ASTM E2603-15(2023)

Standard Practice for Calibration of Fixed-Cell Differential Scanning Calorimeters

SIGNIFICANCE AND USE
5.1 Fixed-cell differential scanning calorimeters are used to determine the transition temperatures and energetics of materials in solution. For this information to be accepted with confidence in an absolute sense, temperature and heat calibration of the apparatus or comparison of the resulting data to that of known standard materials is required.  
5.2 This practice is useful in calibrating the temperature and heat flow axes of fixed-cell differential scanning calorimeters.
SCOPE
1.1 This practice covers the calibration of fixed-cell differential scanning calorimeters over the temperature range from –10 °C to +120 °C.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 7.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Jul-2023
• ICS: 17.200.10 - Heat. Calorimetry
• Technical Committee: E37 - Thermal Measurements
• Drafting Committee: E37.09 - Microcalorimetry

Relations

• Refers: ASTM E473-23b - Standard Terminology Relating to Thermal Analysis and Rheology
• Effective Date: 01-Oct-2023
• Refers: ASTM E1142-23b - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 01-Oct-2023
• Refers: ASTM E1142-15 - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 01-May-2015
• Refers: ASTM E1142-14b - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 15-Aug-2014
• Refers: ASTM E473-14 - Standard Terminology Relating to Thermal Analysis and Rheology
• Effective Date: 15-Aug-2014
• Refers: ASTM E1142-14a - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 01-Apr-2014
• Refers: ASTM E1142-14 - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 15-Feb-2014
• Refers: ASTM E691-13 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-May-2013
• Refers: ASTM E1142-12 - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 01-Sep-2012
• Refers: ASTM E691-11 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2011
• Refers: ASTM E1142-11b - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 01-Aug-2011
• Refers: ASTM E1142-11a - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 15-Jun-2011
• Refers: ASTM E473-11a - Standard Terminology Relating to Thermal Analysis and Rheology
• Effective Date: 15-Jun-2011
• Refers: ASTM E1142-11 - Standard Terminology Relating to Thermophysical Properties
• Effective Date: 01-Apr-2011
• Refers: ASTM E473-11 - Standard Terminology Relating to Thermal Analysis and Rheology
• Effective Date: 01-Apr-2011

Overview

ASTM E2603-15(2023) – Standard Practice for Calibration of Fixed-Cell Differential Scanning Calorimeters provides a comprehensive methodology for the calibration of fixed-cell differential scanning calorimeters (DSCs). This practice is essential for ensuring the accuracy and reliability of measurements related to transition temperatures and energetics of materials in solution. It outlines procedures for both temperature and heat flow calibration within the range of –10 °C to +120 °C. Adhering to this standard enhances confidence in analytical results, fostering quality assurance in laboratory settings.

Key Topics

• Calibration Scope: Covers both temperature and heat flow calibration for fixed-cell DSCs operating between –10 °C and +120 °C.
• Equipment Requirements: Specifies the use of DSCs with appropriate sample and reference cells, temperature controllers, and data collection devices capable of precise measurement.
• Calibration Procedures:
  • Two-point and one-point temperature calibration using certified materials such as various phosphatidylcholines and chicken egg white lysozyme.
  • Enthalpy calibration involving the measurement of transition energetics in lysozyme solutions.
• Calculation Methodology: Details calculations for determining actual specimen temperature and enthalpy, with strict guidelines for determining slope and intercept values for linear calibration equations.
• Reporting Requirements: Includes documentation of reference materials, instrument specifications, calibration results, and conditions used.

Applications

ASTM E2603-15(2023) is widely applicable across diverse scientific and industrial contexts where accurate thermal analysis is critical. Typical applications include:

• Pharmaceutical Development: Verifying thermal transitions in drug compounds and excipients to ensure consistent quality and stability profiles.
• Biochemistry & Protein Research: Measuring denaturation temperatures and energetics in proteins, facilitating biochemical and structural studies.
• Materials Science: Characterizing polymers, lipids, and emulsions for phase transition behaviors required in product development and quality control.
• Reference Laboratories: Providing traceable calibration for DSC instruments used in interlaboratory studies, regulatory submissions, and research environments.

Following the calibration procedures in this ASTM standard ensures measurement traceability, quality assurance, and compliance with international testing norms.

Related Standards

ASTM E2603-15(2023) references and is complemented by several important standards in the field of thermal analysis:

• ASTM E473 – Terminology Relating to Thermal Analysis and Rheology
• ASTM E691 – Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• ASTM E967 – Test Method for Temperature Calibration of Differential Scanning Calorimeters and Differential Thermal Analyzers
• ASTM E1142 – Terminology Relating to Thermophysical Properties

Keywords: calibration, differential scanning calorimeter, DSC, temperature calibration, enthalpy calibration, transition temperature, ASTM E2603, fixed-cell DSC, thermal analysis, material energetics

This standard aligns with internationally recognized principles for standards development, supporting broad acceptance and use worldwide. Implementing ASTM E2603-15(2023) enhances the precision and reliability of DSC measurements across research and industry.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E2603 − 15 (Reapproved 2023)
Standard Practice for
Calibration of Fixed-Cell Differential Scanning Calorimeters
This standard is issued under the fixed designation E2603; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
1.1 This practice covers the calibration of fixed-cell differ- 3.1 Specific technical terms used in this practice are defined
ential scanning calorimeters over the temperature range from in Terminologies E473 and E1142, including differential scan-
–10 °C to +120 °C. ning calorimeter, enthalpy, Kelvin, and transformation tem-
perature.
1.2 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
4. Summary of Practice
standard.
4.1 This practice covers calibration of fixed-cell differential
1.3 This standard does not purport to address all of the
scanning calorimeters. These calorimeters differ from another
safety concerns, if any, associated with its use. It is the
category of differential scanning calorimeter in that the former
responsibility of the user of this standard to establish appro-
have generally larger sample volumes, slower maximum tem-
priate safety, health, and environmental practices and deter-
perature scan rate capabilities, provision for electrical calibra-
mine the applicability of regulatory limitations prior to use.
tion of heat flow, and a smaller range of temperature over
Specific precautionary statements are given in Section 7.
which they operate. The larger sample cells, and their lack of
1.4 This international standard was developed in accor-
disposability, make inapplicable the calibration methods of
dance with internationally recognized principles on standard-
Practices E967 and E968.
ization established in the Decision on Principles for the
4.2 This practice consists of heating the calibration mate-
Development of International Standards, Guides and Recom-
rials in aqueous solution at a controlled rate through a region of
mendations issued by the World Trade Organization Technical
known thermal transition. The difference in heat flow between
Barriers to Trade (TBT) Committee.
the calibration material and a reference material, both relative
2. Referenced Documents
to a heat reservoir, is monitored and continuously recorded. A
transition is marked by the absorption or release of energy by
2.1 ASTM Standards:
the specimen resulting in a corresponding peak in the resulting
E473 Terminology Relating to Thermal Analysis and Rhe-
curve.
ology
E691 Practice for Conducting an Interlaboratory Study to
4.3 The fixed-cell calorimeters typically, if not always, have
Determine the Precision of a Test Method
electrical heating facilities for calibration of the heat-flow axis.
E967 Test Method for Temperature Calibration of Differen-
Despite the use of resistance heating for calibration, a chemical
tial Scanning Calorimeters and Differential Thermal Ana-
calibration serves to verify the correct operation of the calibra-
lyzers
tion mechanism and the calorimeter. The thermal denaturation
E968 Practice for Heat Flow Calibration of Differential
of chicken egg white lysozyme is used in this practice for
Scanning Calorimeters (Withdrawn 2023)
verification of the proper functioning of the instrument’s
E1142 Terminology Relating to Thermophysical Properties
systems. The accuracy with which the denaturation enthalpy of
chicken egg white lysozyme is currently known, 65 %, is such
that it should be rare that a calorimeter provides a value outside
This practice is under the jurisdiction of ASTM Committee E37 on Thermal
that established in the literature for this reference material.
Measurements and is the direct responsibility of Subcommittee E37.09 on Micro-
calorimetry.
5. Significance and Use
Current edition approved Aug. 1, 2023. Published August 2023. Originally
approved in 2008. Last previous edition approved in 2015 as E2603 – 15. DOI:
5.1 Fixed-cell differential scanning calorimeters are used to
10.1520/E2603-15R23.
determine the transition temperatures and energetics of mate-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
rials in solution. For this information to be accepted with
Standards volume information, refer to the standard’s Document Summary page on
confidence in an absolute sense, temperature and heat calibra-
the ASTM website.
tion of the apparatus or comparison of the resulting data to that
The last approved version of this historical standard is referenced on
www.astm.org. of known standard materials is required.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2603 − 15 (2023)
5.2 This practice is useful in calibrating the temperature and that calibration points are taken sufficiently close together so
heat flow axes of fixed-cell differential scanning calorimeters. that linear temperature indication may be approximated.
8. Calibration Materials
6. Apparatus
8.1 Phosphatidylcholines: 1,2-ditridecanoyl-sn-glycero-3-
6.1 Apparatus shall be:
phosphocholine (DTPC) CAS Number 71242-28-9; and 1,2-
6.1.1 Differential Scanning Calorimeter (DSC), capable of
ditetracosanoyl-sn-glycero-3-phosphocholine (DLPC) CAS
heating a test specimen and a reference material at a controlled
Number 91742-11-9. Purities are to be 0.99 or better. Addi-
rate and of automatically recording the differential heat flow
tional calibration materials are listed in Table 1.
between the sample and the reference material to the required
8.1.1 Aqueous suspensions of the phosphatidylcholines are
sensitivity and precision.
prepared as follows. Weighed amounts of a 0.01 Molar, pH 7
6.1.2 DSC Test Chamber, composed of:
solution of the buffer Na HPO – NaH PO and DTPC are
2 4 2 4
6.1.2.1 A device(s) to provide uniform controlled heating or
combined so to give a solution of 1 mass percent of the
cooling of a specimen and reference to a constant temperature
phosphatidylcholine. This procedure is repeated for DLPC.
or at a constant rate within the applicable temperature range of
The solutions are heated in a hot water bath to 5 K above the
this method.
transition temperatures. A vortex mixer is used to shake the
6.1.2.2 A temperature sensor to provide an indication of the
solutions at their respective temperatures until the lipid appears
specimen temperature to 60.01 K.
to have been completely suspended. The solutions may be
6.1.2.3 Differential sensors to detect a heat flow (power)
stored in a refrigerator until use for up to a week.
difference between the specimen and reference with a sensi-
8.2 Chicken egg white lysozyme with purity of at least 95 %
tivity of 60.1 μW.
mass percent.
6.1.3 A Temperature Controller, capable of executing a
8.2.1 Weighed amounts of the lysozyme and of a 0.1 M HCl
specific temperature program by operating the furnace(s)
– glycine buffer at pH = (2.4 6 0.1) are combined to obtain a
between selected temperature limits at a rate of temperature
solution of approximately 3 mass percent.
change of 0.01 K/min to 1 K/min constant to 60.001 K/min or
8.2.2 The concentration of lysozyme in this solution is
at an isothermal temperature constant to 60.001 K.
calculated from UV absorbance at a wavelength of 280 nm,
6.1.4 A Data Collection Device, to provide a means of
using a 1 cm cell and the optical density of 2.65 for a 1 mg
acquiring, storing, and displaying measured or calculated -1
mL solution.
signals, or both. The minimum output signals required for DSC
8.2.2.1 Fill a 1 cm optical cell with buffer solution and
are heat flow, temperature, and time.
another 1 cm cell with the lysozyme solution. Follow the
6.1.5 Containers, that are inert to the specimen and refer-
instrument’s directions for establishing baseline, and if needed,
ence materials and that are of suitable structural shape and
calibration of the absorbance scale. Insert both of the filled
integrity to contain the specimen and reference in accordance
cells in the UV spectrometer if the spectrometer is a dual beam
with the specific requirements of this test method. These
instrument. Scan through the 280 nm region and note the
containers are not designed as consumables. They are either an
absorbance at 280 nm. If the spectrometer is a single beam
integral part of the instrument, whether or not user-removable
instrument, the buffer is measured first, then the lysozyme
for replacement or, in some implementations, are removable
solution is measured and the difference in the recorded absor-
and reusable. Container volumes generally range from 0.1 ml
bances is used to calculate the concentration. Concentration is
to 1 ml, depending on the instrument’s manufacture.
calculated as:
6.2 Analytical Balance, capable of weighing to the nearest
c 5 A/ 2.65 mL mg
~ !
0.1 mg, for preparation of solutions.
where:
6.3 UV spectrophotometer or UV/Vis spectrophotometer,
A = absorbance, and
capable of scanning the UV spectrum in a region about 280 nm.
-1
c = concentration in mg mL .
6.4 Reagents:
NOTE 1—Different concentrations may be used between 1 and 10 mass
6.4.1 Phosphatidylcholines, 1,2-ditridecanoyl-sn-glycero-3- percent, the concentration used shall be included in the report.
phosphocholine (DTPC) CAS Number 71242-28-9 and 1,2-
TABLE 1 Melting Temperature of Calibration Material
ditetracosanoyl-sn-glycero-3-phosphocholine (DLPC) CAS
NOTE 1—The uncertainties for the temperatures are ±0.1 K.
Number 91742-11-9 are the minimum required.
6.4.2 Aqueous buffer solutions, 0.01 Molar, pH 7 aqueous Melting
Calibration Material Temperature
solution of Na HPO – NaH PO and 0.1 Molar, pH (2.4 6
2 4 2 4
°C K
0.1) aqueous solution of HCl + glycine.
1,2-ditridecanoyl-sn-glycero-3-phosphocholine (DTPC) 13.25 286.4
1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) 23.75 296.9
6.4.3 Chicken egg white lysozyme.
1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) 41.45 314.6
1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) 54.85 328.0
7. Precautions
1,2-dieicosanoyl-sn-glycero-3-phosphocholine (DAPC) 65.05 338.2
1,2-didocosanoyl-sn-glycero-3-phosphocholine (DBPC) 73.35 346.5
7.1 This practice assumes linear temperature indication.
1,2-ditetracosanoyl-sn-glycero-3-phosphocholine (DLPC) 80.55 353.7
Care must be taken in the application of this practice to ensure
E2603 − 15 (2023)
9. Procedure 9.3.2.1 Fill the sample cell with the lysozyme + buffer
solution and fill the reference cell with the HCl-glycine buffer
9.1 Two Point Temperature Calibration:
solution—taking care that no air bubbles are retained in either
9.1.1 Determine the apparent transition temperature for
of the cells.
each calibration material, as described in Table 1.
9.3.2.2 Equilibrate the calorimeter near room temperature,
9.1.1.1 Fill the clean specimen cell with the phosphatidyl-
following equilibration the temperature of the calorimeter is
choline suspension, according to the usual method specified for
ramped at 60 K/h until a sufficient baseline is established
the instrument. Fill the reference cell with buffer solution that
beyond the transition peak.
was used to prepare the phosphatidylcholine suspension.
9.1.1.2 Equilibrate the calorimeter approximately 10 K to
NOTE 4—Slower scan rates shall not be used in this step due to potential
aggregation of the denatured protein.
15 K below the expected transition temperature from Table 1.
9.1.1.3 Heat each cali
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