tions and reproducibility of duplicate samples were excellent. ACKNOWLEDGMENT
The writers express their appreciation
to the Firestone Tire and Rubber Co. for the Research Fellowship granted them.
E. ll., ASAL. CHEX 28, 1369 (1956). (2) James, .2.T., Martin, A . J. P., Btocheni. J., (London) 5 0 , 679-90 (1952).
LITERATURE CITED
RECEIVED for review September 24, 1956. Accepted December 20, 1956.
(1) Greene, S. A , AIoberg, AI. I,., Kilson,
Quantitative Aspects of Microchromatography SISTER HELENE VEN HORST, VERONICA JURKOVICH, and YOLANDA CARSTENS Department of Chemistry, Marycrest College, Davenport, Iowa
b Chromatography as a quantitative method requires a consideration of some aspects which influence its reliability. These include stability of chromatograms under varying conditions of light, heat, and humidity, and uniformity of filter paper blank, because certain grades of paper may vary as much as 3270 from the total transmittance of the blank paper. Absorbance values are somewhat erratic for chromatograms stored under otmospheric ccmditions, while minimum deviations resulted for those kept in a dark desiccator. Heating the chromatograms for 5 minutes at 60' C. caused a noticeable loss in amino acids as judged from the intensity of color produced by reaction with ninhydrin.
T
of sensitivity attainable in one-dimensional chromatography makes this method desirable in quantitative micronieasurements of amino acids. If the problems ordinarily encountered could be minimized, this method would probably be preferable because of its simplicity and accuracy. This paper presents the results of R study carried on in two areas which proved of sufficient significance to merit special consideration-namely, limitations in the use of the so-called "base line" in the direct reading of absorbance values, and the stability of chromatograms under varying conditions of light, heat, and humidity. A number of investigators have considered problems involved in the choice of suitable filter paper for chromatography (2-3, 6 , IO). It is obvious that the grade of filter paper used is dependent on the method of analysis and the expected accuracy of the results. If a quantitative micromeasurenient is based on absorbance values of the chromatograms, then a n y variation in the zero reading of the blank filter paper will seriously affect the accuracy of the results. HE DEGREE
788
ANALYTICAL CHEMISTRY
Redfield (8) states that the filter paper blank can be well controlled, because the shape of the base line can be taken into account for each strip on the segments n-here no amino acid is present as compared to the adjacent areas occupied by the amino acids. H e found it suitable to use a constant value of 10% transmittance for this base line. The problems encountered in producing reproducible color intensities in amino acid Chromatograms (4, 8, 9, 12) have likewise been investigated. Recause of the qensitivity expected in ahsorbance m e a ~ u r e n i e n t ~variables , in this area nould probably he significant factors in the reliahility of quantitatire measurements. Kellington ( I S ) reports tliat a variation in relative humidity between 40 and 70%, common in laboratories not equipped with air conditioning, is sufficient to explain the d a j to-day variability in the ninhydrin reaction reported by earlier workers. Khile the effect of heating the chromatograms seems of less concern i n quantitative measurements, some studies have been made in this regard (6,1%'). The effects of each of these factors on amino acid chromatogranis TI ill he considered. PROCEDURE
T o determine the extent to which the use of a constant value for the base line could be relied upon, 15 selected grades of filter paper were used. I n each case, the extent to which the absorbance varied for the filter paper blank wa5 determined for both the crosswise and length-wise cuts of the paper. Absorbance measurements were made with a Model 525 Photovolt densitometer consisting of a transmittance unit in series with a microammeter. A 570mp filter was inserted in the transmittance unit, followed by three different sizes of apertures in succession-a 2-mm. diameter circle, and two slits, one 6 X 1 nim. and one 25 X 1 nim. The absorbance values were read at 1-mm. intervals over a 100-mm. length of filter paper. The l o w s t absorbance
observcd over the 100-nini. length of the paper was taken as the zero reading for each particular strip. Deviations from this zero reading are rrcorded in Table I. I n the preparation of chromatograms to be subjected to varying conditions of light, heat, and humidity, the horizontal method of chromatographic analysis was used ( 2 1 ) . The strips were JF'hotman S o . 4, 1 13 inch wide and 23 inches long. The irrigating solvent 15-as 1butanol-glacial acetic acid-huffer (25 to 12 to 12); the buffer was potassium chloride-hydrochloric acid ipII 3.8). If the ninhydrin wa3 applied as a spray, a 0.3% solution in aqueous butanol was satisfactory. If the ninhydrin \vas added to the irrigating solvent, the proportions were 25 to 12 to 6 to 6 of 1-butanol-glacial acrtic acid-buffer-2.5% aqueous solution of ninhydrin. I n this part of the experiment three different types of amino acids were used-namely, histidine, serine, and ornithine. Each filter paper strip was spotted with 1 pl. of an 8mM solution of one of these amino acids. After the irrigating solvent had traveled the length of the strip, which required approximately 14 hours, the strips were dried a t room temperature in a forced air hood. As the ninhydrin had been added to the irrigating solvent, the spots were revealed either hy heating the strips for 5 minutes at 60" C. or maintaining them for 3 hours a t 25" C. Strips containing spots revealed by both these treatments were mounted side hy side on a frame and compared in their reactions to four additional treatments. One set of chromatograms was kept in the dark under atmospheric conditions; another set was kept continuously in artificial light of about 70 cp. and 40" C. A third set was exposed to daylight in a room free of fumes but under atmospheric conditions. In this latter case the illuminance reached a maximum of about 300 cp. and, of course, was reduced to zero during the night. The humidity of the room in which these chromatograms were stored ranged from 48 to 71%. Another set of chromatogram4 was placed in a desiccator
and kept in the dark. The absorbance values of the chromatogranis were read with the densitometer, using the 3 0 nip filter and the 6 X 1-mm. aperture. By marking the strips i t was possible to read thc absorbance of each spot in the identical position from day to day. This
RESULTS AND DISCUSSION
method eliminated any error wliicli might result from a variation in density of the filter paper. Except for the time required to make the readings, which was about 5 minutes, the chromatograms were kept constantly under the conditions described above.
Uniformity of Filter Paper Blank. Table I indicates t h e extent t o which t h e filter paper blank varied in absorbance from t h e zero point. It also gives a measure of t h e thickness of t h e paper. T h e more transparent grades permitted the use of t h e range of 2.00 t o 3.00 units; those of greater density necessitated t h e use of a range of 3.00 units and above. The various grades of filt'er paper are listed in the order of increasing deviation with the use of the 2-mm. aperture. There is some variation in the zero readings of the lengthwise and crosswise cuts of the paper. Eight grades of lengthn-ise cuts have lower deviations from the zero reading while five grades of crosswise cuts were lower. Two grades (lo not vary for either the lengthwise or crosmise cuts. Obviously, the 25 X 1-mm. slit gives the lowest blank readings, but this aperture is too large for microdeterminations. In general, the "fast flow rate" papers vary most significantly. For example, if Whatman S o . 4 is used with the 2-mm. round aperture, the absorbance variation is 0.17 unit for t'lie crosswise cut. This is equivalent to a 68% transmittance or a variation of as much as 32y0 in the blank reading. -41though previous investigators (1, 3, 6) have preferred either Whatman KO.1 (medium) or Yo. 4 (fast) as giving the best results so far as resolving capacity and other factors are concerned. it ~~-oultl
4.c
3.e w V
z a
m
aa 3.6 (r
m
3.4
3.2
TIME IN D A Y S
Figure 1. Effect of daylight on amino acid chromatograms produced by heating for 5 minutes a t 60" C. or for 3 hours at 2 5 " C.
Table 1.
Whatman Papel Grade CUP 7 40 44
31 DT 44 42 50 50 40 5 20 2 ~~
7 31 DT
3 ?VIM 5
11 1
2 42
L L L
c c; c'
L c'
c
L I, I>
c
I.
c c C I.
c
I,
Variation in Zero Reading of Filter Paper Blank
Floiv Rate
Medium Medium Medium slon Fast Medium slon Slon-
Slow Slow Medium Slow SlonMedium slow Xedium Fast Medium Slow Medium slon JIedium IIedium slon Slow
Medium Medium Xedium slorv SlonFast Fast Fast Fast
4 o
C
Fast Fast
2-Mm. Apertut e
3 3 3 3 3 3 3 3 3 3 3 3 3
00-3 05 00-3 05 00-3 05 00-3 05 00-3 0.5 00-3 05 00-3 06 00-3 06 00-3 06 00-3 06 00-3 .06 00-3 06 00-3 06 3 00-3 07 3 00-3 07 3 00-3 08 3 00-3 08 3 00-3 08 a 00-3 08 3 00-3 08 3 00-3 09 3 00-3 09 3 00-3 09 3 00-3 08 3 00-3 10 3 00-3 10 3.00-3.10
3.00-3.12 3,00-3.13 3.00-3 .19
6-Mm. Apertuie 2 00-2 04 2 00-2 04 2.00-2 04 3.00G.3 04 2 00-2 05 2 00-2 05 2 00-2 04 2 0@2 04 2 00-2 06 3 00-3 06 3.00-3 06 2 00-2 07 2 00-2 07 3 00-3 06 3 00-3 06 2 00-2 04 2 00-2 04 2 00-2 05 2 00-2 05
2 3 2 2 3 2 2 2 2 2 2
00-2 05 00-3 05 00-2 05 00-2 06 00-3 06 00-2 07 00-2 08 00-2 11 00-2 07
00-2 10 00-2 10
25-Mm.
Aperture 2.00-2 03 2 00-2 0:3 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 02 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 04 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 03 2 00-2 04 .. .~ 2.00-2.05 2,00-2.03 2.00-2.05 2.00-2.05 2.00-2,05 2.00-2.07 2.00-2.05 2.00-2.05 2.00-2.09 ~
~
L designates lengthwise (:ut; C, crosswise cut.
VOL. 29, NO. 5, M A Y 1957
e
789
Table II.
a
Effect of Humidity on Absorbance Values of Amino Acid Chromatograms Dark Initial AV Range of Magnitude Sto?age AbSorbAbsorbance, Absorbance, of Ahso1 b-
ProAmino dyed Acid at, C. Histidine 25 60 25 60 Serine 25 60 25 60 Ornithine 25 60 25 60 Values taken from 4th day on.
111
Desic Ilesic Atm .Itm Desic. Desic. htm. Atm. Desic. Desic. Atm. Atm.
ance 3 45 3 63 3 53 3 60 3.34 3.62 3.38 3.75 3.54 3.74 3.62 3.66
28 L>a>sa 3 67 =t 0 013 3 53 0 015 3 85 zk 0 030 3 62 j= 0 042 3.55 + 0 009 3.59 f 0.013 3.88 =t 0.048 3.90 + 0.035 3.62 & 0 012 3.72 & 0.016 4.01 3z 0.031 3.67 & 0.039
*
4.0
3.8
LL;
z 0
2 3.6 (r
v 0)
m
a
3.4
3.2
2
6
10
14 TIME IN DAYS
18
22
Figure 2. Replicate chromatograms of serine heated for 5 minutes at kept under four different storage conditions
seem that either of these grades would not be entirely suitable for use in quantitative measurements based on absorbance values. Washing the filter paper in acid did not seem to lower the blank reading to a n y significant extent. Application of Chromogenic Reagent. T h e method of combining t h e ninhydrin directly with t h e irrigating solvent, as proposed by Nicholson (7), gave results more readily reproducible than the usual spraying technique. When the ninhydrin added to the irrigating solvent produced a solution of the same concentration as that applied by spraying (in this case, a 0.3% solution), the resulting colors and R/ values were identical with those obtained b y spraying the chromatograms. The areas of color were distinct and well defined. Effects of Light, Heat, and Humidity. Figure 1 gives the results of t h e
790
ANALYTICAL CHEMISTRY
26
60" C. and
chromatograms exposed t o daylight. T h e increased absorbance during the first two days indicates t h a t t h e chromatograms were not fully revealed until that time. Dent (4) has shown that full color production takes place if the sprayed papers are dried for 24 hours at room temperature. For serine and histidine the absorbance is somewhat greater for those chromatograms kept a t room temperature than for those heated a t 60" C. This difference is probably due to a loss as a result of heating, and is in agreement with the work of Fowden and Penny ( 5 ) . The unevenness in fading is partly accounted for b y the change in intensity of the sunlight from d a y to day as well as by the variation in humidity. Figure 2 is the comparison of replicate chromatograms of serineproduced at 60" C. for 5 minutes and subjected to each of the four conditions described
28 Daysg 3 65 to 3 70 3 49 to 3 54 3 81 to 3 92 3 52 t o 3 74 3 . 5 2 t o 3 57 3.57to3.62 3.81 t o 3 . 9 8 3.83t03.98 3.59t03.64 3.68t03.77 3.94 to 4.08 3.59 to 3 . 7 5
ance Range 0 05 0 05 0 11 0 22 0.05 0 05 0 17 0 15 0.05
0.09 0.14 0.16
earlier. Variations in initial absorbance may be accounted for by the variation in time interval in reading each of the chromatograms immediately after removing them from the oven. It is evident that atmospheric conditions affect the intensity of the chromatograms. The curve for the chromatograms subjected to artificial light is less erratic than the curve for those stored under atmospheric conditions. This is probably due to the higher temperature of 40" C. produced by the artificial light, Table I1 is a comparison of the chromatograms stored in the dark under atmospheric conditions and those kept in the dark in a desiccator. The initial absorbance reading is given in column 4 ; however, in computing the average for the 28-day period, the readings were taken after the fourth day when maximum production had been reached. All chromatograms produced a t 25" C. show an increase in absorbance. Those produced a t 60' C. and stored in the desiccator show a decrease in absorbance. Furthermore, the deviation and range of those chromatograms stored in the desiccator are considerably less than for those stored in the dark under atmospheric conditions. T h e desiccant inhibits the production of t h e color of the spot, although it also stabilizes the intensity of the fully revealed chromatogram. LITERATURE CITED
(1) Akerfeldt, S., Acta Chem. Scand. 8,.
521 (1954). (2) Block, R. J., Durrum, E L., Zweig, G., ''Manual of Filter Paper Chromatography," p. 37, Academic Press, New York, 1955. (3) Burma, D. P., J . Indian, Chem. SOC. 29, 567 (1952). (4) . . Dent, C. E., Biochem. J . 43, 169 (1948). (,5,) Fowden. L.. Penny, " , J. R..Nature 165, 846 (1950). (6) Kowkabany, G. N., Cassidy, H. G., ANAL.C H E h l . 22, 817 (1950). ( 7 ) Nicholson, D. E., Nature 163, 954 (1948).
(8) Redfield, R. R., Barron, E. S., Arch. Biochem. & Biophys. 35, 443 (1952). (9) Thompson, J. F., Zacharius, R. hf., Steward, F. C., Plant Physiol. 26, 375 (1951). (10) Underwood, J. C., Rockland, L. B., ANAL.CHEM.26, 1553 (1954).
(11) Ven Horst, Sr. H., Carstens, Y., J . Chem. Educ. 31,576 (1984). (12) Wellington, E. F., Can. J . Chem. 30, 581 (1952). (13) Ibid., 31, 484 (1953).
Accepted January 26, 1957. Supported by Grant DRG-334 (T) from the Damon Runyon Memorial Fund for Cancer Research. Presented a t the Section of Inorganic and Physical Chemistry, Iowa Academy of Scienre Meeting, Grinnell, Iowa, April 21, 1956.
RECEIVEDfor review May 16, 1956.
Chromatography of Organic Acidic Compounds on Multibuffered Paper MORTON SCHMALL, E. G. WOLLISH, REM0 COLARUSSO, C. W. KELLER, and E. G. E. SHAFER Analyfical Research laborafory, Hoffmunn-la Roche Inc., Nufley, N. 1.
b
Acidic organic compounds have been separated by a paper chromatographic technique similar to one applied to organic bases. Many acidic compounds form a salt a t a particular pH. level with alkaline buffers, applied in sequence of their ascending pH on a filter paper strip in marked zones. Upon equilibration, chloroform is used as the single mobile phase for descending chromatography. As the stronger acidic compounds are often immobilized at lower pH levels than the more weakly acidic ones, it was possible to separate benzoic acid, several barbiturates, and some phenolic compounds from each other. These compounds could b e visualized on the paper under short wave ultraviolet light. HE literature of paper chromatography, including paper chromatography of acidic compounds, has been covered b y Block, Durrum, and Zweig
( I ) , Lederer (4, Turba (IO),and Hais and LIacek ( 3 ) . The most recent developments were reviewed b y Strain and Sat0 (9). While filter papers completely impregnated with buffers have been widely used for the separation of compounds of related structure, the technique of multibuffered paper Chromatography has been developed for the separation of organic basic compounds. The procedure, employed for the separation of acidic compounds, was somewhat similar to the one described for organic bases ('7).
Isopropylbarbituric acid Barbital Phenobarbital Allylbarbituric acid Diallylbarbituric acid Probarbital Aprobarbital Butabarbital C yclopal sec-Bromallylbarbituric acid Amobarbital Pentobarbital Secobarbital Hexobarbital
The paper was prepared by application of buffers at varied p H levels in marked zones (7). For the separation of acidic compounds, Clark and Lubs buffers were applied at 0.2 p H intervals for the p H range of 7.8 to 9.4, while Soerensen buffers were used for the p H range 9.8 to 12.6, spaced at 0.3 p H intervals. A solution of the sample, equivalent to about 15 7,was placed a t the starting line and the strip was introduced into the chromatographic chamber containing water at the bottom and, in addition, a n open beaker with chloroform. T o hasten saturation with vapor, the chamber was partially lined with filter paper which dipped into the water. It was allowed to come to equilibrium overnight. Chloroform, as the single mobile phase, was then placed in the solvent trough for descending chromatography. When the solvent front had moved down almost to the end of the paper, which usually required about 3 hours, the strip was removed and air-dried. It was inspected under short wave ultra-
METHOD
Apparatus. Usual equipment for descending paper chromatography. Short-wave hIineralight (maximum emission at 2553 A.). Reagents. Chloroform, reagent grade. Clark and Lubs bufkers, double strength ( 2 ) . 3IacIlvaine buffers, double strength ( 5 ) . Soerensen buffers, double strength (8).
Table I.
Compound Barbituric acid
PROCEDURE
pH of Immobilization of Barbiturates
pH Range of Buffered Paper 8,3-12.6 Unbuffered 8.3-12.6 Unbuffered 7.8- 9 . 4 7.8- 9 . 4 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6
pH of Immobilization 8.3 8:3
912 9.2 10.2 10.2 10.5 11.o
11.7 11.7 12.2 12.5 12.5 12.5 Past 12.6
VOL. 29,
R/ 0 0 0 0 0.7 0.75 0.3 0.3 0.35 0.45 0.6 0.6 0.7 0.75 0.8 0.8 0.95
NO. 5 , MAY 1957
791
