Anal. Chem. 1983, 55,585-587
chromotropic acid concentrations, respectively. The improved reagent is made by placing 20 g of chromotropic acid in a I-L flask. One-hundred milliliters of distilled water is added and the chromotropic acid is dissolved. Technical grade 93% sulfuric acid is added to bring the volume to 1L. The reagent may be used for many months if it is stored in a refrigerator. Reaction Time. Klein and Weissman recommend a 30-min boiling time. However, after 30 min, the reaction is still proceeding rapidly (absorbance changes by 1.1%/min). After 60 min, the reaction ppoceeds more slowly (absorbance changes by 0.3% /mh) thus allowing more reproducibility. Therefore, a 60-min reaction time is recommended. Assay Procedure. Place 0.5 mL of appropriately diluted sample in a screw-top test tube. Add 2.5 mL of reagent and seal the test tube with a cap which has an inert gasket (such m rubber or Teflon). Mix the reagent and sample very well on a vortex mixer and place the tubes in a boiling water bath for 60 min. When the reaction is complete, place the test tubes in an ice bath. Add 2 mL of distilled water for the sensitive assay (0.3 g/L hexose maximum) or 12 mL of distilled water for the standard assay (1.2 g/L hexose maximum). Mix the contents of the test tube and read the absorbance at 570 nm. Cellulose Measurement. The cellulose sample must be finely ground (-140 mesh). Place the cellulose sample in a beaker containing a known volume of water and mix well. Mixing is aided by using a spatula as a baffle. Take a 0.25-mL sample from the most turbulent regions of the beaker. Rinse the pipet with a further 0.25 mL of distilled water. Proceed with the method used for soluble hexoses. Because the cellulose is in suspension and not in solution, it is necesmry to take many (-10) samples from the beaker to get a statistical average. Assay Interference. TJpon acid degradation, lignin produces formaldehyde which will interfere with the assay. Some lignin will be released to the aqueous phase as a result of acid or enzymatic hydrolysis of lignocellulose. This interference is not easily corrected, so the hexose determinations will be slightly greater
585
than the actual hexose concentration. The effect of lignin interference on cellulose determinations may be eliminated by removing the lignin by using standard procedures (9, 10). RESULTS Both the standard and sensitive assays produced linear calibration lines when either glucose or cellobiose were used as standards. The cellobiose calibration line was identical with that for glucose provided the cellobiose concentration was expressed as equivalent glucose. The concentration of pure cellulose (Avicel, FEdC Corp., Newark, DE) in aqueous suspension was determined by using the recommended procedure for cellulose measurements. The measurements agreed wjth the known concentrakions within experimental error. Glucose could be used as a cadibration standard provided the cellulose concentration was expressed as equivalent glucose. Registry No. Glucose, 50-99-7; cellobiose, 528-50-7;celluloee, 9004-34-6. LITERATURE C I T E D (1) Miller, G. L. Anal. Chem. 1959, 31, 426. (2) Nelson, N. J. Bo/. Chem. 1944, 153,375. (3) Somogyi, M. J. B i d . Chem. 1952, 195, 19. (4) Dubois, M.; et al. h a / . Chem. 1956, 28, 350. (5) Brobst, K. M.; Lott, C. E. Cereal Chem. 1966, 43,35. (6) Moore, W. E.; Johnson, D. 8. “Procedures for the Chemical Analysis of Wood and Woad Products”; Forest Products Laboratory, Forest Service, USDA; Washington, DC, 1967. (7) Palmer, J. K. Appl. Polym. Symp. 1975, 28, 237. (8) Klein, 8.;Weissman, M. Anal. Chem. 1953, 25,771. (9) Van Soest, P. J.; Wine, R. H. J. Assoc. Off. Anal. Chem. 1968. 51. 780. (IO) Edwards, C. S. J. !bi.Food Agrlc. 1973, 24, 381.
RECEIVED for review September 8,1982. Accepted November 29,1982. This work was funded in part by DOE Contract No. EY-76-S-02-4070.
Drying procedure for Analysis of Biological Samples by X-ray Fluorescence Spectrometry Adrienne S. Frank andl Ivor L. Preiss” Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 1218 I
The trace element content of biological materials and its relationship to disease have gained import in recent studies (1-5). In the case of trace element analysis of biological specimens, sampling techniques and those used in preparing samples for analysis need to be specially designed so as to avoid contamination, loss of elements through volatilization, and changes in mean elemental composition (6). Since there are no whole tissue standards (e.g., a standard liver) to which biological specimens can be compared, assessment of handling techniques with regard to the probability of the introduction of contaminants or alteration of the state of the specimen becomes a difficult task. Without careful scrutiny of each step in sample handling procedures, impurities can easily ’be incorporated into the sample and later attributed to the absolute make u p of the sample. Conclusions or interferences based on the misinterpretation of these results could seriously affect the validity of the study. When information regarding a specific disease is to be obtained from trace element profiles of tissues affected by that disease, the simplest types of handling procedures should be employed. The more complicated a technique is, the more likely the possibility of sample contamination. A method such as XRF analysis, in which the intrinsic bulk elemental content
is examined, is subject to a variety of geometry effects due to sample size, which can vary according to its moisture content. The varying moisture content can thus adversely affect spectral data to be used in comparative studies,. Therefore, a dried sample may be preferable to one wherle moisture content can be lost during spectral accumulation. The drying method must also take into account that detection limits of 1015atoms, on an absolute basis, have been achieved in trace element studies with XRF (7, 8). With these criteria in mind (i.e., minimum handling, contenit or minimum moisture content, and nonvarying geometry in each sample type) a method of drying biological samples for XRF studies has been devised. EXPERIMENTAL SECTION A variety of autopsy samples from laboratory mice and/or hansters, frozen for storage, were removed from the freezer 12-14 h prior to analysis and allowed to thaw in a closed, vented container. A length of 6-0 black, braided surgical silk (Ethicon),found (via XRF) to contain no detectable trace elements, was threaded in the dorsal-ventral direction of the tissue, approximately 0.3-0.5 cm from a longitudinal edge after the specimen had thawed completely. The sample was then weighed and secured (by a rubber stopper) in the center neck of the drying chamber (Figure
0003-2700/83/0355-0585$01.50/00 1983 Amerlcan Chemical Soclety
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ANALYTICAL
CHEMISTRY,
VOL. 55, NO. 3, MARCH 1983 Th
tu u p
REGUL ATOS so0
[
500
v 3oac
200-
Flgure 1. Apparatus used for drying biological tissue samples prior to analysis by XRF: (A) cold finger trap, (€3)fritted glass filter, (C) inlet, (D) port for suspension of tissue samples, (E) CaSO, filled exit port, (F) cold finger trap.
1
,
I
,
I I
I
I
1
I
Table I. Average Weight Loss Experienced by Tissues upon Drying in the N, Flow System tissue sample
no. of samples dried
liver lungs muscle
4 4
9
av wt loss, % 62.9 t 5 62.5 f 1 2 55.3 i: 14
l),D, which had been previously flushed with nitrogen. This closed, three-necked reaction flask precludes air contamination of the sample during the drying cycle. Throughout the drying period, anhydrous, high-purity nitrogen passed through Tygon tubing t o an empty cold-finger trap, A (Figure l),and a fritted glass filter, B, in order to capture any solid contaminants in the stream. The gas then entered the chamber at inlet C and flowed past the tissue specimen and out the exit port, E, through a tube filled with anhydrous CaS04 (W. A. Hammond Drierite Co.). Moisture that the nitrogen had picked up, which was not irreversibly adsorbed on the CaS04,was prevented from returning to the sample by the positive pressure of the gas. This positive pressure also prevented any particulate matter or gaseous contaminants due t o the CaS04 from contact with the sample. Tissue samples, weighing ca. 0.1 g (wet weight), were permitted to dry for a period of 12 h prior to XRF analysis. In general, this time period was defined as that interval after which no further appreciable weight loss could be observed. The largest samples analyzed, sections of liver weighing significantly more than 0.1 g, had t o be dried for no longer than 16 h before constant weight was achieved. If the tissue sample was large, i.e., >0.1 g, or more than one sample was to be dried at one time, the CaS04nearest to the drying vessel became quickly spent. The drying efficiency was found to be proportional to the surface area of anhydrous CaS04at the tube’s end. Although the sample would dry as long as any active CaSO, remained in the tube, the rate of drying was enhanced if a fresh surface of CaS04 was introduced after the first 2 h of drying. A cold-finger trap, F, filled with pure olive oil (Filippo Berio and Co), was positioned after the drying tube where it was used to monitor nitrogen flow in the system. The flow monitor can be removed and a trap placed in dry ice acetone or liquid nitrogen in order to monitor elements removed from the specimen, and not previously adsorbed on the drying agent. After 12 h had elapsed, the tissue sample was removed from the apparatus and weighed. Tissue samples were found to undergo an average weight loss of 61% over the drying period (Table I). The reproducible weight loss was independent of tissue type. A typical background spectrum showing 40 ng of Cu in a cellulose phantom, dried by using the procedure described is shown in Figure 2. Figure 3 represents the raw spectral data from a liver biopsy sample dried to a weight of 32 mg. Trace element levels typically range from 1 to 20 ppm in this sample. RESULTS AND DISCUSSION The weight loss values obtained with the drying system described are comparable to those reported by Koh for Sam-
Flgure 3. XRF spectrum of 32 mg of liver (dry weight). Biopsy sample was subjected to the drying procedure described In text. This sample is doped with 40 ng of Cu for comparison purposes.
ples dried in thermal and microwave ovens (9). A certain percentage of error associated with the N2 flow system data can be attributed to the presence of freezer burn on some autopsy samples which has been stored for a long period of time. Since these samples were no longer homogenous (Le., portions had changed in structure), a value for weight loss based on homogeneity would have some inherent error associated with it. It is important to note that this drying system has associated with it a source of possible contamination, namely, dust from the Drierite used in the drying tube. XRF analysis of Drierite shows that it contains large amounts of the trace elements Co and Sr, both of which could be sample contaminants in the detectable element region. The effect of Drierite as a contaminant was investigated by using the two kidneys and two lobes of liver from a mouse with a 4 day old neuroblastoma. One kidney and liver lobe were dried in the apparatus illustrated in Figure 1. The other two tissue samples were dried in a static nitrogen-filled chamber which had an exposed layer of Drierite on the bottom in place of the drying tube. Each sample was dried individually. The XRF spectra of the samples do not indicate the presence of any Co or Sr. If the samples are contaminated by Drierite (volatile components, dust) it is to a degree lower than the detection limits of the analysis system. In previously reported drying procedures in Table 11, most samples require extensive handling and manipulation which subjects them to a high probability of contamination and/or a possible change of elemental composition. The drying chamber (Figure 1) was designed to protect the sample from the atmosphere throughout the drying time. In addition,
587
And. Chem. 1983, 55,587-591
-
_ _ _ I
Table 11. Methods for Drying Biological Tissue Samples associated problems method thermal oven
microwave oven freeze-drying
dry ashing wet ashing
high temperatures cause loss of volatile elements and alteration of sample matrix (10) 16-72 h needed to dry samples (10) for effective drying samples must be minced (9), internal high temperature effects probable sample contamination from the metallic freezer housing (esp. Cr) is possible ( 11 ), cellular integrity destroyed high temperatures result in a loss of elements such as As, Co, Cr, and Ni by volatilization (12-1 7 ) loss of Se, Te, and Po and matrix interferences due to incomplete destruction of the organic matrix when samples are treated with HNO,/HCIO, or H,SO,/H,O, (12, 1ti), possible sample contamination from reagents used in the procedure ( 19-2 7 )
elementai losses due to volatilization were held at an absolute minimum by keeping the drying chamber and its contents at ambient temperature and a t atmospheric pressure. Losses, including those resulting from the alteration of the biological matrix upon heating, or the loss, via volatilization, of elemenh or compounds having high vapor pressures are therefore reduced or diminated. By use of this drying scheme, the sample is protected from sample-container interactions such as those encountered during ashing and freeze-drying. The sample is simply suspended in an inert, clean atmosphere without any solvent pretreatment, mincing, or prefreezing. This cost-effective method of drying samples is as efficient as those methods already in existence and has been optimized with respect to maintaining sample integrity. The ability to dry entire organs and large pieces of tissue without significant change in form makes this technique particularly well suited to XRF analysis and preserves the sample for other invasive analytical examinations.
ACKNOWLEDGMENT We wish to thank Murriel K. Schauble, Veterans Administration Medical Center, Albany, NY, for her assistance in supervising the maintenance of the experimental animal tumor lines.
LITERATURE CITED Mertz, W. Blol. Trace Hem. Res. 1979, I, 259. Hoekstra, W. G., Suttle, J. W.; Ganther, H. E., Mertz, W., Eds. "Trace Element Metabolism In Animals -2";University Park Press: Baltimore, MD, 1974. Prasad, A. S. "Trace Elements and Iron In Human Metabolism"; Plenum Medical Book Co: New York, 1978. Underwood, E. J. J . Hum. Nub. 1978, 32, 253. Porles, W. van J.; RII, A. M.; Mansour, E. G.; Flynn, A. €?lo/.Trace Elem. Res. 1979, 1 , 229. Sansoni, B.; Iyengai, G. V. I n "Elemental Analysis of Bloiogical klaterials: Current Problems and Techniques with Special Reference to Trace Elements"; IAEA Technical Report Series No. 97: Vienna,
1980: D 59. Prelss.' I. L.: Robie. S. "XRF in Phllatelv". J and E. C., I n Press, IVov.
1982. Frank, A. S. Ph.D. Dissertation, RPI, 1982. (9) Koh, T. Anal. Chtsm. 1980, 52, 1978. (IO) Iyengar, G. V.; Kasperek, K.; Feindegen, L. E. Scl. Total Envlron. 1978, IO, 1. (11) Sansoni, B.; Iyengar, G. V., ref 6,p 83. (12) Gorsuch, T. T. "The Destructlon of Organic Matter"; Pergamon Press: Oxford, 1970. (13) t44Filton, E. I.; Mlnski, M. J.; Cleary, J. J. Analyst (London) 1967, 92, 1
LOI.
(14) Hlslop, J. S.; Williams, D. R. "Nuclear Activation Techniques In the Life Sciences 1972" (Proc. Symp. Butt., 1972);IAEA, Vienna, 1972;p 51. (15) Jones, G. B.; Buckely, R. A.: Chandley, C. S.Anal. Chlm. Acta 1075, 80,389. (16) Koirtyohann, S. R ; Hopklns, C. A. Analyst (London) 1967, IO, 870. (17) van Raaphorst, J. G.; van Weers, A. W.; Haremaker, H. M. An6llySt (London) 1974, QY9 523. (18) Gorsuch, T. T. NBS Spec. Pub/. 1978, No. 422,491. (19) Debeka, R.; Mykutink, A.; Bermann, S. S.;Russel, 0. S.Anal. Chtm. 1978, 48, 1203. (20) Hamilton, E. I.; Minski, M. J. Envlron. Lett. 1972, 3 , 53. (21) Kershner, N. A.; Joy, E. F.; Bernard, A. J. Appl. Spectrosc. 1971, 25, 542. (22) Kuekner, E. C.; Alwarey, R.; Paulsen, P. J.; Murphy, T. J. Anal. Chevm. 1972, 4 4 , 2050. (23) Mykutulk, A.; Russel, D.; Bokyo, A. Anal. Chem. 1976, 4 8 , 1462. (24) NBS Tech. Note 1987, No. 428,56. (25) ORNL Report 3397,USAEC, 1967. (26) Robertson, D. E, I n "Ultra Purity Methods and Techniques"; Zief, Speights, Eds. Marcel Dekker: New York, 1972. (27) Thlers, R. E. "Trace Analysis"; Wlley: New York. 1957;p 637.
RECEIVED for review August 23, 1982.
Accepted Novemb'er 15, 1982. Partial fuinding for this research was provided by the Society for Nuclear Medicine and the RPI Provost's Fund.
Proton and Carbon-I 3 Nuclear Magnetic Resonance Spectrometry of Formaldehyde in Water D. J. Le Botlan, B. G. Mechln, and G. J. Martin" Laboratoire de Chimie Organique Physique, E.R.A. C.N.R.S. No. 315, Universitg de Nantes, 2 rue de la Houssini&e, 44072 Nantes, France
A number of NMR studies, involving either lH or I3C: spectrometry, have been devoted to aqueous solutions of formaldehyde owing to the great industrial importance of this compound (1-12). In aqueous solutions, formaldehyde reacts with water to give methylene glycol which oligomerizes easily according to the following scheme:
CH20 + 1J20s HOCH20H HOCHZOH + HOCHzOH HO(CH2O),-,H
HO(CH2O)ZH + HzO
+ HOCHZOH s HO(CH,O),H + HzO
The structures of the different species present in the solutions have already been determined, mainly by NMR (2-12),
but no special attention has been paid to the influence of temperature on the oligomeric distribution nor to the chemic,d relaxation rate of dilution, i.e., the time required for the solution to reach equilibrium when the concentration is changed. On the other hand, the quantitative determination of oligomers in solutions, which is performed by gas chromatography (GC), necessitates the substitution of the labile hydrogens by a trimethylsilyl group, and one might expect the distribution measured by GC to be perturbed by this chemical treatment. Carbon NMR spectrometry has been used in this work to obviate this difficulty and good quantitative results have been obtained. Moreover, the I3C NMR spectra already published were not fully assigned, preventing any valuable quantitative application.
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