V O L U M E 2 5 , NO. 8, A U G U S T 1 9 5 3 h d d 5.0 grams of boric acid and transfer the solution t o a 400ml. beaker. Dilute t o 200 ml. A4dd 5.0 ml. of nitric acid. Stir while heating until the solution becomes colorless. Add 0.1 S silver nitrate solution in excess (10.0 ml. is usually sufficient) and stir until precipitate coagulates. Allow to stand in the dark for 2 hours. Filter through a close paper and wash thoroughly with a solution containing 0.05 gram of silver nitrate per liter. Dissolve the recipitate by pouring 50 ml. of ammonium hydroxide (1 to 17 through the filter, catching the filtrate in the original beaker. Pour the ammonium hydroxide through a second time if necessary to dissolve the silver chloride. Wash the paper thoroughly with water. Make the filtrate slightly acid with nitric acid (methyl orange indicator), add 5 ml. of 0.1 .I; eilver nitrate solution, and stir t o coagulate the precipitate. ?illow to stand in darkness for 2 hours. Filter through a tared, fine-porosity,. fritted crucible; policil the beaker and stirring rod and wash thoroughly with the wash solution of silver nitrate and then twice with n-ater to remove
1235 the silver nitrate. Dry the cruci1)lr and c:ontente in an oven a t 130" to 150°C. Cool in a dcsiccatrr :md weigh. Weight of silwr chloride X 0.2474 X 20 = 70chloride LITERATURE CITED
(1) Beeghly, H. F., .%XAL. CHEY.,20,1096 (1952). (2) Corbett, J. -4.. .4?lCllySt, 75, No. 894. 475-80 (1950). (3) Hillebrand, W. F.. and Lundell, G . E. F., "ripplied Iilorganic Analysis," New Tork, John Wley 8- Sons, 1929. (4) Parnas, J. K.. 2.anal. Chem., 114, 261 (1938). ( 5 ) Parnas, ,J. K . . and Wagner, R., Bioehem. Z., 125, 253 (1921). (6) Scott, IT. IT,, "Standard Methods of Chemical -halysis," 5th ed., S e w T o r k , D. Van Nostrand Co., 1939. ( 7 ) Thornton, W >I., Jr., "Titanium," A m . Chem. Soc., Mouo,yraph Sei-., S o . 33, Xew T o r k , Chemical Catalog Co.. 1927. (5) Willard, 11. H., and Greathouse, L. H., J . A m . Them S t c , 39. 2366 (1927). REcmT-En f o r rcrirn. S o v e i i . I ' r r 13, 1952.
.Iccepted M a y 14, 1952,
Effect of Temperature on Movement of a Chromatographic Zone LIE TIEN CH LNG', Louisiana State 17nicersity,Rntori Rouge. La. The nioienient of a chroniatographic zone is generallj expressed h> R value. Although factors affecting the R value have been investigated quantitati\elj by some workers, its temperature dependence is little known. This work was undertaken to study the effect of temperature on the R value within -50' to 200" C. It was found that the R values \+ere affected h> temperature in different wa!s. Some of the R values increased with the increasing temperature, wine decreased, and some remained practically constant. The width of the zone also \aried with temperature. Results were interpreted hj assuming that the adsorption takes place primarilg through hj drogen bonding. Results indicate that w i t h a gi\en sol\ent the resolution of different suhqtances on a chromatographic column would be marLerll? affected by temperature and that with a given suhstance the effect of temperature on its R \uliie depends hoth in magnitude and direction iipoti the eol\cnt used. These findings seem to ha\e significant bearing on chromatographic terhnic~"c.
T
HE rate of moveint~ntof :I throniatographic zone is generally expressed by its I2 valur. which has been defined ( 4 ) by LeRosen as the ratio of the distance t,raversed by a zone on the column to the distuncc~traversed by the developing solvent.. Strain (IO) has ititlit.ate:l that the adsorption sequence of certain organic t~onipounclsis tleperident t o some extent on temperature. LeRosen :uic1 Rivet ( 7 ) have shown t,he dependence of R on teniperat.ure in thts s y s tenis o-nitroaniline-silicic acid-benzene, o-nitroaniline-silicic ac,itl--ohloroform, and I>-copene-calcium hydrositie-benzene within the range of 10" to 70" C. The first objective of the p w e r i t investigation was t o extend the temperature study over a wider range (-50" to 200" C.)>employing selected systems of IOU., medium, and high R values. The work of LeRosen arid coworkers (6),Elder and Springer (I), Schroeder ( 9 ) , and IIoyer ( 2 ) at room temperature has indicated t h a t the adsorption on silicic acid primarily takes place 1
Present address, Union Starch & Refining Co., Granite City, Ill.
through hydrogen bonding. The second objective of this study was t o find out if the discussion in terms of hydrogen bonding was valid over a wider temperature range. APPARATUS
Chromatographic Tube. This was a specially designed tube, consisting essentially of a KO.1 chromatographic tube (inside diameter, 9 mm.; length, 150 mm.) with concentric liquid and vacuum chanibers around the tube. Liquid a t a definite temperature was circulated through t,he liquid chamber by means of a circulating pump (Model 5P56HC37, hp., General Electric Co.), thus keeping the tube a t a ronstant temperature. The vacuum chamber was for insulation. I t was found that a t low temperatures, even down to -50" C., no frost was formed on the outside of the tube; consequently the movement of the zone could be observed without difficulty. -4scale 75 mm. in length, marked in 1-mm. intervals, v a s printed in ink on the outside of the vacuum chamber. Two U-shaped tubes were used for preheating or prccooling solution and solvent. One end of each tube was drawn into a fine tip and bent downward. Each tube was so arranged that its tip was just above the chromatographic tube, so that solution and solvent could be delivered directly to the chromatographic tube. To the other end of each tube, a piece of rubber tubing was attached, so t h a t solution or solvent could be drawn in or forccd out by applying suction or blowing at, t,he end of the rubber tubing. High Temperature Bath. The cont,ainer for the liquid bath was a 4-liter beaker set inside of a crock. The space between the beaker and the crock xms filled wit,h rock wool for insulation. A stirrer, a heating coil, a thermometer, and two U-shapcd ghss tubes for heating solution and solvent ~ w r provided e in the, beaker. RIarcol (light mineral oil) was hcvtted to the desired tcmperature in the Iwaker and circulat,rd through t,he chromatographic tube. The rates of heating, pumping. and stirring were rcgulatetl by separatc p o w w t a t e (Tj.pc 116, Superior Electric Co.. Bristol, Conn. ), Low Temperature Bath. The low tempwature liquid bath was a tin can with a big flange, set insitlo t h r same 4-liter beaker used for the high temperature bath. Inside thc tin can there wcre a stirrer, a thcrmonieter, and two glass tubes for cooling the S O ~ U tion and the solvent. Ethyl alcohol was cooled insido thr tin can by a cooling mixture of ethyl alcohol and dry ice, which was in the space between the tin ran and the beaker. Small pirces of dry ice were introduced through a "hatch" on the fl:inge. The temperat,ure was kept constant by regulating the rate of addition of dry ice. The purpose of the flange on the tin can was t o keep t'he carbon dioxide gas from Iwing absorbcd hy the alcohol. When the alcohol absorbed carbon dioxide gas, it h c a m e opaque, making i t impossible to ohserve the movrnicliit of the zone. When the pumping wa,? stopped, t h r dissolved carbon dioxide gas was lib-
1236
ANALYTICAL CHEMISTRY
erated and produced a positive pressure in the system, which made i t impossible to start the pumping again. fiI4TERIALS
Adsorptives. Azobenzene, c . P . , Fisher Scientific Co. pHydroxyazobenzene, Eastman Kodak Co. Sudan 111, aminoazobenzene-2-naphthol, Eastman Kodak Co. 1,4-DibutyIaminoanthraquinone,source not known. p-Aminoaeobenzene, source not known. Adsorbent, Merck reagent grade silicic acid. Because the adsorption power of silicic acid varies with its water content (II), i t was heated at 200” C. for 1 hour to remove the “free water,” and kept in an air-tight bottle. Solvents. Diphenyl ether, Eastman Kodak Go. %-Butyl ether, Eastman Kodak Co. Isopropyl ether, Eastman Kodak Co., free of alcohols. R VALUE DETERWINATIOV
The chromatographic tube was packed to a height of 75 i I mm. under the full vacuum (about 29 inches of mercury) supplied by the vacuum pump. After circulation of the hot or cold liquid from the temperature bath for 3 minutes, sufficient solution to occupy 1 cm. of the column was introduced. This wvas followed by the solvent a t the moment the top of the column appeared to be dry. T h e developing solvent was the same as t h a t used for making the solution. Both the rolution and the solvent were brought to the temperature of thP bath before they were introduced into the column.
mm. mark. -4second thermometer was placed in the constant temperature bath and temperature differences were determined. The temperatures reported in this study were those for the tube. It was possible to keep the temperature of the chromatographic tube constant within + l o C. The concentration of solutions used mas the lowest concentration which gave a zone of sufficient color intensity t o permit its movement to be observed readily. The Sudan IT1 solution was 0.001 M and the 1,4-dibutylaminoanthraquinonesolution was 0.0001 X. The concentration of the other solutions was 0.01 X . RESULTS ARD DISCUSSIOV
The experimental results are listed in Tables I, 11, and 111. Evidence has been presented by Magnus and Kieffer ( 8 ) that silicic acid is probably composed of units as shonm below: Therefore i t may act as an electron donor and/ 0 or a hydrogen donor in hydrogen bond formation. .4ftcr considering the study of Kiselev ( 3 ) -sf concerning the “free” and “structural” water in \€I silicic acid, it may be postulated that the spatial structure of silicic acid contains many capillary openings which hold the free water. Many of the hydroxyl groups of silicic acid are available at the surface of these capillaries for hydrogen bond formation. I n chromatographic systems employing silicic acid as the adsorbent, the possible hydrogen bond equilibrium may be represented by the following diagram: A, B, C represent hydrogen -4,‘,adsprPtive bonds formed among the constituents. A bonding is related to the Adsorbent ‘c “adsorption affinity” of the adB‘,, sorptive. B-bonding t o the L‘com‘sol;ient petition effect,” and C-bonding to the “solvation effect”; the latter two may be together called “solvent” effect. The competition effect is a competitor for the adsorption sites on the adsorbent, thus decreasing the adsorption of the adsorptive. The solvation effect results from the attraction of the solvent for the adsorptive and tends to pull the adsorptive away from the adsorbent or to keep i t in solution, thereby also decreasing the adsorption of the adsorptive. It is apparent that the tenacity of adsorption of the adsorptive by the silicic acid is dependent on the equilibrium set up by these different effects. This in turn depends mainly on the relative strength of the different hydrogen bonds. If a nonpolar solvent is used, there will be no B and C bondinqs: therefore, the adsorption will be mainly dependent on il. .4s the strength of the hydrogen bond is weak, it will be easily broken ap the temperature is increased. It is to be expected t h a t the R value will increase with increasing temperature in systems uping nonpolar solvents. The oxygen atom of diphenyl ether may offer a possibility for the formation of hydrogen bonds between solvent and adsorbent. H o w v e r , this effect seems to be of little significance. LeRosen ( 5 ) reported an RL value of 0.82 for azobenzene on silicic acid a t room tempprature, using benzene as solvent. I n the present $tudy an RL value of 0.81 was obtained for diphenyl ether a t 30’ C. This similarity would seem t o indicate that diphenyl ether had acted like a nonpolar solvent. All the R values increased with increasing temperatures (Table I ) . Azobenzene was weakly adsorbed, Sudan I11 (aminoazobenzene-2-naphthol) was moderately adsorbed, while p-hydroxyazobenzene was strongly adsorbed in systems using diphenyl ether (Figure 1). Thiq difference may be explained by the fact t h a t the hydrogen bonding formed through the hydroxyl group is stronger than that formed through the azo group. Tn the silicic acid-benzene system, phenol has an RL value of 0.27, while azobenzene has an RL value of 0.82 ( 5 ) . Intramolecular hydrogen bond formation in Sudan I11 between the hydrogen atom of
.:
-
I
I -
0 3 t
,A
P-HYDROXY-AZOJENZENE
I
I 40
Figure 1.
60
80
100 120 T E M P E R A T U - E I’C)
140
160
I80
RL Values 2‘s. Temperature of Phenyl Ether
The movement of the zone and of the solvent was carefully measured against the scale outside the chromatographic tube. Positions of the leading and trailing edges of the zone were recorded when the solvent reached positions of 25, 50, and 75 mm. from the top of the column, and RL and RT vere calculated. For RL,the averao;e of three readings was recorded. Until the solvent had moved past the 25-mm. position, the trailing edge of the zone usually moved very little; consequently, measurement of RT a t this stage was not very accurate. Therefore, RT was averaged from two readings taken after the solvent had moved 50 and 75
mm. Sometimes the zone was irregular and the edge of the zone was diffuse. Consequently the determination of the position of a zone was somewhat arbitrary. When the edge of the zone was irregular, a n average of three readings around the column waq taken. When the edge of the zone was diffuse, the reading was taken at the position where the color was distinct. Usually the trailing edge of the zone was diffuse. There was a definite difference between the temperature in the chromatographic tube and the liquid in the temperature-controlling bath. This was due to the cooling effect of the air in the high temperature determinations and the warming effect of the air in the low temperature determinations. However, these differences remained constant under the experimental conditions and were predetermined in control runs a t each temperature employed in this study. A thermometer was placed in the chromatographic tube, so t h a t the tip of the mercury bulb was even with the 50-
~
09-0-0,
:SO-PROPYL
ETHER
0,
’O‘OD-O 0 7 - x\ w
-
3
5
05-
x \x,x
,N-BUTYL
A
ETHER
,d
‘x-x.x-x--x-x-x
//
./A
-
a/
a/
03A’
01
-
A/
-PHENYL
ETHER
the solvent effect, the R value was increased (Table I). If both were weakened to the same extent, the R value was independent of the temperature (Table 11). At low temperature, the solvent became viscous and the diffusion rate of the adsorptive molecules in the solvent was decreased. Because of the low diffusion rate, a molecule desorbed a t the trailing edge of the zone migrated slowly into the bulk of the solution. When the R value reading was taken, this molecule was still in the immediate neighborhood of the last adsorption position. This resulted in a widening of the zone (Tables I1 and 111).
A ’ ~~~~
