1、Designation: E 2603 08Standard Practice forCalibration of Fixed-Cell Differential Scanning Calorimeters1This standard is issued under the fixed designation E 2603; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last re
2、vision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 1.1 This practice covers the calibration of fixed-celldifferential scanning calorimeters over the temperature rangefrom 10 to
3、 +120C.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.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 establi
4、sh appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. Specific precau-tionary statements are given in Section 7.2. Referenced Documents2.1 ASTM Standards:2E 473 Terminology Relating to Thermal Analysis and Rhe-ologyE 967 Test Method for
5、Temperature Calibration of Differ-ential Scanning Calorimeters and Differential ThermalAnalyzersE 968 Practice for Heat Flow Calibration of DifferentialScanning CalorimetersE 1142 Terminology Relating to Thermophysical Properties3. Terminology3.1 Specific technical terms used in this practice are de
6、finedin Terminologies E 473 and E 1142.4. Summary of Practice4.1 This practice covers calibration of fixed-cell differentialscanning calorimeters. These calorimeters differ from anothercategory of differential scanning calorimeter in that the formerhave generally larger sample volumes, slower maximu
7、m tem-perature scan rate capabilities, provision for electrical calibra-tion of heat flow, and a smaller range of temperature overwhich they operate. The larger sample cells, and their lack ofdisposability, make inapplicable the calibration methods ofPractices E 967 and E 968.4.2 This practice consi
8、sts of heating the calibration mate-rials in aqueous solution at a controlled rate through a region ofknown thermal transition. The difference in heat flow betweenthe calibration material and a reference material, both relativeto a heat reservoir, is monitored and continuously recorded. Atransition
9、is marked by the absorption or release of energy bythe specimen resulting in a corresponding peak in the resultingcurve.4.3 The fixed-cell calorimeters typically, if not always, haveelectrical heating facilities for calibration of the heat-flow axis.Despite the use of resistance heating for calibrat
10、ion, a chemicalcalibration serves to verify the correct operation of the calibra-tion mechanism and the calorimeter. The thermal denaturationof chicken egg white lysozyme is used in this practice forverification of the proper functioning of the instrumentssystems. The accuracy with which the denatur
11、ation enthalpy ofchicken egg white lysozyme is currently known, 65%, is suchthat it should be rare that a calorimeter provides a value outsidethat established in the literature for this reference material.5. Significance and Use5.1 Fixed-cell differential scanning calorimeters are used todetermine t
12、he transition temperatures and energetics of mate-rials in solution. For this information to be accepted withconfidence in an absolute sense, temperature and heat calibra-tion of the apparatus or comparison of the resulting data to thatof known standard materials is required.5.2 This practice is use
13、ful in calibrating the temperature andheat flow axes of fixed-cell differential scanning calorimeters.6. Apparatus6.1 Apparatus shall be:6.1.1 Differential Scanning Calorimeter (DSC), capable ofheating a test specimen and a reference material at a controlledrate and of automatically recording the di
14、fferential heat flowbetween the sample and the reference material to the requiredsensitivity and precision.1This practice is under the jurisdiction of ASTM Committee E37 on ThermalMeasurements and is the direct responsibility of Subcommittee E37.09 on Biologi-cal Calorimetry.Current edition approved
15、 Sept. 1, 2008. Published October 2008.2For 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 standards Document Summary page onthe ASTM website.1Copyright ASTM Intern
16、ational, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.1.2 DSC Test Chamber, composed of:6.1.2.1 A device(s) to provide uniform controlled heating orcooling of a specimen and reference to a constant temperatureor at a constant rate within the applicable temper
17、ature range ofthis method.6.1.2.2 A temperature sensor to provide an indication of thespecimen temperature to 60.01 K.6.1.2.3 Differential sensors to detect a heat flow (power)difference between the specimen and reference with a sensi-tivity of 60.1 W.6.1.3 A temperature controller, capable of execu
18、ting aspecific temperature program by operating the furnace(s)between selected temperature limits at a rate of temperaturechange of 0.01 K/min to 1 K/min constant to 60.001 K/min orat an isothermal temperature constant to 60.001 K.6.1.4 A data collection device, to provide a means ofacquiring, stori
19、ng, and displaying measured or calculatedsignals, or both. The minimum output signals required for DSCare heat flow, temperature, and time.6.1.5 Containers, that are inert to the specimen and refer-ence materials and that are of suitable structural shape andintegrity to contain the specimen and refe
20、rence in accordancewith the specific requirements of this test method. Thesecontainers are not designed as consumables. They are either anintegral part of the instrument, whether or not user-removablefor replacement or, in some implementations, are removableand reusable. Container volumes generally
21、range from 0.1 mlto 1 ml, depending on the instruments manufacture.6.2 Analytical Balance, capable of weighing to the nearest0.1 mg, for preparation of solutions.6.3 UV spectrophotometer or UV/Vis spectrophotometer,capable of scanning the UV spectrum in a region about 280 nm.6.4 Reagents:6.4.1 Phosp
22、hatidylcholines, 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (DTPC) CAS Number 71242-28-9 and 1,2-ditetracosanoyl-sn-glycero-3-phosphocholine (DLPC) CASNumber 91742-11-9 are the minimum required.6.4.2 Aqueous buffer solutions, 0.01 Molar, pH 7 aqueoussolution of Na2HPO4 NaH2PO4 and 0.1 Molar, pH (
23、2.4 60.1) aqueous solution of HCl + glycine.6.4.3 Chicken egg white lysozyme .7. Precautions7.1 This practice assumes linear temperature indication.Care must be taken in the application of this practice to ensurethat calibration points are taken sufficiently close together sothat linear temperature
24、indication may be approximated.8. Calibration Materials8.1 Phosphatidylcholines: 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (DTPC) CAS Number 71242-28-9; and 1,2-ditetracosanoyl-sn-glycero-3-phosphocholine (DLPC) CASNumber 91742-11-9. Purities are to be 0.99 or better. Addi-tional calibration mat
25、erials are listed in Table 1.8.1.1 Aqueous suspensions of the phosphatidylcholines areprepared as follows. Weighed amounts of a 0.01 Molar, pH 7solution of the buffer Na2HPO4 NaH2PO4and DTPC arecombined so to give a solution of 1 mass percent of thephosphatidylcholine. This procedure is repeated for
26、 DLPC.The solutions are heated in a hot water bath to 5 K above thetransition temperatures. A vortex mixer is used to shake thesolutions at their respective temperatures until the lipid appearsto have been completely suspended. The solutions may bestored in a refrigerator until use for up to a week.
27、8.2 Chicken egg white lysozyme with purity of at least 95%mass percent.8.2.1 Weighed amounts of the lysozyme and of a 0.1 M HCl glycine buffer at pH = (2.4 6 0.1) are combined to obtain asolution of approximately 3 mass percent.8.2.2 The concentration of lysozyme in this solution iscalculated from U
28、V absorbance at a wavelength of 280 nm,usinga1cmcell and the optical density of 2.65 fora1mgmL-1solution.8.2.2.1 Fill a 1 cm optical cell with buffer solution andanother 1 cm cell with the lysozyme solution. Follow theinstruments directions for establishing baseline, and if needed,calibration of the
29、 absorbance scale. Insert both of the filledcells in the UV spectrometer if the spectrometer is a dual beaminstrument. Scan through the 280 nm region and note theabsorbance at 280 nm. If the spectrometer is a single beaminstrument, the buffer is measured first, then the lysozymesolution is measured
30、and the difference in the recorded absor-bances is used to calculate the concentration. Concentration iscalculated as:c 5 A / 2.65 mL mg21!where:A = absorbance, andc = concentration in mg mL-1.NOTE 1Different concentrations may be used between 1 and 10 masspercent, the concentration used shall be in
31、cluded in the report.9. Procedure9.1 Two Point Temperature Calibration :9.1.1 Determine the apparent transition temperature foreach calibration material, as described in Table 1.9.1.1.1 Fill the clean specimen cell with the phosphatidyl-choline suspension, according to the usual method specified for
32、the instrument. Fill the reference cell with buffer solution thatwas used to prepare the phosphatidylcholine suspension.9.1.1.2 Equilibrate the calorimeter approximately 10 K to15 K below the expected transition temperature from Table 1.9.1.1.3 Heat each calibration material at the desired scanrate
33、through the transition until the baseline is reestablishedabove the transition. Record the resulting thermal curve.TABLE 1 Melting Temperature of Calibration MaterialNOTEThe uncertainties for the temperatures are 60.1 K.Calibration MaterialMelting TemperatureC K1,2-ditridecanoyl-sn-glycero-3-phospho
34、choline (DTPC) 13.25 286.41,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) 23.75 296.91,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) 41.45 314.61,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) 54.85 328.01,2-dieicosanoyl-sn-glycero-3-phosphocholine (DAPC) 65.05 338.21,2-didocosanoy
35、l-sn-glycero-3-phosphocholine (DBPC) 73.35 346.51,2-ditetracosanoyl-sn-glycero-3-phosphocholine (DLPC) 80.55 353.7E2603082NOTE 2Temperature scale calibration may be affected by temperaturescan rate and by time-constant of the instrument.9.1.2 From the resultant curve, measure the temperature forthe
36、maximum of the heat flow, Tp. See Fig. 19.1.3 Using the apparent transition temperatures thus ob-tained, calculate the slope (S) and intercept (I) of the calibra-tion Eq 1 (see Section 10). The slope and intercept valuesreported should be mean values from duplicate determinationsbased on separate sp
37、ecimens.9.2 One-Point Temperature Calibration :9.2.1 If the slope value (S) previously has been determinedin 9.1 (using the two-point calibration calculation in 10.2)tobesufficiently close to 1.0000, a one-point calibration proceduremay be used.NOTE 3If the slope value differs by only 1 % from linea
38、rity (that is,S 1.0100), a 0.5 C error will be produced if the testtemperature differs by 50 C from the calibration temperature.9.2.2 Select a calibration material from Table 1. The cali-bration temperature should be centered as close as practicalwithin the temperature range of interest.9.2.3 Determ
39、ine the apparent transition temperatures of thecalibration material using steps 9.1.1.1 9.1.1.1.9.2.4 Using the apparent transition temperature thus ob-tained, calculate the intercept (I) of the calibration equationusing all available decimal places. The value reported shouldbe a mean value based up
40、on duplicate determinations onseparate specimens.9.3 Enthalpy Calibration:9.3.1 If recommended by the instrument manufacturer,perform an electrical calibration per the manufacturers direc-tions.9.3.2 Determine the enthalpy of transition for the lysozymesolution.9.3.2.1 Fill the sample cell with the
41、lysozyme + buffersolution and fill the reference cell with the HCl-glycine buffersolution taking care that no air bubbles are retained in eitherof the cells.9.3.2.2 Equilibrate the calorimeter near room temperature,following equilibration the temperature of the calorimeter isramped at 60K/huntil a s
42、ufficient baseline is establishedbeyond the transition peak.NOTE 4Slower scan rates shall not be used in this step due to potentialaggregation of the denatured protein.9.3.2.3 The enthalpy of the denaturation is calculated byintegration, using a two-state transition baseline. This enthalpyis then di
43、vided by the mass of sample in the cell. The mass ofsample in the cell, m, is calculated as:m 5 vcwhere:v = is the volume of the measuring cell in milliliters.NOTE 5A two state model refers to a model that assumes thedenaturation reaction proceeds from a single native state to a singledenatured stat
44、e. Although the denaturation reaction involves a transitionbetween one manifold of states to another manifold of states, the two-statemodel adequately represents the average behavior for this protein. Theheat capacity of the solution with the native state protein is oftensignificantly different from
45、 the heat capacity of the solution with thedenatured protein. A two-state transition baseline is one that employs aheat capacity calculated from the thermodynamic progression from onestate to the next and the heat capacities of the aqueous solution of the twostates of the protein.9.3.2.4 A second en
46、thalpy of denaturation is calculatedusing a two-state model and the vant Hoff equation, which isbuilt into the software packages of most fixed-cell calorim-eters.NOTE 6Using the two state model, the equations: Q(T) = DHx(T)K(T) = x/(1-x) define the temperature dependence of the observed curve,if the
47、 enthalpy is defined by the vant Hoff relation: dlnK/dT = DH/RT2.where Q is the integrated enthalpy observed, DH is the enthalpy changefor the two-state reaction, K is the equilibrium constant for the reaction,R is the gas constant, x is the fraction of reactant converted to product andT is temperat
48、ure. The model can be fitted to the curve of apparent heatcapacity against temperature. Failure of the two state model occurs fromprecipitation reactions or other reactions that inhibit a reverse reaction inthe thermodynamic equilibrium.9.4 If practical, adjustment to the temperature scale of theins
49、trument should be made so that temperatures are accuratelyindicated directly.10. Calculation10.1 For the purposes of this procedure, it is assumed thatthe relationship between observed temperature (TO) and actualspecimen temperature (T) is a linear one governed by thefollowing equation:T 5 TO 3 S 1 I (1)where:S and I = the slope and intercept, respectively. (See 10.2for the values for S and I, used in Eq 1.)NOTE 7For some instruments, the assumption of a linear relationbetween observed and actual specimen temperature may not hold. Undersuch conditions, cal
