water was strongly adsorbed, upsetting the asorbotrope, and forming a more nearly ideal mobile phase consisting of isopropyl alcohol and acetone. The acetone-ether asorbotrope was not upset by ethyl alcohol or water because acetone and ether form the most nearly ideal solution of any binary made up of sample and eluent components.
and B, but not by B. This means that B must be present in high enough concentration to appear a t the front; otherwise the dye will not show any separation. I n other words, B must be present in excess of its concentration in the asorbotrope. I n special cases where a dye is available which is desorbed by the asorbotrope but not by either component alone, this dye may be employed t o show any separation that takes place, and a second dye which is displaced by .4 but not by B may be added to indicate which material is a t the liquid front. a0
.
I
I 7 0 I
Table 111. Sjstems Analyzed by Displacement Development and Dyes Employed Accuracy, Sample Componenta Isopropyl alcoholC h s o g r o p y l ether Isupropyl c21Im:~l Diisobutylene
Dye Employedb Plasto Orange RP .4nthraquinone Yiolet Base FIA dye. aromatic marker FI.4 dye, olefin marlier
IfJopropyl alcohol
Isopropyl alcohol C Diisopropyl ether Waterd
Plasto Orange R S
Ethyl alcohol
Ethyl alcohol C Ethyl acetate, acetic acid
Plasto Orange RS
Eluent Isopropyl alcohol
Methanol
tert-Butyl alcohol
sec-Butyl alcohol see-Butyl ether ( 5 7 ) terf-Butyl alcohol tert-Butyl chloride ( 0 3 % )
v.
, . .
1 1 1
0
50
150
100
200
250
300
Zone Length of More Weakly Adsorbed Component, m m .
...
Figure 2.
1
Frontal Analysis of Solvent Blends
...
L u x o l Fast Red B Oil Scarlet ZBL
2
'J'able 1V. Systems Anal?zed by Frontal Analysis and Dyes Employed Cornposition.a '7 Dyc Employed b
...
...
2
1
Oil Scarlet ZBL
Plasto Orange RS Ethyl ether Ethyl alcohold Waterd I n order of decreasing adsorbahility. b To inark Component a t left: no dye needed a t liquid front. c Determined by difference. d N o t determined; bypassed. Determined together. f Special tPchniquc employed.
Acetone
+
%
...
0.11
... ...
0.2
...
Q
Frontal Analysis. The problem of finding a suitable eluent may be avoided by employing frontal analysis, foi which no eluent is required. The moit weakly adsorbed component appears a t the front, anti the extent of the separation may be followed by employing an indicator dye. Only one component can be determined, because all other zones are contaminated lyith this material. The zone occupied by this conlponent is a function of the total quantity of grl used, and hence the accuracy may be improved by redesigning the column to increase its gel capacity, which increases the zone length for an analyzer section of given diameter. Thus the accuracy is limited only by the column dimensions. which afl'ect the sample size and time requirements. It was stated earlier that in a nonideal mixture of -4arid B, either may appear a t the liquid front during frontal analysis, depending on the composition. Usually the indicator dye selected will be displaced by component A, which is more strongly adsorbed according to the eluotropic series, or by a mixture of A
hIethyl ethyl ketone sec-Butyl alcohol
45-70
Capracyl Red B
Acetone Isopropi-l alcohol
40-63
S i l e Blue RB Ind
Diacetone alcohol A1esity-l oxide
20-40
Xile Blue H B Ind
Ethyl alcohol Ethyl ether
10-30
Plasto Orange RS
Benzene Iso-octane FIA dye 0-30 a Component determined is given second together with concentration range in which method is applicable. b Dye indicates mixture front in each case. -
Usu,tlly then, B must be present in a concentration higher than t h a t i n the asorbotrope, whicli sets a lower limit on the amount of B that can be determined. An upper limit is set by the column dimensions, because in order to assure a sharp dye zone, the zone occupied by B shoul( not be longer than about 250 mm. If the fiample to be analyzed is outside the suitable range, its composition may, of course, be adjusted with Eome loss in accuracy. I n frontal analysis the relation betn-een zone length and sample composition must be established empirically as discussed in connection with procedure. Data for some binary mixtures are summarized in Table IV and shown graphically in Figure 2. The lines would presumably intersect the composition axis at the asorbotrope composition, but for some mixtures the dye zones became more diffuse as the axis was approached and the point of intersection could not be determined. Many systems that do not
V O L U M E 26, NO. 10, O C T O B E R 1 9 5 4 separate completely can probably be analyzed by displacement development by correlating the results empirically as is done in frontal analysis. Systems that do separate can also be analyzed by frontal analysis, as shown by the data for benzene and isooctane, which form a straight line through the origin.
1553 components in one operation. By careful eluent selection it is often possible to analyze even nonideal systems by the displacement development technique. Frontal analysis can be employed to determine one component of many systems. Commercially available oil-soluble dyes serve as indicators for both prccedures.
ACCURACY
The accuracy obtainable by displacement development depends on the system and on the concentration of the component to be determined. The accuracy for major components is about +2 to 4% of the value. The better degree of accuracy is obtained when all the components can be determined directly without the use of the factor F . For minor components the accuracy improves to h l Y C or better. By employing a special technique, an accuracy of + O . l % was obtained in the tert-butyl chloride-tertbutyl alcohol system. In frontal analysis the accuracy is about &2%, SUMMARY
Indicator chromatographic analysis of organic mixtures is simple mechanically and can provide analytical results for several
ACKNOWLEDGMENT
Special thanks are due to Frank B. West for suggesting the study of the effect of nonideality on adsorption analysis, to Cline Black for supplying activity coefficients calculated from data from various literature sources, and to F. H. Stross for proposing the term “asorbotrope.” LITERATURE CITED (1)
(2) (3) (4) (5)
Am. SOC. Testing JIaterials, “ASTJl Standards on ‘Petroleum Products and Lubricants,” Appendix IV, Kote 2, 1952. Conrad, -1.L., -1s.~~. CHEM.,20, 7 2 5 (1948). Criddle, D. W., and LeTouriieau, R. L., Zbid., 23, 1620 (1951). Ellis, W. H., and LeTourneau, R. L., Zbid., 25, 1269 (1953). Kipling, J. ,J., and Tester, D. A , J . Chem. Soc., 1952, 4123.
RECEIVED for review February 3, 1954. Accepted June 28, 1954.
Small-Scale Filter Paper Chromatography Factors Affecting the Separation and Sequence of Amino Acids 1. C. UNDERWOOD and LOUIS B. ROCKLAND fruit and Vegetable Chemistry Laboratory, Agricultural Research Service,
Studies were conducted to determine the relationships between solvent character, water content of the solvent, acidic and basic solvent supplements, filter papers, and the separation and sequences of the amino acids on small-scale filter paper chromatograms. With few exceptions the sequence of the amino acids within the acidic, basic, neutral, and cyclic groups was independent of the nine solvents and three filter papers examined. Considered as independent groups, the sequences of the four arnino acid groups were influenced to the greatest degree by the acidity or basicity of the solvent. Solvents supplemented with acidic or basic materials, such as formic acid or ammonia, tended to improve the separation and reproducibility of all the amino acids and particularly, the acidic and basic amino acids. Most satisfactory separations of individual amino acids were obtained with tert-butyl alcohol-waterformic acid and phenol-water-ammonia.
T
HE usefulness of small-scale, trst-tube chromatography for
both qualitative and quantitative investigations has been referred to in earlier publications ( 7 , 8, 16, 20, 23, 24). In a preliminary study, the present authors attempted to apply the tesbtube procedure (24) to the identification of the nitrogenous constituents in citrus juices. Employing the buffered filter papers and solvents suggested by McFarren (18, 19) it appeared from studies on test mixtures and protein hydrolyeates that the small-scale, one-dimensional test-tube technique would be applicable. However, the presence in citrus juices of nitrogenous constituents other than the naturally occurring amino acids interfered with the interpretation of the resulting chromatograms. Therefore, it appeared appropriate to attempt to develop a satisfactory small-scale, two-dimensional procedure. The studies reported in the present paper were initiated as a preliminary step in the elaboration of a procedure for small-scale, two-dimensional filter paper chromatography.
U. S. Department o f Agriculture,
Pasadena 5, Calif.
EXPERIMENTAL
Materials. FXLTER PAPERS.Whatman No. 1, H. Reeve Angel & Co. S & S No. 507, Schleicher Br Schuell Co. S & S No. 589 Blue Ribbon, Schleicher & Schuell Co. SOLVENTS AXD CHEMICALS. tert-Butyl alcohol, Eastman Kodak Co., White Label, redistilled, 81.3’ C. a t 743 mm. of mercury. 2 4,6-CollidineJ Paragon Division, The Matheson Co., used without purification. 1,4-Dioxane. Eastman Kodak Co., White Label, used without purification. 2,4-Lutidine, Eastman Kodak Co., White Label used without purification. Phenol, Baker’s, c.P., purified according to the procedure of DraDer and Pollard (. I S ..) , 113.5’ to 114.5’ C. at 75 mm. of mercury. n-Propyl alcohol, East.man Kodak Co., White Label, redistilled, 97.0 to 97.1 O C. a t 745 mm. of mercury. Pyridine, Eastman Kodak Co., White Labe!, redistilled, 113.0’ to 114.0’ C. a t 745 mm. of mercury. General Procedure. The “museum jar” arrangement of Rockland et al. ( 2 3 ) was used in the present studies. Eighteen No. 4 American Medical museum jars (inside dimensions 15 X 9 x 15 cm.) were employed to obtain a daily capacity of 54 chromatograms, 5 inches square. Soft rubber gaskets were mounted with rubber cement on the underside of each jar cover to prevent excessive evaporation of solvent. Filter paper sheets, 5 X 5 inches, were cut from commercially available sheets, approximately 22 X 22 inches, with a large paper cutter and individually mounted on l / , X 6 inch wooden dowels with pressure adhesive tape. Amino acid solutions (0.06.M) were spotted at’0.5inch intervals approximately j/8 inch above the bottom edge of the filter paper by means of a Gilmont ultramicroburet (0.01 ml. in capacity) or self-filling micropipets ( 6 4 ) . Most satisfactory chromatograms were obtained a t amino acid levels between 0.1 and 1.0 7 , corresponding to the application of 0.1 pl. (a wett.ed area approximately 1 to 2 mm. in diameter) of 0.06M solutions of amino acids. Because of their low solubility in water, nearly saturated aqueous solutions of cystine, tyrosine, and tryptophan were applied to t.he filter papers by replicate additions of 0.1-pl. aliquots, using a stream of warm nitrogen gas to accelerate drying of the wetted areas (20). Approximately 75 ml. of solvent was placed in each museum jar, permitting the bottom edge of the filter papers to be immersed
