Salting-out chromatography

VOL.

18 (x958)

ANALYTICA

SALTING-OUT III. ALIPHATIC

AND

CHIMICA

ACTA

I97

CHROMATOGRAPHY

POLYGLYCOL

ETHERS,

CARBOXYLIC

ACIDS

bY ROGER Ralph

G.

CVrtghi

Chetttical

SARGENT

Laboralory,

AND

Rrrlgers

\Vnt. RIEMAN, Untverstly,

New

III Brtrnswrck,

N.J.

(U.S.A.)

The authors have shown that the analysis of complex mixtures of alcohol9, polyglycols2, and amines is facilitated by salting-out chromatography, a process whereby water-soluble nonelectrolytes are separated by clution through a column of ion-exchange resin with a concentrated salt sdution as eluent. The logarithm of the distribution ratio of the nonelectrolyte wti shown1 to increase linearly with the molarity of salt in the eluent solution, and the slope of the line was found to be identical with the salting-out constant obtained from solubility measurements. In this report, salting-out chromatography is agplicd to the separation of aliphtitic and polyglycol ethers. A brief description of the attempt to utilize this technique for the separation of the aliphatic acids is included. EXPEiW+lENTAL The details for the pruparation of columns of the ion-exchange resin and the proccdurcs for tho ulution arc glvcn 1x1rcf l. EffIucnt fractions of 6.0 ml were analyzed by oxidation with dichromato in 50% sulfuric acid with subsaquont mc,asurumcnt of th&absorbancc of C~F(ISI)‘*~. Ittlersiiltal voiumes The lntcrstltinl or void volume 1’ of a column of ion-cxchangc resin was clctcrmincd by the procodurc which ZollowzP. Slurry the hydrogen form of the resin with water and pour into a glass tube’ whose mtcrnal cross-sectional area is known. After 5 minutes, drain off the excess liquid to the top of the bed. After percolation of at least I 1 of o.rooN hydrochloric acid through the rosin, again drain to the top of the bed and record the height of tltc column. Hcmova the stopcock at the bottom of the column and wash the bottom of the srntcred glass disk to remove any hydrochloric acid that 1s not prcscnt in the bed of rosin. Wash the acid from the resin bed with a sufficient quantity of water and coltcct the effluent m a suitable volumetric flask. The milIicquivaionts of acid in thccfflucnt, found by titration with sodium hydroxldc, is then divided by the normality of the acid to find the intcrstitlal volume?. Ethers The ethers used in this investigation arc listed in column z of Table I by their structural formulas and will be refctrcd to by the corresponding number m column I. Many of the ethers could nat be obtained in pure form and no attempt was made at purification. Ethylene glycol (I) was included in this study because it was a major Impurity tn (9). Ethanol. a common Impurity in (IO), was incfudccl for the comparison of this study with a previous investigation’. Small amounts of (13) and an impurity that IS probably Tr-propyl alcohol were detected in (~4). RESULTS

E&&on variables Fig. I represents the elution graphs for (I), (2) and (9) obtained by eluting the mixture through the vkrious resins with a flow rate of 0.5 cm per minute of z.oikd ammoReferences p. 203

VOL. I8

198

1.32

1.60

f-10-c-c-0-c-c-01-I

I.37

2

3

c-0-c-c-011

* *34

4

o/c-\O

I

HO-C-C-O

2

I-L

‘q-c/ 5

c-c-0kx.z-0i1 01-K

6

C-O-&C.. 01-I

01-I

.CJG

(1958)

2.37

2.98

J 32

o.oRg

3 40

5.30

8.84

‘-33

0.205

2 . ‘1 I

3.80

6 25

I ..IG

0

l&I, .e

3.5:

G&3

IL ‘t

24.7

0.~72

I.‘f’

2.8G

5.83

1’

23.7

Q.303

1937

2.81

5.71

I I.3

I

IX

a.5

10.0

24.5

209

I 3H

0.318

‘833

11.358

,

&c_*_c_~_c

I .32

3 10

(1.H I

1

8

c-0-c-c-0-c

1.36

3.36

7.10

X6.1

I *.# I

0.3.57

0

c-c-o-c-c-o-c-c-of~I

I.37

3.8s

g.20

23 9

I

.tt

o.+t

IO

c-c-o-c-c

1.68

*t.p

i 1.‘)

I.

70

O.‘i &B

I I t2

c-c-c-c-o-c-c-oI~I

7.28 0 2.t b 90

LO

13

c-c-o-c-c-o-~:-(3-o-c-c

ci,_,_,/~ \C

C’ 14

c-c-c-o-t.x.-.-c:

15

C-C-C-C-0-C-C-O-t’-(.‘-l’-C

3.“1 5.33

16

~,-C-C-~-~-~-~-c’-(;

G.g0

-

C-C-Of-I

I

-_

_-__.

I2

-

.gG

--

..-.-

s-8

-_.__

CJ

23.0 --.

. __ --

I

_I

--

1.25

3..t”

-_-

I *70

0.461 0.560

L.V,I

0.52

3-14

V‘.isi

2.50

__

5.33 --

-I

p3Y

-_

- _-.-..

I

0.178

7 75

_ ._.

_--__

.-_ -_ ._

nium sulfate. ‘I’hc anion-cxchangc resins, Dowcx r-X4, and -US, fail to scparatc (I) from (2). Dowex so-S4 was chosen in prcfcrencc to 13owcx so-S8 because the resin of lower cross-linkage is more porous to the large molcculcs encountered in this investigation. The effect of flow rate, Pig. za, and sample size, Fig. zb, are shown by clutions from Dowcs 50-lS4, zoo-400 mesh, with 2.oM ammonium sulfate. The cffccts of these variables and the effect of resin mesh size arc similar to those found carlicrra3. Ammonium sulfa& solutions were also found to bc suitable as cluentsr. Distrihrtimt

ratio artd saltiqpxst

cm&ants

For each clution performed, the distribution means of the ocluatiorP u* =cv+v...........

ratio C of the ether was calculated .*,....*,*..

by (I).

where U* is the volume of effluent at the peak of the clution graph, tncasurcd in milliliters. The interstitial volume of the colutnn ‘c; was dctcrmined to be 33.0’7; of the bed f&?ftm~rccsp.

203

SALTlNG-OUT

. III.

CHROMATbGRAPHk

1x6 cm Dowex

SO-

17.6 cm

50 -X6

Dowax

199

X4

18.0 cm Dowcx

17.1 cm Dowcx

too

1 -X6

&?i%nt , ml

Pig, I. Effect of resin and cross-lInkage. Elation of (I), (CC)and (9) on columns exchangcl resins of diffcront cross-linkage. Each column hacl R cross-scctionnl

of cation- ancl iLIIiOtIarea of 3.90 q&m.

volume for Dowex 50-X4, 200-400 mesh. Columns 3-7 in Table I fist the va.hses of C for various ammonium sulfate eluents. Plots of log C VS. M for several ethers are showr; in Figs. 3 and 4. These straight lines follow the cquationl log c =

log C, +

k&f . . . . . . . . . . . . . . . . , . , (2)

The data in Table I were used to calculate the conditions necessary1 for the separation of synthetic mixtures which contained 0.05 mmol or less of each ether dissoivcd in r RefereWes

~9. 203

R.

SARGENT,

W.

RlEMAN

III

VOL.

18

(x958)

a

0.33 cm/minute

i

0.8

Fig. 2, The effect of various flow rates (a) and sample .Dowcx 50-X4,200-400 mash, 17.6 cm x 3.90 sq.cm.

SIZCY (b) in salting-out

chromatogmphy.

oa Ammontum Wfote aluent thongs8 i_

0 Liters

MofurltV of ammcnium Sulfate Fig, 3. Log C ph-ts and tho soparntion cm x 3.90 sq.cm, 0.7 cm por minuttt. Refwemes

$.

a03

of a mixture

of effluent

of ethers on Dotvex 50-X4.200-400

mesh, 12.0

VOL. 18 (1958)

SALTING-OUT

CHROMATOGRAPHY.

III.

Ammonium

I’lg. 4. Log C plots and the scparataon cm x 3.90 sq.cm, u.7 cm per mmutc.

of a mlxturc

of cthcrs

sulkte

on Dowcx

201

eluent

chonp~

50-X4.200-400

mesh,

14.0

ml of water. For example, the values of U* for the ethers separated in Fig. 3 on Dowex 50-X4, were calculated as 52.1, 99.4, 195, 311 and 406 ml. The observed values were 51.6, 97.9, rgz, 306 and 394 ml. Fig. 4 illustrates the separation of another mixture of ethers under the same conditions. DISCUSSION

Quantitative analysis of complex ether mixtures is readily accomplished by the chromatographic procedure given above followed by oxidation with dichromate4. The hydroxy ethers have high reduction capacities in their reaction with dichromate in 50% sulfuric acid and therefore can be determined accurately in trace quantities. Some difficulty is likely to be encountered in the analysis of the simple aliphatic ethers. Low solubility in aqueous solution, low reduction capacities in the oxidation procedure and volatility combine to make the analysis difficult. Gas-liquid partition chromatography, although not as effective for the high-boiling hydroxy ethers, would probably be prefcrable for the separation of the volatile aliphatic ethers.

The extrapolated C, and the K value of equation 2 for each compound is given in the last two columns of Table I. Although straight lines were obtained with Dowex 50-X4, the slope k for ethanol has a lower value than the salting-out constants (K in Table II) obtained’ with the higher cross-linked resins, Dowex x-X8, and Dowex 50-X8. TABLE COMPARISON

Resin

OF

CO

ELUTION

DATA

C 4JI (NN,).SO.

OF

II ETHANOL

h

WITH

ShvinkaRe 0.0 to 4.oM (NH&O,

Y.

Dowcx

50-X4

1.48

7.75

0.178

29.2

Dowcx Dowex

50-X8

0.836

7*77

0 243

15.9 68

r-X8

0.619

-

References

p. 203

7.08

---_-

0.25G

--

VARIOUS

-

RESINS

_ ^

g iWwnaL H,O g dry NH,R _ .--__-__

per

._ _.

H.0

1 50

0.773 -

*.\I (NH,)&%

0.908

0.574 -

_.-_

_

R. SARGENT,

202

W.

RlEMAN

III

VOL.

18 (x958)

Table II shows that the lowest value of k is obtained by use of Dowex 50-X4 which undergoes the greatest variation in volume with cluent concentration. Consider the distribution of ethanol between the resin phase and an external aqueous phase. As ammonium sulfate is added to the mixture, the ethanol will be salted out of the external phase into the resin phase. A resin of low cross-linkage will offer increasingly greater resistance toward the alcohol because shrinkage increases the ionic concentration within the resin. This view is in accord with the high value of C, for Dowex 50-X4 whose internal water content in the presence of an aqueous external phase is nearly double that of Dowex 50-X8 (Table II). The relationship of the salting-out constant to the concentration of ions in the resin phase is being investigated further in this laboratory. Carboxylic acids Methods analogous to those used for the separation of the amines were employed in an attempt to separate carboxylic acids by salting-out chromatography. Solutions of tripotassium phosphate made excellent elucnts because, by hydrolysis of the phosphate ion, the solutions were sufficiently basic to prevent ion formation by the amine. Similarly, it is necessary to use elucnts that arc sufficiently acidic to repress the ionization of the carboxylic acids if one is to avoid ion-exchange reactions on the resin. The ionization of propionic acid may be repressed to a negligible extent in o.rN hydrochloric acid, A major difficulty arises at this point. Consider the addition of an anion exchanger (I) and a cation exchanger (2) to this mixture : (I) Although propionic acid is virtually unionized in the external solution, upon entrance to the resin phase, it ionizes because the DONNAN membrane cquilibrium7 excludes hydrogen ions from the resin phase. Ionization is facilitated by the exchange of the propionate ion with the chloride form of the resin. The elution of a carboxylic acid from a column of Dowex r with o.rN hydrochloric acid always products an elution graph with two peaks when the effluent is analyzed for total acidity. The first peak, which immediately follows the interstitial volume of eluent, is due to the exchange reaction described above. The same is true also for other cluents used, e.g., sodium or calcium chloride solutions which were O.IIN in hydrochloric acid. (2) A cation exchanger in a form other than hydrogen would lead to ion-exchange reactions. Solutions of hydrochloric acid were therefore used for the elution of carboxylit acids from Dowex 50. Fractions of the effluent were analyzed by two methods: Fatty acids were extracted with chloroform and titrated with sodium hydroxide. Fruit acids and dicarboxylic acids were evaporated to dryness on a steam bath with subsequent titration. It was found that hydrochloric acid was poor as’s salting-out agent and failed to give the large differences in the log C plots that are necessary for the separation of mixtures. Only slight improvement was found for sulfuric acid eluents. Linear log C plots were not obtained (for the fruit acids which could be analyzed by dichromate oxidationa) because of bisulfate formation in the eluent. The presence of sodium or calcium chloride in o .I N hydrochloric acid and ammonium sulfate in sulfuric acid (the ratio of bisulfate to sulfate maintained constant at 0.1) increases the difference in C values, but in all cases, the separation of a mixture of carboxylic acids was not improved in comparison with the salt-free eluent because of an increased spread of the elution graphs. Because of ion exchange, the distribution ratios were higher on the anion exchanger than on the cation exchanger especially for the more highly ionized carboxylic acids. References

p.

a03

VOL. 18 (x958)

SALTING-OUT

CHROMATOGRAPIIY.

203

III.

The use of low concentrations of sodium or calcium chloride in o.xN hydrochloric acid as eluent therefore decreased the values of C for the acids whose ionization constants were larger than IO -3. With eluents of higher salt concentration, the C values increase by virtue of the salting-out effect. No advantage was found in the use of acidified eluents, again because of increased spread of the elution graphs. The elution data in Table III were obtained for the chloride form of Dowex r-X8, zoo-_coo mesh, with an eluent of o.rN hydrochloric acid. Under these conditions there is no salting-out effect and differences in C are due only to ion exchange. TABLE VALUES

fAct1c

Tartaric Malic Citric Fumarrc Maleic

1.45 2.09 2.57 3.92 5.84 7 90

OF

c

FOR

III

CARI3OXYLlC

Formic AcetIc Propionic tr-Butyric Isobutyric rt-Valcric

ACIDS

2.19 I .G2 3.34 6.39 12.4 15.1

Oxalic Malonlc Succinic Glutaric Adlpic

5.70 4.86 467 6.90 II.4

The data show that the followin mixtures may be scparatcd: (I) acctrc, formic, propionic, succinic. n-butyrrc and tsobutyric acids: (2) lactic, malic, malonic. glutanc, and adipic acids. ACKNOWLEDGEMBKT

T!le authors cxprcss thclr gratltudc of this invcstigatlon.

to the Dow Chemical Company

for gcncrous financral support

SUMMARY Distribution ratios of 15 others wcrc dctcrmincd with various ammonium sulfate cluents and were used to determine the conditions ncccssary for the separation of five- and seven-component mixtures by salting-out chromatography. The application of this tcchniquc was found to bo less successful than ion-exchange chromatography for the separation of carboxylic acids. REFERENCES 1 R. SARG~TAND W. RIEMAN, J. Pkys.Ckem., 61 (x957)354. z R. SAHGZNT AND W. RIEMAN, J. Org. Ckem., ZI (rg57) 594. a R. SARGENT AND W. RIEMAN, Anal. GJtim. Ada, 17 (x957) 408. 4 R. SARGENT AND W. RI&MAN, Anal. Clratn. Acla, 14 (rg56) 381. * A. C. BRICYER, privato communication. s J. BEUKENKAMP, W. RIEMAN. AND S. LINDENRAUM, Anal. Ckcm., 26 (1954) 505. 7 F. DONNAN AND E. Gu~cxcNH~1nf, Z. pkystk. Cketn., A162 (1932) 346. Received

July

3rd.

1957