V O L U M E 24, NO. 5, M A Y 1 9 5 2 Table \-I.
773
Relative Efficienc?;for Extracting Iodine from .iqneous Triiodide
(‘arbon tetrachloride Carbon disulfide Bromoform Ethylene bromide Nesitylene
Solubility of Ia Nole Vo Vol. pc 1 15 0.70 5 58
g.16 I .82 10 72
5 61 4.23 5 46 4 86
Relative Efficiency 1.0 8 0 6.0
7.8 6.Y
~~
~~~~
-in ion may be extracted from aqueous solution into another solvent immiscible with water if it can be transformed into a relatively un-ionized form. W t h certain polyvalent ions, this may be brought about by convert’ing into a halide, with iodide more effective than chloride, etc. For example, stannic iodide is such a weak salt that, unlike stannic chloride, it can be formed unliydrolyzed in water solution ( 7 ) and extracted, being quite nonpolar, wit,h carbon tetrachloride, or, better, with carbon disulfide since it is much more soluble in the latter, as the three solubility parameters would permit one to predict. Osmium tetroside is another t’etrahedral, nonpolar inolecule, and its solubility parameter is 12.6; therefore, it should be easily extractable with any nonpolar solvent with a high solubility paranietei. The central atom is leas completely buried in ferric chloride than in stannic iodide, and it cannot be estracted by an insert solvent, but ether is effective by the aid, doubtless, of its dielectric constant, but more by acid-base interaction. The question naturally arises, in ewe of a substance estractable vith diethyl ether, ivhether n diffci,ent ether might be more effec-
tive. guides to the ans\vcr, onf’ would do well to consider the relative basic strengths of the different ethers, and their mutual solubility with wat’er, bec,:iuae the niore soluble the two liquids are in each other the less the difference between them in solvcnt power. l l x n y metal ioiis can lie estracted as chelates. The best solvent to use \vould then depend upon the peripheral atoms and groups of the chelate struct,ure. ils most chelating substances contain amino or hydrosyl groups, hydrogen bonding and acidbase interartion arc. obvious factors t o consider. LITERATURE CITED Heiiesi, H . .I.. a n d Hildehraiid, .J. IT., J . .4m.Chenb. Soc., 71, 2703 (1949); 72, (370 (1950). Causidy, H. C.. “.idsorption a n d C h r o m a t o g r a p h y , ” S e w r o r k , I n t e r s c i e n c e Publishers. 1981. (’legg. D. L . , A N ~ I . .c H A . , 22, 48 (1950). H i l d e b r a n d . .I. H.. and Cochraii, D. R. F.. J . A m . Chem. Soc., 71, 22 (1949). Hildebrand, .J. H.. Fisher, R . R.. and Benesi, H . A , , Ibid.. 72,
4348 (1950).
FT.. a n d S c o t t , R . L.. “Solubility of Soiielectrolytes,” S e w T o r k . Iieinhold P u b l i s h i n g Corp., 1950. P i t a e r , Iedsol\ ents that progressively increased in polarity during the course of the separation. The literature lacks any approach for calculating chromatographic positions ( R values) for such a method. A technique is described for computing these chromatographic positions and the migration of several organic acids in seteral solvent s)stems is analyzed by the simple equation derived. Trial and error testing is eliminated when the previouslj described chromatographic procedure is sealed upward or downward. The broad possibilities of this newer type of chromatograph: are illustrated b> the experimental data.
I
S -1previous report (1) an iniproved chroniatographic proce-
duw was described, which employed a mobile solvent that incrrarcti in polarity during the course of the separation. This increase in solvent polarity \vas obtained by gradually and automatically raising the concentration of alcohol in a chloroformbutanol influent. The apparatus employed introduced butanol cont,inuously into the chromatographic reservoir containing chloroform and the two liquids mixed before entering the column because of the difference in densities. Although the extent and nature of the mixing obt,ained in the process were shown, no data \yere presented to show the effect of varying the rate of increase of solvent polarity on the resolving power of the gel column. Furthermore, the literature lacked methods for computing the chromatographic position of a solute delivered by an influent progressively changing in chemical nature. Consequently, trial and
8
error test,ing was necessary to ascertain resolving power when altered mixing rate of the influent with respect to gel mass was encountered or column size increased or decreased to accommodate larger or smaller analytical sample^. The present report describes relationships between rate of increasp in polarity, resolving poiver, and silica gel mass. I,actic, succinic, a-ketoglutaric, and cis-aconitic acids were selected for the study because of thcir phj-siological importance anti their positmiorison the silica gel chromatogram. Additional acids (Figure 1, C) extend the broad applicability of the previously developed procedure for the separation of acids in mixtures on a single column without niauual manipulation of the solvent once the chromatographic separation has begun. Ideally one quantity of alcohol per unit mass of nonmobile phase would be required to release the maximal concentration
ANALYTICAL CHEMISTRY
774
Then substituting in Equation 1
(peak) of the zone of each acid from the column ( 2 ) . Rate of increase of the solvent with respect to alcohol concentration is described by constants A and b of Equation 1 and these are related to resolving power and gel mass (nonmobile phase) by the argument which follows. dlthough the basic equation ( p = Ae-vb) for the argument followed from the previous report, it is well t o mention that it describes the changes in density of the chloroform-alcohol solvent delivered to the column by the reservoir of described design ( 1 j.
Then for any chromatographic system releasing an acid with any one arbitrarily selected degree of resolving power, the quantity of any single alcohol required a t the peak would be given by P
P
X
K density = constants = fraction number = fraction number a t the peak = slope for the density-composition plot of chloroform alcohol mixtures = intercept for the density-composition P
=
- Jf
K
B
(Ae-VD - ;ir)
(4)
p =
S, A , b y
P M B
$
plot of a chloroform-alcohol mixture Q = gel mass K = fraction volume X = concentration of alcohol
In three different chromatograms of different gel mass showing one degree of resolving power and released by approximately the same volume of alcohol per gram of gel: P2
[-AI
-
$
= _ie-gb
(I)
and from density composition plots for alcohol-chloroform milture
s
= --.up
-
(.12e-”lt2
E ) ] K2 *If
=
P
Since p
[-JI
=
[-Ill (A3e-””lt3 - M E ) ] K S (5)
Consider for each of the three separations that the volume of alcohol required to present the peak of the acid per gram of gel is a single chromatographic fraction (Thatever its size), then
+B
B Aconitic
Succinic
E
1.0
3
Fumaric
5
3; 0.5 z z 5 9 c
3
/4/’
0‘ 4 04
x 50
80 80
;O ;O
80 80
f E
1.5
90 90
100 100
110 110
120 120
140 140
130 130
2 0.5
/\
Oxalacetic
*
I\
v,
5
0 0
170 170
I\
.2
’
160 160
M$c
.
’0 0 1.0
150 150
Fraction Number
u 0.
C
A
n
j -*
‘25
i5
45
145
ti5
tis
t i s iiis 1;s
20s
2is 2 i 5
Fraction Number Figure 1. Chromatograms w i t h Varied Increase in Polarity per Unit Gel Mass Ordinate = ml. of alkali for titrating effluent fraction. Abscissa = fraction number which permits calculation of observed effluent volume from fraction volumes given below. System identifications are given in text. A . System 1. Fraction number = ml. B . System 4. Fraction number X 0.9 = ml. C. System 2 . Fraction number X 0.3 = ml. Hate of change per gram of gel represented in C was less than that of A but greater than B . Volume of each fractlon collected was proportional to gel mass in all experiments. C illustrates possibilities of a progressively changing ohromatographic influent. The 8 acids were separated on a single column without manual manipulation of solvent after separation had begun. Acidity between fractions 75 and 85 was due to impurities in oxalacetate.
V O L U M E 24, NO. 5, M A Y 1 9 5 2 Table I.
775
Experimental .Alcohol Volume, Effluent Volume, and S" Value for Six Chromatographic Systems lb A = 1.74 b = 0.040C
2b A = 1.71 b = 0.057 .41cohol, Peak, ml. ml. 0.83 18.0 1.90 23.6 2.60 27.2 3.40 30.0 5.40 34.6
System Number and Constants 3 4b 9 = 1.49 A = 1.52 b = 0.015 b = 0.007 , AlcoAlcohol, Peak, hoi, Peak, ml. ml. S mi. ml. S 0.45 21.6 71 33 1.15 25.5 18.1 22 2.23 27.3 1.99 29.3 32.0 19 3.46 13.7 2.70 33.0 39.3 5.07 11.5 3 . 4 0 3 7 . 0 16 8.7 6.93 40.8 4 50 41 7 14
5
6 A = 1.50 b = 0.011 Alcohol, Peak, S ml. ml. 27.0 1.00 16.6 21.0 2.20 20.6 17.0 2.60 22.6 14.0 3.00 24.0 11.0 3.80 26.5
AlcoAicohol, Peak, hol, Pealid, .4cid ml. ml. S S ml. ml. S Fumaric 0.77 1 6 . 0 36 1.00 18.0 35 25 1.60 22.6 2.40 Lactic 21.6 15 20 14 2.10 23.3 17 Succinic 2.40 22.0 13 15 2 90 28.0 13 14 a-Ketoglutaric 3.20 26.0 12 4.60 34.0 11 Aconitic 3.60 26.0 12 12 a Equation 7 , defines S. b Quality of these separations shown in Figure 1. e A and b are constants obtained by studying composition of effluent with respect to alcohol for each systein-i.e., system 1, system 2, etc. Constants would fit general equation (text, Equation 1) p = Ae-ub. d Peak ml. effluent volume at peak divided by gel mass. This would be equal t o K/Q of Equation 7 of text, since "one fraction" presented peak of each acid.
-
and since '1.1and B , by chemical nature, are the Eame throughout, they can be removed algebraically and
or for any one arbitrarily selected degree of resolving power for each gram of gel
(y)
K \Till be another constant, say S
anti
(7) .In examination of Table I and Figure 1 indicates that there is an approximate quantity of alcohol per gram of gel associated with the release of the middle of the acid zone (peak) for each acid for the several systems which show the best resolving power. For systems that release acids quickly with poor resolution, less alcohol per gram of gel is required. The slow release of these acids requires a larger volume. Systems 2 (Figure 1, C) and 5 showed similar resolving power and the S value for each acid in 2 (Table I), excluding fumaric, is nearly identical to the corresponding one in system 5. Chromatograms from systems 1 and 6 (Figure 1, A ) showed sharper peaks than any of the syst,ems represented and the S values, from lactic to aconitic acids, show the smaller range. Deviations for the one acid, fumaric, suggest that Equation 7 is not applicable to acids showing mobility in chloroform. This would he expected because the argument presented above assumes t h a t the chloroform is essentially a vehicle for the effective mobile solvent, the alcohol. The data for the other four acids permit the assumption, however. Equation 7 will allow calculation for peak positions and thereby resolution on a chromatographic column of any size. For example, given the characteristics of a new reservoir (values A and b, which are experimentally determined) and the S and K values (Table I ) for, let us say, aconitic acid, the ideal amount of gel (&) could he computed. From this Q value, the product (& X K ) would provide the position of aconitic acid in the effluent for t,he column used. It is of further interest that the data for system 6 (Table I ) suggest that partition alone (between the aqueous and nonaqueous phases) does not fully explain the behavior of the solutes for this t>-peof procedure, a t least. The water to gel ratios in systems 5 and 6 were about one half t,hose in the other experiments, hut the fluctuations in S values t,hroughout.for any acid, except fumaric, are not related to these water-gel ratios. EXPERIMENTAL
The experimental details for this study were similar to those presented in the first report (1). Ratios of diameter of the column
to the height of the column and rolunie of the aqueous phase to the mass of the gel were approximately constant except where mentioned above. For the measurement of the effluent organic acids 0.001 S base was employed. Four milligrams of thymol blue were dissolved in each 100 ml. of the base to provide the solution with its own indicator. This volumetric solution was prepared immediately before use. Varying rates of change in polarity were obtained by altering the alcohol concentration in a chloroform-alcohol solution which mixed with a fixed volume of chloroform containing initially no alcohol. Gel mass was varied randomly throughout the experiments. The particular chromatographic systems used in this study, previously referred to by number, are described below: System 1. A 1 to 1 mixture of 30% n-amyl alcohol (l-pentanol) in chloroform with 30% tert-amyl alcohol in chloroform was delivered into 50 ml. of chloroform. Gel mass was 3.0 grama. Aqueous volume was 2.0 ml. Column diameter was 9 mm. System 2. A 1 to 1 mixture of 3001, n-amyl alcohol in chloroform with 30% tert-amyl alcohol in chloroform was delivered into 50ml. of chloroform. Gel mass was 1 1grams. Aqueous volume was 0.8 ml. Column diameter was 6 mm. System 3. tert-Amyl alcohol, 40%, in chloroform was delivered into 175 ml. of chloroform. Gel mass was 2.7 grams. Aqueous volume a a s 1.9 ml. Column diameter was 6 mm. System 4. tert-.Imyl alcohol, 40%, in chloroform was delivered into 220 ml. of chloroform. Gel mass was 3.00 grams. .%queous volume was 1.9 ml. Column diameter was 9 mm. System 5. A 1 to 1 mixture of 60% n-amyl alcohol in chloroform with 30y0 tert-amyl alcohol in chloroform was delivered into 175 ml. of chloroform. Gel mass was 3.0 grams. Aqueous volume was 1.0 ml. Column diameter mas 9 mm. System 6. A 1 to 1 mixture of 60% n-amyl alcohol in chloroform with 60% tert-amyl alcohol in chloroform was delivered into 175 ml. of chloroform. Gel mass was 4 grams. Aqueous volume was 1.5 ml. Column diameter was 9 mm. I n blank determinations, the densities of 10-ml. fractions plotted against fraction number described curves which were fitted by Equation 1, the constants of xhich described mixing rate. The chloroform used throughout the study contained 0.8% ethyl alcohol. ACKNOWLEDGMENT
This investigation was supported in part by a grant from the Sational Cancer Institute of the Sational Institutes of Health, Public Health Service. LITERATURE CITED
(1) D o n a l d s o n , K O., T u l a n e , V. J . , a n d Marshall, L. bI., ANAL. CHEM.,24, 185 (1952). ( 2 ) M a r t i n , A. J. P., a n d Synge, R. L. lI.,Biochern. J . , 35, 1358
(1941). RECEITED for review November 14, 1951. .4ccepted March 1, 1952.
