1、Designation: D 4638 03Standard Guide forPreparation of Biological Samples for Inorganic ChemicalAnalysis1This standard is issued under the fixed designation D 4638; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last r
2、evision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope*1.1 This guide describes procedures for the preparation oftest samples collected from such locations as streams, rivers,ponds,
3、 lakes, estuaries, oceans, and toxicity tests and isapplicable to such organisms as plankton, mollusks, fish, andplants.1.2 The procedures are applicable to the determination ofvolatile, semivolatile, and nonvolatile inorganic constituents ofbiological materials. Analyses may be carried out or repor
4、tedon either a dry or wet basis.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitati
5、ons prior to use. For a specifichazard statement, see 9.3.3.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent Water3. Terminology3.1 DefinitionsFor definitions of terms used in this guide,refer to Terminology D 1129.4. Summary of Guide4.1
6、 Samples are collected, where possible, with nonmetallicor TFE-fluorocarbon-coated sampling equipment to preventcontamination, stored in plastic containers, and kept either at4C or frozen until returned to an adequate facility for analysis.4.2 Before analysis, samples are allowed to return to roomte
7、mperature. Large foreign objects are mechanically removedfrom the samples based upon visual examination; smallerforeign objects are also removed mechanically, with the aid ofa low-power microscope.4.3 Wet samples of small organisms such as plankton, aremixed for preliminary homogenization, then allo
8、wed to settle,to remove most of the occluded water. Larger organisms, suchas fish, should be patted dry, using paper towels.4.4 Where less than a whole organism is to be analyzed,tissue excisions are made with nonmetallic tools such as plasticknives or TFE-fluorocarbon-coated scalpels.4.5 Moisture d
9、eterminations are made on separate samplesfrom those analyzed for volatile or semivolatile constituents.4.6 Analyses for volatile constituents are made using wetsamples from which supernatant liquid or occluded water hasbeen removed (see 4.3). The results may be calculated to thedry, original-sample
10、 basis, using the results of a moisturedetermination carried out on a separate sample.4.7 Analyses for semivolatile constituents are made on wetsamples or samples previously dried at a temperature (depen-dent on constituents of interest), or using a procedure, found tobe adequate for the purpose, an
11、d specified in the correspondinganalytical procedure.4.8 Analyses for nonvolatile constituents are made onsamples previously dried at a temperature (dependent onconstituents of interest), or using a procedure found to beadequate for the purpose, and specified in the correspondinganalytical procedure
12、.4.9 Digest the samples according to the procedures outlinedin Section 9.4.10 A flow diagram outlining typical procedures is shownin Fig. 1.5. Significance and Use5.1 The chemical analysis of biological material, collectedfrom such locations as streams, rivers, lakes, and oceans canprovide informati
13、on of environmental significance. The chemi-cal analysis of biological material used in toxicity tests may beuseful to better interpret the toxicological results.5.2 Many aquatic biological samples, either as a result oftheir size, or their method of collection, are inherently hetero-geneous in that
14、 they may contain occluded water in varyingand unpredictable amounts and may contain foreign objects or1This guide is under the jurisdiction of ASTM Committee D19 on Water and isthe direct responsibility of Subcommittee D19.05 on Inorganic Constituents inWater.Current edition approved Dec. 1, 2003.
15、Published January 2004. Originallyapproved in 1986. Last previous edition approved in 1999 as D 4638 95a (1999).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the
16、standards Document Summary page onthe ASTM website.1*A Summary of Changes section appears at the end of this standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.material (for example, sediment) not ordinarily intended foranalysis
17、, the inclusion of which would result in inaccurateanalysis.5.3 Standard methods for separating foreign objects, tofacilitate homogenization, will minimize errors due to poormixing and inclusion of extraneous material.5.4 Standardized procedures for drying provide a means forreporting analytical val
18、ues to a common dry weight basis, ifdesired. Analyses may also be carried out or reported on a wetweight basis.6. Preliminary Treatment of Samples6.1 Treat small heterogeneous samples, such as plankton, asfollows:6.1.1 Allow for the sample to return to room temperature.6.1.2 Remove foreign objects,
19、such as leaves and twigs,mechanically, using nonmetallic instruments. Use a low-powermicroscope to facilitate removal of smaller foreign objectssuch as paint chips.6.1.3 Transfer the sample to a beaker and thoroughly mix itwith a glass stirring rod or equivalent, and allow it to settle sothat most o
20、r all of the occluded water can be decanted.6.1.4 If chemical analyses are to be carried out on a wetsample, and a large amount of material is available, remove anumber of small portions (at least 5) from random locations inthe beaker, and composite them to obtain a representativesample of a size su
21、fficient for chemical analysis and a separatemoisture determination. Using a tissue disrupter, blender, orequivalent, homogenize the sample or composite (to ensurelack of contamination, carry a standard or blank, or both,through this procedure). Remove a subsample for moisturedetermination and proce
22、ed to Section 7. Retain the remainderand proceed to Section 9.6.1.5 If chemical analyses are to be carried out on a drysample, and a large amount of material is available, remove anumber of small portions (at least 5) from random locations inthe beaker, and composite them to obtain a representatives
23、ample of a size sufficient for the analysis. Using a tissuedisrupter, blender, or equivalent, homogenize the sample, orcomposite (to ensure lack of contamination, carry a standard orblank, or both, through this procedure), and proceed to Section7.6.2 Treat large samples such as fish as follows:6.2.1
24、 Allow the sample to return to room temperature.6.2.2 Pat the sample dry with paper toweling to remove asmuch water as possible.6.2.3 Transfer the sample to a nonmetallic surface, such asa flat glass plate, and excise a sufficient quantity of material, orspecific organs, to obtain sufficient materia
25、l for analysis. Makeexcisions with plastic knives or TFE-fluorocarbon-coated scal-pels.6.2.4 If chemical analyses are to be carried out on a wetsample, use a tissue disrupter, blender, or equivalent, tohomogenize the material (to ensure lack of contamination,carry a standard or blank, or both, throu
26、gh this procedure).FIG. 1 Flow Diagram for the Preparation of Biological Samples for Inorganic AnalysisD4638032Remove a subsample for moisture determination and proceedto Section 7. Retain the remainder and proceed to Section 9.6.2.5 If chemical analyses are to be carried out on a drysample, use a t
27、issue disrupter, blender, or equivalent, tohomogenize the material (to ensure lack of contamination,carry a standard or blank, or both, through this procedure) andproceed to Section 7.7. Drying Procedures7.1 Use a sample or subsample prepared in accordance withthe directions given in Section 6.7.2 T
28、reat subsamples from biological materials that are toundergo chemical analysis without drying for moisture deter-minations as follows:7.2.1 Accurately weigh 5 to 10 g 6 1mgor10to25g610 mg of material into a nonmetallic container which has beenpreviously tared, and weighed with the same accuracy.7.2.
29、2 When a limited amount of material is available,determine the moisture ona1to2-gsample, and weigh with anaccuracy of6 0.1 mg. The use of samples smaller than 1 g isnot recommended for moisture determination.7.3 When an entire sample is to be dried prior to chemicalanalysis, a moisture determination
30、 is also required. Transfer theaccurately weighed material (1 to 2 g 6 0.1mg,5to10g61 mg, 10 g 6 10 mg) into a dry nonmetallic container whichhas been previously tared, and weigh with the same accuracy.7.4 If a moisture determination (or sample drying) is to bemade using an oven, treat as follows:7.
31、4.1 Transfer the containers holding the material to an ovenanddryfor2hatoneofthefollowing temperatures:7.4.1.1 For the determination of semivolatile constituents,use the temperature specified in the analytical procedure for theconstituents(s).7.4.1.2 For determination of nonvolatile constituents use
32、105 6 2C.7.4.2 Cool in a desiccator, then weigh the dried sampleswith the same accuracy as the wet samples.NOTE 1Biological materials tend to be very hygroscopic. Keepweighing times to a minimum.7.4.3 Repeat drying at hourly intervals, to attain a constantweight.7.5 If a moisture determination (or s
33、ample drying) is to bemade at room temperature, treat as follows:7.5.1 If drying is to be done in a desiccator, ensure that thedesiccant in the bottom is fresh, and some means is available toindicate when the desiccant loses its drying capacity (forexample, color change). A vacuum desiccator may als
34、o beused.NOTE 2If a vacuum desiccator is used, bear in mind that this maycause the loss of volatile or semivolatile inorganics such as mercury, if thedried sample is to be subjected to chemical analysis.7.5.1.1 Transfer the containers holding the material to adesiccator.7.5.1.2 Leave the material in
35、 the desiccator for 48 h, thenweigh the dried samples with the same accuracy as the wetsample.7.5.1.3 Repeat weighings at 4-h intervals, to attain a con-stant weight (see Note 1).7.5.2 Alternatively, sample drying or moisture determina-tions may be carried out in a laminar flow hood; treat asfollows
36、:7.5.2.1 Transfer the containers holding the material to anappropriate hood and turn it on.7.5.2.2 Leave the material in the hood for 48 h, then weighthe dried samples with the same accuracy as the wet sample.7.5.2.3 Repeat weighings at 4-h intervals, to attain a con-stant weight (see Note 1).NOTE 3
37、Air-drying in the open is strongly discouraged unless it iscarried out in a clean room, where possible contamination from airborneparticulates can be controlled.7.6 If a moisture determination (or sample drying) is to bemade using a freeze dryer, treat the determination as follows:7.6.1 Transfer the
38、 containers holding the material to thefreeze dryer.7.6.2 Follow the manufacturers instructions for the particu-lar unit in use. Make certain that a trap is placed between thevacuum pump and the drying chamber to prevent pump oilfumes from possibly contaminating the sample. Drying isusually complete
39、 when the internal pressure in the dryingchamber reaches 50 millitorrs or less.7.6.3 Transfer the freeze-dried samples to a desiccator forstorage, and weigh them with the same accuracy as the wetsamples (see Note 1).NOTE 4Because freeze drying occurs under vacuum, this may causethe loss of volatile
40、or semivolatile inorganics such as mercury, or both, ifthe dried sample is to be subjected to chemical analysis.7.7 The possibility of loss of volatile constituents dictatesthe drying procedure to be used, prior to chemical analysis.Determine volatile constituents using undried samples. Deter-mine s
41、emivolatile constituents using samples dried at a tem-perature at which no significant losses occur.7.8 Analytical data reported on a dry weight basis shouldinclude percent moisture so that wet weight values can beobtained. Likewise, wet weight analytical data should includepercent moisture to permi
42、t recalculation to a dry weight basis.7.9 Use the following equations to calculate percent mois-ture and to correct analytical results from samples analyzedwhen wet.7.9.1 Calculate percent moisture as follows:moisture, % 5 Ww/Wd!100 (1)where:Ww= wet weight, g, andWd= dry weight, g7.9.2 To calculate
43、concentrations on a dry weight basis,when determinations have been made on an undried sample,use the following equation:Cd5Cw100!100 2 % moisture(2)where:Cd= concentration on a dry weight basis, andCw= concentration on a wet weight basis.D46380338. Reagents8.1 Purity of ReagentsReagent grade chemica
44、ls shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.3Other grades may be used,provided it is first ascertained that the r
45、eagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.8.2 Purity of Water Unless otherwise indicated, refer-ences to water shall be understood to mean reagent waterconforming to Specification D 1193, Type I. Other reagentwater types may be used, pr
46、ovided it is first ascertained that thewater is of sufficiently high purity to permit its use withoutadversely affecting the bias and precision of the test method.Type II water was specified at the time of round robin testingof this method.8.3 All of the following reagents may not be required for ap
47、articular procedure. Check the digestion procedure(s) ofinterest (Section 9) prior to preparing any reagents.8.3.1 Amyl Alcohol.8.3.2 Hydrochloric Acid (1 + 1)Mix 1 volume of hydro-chloric acid (HCl, sp gr 1.19) with 1 volume of water.8.3.3 Hydrogen Peroxide Solution (30 % H2O2w/v)Commercially avail
48、able.8.3.4 Magnesium Nitrate Solution (7 g/L)Dissolve7gofmagnesium nitrate Mg(NO3)26H2O in water and dilute to1000 mL.8.3.5 Nitric Acid (sp gr 1.42)Concentrated ultra-purenitric acid (HNO3).8.3.6 Nitric Acid (1 + 9)Mix 1 volume of nitric acid(HNO3, sp gr 1.42) with 9 volumes of water.8.3.7 Nitric-Pe
49、rchloric Acid Solution (3 + 1)Mix 3 vol-umes of ultra-pure concentrated nitric acid (HNO3, sp gr 1.42)with 1 volume of ultrapure concentrated perchloric acid(HClO4, sp gr 1.67).8.3.8 Sulfuric Acid (sp gr 1.84)Concentrated ultra-puresulfuric acid (H2SO4).8.3.9 Sulfuric Acid (1 + 9)Mix 1 volume of sulfuric acid(H2SO4, sp gr 1.84) with 9 volumes of water.9. Digestion Procedures9.1 Many procedures are available for the destruction ofbiological material prior to inorganic analysis, but almost allthe methods fall into one of two main classes: dry ashing andwet digestio
