Tetraphenylphosphonium and Tetraphenylstibonium Chlorides as Analytical Reagents HOBART H. WILLARD AND LOWELL R. PERKINS' University of Michigan, .4nn Arbor, Mich.
The extensive use of tetraphenylarsonium chloride as an analytical reagent prompted this investigation of the corresponding phosphonium and stibonium compounds, which behave in a similar manner. They form insoluble crystalline salts with permanganate, perrhenate, pertechnetate, perchlorate, periodate, fluoborate, and certain complex metal chlorides such as those of mercury, tin(IV), cadmium, thallium(III), and tellurium(1V). Some of these can be weighed directly; the others can be determined titrimetrically by adding an excess of the reagent and titrating the excess with iodine to form a periodide. These salts are all extractable from aqueous solution by chloroform. The peculiar properties of these salts make them valuable analytical reagents-for example, tetraphosphonium permanganate is completely insoluble and completely extractable.
T
H E use of tetraphenylarsonium chloride as an analytical reagent was applied by Killard and Smith (9) to the determination of rhenium, mercury, cadmium, zinc, and tin. Its use for the determination of thallium was described by Smith (6), for platinum by Bode ( 2 ) ,for tellurium by Bode ( S ) , for cobalt by Potratz and Rosen ( 4 ) and Affsprung, Barney, and Potrata ( I ) , for technetium by Tribalat and Beydon (7), and for the separation of rhenium from molybdenum by Tribalat (6). Because i t appeared probable that the corresponding derivatives of phosphorus and antimony would also be useful reagents, these compounds were prepared and their analytical properties investigated. Potratz and Rosen ( 4 ) investigated qualitatively the use of a number of onium compounds for the detection of bismuth and cobalt. The preparation of tetraphenylphosphonium and tetraphenylstibonium chlorides was described by Willard, Perkins, and Blicke (8). The former can be prepared more easily and a t a lower cost than tetraphenylarsonium chloride. In general, the ions precipitated by the phosphonium and stibonium salts are much the same as with the arsonium. Tetraphenylphosphonium chloride is more soluble and the corresponding stibonium compound is less soluble than the arsonium salt. The precipitates obtained in some cases follow the same order. However, the solubility of tetraphenylstibonium chloride is so low that its use as a reagent is extremely limited. The presence of considerable sodium chloride is essential for complete precipitation in nearly all cases. The procedures using tetraphenylphosphonium chloride are so similar to those already described (9) for tetraphenylarsonium that in many cases only the necessary variations are listed here. The interfering ions are also similar. I n most cases the direct weighing of the precipitate is not feasible, because the wash solution must contain sodium chloride, although in a few cases the precipitate is so insoluble that it can be washed with water. Titrimetric methods are based upon the addition of a known amount of the reagent, and titration of the excess by iodine to form the triiodide. The precipitates, like 1
those with tetraphenylarsonium. are readily soluble in chloroform, thus making extractions very convenient] especially for spectrophotometric methods. TITRATION WITH IODINE
The potentiometric titration curve is similar to that found by Willard and Smith (Q), and the precipitate is the triiodide, (CBH&PI~. The best concentration of sodium chloride is 4.0 .If, and if more than 50 mg. of the phosphonium chloride are present it is best to add the sodium chloride after about half the iodine solution has been added. At the end point the temperature should not be over 30" C. Moderate amounts of hydrochloric acid were without effect. A visual end point is possible, using starch as indicator and chloroform to dissolve the triiodide, but in this case sodium chloride should not be added. The precision is less than for the potentiometric method. Oxidizing agents, such as nitric acid, give low results, aa do large amounts of organic acids. Nitrate in a neutral solution does not interfere. The interfering ions are the same as in the titration of tetraphenylarsonium chloride. The average error for 2 to 100 mg. of tetraphenylphosphonium chloride is izO.07 mg. DETERMINATION OF MERCURY
A4nalyticalreagent mercury was dissolved in nitric acid, the excess neutralized with sodium hydroxide, and the solution diluted to 1 liter. One milliliter contained 5.00 mg. of mercury. The solution of tetraphenylphosphonium chloride was standardized by titration with an iodine solution, which was standardized against arsenious oxide. Direct titration of mercury with tetraphenylphosphonium chloride was impossible because of a lack of any suitable method of determining the end point. An amperometric method might be possible for small amounts. A gravimetric determination by weighing the precipitate of [( CsHs)rP]2HgClawas impossible because no suitable wash solution could be found to remove the sodium chloride. The titration of excess of the reagent, however, gave good results. The volume of the mercuric solution was adjusted to about 100 ml. after the reagent had been added. A 2.0 to 3.0 M concentration of sodium chloride was most suitable. An excess of about 10 to 20 ml. of 0.01 M tetraphenylphosphonium chloride was added to the warm solution and the solution allowed to stand with occasional stirring for 5 minutes to 2 hours, the longer time being required for amounts of 1 mg. The solution must cool to room temperature before it is filtered through a filtering crucible and -
~-
Table I. XaCl Concn.,
M
3.0 3.0 3.0 2.0 1.5 1.5
Present address, E. I. du Pont de Semours & Co., Orange, Tex.
1634
.~
Determination of Mercury
10-ml. excess of 0.01 -11' (C6Ha)aPCI Total Volume, Hg Present, Hg Found, Ml. hZg. .Mg. 40 0.50 0.5 60 5.00 4.98 15.00 15.02 100 100 50.02 50.07 150 100.04 100.08 200 150.06 150.8
Error, Mg. 0 -0.02 +0.02 fO.05 +0.04
+0.7
V O L U M E 25, N O . 11, N O V E M B E R 1 9 5 3 washed with saturated sodium chloride solution. Free acid can be present up to 1.0 M , except that free nitric acid must be neutralized. Table I shows the results obtained. Interfering ions have been mentioned (9). DETER.MIN.4TION OF TIN
Stannic tin is precipitated by tetraphenylphosphonium chloride as the chlorostannate, [(C&)J']zSnCle. Only a titrimetric method is possible. A standard tin solution was prepared by dissolving reagent grade tin in concentrated hydrochloric acid through which chlorine gas was bubbled. One milliliter of the solution contained 3.96 mg. of tin and about 0.1 ml. of concentrated hydrochloric acid.
Table 11. Determination of Tin 100-ml. total volume 10-ml. excess of 0.01 Af (CaH5)4PCl 2 ml. of HC1 (sp. gr. 1.18) XaCl Concn., Tin Present, T i n Found, M AI& Mg. 3.5 1 98 1 96 3.0 7 92 7.85 3.5 39 62 39.64 3.0 99.05a 98.97 2.0 1-18..>7a 148.90 a Final volume I50 ml.
Error, Mg.
-0.02 -0.07
+0.02 - 0 08
-t0.33
1635 DETERMINATION OF PERRHENATE
Tetraphenylphosphonium perrhenate, like the corresponding arsonium salt, is sufficiently insoluble to permit both gravimetric and titrimetric determination of rhenium. The procedure was the same as that recommended by Willard and Smith ( 9 ) , escept that a sodium chloride concentration of 2.5 to 3.0 M is necessary for the gravimetric, but only 1.5 AI for the titrimetric method.
Table I\'.
Determination of Rhenium
Total volume, 100 ml. 10-ml. excess of .0.012 .Vf (CsHs)rPCl KaC1 ReO4Concn., Present, M hlg. 3 .on 0.38 2.5 7 63 2.5 15.25 2.5 76.27 2.5 129.67b a Volume was 50 ml. b Volume was 150 rid.
ReOc Found Grav., Mg. 0.31 7.56 15.33 76.17 129.60
Error, Mg. -0.07 -0.07 +o 08 -0.10 - 0 07
ReO4Found, Titr., Mg. 0.33 7.60 15.30 76 22 129 69
Error, Mg.
-0.05 -0.03 +0.05 -0.05 +0.02
The standard perrhenate solution, prepared by dissolving recrystallized potassium perrhenate in water, contained 7.63 mg. of perrhenate, Reo,-, per milliliter. Hydrochloric acid up to 3.0 .lI was not harmful. The results are shown in Table I\-. DETERVINATION OF PERCHLORATE
The procedure is the same as for mercury, escept that the optimum concentration of sodium chloride is 3.0 to 3.5 J I , not much more than 10 ml. excess of reagent should be added, the hydrochloric acid Concentration should be 0.1 to 1.0 d/, and it is unnecessary to precipitate the tin in a warm solution. Table I1 show. the results obtained. DETERMINATION OF CADMIUM
Cadmium is precipitated as [(CsH,),P]2CdCI,, but the precipitate is more soluble than those of mercury and tin. The cadmium solution was prepared by dissolving pure electrolytic cadmium in nitric acid, neutralizing with sodium hydroxide, and adding a few millilit.ers of hydrochloric acid. One milliliter contained 4.00 mg. of cadmium.
Table 111. Determination of Cadmium 100-1111. total volume 10-nil. excess of 0.012 M reagent Solution neutral t o methyl orange
NaCl Concn., lU 4 5 ' 4 5 3 5 a
3 5 3 5 Total volume 150 ml.
Cd Pre3ent. Mg. 0 40 12 00 40 01 80 02a 120 04a
C d Found, blg. 0 43 12 04 40 04 79 95 120 12
Error,
>Ig.
+ O 03 + O 03
+0 03 - 0 07 f O 08
The procedure is the same as for tin, but the optimum range of concentration of sodium chloride is 3.5 to 4.5 -41, and a 30 to 40% excess of reagent is needed. Precipitation is best made in a warm solution, but the solution must be allowed to cool before filtering. The concentration of hydIochlorir acid should be no greater than 0.7 .ll. Table I11 shows the results. DETERMINATION OF ZINC
The precipitate of [(C6Hs)aP]zZnC14 is too soluble to permit a satisfactory separation of zinc.
Tetraphenylphosphonium perchlorate is so insoluble that it may be employed in both a gravimetric and a titrimetric method. As with rhenium, both methods may be carried out simultaneously by weighing the precipitate after drying at 110' C. and by titrating the filtrate and aashings with iodine. Potassium perchlorate was prepared by neutralizing analytical reagent potassium carbonate with perchloric acid and recrystallizing the salt. A standard solution was prepared by dissolving the salt in mater. This solution contained 3.61 mg. of Clod- per milliliter. To the neutral or acid solution of the perchlorate, 2.0 to 3.0 IbI in sodium chloride, mas added a measured amount of approximately 0.01 M tetraphenylphosphonium chloride, about 10 ml. in excess. The precipitate was rapidly stirred for several minutes and then allowed to stand for 2 hours or longer. It mas filtered through a filtering crucible, washed several times with ice water, dried a t 110" C., and weighed as tetraphenylphosphonium perchlorate, (CsH&)4PClOa. The ewes8 of reagent in the filtrate and wmhings can be determined by the usual potentiometric titration with iodine. The presence of sodium chloride caused the precipitate to be more crystalline and to adhere less to the walls of the beaker. Heating the solution before precipitation had a similar effect. Concen' trations of hydrochloric acid up to 2 Jf had no effect. Table 5 shows the results obtained with pure perchlorate solutions The presence of chlorate interfered 15 ith both the precipitation of tetraphenylphosphonium perchlorate and the titration with iodine. The interference \%aseffectively removed for as much as 500 mg. of chlorate bv reduction of the chlorate in acid solution
Table Y. Determination of Perchlorate Total volume, 100 ml. Excess of 10 ml. of 0.012 .M (CsHdrPCl SaCl c104 C104ClO. Found, Error, Found, Concn., Present, Grav., Mg. Mg. ,M hlg. Titr,, l f g . 0 59 +o 05 0 48 3 oa 0 54 10 74 - 0 08 10 77 2 5 10 82 10 83 10 80 +o 01 3 0 10 82 54 18 54 20 285 84 12 f O 06 -0 06 126 27 126 22 2 Ob 126 28 Total volume 50 rnl. b Total volume 150 rnl.
Error, >Zg.
- 0 06
- 0 05 -0 02
+o os -0
01
ANALYTICAL CHEMISTRY
1636 with sodium bisulfite. was as follows:
To the cold, slightly acid solution, an amount of sodium bisulfite was added equal to about five times the weight of chlorate present. The solution was allowed to stand 30 minutes and gently
boiled until no more sulfur dioxide could be detected; the solution was kept slightly acid throughout this period. An excess of standard tetraphenylphosphonium chloride solution was added to the rapidly stirred hot solution and after standing 2 hours the cold solution was treated as described above. The results are shown in Table VI.
Table VI. Determination of Perchlorate in Presence of Chlorate 2.5 M sodium chloride
ClOa, Mg. 50 100 100 200 500 1000
NaHSOs Sdded, G. None None 0 5 1 0 2 5 5 0
DETERMINATION OF P E R M 4 S G A N A T E
In the presence of chlorate the procedure
Total volume 100 ml. 18.04 mg. of Clod present ClopFound Error, Grav., JIg. Mg. 20 0 +2 0 21.9 $3.9 18.08 f0.04 17,95 -0.09 18.15 +O.ll 18 37 +o 33
-
(2104 Found, T i t r . , Mg. 19.9 22.3 18.09 17.98 18.12 18.26
Error, Mg.
+19 +4 3 +o 05 -0 06
+o os
+o
22
Tetraphenylstibonium chloride, like the corresponding arseiiic and phosphorus compounds, reacts lvith perchlorate ion to form the insoluble perchlorate, (CGHj),PSbC14. This reaction permits only the gravimetric determination of perchlorate, because tetraphenylstibonium chloride cannot be successfully titrated n4th iodine. The gravimetric determination was successful, but because of the low solubility of the reagent in 1.5 to 2.0 M sodium chloride solution the very small excess that could be present made the method impractical, unless the amount of perchlorate present was known to within 1 mg.
Permanganate forms very insoluble purple precipitates of (COH&PMnO4 and (C~H,),SbMn04 with tetraphenylphosphonium chloride and tetraphenylstibonium chloride, and may be determined gravimetrically with either reagent, but titrimetrically only with the phosphonium salt. 2 . direct titration was not successful with either.
A standard permanganate solution was prepared in the usual way, and standardized against arsenious oxide. It contained 3.58 mg. of ?vlnO4 per milliliter. A definite excess of tetraphosphonium chloride was added to the hot permanganate solution containing sufficient neutral salt, preferably not a chloride, to provide a salt concentration of 0.5 to 1.0 M ; this was necessary to cause complete precipitation, and sodium sulfate or nitrate is preferred. The solution was alloa-ed to stand until it had reached room temperature, filtered, washed several times with water, and dried in a desiccator to constant weight, which usually required 2 or 3days. Heatingof the dry salt a t 100" C. causes decomposition. The filtrate and washings were titrated potentiometrically with standard iodine. When nitrate was added it was necessary first to make the solution just alkaline to methyl red. The results are shown in Table T'III. Sulfuric, nitric, and phosphoric acids up to 1.0 -11had no effect. Hydrochloric acid reduces the permanganate.
Table VIII.
.\InOl- Present, Ng.
0 18 1 79 7 17 53 76 71 68 107 52
Determination of Permanganate
Total volume 100 ml. 1.0 .M NaXOs 10-ml. exce-s of 0.012 .1/ Jln04- Found, xg. Error, (Grav.) 3Ig. -0 03 0 15 + O 03 1 81 7 16 -0 01 53 82 1 0 06 71 60 -0 08 107 64 -0 12
-
(CsHs)aPCl 3InO4- F o u n d , Erior, 3Ig. -0 01 + O 06 -0 05 ' 0 08 + O 04 + O 07
Mg.
(Titr.) 0 17 1 85 7 12 53 84 71 72 107 59
~
_
_
D E T E R M I S A T I O Y OF P E R I O D A T E
Tetraphenylphosphonium periodate, (CeHj)rPIO4, is sufficiently insoluble to make possible a titrimetric but not a gravimetric determination, as the precipitate must be washed with sodium chloride solution. The standard periodate solution was prepared by dissolving recrystallized potassium periodate, KIO,, in water. One niilliliter of this solution contained 1.20 mg. of periodate. Excess of standard tetraphenylphosphonium chloride TI as added to a cold, slightly acid solution of periodate containing sufficient sodium chloride to make the final concentration 2.0 111. The precipitate was allowed to stand about 2 hours, filtered, and washed with saturated sodium chloride solution. The combined filtrate and washings were titrated as usual. If the solution was heated before precipitation, the results were a h a y s low. Only a drop or two of concentrated hydrochloric acid in excess may be present, and if the solution was alkaline to methyl red, the results were inaccurate. Iodate interfered with the titration in acid solution, but interference of the acid was reduced if the solution was saturated with sodium bicarbonate. The results are s h o m in Table VII.
Table VII.
Determination of Periodate
Total volume 100 ml. 10-ml. excess of 0.12 -11 (CsHa)rPCl KIOa Present, 104- Present, 1 0 4 - Found, h'sC1 Canon., M Rlg. Jlg. Mg. 3.0a 0 0.90 0.95 3.0a 0 9.02 8.96 3.0 15.03 0 15.01 2.0 90 21 0 90.30 2.0 102,24 102.35 0 2.0 25 30.07 30.01 2.0 30 07 30.14 100 30.07 2.0 150 30,54 0 T o t a l volume 50 ml.
-~ _____
~
Error, Rlg. +0.05 -0.06
-0.02
+0.09 +o. 11 -0.06
+0.07 +0.47
For the determination of manganese in a manganous salt, the best oxidizing agent is sodium bismuthate, in a solution contaiiiing 5 to 10 grams of metaphosphoric acid to form a soluble coniplex with the trivalent bismuth. The excess of bismuthate mas filtered off. If the amount of soluble bismuth was not over 350 mg., 1.2 31 nitric acid solution was also satisfactory. The errors xere about the same as in Table VIII. The same procedure with tetraphenylstibonium chloride gave results in the presence of 0.1 to 0.8 J1 sodium nitrate or 0.1 to 0.5 A1 sodium sulfate similar to those in Table VIII, but the largest amount determinedwas 18 mg. of IrInOa-. An excess of 10 ml. of 0.01 M stibonium chloiide was added, The tetraphenvlstibonium chloride was standardized gravimetrically by precipitation as the perchlorate in 1.5 31 sodium chloride solution containing an excess of perchlorate. DISCUSSION
I t may be predicted that tetraphenylphosphonium chloride can also be used to precipitate platinum, tellurium, thallium, and technetium, for which the arsonium compound has already been employed. Interfering substances have been listed (9), but there have been some additions and modifications. The alkali metals, alkaline earths, aluminum, manganese(II), chromium (111), nickel, cobalt, zirconium, sulfate, borate, phosphate, carbonate, acetate, tartrate, and citrate are not precipitated and do not interfere. The following ions are precipitated either completely or partially: permanganate, perrhenate, periodate, perchlorate, persulfate, borofluoride, chromate, molybdate, tungstate, thiocyanate, iodide, stannic, cadmium, zinc, platinum, ferric, gold, antimony, bismuth, titanium, thallic, tellurous, arid uranyl. The interference of tin was eliminated by complexation
_
V O L U M E 25, N O . 11, N O V E M B E R
1953
with bitartrate, of ferric ion by phosphate and phosphoric acid, and of titanium by metaphosphoric acid. The influence of c o p per ixi the iodine titration was eliminated by the addition of citrate. Tribalat (6) precipitated perrhenate from alkaline solution to separate it from molybdate. The use of fluoride as a complexing agent is restricted because of the l o v solubility of the arsonium, phosphonium, and stibonium salts. Although the borofluoride ion, B R - , is quantitatively precipitated, an attempt to use this as a method for fluoride failed because of the rapid hydrolysis of this ion,
1637 metric determination of permanganate and perchlorate, but in the latter case the low solubility of the reagent makes the method impractical. The salts are soluble in chloroform. The average error under recommended conditions is about zkO.06 mg. LITERATURE CITED
Affsprung, H. E., Barney, S . A., and Potrate, H. b.,AXAL. CHEM.,23, 1680 (1951). Bode, H., 2.anal. Chem., 133, 95 (1951). Ibid., 134, 100 (1951). Potratz, H. A., and Rosen, J. M., ANAL.CHEM.,21, 1276 (1949). Smith, W. T., Jr., Ibid., 20, 937 (1948). Tribalat, S., Ann. Chim., 4, 289 (1949); Anal. Chim. Acta, 3, 113 (1981). Tribalat, S., and Beydon, J., Ibid., 6, 96 (1952). Willard, H. H., Perkins, L. R., and Blicke, F. F., J . Am. Chem. SOC.,70, 737 (1948). Willard, H. H., and Smith, T. LI., IXD.EXG.CHEM..A N ~ LED., . 11, 186, 269, 305 (1939).
SUhIMARY
Tetraphenylphosphonium chloride precipitates quantitatively, in the presence of sodium chloride, complex chloride salts of tin, cadmium, and mercury such as [(C6H&PI2SnC16. These cannot be weighed as such, because sodium chloride must be used in the wash solution. The excess of reagent may be titrated potentiometrically with standard iodine to form (CnHJdPI?. Tetraphenylphosphonium permanganate, perrhenate, and perchlorate are insoluble i n cold water and therefore both gravimetric and titrimetric methods are possible. For periodate the titrimetric method only can be used. Tetraphenylstibonium chloride may be used for the gravi-
RECEIVED for review May 15, 1953. Accepted August 10, 1953. From a thesis submitted by Lowell R. Perkins t o t h e Graduate School of t h e Unirersity of Michigan in partial fulfillment of the requirements for t h e degree of doctor of philosophy.
Determination of Malic, Tartaric, and Citric Acids in Fruit by Ion Exchange Chromatography HENRY H. SCHENKER'
AND
WILLIAM RIEMA.\: I11
School of C h e m i s t r y , Rutgers University, New Brunswick, N. J . The methods for the determination of malic, tartaric, and citric acids in fruit are very lengthy. This paper describes a procedure in which the acids are separated by ion exchange chromatography and then determined by oxidation with permanganate. All three acids can be determined in a fruit in 8 hours by the proposed method, and the error in each determination is usually ahout *0.1 mg. of acid. The sample may contain a maximum of 24 mg. of any one acid. The method is applicable to fruit, fruit juices, and fruit prodncts.
T
111: methods gene] ally used for the determination of malic, taitaric, and citric acids in fruit are those described by the .lssoc*iation of Official Agricultural Chemists (1). These proceclures are very time-consuming. TT'ieland and Feld ( 5 ) suggested the use of paper chromatography for the separation and determination of the fruit acids; relative errors of about *lo% nre frequently incurred by this method. Isherwood ( 3 ) applied pnrtition chromatography to this prohleni by a procedure that involves a tedious extraction and then concentration of the fruit : i d s . These steps alone take 48 hours and fail to evtract the ncids qunntitatively. RECORIMENDEI) PROCEDURES
Reagents. ,411 reagents were of reagent grade. Eluant A was 0.0800 J f with sodium nitrate, 0.0013 .If with sodium t,etraborat,e,and 0.30 -21 with boric acid. Eluant B was 0.1G00 .lf with sodium nit,rate and had the same concentrations of sodium tetraborate and boric acid as eluant A. Both eluants viere prepared by weighing the required amounts of solutes (on an analytical balance i n the cases of sodium nitrate and tetraborate) and diluting ill a volumetric flask. The pH lyas then checked with a Beckman pH meter and, if necessary. was adjucted to 6.15 by the dropwise addition of 1 -1fsodium hydroxide. 1 Present address, Carothers Research Laboratory, Experiment Station, E . I , d u P o n t de Semours & C o , , Kilmington. Del.
Potassium permanganate, about 0 1 .\; was prepaied and standardized by the usual method. Standard oxalic acid of exactly the same normality as the permanganate was prepared. The normality was checked by titration with the permanganate. Sulfuric acid, 9 M , was prepared. Apparatus. A glass tube 3.8 sq. em. in cross-sectional area, provided with a sintered-glass filter disk of medium porosity, was filled t o a depth of 25 em. with Dowex-1, a strong-base anion exchange resin. The flow rate through this column waa controlled by a Hoffman clamp attached to a piece of rubber tubing fitted to the lower end of the filter tube. Excessively fine particles of resin had previous1 been removed by decantation, As the resin was received in the cxloride form, i t was converted to the nitrate form by passing 0.5 M sodium nitrate through the column until the effluent gave a negative test for chloride. Then 1 liter of eluant -4was passed through the column. -1constant-temperature bath was maintained a t 30" zk 1'. Preparation of Juice. The juice was obtained by pressing or squeezing the fruit and was centrifuged and filtered through Whatman KO.41 paper. One milliliter of the juice was then titrated with 0.1 S sodium hydroxide to find the total concentration of fruit acid as a guide to the selection of the proper size of sample for the elution. Suffirient solid boric acid was added to the juice to give a 0.30 solution. The juice was stored in a refrigerator. Procedure for Elution. The column \vas prepared for use by the passage of 500 ml. of eluant A. A sample of 5 ml. or less, containing not over 24 mg. of any one acid, was pipetted into the column, and the inside w A l was rinsed with a small amount of eluant A. The column was drained until the meniscus just reached the resin. Then 627 ml. of eluant A were passed
