Advances in Analytical Methods for Plant Growth Substance Analysis

8 Advances in Analytical Methods for Plant Growth Substance Analysis MARK L. BRENNER

Downloaded by UNIV LAVAL on May 10, 2016 | http://pubs.acs.org Publication Date: September 27, 1979 | doi: 10.1021/bk-1979-0111.ch008

Department of Horticultural Science and Landscape Architecture, 324 Alderman Hall, University of Minnesota, Saint Paul, MN 55108

There has been substantial progress in analytical methods for plant growth substance (PGS) research during this past decade. Previously, plant scientists had to primarily rely on the use of biological assays to determine the presence of the various compounds in which they were interested. Bioassays have helped in establishing the presence of PGS in plants. However, they have many inherent limitations in sensitivity and reliability. Bioassays may be somewhat imprecise in identifying active compounds, due to a test tissue's ability to perhaps alter an unknown compound to a form that would elicit the biological response even though the originally added compound was inactive. Bioassays often lack specificity in that an observed response can be the net result of inhibitory and promotive substances. Bioassays often take days to complete. Another limitation of bioassays is that their response curve usually extends over a logarithmic range; levels must differ by a factor of at least five to ten for the bioassay method to detect a significant separation. Since many of the plant growth substances now have been structurally characterized, it is no longer necessary to rely on bioassays for PGS identification. In fact, physico-chemical techniques have become the methods of choice. There have been a number of reviews discussing some of the aspects of PGS analysis by physico-chemical procedures (1, 2, 3, 4, 5). This report will briefly survey the previous literature with special attention given to the advantages and disadvantages of the available approaches. Special emphasis will be given to the potential uses of high performance liquid chromatography (HPLC). Qualitative vs.

Quantitative Analysis.

Q u a l i t a t i v e Analyses. A c l e a r d i s t i n c t i o n must be made between q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s e s . Qualitative analysis merely demonstrates the presence of a compound i n the extracted sample while q u a n t i t a t i v e a n a l y s i s determines the a c t u a l amount 0-8412-0518-3/79/47-lll-215$07.50/0 © 1979 American Chemical Society

Mandava; Plant Growth Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


216

PLANT GROWTH

SUBSTANCES

of the s p e c i f i c compound being analyzed. B i o l o g i c a l assays permit an assessment of the presence of compounds with a c t i v i t y " l i k e " that of a given standard but f o r the reasons described above, they do not provide d e f i n i t e , q u a l i t a t i v e or q u a n t i t a t i v e proof f o r the presence of a s p e c i f i c compound. They do provide proof that a compound with a c t i v i t y " l i k e " that of the c l a s s being analyzed i s present. One of the key l i m i t a t i o n s to s u c c e s s f u l PGS a n a l y s i s i s the i n h e r e n t l y low l e v e l of PGS found w i t h i n p l a n t t i s s u e . T y p i c a l l e v e l s of many of these compounds range from 10 pg to 10 yg per gram f r e s h weight of t i s s u e , with l e v e l s most o f t e n l e s s than 10 ng per gram f r e s h weight. The recovery of the PGS during sample p r e p a r a t i o n thus becomes very c r i t i c a l . Instrumental q u a l i t a t i v e a n a l y s i s of PGS i n the past has p r i m a r i l y been attempted by gas l i q u i d chromatography (GLC) f i t ted with e i t h e r a flame i o n i z a t i o n d e t e c t o r (FID) or an e l e c t r o n capture detector (ECD). However, the FID i s e s s e n t i a l l y a nons e l e c t i v e detector and the ECD responds only to e l e c t r o n e g a t i v e compounds. Thus, i n the use of GLC-FID, one t o t a l l y r e l i e s on p r i o r cleanup, the gas chromatographic s e p a r a t i o n , and i d e n t i f i c a t i o n by co-chromatography f o r i d e n t i f i c a t i o n . When attempting to i d e n t i f y PGS that occur at t r a c e l e v e l s , the l i k e l i h o o d of having m u l t i p l e compounds w i t h i n a given observed peak i s very great (6). However, when one couples a very s e l e c t i v e d e t e c t o r such as a mass spectrometer (MS) to the gas chromatography then the l i k e l i h o o d of v a l i d i d e n t i f i c a t i o n of a given compound i s g r e a t l y enhanced. One documented example i n which peaks (presumed to be g i b b e r e l l i n s ) detected by an GLC-FID proved to i n c o r r e c t l y estimate the q u a n t i t y of a PGS has been reported by Williams et^ a l . (26). T h e i r use of a GLC-MS provided s u b s t a n t i a l proof of the presence of GA^ and GAg when the t i s s u e e x t r a c t was p u r i f i e d by t h i n l a y e r chromatography. GLC-MS has been used to provide unequivocal, q u a l i t a t i v e proof of the presence of many of the major PGS. Examples of some of the compounds i d e n t i f i e d by t h i s method are l i s t e d i n Table 1. Although the use of GLC-MS remains one of the best methods of i d e n t i f y i n g t r a c e b i o l o g i c a l compounds, r e l a t i v e l y l a r g e amounts (0.1 to 1.0 kg) of t i s s u e are r e q u i r e d f o r a n a l y s i s (Table 1) using f u l l MS scan. For GLC-MS, one would i d e a l l y des i r e more than 1.0 yg of compound f o r p o s i t i v e i d e n t i f i c a t i o n . T h i s amount i s p a r t i c u l a r l y necessary f o r t r i m e t h y l s i l y l ethers (TMS) d e r i v a t i v e s of z e a t i n , which have unstable fragmentation patterns (22). However, the d e t e c t i o n l i m i t may be extended down to the ng range by use of m u l t i p l e i o n d e t e c t i o n (MID). Detect i o n of c y t o k i n i n s by MID may be f u r t h e r aided by permethylation of the compounds (22) due to the greater s t a b i l i t y of the methylated cytokinins. S i n g l e i o n d e t e c t i o n (SID) a l s o allows d e t e c t i o n of PGS down to 10 ng or even lower (27, 28, 29, 30). However, f o c u s i n g on one i o n may introduce e r r o r s . The amount of the p a r t i c u l a r i o n


Mandava; Plant Growth Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979. (12

(13)

GLC-MS

GLC-MS ME METMS

68 g f r wt

6

METMS

5

25.8 kg f r wt

2

GA o>

l 5

G A

2 9

Pisum sativum

Phaseolus v u l g a r i s GA GA , GA , GA , GA , GA , GA , GA 8 17 19 21

3

(ID

GLC-MS ME METMS^

25.8 kg t i s s u e

Phaseolus coccineus

+

GA -GA i

2

(10)

GLC-MS

ME

20 kg f r wt

Humulus lupulus L .

ABA

1

(9)

GLC-MS

ME

1 kg f r wt

Pisum sativum L , chloroplasts

ABA

(8)

GLC-MS

ME TMS

315 g f r wt 3

Ref. (7)

Detector d i r e c t probe MS GLC-MS

Ceratonia s i l i q u e L .

ME

2

Type of derivative made

ABA

Amount of tissue extracted

standards

1

Source of PGS

Examples of p l a n t growth substances q u a l i t a t i v e l y i d e n t i f i e d by gas chromatography-mass spectrometery,

ABA

Compound

Table I .


to

g §

po


1

Pseudotsuga

standards

Zea mays

standards

malt

IAA

IAA-amino a c i d conjugates

di-o- & t r i - o IAA-myo-inositols

Z, ZR

Ζ extract

Zea mays

IAA

menziesii

Zea mays

IAA

Source of PGS

Pharbitis n i l C y t i s u s scoparius Phaseolus v u l g a r i s

continued

GA glucosides and glucosyl esters

Compound

Table I .

METMS


none

ME METMS

none

TMS

10.5 kg k e r n e l s TMS

300 g f r wt

25-255 g f r wt TMS

d i f f u s a t e from none 15,000 c o l e o p t i l e tips

Amount of tissue extracted Detector

d i r e c t probe MS

GLC-MS

GLC-MS


MS

d i r e c t probe MS a f t e r GLC




(21)

(20)

(19)

(18)

(17)

(16)

(15)

(14)

Ref.

to

oo


TMS = t r i m e t h y l s i l y l

ethers,

METMS = methyl e s t e r t r i m e t h y l s i l y l e s t e r .

14

M e r c u r i a l e s ambigua

——

1 sap permethylated TMS

TMS

a c i d ; Ζ - z e a t i n ; ZR = z e a t i n

GLC-MS

GLC-MS

GLC-MS

GLC-MS

permethylated

150-250 g f r wt TMS

2.5

90 g dry wt

GLC-MS

Detector

TMS


ABA = a b s c i s i c a c i d ; GA = g i b b e r e l l i n ; IAA - i n d o l e - 3 - a c e t i c riboside.

ME = methyl e s t e r .

3

2

X

2

Z, ZR Δ - isopentyladenosine

g l y c o s y l ZR

Vinca rosea

glycosyl Ζ

pseudoplatanus

Acer

cerasus

ZR

Prunus

90 g dry wt

Actinidia

Z, ZR

chinensis

20 kg

Humulus l u p u l u s L*

1

Ζ

Compound

Amount of tissue extracted

continued

Source of PGS

Table I .


(25)

(24)

(23)

(22)

(10)

Ref.

PLANT GROWTH

220

SUBSTANCES

of i n t e r e s t may be due to the PGS or to i m p u r i t i e s . The a p p l i c a t i o n of MID reduces the p o s s i b i l i t y of e r r o r i f one matches the r a t i o of i n t e n s i t y of s e v e r a l ions w i t h i n the standard compound to the unknown compound.


Q u a n t i t a t i v e A n a l y s i s . A number of s c i e n t i s t s have claimed that PGS a n a l y s i s by GLC-MS with SIM or MID d e t e c t i o n facilitates quantitative analysis. T h i s i s only true i n the sense that one i s able to o b t a i n a r e l i a b l e estimate of the quantity of the compound that i s a c t u a l l y detected by the MS system. However, part of the sample may be l o s t during e x t r a c t i o n and p u r i f i c a t i o n , before the sample reaches the f i n a l d e t e c t o r . The only apparent means of c o r r e c t i n g for such l o s s i s the a d d i t i o n of an i n t e r n a l standard to the sample at the s t a r t of sample p r e p a r a t i o n . Use of i n t e r n a l standards. Mann and Jaworski (31) reported that when the recovery of { 1 - C } IAA i s monitored during a samp l e p u r i f i c a t i o n procedure, considerable l o s s of IAA can be detected. Bandurski and Schulze (32) suggested the use of reverse isotope d i l u t i o n to help quantify the a c t u a l l o s s of IAA during sample a n a l y s i s . In t h i s procedure, one adds a t r a c e amount of r a d i o - l a b e l e d compound which i d e a l l y i s i d e n t i c a l to the compound being monitored. High s p e c i f i c a c t i v i t y i s r e q u i r e d so that s t a t i s t i c a l l y s i g n i f i c a n t amounts of isotope can be detected without having to add an excessive quantity (mass) of i n t e r n a l standard. The amount of i n t e r n a l standard must be l e s s than the amount of PGS. One may then a c c u r a t e l y determine the recovery e f f i c i e n c y of the i n t e r n a l standard and thus of the PGS (32). L i t t l e et^ a l . (30) found that the recovery e f f i c i e n c y of both IAA and ABA could vary up to f i v e f o l d . We have demonstrated the same type of v a r i a b i l i t y (Table 2). Examination of the data from L i t t l e et a l . (30) as w e l l as from my research group (Table 2) shows that recovery v a r i a b i l i t y f o r ABA i s not as great as f o r IAA. Thus when a n a l y z i n g f o r both compounds from the same samp l e , one needs to use two i n t e r n a l standards. The number of i n t e r n a l standards r e q u i r e d when monitoring s e v e r a l compounds should be c a r e f u l l y considered. For example, Cohen and Bandurski (33) demonstrated that the IAA conjugates are s t a b l e to o x i d a t i o n by peroxidases, while f r e e IAA i s n o t . Thus, the a d d i t i o n of {l- C} IAA would not adequately monitor recovery of IAA conjugates. Another example i s the use of k i n e t i n as an i n t e r n a l standard for the e s t i m a t i o n of z e a t i n r e covery (34). The s e l e c t i o n of k i n e t i n for z e a t i n recovery e s t i mation must be questioned on the b a s i s of l a r g e d i f f e r e n c e s i n t h e i r p a r t i t i o n c o e f f i c i e n t s (35). Another approach has been the use of nonlabeled isomers of a compound such as 6-(hydroxybenzyla m i n o ) - 9 - 3 - D - r i b o f u r a n o s y l p u r i n e to estimate the recovery of 6(o-hydroxybenzylamino)-9-$-D-ribofuranosylpurine (36). Saunders group (37) has added 2-trans-ABA as a n o n r a d i o a c t i v e i n t e r n a l standard. E i t h e r i n t e r n a l standard must be shown to p a r t i t i o n 14

lk

1



li+

1L+

C} ABA

14

1 4

C } IAA

{ 1 - C } IAA

U-

{2- C} ABA

{2-

corms

Freesia hybrida

esculentum stems

Lycopersicon

leaves

Cornus s t o l o n i f e r a

G l y c i n e max. leaves

b . 14

a . 16

45

27

12

Samples

added

Tissue

of

Number

28-90 17-72

60

10-85

64

46

61-93

59-77

%

Range

72

68

Mean

+0.187

+0.158

+0.182

+0.078

+0.042

S t d . dev.

Recovery

Net recovery e f f i c i e n c y of i n t e r n a l standards of plant growth substances a f t e r e x t r a c t i o n and p u r i f i c a t i o n .

Compound

Table I I .



222

PLANT GROWTH

SUBSTANCES

e x a c t l y l i k e the compound being followed f o r absolute confidence i n i t s use. What may prove to be the u l t i m a t e choice f o r an i n t e r n a l standard when using an MS (37) i s the a d d i t i o n of a PGS standard as a deuterated compound to the i n i t i a l sample p r e p a r a t i o n . The deuterated compound i s q u a n t i f i e d d i r e c t l y on the MS r a t h e r than having to subsequently subject the sample to conventional r a d i o isotope d e t e c t i o n methods. T h i s procedure has been a p p l i e d to ABA (29) and IAA. (38, 39) a n a l y s e s . A high deuterium content ( l a b e l e d at f i v e or more p o s i t i o n s ) should be sought to avoid confusion with n a t u r a l l y "heavy" i s o t o p i c compounds (39). As summarized i n Table 3, there are a number of examples i n which i n t e r n a l standards have been e f f e c t i v e l y used f o r the quant i t a t i v e a n a l y s i s of PGS by GLC-MS. The prime l i m i t a t i o n of t h i s approach has been the high cost of the i n s t r u m e n t a t i o n . As w i l l be discussed i n a l a t e r p o r t i o n of t h i s manuscript, other s e l e c t i v e d e t e c t o r s used on e i t h e r a GLC or an HPLC should o f f e r other choices for PGS a n a l y s i s , but they are not as s p e c i f i c as GLC-MS. Use of HPLC f o r PGS A n a l y s i s . P r e p a r a t i v e HPLC. Most current PGS a n a l y t i c a l procedures have been optimized to examine a s p e c i f i c c l a s s of PGS. Theref o r e , s e v e r a l d i f f e r e n t procedures must be developed to analyze the major c l a s s e s of PGS i n a s i n g l e p l a n t sample. Exceptions to t h i s approach have been reported by Shindy and Smith (44) and by Wightmann et a l . (45) who attempted to i d e n t i f y the four major c l a s s e s of PGS from the same p l a n t sample with a s i n g l e procedure. However, they o f f e r e d no p o s i t i v e proof of p u r i t y of the compounds which were i d e n t i f i e d by GLC-FID. {Shindy and Smith confirmed the a c t u a l presence of s e v e r a l PGS by GLC-MS but d i d not determine the p u r i t y of the peaks (44).} The time consumed i n the m u l t i s t e p processes necessary to o b t a i n s u f f i c i e n t l y p u r i f i e d e x t r a c t s s u i t a b l e f o r a n a l y t i c a l chromatographic procedures has been a great l i m i t a t i o n on PGS r e s e a r c h . A d e s i r a b l e goal would be to reduce to a minimum the number of steps i n v o l v e d f o r quant i t a t i v e recovery of m u l t i p l e PGS from a s i n g l e p l a n t sample. P r e p a r a t i v e HPLC (prep-HPLC) used to t h i s end by a number of r e searchers (Table 4 ) , g r e a t l y improves the s e p a r a t i o n and recovery e f f i c i e n c y of many PGS and s u b s t a n t i a l l y reduces the s e p a r a t i o n time compared to c l a s s i c a l procedures. A p p l i c a t i o n of Prep-HPLC to PGS A n a l y s i s . Reverse phase l i q u i d chromatography has proven to be w e l l s u i t e d for cleanup of p l a n t e x t r a c t s by prep-HPLC (4, ^6, 47, 48). When the mobile phase i s i n i t i a l l y an aqueous b u f f e r at pH 2.8, a l l but the h i g h l y charged ( e . g . , z e a t i n r i b o t i d e with 5 AMP used as a r e p r e s e n t a t i v e compound f o r z e a t i n r i b o t i d e ) p l a n t hormones are r e t a i n e d at the head of the column ( F i g . 1). Since the PGS are r e t a i n e d , samples can be i n j e c t e d onto the column i n a d i l u t e form. In1


Mandava; Plant Growth Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 2

6

ME

{2-

DPA

Populus

Rhaphanus

(o-OH BAPriboside)

Raphanatin 2

2

2

2

5

Z = zeatin.

ether.

chromatography-electron capture

SIM = s i n g l e i o n m o n i t o r i n g on GLC-MS.

GLC-EC = gas l i q u i d

5

4

(43) SIM TMS

{ H } raphanatin ί Η - ) Z

2

(36) SIM

TMS

p-OH BAP r i b o s i d e

C-} IAA

detector.

ABA = a b s c i s i c a c i d ; DPA = 4'-dihydroxyphaseic a c i d ; PA= p h a s e i c a c i d ; o-OH-BAP r i b o s i d e = 6-(ohydroxybenylmino)-9-3-D r i b o f u r a n o s y l p u r i n e ; Raphanatin = 7-3-D glucopyranosylzeatin.

sativa

(30) SIM

ll+

SIM TMS

U-

(42) scan

full

TMS

3

{5- H-} IAA

14

14

(32)

(41)

(40)

scan

4

scan

GLC-EC

full

full

DPA

TMS

C-}

(30)

(29)

ME

K

3

Ref.

{1- C} IAA

L

SIM

f u l l scan dual i o n monitoring

Method o f a n a l y s i s on GLC-MS

{2- C} PA

ME = methyl e s t e r ; TMS = t r i m e t h y l s i l y l

3

2

vulgaris

sitchensis

Picea

mays

IAA

Zea

IAA

1

vulgaris

Avena s a t i v a

Phaseolus

IAA

PA

ME

{2- C-} ABA

Lactuca

14

{2- C-} ABA

ME

Phaseolus

sitchensis

li+

ABA

ME

Picea

{ H -} ABA

Type of derivative

ABA

mays

Internal standard

of PGS u s i n g gas chromatography-mass s p e c t r o s c o p y .

Zea

1

Source of PGS

Examples of q u a t i t a t i v e a n a l y s i s

ABA

Compound identified

Table I I I .



coccineus

Phaseolus

bicolor

Sorghum

sample

GPC

2

partition

sephadex-G10

partition

none

none

cleanup

Prior

phase

phase

silica

adsorption

" 18 C

reverse

~C18

reverse phase

Q 1 Ο

—C 1

reverse

Column

counting

a n a l y t i c a l HPLC

MS

scintillation

GLC

HPLC

analytical

bioassay

GLC-EC

identification

Analytical

(49,

(48)

(47)

(46)

Ref.

GPC = g e l permeation chromatography.

*ABA = a b s c i s i c a c i d ; IAA = i n d o l e - 3 a c e t i c a c i d ; DPA = dihydrophaseic a c i d ; PA = phaseic a c i d .

gibberellins

DPA, PA

IAA, ABA,

Lycopersicon

Cytokinins

esculentum

G l y c i n e max.

PGS

ABA

1

Source of

Example of the use of p r e p a r a t i v e HPLC f o r PGS p u r i f i c a t i o n .

Compound(s)

Table IV.


50)

η w en


8.

BRENNER

225

Plant Growth Substance Analysis

INJECTION

PREPARATIVE SEPARATION

-1U

Β

SAMPLE CONCENTRATION

GRADIENT ELUTION

Figure 1. Diagram of sample concen tration on a reverse-phase LC column (right) followed by separation when the solvent strength of the mobile phase is increased. Column: 018, 10 mm X 25 cm; solvent: 0.1N HAc to 50% ETOH in 0.1N HAc in 20 min;flowrate: 50 mL/min; detector: UV 254 nm. μ



226

PLANT GROWTH

SUBSTANCES

j e c t i o n volumes as l a r g e as 4.0 ml are q u i t e p r a c t i c a l because l a r g e q u a n t i t i e s of s o l u t e may be i n j e c t e d without s o l u b i l i t y problems. In a d d i t i o n , the t r a n s f e r of s o l u t e to the column i s more e f f i c i e n t when made i n a l a r g e i n j e c t i o n . The PGS are separated and eluted from the column by the a d d i t i o n of a w a t e r - m i s c i b l e organic solvent to the mobile phase. We have found that ethanol works w e l l f o r t h i s purpose. When m i c r o p a r t i c l e - p a c k e d HPLC columns are used, the solvent change must be accomplished as a g r a d u a l , continuous g r a d i e n t . Dis continuous increments (step gradients) tend to cause r a p i d v i s c o s i t y and thermal changes along the column bed; these are known to cause d e s t r u c t i o n of the bed. The gradient p r o f i l e d r a m a t i c a l l y a f f e c t s the s e p a r a t i o n on the column. A l i n e a r gradient of 0.1 Ν aqueous a c e t i c a c i d to 0.1 Ν a c e t i c a c i d i n 50% (v/v) ethanol/water d e l i v e r e d i n a 25 minute p e r i o d has been used to separate many PGS standards ( F i g . 2). Ciha et a l . (46) have e s t a b l i s h e d that a s i m i l a r gradient sequence permits r a p i d recovery of a f r a c t i o n c o n t a i n i n g ABA from crude p l a n t e x t r a c t s . The recovery e f f i c i e n c y of {2- C}-ABA and of the endogenous p l a n t ABA was g r e a t e s t when a l l conventional p a r t i t i o n i n g was by-passed and the crude p l a n t e x t r a c t was i n j e c t e d d i r e c t l y onto the chromatograph. We have extended that s e p a r a t i o n technique to allow recovery of m u l t i p l e PGS from a s i n g l e sample. Now, from the same p l a n t e x t r a c t , s p e c i f i c f r a c t i o n s containing z e a t i n , z e a t i n r i b o s i d e , IAA, I A A - a c e t y l - a s p a r t a t e , ABA, phaseic a c i d and dihydrophaseic a c i d can be r e c o v ered . The r e s o l u t i o n shown i n F i g u r e 2 was accomplished by a h i g h l y e f f i c i e n t column which we packed with 10 ym diameter Bondapak ]IC\Q p a r t i c l e s (Waters A s s o c i a t e s ) . This column (10 mm I . D . χ 25 cm) has over 5000 t h e o r e t i c a l p l a t e s as compared to 600 for the two 1.0 m columns packed with 35 to 75 ym diameter p a r t i c l e s p r e v i o u s l y used (46, 47). To p r o t e c t the column from compounds that i r r e v e r s i b l y a d here to or p a r t i t i o n i n t o the column packing, a precolumn may be used. A p e l l i c u l a r packing coated with material ( C Corasil I I , Waters A s s o c i a t e s ) has proven b e n e f i c i a l , yet has a m i n i m a l l y d e t r i m e n t a l e f f e c t on compound r e s o l u t i o n . F r a c t i o n s are c o l l e c t e d on the b a s i s of the r e t e n t i o n times of the r e s p e c t i v e PGS standards. The remaining p o r t i o n s of the column e f f l u e n t are d i v e r t e d to waste. Figure 3 diagramatically represents the sequence of events used f o r prep-HPLC of PGS sam ples. The a d d i t i o n of an a c i d i c b u f f e r (0.1 Ν acetate) that serves as a p o l a r m o d i f i e r i n the mobile phases ( i n both water and e t h anol) i s r e q u i r e d for c o n s i s t e n t r e s u l t s . I t serves to protonate a l l of the PGS and helps to minimize adsorptive p r o p e r t i e s of the column, thereby f a c i l i t a t i n g r e p r o d u c i b l e r e s u l t s (47). The b u f f e r accomplishes t h i s by s a t u r a t i n g the exposed s i l i c i c a c i d sites. E t h a n o l , r a t h e r than methanol, i s the p r e f e r r e d organic ll+

1 8



8.

BRENNER


10 TIME

227

15 (min)

Figure 2. Separation of PGS standards on a preparative HPLC column. Note that the retention time of 5' AMP would be representative of cytokinin ribotides.




to to

00

8.

BRENNER


229


mobile phase because i t more e f f e c t i v e l y reduces peak t a i l i n g . A c e t o n i t r i l e a l s o works w e l l but much greater care must be taken to avoid operator exposure to t o x i c solvent vapors. A f t e r the compounds of i n t e r e s t are eluted from the column, the c o n c e n t r a t i o n i s l i n e a r l y increased to 0.1 Ν a c e t i c a c i d i n 95% (v/v) ethanol/water to remove the more nonpolar components r e t a i n e d on the column. Following an adequate e q u i l i b r a t i o n (greater than 10 column volumes) at maximum solvent strength (95% e t h a n o l ) , the solvent i s l i n e a r l y programmed to r e t u r n to the i n i t i a l c o n d i t i o n s of 0.1 Ν aqueous a c e t i c a c i d . Automation of Prep-HPLC. Reverse phase prep-HPLC s e p a r a t i o n has proven to be a very r e p r o d u c i b l e technique. For t h i s reason, the process can be automated. In a d d i t i o n to the standard com ponents of an HPLC system, the f o l l o w i n g are r e q u i r e d f o r auto mation: an a u t o i n j e c t o r capable of l a r g e volume i n j e c t i o n s (2.0 to 5.0 m l ) , a programmable c o n t r o l l e r (a microprocessor c o n t r o l l e r ) , a f r a c t i o n c o l l e c t o r , and a waste v a l v e (3-way valve) con t r o l l e d by a s o l e n o i d ( F i g . 4 ) . The microprocessor should allow programming of the solvent flow r a t e , the sequence for solvent gradient formation, the time of i n j e c t i o n , advancement of the f r a c t i o n c o l l e c t o r (to c o l l e c t s p e c i f i c f r a c t i o n s r a t h e r than j u s t uniformly incremented advancement), and c o n t r o l of a waste valve. Automation of the prep-HPLC system o f f e r s s e v e r a l advantages over manual o p e r a t i o n . Time use e f f i c i e n c y increases s e v e r a l fold. For example, we have been able to quadruple the number of samples separated per day. Greater p r e c i s i o n i s a l s o obtained due to the accurate timing of the m i c r o p r o c e s s o r - c o n t r o l l e d f u n c tions. The other obvious advantage i s more e f f e c t i v e use of labor. However, automation without the proper c o n t r o l s has def inite limitations. That i s , i f one of the components of the system f a i l s while everything e l s e continues to operate, then a l l of the samples i n j e c t e d while the system i s malfunctioning may be lost. From our experience the f o l l o w i n g functions must be moni tored f o r unattended operation (closed-loop c o n t r o l ) : high p r e s sure l i m i t (to detect a plugged l i q u i d p a t h ) , low pressure l i m i t (to d e t e c t a l e a k ) , pressure p u l s a t i o n (to monitor uniform s o l vent f l o w ) , sample i n j e c t i o n (to v e r i f y sample i n j e c t i o n ) , l i q u i d l e v e l sensors (to v e r i f y adequate reserve of s o l v e n t s ) , and a d vancement of the f r a c t i o n c o l l e c t o r s (to v e r i f y that the f r a c t i o n c o l l e c t o r a c t u a l l y advances and that a new tube i s ready to c o l l e c t the next sample). Thus, i f any of the monitored items i n d i cates a f a u l t y system, the microprocessor should e i t h e r c o r r e c t the problem or should shut the system down. A n a l y t i c a l HPLC of PGS. A number of r e p o r t s are c u r r e n t l y a v a i l a b l e on the use of HPLC f o r the a n a l y t i c a l i d e n t i f i c a t i o n of n a t i v e PGS (Table 5 ) . These techniques have p r i m a r i l y r e l i e d on p u r i f i c a t i o n by p r i o r p a r t i t i o n i n g and chromatographic s e p a r a t i o n


Mandava; Plant Growth Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979. c a t i o n exchange partition

none

Standards

Sorghum b i c o l o r

Cytokinins

Zeatin

Zeatin

riboside

reverse phase-C.

TLC

Agrobacterium tumefaciens

N^-(A -isopentenyl) -adenine

Sephadex-G10

p a r t i t i o n ; PVP

c a t i o n exchange

none

Standards

Cytokinins (multiple)

Z

reverse phase-C.

p a r t i t i o n and Sephadex-G10


ABA

0

Q

reverse phase-C^g

adsorption p a r t i t i o n NH^

prep HPLC

G l y c i n e max

c a t i o n exchange

p a r t i t i o n & TLC anion exchange

ABA

vinifera

Column

p a r t i t i o n and Sephadex-G25

Vitis

Prior Sample Clean Up

Malus Gossipium

1

Source of PGS

Examples of the use of a n a l y t i c a l HPLC f o r PGS.

ABA

ABA

Compound

Table V.


UV-254 nm

UV-254 nm

UV-254 nm

(58)

(57)

(56)

(55)

(47)

UV-254 nm bioassay UV-254 nm

(45)

UV-254 nm

(54)

(52,

UV-254 nm UV-254 nm

Ref

Detector

53)


G l y c i n e max partition Gossypium hirsutum Sephadex-G10 Phaseolus v u l g a r i s others

IAA

f o r Table I .

(61)

fluorescence electro chemical

See a b b r e v i a t i o n s

(47) UV-254 18

reverse

partition Sephadex-G10


IAA

anion exchange reverse phase-C 18 adsorption

phase-C

(53)

UV-254

anion exchange

p a r t i t i o n TLC

Vitis vinifers

?

IAA

5

GA , GA , GA

2 Q

(60)

4

Standards

3

GA , GA , GA ,

1

UV-265 nm

Ref.

none-all d e r i adsorption v a t i z e d as AgNO^-silica ρ - n i t r o b e n z y l esters

Detector

(59)

Column

UV-260 nm

Prior Sample Clean Up

p a r t i t i o n ; LH-20 reverse phase-C^

Source of PGS

Phaseolus vulgaris

continued

Cytokinins (multiple)

Table V .


Co

S.

C5

S-

&

Co

ο*

CO

S?

M

2


I

L

Figure 4.

Microprocessor Interactive Controller

Pressure Monitor

Pump A

Liquid

path: Output control lines Input control lines Detector signal lines

Schematic diagram of an automated preparative HPLC system.


>

Η

Η

Ο S3 Ο

H

F

to Co to

8.

BRENNER


233


on one or two HPLC columns. I d e n t i f i c a t i o n has g e n e r a l l y been accomplished with a UV detector at 254 nm. However, t h i s method i s r e l a t i v e l y n o n s e l e c t i v e , s i n c e most aromatic compounds absorb r a d i a t i o n at 254 nm. The use of a UV detector on an HPLC system i s only s l i g h t l y more s e l e c t i v e than a flame i o n i z a t i o n detector on a GLC system. S e l e c t i v e Detectors f o r A n a l y t i c a l HPLC. Sweetser and Swartzfager (61) demonstrated that e i t h e r fluorescence or e l e c trochemical d e t e c t o r s are e f f i c i e n t f o r s e l e c t i v e i d e n t i f i c a t i o n of IAA. Fluorescence d e t e c t i o n i s much more s e l e c t i v e than UV d e t e c t i o n since fewer compounds f l u o r e s c e than absorb UV r a d i a tion. E l e c t r o c h e m i c a l d e t e c t i o n i s a l s o s p e c i f i c because only compounds that may be o x i d i z e d or reduced are d e t e c t e d . IAA i s o x i d i z e d at a low voltage p o t e n t i a l (0.7 to 0.9 V) r e l a t i v e to other compounds. In our hands, these two methods of d e t e c t i o n appear to be q u i t e a c c u r a t e , s i n c e the same p l a n t sample y i e l d s the same q u a n t i t a t i v e data by both methods. Since they are q u i t e d i f f e r e n t , using both d e t e c t i o n method adds c r e d i b i l i t y to the assays. Future S e l e c t i v e A n a l y t i c a l HPLC Methods f o r PGS. The IAA a n a l y t i c a l procedure developed by Sweeter and Swartzfager (61) i s an approach that should be extended to the other PGS. Several other s e l e c t i v e a n a l y t i c a l techniques are promising but s t i l l need to be proven s u i t a b l e f o r PGS a n a l y s i s . One t e c h nique i s the simultaneous monitoring of UV absorbance at s e v e r a l d i f f e r e n t wavelengths. The r a t i o of absorbance at the r e s p e c t i v e wavelengths has proven to be unique f o r many compounds (62, 63). As with MID on MS, the more wavelengths that are simultaneously monitored, the greater i s the l i k e l i h o o d of v a l i d i d e n t i f i c a t i o n . Another a n a l y t i c a l technique i s the formation of d e r i v a t i v e s which are f l u o r e s c e n t or absorb UV r a d i a t i o n at unique wavelengths. The compound of i n t e r e s t may be d e r i v a t i z e d and i n j e c t e d onto the HPLC system; the column separates the reactants and then passes them through the d e t e c t o r . The compound may a l s o be der i v a t i z e d "post column" as done by amino a c i d a n a l y z e r s . The d é r i v â t i z i n g r e a c t a n t i s metered to mix with the column e f f l u e n t and i s then sent to the d e t e c t o r . I d e a l l y , only the d e r i v a t i z e d products should be d e t e c t a b l e . D e r i v a t i v e formation i s e s s e n t i a l f o r a n a l y s i s of g i b b e r e l l i n s because they only absorb r a d i a t i o n below 230 nm, which i s an extremely n o n s p e c i f i c r e g i o n . Benzyl e s t e r s (49) and p - n i t r o b e n z y l e s t e r s (60) of g i b b e r e l l i n s have s u c c e s s f u l l y been synthes i z e d p r i o r to i n j e c t i o n to permit t h e i r d e t e c t i o n as they e l u t e from HPLC columns. U n f o r t u n a t e l y , these d e r i v a t i v e s have added l i t t l e s e l e c t i v i t y to the a n a l y t i c a l procedure. The d e r i v a t i v e s are monitored at 254 or 265 nm which, as p r e v i o u s l y mentioned, i s a nonspecific region. A new method of d e t e c t i o n of PGS by HPLC has been introduced



234

PLANT GROWTH

SUBSTANCES

with the development of o n - l i n e l i q u i d s c i n t i l l a t i o n counters that now are commercially a v a i l a b l e . Reeve et a l . (49) and Reeve and C r o z i e r (50) described a system i n which the s c i n t i l l a t o r i s added to the column eluant which then passes through a l i q u i d s c i n t i l l a t i o n counter. T h i s approach i s d e s t r u c t i v e and thus only a p o r t i o n of the column e f f l u e n t should be d i v e r t e d to the s c i n t i l l a t i o n counter i f f u r t h e r work i s to be done on a p o r t i o n of the e f f l u e n t . Another approach i s the use of a s c i n t i l l a t i o n counter with a s p e c i a l flow c e l l packed with s c i n t i l l a t o r beads. T h i s new technology i s advantageous because i t i s nondestructive to the sample, yet o f f e r s d e t e c t i o n e f f i c i e n c y comparable with the conventional l i q u i d s c i n t i l l a t i o n system. For a v a l i d com p a r i s o n of e f f i c i e n c y , one should recognize that the system that adds s c i n t i l l a n t must d i l u t e the sample by a f a c t o r of 4 to 10, while the packed c e l l system with s o l i d s c i n t i l l a n t does not d i l u t e the sample. T h i s normalizes the d i f f e r e n c e between the two types of s c i n t i l l a t i o n counting since adding the s c i n t i l l a t o r i s u s u a l l y three to four times more e f f i c i e n t than using the system packed with a flow c e l l s c i n t i l l a t o r . R a d i o - l a b e l e d PGS can be detected i n the presence of many other compounds and thus can be f r a c t i o n a t e d from crude samples ( F i g . 4) on the b a s i s of r a d i o a c t i v i t y of e l u t i n g peaks. This procedure i s i d e a l for the s e p a r a t i o n and i d e n t i f i c a t i o n of PGS metabolites and f o r a n a l y s i s of PGS recovery e f f i c i e n c y . Other S e l e c t i v e

D e t e c t i o n Procedures f o r PGS.

The use of GLC-EC has become a w e l l accepted method for the a n a l y s i s of ABA as d e s c r i b e d by Saunders (37). The p u r i t y of ABA (methyl e s t e r ) detected on t h i s system may be confirmed by form ing the t r a n s - Α Β Α isomer methyl e s t e r i n s u n l i g h t while i n ace tone and rerunning the sample. We have found that prep-HPLC i s u s e f u l i n the p u r i f i c a t i o n of plant e x t r a c t s f o r ABA a n a l y s i s by GLC-ED (45). Another unique i d e n t i f i c a t i o n method takes advan tage of the extreme cotton e f f e c t that ABA e x h i b i t s . The degree of o p t i c a l r o t a t i o n can be used f o r q u a n t i f i c a t i o n of ABA i f the sample i s h i g h l y p u r i f i e d (37). S p e c i f i c monitoring of n i t r o g e n or phosphorus c o n t a i n i n g com pounds may be accomplished with an a l k a l i n e - f l a m e i o n i z a t i o n de t e c t o r on a GLC w i t h s u b s t a n t i a l l y greater s e n s i t i v i t y than an FID. The a l k a l i n e - F I D has r e c e n t l y been reported (64) to detect IAA-raethyl e s t e r s from p l a n t samples. Another a n a l y t i c a l procedure that has drawn considerable a t t e n t i o n i s the conversion of IAA to indole-a-pyrone (65, 66, 67, 68). The l i m i t a t i o n s of t h i s technique are that i t i s s p e c i f i c to f r e e IAA, the assay i s d e s t r u c t i v e , and the l i m i t of d e t e c t i o n i s approximately 1 ng. However, for those s t u d i e s that only r e q u i r e q u a n t i f i c a t i o n of IAA, the procedure should be s e r i o u s l y considered. As Bandurski documented i n t h i s volume, there are many other forms of IAA that occur i n s u b s t a n t i a l amounts. The


8.

BRENNER


i n d o l e - a - p y r o n e procedure would f a i l

235

to detect these other auxins.


Futher Refinement of PGS A n a l y s i s . S e l e c t i o n of A p p r o p r i a t e Method of Sampling. As reviewed by Dennis (69), the a p p r o p r i a t e sampling of t i s s u e represents a s i g n i f i c a n t challenge when attempting to r e l a t e PGS l e v e l with f u n c tion. Even i f the c o r r e c t t i s s u e ( c e l l s or o r g a n e l l e s ) i s sel e c t e d f o r e x t r a c t i o n , the problem of determining the best s o l vent f o r PGS e x t r a c t i o n s t i l l e x i s t s . Methanol has most commonly been used (1). However, n a t u r a l PGS e s t e r s can undergo t r a n s e s t e r i f i c a t i o n such as the formation of methyl a b s c i s a t e (70). When the e x t r a c t i o n i s done w i t h acetone, no methyl a b s c i s a t e i s recovered. Acetone has a l s o been used f o r recovery of n a t u r a l IAA e s t e r s (18, 32). Another approach to minimize formation of methyl e s t e r s has been to e x t r a c t with hot water (53). Dichloromethane has been used (71) i n preference to methanol or ethanol to minimize the conversion of i n d o l e - 3 - p y r u v i c a c i d to IAA. The use of T r i t o n X-100 to d i s p e r s e T r i t i c u m c h l o r o p l a s t membranes has been reported to increase the recoverable y i e l d of GAg by 1000 as compared with methanol e x t r a c t i o n . The authors suggest that the methanol causes i r r e v e r s i b l e b i n d i n g of GA to the p l a s t i d membrane (72). However, the enhanced recovery using T r i t o n X-100 has been disputed (73) and was not b e n e f i c i a l i n the e x t r a c t i o n of c h l o r o p l a s t s of Pisum. Now that physico-chemi c a l procedures are a v a i l a b l e f o r many of the PGS, more a t t e n t i o n should be d i r e c t e d at improving the e x t r a c t i o n procedures for PGS. S

M i n i m i z a t i o n of I m p u r i t i e s . The presence of solvent i m p u r i t i e s may be one of the most common sources of a n a l y t i c a l e r r o r . Even high p u r i t y s o l v e n t s have been documented to contain p l a s t i c i z e r s (74) and c a r e f u l p u r i f i c a t i o n i s r e q u i r e d before use (75). The p u r i t y of the s o l v e n t s should be examined by the d e t e c t i o n procedures that w i l l be used for the PGS (75). For reverse phase HPLC, removal of v o l a t i l e organic compounds from water can be e s p e c i a l l y troublesome. The problem may be d i minished by f i l t e r i n g the water through a c t i v a t e d c h a r c o a l . Pumping the aqueous mobile phase through a scrubber column (packed with reverse phase m a t e r i a l ) l o c a t e d between the pump and i n j e c t o r a l s o has been u s e f u l . Maximization of HPLC Column E f f i c i e n c y . Many of the a v a i l able HPLC bonded phase column packings are s o l d as s i n g l e f u n c t i o n packings, such as c a t i o n exchange m a t e r i a l s . However, these packings often have s e v e r a l types of f u n c t i o n s . Many i o n exchange packing m a t e r i a l s were made by c o v a l e n t l y a t t a c h i n g the i o n exchange group to the s i l i c support by means of an organic l i n k (Fig. 5). The organic phase a l s o serves to p r o t e c t the s i l i c a support from s o l u b i l i z a t i o n by aqueous b u f f e r s . However, charged molecules that a l s o are nonpolar, such as most PGS, w i l l be sep-


236

SUBSTANCES


PLANT GROWTH

Figure 5.

Diagram of the dual functionality of ion exchange and liquid-liquid partition of HPLC column packing



8.

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237

arated both by p a r t i t i o n i n g i n the organic phase and by i o n exchange on the same column. We have found that the organic phase of Vydac anion exchange m a t e r i a l has a higher a f f i n i t y than the anion exchange s i t e s f o r IAA. Thus, at low pH which maintains IAA i n the protonated form, most of the IAA p a r t i t i o n s i n t o the column packing and r e s u l t s i n a n o n l i n e a r c o n c e n t r a t i o n response curve ( F i g . 6 ) . However, the p a r t i t i o n i n g may be minimized by i n c r e a s i n g the pH to 6, which converts IAA to the charged, more p o l a r form, r e s u l t i n g i n a l i n e a r c o n c e n t r a t i o n response curve ( F i g . 6). Another method to reduce the dual s e p a r a t i o n process i s the a d d i t i o n of a m i s c i b l e organic solvent to the mobile phase to overcome p a r t i t i o n i n g i n t o the column packing. A c e t o n i t r i l e added to the mobile phase subs t a n t i a l l y increases the e f f i c i e n c y of the IAA s e p a r a t i o n even though i t a l s o reduces the k ( F i g . 7 ) . The s e p a r a t i o n may be optimized by decreasing the b u f f e r strength i n the presence of the a c e t o n i t r i l e . S i m i l a r d u a l f u n c t i o n a l i t y e x i s t s for many of the reverse phase packing m a t e r i a l s ( F i g . 5 ) . Due to s t e r i c hindrance, the s i l i c a support i s incompletely coated with the o c t a d e c y l molecules. The exposed s i l i c a groups serve as strong adsorption s i t e s but the e f f e c t may be minimized by the a d d i t i o n of an o r ganic a c i d to the mobile phase (46). T

Determination of C o r r e c t i o n for Sample Loss Due to Adsorption. Adsorption of the PGS to glassware can be a cause of s i g n i f i c a n t l o s s e s of samples. The problem i s p a r t i c u l a r l y p r o nounced for r e l a t i v e l y pure samples of PGS. The adsorption p r o cess described by the Langmuir isotherm i n d i c a t e s t h a t , at very low c o n c e n t r a t i o n s , most m a t e r i a l i s adsorbed to the glassware. As the c o n c e n t r a t i o n i s i n c r e a s e d , the a d s o r p t i o n s i t e s are s a t urated and thus, a greater percentage of sample i s recovered. The recovery of PGS at the nanogram l e v e l i s s e n s i t i v e to l o s s e s by a d s o r p t i o n , while at the microgram l e v e l a d s o r p t i o n i s i n s i g nificant. T h i s a l s o i n d i c a t e s that estimation of recovery by use of i n t e r n a l standards r e q u i r e s that the standard should be added at l e v e l s approximating those of the sample. The s i l y l a t i o n of a l l glassware that contacts the p l a n t ext r a c t has proven to e f f e c t i v e l y reduce adsorption l o s s e s . As diagrammed i n F i g u r e 8, the hydroxy1 adsorption s i t e s on the s i l i c a surface can be coated with d i c h l o r d i m e t h y l s i l a n e . The unreacted c h l o r i d e groups are then d i s p l a c e d with methanol i n a substitution reaction. A secondary advantage of the s i l y a t i o n process i s that water w i l l not adhere to the g l a s s s u r f a c e . Aqueous residues bead together, which allows more e f f i c i e n t sample transfers.



238

PLANT GROWTH

SUBSTANCES

Figure 6. Effect of pH on anion exchange of IAA. Column = Vydac AX — TP (10μm); 3.9 mm ID χ 20 cm,flow= 2.0 cm min' , mobile phase = 0. 'ΝαΗ ΡΟ — Ήα ΗΡΟ, buffer. Pump = Waters Associates 6000A, solvent pro grammer, Waters Associates 600. 3

2

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2

1

(


8.

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239

1 Downloaded by UNIV LAVAL on May 10, 2016 | http://pubs.acs.org Publication Date: September 27, 1979 | doi: 10.1021/bk-1979-0111.ch008

CUD

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ο

o

Kl Su



Figure 8. Diagrams of the silylation reaction of glassware


H > 5!

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8.

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241


Conclusion A n a l y t i c a l methods f o r PGS research have been g r e a t l y im proved during t h i s past decade. GLC-MS a n a l y s i s has proven to be the method of c h o i c e , p a r t i c u l a r l y when appropriate i n t e r n a l s t a n dards are used for accurate assessment of PGS recovery. HPLC, the most r a p i d l y developing form of separation s c i e n c e , should sub s t a n t i a l l y enhance present PGS a n a l y t i c a l e f f o r t s . One advantage of HPLC i s the s u b s t a n t i a l p u r i f i c a t i o n obtained f o r PGS compounds from crude plant e x t r a c t s . For a n a l y t i c a l i d e n t i f i c a t i o n by i n strumentation, scrupulous p u r i f i c a t i o n i s r e q u i r e d , along with s e l e c t i v e i d e n t i f i c a t i o n of the PGS. P r e f e r a b l y , two d i f f e r e n t a n a l y t i c a l procedures should be u t i l i z e d for p o s i t i v e i d e n t i f i c a t i o n of a given compound.

Manuscript no. 10,818 of the A g r i c u l t u r a l Experiment U n i v e r s i t y of Minnesota, Saint P a u l , MN 55108.

Station,

Literature Cited. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Hillman, J. R., Ed. "Isolation of Plant Growth Substances"; Cambridge University Press: London, 1978; p. 157. Sweetser, P. Β., in "Proceedings of the Fifth Annual Meeting of the Plant Growth Regulator Working Group", Abdel-Rahman, M., Ed., 1978; pp. 1-19. Hedden, P., in "Proceedings of the Fifth Annual Meeting of the Plant Growth Regulator Working Group", Abdel-Rahman, M., 1978; pp. 33-44. Brenner, M. L . , in "Proceedings of the Fifth Annual Meeting of the Plant Growth Regulator Working Group", Abdel-Rahman, M., Ed., 1978; pp. 20-32. Russel, S., in "Gibberellins and Plant Growth", Krishnamoorthy, N. J., Ed., John Wiley and Sons: New York, 1978; pp. 1-34. Cram, S. P.; Risby, T. H. Anal. Chem., 1978, 50:213R-243R. Gray, R. T.; Mallaby, R.; Ryback, G.; Williams, V. J. Chem. Soc. Perkin II, 1974:919-924. Most, Β. H.; Gaskin, P.; MacMillan, J. Planta (Berl.), 1970, 92:41-49. Railton, I. D.; Reid, D. M.; Gaskin, P,; MacMillan, J. Planta (Berl.), 1974, 117:179-182. Watanabe, N.; Yokota, T.; Takahashi, N. Plant and Cell Physiol., 1978, 19:1263-1270. Binks, R.; MacMillan, J.; Pryce, R. J. Phytochem., 1969, 8: 271-284.


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12. 13. 14. 15. 16.


17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

P L A N T G R O W T H SUBSTANCES

Durley, R. C.; MacMillan, J.; Pryce, R. J. Phytochem., 1971, 10:1891-1908. Frydman, V.M.; MacMillan, J. Planta, 1973, 115:11-15. Yokota, T.; Hiraga, K.; Yamane, H. Phytochem., 1975, 14: 1569-1574. Greenwood, M. S.; Shaw, S.; Hillman, J. R.; Ritchie, Α.; Wilkins, M. B. Planta, 1972, 108:179-183. Bridges, I. G.; Hillman, J. R.; Wilkins, M. B. Planta, 1973, 115:189-192. DeYoe, D.; Zaerr, J. B. Plant Physiol., 1976, 58:299-303. Feung, C.; Hamilton, R. H.; Mumma, R. D. J. Agri. Food Chem., 1975, 23:1120-1124. Ehmann, Α.; Bandurski, R. S. Carbohyd. Res., 1974, 36:1-12. Upper, C. D.; Helgeson, J. P.; Schmidt, C. J., in "Plant Growth Substances 1970", Carr, D. J., Ed.; Springer-Verlag: New York, 1972; pp. 798-807. van Staden, J.; Drewes, S. E. Plant Sci. Lett., 1975, 4: 391-394. Young, H. Anal. Biochem., 1977, 79:226-233. Horgan, R.; Hewett, E. W.; Purse, J. G.; Horgan, J. M.; Wareing, P. F. Plant Sci. Lett., 1973, 1:321-324. Morris, R. O. Plant Physiol., 1977, 59:Mass spectroscopic identification of cytokinins, pp. 1029-1033. Dauphin, B.; Teller, G.; Durand, B. Planta, 1979, 144:113119. Williams, P. M.; Bradbeer, J. W.; Gaskin, P.; MacMillan, J. Planta (Berl.), 1974, 117:101-108 Gaskin, P.; MacMillan, J., in "Isolation of Plant Growth Sub stances", Hillman, J. R., Ed.; Cambridge University Press: London, 1978, pp. 79-96. McDougall, J.; Hillman, J. R., in "Isolation of Plant Growth Substances", Hillman, J. R., Ed.; Cambridge University Press: London, 1978; pp. 1-26. Rivier, L.; Milon, H.; Pilet, P. E. Planta, 1977, 134:23-27. Little, C. Η. Α.; Heald, J. L.; Browning, G. Planta, 1978, 139:133-138. Mann, J. D.; Jaworski, E. G. Planta (Berl.), 1970, 92:285291. Bandurski, R. S.; Schulze, A. Plant Physiol., 1974, 54:257262. Cohen, J. D.; Bandurski, R. S. Planta, 1978, 139:203-208. Dekhuijzen, H. M.; Gevers, E. C. T. Physiol. Plant., 1975, 35:297-302. Letham, D. S. Planta (Berl.), 1974, 118:361-364. Thompson, A. G.; Horgan, R.; Heald, J. K. Planta, 1975, 124: 207-210. Saunders, P. F., in "Isolation of Plant Growth Substances", Hillman, J. R., Ed.; Cambridge University Press: London, 1978; pp. 115-134.


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38. 39. 40. 41. 42. 43.


44. 45. 46. 47. 48. 49. 50.

51. 52. 53. 54. 55. 56. 57.

58. 59. 60. 61. 62. 63.


243

Caruso, J.; Smith, R. G.; Smith, L. M.; Cheng, T.; Daves, G. D., Jr. Plant Physiol., 1978, 62:841-845. Magnus, V.; Bandurski, R. S. Plant Physiol., 1978, 61 (Suppl.):63. McWha, J . Α.; Hillman, J . R. Z. Pflanzenphysiol., 1974, 74: 292-297. Walton, D. C.; Dorn, B.; Fey, J. Planta, 1973, 112:87-90. Hillman, J . R.; Math, V.B.; Medlow, G.C. Planta, 1977, 134: 191-193. Summons, R. E.; McLeod, J . K.; Parker, C. W.; Letham, D. S. FEBS Let., 1977, 82:211-214. Shindy, W. W.; Smith, O. E. Plant Physiol., 1975, 55:550554. Wightman, F., in, "Plant Growth Regulation", P i l e t , P. E., Ed.; Springer-Verlag: Berlin, 1977, pp. 77-90. Ciha, Α.; Brenner, M. L.; Brun, W. A. Plant Physiol., 1977, 59:821-826. Carnes, M. G.; Brenner, M. L.; Andersen, C. R. J . Chromatogr., 1975, 108:95-106. Durley, R. C.; Kannangara, T.; Simpson, G. M. Can. J. Bot., 1978, 56:157-161. Reeve, D. R.; Yokota, T.; Nash, L.; Crozier, A. J . Exp. Bot. 1976, 27:1243-1258. Reeve, D. R.; Crozier, Α., in, "Isolation of Plant Growth Substances", Hillman, J . R., Ed.; Cambridge University Press: London, 1978; pp. 41-78. Suttle, J.; Zeevart, J. A. D. Plant Physiol., 1978, 61 (Suppl.):49. Düring, H.; Bachmann, O. Physiologia P l . , 1975, 34:201-203. Düring, H. Experentia, 1977, 33:1666-1667. Sweetser, P. B.; Vatvars, A. Anal. Biochem., 1976, 71:68-78. Challice, J . S. Planta (Berl.), 1975, 122:203-207. Hahn, H. Plant Cell Physiol., 1976, 17:1053-1058. Cole, D. L.; Leonard, N.; Cook, J . C., Jr., in, "Recent Developments i n Oligonucleotide Synthesis and Chemistry of Minor Bases of tRNA," International Conference, Poland, 1974, Uniwersytet Im. Adama Mickiewicza Press: Poland, 1974, pp. 153-174. Kannangara, T.; Durley, R. C.; Simpson, G. M. Physiol. Plant., 1978, 44:295-299 Holland, J . Α.; McKerrell, E. H.; Fuell, K. J.; Burrows, W.J. J. Chromatogr., 1978, 166:545-553. Heftmann, E.; Saunders, G. Α.; Haddon, W. F. J. Chromatogr., 1978, 156:71-77. Sweetser, P. B.; Swartzfager, D. G. Plant Physiol., 1978, 61:254-258. Baker, J . K.; Skelton, R. E.; Ma, C. J. Chromatogr., 1979, 168:417-427. L i , K.; Arrington, J . Anal. Chem., 1979, 51:287-291.


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64.

Swartz, H. J.; Powell, L. E. Plant Physiol., 1978, 61 (Suppl.):63. 65. Stoessl, Α.; Venis, M. A. Anal. Biochem., 1970, 34:344-351. 66. Knegt, E.; Bruinsma, J . Phytochem., 1973, 12:753-756. 67. Mousdale, D. Μ. Α.; Butcher, D. H.; Powell, R. G., in, "Iso lation of Plant Growth Substances," Hillman, J. R., Ed.; Cambridge University Press: London, 1978, pp. 27-40. 68. Kamisaka, S.; Larsen, P. Plant and C e l l Physiol., 1977, 18: 595-602. 69. Dennis, F. G., Jr. HortScience, 1977, 12:217-220. 70. Milborrow, Β. V.; Mallaby, R. J . Exp. Bot., 1975, 26:741748. 71. Atsumi, S.; Kuraishi, S.; Hayashi, T. Planta (Berl.), 1976, 129:245-247. 72. Browning, G.; Saunders, P. F. Nature, 1977, 265:375-377. 73. Railton, I. D.; Rechav, M. Plant Sci. Lett., 1979, 14:75-78. 74. De Zeevw, R. Α.; Jonkman, J . H. G.; van Mansvelt, F. J . W. Anal. Biochem., 1975, 67:339-341. 75. Martin, G. C.; Nishijima, C. HortScience, 1977, 12:212-216. RECEIVED June 19, 1979.


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