COMPARISON OF QUANTITATIVE DATA FROM ATOMIC ABSORPTION SPECTROMETRY AND X-RAY MICROANALYSIS FOR INORGANIC AND BIOLOGICAL SAMPLES

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COMPARISON OF QUANTITATIVE DATA FROM ATOMIC ABSORPTION SPECTROMETRY AND X-RAY MICROANALYSIS FOR INORGANIC AND

BIOLOGICAL SAMPLES

P. Mery, D. Chriqui, D. Gallant, B. Bouchet, B. Tripathi

To cite this version:

P. Mery, D. Chriqui, D. Gallant, B. Bouchet, B. Tripathi. COMPARISON OF QUANTITATIVE

DATA FROM ATOMIC ABSORPTION SPECTROMETRY AND X-RAY MICROANALYSIS FOR

INORGANIC AND BIOLOGICAL SAMPLES. Journal de Physique Colloques, 1984, 45 (C2), pp.C2-

473-C2-479. �10.1051/jphyscol:19842108�. �jpa-00223775�

JOURNAL DE PHYSIQUE

Colloque C2, supplément au n°2j Tome 45

?

février 1984 p3-ë^ C2-473

COMPARISON OF QUANTITATIVE DATA FROM ATOMIC ABSORPTION SPECTROMETRY AND X-RAY MICROANALYSIS FOR INORGANIC AND BIOLOGICAL SAMPLES

P. Mery, D. Chriqui, D . J . G a l l a n t * , B. Bouchet* and B.K. T r i p a t h i * *

Vniversite P. et M. Curie, Laboratoire de Cytologie et Morphogenese Vegetates, E.R.A. C.N.R.S. n° 616, 75230 Paris, France

*Institut National de la Recherche Agronomique, Laboratoire de Technologie des Aliments des Animaux, Unite de Microscopie, Rue de la Geraudiere,

44072 Nantes Cedex, France

**Universite P. et M. Curie, Laboratoire d' Histophysiologie Vegetate, 75230 Paris, Franee

RESUME

Une comparaison entre l'analyse quantitative d'échantillons par absorption atomique de flamme (AAS) et par microanalyse en sélection d'énergie (EDS) a été effectuée en étudiant un complexe inorganique (milieu de MURASHIGE et SKOOG) ou des explants foliaires de Datura innoxia en culture in vitro sur ce même milieu en pré- sence de saccharose et/ou de kinétine.

En ce qui concerne le complexe inorganique (poudre minérale), l'absorption atomique donne de meilleurs résultats approchant les valeurs théoriques à moins de 1% près; la microanalyse donne des résultats avec une erreur de 4% à 25KeV.

Dans les mêmes conditions opératoires, la microanalyse de cendres des explants foliaires n*a pu permettre de mesurer significativement les pertes ou gains de K+.

Une quantification plus effective a cependant pu être obtenue par l'analyse en EDS des échantillons biologiques soigneusement lyophilisés.

ABSTRACT

Quantitative data from Atomic Absorption Spectroscopy (AAS) and X-rays Energy Dispersive System (EDS) have been compared using either an inorganic complex

(MURASHIGE and SKOOG's medium) or Datura innoxia leaf explants grown in vitro on this medium with an addition of sucrose and/or kinetin.

For the inorganic complex (mineral powder) AAS gave mean results approaching the theoretical values with an error lower than 1% although EDS results showed an error near 4% at 25KeV. Used in the same experimental conditions on their ashes, EDS of the biological samples was not able to account significantly the K+ efflux and uptake of the leaf explants. But more effective quantification was obtained when analysis were directly effectued on biological samples carefully freeze-dried.

1. INTRODUCTION

Although qualitative results obtained by X-ray microanalysis are now undoubt- less, very few informations about their quantitative validity are still available.

The aim of this work was to compare the quantitative data obtained by both X-ray microanalysis and atomic absorption spectrometry on samples of known and unknown compositions. Such preliminary studies were attempted in order to prepare further in situ evaluations of K+ and Ca++ in a case of cell proliferation induction.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842108

JOURNAL DE PHYSIQUE

2. MATERIAL AND METHODS 2.1. MATERIAL

Datura innoxia leaf explants are grown in vitro on MURASHIGE and SKOOG's basic medium added with sucrose and kinetin (BMSK). In this case, mitosis of reactivation occur at the 48th hour, followed by a callus and bud regeneration. Both sucrose and kinetin are necessary for such a process

;

no regeneration occur on basic medium (BM) alone. Before this studies, ion analysis were performed for inorganic samples of known composition (MURASHIGE and SKOOG's medium powder) and organic samples of unknown composition (control and treated leaf explants), both on the freeze-dried material or on their ashes.

2.2. METHODS

2.2.1. Atomic Absorption Spectrometry (AAS)

K+ and Ca++ contents were determined after dehydration of the samples in a drying oven (24 h at llO°C) then mineralization by the classical procedure HN03- HC104 solutions were analyzed using a Varian-Techtron AA-4 type atomic absorption spectrometer. Results are given in mg of K+ or Ca++ per g of dry matter.

2.2.2. X-ray microanalysis procedures (EDS)

Two ways of sample preparation (ashes and freeze-dried explants) were tested

;

ashes were covered by a carbon film (20 nm) to avoid charges phenomena. Element analysis was performed using a JEOL JSM 50A S.E.M. fitted with an EDAX 711 EDS System.

Preliminary studies for checking best conditions of analysis were made with various counting times and accelerating voltages; further studies used the following data

:

detector tilting

:

45O

;

X-ray emergency angle

:

34O3

;

sample tilting

:

O 0

;

acceleration voltage

:

25 KeV

;

analyzed area

:

3 x 3 cm at 300 X

;

counting time

:

200 s (one scan per s)

;

permanent control of the electron beam stability

;

resolu- tion given for Mn, Ka peek

:

148 eV

;

standard

:

BaC12

;

Z.A.F. correction.

3. RESULTS

3.1. QUANTITATIVE DATA FROM A COMPLEX INORGANIC SAMPLE OF KNOWN COMPOSITION (MURASHIGE and SKOOG's medium)

3.1.1. Real values

I I I I I I I I I

I Elements I Na I Mg I P I S ( C1 I K I Ca I I

I I I

I I I I I I I I

I XI

:

mg/g I I I I I I I I I

I of powder 10.97 17.8 18.3 111.1 145.0 1166.1 125.7 1

I I I I I

I I x2

:

I I I I I I I I

I I I I I I I I

I relative % 1 0.37 1 2.95 1 ^3.13 1 4.19 1 16.99 1 62.66 1 ^9.70 1

1 I I I

I I x3

:

I I I I I I I I

I I I I I I I I

I absolute % 1 ^0.097 1 ^0.78 1 ^0.83 1 ^1.11 1 ^4.50 1 16.60 1 ^2.57 1

I I I I

3.1.2. AAS results for K+ and Ca++

I I I I I

I I ^Kc 1 ^Ca++ I ^K/Ca I

3.1.3. Microanalysis-X assays

I

1 AAS results I I / ^between I

I (B +- 2 SE) 1 163.2 +- ^4.2 1 21.2 +- 0.9 / 7.2 and 8.2 1

3.1.3.1. Results for various accelerating voltages compared to real contents x3

I

I

T I I I I I I I I

I ^voltage I ^{I N a I M g l P I S I C 1 1 K I C a I}

I I I I I

I I I I I I I I I I

I I R I - 1 0.4051 0.3531 1.687112.8881 3.8941 3.9861

1 ^{15 KeV} 1 ^x3 - ² 1 ^-0.097 1 ^-0.375 1 ^-0.477 ( +0.577 1 ^+8.388 1 ^-12.716 ( ^+1.416 1

I I I 1 I I I I

I I I I I I I I I I

I

i I X I 1 0.226 1 0.189 1 ^1.172 1 7.66 1 6.174 I ^2.848 1

1 20 KeV I ^x3 - R 1 ^-0.097 1 ^-0.554 1 ^-0.641 1 +0.062 1 +3.16 1 -10.436 1 ^+0.278 1

I I I I I

I real contents I I I I

1 ^(xl) 1 ^166.1 1 ^25.7 1 ^6.46 1

I I I I I

I ^variation I I 1 1

I (xl - X) 1 ^2.9 1 ^4.5 1 ^1.24 1

I I I I

I I I I I I I I I

I ^X ( 0.0381 0.5261 0.2181 0.5541 4.1221 15.6551 1.9231 25 KeV ( ^x3 - E 1 ^-0.060 1 ^-0.254 1 ^-0.612 1 ^-0.556 1 ^-0.378 1 - 0.955 1 ^-0.647 1

I I I I

I I I I I I I I I

I

%

I - 1 0.192 1 ^0.150 1 0.256 1 4.263 ( 3.564 1 1.230 1

30 KeV I x3 - ² 1 -0.097 1 -0.588 1 -0.680 1 -0.854 1 -0.237 1 -13.046 1 -1.340 1

I

3.1.3.2. Effects of counting times

I

Comparing the concentration rates after differents counting times 2 50, 100, 150 and 200 sec), we saw slight but regular increases of S, P , Ca and K, and slight decreases and then increases of C1 and Mg. The Mg concentration rate was very irregular below 50 sec. According to a better ratio peak/background, counting time was choosen at 200 sec.

I I I I I

C2-476 JOURNAL DE PHYSIQUE

3 . 2 . QUANTITATIVE DATA FROM

AN ORGANIC

SAMPLE OF

UNKNOWN

COMPOSITION (DATURA

I N N O X I A

LEAVES)

3.2.1. Microanalysis

X

3.2.1.1. On a s h e s

Mean v a l u e s

:

i ^{65.8 %} i ^10.4 %i I 6.3 I

I I I I I I I I I I

I I

I ^Na I ^Mg I

P

I ^S I C 1 I

^K

I Ca I T o t a l I ^K/Ca I

3.2.1.2. On f r e e z e - d r i e d samples ( l e a f d i s c s )

I

I I I I I I I I I I

1 Na I Mg 1

^P

I ^S I

^{C 1}

^]

^K

I Ca 1 T o t a l I K / C a ]

I I I I I I I I

I I I I I I I I I I I

1 0 . 2 7 1 / 0.173 1 0 . 2 2 9 1 1 . 1 2 0 / 2.358 1 7.898 1 0 . 5 2 9 1 12.578 1 14.9 1

Line

I

I I I I I I = \ = I

I I I I 1 62.8 % 1 4 . 2 % I I I

I I I I I I I I I ^I I ^I

I I I I I I I I I I I

1 0.072 1 0.076 1 0.091 1 0.399 1 0.834 1 3.601 1 0.118 1 5.191 1 30.2 1

Line 2 1 I I I I I = I = I

I I I I 1 1 69.4 % 1 2 . 3 % I 1 I 1 I

I I I I I

Mean v a l u e s

:

1 66.1 % 1 3 . 2 5 % I 1 20.3 1

3.2.2. AAS r e s u l t s f o r K+ and Ca++

I I I

Mean v a l u e s (+-SE) a f t e r 7 l i n e s o f a n a l y s e s ( t o t a l e f f e c t i v e

:

40 f o l i a r d i s c s o f

1

cm d i a m e t e r ) each l i n e being analysed t w i c e . d.m.

:

dry m a t t e r

;

f.m.

:

f r e s h m a t t e r .

I I

I I

I I

I I I I I I I I

I 1 ^0.114 1 1.129 1 2.821 1 4.041 1 11.976 1 49.888 1 6.189 1 76.158 1 8 . 0 5 1

1 Line

I

I I I I I I = [ = I

1 ^{6 5 . 5 %} 1 ^{8 . 1 %} I ^I

I I I I I I I I ^I

I Units 1 K+ c o n t e n t I Ca++ c o n t e n t 1 K/Ca 1

I I I I I

I I I I I

I I I I I I I I I I ^I I

I 1 0 . 1 8 9 1 0 . 9 2 0 1 1 . 9 8 1 1 1 . 2 1 4 1 6 . 0 2 5 1 2 8 . 8 2 7 1 4 . 2 1 5 1 4 3 . 3 7 1 1 6 . 8 4 1

I Line 2 1 I I I I I = = I

I I 1 I I I 1 66.5 % 1 ^{9 . 7} % I 1 I I

I I I I ^I

I I I I I

I I I I I I I I

I 1 0 . 1 5 9 1 0 . 6 2 1 1 1 . 0 7 4 1 0 . 8 2 2 1 4 . 7 3 7 1 2 2 . 9 9 ( 4 . 7 1 1 1 3 5 . 1 1 4 1 4 . 9 ^I 1

/ Line 3 1 I I I I I = I = / I I

1 65.5 % 1 13.4 % I

I I I I I

I I

I I

I ^{I I I}

I I I I I

I

3.2.3. Conclusions

- Comparison of the K/Ca ratio by the two methods of analysis

:

mean ratios of 4.6 and 6.3 are respectively given by AAS and microanalysis on ashes, with larger fluctuations in the second case.

- Comparison of the two methods of sample preparation for microanalysis

:

K content is not strongly affected by this factor and varies from 65.8 % for ashes to 66.1 % for freeze-dried specimen. On the contrary, Ca content is largely dependent of this factor and varies from 10.4 % for ashes to 3.25 % for freeze-dried samples.

3.3. APPLICATION TO PLANT PHYSIOLOGY (evolution of K+ content of leaf discs during the first steps of the culture)

3.3.1. AAS results

K + relative content per

explant @

t

1 : +

1 6 12 24 hours 48

R N A D N A 0

M i t o s i s ^First

I

j

^Synthesis

_{10 hours 48}

Fig.1

: AAS

results -

A :

Evolution in function of time of the K+ relative content

of leaf explants grown in vitro on basic medium (BM) and basic medium with sucrose

and kinetin (BMSK); Control (see the arrow); B

:

Evolution of the same samples dry

matter; Control (see the arrow); C

:

First biological steps.

JOURNAL DE PHYSIQUE

3.3.2. Microanalysis-X results 3.3.2.1. On ashes

K+percentage (mean values)

BM

60.

*

0 6 hours 24

F i g .

2 :

EDS results

:

evolution in function of time of K+ relative content in ashes of leaf explants grown in vitro on basic medium (BM) and basic medium with sucrose and kinetin (BMSK); Control (see the arrow).

3.3.2.2. On freeze-dried leaf discs

I I I I I

I 1 CONTROL 1 ^BM I ^BMSK I

3.3.2.3. Conclusions

i ^{I I I}

1 0

I 1 ^{1 h 1} 2 4 h 1 7 2 h / l h 1 2 4 h 1 7 2 h I

I ^Distal I 1 I I I I I 1

[ pole Dp 1 ^3.443 1 2.555 1 2.028 1 1.334 1 0.493 1 ^1.446 1 0.497 1

- AAS reveals an efflux of K+ around the 3rd hour followed by an influx at the 48 th hour, especially on BMSK. X-ray microanalysis on ashes is not able to account such slight variations.

I

- X-ray microanalysis on freeze-dried samples is although able to indicate a migration of K+ from the distal pole to the proximal pole of the explants since the first hour such an accumulation at the basal end is more intense on BMSK than at the 72th hour.

I

4. CONCLUSIONS

4.1. ABOUT METHODS

I Proximal ( I

I I I I I I I

I pole Pp 13.984 11.569 13.980 12.004 10.598 12.426 11.064 1

Comparison of the results obtained by the two methods on spec.imens of known and unknown composition reveals that quantitative data with X-ray microanalysis depend on numerous parameters which must largely be used and interpreted with great caution. Preliminary tests are necessary to determine the best acceleration voltage,

I I I

I I I I I I

I Pp/Dp 11.16 10.61 11.96 11.50 11.21 11.68 12.14 1

I I

best sample orientation and minimal counting time

;

the other conditions must be constant. The sample preparation methods (ashes or freeze-drying) influence also the quantitative data

;

the results obtained with ashes seem to be more homogeneous than with freeze-dried saoples. Respectful 07 Chest cautions, it romains actually a more greater lack of precision with quantitation by X-ray microanalysis than by AAS, fro21 which the data - for complex samples of known composition - are always nearer to the real data and with lower variance. Thus it seems that X-ray microanalysis could not account for slight variations of a given element in total samples (ashes or freeze- dried) and therefore could be difficultly used as a tool for minute in situ quanti- tative analysis. AAS could be used as a complementary method for some controls on total samples but it remains actually a need of other controls (internal standards) for in situ analysis.

ABOUT K+ EVOLUTION ON THE EXPLANTS IN INDUCTIVE OR NON-INDUCTIVE CONDITIONS

The two culture conditions are inductive of an early and strong leakage of K+

from the explants towards the media, followed after the 3rd hour by an influx of

this ion. Such observations are likely relative to the membrane disorganisa-

tion/reorganisation arising during the first hours in vitro, as previously shown, by

ultrastructural examinations. It must be pointed out that the influx of K+ is clear-

ly demonstrated on BMSK between the 24th and the 48th hour (period preceding the

first mitosis). It seems therefore that a relation between the K+ content and the

nucleic acid metabolism reactivation can be expected. Microanalysis-X should give a

suitable complement of information about the microlocalization of K+ during this

process, rather by analysis of X-ray images for each cell compartment on ultrathin

sections than by semi-quantitative in situ analysis.