1、Designation: E2603 15Standard Practice forCalibration of Fixed-Cell Differential Scanning Calorimeters1This standard is issued under the fixed designation E2603; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revi
2、sion. 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 This practice covers the calibration of fixed-cell differ-ential scanning calorimeters over the temperature range from10 to +12
3、0C.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 establish a
4、ppro-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:2E473 Terminology Relating to Thermal Analysis and Rhe-ologyE691 Practice for Conductin
5、g an Interlaboratory Study toDetermine the Precision of a Test MethodE967 Test Method for Temperature Calibration of Differen-tial Scanning Calorimeters and Differential Thermal Ana-lyzersE968 Practice for Heat Flow Calibration of DifferentialScanning CalorimetersE1142 Terminology Relating to Thermo
6、physical Properties3. Terminology3.1 Specific technical terms used in this practice are definedin Terminologies E473 and E1142, including differential scan-ning calorimeter, enthalpy, Kelvin, and transformation tem-perature.4. Summary of Practice4.1 This practice covers calibration of fixed-cell dif
7、ferentialscanning calorimeters. These calorimeters differ from anothercategory of differential scanning calorimeter in that the formerhave generally larger sample volumes, slower maximum tem-perature scan rate capabilities, provision for electrical calibra-tion of heat flow, and a smaller range of t
8、emperature overwhich they operate. The larger sample cells, and their lack ofdisposability, make inapplicable the calibration methods ofPractices E967 and E968.4.2 This practice consists of heating the calibration mate-rials in aqueous solution at a controlled rate through a region ofknown thermal t
9、ransition. The difference in heat flow betweenthe calibration material and a reference material, both relativeto a heat reservoir, is monitored and continuously recorded. Atransition is marked by the absorption or release of energy bythe specimen resulting in a corresponding peak in the resultingcur
10、ve.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 calibration, a chemicalcalibration serves to verify the correct operation of the calibra-tion mechanism and the calorimeter.
11、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 denaturation enthalpy ofchicken egg white lysozyme is currently known, 65%, is suchthat it should be rare that a calorimeter
12、 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 the transition temperatures and energetics of mate-rials in solution. For this information to be accepted withconfiden
13、ce 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 useful in calibrating the temperature andheat flow axes of fixed-cell differential scanning calorimeters.6. Apparatus6.1
14、 Apparatus shall be: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 May 1, 2015. Published August 2015. Originallyapproved in 2008. Last previous edition
15、approved in 2008 as E2603 08. DOI:10.1520/E2603-15.2For 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.Copyright
16、ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.1.1 Differential Scanning Calorimeter (DSC), capable ofheating a test specimen and a reference material at a controlledrate and of automatically recording the differential heat flowbetween the s
17、ample and the reference material to the requiredsensitivity and precision.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 temperature range ofthis
18、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 executing aspecific temp
19、erature 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, storing, and displaying
20、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 reference in accordance
21、with 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 range from 0.1 mlto
22、 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 Phosphatidylcholines, 1,
23、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 NaH2PO4and 0.1 Molar, pH (2.4 60.1) aqueous so
24、lution 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 indication may be app
25、roximated.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 materials are listed in
26、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 DLPC.The solutions a
27、re 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.8.2 Chicken egg white
28、 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 UV absorbance at a wav
29、elength 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 absorbance scale. In
30、sert 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 and the difference in
31、 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 included in the report.9.
32、 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 forTABLE 1 Melting Temperat
33、ure of Calibration MaterialNOTE 1The uncertainties for the temperatures are 0.1 K.Calibration MaterialMeltingTemperatureC K1,2-ditridecanoyl-sn-glycero-3-phosphocholine (DTPC) 13.25 286.41,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) 23.75 296.91,2-dihexadecanoyl-sn-glycero-3-phosphocholine
34、(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-didocosanoyl-sn-glycero-3-phosphocholine (DBPC) 73.35 346.51,2-ditetracosanoyl-sn-glycero-3-phosphocholine (DLPC) 80.55 353.7E2603 152the instrument.
35、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 through the tran
36、sition until the baseline is reestablishedabove the transition. Record the resulting thermal curve.NOTE 2Temperature scale calibration may be affected by temperaturescan rate and by the time-constant of the instrument.9.1.2 From the resultant curve, measure the temperature forthe maximum of the heat
37、 flow, Tp. See Fig. 1.9.1.3 Using the apparent transition temperatures thusobtained, calculate the slope (S) and intercept (I)ofthecalibration Eq 1 (see Section 10). The slope and interceptvalues reported should be mean values from duplicate deter-minations based on separate specimens.9.2 One-Point
38、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 linearity (that is,S 1.0100)
39、, a 0.5 K error will be produced if the testtemperature differs by 50 K 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 Determine the apparent transi
40、tion temperatures of thecalibration material using steps 9.1.1.1 9.1.1.3.9.2.4 Using the apparent transition temperature thusobtained, calculate the intercept (I) of the calibration equationusing all available decimal places. The value reported shouldbe a mean value based upon duplicate determinatio
41、ns 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 lysozyme + buffersolution
42、 and fill the reference cell with the HCl-glycine buffersolutiontaking 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 60 K/h until a sufficient baseline is es
43、tablishedbeyond 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 divided by the mass of sam
44、ple in the cell. The mass ofsample in the cell, m, is calculated as:m 5 vcwhere:v = 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 state. Although the denaturatio
45、n 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 the heat capacity of the s
46、olution 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 enthalpy of denaturation is c
47、alculatedusing 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) = Hx(T)K(T) = x/(1-x) define the temperature dependence of the observed curve,if the enthalpy is defined by the
48、vant Hoff relation: dlnK/dT = H/RT2.where Q is the integrated enthalpy observed, H 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 temperature. The model can be fitted t
49、o 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 theinstrument 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
