studies in the relationship between molecular structure and chromatographic behaviour—III

Talmta.

1966. Vol.

STUDIES

13. pp. 1281 to 1288.

Pcqamon

Prcas Ltd.

Printed

in Northun

Irehnd

IN THE RELATIONSHIP BETWEEN MOLECULAR STRUCTURE AND CHROMATOGRAPHIC BEHAVIOUR-III

MECHANISM

OF THE REVERSED-PHASE CHROMATOGRAPHY OF SOME ALKYL PHENOLS L. S. BARK and R. J. T. GRAHAM

Department

of Chemistry, Royal College of Advanced Technology, Salford 5, Lancashire.

(Received 9 December, 1965. Accepted 6 April, 1966) Summary-It can be shown that thin-layer chromatography by the reversed-phase technique gives results which are suthciently reproducible for correlation of chromatographic behaviour with chemical structure to be attempted. To a first approximation the Martin additivity principle is valid, but variations in the intermolecular forces between solute molecules and solvent molecules, caused by the easy transmission of electronic effects through an aromatic system, result in deviations from ideal conditions.

THE factors determining the chromatographic behaviour of a compound are its fundamental molecular properties and the extra-molecular factors of the system in In any study of chromatographic behaviour and which it is chromatographed. structure it is necessary that these latter factors be studied so that any variation in the chromatographic behaviour of a series of compounds, caused by these factors, can be attributed to them and not be confused with variations caused by molecular changes in the series. Of the extra-molecular factors which are of major importance in thin-layer chromatography (TLC), the nature of the layer is the most important. When adsorption chromatography is being considered, the composition and nature of the layer is controlled largely by the activation processes employed, and the treatment of the layer during loading. l When reversed-phase chromatography (RPC) or liquid-liquid partition chromatography is being considered, then the preparation of the layer is important. In RPC on paper, the paper is dipped into the impregnation mixture consisting of a volatile solvent and the relatively non-volatile liquid which is to serve as the stationary liquid phase; the volatile solvent is then allowed to evaporate. Green et aLz have stated that in order to obtain R, values of sufficient reproducibility for comparative studies, the papers must be kept horizontal and simultaneously flattened by pressure, and the solvent allowed to evaporate only from the exposed edges of the paper. In this way directional concentration gradients in the solvent layer are claimed to be avoided. Thin layers prepared for RPC have been prepared by dipping previously prepared layers (on plates) into the impregnation mixture and allowing volatile solvent of the stationary phase to evaporate freely. The authors indicate that the whole operation must be done slowly and at room temperature in order to avoid crumbly or uneven layers3 Purdy et al4 and Wagner et al6 have prepared plates and impregnated them by capillary action, the thin layer itself acting as a wick. Impregnation of a prepared layer by spraying it with the non-volatile reverse-phase has also been reported.6 In the investigation of the effect of structural factors on the chromatographic 1281 6

-0.50 -0.18 -0.24 -0.24 +0.21 +0.08 +0.11

0.76 060 0.63, 0.635 0.38 0.455 044

-0.59 -0.22 -0.32 -0.29 +0.18 + 0.027 -to.079

0.79, 0*626 0.676 0.66 0.40 O*4g1 0.45,

0.69 0.45 0.52 0.52 0.25, 0.34, 0.33,

RF

2.0

-0.35 + 0.08 -0.04 -0.04 + 0.47 $028 +0.30

RI -0.18 $033 +0.18 + 0.20 +0.66 +0.47 to.50

060

0.32 0.40 0.39 0.18 0.255 0.24

RP

3.0 RF

Stationary phase obtained from ethyl oleate dissolved in diethyl ether. Mobile phase ethanol: water (1:3 v/v). + RH values are calculated in accordance with the equation of Bate-Smith and Westall.

Phenol 2-Methylphenol 3-Methylphenol bMethylpheno1 2-Ethylphenol 3-Ethylphenol CEthylphenol

%I

RF

RY

I.0

RF

0.75 RF

R&l

-0.114 0.389 0.213 0.241 0.704 0.550 0.616

4.0

PHASE

0.56, 0.29 0.38 0.36, 0.16, 0.22 0.19,

OF STATIONARY

of ethyl oleate, %

VALUES‘WITHVARIATION

Original concentration

TABLEI.-VARLAI?ONS IN RF AND RI*

0.50 0.29 0.32 0.32 0.15 0.20 0.19

RF

5.0

-to*39 +0.33 $0.33 +0.75 t-O.60 $0.62

0.00

RX

1283

Relationship between molecular structure and chromatographlc bohavlour-III

behaviour of a series of homologues, it is essential to obtain RF values which are reproducible to within fine limits. The methods described above do not give such values, and it was thus felt that further explorations of technique were necessary. We have investigated the effect of incorporating the reverse-phase solvent in the liquid employed in the preparation of the slurry used to coat the plates. EXPERIMENTAL Solutions of freshly distilled ethyl olaate in diethyl ether (70 ml of various concentrations) were slurrled with cellulose powder (15 g of Camlab M.N.300* sieved to pass a B.S. 200-mesh sieve). The resulting slurries were coated on to clean glass plates (20 x 20 cm) with a commercial spreading apparatus (Shandon scientific Co. Ltd.). The plates were air-dried (15 min at 25”). Solutions of chromatographically pure phenols (0.25 % v/v in cyclohexane) were spotted on to a plate with the spotting device provlously described. 1 The loaded plates were placed in a double saturation chamber’ and the chromatograms developed with various mixtures of super-dry ethanol’ (10,25,30, and 37.5 %) and deionized water. After development of the chromatograms for a standard time (that necessary to give a run of 14 f 0.5 cm; cu. 2) hr) the plates were removed from the saturation chamber, allowed to partly dry (10 min at 25”) and were then sprayed with a (1 + 1) mixture of aqueous potassium permanganate (0.5% w/v) and aqueous sodium carbonate (O-375‘Aw/v anhydrous sodium carbonate). The phenols were made visible aa yellow spots on a pale purple background. Summary of the results

Each result for a particular compound is the mean of at least 4 results varying by not more than 0.01 Rx units from the arithmetic mean. The result for phenol ia the mean of approximately 100 results varying by not more than 0.01 Rx units from the arithmetic moan. The results are given in Tables I and II. TABLE

II.-VARIATIONS

IN

Rx

AND

OF ETHYL

Rn

VALUES

ALCOHOL

IN

WITH THE

VARIATION

MOBILE

IN

THE

CONCENTRATION

PHASE

Composition of mobile phase, % v/v ethanol 10

Compound RF

Phenol 2-Methylphenol 3-Methylphenol CMethylphenol 2-Ethylphenol 3-Ethylphenol rtEthylpheno1

0.76 0.49, 0.58 0.56 0.30 0.39 0.37

25 RY

RF

-0.50 +0*01 -0.14 -0.11 +0.37 + 0.20 +0*22

0.79 0.62 0.67 0.66 040 0.48 0.45

30 hi

-0.59 -0.22 -0.32 -0.29 +0*18 +0.03 $0.08

RF

0.83 0.68 0.72 0.71 0.49 0.58 0.56

37.5 RP

-0.70 -0.33 -0.42 -040 +0*01 -0.14 -0.11

RF

R?d

090 0.78 083 0.83 0.61 0.70 0.69

-0.99 -0.56 -0.69 -0.69 -0*20 -0.37 -0.35

Stationary phase obtained from 0.75 % v/v ethyl oleate in diethyl ether. DISCUSSION

OF THE RESULTS

The reproducibility of the 100 or so results for phenol shows that the technique of incorporating one of the phases for RPC in the slurry used to coat the plates, does give values acceptable for comparative studies. Comparison of the results obtained for some phenols with those obtained by Green et ~1.~ with an apparently identical phase system for RPC on paper shows marked differences (Table III). To obtain RF values similar to those obtained by Green et al. it is necessary to alter the composition of the phases. Since we consider the mechanism of chromatography of these phenols by RPC to be essentially partition of the phenols between the ethyl oleate and the polar mobile * Camlab Laboratory Supplies, Cambridge, England.

1284

L. S. BARK and R. J. T. GIUHAM

phase, the difference in results obtained by RPC on paper and on thin layers, using the same loading of stationary phase, may be due to the differences in the availability of the phase on the two systems. We suggest that in TLC, almost all the very fine particles of cellulose are coated with a layer of the stationary phase, to give in effect an unbroken layer of stationary phase, whereas if fibrous paper is used much of the TABLE

III RB values

Compound System A*

System At

System Bt

0.92 0.78 0.85 0.85 0.70

0.50 0.29 0.32 0.32 0.19

0.90 O-78 0.83 @83 0.69

Phenol 2-Methylphenol 3-Methylphenol 4-Methylphenol ~Ethylphenol Stationary System A: 5 % ethyl System B: 0.75 % ethyl * Values from Green et t Our values

phase : Mobile phase ; oleate: 25 % v/v ethanol/water; oleate: 375% v/v ethanol water al.@

stationary phase is absorbed into the fibres and is not readily available for use in the fairly rapid chromatographic exchange, which probably takes place across the interface of the mobile and stationary phases. The fact that the TLC layers so treated are more easily removed from the plates than are similar layers which have just been wetted with water by capillary action, may mean that only a small amount of the cellulose is bound to the glass surface by the normally occurring forces. The time taken for the mobile phase to traverse the plate is comparatively long, indicating that most of the cellulose cannot be used for the normal capillary action, which is probably accelerated by hydrogen bonding between the cellulose hydroxyl groups and the water or ethanol. It is probable that the mechanism of the process on paper is one of both adsorption (on to the exposed cellulose) and partition (between the two phases),lblp whereas on thin layers it is probable that the mechanism is more nearly the ideal of continuous and successive liquid-liquid extraction. Thus the results obtained on thin layers should more nearly approach ideality. Martini5 suggested that there existed a relationship between the change in standard chemical potential of a compound and its partition coefficient, such that all isomeric compounds containing the same groups would be expected to have the same partition coefficient between the same pair of phases, provided that the degree of ionisation etc. is unchanged. He further suggested that the addition of a particular substituent to a molecule would alter the partition coefficient of that molecule by a fixed amount, depending only on the nature of the substituent and the phases used. Let us consider a reversed-phase chromatograp~c system of two liquid phases in equilibrium, the two phases being designated by the subscripts S for the stationary phase and L for the mobile phase. Let each phase be a binary solution, the components for the stationary phase being the solution of the phenol in the non-volatile liquid used in the preparation of the thin layer, and the components for the mobile phase

Relationshipbetweenmolecularstructure and chromatographicbehaviour--III

1285

being the solute (phenol) and the liquid eluent system. Then according to the conditions of heterogeneous equilibrium, derived from thermodynamics, we have: sP1 =

(1)

LPl

where ,u~is the chemical potential of component 1 (the solute). For an ideal solution (ignoring the interaction between the phenol and the aqueous ethanol) by definition ,ul = ~1” + RflnN, (2) where N1 is the mole fraction of the solute of the solute, (Le., the chemical potential state usually taken to be an infinitely dilute becomes equal to its mole fraction). Then

and rlo is the standard chemical potential of the solute in some arbitrarily defined solution in which the activity of the solute substituting equation (2) into (1) gives:

s,ulo + RT In sN, = @I0 + RT In LN1 or

sP1O- L~lo = RTln LN,/,NJ. Since

(3)

API” = sccl” - LPI’,

equation (3) becomes

A,u10= RT In (LN1/sNd ApI = RTln u

or

(4)

where u is the partition coefficient of component 1 between the mobile and stationary phases. Consden, Gordon and Martini* note that the relation of the partition coefficient to the R, value is Cc= where A, = thickness of the mobile phase and A, = thickness of the stationary phase. If we consider any particular pair of phases and assume uniform distribution of a particular phase within the common boundaries of the two phases, then AL/A, may be assumed to be constant. Thus for two solutes (1 and 2) differing only by one methylene group we have from equations (4) and (5) Ap:=RTln($(&--1))

(6)

and Aruzo= RTln($(&--

1)).

Martin15 makes the assumption that A/J,’ = API” + A,&,,

(8)

i.e., Ap,O remains unaltered by introduction of a methylene group into the molecule; if this be so, subtraction of equation (6) from (7) gives -&&B

= RTln (k-1)

-RTln(-&-

1)

1286

L. S. BARK and R. J. T. GRAHAM

or - A,ulO,uz= constant.

A In 1 (i&

(9)

4).

Since by definition RM = log ((1 then introducing

this into equation

RJR&

(9) we obtain:

-A&,

= 2.303RT AR,.

If Martin’s assumptiorP is valid, then there should be the same change in RM between phenol and each of the isomeric mono-methyl phenols, and between the mono-methyl and the mono-ethyl phenols. This ARM should also remain constant for changes in the composition of the liquids used for the stationary and mobile phases, since the partition coefficients of the phenols are dependent only on the changes in chemical potential. TABLE IV

Partition coefficient, 1/k [Phenol in water] Compound

[Phenol in cyclohexane]

Dissociation constant, p& at 28°C

Phenol 2-Methylphenol 3-Methylphenol 4-Methylphenol 2-Ethylphenol 3-Ethylphenol 4-Ethylphenol

5.26 0.75 1.43 1.25 0.14 0.37, 0.36

9.98* 10.28* 10.087 10.19* 10.20: 9.90: 100l::

* G. R. Sprengling and C. W. Lewis, J. Am. Chem. Sot. 1953, 75, 5709. t F. G. Bordwell and G. D. Cooper, ibid. 1952,74,1058. $ Reference 17.

Using a similar reversed-phase system, Green et al9 found that AR, was reasonably constant for phenols not containing a group orrho to the phenolic group; alkyl groups ortho to the phenolic group have a different value. Since they considered only one system (5 % ethyl oleate/25 % aqueous ethanol) only one value obtained from Table I in the 2-position they may be compared with theirs. For phenols not substituted obtained a ARM value of 0.29,; for the similar system with the phenols they used and some others, we also obtained a AR, value of 0.29. It must however be noted that the RF values obtained by these workers differ from ours (see Table III). For the two systems giving the same RF values for the same phenols, the AR, values are: ours O-31,, Green et al. O-29,. This difference in AR, corresponds to RF differences of less than O-01 RF units at RF values 0.80 and O-20, and less than O-015 Rrr units at REYvalues about 0.50. Even with rigorously standardised conditions we accept RF values differing by 0.01, so we consider that these differences in ARM are not significant. However, we consider that the variations in the ARM values shown in Tables I and II indicate that there are variations in the chemical potentials of the various isomers in the various systems. The most probable cause of the variations is the deviation of the solutions

Relationshipbetweenmolecularstructure and chromatographicbehaviour-III

1287

from idea&y, which may be brought about by various factors, among which is the alteration in the amount of dissociation or ionisation of the phenol in either or both of the solvent phases. This will alter the partition coefficient. A comparison of the results obtained by Golumbic, Orchin and Weller,l’ from partition of the phenols between cyclohexane and water (Table IV), and the RF values obtained (Table I) shows that there is a close parallelism between the Rs. values and the partition coefficients even though the systems are different. The ionisation constant of the phenols in most cases varies within small limits, so that differences in extractability are seldom due to differences in acid strength. The mechanism of the dissolution of the polar phenols in the two phases is governed by the intermolecular forces operating. These include polar contributions from dipoles and induced dipoles, dispersion forces and hydrogen bonding. When these forces exist between solute and solvent they may be considered as acting as solvation forces, the amount of the solvation by a particular solvent being a measure of the dissolution of the solute by that solvent. The dielectric constant of ethyl oleate is 3.17 units at 25”, of water 78-54 units, and of ethanol 24.3 units .l* It is probable that even in a 37.5 % v/v ethanol/water mixture the dielectric constant will be 50-60 units. Thus whilst ethyl oleate may be regarded as a polar substance, the dissolution of the phenol in the mobile phase will be governed mainly by the solvation of the phenol through hydrogen bonds formed between this phase and the hydrophilic phenolic group. The authors consider that solubility factors are important in determining R,; the greater is the RF value of a compound then the greater is the dissolution in the mobile phase. We do not suggest however that the increase in the RF of a particular compound with increase in the amount of ethanol in the mobile phase is due in any way to any deterioration in the amount or strength of the hydrogen bonding between the phenol and water. Working with dioxan and phenols dissolved in cyclohexane, Bala and SuzukP have shown that only very small amounts of the polar phase (dioxan) are required to give complete hydrogen bonding with phenols at concentrations similar to those used here. We suggest that the increase in the Rr of any particular phenol with increase in the alcohol content of the mobile phase is caused by increased solubility in the mobile phase. For a given composition of the mobile phase, the decreased polarity caused by alkyl substitution in the phenol results in an increase in the solubility in the stationary phase and a decrease in Rr. The relatively larger effect of the substituent in the 2-position may be due to an electronic effect on the phenolic group. This ‘ortho effect’ has been previously reported for phenols9~17*20 and though with some substituents in the 2-position internal hydrogen bonding occurs, in the case of the Zalkyl phenols the evidence for hydrogen bonding is not conclusive. Studies of molecular models of such phenolP indicate that there is probably only small steric inhibition of rotation of the unsolvated phenolic group by a Zmethyl group, and hydrogen bonding is not probable. The transmission of the electron donor properties of the methyl group to the phenolic group, through the +electron system of the nucleus, probably accounts for the decrease in the polarity of the oxygen-hydrogen bond in the phenolic group. This would result in a decrease in the amount of solvation of the phenol and hence a lower solubility. Infraredzl and NMR22 studies also suggest that this or& effect is caused by electronic transmission between the substituent and the functional groups. This

1288

L. S. BARK and R. J. T. GRAHAM

results in a further deviation from ideality and would indicate that in systems where the electronic effects are more pronounced the deviations would be still greater. The substitution of groups having marked electron attracting or releasing properties should have a very noticeable effect. Such systems are under investigation and will be reported later. Zusamme&saur@-Es kann gezeigt werden, da0 die Diinnschichtchromatographie mit umgekehrten Phasen Resultate zeitigt, die geniigend reproduzierbar sind, urn den Versuch zu rechtfertigen, Beziehungen zwischen chromatographischem Verhalten und chemischer Struktur aufzusuchen. In erster N&herung ist das Martin’sche Additivitgtsprinzip giiltig, aber hderungen dir intermolekularen Krtifte zwischen den Molekiilen von Liisungsmittel und Gel&tern. die durch die leichte Fortleitung elektronischey Effekte durch ein arbmatisches System verursacht werden, ergeben Abweichungen von den idealen Bedmgungen. R&urn-n peut montrer que la chromatographie en couches minces par la technique des phases inver&s donne des rksultats sfiamment reproductibles pour que soit tend un rapprochement entre le comportement chromatographique et la structure. En premibre approximation, le principe d’additivitt de Martin est valable, mais des variations dans les forces intermolt?culaires entre les mol&ules de solutC et celles de solvant, caudes par une transmission ais& des effets tlectroniques ii travers un systhme aromatique, ont pour consequence des dCviations par rapport aux conditions id&ales.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

REFERENCES L. S. Bark, R. J. T. Graham and D. McCormick, Tufuntu, 1965, 12, 122. J. Green and S. Marcinkiewicz, J. Chromatog., 1963, 10, 35. H. K. Mangold and D. C. Malins, J. Am. Oil Chemists Sot., 1960,37, 576. S. J. Purdy and E. V. Truter, Analyst, 1962, 87, 802. H. Wagner, L. Horhammer and B. Dengler, J. Chromatog., 1962, 7,211. J. Vaedtke and A. Gajewska, ibid., 1962, 9, 301. A. I. Vogel, A Textbook of Practical Organic Chemistry, 3rd. Ed., p. 168. Longmans, London, 1956. E. C. Bate-Smith and R. J. Westall, Biochem. Biophys. Actu, 1950, 4,42. J. Green, S. Marcinkiewicz and D. McHale, J. Chromatog., 1963, 10,42. S. Marcinkiewicz and J. Green, ibid., 1963, 10, 372. L. S. Bark and R. J. T. Graham, Analyst, 1960,85,663. Idem, ibid., 1960, 85, 904. I&m, ibid., 1960, 85,907. J. Green, S. Marcinkiewicz and D. McHale, J. Chromatog., 1963, 10, 158. A. J. P. Martin, Biochem. Sot. Symposia, 1950,3,4. R. Consden, A. H. Gordon and A. J. P. Martin, Biochem. J. 1944, 38, 224. G. Gohunbic, M. Orchin and S. Weller, J. Am. Chem. Sot., 1949, 71, 2624. National Bureau of Standards, Washington, Circular 514. H. Bala and S. Suzuki, J. Chem. Phys., 1961,35,118. L. S. Bark and R. J. T. Graham, Tufunta, 1964,11, 839. A. W. Baker and W. W. Kaeding, J. Am. Chem. Sot., 1959,81,5904. I. Granacher, Helv. Phys. Actu, 1961,34,272.