Histochemistry 62, 337-345 (1979)

Histochemistry 9 by Springer-Verlag 1979

Elemental Analysis of Solid Microscopic Samples in the Flameless Atomic Absorption Cuvette Markus Lezzi Institute for Cell Biology, Swiss Federal Institute of Technology,H6nggerberg, CH-8093 Zfirich, Switzerland

Summary. Solid microscopic samples are precisely located by means of a newly developed applicator under microscopic control in the center of a flameless graphite tube cuvette. The parts of the applicator that reach into the cuvette are made of quartz which can be cleaned by heating. The values for the K and Na content of samples such applied are highly reproducible. No contamination could be detected. Solid samples yield higher signals than liquid samples. A method of calibration is described which uses lyophilized pieces of egg albumin containing known amounts of K and Na as standards. Introduction By the introduction of the Massmann (1968) cuvette an electrically heated graphite tube - the quantitative determination of picogram amounts of Na, K, Mg and other physiologically important elements by atomic absorption spectrometry became possible. Usually, the samples are introduced into this cuvette (or modifications thereof) in a liquid form by means of a micropipette. Therefore, if solid samples are to be measured they must first be dissolved. However, this is not always possible or desirable either because the respective samples are insoluble or because they occur in such limited amounts that the resulting solutions would be very dilute or very small in volume. The problems of preparing and handling such solutions without contamination, particularly in the case of alkaline and alkaline earth elements, are immense. If solid samples could be applied directly, i.e., without further preparations, these problems could be circumvented and more reproducible results could be obtained. With solid samples, on the other hand, the mode of application into the graphite tube is not trivial especially when the samples are very small. The introduction and exact positioning of the sample into the cuvette has to be accomplished by an instrument which does not contaminate the interior of the cuvette. The known methods of solid sample application are not satisfactory

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338

M. Lezzi

in this respect: the graphite microboat (provided by Instrumentation Laboratory, Lexington, Mass. USA) which stays inside the graphite tube during atomization is r e l a t i v e l y l a r g e (5 x 5 m m ) so t h a t t h e d a n g e r o f c o n t a m i n a t i o n d u r i n g l o a d i n g ( f o r w h i c h it h a s t o b e r e m o v e d f r o m t h e c u v e t t e ) c a n n o t b e e x c l u d e d ; t h e t o o t h p i c k s u s e d b y B e r g g r e n et al. ( 1 9 7 8 a n d p e r s o n a l c o m m u n i c a t i o n ) f o r d e p o siting the sample in the cuvette need no further discussion in terms of contamination hazards. In the present paper a new method of application of solid microscopic s a m p l e s is d e s c r i b e d w h i c h a v o i d s c o n t a m i n a t i o n a n d w h i c h g i v e s h i g h l y r e p r o ducible results.

Materials and Methods The present investigations were performed with an atomic absorption spectrometer 300 S from PerkinElmer (Ueberlingen, Germany) equipped with the flameless attachment HGA 72. It has to be emphasized that the same method of solid sample application can be used with other instruments, as well, after slight modifications of the applicator (Fig. 1) and the cuvette. (With IL 751 plus 555 from Instrumentation Laboratory, for example, the applicator shown in Fig. 1 a may be used and a second hole ( ~ 1 mm) has to be bored into the cuvette for viewing.) K and Na were measured at 766 or 589 nm with a slit setting of 2 or 7, respectively. The energy meter was always set to 35-40 gAmp. Heating program A was normally chosen for K, program B (see Table 1) for Na. The signals were recorded at 2, 5 or 10 mV sensitivity and their peak heights measured with a ruler. In some instances the integrals of the signals were also determined with an integrator (constructed by Dr. P. Wuhrmann). In each cycle the instrument was autozeroed at the ashing step. The applicator used for depositing the solid microsamples in the cuvette is depicted in Fig. 1 b. It consists in its core part of a capillary with a tip diameter smaller than that of the sample (~50-100 gm) which, thus, is held at the capillary's tip by aspiration when the applicator is connected to vacuum via a three-way stopcock. The capillary is made of quartz such that it can be cleaned by heating in a gas flame. Depending upon the flameless equippment used and the path of access to the cuvette the tip of the capillary is bent. This is in order to reach better the microdepression (0.2 x 0.2 mm) drilled in the bottom of the cuvette (to prevent sample dislocation by the purging gas). The other parts of the applicator serve 1) to protect the capillary plus sample during the transfer and application process and 2) to allow a controlled deposition of the sample in the cnvette. The capillary can be retracted very smoothly into a quartz nozzle. The nozzle's tip is wider than the opening in the cuvette through which the sample is to be applied. Furthermore, it is rounded and polished such that the applicator resting firmly on the opening of the cuvette can be tilted in every direction much like a ball joint in its socket. With the type shown in Fig. 1 b the mouth of the nozzle is wide enough that the capillary plus sample does not touch the edge of the nozzle (provided the capillary holder moves without any play in the mantle of the applicator); with the type shown in Fig. 1 a the capillary is guided centrically by a constriction in the nozzle's tip. The deposition of the sample inside the graphite cuvette is monitored with a stereo-microscope (magnification 30 x) through a hole in the center of the graphite tube. The cuvette's interior is illuminated either transversely through the opening opposite to the applicator's entrance or perpendicularly through the optics of the microscope. All solutions were made up with quartz bidistilled water in 40 ml polyethylene bottles which were soaked once in 1 N HC1 and 5 times in quartz bidistilled H20 for 2 days each. NaC1 and KC1 (p.a. quality) were from Merck (Darmstadt). Chicken egg albumin (5 x recrystallized, NaC1 content < 0.1%) was from Serva (Heidelberg). The stock solutions contained Na plus K in concentrations corresponding to those found in nuclear sap of Chironomus salivary glands, i.e., 20 and 100 mM, respectively, plus 0% or 20% (weight per weight) egg albumin. The final dilutions were made to attain a Na and K content per 10 or 20 p.1 sample in the range of that present in the solid samples (see Fig. 3). The actual concentrations in the stock solution and dilutions were

Atomic Absorption of Solid Samples

339 4

3

1

2

a)

b)

113 9

14

15

11

14

2

c) Fig. 1. Applicator for controlled deposition of solid microsamples in the graphite tube cuvette of the flameless atomic absorption apparatus, a Retracted state. Nozzle type for narrow access openings, h Extended state. Nozzle type for wide access openings, e Explode view of capillary holder. J: silicone vacuum tubing connected via a three-way-stopcock to an aspirator. 2: shaft of capillary holder, brass. 3: mantle of applicator, brass. 4. adjustment screw fixed to the hoIder for confining the span o f extension and retraction of the capillary. 5. rim to prevent sliding back of nozzle. 6: O-rings for firm mounting of the nozzle. 7: quartz nozzle, type for narrow access openings; note the constriction for centric guidance of the straight capillary (8) pulled from a 0.9 mm quartz tube. 9: tightening screw for capillary mounting. 10: quartz nozzle type for wide access opemngs. 11." quartz capillary type with bent tip. 12. graphite tube cuvette; the cuvette's interior is viewed through the upper hole with a stereomicroscope, through the left opening (opposite to the access opening) it is illuminated with a 12 V microscope lamp. 13. microdepression (0.2 x 0.2 mm) for the sample. 14." O-rings and spacer (15) for capillary mounting. Total length of applicator: 19.5 cm Table 1. Heating programs of the flameless cuvette Program

A B " b

Drying

Ashing

Atomizing

Cleaning

T~

tb

T

t

T

t

Purge gas

T

t

85 85

15 15

680 680

15 15

2300 2550

5 6

off on

2600 -

3 30

Nominal temperature in ~ Seconds

calculated on the basis of gravimetric measurements (for correction of the Na content of solutions containing egg albumin, see Results). The liquid samples were delivered into the cuvette with an Eppendorf micropipette. Solid standards were prepared by two different methods. Method I." Droplets (approximately 10gl) of the stock solution containing egg albumin were

frozen on a liquid nitrogen cooled copper plate, lyophilized and then soaked in silicone oil. They were broken in small pieces which after removal of the silicone oil were stored over P;O5 until weighing on an evacuated quartz fiber balance (Lezzi, 1979) and elemental analysis. This procedure follows essentially that for preparing lyophilized cell components, e.g., of larval Chironomus salivary glands and will be described in detail elsewhere.

340

M. Lezzi

MethodH: 0.1 ml of the stock solution containing egg albumin were pipetted into thoroughly cleaned and precalibrated 1 ml vials, weighed and frozen in liquid nitrogen. After lyophilization the vials wereclosed,weighed,and stored over PzOs. The weight loss of the sample during lyophilization was calculated and corrected for that of empty vials; it corresponded very precisely to the amount of water originally added for making up the stock solution (expected 80%, determined 79.7%). Small pieces of the dried materiat were weighed and analysed as described above.

Results

The Application Procedure Before each measurement the quartz capillary of the applicator is cleaned by moving it through a gas flame until the flame remains colorless. Care must be taken that thereby the capillary is not bent. Since electrostatic interactions make a controlled handling of the dry sample impossible the capillary and the sample are treated with an antistatic pistol (Zerostat Instruments Ltd., England). Then, the sample is aspirated onto the tip of the capillary under a stereomicroscope (magnification 30 x ) and the capillary is retracted into the applicator. The applicator is placed with its nozzle onto the right hand opening of the cuvette's case which is lifted above the spectrometer for loading. The capillary is slowly extended horizontally until it becomes visible in the viewing hole under the stereomicroscope. At this step of the procedure the vacuum is shut off by turning the three-way stopcock in the vacuum tubing. The applicator is tilted and rotated so that the sample can be deposited into the depression in the graphite tube. Thereafter the capillary is retracted, the applicator removed from the cuvette's case, the cuvette's case-closed and the measuring cycle started. Despite the antistatic treatment the detachment of the sample and its controlled placement into the depression is not always easy; therefore, it is sometimes advisable to remove the sample from the cuvette and to repeat the procedure starting with the antista}ic treatment, in any case the exact location of the sample has to be monitored carefully. Immediately after each measurement the blank value is determined by repeating above procedure without sample and by omitting the initial cleaning of the capillary. As a rule the blank value does not exceed that of air; otherwise, it is added to the value of the preceding sample and a second blank value is determined with the cleaned capillary.

K and Na Determination in Solid Samples The reproducibility of the present method and the possible involvement of systemic errors due to contamination, losses, weighing errors etc. was tested with solid standards of known K and Na content. Since most biological samples consist predominately of protein a proteinaceous matrix was chosen with a low content of metal ions. The egg albumin (5 • crystallized) from Serva has a nominal Na-contaminatiom of less than 1 mg per gram. Its exact content of K and Na was determined by atomic absorption measurements of a series

Atomic Absorption of Solid Samples

Fig. 2. Correlation between weight (as expressed by the deflection of the quartz fiber balance) and K content (as expressed by the peak height of the signal) of lyophilized egg albumin standards prepared by Method II. Deflection is given in arbitrary units (1 unit=0.155 ng). The signal is given in mm at 5 mV setting of the recorder (concerning the problem of converting it into gram K, see Text). Heating program A (Table 1). Open and closed circles : values of two independent series of determinations, a, b: albumin pieces jumped away during deposition (i.e., they could not be deposited into the microdepression of the cuvette)

34l

./

30o-

y ~o

Y

./"

2oo-

O lOO-

/~

.'>

o

J

i

i

400

600

800

Deflection

/

0.8

9

/

/ /

e/

0.~.

/ //

~/ /I 9

Fig. 3. The apparent K content of solid samples (egg albumin standards prepared by Method I). K contents calculated from the height (closed circles) or the integral (open circles) of the signals. Broken line: theoretical correlation calculated from the values of liquid standards. The aberrant value probably originates from a sample which was dislocated after deposition (see Fig. 2)

.~ 0.4"

o

/

o/

~

// 0.2-

o

// /

/ 20 Dry

4'0 Weight

6'0

(ng)

o f dilutions. The K a n d N a c o n t e n t s o f egg a l b u m i n were f o u n d to be 0.02 m g a n d 0.66 m g p e r g r a m d r y weight, respectively. Thus, the K c o n t a m i n a t i o n i n t r o d u c e d by the a l b u m i n to the l y o p h i l i z e d s t a n d a r d s is negligible w h e r e a s the p r e c a l c u l a t e d N a c o n t e n t o f the s t a n d a r d s has to be c o r r e c t e d b y a f a c t o r o f 1.44. A s evident f r o m Figs. 2 a n d 3 t h e r e is a g o o d c o r r e l a t i o n b e t w e e n weight a n d K - s i g n a l o f the solid a l b u m i n samples. T h o s e m e a s u r e m e n t s w h i c h y i e l d e d a clearly r e d u c e d K signal were d i s c a r d e d since they p r o b a b l y a r o s e f r o m s a m p l e s w h i c h were n o t l o c a t e d in the m i c r o d e p r e s s i o n o f the cuvette d u r i n g a t o m i z a t i o n . T h a t s o m e o f these s a m p l e s j u m p e d a w a y d u r i n g a p p l i c a t i o n was o b s e r v e d in the s t e r e o m i c r o s c o p e (see Fig. 2); o t h e r s m a y have been d i s l o c a t e d by the p u r g e gas s t r e a m d u r i n g the d r y i n g o r a s h i n g step. S a m p l e d i s l o c a t i o n h a p p e n s when the s a m p l e s are t o o large or i n c o r r e c t l y d e p o s i t e d o r when the d e p r e s s i o n is t o o wide o r t o o shallow.

342

M. Lezzi

Table 2. Determination of K and Na in lyophilized samples of various origin at different atomization temperature Type of sample

Method of preparation

Heating program

Number of determinations

Element content (rag per g dry weight) mean + standard deviation "apparent '""

actual b

14.90 • 0.7 15.00 +_1.1

13.0 13.0

a) Determination of K in solid standards Egg albumin Egg albumin

I II

A A

5 7

b) Determination of K in cellular components of Chironomus salivary glands Nuclei Cytoplasm Secretion

I I I

A A A

4 4 4

12.29• 6.29• 7.45•

~ c c

c) Determination of Na in solid standards, Egg Egg Egg Egg

albumin albumin albumin albumin

II II II I

A Ad B B

7 3 7 5

2.78 • 0.40 2.81 +0.25 2.29 + 0.23 2.20 • 0.13

2.17 2.17 2.17 2.17

Calculated by using the conversion factor (from signal to element content) determined with

liquid samples b c

Na determinations corrected for Na contamination of egg albumin (see Text) Calculated by using the conversion factor determined with solid samples Purge gas flow turned off during atomization

Solid samples consistently yielded K signals which were 15% higher than those of liquid samples of the same K content (see Fig. 3 and Table 3). This was true whether the samples were prepared according to Method I or Method II (see Table 2) indicating that contamination by K or loss of dry weight material during sample preparation is not the cause for this difference. The fact that the integrals of the K signals approximate more closely the theoretical values than their heights suggests that the difference between solid and liquid samples in terms of their K signal is due to a difference in their atomization kinetics. This notion is substantiated by the finding that the observed difference is dependent upon the atomization temperature as could be demonstrated with Na measurements (see Table 2): at 2300 ~ C, the Na signal of solid samples is 30% higher than that of liquid samples whereas at higher temperatures (2550~ C) this difference becomes insignificant. The purge gas flow rate does not seem to influence this effect. The applicator clearly does not contaminate the sample or the cuvette since the value of " b l a n k " measurements are indistinguishable from those of air (i.e., for K they are zero). As an illustration the record of a series of Na measurements is given in Fig. 4. The data in Table 2 show that Na determination with solid samples is also very reproducible (provided the values of determinations in which the sample could not successfully be deposited are omitted).

Atomic Absorption of Solid Samples

343

mm 150-

100-

10

503 5 2

b

~ 'a:_: t....J 5sec

Fig. 4. Recorder tracings of determination of Na in egg albumin standards prepared by Method II. Heating program A. Recorder set at 5 mV, automatic recording during atomization step. l 10, egg albumin pieces; 4: lost; 9': broken-off piece from sample 9. a: air (=idle run). b. " b l a n k s " ( = application procedure without sample). - The applicator's quartz capillary was cleaned by heating after sample 4 and between samples 7 and 8

This finding reemphasizes that even with Na the hazard of contamination or losses is minimal. In order to test the described method of solid sample application with biological material, K was determined in isolated nuclei, pieces of cytoplasm, and secretion of lyophilized salivary glands of oligopausing Chironomus tentans larvae. The values were standardized with solid egg albumin standards (Method H). The results given in Table 2 show that nuclei contain significantly more K per dry weight than cytoplasm or secretion (probability, P, for difference being zero: 40.01). The K content of nuclei corresponds to that previously published for the same stage (Wuhrmann et al., 1979).

Discussion

The present paper demonstrates that it is possible to analyze reproducibly by flameless atomic absorption spectrometry the elemental content of solid micro-

344

M. Lezzi

samples weighing less than 10 ng. For reproducible measurements the samples have to be precisely located in the center of the cuvette. By use of the special applicator described in this paper this can be accomplished without contaminating the cuvette or the sample. Clearly, reproducibility also depends upon the method of standardization of the values obtained by atomic absorption measurement. In the present work this was done by determining~the sample's dry weight on an evacuated quartz fiber balance (Lezzi, 1979). The accuracy and comfort of handling of this newly developed balance is better than that of conventional quartz fiber balances thus rendering standardization by dry weight a feasible and reliable method. Most unexpectedly it was found that solid samples yield higher signals than corresponding liquid samples. While the theoretical basis of this phenomenon cannot be discussed here its practical implication is of eminent importance. It makes indispensible the use of solid rather than liquid standards to calibrate the signals of solid samples. The present paper demonstrates that lyophilized pieces of egg albumin containing a known amount of K and Na are useful solid standards once the amount of Na in egg albumin has been determined. The difference between the signals of solid and liquid samples is temperature dependent indicating that, especially with solid samples, the temperature of atomization is very critical. Since most flameless graphite tube cuvettes exhibit a rather steep temperature gradient fi'om the center towards the ends even a slight deviation from the center in the position of the sample will perforce influence the yield of the signal. (This is particularly true when the peak height rather than the integral of the signal is measured.) Therefore, it is an absolute requirement that the exact location of the solid sample in the cuvette be carefully monitored with a microscope and that the sample be protected from dislocation in the purge gas stream by depositing it into a microdepression, The data on the K content of nuclei, cytoplasm, and secretion of larval Chironornus salivary glands illustrate the applicability of the described method. The difference between nuclei and cytoplasm in terms of K per dry weight is very large and highly significant. Since the electrochemical activity of K is the same in both compartments (Wuhrmann et al. 1979) this difference must be due to a higher water content and/or more extensive K binding in nuclei than in cytoplasm. The findings published by Kroeger et al. (1973) and by Wuhrmann et al. (1979) suggest that both contribute to the difference. Acknowledgments. The skilful construction of the applicator by Mr. K. Evers and Mr. R. Florin is gratefully acknowledged. I thank Mr. C. Hauri of the "Kunstgewerbeschule der Stadt Ziirich", class for Scientifique Drawing, for preparation of Figure 1. I am grateful to Dr. P. Wuhrmann and Mrs. U. Riesen-Willyfor help and advice. I thank Prof. Dr. H. Zuber, Institute for Molecular Biology and Biophysics,for use of the atomic absorption spectrometer. This work was supported by the Swiss National ScienceFoundation.

References

Berggren, P.-O., Berglund, O., Hellman, B. : Determination of calcium in microgram amounts of dried biological material by flameless atomic absorption spectroscopywith special reference to the pancreatic islets. Anal. Biochem.84, 393 401 (1978)

Atomic Absorption of Solid Samples

345

Kroeger, H., Tr6sch, W., M/iller, G. : Changes in nuclear electrolytes of Chironomus thummi salivary gland cells during development. Exp. Cell Res. 80, 329 339 (1973) Lezzi, M. : A gravimetric system for lyophilized samples in the submicrogram range. Histochemistry 59, 287-294 (1979) Massmann, H. : Vergleich von Atomabsorption und Atomfluoreszenz in der Graphitkuvette. Spectrochim. Acta 23B, 215-226 (1968) Wuhrmann, P., Ineichen, H., Riesen-Willi, U., Lezzi, M. : Change in nuclear potassium electrochemical activity and puffing of potassium-sensitive chromosome regions during Chironomus development. Proc. Natl. Acad. Sci. USA 76, 806-808 (1979)

Received June 6, 1979

Elemental analysis of solid microscopic samples in the flameless atomic absorption cuvette.

Histochemistry 62, 337-345 (1979) Histochemistry 9 by Springer-Verlag 1979 Elemental Analysis of Solid Microscopic Samples in the Flameless Atomic A...
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