pH-Chromatography of chlorinated phenols

JOURNALOP CHROMATOGRAPHY

572

pH-CHROMATOGRAPEY

OF CHLORINATED

2. VACEK, 2. g*OTA AND J. STAN&K S$olana, Nevalrovice and Research I&titule (Received January 8th, 1965)

ofAgrocltemicel!

PHENOLS

Tschno~ogy,

Dl(atisZava

(CzecJzoslovakia)

Paper chromatography of some chloropherrols has been studied by several authors’. The separation of o- and +chlorophenols was described by CI-IOGULLLAND BISSING~. A more exhaustive review was published by SCHL~GL AND SIEGEL~, who studied the separation of nine isomeric chlorophenols or of their derivatives (azo dyes and chlorophenoxyacetic acids). These authors draw attention to the tailing of free chlorophenols and azo dyes as well as to other complications; with chlorophenols the situation is even more complicated because of the volatility of the lower chlorinated derivatives. In the case of chlorophenoxyacetic acids a smooth separation of the analyzed mixture into fractions according to the number of chlorine atoms per molecule is possible by chromatography, but the separation of isomers by this method is feasible only in exceptional cases. Separation of the six chlorophenol isomers in a system with stationary formamide and xylene as mobile phase has been described by GREBENOVSKQ~, who solved the problems arising from the volatility of the lower isomers by using chromatography between glass plates according to the method of WUDECEK~. In the present communication a description is given of the separation of all 19 isomeric chlorophenols using the principle of pH-chromatography0. Findings on the relationship between chromatographic behaviour and structure are also presented and compared with theoretical assumptions as developed by SOCZEWI~~SKL AND WAICSMUNDSKL7-“0. EXPERJMENTALAND RESULTS The method of preparation of the individual compounds and their physicochemical constants are given in Table I. All the chromatographic data presented here were obtained on Whatman No. 3 paper impregnated with IO y0 olive oil in benzene using universal Britton-Robinson buffers of different pH as the mobile phase. In addition to this basic system, complementary data were obtained for the systems shown in Table II. I The compounds were placed on the paper in rt-butanol solution. Phenol, monochlorophenols and dichlorophenols were chromatographed between glass plates as described by GREBENOVSK~. 4-Aminoantipyrine, the Folin-Ciocalteau reagent and U.V. light were used for detection. After detection with 4_aminoantipyrine, most chlorophenols appear in different hues of red (from orange to purple blue) ; only the derivatives with positions 3 and 5 substituted yield a blue or blue-green colour (3,4,5trichlorophenol,’ tetrachlorophenols and pentachlorophenol change eventually to yellow-brown, cf. ref. 24). J.‘Chvomatog.,

29

(1965)

572-579

p&CHROMATOGRAPHY. TABLE

PHENOLS

573

I

PREPARATION

No.

OF CHLORINATED

OF THE

INDIVIDUAL

COMPOUNDS

AND

P@aration*

Compound

THEIR

CONSTANTS

Melting

point **

P? I

: 4 2 g 9 10 11 12 =3 =4

=5

16 =7 18 19 20

Phenol 2Chlorophonol 3Chlorophcnol 4Chlorophenol 2,3-Dichlorophenol 2,4-Dichlorophenpl ’ 2,5-Dichlorophenol z,G-Dichlorophenol 3,4-Dichlorophenol 3,5-Dichlorophenol 2,3,4-Trichlorophenol 2,3,5-Trichloropheuol z,3,6-Trichlorophenol 2,4,5-Trichlorophenol 2,4,&Trichlorophenol 3,4,_+Trichlorophenol 2,3,4,5-Tetrachlorophenol 2,3,4,6-Tetrachlorophenol 2,3&G-Tetrachlorophenol Pentachlorophenol

40.2~4I.0,

,,

8.0-8.4 3=.5-32-S

ii 11 a 22 a 12 a =3 C =4 15 1G a’ a 17 18 19 20 d

42.0-43.0

57.0-58.0 44.0-45.0 57.5-58.5 G6,0-67.0 66.5-67.5 67.0-68.0 81.0-82.0 55.8-61.0 56.o-57.5 65 *o-66.5

.J

‘)

68.5-69.5 gg.o-X00.5 11o.o--116,o

68.o-69.0 114.0-115.0 18g.o-190.0

product, purified by crystallization * The numbers are reference’numbers. a = commercial from petroleum ether; b = commercial product, rectified, acetylated, the acetate rectified, from 1,3,5-trichlorobenzenea3 ; d = hydrolyzed and again repeatedly rectified ; c = prepared commercial product, recrystallized from benzene, ** The melting points were determined using KOFLER’S hot stage.

The pH gradient during chromatography with stationary olive oil and mobile buffer was determined by measuring the pH of a water eluate of a chromatogram strip cut out perpendicularly to the direction of flow of the mobile phase immediately after removing the paper sheet from the chromatographic jar. Relationships bet.yeen the RF values of the individual compounds and the pR of the mobile phase in the olive oil system are shown in Figs. I, 2,3 and 4, from which it follows that undissociated chlorophenols are. separated according,to the number of chlorine atoms in their molecule. The. most mobile is unsubstituted phenol, the mobility decreasing. with .the number ,of chlorine substituents. Undissociated tri-, tetra- and pentachlorophenols do not differ in their ‘mobilities and hence cannot be resolved. The chlorophenol anions, however, are separated according to the position TABLE

II

SYSTIWS FOR THE

Stdtionary -’

SEPARATION

OF. ISOMERIC

M$Gle, phase

phase

4d% kerosene in n-hex&e IO O/Opolyethyleneglycol succinate Britton-Robinson buffer .Britton-Robinson buffer Britton-Robinson buffer

,!. ,:

CHLOROPHENOLS

I’

‘. Britton-Robinson ‘buffer Bitt&-Robinson buff or Ethyl acetats Iso-amyl acetate 40 O? *kerosene iti n-hexane

.

, J. Ghvomatog.;

rg (1965) 572-579

Z. VACEK,

Z. %O+A,

‘Jo, STANtiK

as 0.6 0.4

%3 02

af Fig. z. Relationship between the R F x~lues of phenol and monochlorophenols and the tiH of the mobile phase. Q phenol: 0 2-chlorophcnol; 0 3-chlorophenol; 0 4-chlorophonol. Fig. 2. Relationship between the Rp values of dichlorophenols and the pH of the mobile phase. 9 2,5-dichlorophenol; 0 z,4-dichlorophenol; Q 2,5-dichlorophenol; 0 2,Sdichlorophenol; @ 3,4dlchlorophenol and 3,5-dichlorophenol,

I

Fig. 3. Relationship between the Rn &+es of, trichloroljhcnols and the pH of the mobile phase, 0 2,3,4-trichlorophenol: d ‘2,3..5-trichlorophenol and 2,4,5-trichlorophenol; Q 2,3,6-lrichlorophenol; ‘0 2,4,f%.richlorophenol; 0 5,+,5&ichlorophenol. Ffg. 4. &el&iokliip between the RF vilues df the tetrachlorophenols and pentachlorophenol and the pH of thd mobile phase. 0 i,3,4,6-l+achlorophenol; o 2,3,4,5-tetrachlorophenol &cl 2,3,5,6tetrachlorophenol ; 0 pentachlorophenol. , .’

of the substituents in the nucleus with respect to the hydroxyl group. The fastest derivatives are those with both ortho-positions occupied, those with one orttzo-position free are somewhat less mobile. and ,the derivatives with both &ha-positions free are the slowest. It can also be seen from the figures that the technique described here yields relatively good results. and that the “selection of a suitable pEI for ‘the’ mobile phase results in the separation .of ,most .of ,the compounds examined. The following pairs form ,exceptions: 3,4- and 3,5-dichlorophenol, 2,3;5- and z,4,+trichlorophenol, and 2,3,4,5- and 2,3,5,6-tetrachlorophenol:These behave practically identically over the whole pEf range and cannot ,be~distingui&ed~m the system used,” ‘, : In contrast to the basic system (stationary olive, oil nnd,mobile buffer), in’which J.. CkYomatog.,

zg (1965)

572-579

pEJ[-CJZROMATOGRAPHY OF’CHLORINATEDPHENOLS

575

the chlorophenol spots are regular in shape, the fixed buffer systems give irregular and elongated spots. DISCUSSION Relationships between the chromatographic behaviour and the pH of the mobile phase were derived mathematically by SOCZEWI~SKLAND WAKSMUNDSICI~-lo. They arrived at the following equations for the Rp and RM values: .<

ar ----

RF =

(1)

--A-

and RM

= log

mlTK;

CHJ-I

-

log

“Y

where a is the partition coefficient of .the undissociated acid, Y the ratio of the areas of the cross sections of both phases in paper, and KA the dissociation constant. These equations are graphically represented in Fig. 5. It can be seen from Figs. r-4. that the m,easured relations between RF and pH of the mobile phase differ from the theoretical values mainly in the region where pH > @ pH$ (the pH corresponding to the inflexion point of the curve), and the individual compounds could not be separated chromatographically under these conditions. One of the assumptions from which the equation was derived is a complete lack of solubility of the anion in the organic phase; the substances would have to migrate with the front of the mobile phase. However, the behaviour of chlorinated phenols does not agree with this prediction (Figs. r-4). The deviations can probably be explained by an affinity of the phenolate anion for the stationary phase or its carboxyl group. This assumption is supported by the effect of steric hindrance. On substituting.the ortko-positions of phenol with chlorine, the mobility decreases as follows OH

OH

OH

‘yt= 1-3 irrespective of the position of ,other substituents. Th.e same relative mobilities were succinate as the stationary phase. Then the :,,,,obtained by using polyethyleneglycol &P values dropped to about one-half of the expected values. With kerosene as the stationary phase this effect was not observed.

,*’

J. CAvomtzZog.,

ig (1965)

572-57g+

ZVACEK,

596

Z. ;+OTA, J. STAN&C:

0.9

RF0.0

0.9 RF

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.X ,

4

5

6

7

8

9 LO l.L 12pH

Fig. 5. Graphical representation of cqns. (I) and (2). (I)plcn = 7, NY = 1; (III) pICn = 7, QY = x0-“; (IV) pI
IO;

(II) PK~

=

7, W

Fig. 6. Graphical representation of eqns. (3) and (4). (I)pl<,,= 7, w = 10-1, ~3 = 102; (II) plc,, 7, W = 10-1, /g = 10; (III) plCn = 7,cW = IO-1,p = 1; (IV) pl
L

--e--5

7

8

=

=

9

Fig.’ 7. Relationship between the Rw values of some !%lorophenols and the pH of ‘the mobile phase. (I) pentachlorophenol; (II) 2,3,t%richlorophenol; (III) 3,4,3+ichlorophenol; (IV) 2,3,4trichlorop+xxol.

.I. Chromalog.,

19

(1965)

spz-57g

577

pH-CHROMATOGRAPEEY OP. CRLORLNATED PHENOLS

This explanation was confirmed also in cases where buffer-impregnated papers were used with esters as the mobile phase. In the region of pII >.$%A the 2,6-dichloro-derivatives are slowest while compounds with both ortho-positions free move with the so1ven.t front. With kerosene this effect again is not apparent., The discussed affinity, of the phenolate anion for the organic’ phase has been expressed quantitatively. In order to calculate the. new function characterizing the relationship between Rp values and the pH of the mobile phase, we used the partition coefficient of the undissociated acid,

[HA] mob --[HAI stat as awe11 as the distribution B =

[A-l -w

stat

By substituting

it follows

for the anion,

mob

CA-1

~~=----

function

in the expression

for apparent

partition

coefficient

WA1 nq +----CA-h WAIorg -I- CA-lore

that

1-n +---w+1 K = a/!3 aKn + PIP’-1 For very high values of @, i.e. when the anion possesses a minute organic phase, a& will be much smaller than @[H+] and the fraction

af%nity for the simplifies to

which is actually the original expression of GOLUMBIC AND ORCHLN~~ as used SOCZEWL&SKL AND WAKSMUNDSKL in their calculations*. Using the well-known formulae for the partition coefficients Rp and RM, KY

RF=--

KY +

and RM = -

log Kr

I

where Y is the ratio between the areas of the cross-sections following expressions are derived : RM=

106 (a%

+

PW”I) -

log (Kn + [Elf]) -

of the two

log a/%

I

RF

=

by

phases,

the

:

(3) (4)

---

~KA + PW+l --I + o$r(Kn + IF+11 J. Clwomatog.,

19 (W-55) 572-579

2. VACEK,

578

2. %OTA,

J. STAN&K

..

Fig. 6 shows the graphical representation of these expressions, It'appears that various anions can be separatediftheydifferin the size of the distribution function& It can also be seen that the dissociated and undissociated forms of the electrolyte being separated will differin'theirmobilities only when the partition coefficientsof the two forms differ,i.e.a,+ fi.In other words, the chromatographic behaviour of undissociated acids is defined ,by the partition coefficientsa; dissociated acids are resolved, according to the distribution function /3. In contrast to existing views (eqn. 2, Fig. 5) the RM = f (pH) function is also sigmoid in character (Figs. 6 and 7). Its value does not decrease with increasing pH without limit but stops at a value

equal to -log log (GV

@. The distance between the two parallel parts of the curve is given by

l

In agreement with the results of analyzing eqn. 3 the $$
phenols will lie near the breaks in the curves constructed for each compound from the measured values. Only in a few cases is the break in the curve shifted with respect to the corresponding p& toward the alkaline region: for 3,5-dichlorophenol by 2.5, for 3,qdichlorophenol by 2, for pentachlorophenol by 1.5, and for z,3,5,6-tetrachlorophenol by I pl3 unit. It follows from eqns. (3) and (4) that the PHI value is shifted with respect to the p& value.toward the a.lkaline region depending on the magnitude of CQJ (ref. 26). With increasing lipophilic character (i.e. with decreasing ar) this difference increases. Comparison of pHc values with p&l values shows that this shift increases roughly in proportion to the number of chlorine atoms in the molecule. This is in agreement with the observation that the RF value decreases in this order (with increasing lipophilic character of undissociated chlorophenols) . ACKNOWLEDGEMENTS

The authors are indebted to Mr. 0. SCHIESSL for help with the preparation and application of the compounds used and to Dr. J. DRAI-IO~~OVSKS for valuable comments and discussion of problems. SUMMARY

The chromatographic separation of all isomeric chlorophenols is described and relationships between chromatographic behaviour ,and structure are discussed. A new equation for the relation between’the RF value of the organic electrolyteand the pH value of the buffered phase has been derived on the assumption dissociated form of this electrolyteto the stationary phase. REFERENCES

of affinityof the

1

6o’(Ig55) H. G. 'BRAY,~. M. CRADDOCIC AND W.V.TNORPE, Biocltcm.J., H.S. CWOGUILL AND D. F. BISSLNG, Anal. Chcm., 32 (1960) 440. 3 I<.SCSL~GL AND A..SIEGEL, Mona&k. CAem.. 84 (1953)686.

I

225.

2

4 E.GRLBENOVSK~,Z.

A+zaZ. Chem., 185 (1962) 2go.

'5 S, HUDJZ~XIC,Ckem. Lisly,4g (rg33) 60. 6 7 8 g

V. RETINA, Nature, 182 (1938) 796. A.~AKSMUND~KL AND E. SOCZEWIRSKI,N&~Y~, 184 (Igsg) 977. E. SOCZEWI~~SICIAND A. WAKSMUNDSKI, BUZZ. Acad. Polon. Sci., SW, Sci. Cl&n., g (1961) 445. E. SOCZEWI~~SKI, Some Genera2 Problems of Pafier Ckromatopap78y, Czech. Acad. Sci.,Prague, 1962, pq 57.

J. Chvomalog.; Ig (1965)572-57g

PH-CHROMATOGRAPHY

OF CHLORLNATED

PRENOLS,

579

zo E. SOCZEWI~+SKL, Rocznihi Chsm., 37 (x963) 467. 32 E. H. J~~~~TREss, Tlce .Z%$avaliora, &opt&es, ClsamiGaZBehavior and Idelztification of Organic Chlorine Compounds. Tables of Data on Sslectsd Compounds of Order III, Wiley, New Stork,1948, :3: 255 (I)." ’ ‘. 12 E. H. HTJNTRE~S, ibid., 3: x&o (2)(7). r3 E. H. HuNTREss,~~~~., 3: ~460 (2). ~4 E. H. HUNT~SS, $bid., 3: 2x85 (4). r5 E. H. HUNTRESS, ibid., 3: x.340 (x). 16 E. H. HUNTRESS, ibid., 3: 1160 (3). z7 E. H. HUNTRESS, ibid., 3: 2885 (I). 18 E. H. HUNTRBSS, ibid., 3: 3523 (x). rg E. H. HUNTRBSS, ibid., 3: 1687 (r). ao E. H. HUNTRZSS, ibid., 3: 3460 (z). 21 C. GOLUMBICANDM.ORCXCN, J.Am.Chem.Soc., 72 (xg5o)4x45; AnaZ.Ckem.,23 (1951)1210. 22 2. S%OTA. AND 0. SCBZISSL, Czech.Pat., 105281 (x962). 23 M.L~~~#,O.SC~IESSL,T.ZAWORSK~ ANDV.MAR~EKOVA, Sb.Prdci Vysh. K?st.Agvockem. Tech., 2 (x96x) ZOL. 24’K. Bz~cz1~,Analyst,'88 (tg63)622.' 25 J. D~~ao~ovs~ir,unpublishecl resulti.. 26 E, SOCZEWI&SKL AND A. WAECSMUNDSKL, Ann. Univ. Maviaa C&e-Skloddwsha, Lzlblin-Polonia,

Sect. AA; 1.4 (1961) ~17. .

J. Chromatog., xg (1965) 572-579