THE JOURNAL OF BIOLOGICAL CHEVI~TRI Vol. 238, No. 3, March 1963

Printed in U.S.A.

The Biosynthesis of Chlorogenic Acid and Related Conjugates of the Hydroxycinnamic Acids*

CHROMATOGRAPHIC SEPARATION AND CHARACTERIZATION

KENNETH R. HANSON AND MILTON ZUCKER

From the Departments of Biochemistry and of Plant Pathology and Botany, The Station, New Haven, Connecticut

(Received for publication, August 2, 1962)

Connecticut Agricultural Experiment

The widespread occurrence in plant tissues of conjugates with quinic acid1 and with glucose of p-coumaric acid., caffeic acid, and ferulic acid (Fig. 1) is well established (2-6). Many of these compounds occur as only trace components in extremely com- plex mixtures of phenolic substances. Uncertainty exists as to the exact chemical structure of most of them, and relatively little is known about their biosynthesis.

The primary concern of the present investigation is with the biosynthesis of the most familiar of these conjugates, chlorogenic acid, the structure of which was established as 3-0-caffeoylquinic acid by Fischer and Dangschat in 1932 (7,8). It has been shown previously (9) that this compound, along with possible biosyn- thetic intermediates, accumulates in potato tuber tissue when disks of the pulp are cultured on a variety of substrates, viz. glucose, quinic acid, phenylalanine, and cinnamic acid. How- ever, the paper chromatographic methods employed in that work were not well suited to the purification and estimation of the minute quantities of the compounds related to chlorogenic acid that were encountered. This paper describes a procedure for the separation of conjugates of the above type by partition chromatography on silica gel. Sondheimer has shown (5) that chlorogenic acid can be separated in this way from its isomers and from caffeic acid by the procedure of Bulen, Varner, and Burrell (10). The present system differs from theirs principally in the use of a linear solvent gradient rather than of a stepwise elution regime, and is well suited to the quantitative analysis of complex mixtures of conjugates.

With the aid of the analytical silica gel column, it has been possible to study the mixtures of conjugates present in the potato tuber as harvested or in slices of pulp tissue cultured on a solution containing n-phenylalanine and quinic acid. The bear- ing of these findings on chlorogenic acid biosynthesis is discussed. Among other things, the results establish that the potato tuber is capable of synthesizing a far greater variety of phenolic con- jugates than has generally been realized. Evidence has been found for 17 or 18 such compounds, among which are previously unknown conjugates of shikimic acid.

* Support of part of this work by the National Science Founda- tion is gratefully acknowledged.

1 The quinic, shikimic, and chlorogenic acids frequently are represented as the mirror images of their known structures. The chemical evidence establishing the absolute configurations shown in Fig. 1, has been reviewed by one of us elsewhere (1).

EXPERIMENTAL PROCEDIJRE

Apparatus for Column Chromatography-A device for producing a linear gradient (ll), consisting of a reservoir and a mixing vessel of identical shape (cylindrical) and capacity (1 liter), was constructed with a syphon of l-mm glass capillary tubing between the two vessels; a similar tube led from the mixing vessel to the chromatography column. A Teflon-coated mag- netic stirring bar was used in the mixing cylinder, and neoprene stoppers were used to close the reservoir and mixing vessel, since these came into contact only with the solvent vapors. Effluent from the tapered capillary tip of a chromatography tube, 30 X 0.8 cm, was conducted through medical grade poly- ethylene catheter tubing, 0.034 to 0.060 inch (Clay-Adams, Inc.), to the light cell of an ultraviolet light absorption meter (Gilson Medical Electronics), and thence to a drop-counting fraction collector. The absorption meter, which recorded percentage transmission, was fitted with a dual interference filter having a peak transmittance of 280 rnp.

Reagents for ilnalytical Column-Dehydrated silica gel was prepared from commercial “silicic acid for chromatography” (Mallinckrodt) as described by Bulen, Varner, and Burrell (10). The batch of gel employed for most of the experiments was suitably hydrated when the powder had taken up one-half its weight of 0.5 N sulfuric acid.

The solvent mixtures for elution were prepared from cy- clohexane (practical grade), t-butyl alcohol (m.p. 24-25”), and chloroform (reagent grade). The last solvent was washed three times with water before use. The cyclohexane-chloroform mixture (10% by volume) was equilibrated with 0.5 N sulfuric acid, and the t-butyl alcohol-chloroform mixtures (30 and 40 y0 by volume) were equilibrated with 0.25 N sulfuric acid, viz. 800 ml of the 30% mixture with 15 ml of acid, and 300 ml of the 40% mixture with 10 ml of acid.

Analytical Column (Standard Conditions)-A column of silica gel dispersed in cyclohexane-chloroform (25% by volume) was prepared from 7 g of dried gel and 3.5 ml of 0.5 N sulfuric acid, and the gel was packed under 4 lb in2 pressure of air to a final length of approximately 17 cm.

The sample of partially purified plant extract or other ma- terial to be applied to the column was concentrated nearly to dryness with a rotary evaporator (bath temperature, 30”). Portions of dried gel were added to the sample, and the mixture

1105

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1106 Conjugates of Hydroxycinnamic Acids Vol. 238, No. 3

6H Quinic Acid

Shikimic Acid

was ground together until a freely flowing powder was obtained. This stage was reached when approximately 0.5 g of gel had been added. If the sample had not been acidified previously, the gel was partially hydrated with 0.5 N sulfuric acid (0.2 ml to 1 g) before use. The powder was then suspended in cyclohexane- chloroform (25% by volume) and transferred to the column as described by Bulen, Varner, and Burrell (10).

The column was developed overnight at a flow rate of ap- proximately 75 ml per hour (2 to 4 lb in-2 pressure). The mix- ing vessel of the gradient device initially contained 800 ml of cyclohexane-chloroform (10 $& by volume), and the reservoir, the same volume of t-butyl alcohol-chloroform (30 To by volume). These conditions produce an increase of 1.87 ml of t-butyl al- cohol for each successive 100 ml of solvent delivered 60 the col- umn. The form of the gradient generated was examined by performing a blank run in which dye was present in the reservoir solution. During development, the excess water separating in the mixing vessel collected on the walls or remained at the surface and did not pass to the column. The fractions corresponding to the various peaks of the continuously recorded percentage transmission curve were combined, and the total number of optical density units2 associated with each peak was determined. The pooled fractions were stored at - 10” for future examination.

Examination of Potato Tuber Tissue-The cortex of the tuber is defined as the layer of tissue (5 to 8 mm) between the vascuIar cylinder and the skin. The tissue within the vascular cylinder is termed pulp (12).

Cultured pulp tissue: Slices 1 mm thick were cut from tubers (Solarium tuberosum L. ‘Kennebec’), and sets of washed slices (100 g each) were cultured in the dark at 25” on 25 ml of a solu- tion containing n-phenylalanine (0.05 M) and quinic acid (0.05 M)

adjusted to pH 6.5 with potassium hydroxide. The total period of exposure of the tissue to daylight before culturing was ap- proximately 20 minutes. At the end of the culture period (16 or 40 hours), the slices were washed with water and extracted twice with 95% ethanol (500 + 300 ml). After the absorption spec- trum of the filtered extract (840 ml; alcohol concentration greater than 80 %) had been determined, the solution was divided into two equal parts. One portion, after being concentrated, was stored at -10” for future examination; the other was con-

2 An optical density of 1 (l-cm light path, wave length of maximal absorption unless otherwise specified) is observed for a solution with a concentration of 1 optical density unit per ml. The number of units present in a solution is numerically equal to the product of the optical density and the volume (milliliters).

centrated until a sticky brown gum was obtained. When such extracts were examined with the analytical column, a multitude of false peaks was observed in the effluent record. A preliminary separation of the phenolic components, therefore, was performed according to the method of Whiting (13).

A short column, 10 X 0.8 cm, was prepared from 4 g of dried gel. The sample was added to the column in association with 4 g of dried gel, and the column was developed with t-butyl alcohol- chloroform (40% by volume) at the rate of 100 ml per hour until the optical density (l-cm light path) of the effluent fell to ap- proximately 0.3 at 280 mp (about 150 ml). A sample containing approximately 800 optical density units at 320 rnp was con- centrated and applied to the analytical column.

Untreated tissue: Pulp tissue, 500 g, was extracted twice with 95% ethanoI (1800 + 900 ml), and the extract was concentrated to a stiff yellow-brown gum. Dried gel, approximately 7 g, and 0.5 N sulfuric acid, 1 ml, were added to the gum and the resulting material was treated as described above, except that a larger preliminary gel column was used (2-cm diameter, 8 g of dried gel). The effluent volume was approximately 400 ml. The same procedure was used for the cortical tissue, and for slices of pulp tissue that had been maintained on water alone for 16 hours (water control sample).

Detection and I&&cation of Components

Paper Chromatography-Separations were performed by ascending chromatography on sheets of Whatman No. 1 paper. In general, 1 to 2 optical density units of material were applied to the chromatograms, i.e. 0.05 to 0.1 pmole. No period of equilibration was employed.

The solvent systems used were: System A, n-butyl alcohol- glacial acetic acid-water (4 : 1: 5 by volume, upper organic phase) ; System B, acetic acid (5%) ; System C, benzene-acetic acid- water (6 :7 : 3, upper phase) (14) ; System D, n-butyl alcohol- ammonium carbonate solution (7.6% = 0.8 M)-ammonium hy- droxide solution (1: 10 dilution of concentrated reagent = 1.5 N) (2: 1: 1, upper phase employed for irrigation, aqueous phase placed in an open cylinder in the center of the chromatography jar) ; System E, n-butyl alcohol-88% formic acid-water (4: 1:5, upper phase; several hours are required for the two phases to separate).

Alkaline and Enzymatic Hydrolysis-Hydrolyses were per- formed essentially as described previously (9), except that anthocyanase B (Rohm and Haas) was used in addition to pectinase (Nutritiona BiochemicaIs). Anthocyanase B ex- hibited a strong ,&glucosidase activity toward salicin.

Qualitative IdentiJicationsThe hydroxycinnamic acids and their conjugates were detected and characterized by their absorption spectra in acidified and alkaline ethanol solution and, on paper chromatograms, by their RF values, their fluorescence under ultraviolet light, and their reaction in the Hoepfner test

(15). The carbohydrate, i.e. nonphenolic, moieties from the various

ester fractions were detected and characterized as follows: (a) Quinic and shikimic acids were detected on chromatograms as described by Cartwright and Roberts (16). (b) Shikimic acid was also detected by spraying the chromatogram with 1 y0 aniline hydrocholoride in ethanol, drying the paper in air, and then spraying with potassium periodate (0.2 %)-potassium hydroxide (0.05 N) solution. Shikimic acid gave a bright red coloration almost immediately, whereas quinic acid did not react or, if

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March 1963 K. R. Hanson and M. Zucker

present in high concentration, gave a red color after a period of 20 minutes or more (17). (c) Glucose and other reducing sugars were detected by the aniline-oxalate spray described by Zim- merman (18) and Partridge (19). (d) Glucose was detected enzymatically with glucose oxidase by the method of Saloman and Johnson (20).

Stoichiometry of Hydroxycinnamic Acid ConjugatesIn deter- mining the ratio of hydroxycinnamic acid to carbohydrate in the several esters, a molar extinction coefficient of 20,000 for the phenolic moiety was assumed. Quinic acid was determined by the periodate-thiobarbituric acid method described previously (9), except that before assay the alkaline hydrolysate was added to a column (0.8 X 2.5 cm) of Dowex 2 (200 to 400 mesh) in the acetate form and the quinic acid was eluted with 6 N acetic acid (10 ml). Shikimic acid was determined by the periodate- thiobarbituric acid method of Saslaw and Waravdekar (21).

Ionic Charge-Electrophoretic mobilities were determined in 0.02 M phosphate buffer, pH 7, on Whatman No. 3MM paper with a potential gradient of 10 volts per cm. Compounds with approximately the mobility of chlorogenic acid were ranked as having a charge of - 1.

Enzymatic Hydroxylations-An enzyme fraction with a high polyphenoloxidase activity was obtained by preparing an acetone powder from an extract of potato tuber cortical tissue and treating the powder with ice water. Enzymatic hydroxylations were performed as described previously (9), except that the pH of the reaction was 5 and ascorbic acid was added at the end of incubation.

Reference Compounds

3-O-p-Coumaroylquinic acid, isolated from apples (22), was the gift of Dr. A. H. Williams, Long Ashton Research Station, University of Bristol, Bristol, England; neochlorogenic acid and “band 510” substance, isolated from green coffee beans (23), were the gift of Dr. E. Sondheimer, Department of Biochemistry, State University College of Forestry, Syracuse University, Syracuse, New York; Hauschild’s substance, isolated from treated leaves of Ilex paraguariensis St. Hl. (24), was the gift of Dr. V. Deulofeu, Facultad de Ciencias Exactas y Naturales, Laboratorio de Quimica Organica, Buenos Aires, Argentina. Chlorogenic acid and partially purified isochlorogenic acid were obtained from Mann Research Laboratories, Inc.

RESULTS

Analytical Silica Gel Column: R,, Value-The ability of the analytical column to resolve complex mixtures of phenolic and nonphenolic acids is shown in Fig. 3. Recoveries of applied reference compounds were essentially quantitative (+3%), and under standard conditions the maximal observed variation in the peak effluent volume3 (650 ml) for chlorogenic acid was ~20 ml.

The effects of varying the solvent gradient and the length of the column are shown in Fig. 2. The properties of the system are such that the changes in the peak effluent volume of a given compound produced by unintentional variations in the length of the column, or in the gradient, are minimized. The ratio of the peak effluent volumes for the compounds examined was inde- pendent of the applied gradient and the length of the column

3 The “peak effluent volume” is “that volume of effluent col- lected while a given compound moves from the top of the column to the bottom and is measured at. the point at which the greatest concentration of the compound is eluted” (25).

EFFLUENT VOLUMES STANDARD RUN

FIG. 2. Variation of peak effluent volumes with change in col- umn length and solvent gradient for various components of cul- tured potato tuber pulp tissue. Components are numbered as in Table II and Fig. 3B. For a standard run, 7 g of dried silica gel were used; the gradient was 1.87 ml of t-butyl alcohol per 100 ml of solvent delivered (as in Fig. 3). Curve A, 7 g of gel, gradient one-half of standard (0.94 ml per 100 ml); B, 11 g of gel, standard gradient; C, 4 g of gel, standard gradient; D, 7 g of gel, gradient twice standard (3.75 ml per 100 ml).

within the rage of conditions investigated. It follows that a given compound. may be characterized by the ratio of its peak effluent volume to that of chlorogenic acid. The term R,, is employed to designate this ratio. Although solvents from different sources and silica gel purchased at different times were employed, R,, values remained constant.

Increase in the gradient sharpened the peaks, but some loss in resolution was noted. The gradient chosen for the standard run represents a compromise between the need to obtain sufficient resolution and the need to avoid excessive dilution of minor components present in extracts of plant tissues.

Separation and Properties of Reference Compounds-The effluent peaks for several hydroxycinnamic acid conjugates ob- tained either commercially or through the generosity of other investigators are recorded in Fig. 3A, and various properties of these conjugates are listed in Table I. When commercial isochlorogenic acid was examined, three major components, termed a, b, and c, were found in addition to a small amount of chlorogenic acid and a number of minor ultraviolet light-absorb- ing impurities. On subsequent chromatography of the three peak substances, no changes in peak effluent volumes were ob- served. Paper chromatography of the hydrolysates of all three components gave no evidence for the presence of glucose.

Repeated chromatography on the analytical column of chloro- genie acid in amounts varying from barely detectable to greater than the useful capacity of the column failed to show that cis and trans isomers could be resolved by the system. The other reference caffeoyl esters similarly gave rise to only one peak. When, on the other hand, small quantities of 3-O-p-coumaroyl- quinic acid were applied to the column, two peaks, R,, 0.69 and 0.76, were obtained. Chromatography of either the first or the second peak gave rise to the same double peak pattern.

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Conjugates of Hydrmycinnamic Acids Vol. 238, No. 3

OX0 ;

ii ; 40 it

.- 5 Ly

Shi i i 9 n i : :-t*

] : ; :

j . . ...’ i. -._.. ,’ f . . ...“” . . . . “” . . . . -

, * -. .-. , . . .

1 I I 6 ‘EFFLUENT VOLUME (ml.) ’ 500 ’ I I I Il.000 ’ I I I I I

FIG. 3. Separation of compounds on the analytical silica gel eluted at the termination of the standard run. The dashed lin column under standard run conditions. Optical densities were was constructed from titration values (fraction volumes of 10 ml measured in a cell of l-cm light path. A, Mixtures of reference (10). B, Phenolic components of potato tuber pulp tissue cul- compounds (composite record): Gin, cinnamic acid; Fer, ferulic tured for 16 hours on n-phenylalanine and quinic acid; each ali- acid; Cou, p-coumaric acid; Cuf, caffeic acid; Fum, fumaric acid; quot was equivalent to 50 g of tissue. Components are numbered Wi, 3-0-p-coumaroylquinic acid; Ha, Hauschild’s substance; SW, as listed in Table II. The dashed line records the optical density succinic acid; I,, Ia, I,, components of commercial isochlorogenic at 257 rnp and serves to indicate that Components 7, 8, and 12 are acid ; Chl, 3-0-caffeoylquinic acid (chlorogenic acid) ; Son, “band p-coumaroyl esters. C, Phenolic components of uncultured pulp 510” substance; Neo, neochlorogenic acid; Oxa, oxalic acid; Shi, tissue ; 500 g of tissue were examined. Components are numbered shikimic acid. The quinic acid and citric acid had not been as listed in Table III.

At all times the leading peak was the larger, but the relative

amounts of substance in the two peaks varied. It seems probable that equilibration between the cis and trans forms of the caffeoyl esters takes place on the column during chromatography, but that the two forms of the p-coumaroyl ester are sufficiently stable to be separated partially. A similar phenomenon is observed on paper chromatography of the hydroxycinnamic acids and their conjugates. These compounds give rise to double spots in dilute acetic acid (26, 27) and in formic acid- sodium formate systems (14). The ease of cis-bans intercon- version increases in the order: cinnamic (28), p-coumaric, and caffeic acids (26).

The various reference compounds were stable under the acidic conditions employed for chromatography. When the fractions

20

IO

corresponding to them were concentrated and again chromato- graphed, no hydrolysis to caffeic acid or p-coumaric acid was detected, and the only isomeriaation observed was that of the p- coumaroyl ester. Samples of neochlorogenic acid and “band 510” substance that had been chromatographed and stored at -10” in acidic chloroform solution for 2 months were subjected

to electrophoresis in pH 7 buffer. No material of zero charge was detected, which indicated that lactone formation had not taken place.

Characterization of Tissue Components-Typical elution records for extracts of tissue cultured for 16 hours on n-phenylalanine and quinic acid, and of untreated pulp tissue are shown in Fig. 3, B and C. Various properties of the ultraviolet light-absorbing

compounds of the cultured pulp (16 and 40 hours) are listed in

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March 1963 K. B. Ha&on and M, Zucker 1109

TABLE I

Properties of reference compounds - -

1

Absorption spectra” Molar ratio of quinic acid to caffeic acid

Ionic charge at pH 7 RF in System B Reference compound

3-0-Caffeoylquinic acid, i.e. chlorogenic acid ..................................

Neochlorogenic acid .................... “Band 510” substance. ................. Hauschild’ssubstance .................. Isochlorogenic acid a. .................. Isochlorogenic acid b. .................. Isochlorogenic acid c ................... 3.0-p-Coumaroylquinic acid ............

% RF in System 1

Amin I I

hl.sx hll%i (alkali)

265 328 52 265 328 50 265 328 49 265 328 50 265 328 70 265 328 70 265 328 70 250 315 50

0.58, 0.70 1.0 0.61, 0.69 1.2 0.58, 0.70 0.8

0.9 1.0 1.1

-1 -1 -1

0 -lb --I* - 1”

-1

1 0.60 1.56 0.55 1.22 0.63 0.71 0.80 0.86 0.70 0.94 0.71 1.10 0.66

0.69, 0.76 0.78

0.19,0.29 0.20,0.29 0.30,0.41 0.70, (0.80)

Q The absorption spectra exhibited prominent shoulders at 300 mp. * Moved more slowly than chlorogenic acid on electrophoresis.

TABLE II

Phenolic components of potato tuber pulp tissue cultured on L-phenylalanine and quinic acid: fractions eluted from analytical silica gel column

-

e- Absorption spectraa Tissue contentb

ho time: 16 hours: 40 hours: maximal

I I

minimal minimal VdUeS V&ES WlUeS

m~~moles,lg fresh lirsue

Peak (see Fig. 3R)

Probable identity, or products of hydrolysis

Carotenoids Ferulic acid p-Coumaric acid Unknown Caff eic acid (Caffeic acid, quinic acid)d 0-p-Coumaroylshikimic acid 3-0-p-Coumaroylquinic acid (Caffeic acid, shikimic acid) 3-O-Caffeoylquinic acid, i.e. chloro-

genie acid (Ferulic acid, quinic acid) 0-p-Coumaroyl glucose 0-Glucosylcaffeic acid Unknown (Caffeic acid, glucose, fructose)

onic chargs atpH7

-1 -1

0 -1

0 -1 -1 -1 -1

0 0

-1

0, -1

Amin ALllitX (alkali)

%l 2

-_

265 260 260 265 265 260 260 265 265

mr E

(294)c 318 310 312

(296) 323 325 315 315 328 328

20 12 24 27 49 50 50 48 52

265 328 46 260 318 56 265 322 21

265 328 41

1 0.01 2 0.09 3 0.23 4 0.33 5 0.42 6 0.52 7 0.56 8 0.67 9 0.78

10 1.00

11 1.09 12 1.28 13 1.49 14 1.64 15 1.81

Trace Trace 132 159

21 21 113 67

15 42 53 61 16 36 55 50

127 200

11 27 77 23 27 40

Trace Trace 7 Trace

- - -

>lO 20

- - -

40

- - - - -

0.9 1.2 0.7 1.5 0.9

- a The absorption spectra of peak Components 3, 4, 6 to 13, and 15 exhibit.ed prominent shoulders at 300 mp b Minimal values were calculated from the absorption spectra of the pooled peak fractions. The true concentrations, corrected on

the basis of uniform losses, are 1.6 times the recorded values. Maximal values (untreated pulp tissue) are the values of Table III corrected for an estimated loss of 75%. The dashes signify that the component was present in too low a concentration to detect.

c Major (leading) peak: Amax, 380, 410, 430, 452, and 480 rnp; minor (following) peak: A,,,, 385, 415, and 435 rnp. d Parentheses indicate products of hydrolysis.

Table II. Since the phenolic components of untreated cortical tissue closely resembled those present in untreated pulp and in control slices of pulp maintained for 16 hours on water, their properties are listed together in Table III.

The RF values for the principle components of cultured pulp tissue are presented diagrammatically in Fig. 4. Single spots were obtained for Components 2 to 12 when systems containing a nonmiscible organic solvent were employed (A, C, and D). Only when the chromatogram was heavily loaded and the sample was applied as a streak to the paper could traces of other fluores- cent materials be detected. Therefore, within the limits of the

resolving power of silica gel and paper chromatography, the effluent Peaks 2 to 12 were each held to correspond to one principal phenolic compound.

The RF values for the various conjugated peak components present in untreated tissue are listed in Table III. Component j gave rise to a spot (RF 0.44) in addition to the two expected in System B.

Components with properties which remained unchanged after being subjected to hydrolytic conditions (alkaline and enzymatic) were assumed to be unconjugated. The ultraviolet absorption properties, fluorescence, and the Hoepfner reactions of the

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1110 Conjugates of Hydroxycinnamic Acids

TABLE III

Vol. 238, No. 3

Phenolic components of potato tuber pulp and cortical tissue: fractions eluted from analytical silica gel column - -

-

) -

-

1

-

Probable identity or products of hydrolysis

-

N

c

--

Mar ratio of quinic acid to

:affeic acid

Ionic :har e

If; tP

-

I -

_-

Tissue content: minimal valuesb

Peak (see Fig. 3Cj Pp in System B

Pulp Cortex

10

.- fw n

a 0.01 Carotenoids a b+c 0.41,0.43 0.79 0.34, 0.63, Caffeic acid + conjugate: 265 325 53 4

0.71 (caffeic acid, quinic acid)c d 0.48 0.70,0.78 (Caffeic acid, quinic acid) 0.8 265 325 52 2 e 0.55 0.78 0.64, 0.71 (Caffeic acid, quinic acid) 1.0 265 325 50 5

identical with Peak 6 Table II

f 0.73 0.76 0.64, 0.75 (Caffeic acid, quinic acid) 1 g 1.00 0.60 0.58,0.70 3-O-Caffeoylquinic acid, i.e. 0.9 265 328 49 10

chlorogenic acid h 1.22 0.65 0.56, 0.69 (Caffeic acid, quinic acid), 1.2 265 328 59 1

probably “band 510” sub- stance

i 1.37 Unknown Trace

j 1.52 0.55 0.44, 0.60, (Caffeic acid, quinic acid), 265 328 Trace 0.69 probably neochlorogenic

acid k 1.84 Unknown 260, 328 Trace 1 2.12 Unknown Trace

a Carotenoid maxima as for Table II. b Minimal values were calculated from the absorption spectra of the pooled peak fractions. The true concentrations, corrected on

the basis of uniform losses, are approximately 3 to 4 times these values. c Parentheses indicate products of hydrolysis.

0

-1

-1

7

3 50

6

4 3

Trace Trace

The evidence for the presence of shikimic acid, quinic acid, and glucose in the hydrolysates is summarized in Table V. When sufficient material was available, the ratio of phenolic acid to quinic or shikimic acid was determined by carrying out the periodate-thiobarbituric acid reaction under the appropriate experimental conditions. In all of the quinic acid conjugates, including the reference compounds, the ratio approximated 1: 1. Component 9 gave a ratio of caffeic to shikimic acid of 1:1.5.

TABLE IV

Properties of free hydroxycinnamic acids and their conjugates on paper chromatograms D

1 - I Hoepfner test Ultraviolet fluorescenceb

1 Standards and components”

0. 00

7

+ NHa vapor H%?&f

None

Orange

Red

Orange

Light yellow

Ned& and

Quench- ing

Blue- white

Blue- white

Faint blue

Blue

HNOt

None

Red

Yellow

Yellow

Light yellow

.-

-

Bright blue

Blue- green

Blue- green

Blue

Bright blue

1 p-Coumaric acid and its esters

e Caffeic acid 2 3 1 5 6 7 8 9 lo 11 12 a 15

FRACTIONS Conjugates of caf- FIG. 4. Paper chromatography: representation of RF values feic acid

for the phenolic components of potato tuber pulp tissue cultured Peak 13 on n-phenylalanine and quinic acid. The components are num- bered as listed in Table II. System A, n-butyl alcohol-acetic acid- water; B, 5% acetic acid; C, benzene-acetic acid-water; D, n-butyl

Ferulic acid and

alcohol-ammonium carbonate-ammonium hydroxide. Peak 11

conjugates and of the phenolic moieties released upon hydrolysis Q Conjugates are as listed in Tables I, II, and III. 6 Colors were observed with an ultraviolet lamp having a

were as indicated in Table IV. Although the material associated maximal output of 366 mp. Variation in the quality of color was

with Peak 15 appeared to contain at least two compounds, observed according to the amount of compound applied to the

caffeic acid was the only phenolic acid detected on hydrolysis. chromatogram: normally 1 to 2 optical density units were applied.

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March 1963 1111 K. R. Hanson and M. Zucker

TABLE V

Properties of carbohydrate products from hydrolysis of conjugates

Components 7 through 11, (b + c) to h, and j were hydrolyzed both enzymatically (pectinase) and with alkali. The sugar moieties released from Components 12, 13, and 15 were detected after the conjugates had been hydrolyzed by anthocyanase B. Component 13 was stable to alkaline but hot’to acid hydrolysis.

Standards and components hydrolyzed RF in System A

Shikimic acid.. Peaks 7 and 9”. Quinic acid.. Peaks 8, 10, and 11. Peaks (b + c) to h and j . Glucose.................... Peaks 12, 13, and 15*. .

0.40 0.40

0.28 (0.13) 0.28 (0.13) 0.28 (0.13)

0.17 0.17

RF in System

E

0.48 0.48 0.36 0.36

I-

_-

-

Periodate and thiobarbituric acid assays

(9) (21)

Yellow Yellow Red Red Red Colorless Colorless

Blue Blue Colorless

Colorless

Chromatographic spray reagents

Periodate, nitroprusside, Gperazine (16)

Yellow Yellow Yellow Yellow Yellow Colorless Colorless

Aniline, periodate

Red Red Colorless Colorless Colorless

Aniline, oxalate (18, 19)

Brown Brown

a On hydrolysis, Component 9 gave rise to a trace of quinic acid so that an orange, rather than a yellow, periodate-thiobarbituric acid reaction was observed.

b A second sugar, which slowlv formed a red color with the aniline-oxalate spray, was also detected in the hydrolysate of Peak 15 material. Its %F value (0.24) was similar to that of fructose.

Standards were not available for comparison, and it is possible, therefore, that the correct ratio for Component 9 (and also Component 7) is 1: 1.

I f it is assumed that the various conjugates are composed of the identified hydroxycinnamic acid and carbohydrate moieties only, then certain conclusions may be reached about the mode of linkage between the two portions. Thus Components 7, 8, and 12 all show a bathochromic shift of their absorption maxima in alkali (Ah,,, (alkali)) of about 50 mp, and give rise to p-cou- maric acid on hydrolysis; these findings indicate that the phenolic hydroxyl group is unsubstituted and that the compounds are p-coumaroyl esters. 3-0-p-Coumaroylquinic acid (Table I) exhibits a similar displacement. Similarly, Component 11 shows a shift of 46 rnp and gives rise to ferulic acid on hydrolysis, which indicates that this compound is a feruloyl ester. Since Conjugates 7 and 8 have ionic charges of - 1 at pH 7, the carboxyl groups of quinic acid and shikimic acid must be free in these compounds. The zero charge of Component 12 is in agreement with the proposed 0-p-coumaroylglucose structure. The natural occurrence of I-O-p-coumaroylglucose in the genus Xolanum has been reported by Harborne and Corner (2) (alkali shift of 52 mp). Since the feruloyl ester 11 carries a zero charge at pH 7 and gives rise to quinic acid on hydrolysis, this compound must be an ester of quinide (the 1 ,3-lactone of quinic acid), if it is true that only two components are present in the conjugate.

Of the various caffeic acid conjugates, Component 13 is un- usual in that it shows a bathochromic shift of 21 rnp instead of 50 mp. Since the other moiety is glucose, and since the com- pound carries a charge of -1 at pH 7 and, moreover, is hy- drolyzed by acid and anthocyanase but not by alkali, this com- pound presumably is a glucoside of caffeic acid. The magnitude of the alkali displacement, but not the position of the absorption maximum, corresponds to the 3-0-P-glucoside reported by Harborne and Corner to be present in berries of a member of the genus Solarium (2). An alkali shift of 43 rnp is reported for the 4-0-@-glucoside by these authors.

Since the various other conjugates give rise to caffeic acid on hydrolysis and show alkali shifts of the order of 50 mp, at least one hydroxyl group is unsubstituted. Compound 6, apparently

identical with Compound e, is not ionized at pH 7 and could therefore be an 0-caffeoyl ester of quinide. It differs from Hauschild’s substance in its R,, value, but resembles it in other respects. Compounds h and j have properties resembling the reference compounds, “band 510” substance and neochlorogenic acid, but the amount of material available for comparison was small. To establish that these compounds are indeed caffeoyl esters, methylation studies would be necessary.

The properties of the compounds identified as 3-O-p-cou- maroylquinic acid, Peak 8, and chlorogenic acid, Peaks 10 and g, were examined with particular care. The former was chro- matographed repeatedly on the analytical column. As with the reference compound, chromatography of the leading peak gave rise to two peaks. The component in Peaks 10 and g resembled chlorogenic acid in all respects.

In agreement with the behavior of 3-0-p-coumaroylquinic acid, it was found that the p-coumaroyl esters 7 and 12 on chromatography exhibited double peaks. In each case further chromatography of the leading peak gave rise to two peaks.

Enzymatic Hydroxylations-When a sample of the reference compound, 3-0-p-coumaroylquinic acid, was oxidized with an enzyme fraction from the potato cortex and the products of the reaction were separated on the analytical silica gel column, only three peaks were detected. The first two corresponded to the starting material both in their peak effluent volumes and in

absorption spectra. The third peak exhibited an R,, value of 0.98 (the first p-coumaroyl ester peak R,, value being used as a reference point) and an absorption spectrum corresponding to chlorogenic acid, and had all of the properties of that compound on paper chromatography. The chlorogenic acid peak repre- sented approximately 10% of the recovered material, but only about 3% of the ester taken.’ Component 8, believed to be identical with the above reference compound, gave rise on oxida- tion to a compound indistinguishable by paper chromatography from chlorogenic acid.

Component 7, 0-p-coumaroylshikimic acid, gave rise to three peaks under similar oxidation conditions. The last of these peaks, R,, 0.75, corresponded to Component 9 (caffeoylshikimic acid) and was indistinguishable from it on paper chromatography.

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1112 Conjugates of Hydroxycinnamic Acids Vol. 238, No. 3

Analysis of Tissue Extracts: Quantitative Aspects--Various losses of phenolic materials were encountered in the analysis of tissue extracts, although the preliminary column and analytical column steps were reproducible (spread less than 3% for the former and less than 10% for each of the various peaks in the latter). The recovery of material from the preliminary column was of the order of 70%, and from the analytical column 80% for the cultured pulp tissue and 40% for the cortex and the untreated pulp (measured at 320 mp). Part of these losses are accounted for as material remaining on the preliminary column, but the major loss arose from the oxidation and polymerization that took place when the acidic eluate from the preliminary column was concentrated. The amount of hydrolysis of the components believed to contain glycosidic linkages was probably small. Although these losses were considerable, they were no greater than are normally suffered in paper chromatography. Some alternative preliminary step is clearly desirable.

Effects of Culture on Phenolic Cwnposition of the Tuber-The only hydroxycinnamic acid conjugates found in the untreated tuber, whether outer cortical or inner pulp tissue, were those containing caffeic and quinic acids (Table III). In the cortex, which was much richer in phenolic substances than the pulp, chlorogenic acid accounted for 60 ‘% of the phenolic content. Its concentration was estimated to be 0.2 pmole per g of fresh weight of cortical tissue (maximal value; 0.1 y0 of the dry weight), which is 5 times greater than that in the pulp. These values agreee well with estimates based on direct paper chromatographic examination of t,uber extracts (29). In comparison, the maxi- mal concentration of chlorogenic acid is appreciably less than that of phenylalanine reported for potato tuber (var. Sebago, 0.84 pmole per g) and very much less than the reported concen- trations of asparagine, glutamine, and citric acid (30, 31). Caf- feic acid occurs in tuber tissue in about one-tenth of the concen- tration of chlorogenic acid. Its presence has been report,ed (29). Peak substance e was the second most abundant compo- nent detected in untreated tissue. The presence of this sub- stance has not been reported previously.

When slices of pulp tissue were kept moistened with distilled water for 16 hours in the dark (water control sample), an in- crease of slightly more than 2-fold was observed in the total content of phenolic substances as estimated from the absorption maximum at 326 rnp of the alcoholic extract. Analyses revealed, however, that no phenolic components other than those found in the untreated pulp were present, and that the relative pro- portions were similar.

I f the pulp slices were cultured on L-phenylalanine and quinic acid instead of water, major qualitative and quantitative changes occurred in the composition of the phenolic fraction (Table II). A 16-hour period of culture produced an increase of more than IO-fold in the absorption at the maxima of crude alcoholic ex- tracts (319 mp). Of the components found in untreated pulp and water control slices, only caffeic acid, peak substance e (Peak 6), and chlorogenic acid were readily detected in cultured tissue.

The most striking qualitative changes produced were the accumulation of free p-coumaric acid and its esters with shikimic acid (Peak 7), quinic acid (Peak 8)) and glucose (Peak 12). Neither free p-coumaric acid nor any of its conjugates were detected either in the water control or in tissue before culture, even though up to 10 times the quantity of untreated tissue was

processed. Consequently, the natural steady state concentra- tions of these phenols must be one or more orders of magnitude less than the levels attained in cultured tissue, and their ac- cumulation represents a specific response to the exogenously supplied phenylalanine and quinic acid.

The concentrations of the various components of cultured tissue recorded must be considered to be the difference between the amount synthesized and the amount further metabolized per g of fresh tissue. Oxidation of phenolic material causes the tissue to turn light brown on culture. Preliminary experi- ments indicate that the nonhydrolyzable polymers formed may be as much as, or several times greater than, the amount of phenolic material present in the cultured tissue.

DISCUSSION

Silica Gel Column-The separations of complex mixtures of hydroxycinnamic acid conjugates described above demonstrate the exceptional resolving power of the silica gel partition chro- matography system. Only a single caffeic acid-quinic acid conjugate, chlorogenic acid, had been detected previously in potato tubers by means of two-dimensional paper chromatog- raphy (29), whereas column analysis has indicated that as many as seven such conjugates are present. Although countercurrent distribution has been used to separate mixtures of these com- pounds (32-34), the column method possesses the advantage that a short column may be equivalent in its resolving power to a large countercurrent train (35). Fully automatic analytical systems employing silica gel chromatography have been described recently (36, 37).

The fact that the ratio of the peak effluent volume for a given substance to that of chlorogenic acid (& value) is independent of the variables of the system (applied gradient, column di- mensions, gel hydration) is of service in the identification of compounds. Although the R,, relationship is not simply related to the RF relationship (see “Appendix”), it possesses all of the practical usefulness of that ratio.

Reference CompoundsThe identification of the various conjugates present in the potato rests, in part, on a knowledge of the similar compounds isolated from other sources and studied by the classical methods of organic chemistry. The available information concerning the reference compounds employed may be summarized as follows: Chlorogenic acid (3-O-caffeoylquinic acid) has been investigated extensively (7, 8) and recently has been synthesized (38). 3-0-p-Coumaroylquinic acid has been synthesized, and the synthetic material shown to be identical by direct comparison with the compound isolated by Williams (39). I-O-Caffeoylquinic acid has been synthesized (40,41), and its properties are reported to be identical with those of neochloro- genie acid (41). The properties of “band 510” substance re- semble those of chlorogenic acid and neochlorogenic acid in many respects, and it has been suggested, therefore, that this compound is an 0-caffeoylquinic acid (23).

Isochlorogenic acid, isolated from coffee, is also considered to be an 0-caffeoylquinic acid and, as such, would represent the one remaining position isom’er (32, 23). Although the molecular weight by the cryoscopic method (32) is in agreement with the above assignment, the minimal molecular weight found by titra- tion was almost twice that of chlorogenic acid (32, 23). The present finding (Table I and Fig. 3A) of three closely related components in commercial isochlorogenic acid raises further problems. One of the peaks termed Z,, IJ,, or I, (Fig. 3.4) may

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correspond to pseudochlorogenic acid, a compound resembling isochlorogenic acid and found in black rot-infected sweet potato (33). Clearly all of these compounds cannot be simple position isomers of chlorogenic acid. If the cryoscopic molecular weight determination for the isochlorogenic acid first isolated were in error, then the compounds in question may be dimers in which two members of the chlorogenic acid series are joined by ester linkages. A fourth position isomer of chlorogenic acid would then remain to be discovered.

Evidence has been presented (24) that Hauschild’s substance is a caffeic acid ester of quinide (the 1,3-la&one of quinic acid); there are three possible position isomers of this type. In ethanol, the isolated material exhibited the rotation [(Y]. +49” as com- pared with [LY]~~ -17” reported for I-O-caffeoylquinide syn- thesized from natural quinic acid (40). As indicated above, peak substance 6 (equivalent to e) may be an 0-caffeoylquinide. In addition to these compounds, 1,4-0-dicaffeoylquinic acid (cinarine) has been isolated from artichokes and has been syn- thesized (42).

Metabolism---The findings of the present study complement the investigations of the biosynthesis of chlorogenic acid pre- viously reported from this laboratory (9). It was postulated that phenylalanine is first converted to cinnamic acid and that the formation of chlorogenic acid proceeds through the stages, 3-0-cinnamoylquinic acid and 3-0-p-coumaroylquinic acid.

The identification of the p-coumaroylquinic acid ester in tissue cultured on n-phenylalanine and quinic acid by comparison with a sample of the 3-isomer confirms the previous finding. The former inability to obtain consistent ratios of quinic to p-coumaric is now accounted for. Since paper chromatography does not satisfactorily resolve the quinic ester from O-p-coumaroylshi- kimic acid and 0-p-coumaroylglucose (Peaks 7 and 12), the samples of the ester obtained undoubtedly were contaminated with the last two compounds.

The enzymatic hydroxylation of 3-0-p-coumaroylquinic acid to chlorogenic acid by a polyphenoloxidase preparation from the potato tuber is now clearly demonstrated. The small amount of substrate available precluded any attempt to dis- cover whether this “cresolase” type of activity was separable from the activity responsible for the further oxidation of chloro- genie acid. Mason (43) has proposed that the polyphenol- oxidases function solely as cresolases in vivo. If this hypothesis is correct, then the normal metabolic role of these enzymes would be in the biosynthesis rather than the destruction of polyphenols. A number of such “cresolase”-type enzymes, showing various specificities under physiological conditions, recently have been found in mushrooms (44).

No cinnamoyl esters were detected in extracts of cultured tissue by the analytical silica gel column. However, the cin- namoyl ester isolated in the previous investigation and the recently detected cinnamoylglucose ester (2) were present in very small concentrations, and the failure to observe these compounds on direct analysis is not surprising. The chemical synthesis of compounds in the 0-cinnamoylquinic acid series and their behavior on the analytical column will be reported later.

The first stage in the proposed pathway, the direct conversion of n-phenylalanine into trans-cinnamic acid, is in all probability catalyzed by the enzyme, phenylalanine deaminase. This enzyme has been detected in a number of tissues and purified from barley stems (28). The reaction is irreversible.

The occurrence of the previously unknown conjugates of

p-coumaric acid and caffeic acid with shikimic acid is of interest. Weinstein, Porter, and Laurencot (45) have shown that quinic acid is readily converted into shikimic acid in a number of tissues, and it seems probable that the accumulation of these compounds represents a response of the tissue to abnormally high levels of shikimic acid. The possibility that direct transformation of quinic to shikimic conjugates may take place cannot be elimi- nated, however. Similarly, the accumulation of conjugates of glucose not normally detected in tuber tissue may result from the prior accumulation of unusually high quantities of p-cou- maric and caffeic acids. The formation of such conjugates in plant tissues cultured with hydroxycinnamic acids has been studied by Harborne and Corner (2). The free hydroxycinnamic acids under the same conditions do not promote the accumulation of the corresponding quinic acid conjugates (2, 9).

Although the concentration of phenolic substances was greater in cortical tissue and in slices of pulp tissue maintained on water than in untreated pulp tissue, the same conjugates of caffeic and quinic acids were present in each case. The difference in the availability of oxygen to the tissues has been suggested as a factor responsible for these quantitative alterations (46). The changes in metabolism known to occur in tissue slices (47) ap- parently have no qualitative effects on phenolic biosynthesis. Culturing pulp tissue with n-phenylalanine and quinic acid not only produces much larger quantitative effects, but results in the accumulation of six major components, including conjugates of p-coumaric acid, not observed in the original tissue. Thus, synthesis of phenolic substances in the tuber under normal conditions of growth may be limited by lack of substrates as well as by lack of oxygen.

Racusen and Aronoff (48) have demonstrated that synthesis of phenylalanine from Cl402 in soybean leaves takes place only in the light. If, in the potato plant, synthesis were also limited to tissue exposed to light, then leaves, as compared with the tuber, might be expected to have a more generous supply of phenylalanine. It is of interest that leaves contain very high levels of chlorogenic acid (49) and that conjugates of p-coumaric acid have been detected in extracts of this tissue (2).

SUMMARY

1. A system for partition chromatography on silica gel has been developed for the quantitative separation of complex mixtures of phenolic conjugates (compounds related to chloro- genie acid; S-0-caffeoylquinic acid). The method employs a linear solvent gradient of 10 % cyclohexane-chloroform (by volume) to 30% t-butyl alcohol-chloroform (by volume). Its high resolving power is demonstrated by the separation of at least eight conjugates of caffeic acid and quinic acid.

2. The ratio of the peak effluent volumes for any two com- pounds resolved is independent of the applied gradient and of the length of the column over the range of conditions studied. The various phenolic compounds investigated, therefore, have been characterized in terms of their effluent volume ratios relative to chlorogenic acid (R,, values).

3. The analytical method, coupled to a preliminary short silica gel column fractionation step, has been applied to potato tuber cortex and pulp tissue, to pulp tissue slices that had been maintained on water, and to slices cultured on a solution con- taining r,-phenylalanine and quinic acid. The 18 resolved com- ponents were characterized by paper chromatography, electro-

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1114 Conjugates of Hydroxycinnamic Acids Vol. 238, No. 3

phoresis, their ultraviolet absorption and fluorescence, and chemical and enzymatic methods.

4. Culturing the tissue stimulated phenolic biosynthesis greatly and resulted in the accumulation of 3-O-p-coumaroyl- quinic acid, previously unknown conjugates of p-coumaric and caffeic acid with shikimic acid, and also conjugates of these hydroxycinnamic acids with glucose. Chlorogenic acid, the major phenolic component, and a previously unknown conjugate of caffeic acid and quinic acid bearing a neutral charge at pH 7 were present in both cultured and untreated tissue.

5. The role of phenylalanine in phenolic biosynthesis in this tissue and of 3-0-p-coumaroylquinic acid in the biosynthesis of chlorogenic acid is discussed.

AcknowledgmentsAppreciation is expressed to Dr. H. B. Vickery for his aid in the preparation of this manuscript, and to Mrs. Gladys Trepanier for her able technical assistance.

APPENDIX

Concerning R,, and Rp Relationships

It is shown in Fig. 2 that, over a significantly broad range of conditions, the ratio of the peak effluent volume for a given compound to that of chlorogenic acid (or some other internal standard) is independent of the applied gradient and the length of the column. The fact that such a simple relationship is observed requires comment.

Certain of the factors determining the peak effluent volume of a compound may be discussed with the aid of Fig. 5. In this representation, the cross-sectional areas of the column, length I, is the sum of the cross-sectional areas of the mobile solvent phase, A,, and the stationary gel phase, A,. At the start of development, the developer front and the solute are present at the origin. The situation is shown in which the average solute molecule, after some period of development, has migrated through the distance x and the developer front through the distance x + y. The composition of the eluting solvent at the solute maxi- mum M is thus a function of the volume of solute v (= Amy) that has passed the maximum. Since the partition coefficient between gel and solvent, defined as the concentration in the stationary phase divided by the concentration in the mobile phase, is also a function of this composition, it is designated cy(,). The distribution of material at the cross section M is thus (cq,,A,)/A, = t&/&t,, where 6t and 6t, are the average times spent by the average molecules in the stationary and mobile phases, respectively, in the time interval 6t, + at,. When the

0 h4 D + * *

* PARTITION COLUMN * EFFLUENT +

FIG. 5. Idealized representation of a partition chromatography column. A, and A,, cross-sectional areas of the mobile and stationary phases; 0, origin; M, solute maximum; D, developing solvent front. For convenience the developing solvent that has passed through the column is considered to flow down a tube of cross section A,.

molecule is in the mobile phase, it will be carried along with the solvent. I f instantaneous equilibrium is assumed to be reached between the two phases, then it follows (see LeRosen (50)) that in the limit as t approaches zero the average motion of solute relative to the motion of solvent is

dx/dy = dtmldt, = A,/(A,w(,,)

Since A,dy = da, then A,dx = (l/cu~~,) dv, and

(1)

where p8 is the volume of stationary phase in the column and V, the “elution volume,” is the volume of eluent that has passed the peak maximum during the elution process. V is thus less than the “peak effluent volume” by the volume of mobile phase in the column. Provided that a linear adsorption isotherm is involved, Equation 1 may be applied also to a combination of adsorption and partition chromatography. When -no gradient is employed, a(Y) = a = a constant; then V = aVs, in agree- ment with the “theoretical plate” treatment of chromatography (35, 51).

If, for any two compounds a and b separated on the column, the “elution volume” ratio is independent of the length of the column (VJVb = k) then the expressions for l/a(,) for the two compounds are related by the functional equation

l/bcy(“) = kl&oc&) (2)

For the range of conditions investigated, the difference between V and the peak effluent volume is small and may be neglected. Equation 2 thus may be regarded as a corollary of the R,, re- lationship.

The expression for l/al(,) for a given compound must vary with the applied gradient. If, for two compounds a and b at the gradients 1 and 2, the functional Equation 3 applies,

~aar(,)l~~oc(,) = %w,lLe, = c (3)

where C is a function of the gradient but is independent of v, then k( = Va/Vb) will be independent of the applied gradient.

It is clear that many functions of various types will satisfy Equation 2. The following simple expression, Equation 4, has been tested as a first approximation for the system studied, where h depends on the substance and on the gradient and n

is a constant of the system.

Since

l/a<,, = hv” (4)

P, = (h/(n + l))V@+l) (5)

n may be calculated from the changes in peak effluent volume with column length recorded in Fig. 2. The values n = 3.7 and 3.4 found for the increase and decrease of p’, are in reasonable agreement. (It should be noted, however, that when v = 0, l/oc(,) = 0, i.e. the solute. is in the stationary phase entirely, whereas a low, but finite, value for I/LY(~) must in fact occur.) On this estimation, the distance moved by a compound along the length of the column is roughly proportional to the 4.5th power of the effluent volume. For example, when 0.6 of the peak effluent volume has passed, the compound will have moved only 0.1 of the length of the column.

The above discussion provides an account of the separation

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March 1963 K. R. Hanson and M. Zucker

process in terms of the partition coefficients of the compounds 23.

under investigation. A further analysis would require that the

functional Equations 2 and 3 be accounted for in thermodynamic

24

’

terms. The discussion serves to emphasize the differences be- 25.

tween the gradient situation and the no gradient situation (to

which the RF relationship applies). In the latter, the rate of 26.

motion of a compound along the length of the column is uniform 27.

at uniform flow rate, whereas in the former a continuously chang- 28.

ing acceleration occurs. 29.

1. 2.

3.

4. 5. 6. 7.

8.

9. 10.

11. 12.

13.

14.

15.

16.

17.

ii:

20.

21.

22.

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Kenneth R. Hanson and Milton ZuckerCHARACTERIZATION

Hydroxycinnamic Acids: CHROMATOGRAPHIC SEPARATION AND The Biosynthesis of Chlorogenic Acid and Related Conjugates of the

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