Plants (Berl.) 122, 203--207 (1975) 9 by Springer-Verlag 1975

Short Communication

Separation of Cytokinins by High Pressure Liquid Chromatography J a m e s S. Challice Long Ashton Research Station, University of Bristol, Bristol BSI8 9AF, U.K. Received 21 October; accepted 18 November, 1974

Summary. A number of eytokinin reference compounds have been successfully separated by High Pressure Liquid Chromatography using columns of pellicular strong cation exchange resin and of pellieular polyamide. On polyamide, all cytokinins were elated within 10 rain with an aqueous buffer but on the cation exchange resin some cytokinins (generally those with bulky l~8-substituents and in addition lacking an Ng-ribosyl substituent) had excessively long retention times with aqueous buffer eluent. However, addition of methanol to the buffer enabled these cytokinins to be separated and eluted within a reasonable time. As small an amount as 5 nanogram of cytokinin could readily be detected by the procedures described. I n recent years a considerable amount of research activity has been concerned with the nature, distribution and role of cytokinin growth-regulators in plants (recent reviews are b y Skoog and Armstrong, 1970 ; Steward and Krikorian, 1971 ; Hall, t973). The presently accepted criterion for the presence of naturally occurring cytokinins is t h a t of biological activity as measured either by the stimulation of callus-growth or by the more convenient stimulation of betacyanin synthesis in Amaranthus cotyledons and hypocotyls (Biddington and Thomas, 1973). A major drawback of all bioassay procedures is their tediousness, coupled with the fact t h a t the bioassays do not, of course, distinguish between different cytokinins; although different cytokinins give different response-levels in the bioassays mentioned. I t has always been a hope t h a t eventually these bioassay procedures will largely be replaced b y more reliable physico-chemical procedures. The advent of High Pressure Liquid Chromatography (HPLC) now makes this a real possibility. Pool and Powell (1972, 1974) have developed some separations of eytokinins on columns of pellicular strong cation exchange resin (Zipax SCX) using phosphatebuffers of varying p H and ionic strength as eluting solvents; however, their conditions did not allow for the elution of all cytokinins within reasonable times, parines such as N6-benzyladenine and zeatintaking as much as 1 hour or longer to elute from the columns. Experience has shown that for a 1 m • 4 m m H P L C column packed with pellicular material, with an eluent flow rate of 1.5 ml per min at 40 ~ C, substances with retention times much greater than 10 min give peaks which are very much broader t h a n those which are closer to the methanol solvent peak which generally appears between 3.1-3.4 min. Hence for optimum resolution of a complex mixture, the components should be eluted between 3.1-10 min; substances having exees-

204

J.S. Challice 3,7 adenosine

7.1

adenine

8-5 N6-

benzvlad~

retenl:ion t i m e (rains)

atin 3side i.3

l

9 t I I t w 5I 10

solvent peak

1

T

T

l

{ 0

Fig. 1. HPLC separation of purines on a strong cation exchange resin with an aqueous buffer eluent. In this system N~-(A2-isopentenyl) adenosine gave a broad peak at 23.8 rain; zeatin, dihydrozeatin, kinetin, N6-bcnzyladenine and N6-(zie-isopentenyl)adenine had unacceptably long retention times. N~-methyladenosine ran at 4.5 min, kinetin riboside just before zeatin riboside and N~-methyladenine close to N6-(d2-isopcntenyl)adenosine. Operating parameters-column: Zipax SCX (pellieular) 1 metre • 4 mm diam. (glass), eluent: 0.05 hi KH~PO4 (pH4.5) temperature: 40 ~ C, flow rate: 1.5 ml per min, pressure: ~ 500 psi, detector: UV 254 rim, absorbance range: 0-0.01, chart: 1 cm per min, sample loadings: I00 nanograms (200 ng for N~-benzyladenosine), injection vols: 5 F1

sively long retention times will in general not be resolved from each other unless their individual retention times differ greatly from each other. The present investigations were made using the Jobling H P L C modular system with 1 m • 4 m m diam. glass columns (water-jacketed at 40 ~ C, thermostatically controlled), an L D 711 1000 psi variable flow rate reciprocating piston p u m p (constant flow) with pulse-damper, and an L D 1285 high sensitivity U V absorbance detector operating at 254 nm. The eluting solvents were vacuum-degassed at 60 ~ C prior to p u m p i n g t h r o u g h the columns, in order to prevent the formation of air-bubbles in the system. Columns were dry-packed either with D u P o n t Zipax SCX (pellicular strong cation exchange resin) or with Reeve Angel Pellidon (pellieular polyamide). I t was found t h a t b y lagging the region between the b o t t o m of the column and the detector cell, baseline noise at m a x i m u m sensitivity, due to t h e r m a l variation, could be considerably reduced when the system was operated at 40 ~ C; at higher operating temperatures, however, baseline noise proved excessive. Other operating parameters are given with Figs. 1-3, which illustrate some separations which were obtained.

Separation of Cytokinins

205

5,4

7.5

N~-(A~-isopentenu

I

adenosine

kinetir

16-5

r

i

i

retention time(rnins)25 2

I0

i

j

r

15 10

i

i

P

l

~

zeatin

3.2 ;~

I

5

0

Fig. 2. HPLC separation of purines on a strong cation exchange resin with an aqueous buffer/ methanol eluent. In this system Ns-(A~-isopentenyl)adenine gave a broad peak at 46.5 rain; adenosine, zeatin riboside, adenine and N6-benzyladenosine had retention times approximately equal to that of the MeOH solvent peak at 3.2 rain. Other purines ran as follows: NS-methoxypurine (3.4 min), N6-Bis(hydroxyethyl)adenine (3.6 rain), hT6-(hydroxyethyl) adenine (3.8 rain), NS-methyl-adenine (5.1 rain), dihydrozeatin (7.8 min) Operating parameters--column: Zipax SCX (pellicular) 1 metre • 4 mm diam. (glass), eluent: 0.5 M KH2PO4:MeOH (10:1), temperature: 40~ C, flow ra~e: 1.5 ml per min, pressure: ~ 500 psi, detector: UV 254 nm, absorbance range: 0-0.01, chart: 1 cm per rain (10 rains) thereafter 1 cm per 5 mins, sample loadings: 200 nanograms (1O0ng for zeatin), injection vols: 5 ~l

I n practice the actual pressures required in order to generate flow rates of 1.5 ml/min varied with the length of time a particular column had previously been in operation: freshly packed columns under these conditions required pressures as low as 150 psi whilst after continued operation the pressure could rise to a figure as high as 1000 psi. A t this stage it was necessary to remove the top 0.5 cm of column packing and to replace it with fresh material; this resulted in the re-establishment of lower operating pressures. The actual separations remain unaffected b y this pressure variation because the p u m p produces a constant flow of eluent, regardless of minor variations in resistance to eluent flow. Above 1000 psi the flanges on the P T F E connecting pipes blow out, thus preventing dangerous pressures from being reached. The purines which were employed as eytokinin reference standards in the separations shown in Figs. 1-3 were all obtained from either Sigma, Calbiochem or Koch-Light. Stock solutions were prepared in methanol and these were further diluted, generally to give 100 ng per 5 ~l, prior to injection. The methanol used was of normal reagent grade; the corresponding grade of ethanol was unsuitable due to the excessively strong solvent peaks which were obtained. Fig. 1 illustrates an optimised separation of a group of ]?urines on Zipax SCX ; under these conditions a further group of purines, characterised b y the presence of a bulky NS-substituent and in addition the lack of an ~9-ribosyl substituent, exhibited excessively long retention times, due presumably to adsorption onto 14

P l a n t a (Berl.), Vol. 122

206

J.S. Challiee 3.8

I

No-'(A2- isopentenyl adenosine)\

NS-(/12-is~

Izeatin

riboside

I

~~

llll

aOen,n.\fillIII II II IIII[solventpeak 8,5 6~9 IIM III|I(MeOH)

.~

A

// Iitl III/I

,n,eot,o. 1

retention time(rains) 10

Fig. 3. HPLC separation of purines on polyamide with an aqueous buffer eluent. In this system N~-(fl-hydroxyethyl)adenine,Ne-Bis(hydroxyethyl)adenine, adenosine, adenine and N6-methyladenine ran between the solvent peak and zeatin riboside; dihydrozeatin and zeatin ran between zeatin riboside and kinetin riboside; kinetin ran between Ne-(zJ=-isopentenyl)adenosine and ~e-benzyladenosine. Operating parameters--column: Pellidon polyamide (pellicular 1 metre • 4 mm diam. (glass), eluent: 0.05 M KH2POa (pH 4.5), temperature: 40~ C, flow rate: 1.5 ml per min, pressure: ~ 500 psi, detector: UV 254 nm, absorbanee range: 0-0.01, chart: 1 cm per min, sample loadings: 100 nanograms, injection vols: 5 F1

the polystyrene resin rather than to an ion-exchange phenomenon. Addition of 10% v/v methanol to a buffer of ten-fold increased molarity produced a mobile phase capable of eluting the latter group of purines and Fig. 2 illustrates an optimised separation. Reduction of methanol concentration improves the resolution between zeatin and ~T~-(A~-isopentenyl)adenosine but increases the retention times of slower-running purines to inconveniently high values. Continued vacuum de-gassing of the mobile phase gives rise to this effect, due to loss of methanol. When separating complex mixtures of purines in practice it is advisable to run initially with 0.05 M KH2PO 4 in order to obtain separation of the fasterrunning purines, then to change the mobile phase to 0.5 M KH~PO4 :MeOH (10 : 1), running until the column is flushed free of other purines, then to inject the same sample once again. l~ig. 3 illustrates what is in m a n y respects an ideal separation pattern; here it will be noted that there are no complications due to excessively long retention times of some purines. I t will be noted that although purine pairs such as zea~in and dihydrozeatin, zeatin riboside a n d adenosine, adenine and adenosine, kinetin and Nn-benzyladenosine are not resolved on the polyamide column, they are in

Separation of Cytokinins

207

fact well-resolved on one or other of the cation exchange systems as in Figs. 1 and 2. Thus the separation systems described here are collectively of considerable resolving power and should greatly facilitate the identification of new cytokinins in plant extracts. W i t h the U V detector operated at m a x i m u m sensitivity, as in Figs. 1-3, as little as 5 n a n o g r a m of cytokinin could readily be detected. W o r k is in progress on the development of suitable clean-up procedures for leaf extracts, prior to injection into H P L C columns. Preliminary results have shown t h a t a range of eytokinin reference standards are n o t recovered after being added to strawberry (Fragaria • ananassa cv. Ostara) leaf extracts which are cleaned-up b y procedures involving the use of strong cation exchange resins in either H+ or N H + forms. The author is grateful to Dr. S. Shaw for preliminary advice on HPLC separations using Zipax SCX, to Dr. R. Horgan for helpful comments on the use of existing clean-up procedures for leaf extracts and to Prof. L. E. Powell and Mr. R. M. Pool for providing advance details of their work on cytokinin analysis by HPLC.

References

Biddington, N. L., Thomas, T. H. : A modified A~ranthus betacyanin bioassay for the rapid determination of cytokinins in plant extracts. Planta (Berl.) 111, 183-186 (1973) Hall, R. H. : Cytokinins as a probe of developmental processes. Ann. Rev. Plant Physiol. 24, 415-444 (1973) Pool, R. M., Powell, L. E. : The use of pellicular ion-exchange resins to separate plant eytokinins by high-pressure liquid chromatography. Hortseienee 7, 330 (1972) Pool, R. M., Powell, L. E. : Cytokinin analysis by high pressure liquid chromatography. Prec. Int. Cong. Growth Reg. (Tokyo, 1973) (in the press) Skoog, F., Armstrong, D. J. : Cytokinins. Ann. Rev. Plant Physiol. 21, 359-384 (1970) Steward, F. C., Krikorian, A. D. : Plants, chemicals and growth. London: Academic Press 1971

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Separation of cytokinins by High Pressure Liquid Chromatography.

A number of cytokinin reference compounds have been successfully separated by High Pressure Liquid Chromatography using columns of pellicular strong c...
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