TAM: Thermal Activity Monitor

Basic Theory &

Applications Training
2015

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Agenda – Basic TAM Course

•Introduction to calorimetric principles and TAM instruments

•Calibration

•Overview of TAM Assistant

•Experimental part: Calibration

•Sample preparation and experimental considerations

•Experimental part: Setting up an experiment

•Data handling and generating reports

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Thermal Measurements

▪ Virtually all chemical and physical processes result in

either heat production or heat absorption.

▪ Calorimetry quantifies the amount and rate of heat

release in terms of heat flow, heat and heat capacity.

▪ Calorimetry is a non-specific technique making it ideal

for studying almost all kinds of physical and chemical
processes in life sciences, materials sciences and within
pharmaceuticals.

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Thermal Analysis and Calorimetry

▪ Thermal analysis
▪ The change of a property as function of
temperature
▪ DSC (scanning mode), TGA, DMA, TMA (slow
scanning mode)

▪ Calorimetry
▪ The measurement of heat properties, i.e. heat
flow, heat and heat capacity as function of
time and temperature

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Thermal Analysis versus Calorimetry

Thermal Analysis Calorimetry

ITC
TGA Solution
DSC
Calorimetry
TMA
TAM Reaction
DMA Calorimetry

Scanning, Temperature-Induced Scanning or Isothermal

Processes

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 Calorimetric Range

1 nW 1 mW
-11 -9 -7 -5 -3 -1 1 3

-12 -10 -8 -6 -4 -2 0 2 4
1 pW 1 mW 1W

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Specific Sensitivity – a Comparison

Sample Specific
Sensitivity
amount sensitivity
Nanocalorimeter 10 nW 5g 2 nW/g
DSC 200 nW (0.2 mW) 10 mg 20 µW/g

Difference in specific sensitivity X 10,000

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TAM versus DSC

•Complementary techniques
•High sensitivity in the range - nW vs. µW
•High specific sensitivity - g vs. mg
•Measurements in hours vs. minutes
•Absolute heat capacity determinations –
< 0.2 % vs. 1-5 %
•Most TAM experiments are done
isothermally

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INSTRUMENTATION

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TAM – Thermal Activity Monitor

Represents a range of products used for

calorimetric measurements

TAM IV TAM III SolCal TAM Air

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General Features of the TAM

▪ Thermostat
▪ Calorimeters (4mL, 20mL, 125mL)
▪ Sample handling (Ampoules)
▪ Accessories
▪ Software

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TAM – Isothermal and Scanning Thermostat

▪ Temperature range: 4° – 150 °C (TAM III is 15° – 150 °C)

– Isothermal and slow scanning (2 °C/h)
– Temperature stability: < 0.1 mK/24 h
– Temperature accuracy: ±0.1oC
– Temperature precision: < ±0.1 mK

▪ Multi functional calorimeters and accessories

– Different measuring modes

▪ High sample throughput

– Up to 48 individual twin heat flow calorimeters.

▪ Outstanding sensitivity and long-term stability (µW-nW)

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TAM Temperature Modes

•Isothermal
•Step-wise scanning
•Slow scanning (max ±2 °C/hr)

Isothermal Step- Scanning

wise
Heat flow

Time
- Temperature profile
- Example heat flow data

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 TAM Thermostat

An oil based liquid bath system outer

for a continuously circulated heat tank

Calorimeter
sink medium that prevents any
thermal event in a test sample or
from the room environment from inner
altering the constant temperature tank
bath
Peltier
cooler

fine heater
over flow
system

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TAM IV Thermostat Features

•A temperature regulation system utilizing state-of-the-art

electronic thermistor sensors to constantly adjust the
heating, cooling and uniform oil flow speed for a
temperature drift over 24 hours that is less than ±100 μ°C
Bath mean temperature (µK)

150 Error limits

(specifications)
100
50
0
-50
-100
-150
0 6 12 18 24
Time (h)
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Multi-Functional Calorimeters of the TAM III/IV

•TAM is a flexible system which can be configured for a

variety of applications.

•Additional functions or increased measuring capacity is

easily obtained by adding:
▪Calorimeters
▪Sample handling systems (ampoules)
▪Accessories or micro-reaction systems

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The Flexibility of the TAM

Sample size

Nanocalorimeter
Multicalorimeter (4 mL) Microcalorimeter Multicalorimeter (20 mL)
Macrocalorimeter

Absolute Sensitivity
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Nanocalorimeter

•Highest sensitivity twin channel calorimeter

▪Heat flow (or flux)
◆ high sensitivity, longer time scales
▪Dynamic heat flow
◆ Considers the thermal inertia of a calorimeter
▪Power compensation (high resolution, shorter time scales)
◆ Referred to as ‘Feedback’ in software
◆ A constant electric power is supplied to both sample and
reference calorimeters continuously.
◆ Time constant of calorimeter significantly smaller than
heat flow mode.

•Reference accessible by user

▪Up to 4 mL total volume ampoules

•Use with 1 or 4 mL Micro Reaction System(s)

▪Only choice for high sensitivity isothermal titration
calorimetry (ITC)

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Detection System of the Nanocalorimeter

Thermopiles
(two on each side)

Foil Heaters for

Calibration/
Compensation

Heat Sink
Partition wall
Sample

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Nanocalorimeter – Heat Flow Stability over 24hr

30

20 Baseline stability/24h
Heat flow (nW)

Error limits (specifications)

10

0
Drift
-10

-20

-30
0 4 8 12 16 20 24
Time (h)

The absolute temperature in this measurement was 35.0˚C

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Microcalorimeter

▪ Highest specific sensitivity twin channel

calorimeter
– Heat flow (or flux)
▪ high sensitivity, longer time scales
– Dynamic heat flow
▪ Considers the thermal inertia of a calorimeter

▪ Reference accessible by user

– 20 mL total volume ampoules
– For large samples or increased gas phase

▪ Used with 20 mL Micro Reaction System(s)

– Only choice for Micro Solution Ampoule

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 Minicalorimeter (4 and 20 mL versions)

•High Throughput twin channel

calorimeter
◆ Heat flow (or flux)
◆ Dynamic heat flow

•Reference NOT accessible by user

▪Must determine type of plug to be inserted
to minicalorimeter for best heat capacity
balance and performance.
▪4mL size best for temperature ramp
▪20 mL size for vacuum/pressure ampoule

4mL minicalorimeter attached to its computer interface

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Minicalorimeter (4 mL)

Sample
Ampoule
holder Outer
Steel
Cylinder

Thermopiles
Heat Sink

Reference
Ampoule
holder

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Minicalorimeter Measuring Assembly

Sample Side
Calibration Heater

Thermopiles (one each side)

Reference plug is permanently

inserted and must be selected based on
the type of ampoule being used.

Reference Side
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Minicalorimeter (unbalanced) - Baseline Stability

300

200
/ nW

100
P /nW

0
Heat flow

-100

-200

-300
0.00 4.00 8.00 12.00 16.00 20.00 24.00
Time /h
The absolute temperature in this measurement was 50.0 ˚C. Note: must acknowledge
25
that the calorimeter is not balanced due to the permanent reference plug.

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 Multicalorimeter (4 mL)

•For increased measuring capacity and

productivity of TAM

•Consists of six minicalorimeters (4 mL)

▪Also 20 mL multicalorimeters that consist of 3 –
20 mL minicalorimeters (not shown)

•Four multicalorimeters can be used with a

TAM thermostat to provide 24 simultaneous
measurements or alternatively the TAM
48...

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 Minicalorimeters (4 mL) in TAM 48

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Macrocalorimeter

Lifter

Access tube

Thermal shunts

Sample cell with

battery and fixture

Reference cell with

fixture

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Calorimeter Theory

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 Definitions

•Rate of heat production

▪The rate of heat produced (exothermic) or consumed
(endothermic) by the sample

•Rate of heat exchange

▪The rate of heat flow between the sample and the surrounding

Note: During Steady State conditions these properties are equal

Exothermic reactions → Positive Heat flow signal

Endothermic reactions → Negative Heat flow signal

The heat flow is proportional to the rate of the reaction

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Rate Equation in Terms of Heat Flow

dC
= k  f (c )  mol 
 m3 s 
dt
dQ dC  J   mol J 
= H  m3 s  =  m3 s mol 
dt dt
dQ
= H  k  f (c) = Heat flow signal from TAM
dt

Enthalpy → Reaction rate → Concentration →

Thermodynamic Kinetic Analytical
Information Information Information

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 Heat Flow versus Time

18000

16000
flow (µW/g)

14000
Shows how the
Heatflow (µW/g)

12000
f(t, P, Q) reaction rate varies
10000
with time.
8000
Heat

6000

4000

2000

0
0 1 2 3 4

Time(d)
Time (d)

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 Energy versus Time

18000

16000

14000
Shows how the
(J/g)

12000
extent of reaction
Energy (J/g)

10000
varies with time.
Energy

8000

6000

4000

2000

0
0 1 2 3 4

Time (d)
Time (d)

33 TAINSTRUMENTS.COM
 Heat Flow versus Energy

18000

16000
(µW/g)
14000
(µW/g)

12000
flow

10000
Heatflow

8000
Heat

6000

4000
2000

0
0 5000 10000 15000 20000

Energy
Energy (J/g)
(J/g)

Shows how the reaction rate varies with the extent of reaction
(ex. 1 order → Q proportional to P with rate const., k, as proportionality constant)

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 Calorimetric Unit


General Heat Balance Equation
 dQ 
  To
 dt 

dQ   dT 
= + C 
TS dt  dt 
 dT  Rate of Heat Rate of Heat Rate of Heat
C 
 dt  Production = Exchange + Accumulation

The measured property

Rate of Heat Heat flow

After calibration the following holds: Production = Monitored
(dQ/dt) by TAM

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The Heat Balance Equation (in terms of temperature)

dQ dT
= +C Single calorimeter
dt dt

 = k (T − To )
Note: dQ/dt depends on
(Newton's cooling law) the surrounding temperature.

k = Heat Conductance = C/

dQ dT
= k (T − To ) + C
dt dt

The temperature can be monitored by a thermopile as is the case in TAM or

by using thermistors, as is the case of the Solution Calorimeter (SolCal).

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The Heat Balance Equation (in terms of voltage)

dQ dT Substitute Newton’s cooling law and

= +C potential (or voltage) terms [Seebeck]
dt dt

 = k (T − To ) =
k U dT 1 dU
V  T= + To  =
g g dt g dt

dQ k C dV k  C dV   dV 
= V+ = V +  =  V +  
dt g g dt g  k dt   dt 

Tian Equation
g = Seebeck coefficient (V/K)
dQ  dV 
=  V +    = calibration constant (W/V)
dt  dt 
 = time constant (s)

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 Twin Channel Calorimeter (as used in TAM)

Sample cell (S) Reference cell (R)

Surrounding To Surrounding
(heat sink) (heat sink)
kS kR

S R
Sample Sample
holder holder
dQS dQR
TS CS TR CR
dt dt

To

k = C/ = heat transfer coefficient

T=(Ts-To)-( TR-To)=Ts-TR between the sample and the
surrounding or heat conductance
With a twin system the
 = time constant
noise will be reduced!

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 The Heat Balance Equation (Twin System)

Sample side
dQ S dT
= k S (TS − To ) + C S S
dt dt

Reference side
dQ R dTR
= 0 = k R (TR − To ) + C R
dt dt

Subtraction gives
dQS dTS dTR
= k S (TS − To ) + CS − k R (TR − To ) − CR
dt dt dt

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The Heat Balance Equation (Twin System)

dQ S d (TS − TR )
= k (TS − TR ) + C
dt dt
Assumptions:
k = kR = kS
C = CR = CS

Qseries DSC does not make this assumption in (T4 mode). Q20 and 29XX DSC and most
other commercial DSCs also make this assumption. High heat capacity and the thermal
stability within the calorimeters aid in the acceptance of the assumptions for the TAM.

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 Heat Capacity Balance versus Noise Level

4 mL
Mini
calorimeter

Empty Balanced High side

(unbalanced) within 20% of
balance
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 Heat Capacity Balance versus Noise Level

20 mL
Mini
calorimeter

Empty Balanced
(unbalanced)

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 Choosing a Reference

Sample side = Micro Solution Ampoule + 16 Inserted in 4 mL Calorimeter:(s)

mL solvent ( = 240 s) Empty 96

SS circlip 205
Reference side = SS circlip ampoule with SS screw cap 202
thick lid + 16 mL solvent ( = 240 s) Glass circlip 151

Empty 20 mL calorimeter (  60 s)
RH Perf./Titr. 206
1 g Water 41-44
1 g Glass beads 5-10
Heat capacity (C) of common materials:
(measured with TAM 2277 at 25 °C)
Water 4.18 J/K•g
*Use GPT to measure .
Sand (Quartz) 0.8 J/K•g
Glass 0.84 J/K•g
Stainless Steel 0.47 J/K•g Balance heat capacity
Aluminum 0.90 J/K•g (C) or time constant ()*
 = C/k
See also EN 008

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 Heat Capacity of Water Versus Temperature

4
.
2
2

4
.
2
1
HeatCapacity(J/Kg)
4
.
2
0

4
.
1
9

4
.
1
8

Water = 4.18 J/K•g @ 25 °C

0 2
0 4
0 6
0 8
010
0
T
e
m
p
er
a
t
ur
e
(
C
)
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 Choosing a Reference – 4 mL Minicalorimeters

C p ,ri − 0.24 − C p ,a − C p , s  m
 = 100 
C p ,ri + 7.18

Minicalorimeters:

Cp,ri , Cp,a and Cp,s are heat capacities of reference insert, ampoule and the substance
Heat capacity of the ampoule holder (reference side) = 7.42
Difference in heat capacity of the ampoule holders sample and reference side = 0.24
Mass (m) of substance used can be optimised so that  approaches zero.
Maximum deviation for which the signal will lie within specifications is ±20%.

45 TAINSTRUMENTS.COM
 Choosing a Reference – 4 mL Minicalorimeters

Cp,ri /J K-1 Optimized for:

Ref. insert 1/4 6.89 3-ml glass vial with 2g

organic solid material

Ref. insert 2 19.18 4-ml Stainless steel

ampoule with 2 ml of an
aqueous solution
Values for the minicalorimeter balance
Ref. insert 3 8.43 4-ml glass vial with 2g
organic solid material
equation shown on the previous slide.
Ref. insert 5 3.67 3-ml glass vial with 150
mg organic solid material

Ref. insert 6 15.89 4-ml Stainless steel

ampoule with 2g organic
solid material.

Ref. insert 7 9.24 3-ml glass vial with 3g

Product no Cp,a /J K-1 Description

organic solid material

2509-51 2.34 5 ml Heat Seal Glass Ampoule
Ref. insert 8 10.73 4-ml glass vial with 2.8g
organic solid material
95.53.1015 4.32 3 ml Disposable Glass Ampoule

24.20.0400 5.84 4 ml Disposable Glass Ampoule

2502-40 11.9 4 ml St.Steel Ampoule circlip cap

2277-301 11.5 4 ml St.Steel Ampoule threaded cap

3320 10.8
46
4 ml St.Steel Ampoule threaded cap

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Choosing a Reference – 4 mL Minicalorimeters

Please refer to Table 1 that lists the total Cp of all 8 models of the 20 mL minicalorimeters.
For sample Cp calculations, utilize Table 2 for a list of some common sample materials.

4mL minicalorimeter Some common heat capacities

Disposable Disposable Cp, J K-1 g-1
3mL glass 4mL glass 4mL steel 4mL steel 4mL Hastelloy Liquids
95.53.1015 24.20.0401 2277-301 3320-1 3320 Water 4,18
Ampoule Cp (J/K) 4.3 5.8 11.9 10.5 9.2 Ethanol 2,43
Reference Total Cp Propanol 2,40
Sample Cp (J/K) Benzene 1,73
Number (J/K)
Toluene 1,71
X=1 6.9 2.6 ± 1.4 1.1 ± 1.4
Pentane 2,33
X=2 19.2 14.9 ± 3.8 13.4 ± 3.8 7.3 ± 3.8 8.7 ± 3.8 10.0 ± 3.8
Heptane 2,24
X=3 8.5 4.2 ± 1.7 2.7 ± 1.7 DMSO 1,93
X=4 6.9 2.6 ± 1.4 1.1 ± 1.4 Solids Ethylene glycol 2,5
X=5 3.7
X=6 15.9 11.6 ± 3.2 10.1 ± 3.2 4.0 ± 3.2 5.4 ± 3.2 6.7 ± 3.2 Inorganic:
X=7 9.3 5.0 ± 1.9 3.5 ± 1.9 NaCl 0,86
X=8 10.8 6.5 ± 2.2 5.0 ± 2.2 0.3 ± 2.2 1.6 ± 2.2 Quartz (SiO2) 0,76

Organic:
Lactose 1,22
Example 1: Sample of gunpowder loaded into a calorimeter with reference Glycine 1,32
number 6 (15.9 J/K). Urea 1,55
Glucose 1,24
Salicylic acid 1,16
Using a stainless steel threaded ampoule (2277-301) with Cp of 11.9 J/K the Gun powder 1,28
remaining heat capacity to balance the calorimeter is 4 J/K. Knowing the sample is
gun powder that has a Cp of 1.28 J/gK one can calculate that approximately 3.1 g  Table 2: List of Sample Cp
2.5 g of gun powder must be loaded into the ampoule to best balance the
calorimeter to within 20% of the total Cp.

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Choosing a Reference – 20 mL Minicalorimeters

Please refer to Table 1 that lists the total Cp of all 8 models of the 20 mL minicalorimeters.
For sample Cp calculations, utilize Table 2 for a list of some common sample materials.

20 mL minicalorimeter Some common heat capacities

Stainless Stainless Glass Cp, J K-1 g-1
Thread o-ring High Pressure Disposable Liquids
3440-1 3348-1 24.60.2001 Water 4,18
Ampoule Cp (J/K) 34 53 15 Ethanol 2,43
Reference Total Cp Propanol 2,40
Sample Cp (J/K) Benzene 1,73
Number (J/K)
Toluene 1,71
X=1 29 14 ± 5.8
Pentane 2,33
X=2 37 3.0 ± 7.4 22 ± 7.4 Heptane 2,24
X=3 46 12 ± 9.2 31 ± 9.2 DMSO 1,93
X=4 53 19 ± 10.6 38 ± 10.6 Solids Ethylene glycol 2,5
X=5 57 23 ± 11.4 4 ± 11.4 42 ± 11.4
X=6 62 28 ± 12.4 9 ± 12.4 47 ± 12.4 Inorganic:
X=7 66 32 ± 13.2 13 ± 13.2 51 ± 13.2 NaCl 0,86
Quartz (SiO2) 0,76
X=8 71 37 ± 14.2 17 ± 14.2 56 ± 14.2
Organic:
Lactose 1,22
Example 1: Sample of gunpowder loaded into a calorimeter with reference Glycine 1,32
number 6 (62 J/K). Urea 1,55
Glucose 1,24
Salicylic acid 1,16
Using a stainless steel threaded ampoule (3440) with Cp of 34 J/K the remaining Gun powder 1,28
heat capacity to balance the calorimeter is 28 J/K. Knowing the sample is gun
powder that has a Cp of 1.28 J/gK one can calculate that approximately 22 g  9.6 Table 2: List of Sample Cp
g of gun powder must be loaded into the ampoule to best balance the calorimeter to
within 20% of the total Cp.

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 When to Calibrate

Calibration is suggested if...

• TAM has been switched OFF
• Temperature has been changed
• Change experimental conditions
- The time constants of calorimeter are increased when an
ampoule or accessory are loaded.
▪ Choose correct reference and perform calibration with the
accessory in measuring position.
- Routinely at regular intervals due to ageing of the semi-
conductor thermopiles (e.g. once every third month)

49 TAINSTRUMENTS.COM
 Calibration of TAM IV

• The calorimeters of TAM IV have been calibrated at 6

different temperatures so as to diminish the influence
of temperature on the users calibration results.

• When the user makes a calibration, the results are

compared with that obtained from the “factory
calibration” and deviation is calculated.

• The deviation is represented by a unit-less calibration

constant (called the gain constant in TAM Assistant)
and should be close to unity (normally 0.95-1.06).

50 TAINSTRUMENTS.COM
 Purpose of Calibration

•To ensure that the displayed heat flow corresponds to the

true heat flow caused by the sample
▪Conversion of measured voltage to heat flow
▪Account for any heat losses

•Calibration of TAM is performed using inbuilt calibration

heaters.
▪The inbuilt calibration can be validated using external
calibration heaters or chemical test reactions.

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 Two Types of Calibrations

•Heat flow calibration – for ‘slow’ processes

•Dynamic calibration - for ‘fast’ processes

52 TAINSTRUMENTS.COM
 Static or Dynamic Calibration

•Any calibration makes use of an internal electrical heater

(i.e. precision resistor) with known calibration power
applied. The voltage monitored by the thermopiles is
proportional to the heat flow through the module.
▪Calibration is needed in order to convert the voltage to the
corresponding rate of heat production.
◆ For slow processes a static calibration can be performed. The
displayed heat flow corresponds to the rate of heat production.

◆ For fast processes, i.e. when the response of the reaction is less
or in the range of the time constant of the system, a dynamic
calibration should be used. The thermal inertia of the calorimetric
system is taken into consideration. The displayed heat flow
always corresponds to the rate of heat production.
- ITC experiments: utilize Feedback mode or dynamic calibration

53 TAINSTRUMENTS.COM
 Calibration Conditions

•Ampoule Experiments
▪ Empty ampoule lifters (or empty ampoules) should be in position in both sample and
reference side

•RH Perfusion Experiments

▪ The empty RH Perfusion ampoule should be in measuring position to consider heat loss
effects through the ampoule
▪ A reference ampoule should be in position
▪ Calibration can be performed with or without gas flow since the cooling effect can be
negligible below 40 ºC. Humidity should be set to initial RH.

•Titration Ampoule Experiments

▪ The titration ampoule filled with the solvent should be in measuring position to consider
heat loss effects through the ampoule
▪ A reference ampoule should be in position
▪ Optional: Stirrer ON
◆ This can account for any shift in frictional heat flow from the stirrer. Although the

baseline sections of the experiments provide this correction.

54 TAINSTRUMENTS.COM
 Static Calibration - Pulse
Exponential heat exchange

P (µW) t
Calibration Power ON P = Po exp(− )
= set µW pulse 

Calibration
Power OFF

How to obtain 
The time until P/Po = 1/e = 0.37
P/Po = 1/e

Empty calorimeter or Accessory and Reference in position.

Time

55 TAINSTRUMENTS.COM
 Heat Flow Calibration Procedure

• Ensure that the heat flow baseline is stable

• Apply settings:
▪ Pulse calibration and integration(~0.5-1 h)
▪ Steady state calibration (2-3 h)
◆ Not for minicalorimeters

• Start the calibration in TAM Assistant

▪ Keep a running record of the calibration constants

56 TAINSTRUMENTS.COM
 Dynamic Mode

• Heat flow data will not reflect the true response of the sample for reactions with elapsed times
less than 10 min. For reactions where the slope of the heat flow time curve ( f) is changing
rapidly a dynamic correction can be applied to obtain the true response of the sample (P) using
the following formula (Tian’s equation)
df
P =f +τ
dt
• Dynamically corrected data represents the true data of the sample and has been calculated
from Heat flow data using the information about time constants obtained from a dynamic
calibration.
▪ The TAM Assistant software contains functions for considering the effects of the thermal inertia
(dynamic mode). TAM Assistant uses two time constants rather than one to get a better precision in the
correction (cf. Taylor expansion).

df df
P = f + ( 1 +  2 ) +  1  2 2
dt dt
1 and 2 are time constants obtained from dynamic calibration

57 TAINSTRUMENTS.COM
Dynamic Calibration

•Dynamic calibration refer to calibration under non-steady state conditions

•A known electrical calibration power is applied in two steps and the

dynamic of the curvature is analyzed in terms of time constants.

•Dynamic calibration should always be performed if the response time of

a process is less than 15 min

30
Calibration
power
Heat flow (µW)

20

Measured heat
10 flow

0
0 20 40
Time,min
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 Calibration Results Example

After static calibration After dynamic calibration

Electrical Electrical Electrial Electrial
P Power ON Power OFF Power ON Power OFF
Name of signal from TAM 2277
Dynamic
calibration

Raw Corr
(Heat flow (Raw data
monitored recalculated
by the heat using info
flow sensor) about time
constants)

Red curve = known electrical  1 and  2

power applied at the
Time
are determined
resistor below the ampoule

59 TAINSTRUMENTS.COM
 Dynamic Calibration Procedure

• Set measuring principle to “Dynamic mode”

• Introduce the ampoule with sample and/or reference

• Wait until the heat flow signal is stable.

• Start the calibration: there are two options

▪ Time-constant calibration
▪ Full dynamic calibration

• After 30-60 min the dynamic calibration is completed

60 TAINSTRUMENTS.COM
 General Performance Test (GPT)

• General Performance Test

▪ A test to evaluate the performance of a calorimetric system, i.e. thermostat
with a calorimetric unit.
▪ Recommended to perform GPT near ambient temperature
◆ 20 - 40°C

• Calculated parameters (for A and B side)

▪ Time constants and difference between A and B side.
▪ Drift, Fluctuation and Error over 24 hrs
▪ Short term noise

• Method: GPT experimental wizard (Validation folder)

• Evaluation: GPT analysis

▪ The analysis function gives a report with a Yes or No answer as to whether
the calorimeters are within specifications

61 TAINSTRUMENTS.COM
Calorimeter Enthalpy Validation

▪ Non-isothermal experimental wizard.

– Ramp 2°C/hr from 58-80 °C
▪ Accurately weigh biphenyl in TAM ampoule (single use only).
▪ Utilize peak analysis button on tool bar.
▪ Theoretical = 121.41 J/g
– Note that endothermic enthaply is calculated as a negative value in TAM
Assistant and the negative sign should be neglected.

TAINSTRUMENTS.COM
Ampoules and Accessories

TAINSTRUMENTS.COM
TAM IV – sample handling system

The TAM IV offers a complete array of ampoules

in two basic types; open and closed.

• Open ampoules are part of the micro reaction

system for the direct manipulation or
modification of the sample or its surroundings
during the experiment. (examples right)
• Closed, also referred to as static, ampoules
contain the specimen in a static fashion: no
manipulation of the sample is performed during
the measurement. (examples below)

TAINSTRUMENTS.COM
Static measurements

•Stability
•Compatibility
•Reaction kinetics
•Amorphicity
•Polymorphism
•Curing
•Safety assessment
•Microorganism growth
•Etc...

TAINSTRUMENTS.COM
 Closed, Sealed or Static Ampoules

•Disposable glass ampoules

▪3, 4, and 20 mL

•Glass heat seal ampoules

▪5 mL

•Stainless steel and hastelloy

(pH<4)
▪Threaded with Teflon™ or o-ring seal
▪4 and 20 mL

•Circlip cap ampoules in stainless

steel, hastelloy, or glass
▪1, 4 and 20 mL

66 TAINSTRUMENTS.COM
Titration setup
– Possibilities for adding and mixing

• Binding affinity, stoichiometry

and thermodynamics
• Complex formation
• Cmc determinations
• Enzyme kinetics
• Mixing enthalpies
• Dissolution kinetics
• Absorption
• Reaction kinetics
• Swelling
• Drug effect on living
cells
• Etc...

TAINSTRUMENTS.COM
 Titration Ampoule

•For isothermal titration calorimetry (ITC)

▪ Refers to repeated injections of a substrate into a
solvent in order to study ligand binding or other
molecular interactions.

•Dedicated software control and analysis

functions makes the evaluation of ligand
binding simple and straight forward.

•Gold propeller or turbine stirrers available

•Stainless steel, glass or hastelloy

▪ 1 (shown), 4 or 20 mL sizes available

68 TAINSTRUMENTS.COM
Gas or liquid perfusion

• Stability
• Compatibility
• Absorption
• Reaction kinetics
•Oxidation
• Safety assessments
• Hydration
•Swelling
• Metabolism of living
small animals
• Etc...

Could add mixing &

injection possibilities
TAINSTRUMENTS.COM
Matrix Cartridge

•Stainless steel hollow tube, with Teflon

mesh at each end, capped by Teflon
ends.
▪Use with 4 mL perfusion ampoules
▪Attach to the central shaft forcing liquid
through the “column” then around and
out.
▪Mesh has 105 μm openings

•Useful for making sure that the

perfusion liquid flows through the
material to be tested, rather than just
over the top of the material bed.

70

TAINSTRUMENTS.COM
Glass Plate Holder

•Frame made in stainless steel that

can hold two glass plates
▪Use with 4 or 20 mL perfusion or
titration ampoules
▪Attach to the central shaft
▪Can be stirred or rotated

•Useful for studying biological

activity and for studying
immobilized samples

71

TAINSTRUMENTS.COM
Perfusion Insert

•Attaches to the central shaft and

allows gas to flow to the bottom of
the reaction vessel.
▪Use with 4 mL perfusion MRS
▪Attach to the central shaft – 3 holes in
the bottom of the shaft
▪Typically placed in reaction vessel
before sample is placed in the vessel.

•Useful for making sure gas flows

through the sample rather than just
over the top of the sample bed.
72

TAINSTRUMENTS.COM
Relative humidity perfusion

• Amorphicity assessments
• Polymorphism
• Stability
• Compatibility
•Absorption
• Reaction kinetics
• Safety assessments
• Hydration
•Swelling
• Etc...

Equally applicable for other

type of solvents and vapour
pressures

TAINSTRUMENTS.COM
 Verify the Humidifying System

•One Point calibration with Zero

▪ Zero adjustment using an empty RH Perfusion ampoule (0% RH)

▪ A salt solution with a known RH is loaded into the ampoule and heat flow is adjusted to
zero to calculate error in RH.

▪ The difference between the expected RH and calculated RH defines the error

•Two Point Calibration

▪ Zero adjustment with a saturated salt solution in the ampoule

▪ The first salt solution is replaced with a second salt solution and the RH adjusted to
zero to calculate error in RH.

▪ The difference between the expected RH and adjusted RH defines the error

74 TAINSTRUMENTS.COM
Suitable Salt Solutions

(1983)

TAINSTRUMENTS.COM
 RH Perfusion Calibration – Method I

P (µW)
1st Part 2nd Part 3rd Part
600 min 1+6xM
Waiting for Load
stable RH amp.
conditions Wait for
phys.
equilib.
RH Perfusion
empty.
Reference
empty.

Zero adj. if
necessary!!

Pause BL Pause M M M M M M M BL
Time
TAINSTRUMENTS.COM
LiCl at 25°C; RH from 5-15% in 1% steps

16 150

C: -79.1±0.1 µW
C: -74.7±0.05 µW
14 100 C: -57.0±0.04 µW
Relative humidity (%) C: -39.1±0.04 µW
C: -22.7±0.05 µW
C: -6.28±0.05 µW

Heat flow (µW)

C: 10.0±0.04 µW
12 50 C: 28.4±0.05 µW
C: 47.5±0.04 µW
C: 65.3±0.06 µW

10 0

8 -50
C: -63.3±0.04 µW

6 12 18 24 30 36 42
Time (ho ur)

TAINSTRUMENTS.COM
 LiCl at 25°C; RH from 5-15% in 1% steps

RH Calibration Curve

78 TAINSTRUMENTS.COM
 LiCl at 25°C; RH from 5-15% in 1% steps

RH Calibration Curve

Solve for y = 0

187.5 / 16.641
= 11.27%

LiCl at 25°C
Should be 11.3%

Correction factor
= 0.03%

79 TAINSTRUMENTS.COM
Dissolution

•Heat of dissolution
• Heat of wetting
• Amorphicity
• Polymorphisms
•Dissolution kinetics
• Etc...

Solution Calorimeter
(solid or liquid samples)
Microsolution ampoule
(solid sample)
TAINSTRUMENTS.COM
Micro Solution Ampoule

•Designed for monitoring dissolution

▪Available in 20 mL volume only and
used with a Microcalorimeter

▪Up to three repeated injections of a solid

sample

◆ Sample size 1-50 mg

TAINSTRUMENTS.COM
 Micro Solution Calorimeter Experiment

P (µW) 1st Part 2nd Part

Waiting for
stable Dynamic
conditions. Calibration Look at Corr data
is automatically Up to 3 injections, in Screen Plot
Ampoule
repeat factor = 2,
filled with performed in the M+BL and View Results!
solvent in
position. method (max. 50 min)
Reference
filled with Must run blank
solvent in
position.

Zero adj. if
necessary!!

Pause Main M M M Time 82

Pause BL BL BL BL
TAINSTRUMENTS.COM
 Precision Solution Calorimeter

Crushing ampoule (1 mL)

in stirrer

Reaction vessel with

solvent (100 or 25 mL)

Sapphire tip
TAINSTRUMENTS.COM
 Simultanoues pressure and heat flow
measurements

Vacuum / Pressure ampoule

•Gas producing reactions
•Safety assessment
•Absorption
•Etc...

TAINSTRUMENTS.COM
Vacuum / Pressure Ampoule

•Designed for Vacuum Thermal

Stability (VTS) test
–STANAG 4582 and Military standard
1751A
–Available in 4 and 20 mL
–Up to 10 bar pressure
–10-100 mTorr vacuum

85

TAINSTRUMENTS.COM
Large and heterogenous samples

Soil 17 (3-15-10).rslt, Ca(NO3)2 Glu Soil 16 (3-19-10).rslt, Straw+N Glu

•Batteries
Soil25 (3-26-10).rslt, 25 FYM Gluc Soil 23 (3-19-10).rslt, Sludge Glu
Soil 45 (3-19-10).rslt, (NH4)2SO4 Glu

•Environmental science
200

•Food applications

Normalized heat flow (µW/g)

150

100

50

0
0 8 16 24 32 40 48
Time (ho ur)

TAINSTRUMENTS.COM
 Battery testing

•Only method that directly

measures the occurrence of
non-current producing
reactions under load
•Very sensitive method to
assess self-discharge,
sometimes the only method
•Non-destructive
•Simple

TAINSTRUMENTS.COM
Macrocalorimetry Accessories

• P/N 604226.901 Macrocalorimeter Ampoule Lifter (Qty 1)

• P/N 604227.901 Macrocalorimeter Battery Lifter (Qty.1)
• P/N 604334.901 125 mL Glass Ampoule (Qty. 20)
• P/N 604328.901 Start-up Kit 125 mL Glass Ampoule
• P/N 604331.901 Lifting Hook for 125 mL Glass Ampoule (Qty 1)
• P/N 604329.901 18650 Battery Adapter Kit
• P/N 604353.901 C-Cell Battery Adapter Kit
• P/N 604352.901 D-Cell Battery Adapter Kit

125 mL Glass Ampoule 18650 Battery Adapter TAINSTRUMENTS.COM

Battery Fixtures

• Currently available fixtures for the Macrocalorimeter are for C-

and D-cell regular batteries and 18650 Li-ion batteries.

▪To optimise the thermal contact between the battery and

the heat flow detector

▪To make sure the position of the battery in the calorimeter

is reproducible

▪To facilitate the insertion of the battery into the calorimeter

▪To avoid short-circuiting the battery and facilitate

connections to the battery for load measurements

TAINSTRUMENTS.COM
 Accessories

•New accessory interface will control up to

eight independent accessories
▪Mass flow controllers
▪Peristaltic pumps
▪Syringes pumps and stirrer control
▪New Voltage In/Out module
This module can supply or measure voltages for up to
three independent probes/sources. This can be
accessories such as a user-configured pH-probe or a
light source.

TAINSTRUMENTS.COM
Voltage I/O module

•This module can supply or measure voltages for up to

three independent probes/sources.

Input Output
0 to 15 V (1 amp max) 0 to 14 V

User defined Probes Uses

pH Activating pumps
Turbidity Switch selection
solvent lines
Dissolved O2
UV-Vis detection Activating a light in the
sample chamber
Ionic strength UV or visible
Measuring voltage
battery testing

TAINSTRUMENTS.COM
 TAM Assistant

Dedicated software for control of TAM III, TAM IV, or TAM

Air for data collection, data analysis, and report creation.

TAINSTRUMENTS.COM
 TAM Assistant Allows You to:

•Control devices
•Run experiments
•View and edit results
•Perform analysis and calculations
•Create and edit reports
•21 CFR 11 compliant version available

93 TAINSTRUMENTS.COM
Temperature Watchdog

•When loading a cold ampoule into a warm thermostat it

may be necessary to deactivate the watchdog
temporarily.

TAINSTRUMENTS.COM
Precision Solution Calorimeter
(SolCal)

TAINSTRUMENTS.COM
Precision Solution Calorimeter

•Semi-adiabatic - some of the heat

formed will be exchanged with the
surroundings.
▪Surrrounding is an air jacket held isothermal by
the TAM thermostat

•The temperature of the sample will

change during the experiment.

•The temperature of the solution is

measured by means of a thermistor.

•Heat of dissolution
▪amorphicity

TAINSTRUMENTS.COM
 Precision Solution Calorimeter

•Solvent volume of 25 or 100 mL

▪Available with titration configuration

•Temperature range 15-90 °C

Bridge plug
▪Default temperatures 25, 35, and 45 °C
Sample in crushing
•Crushing ampoules with sample volume ampoule
(solid or liquid) up to 1.1 mL

•Highest accuracy and versatility in Thermistor

sample concentration

•Separate SolCal software for complete

experimental control, data acquisition, Calibration
heater
data analysis and reports
Sapphire tip

97 TAINSTRUMENTS.COM
 Heat Balance Equation - SolCal

+ k  (T − Ts )
dQ dQF dT
Temperature of
surrounding
− − =C (1)
and in calorimeter
dt dt dt
Heat of Solution
Ts Heat exchanged
with surrounding
Stirring, heating Heat accumulated
T from termistor by the system

Refer to Ch. 2 Precision

C Heat Capacity Solution Calorimeter
k Instruction Manual
Heat exchange coefficient

98 TAINSTRUMENTS.COM
 Heat Balance at Baseline

+ k  (T − Ts )
dQF dT
− =C (2)
dt dt
dT
At t  C →0
dt
T T

= k  (T − Ts )
dQF
Tsurr − (3)
dt
Substitution of eq. 3 into eq.1 gives
Time

− k  (T − T )
dQ dT
− =C (4)
dt dt
dQF
99
dt is eliminated

TAINSTRUMENTS.COM
Heat of Solution

 dT 1 
−  (T − T )
dQ
− = C  (4b)
dt  dt  
T
C
where =
End k

T Integration of 4b
E
dQ
Heat of Solution = −Q = −  dt
Start S
dt

 E

− Q = C  Tobs +   (T − T )  dt 
tS tE Time 1
  S 

Tobs = TEnd − TStart

− Q = C  Tcorr
100

TAINSTRUMENTS.COM
Heat of Solution

T − Q = C  Tcorr
End

T
Tobs Tcorr = Tobs − Tadj

Start E
Tadj =   (T − T )  dt
1

tS tE Time
S

In order to calculate Tadj, t and T have to be obtained

101

TAINSTRUMENTS.COM
Baseline

+ k  (T − T )
dT
T 0=C (5)
dt
by differentiation

T −t
T = T − Tnorm  e  (6)
−t
T0 T = T − Tnorm  e 

where

Tnorm = T0 − T
t0 (7)
Time

The exponential temperature function for the baseline gives us

T, 
102

TAINSTRUMENTS.COM
Break Experiment
Electrical Break
Calibration Experiment

Input QCal ?

Output Tobs
Cal
Tobs
Break

- -
Baseline Tadj
Cal
Tadj
Break
Analysis, T, t

Tcorr
Cal
Tcorr
Break

QCal
C=  Q = C  T Break

Tcorr
Cal reaction corr
103

TAINSTRUMENTS.COM
 Isothermal versus Adiabatic Calorimetry

• Isothermal calorimeters directly measure the rate of heat

production, which is proportional to the overall reaction rate. The
heat capacity of the sample may be unknown.
• Adiabatic calorimeters measure the change in temperature of a
sample, which is used to calculate the heat produced. The heat
capacity of the system must be known.
• Isothermal calorimeters are very stable and need not be
calibrated more than a few times a year. In contrast, adiabatic
calorimeters must be calibrated often and are typically done
before each run.
• The temperature in an isothermal calorimeter doesn’t increase to
unrealistic temperature whereas the final temperature in adiabatic
calorimeters can be very high.
• Semi-adiabatic calorimeters are a better choice for faster
reactions, but isothermal calorimeters exhibit long term stability.
• Isothermal calorimetric experiments are easy to perform.

10
4
TAINSTRUMENTS.COM
 Verify Performance of SolCal

• 100 mL water
• 3 - 50 J electronic calibrations
• Cp of system = 445 8 J/K
• Average of three within 0.2 J/K
•  = C/k  8.200 ks

Use 10 J calibrations for 25 mL vessel

Cp estimated at 115-118 J/K

10
5
TAINSTRUMENTS.COM
Verify Performance of SolCal

Calculate heat capacity of water and vessel.

SYSTEM/Check Exponential Fit less than 10 K.

TAINSTRUMENTS.COM
 Chemical Calibration

•Exothermic heat reaction

▪Dissolution of 0.5 g TRIS in 0.1 M HCl
At 25 ºC RHº = -29.75 ±0.02 kJ/mol

•Endothermic heat reaction

▪Dissolution of 0.5 g TRIS in 0.05 M NaOH
At 25 ºC RHº = +17.19 ±0.02 kJ/mol
TRIS - molecular weight: 121.137 g/mol, Density: 1.35 g/cm3, Cp: 124 JK-1mol-1

•Endothermic heat reaction

▪Dissolution of KCl in water
At 25 ºC RHº = +17.58 ±0.02 kJ/mol

10
7
TAINSTRUMENTS.COM
Example Report – SolCal experiment (page 1)

TAINSTRUMENTS.COM
Example Report – SolCal experiment (page 1)

TAINSTRUMENTS.COM
 Example Calculation for KCl Experiment

Before and After Break

Molality of KCl = m = 0.00465 mol/0.1 kg = 0.0465 mol/kg
Hsol(KCl) (25 °C) from Equation 1 = 235.032 J/g
MKCl = 74.5513 g/mol
Cp from Equation 2 = -158.85 J/mol·K Corrected Mass of KCl = 346.602 mg = 0.00465 mol
Mass of Water = 100.0 mL = 0.1 kg
Hsol(KCl)(T) from Equation 3: Assume: water = 1.00 kg/dm3

Before break - T = 24.95 °C - Hsol(KCl)(T) = 235.14 J/g

After break - T = 25.127 °C - Hsol(KCl)(T) = 234.76 J/g

Error Calculation:
Before break - Hsol(KCl)(T) = 234.77 J/g (from report)
Error = [(234.77 - 235.14)/235.14]100 = -0.16%
After break - Hsol(KCl)(T) = 234.78 J/g (from report)
Error = [(234.78 - 234.76)/234.76]100 = 0.001%

TAINSTRUMENTS.COM
 Comments on KCl Experiment

▪ Usually calculation using the calibration before or after the break give
slightly different results of H. One reason is hydrodynamic stirring
changes after breaking the ampoule and may result in different
estimations of t and , which are essential for calculation of the
calibration constant. Secondly, the system may not have achieved
steady-state when the first calibration begins (check standard
deviation). For this reason the calculation that utilizes the calibration
after break is typically a better representation.
▪ Corrections for evaporation and condensation effects when breaking
the ampoule can be taken as negligible in this case.
▪ For additional sources of error, see reference by I. Wadso in the
Appendix of the Precision Solution Calorimeter Instruction Manual.

11
1
TAINSTRUMENTS.COM
Calculation – Heat of Solution for KCl

Concentration Dependence
Conditions:
Molecular weight: 74.5513 g/mol (KCl: NIST 1655)
Temperature = 25 °C = 298.15 K
KCl Molality = mKCl = 0.05551 - 0.15 mol/kg
Deviation from NIST certificate using the formula below should be less than 0.3 %
when concentrations are in the given range. Concentrations outside this range should
be regarded as an extrapolation and may result in an increase in error.

Hsol(KCl) = A·mKCl3 + B·mKCl2 + C·mKCl+ D [1]

Definitions: Hsol(KCl) in J/g

A = 203.7205 J/g·kg3·mol3 B = -144.7988 J/ g·kg2·mol2
C = 31.5119 J/ g·kg·mol D = 233.8599 J/g

TAINSTRUMENTS.COM
Calculation – Heat of Solution for KCl

Temperature Dependence

Semi-adiabatic calorimeters by definition measure change in

temperature. Therefore, the heat of solution should be calculated at the
temperature to which the dissolution process is measured by using the
change in heat capacity (Cp).

Cp = (-114.1 + 28.95·m1/2 + 6.7·m)* - 51.30 [2]

Hsol(KCl)(T) = Hsol(KCl) (25 °C) + Cp (T - 25.00)/MKCl [3]
*Apparent Cp of solution
Definitions:
Hsol(KCl) in J/g Cp of crystalline KCl = 51.30 J/ mol·K
Cp in J/mol·K MKCl = 74.5513 g/mol
TAINSTRUMENTS.COM
 Example of Bouyancy Correction

Wv = Wa + (Vair - Vcwair) Definitions:

Weight of KCl in air = Wa
Substitute… Weight of KCl in vacuum = Wv
Volume of KCl = V
V = Wv/KCl Volume of Counter weights = Vcw

Then…

1 − a cw
Wv = Wa 
1 − a
 ( )
= Wa  1 + a 1 KCl − 1 cw  Wa  (1.000455)
KCl

Assume:
Concentration of KCl and
Ambient Temperature = 22 ± 1 C Temperature are also important
Ambient RH = 35 ± 15% for calculation of H!
Ambient Pressure = 750 ± 10 mm Hg
Density of Air = air = 0.0012 g/cm3
Density of KCl = KCl = 1.98 g/cm3
Density of Counter Weights = cw = 7.95 g/cm3 (e.g. brass  7.8 g/cm3, ss  8.0 – 8.4 g/cm3)

TAINSTRUMENTS.COM
Ideal experimental tips

Make sure that

calibration heats are
as close as possible
to break heat.

Ideally, heats should

be around the
equilibration point or
start at the same
point away from
equilibration.

TAINSTRUMENTS.COM
Ideal experimental tips

TAINSTRUMENTS.COM
Ideal experimental tips

Wait until the standard deviation of

the baseline is less than 10 uK and
that the fit status is in Decay before
starting the run.

TAINSTRUMENTS.COM
 Experimental
Considerations

TAINSTRUMENTS.COM
 TAM is a Non-Specific Technique

▪ TAM is sensitive to all physical and

chemical processes associated with
a heat flow. Thus, the monitored heat
flow may contain contributions from Process 1+2
several processes.

Process 2
▪ Individual contributions may be

Heat flow
distinguished by varying the Process 1
experimental conditions.

▪ Consider a blank experiment Time

11
9
TAINSTRUMENTS.COM
 Designing an Experiment

•Choice of sample handling system

•Handling of ampoules
•Sample considerations
•What to use as reference

12
0
TAINSTRUMENTS.COM
 Sample Handling Systems

•Closed or sealed (static) Ampoules

•Open ampoules - Micro Reaction
System
•Micro Solution Ampoule

12
1
TAINSTRUMENTS.COM
Tools Required - Disposable Glass Ampoules

•Crimping tool •Ampoules and Caps

▪Used to seal the cap on the ▪Aluminum, Butyl rubber and
glass ampoule a Teflon gasket
•Adjustment tool (not shown) ▪May introduce initial
disturbances!
▪Adjust the dimension of the
caps when in position •Lifting eyelets
•Centring tool
▪Used to make a mark for the
lifter eyelet

3mL lifting eyelets shown here

TAINSTRUMENTS.COM
 Sealing Disposable Glass Ampoules (3 & 4 mL)

Sealing the Ampoule:

•Weigh ampoule and sample. Place an aluminum cap and
seal onto a clean ampoule rim (Figure 1).
•Crimp the cap with the tool provided. Rotate the crimping tool
90° and crimp again (Figure 2). Verify the seal by trying to
rotate the cap.
1 2 3
• Alignthe cap with the PEEK alignment tool. Rotate the
alignment tool 90° and crimp again (Figure 3). Verify the seal
again by trying to rotate the cap.
•Use centering tool to make an indention in the cap (Figure 4).
This indention will be a guide for the lifting eyelet (Figure 5).
5
• Thread the lifting eyelet into the cap (Figure 6).
4
•Make sure to wipe off all sample and fingerprints from the 6
outside of the ampoule before loading into the calorimeter.
•Do not to throw away the lifting eyelet after the experiment is
completed.

TAINSTRUMENTS.COM
 Disturbances with Disposable Glass Ampoules

•These ampoules may be associated with a disturbance in the

1-5 µW-range during the first 10 hours due to:
▪the sealing procedure introducing stress and subsequent relaxation
phenomena
▪sorption/desorption phenomena

•Can be minimized by
▪pre-storing the ampoules and lids at the operating temperature for 24 hours
▪Preparing the sample and the reference ampoule at the same time (use a
new reference for each measurement)

•Always consider the risk for interaction between the sample

and the ampoule
▪Steel  peroxides, HCl
▪Basic solvents  glass
▪Pressure build up

TAINSTRUMENTS.COM
 Heat Seal Ampoules

•Completely sealed
•Heat seal ampoules of glass
•Special ampoule lifters required

Water circulation
inlet/outlet

Note: precautions should be

taken to protect the sample
towards the heat during the
sealing procedure
TAINSTRUMENTS.COM
 Threaded Cap Ampoules

•Stainless steel
▪resistance towards corrosion
▪should not be used for solvent with pH < 4

•Hastelloy
▪Improved resistance towards corrosion and acids
▪excellent for use with organic solvents

•The cap is sealed with a disposable Teflon

disc and/or o-ring (not shown)
•Stable for most applications
•Stands pressures up to at least 2 bar

TAINSTRUMENTS.COM
Circlip Cap Ampoules

•Stainless steel
▪resistance towards corrosion
▪should not be used for solvent with pH < 4

•Hastelloy
▪Improved resistance towards corrosion and acids
▪excellent for use with organic solvents

•Glass with stainless steel or Hastelloy collar

•O-sealing made in Nitrile, EPDM, Viton® or Kalrez ®

•Stands 8 bar pressure (precautions must be made)

TAINSTRUMENTS.COM
Equilibrium

•Thermal equilibrium
▪Within 60 min after loading
◆ depends on sample size – 20mL ampoules may take longer to
fully equilibrate
•Physical equilibrium
▪Depends on the sample and the pre-history
▪Might depend on the ampoule itself
•Chemical equilibrium
▪Slow/fast reactions

TAINSTRUMENTS.COM
Pre-history of the Sample

•The sample should be stored under controlled conditions

for at least 24 hours before a measurement
▪relative humidity
▪temperature
▪atmosphere (e.g. nitrogen, air, oxygen)
•The time to reach physical equilibrium must be considered

TAINSTRUMENTS.COM
Sample Geometry and Surface Area

•Powder (small particle size)

▪Chemical processes will occur homogeneously in the sample

•Bulk samples (large particle size)

▪May show a heterogeneous response
◆ diffusion limited oxidation

◆ pressure build-up by volatiles formed

•Try powder, films, granules or, specimens with different

thickness. The influence of geometry can be studied using
different particle size with the same amount of sample.
▪If the specific heat flow (µW/g) is the same for two different sizes,
this effect is not important. Otherwise it must be considered.

TAINSTRUMENTS.COM
Sample Amount

•The response in heat flow may be dependent on the

amount of samples (different bed volumes) in the ampoule
▪If the specific heat flow (µW/g) is the same for different amounts of
samples either there is possibly a layering (or caking) effect or the
effect is not important (heat flow/g consistent).

TAINSTRUMENTS.COM
Kinetic Evaluation

•Be sure the response in heat flow reflects the kinetics of

the process of interest
•In many case the first 5-10 hours should be excluded
because of a non physical equilibrium
▪other process(es) contribute to the heat flow
◆ Examples: Evaporation from hygrostat and adsorption on the walls of
the ampoule

TAINSTRUMENTS.COM
Choice of Reference Materials

•A reference material is used to balance the heat capacity

of the sample and the reference ampoule.
•With a good balance in heat capacity the short-term noise
will be reduced. However, if the system is not well-
balanced the average heat flow values is not affected.
•A proper balancing of the ampoules is needed when the
response in heat flow is low, e.g. during titration
experiments.
•Example of reference materials: sand, glass pearls, water

TAINSTRUMENTS.COM
 Baseline collection

•When looking at absolute energies or comparing two curves, it is essential that

you have a baseline that is set to zero. There should be no heat being
produced which means an empty calorimeter and empty reference.

•Collecting a good baseline means:

▪Waiting until the baseline is stable before starting collection
◆ Set “Signal Stability Conditions” and let the instrument wait until the signal is stable
◆ Set a “Maximum time to wait” if time is critical

TAINSTRUMENTS.COM
Baseline collection

TAINSTRUMENTS.COM
 Baseline collection

•When looking at absolute energies or comparing two curves, it is essential that

you have a baseline that is set to zero. There should be no heat being
produced which means an empty calorimeter and empty reference.

•Collecting a good baseline means:

▪Waiting until the baseline is stable before starting collection
◆ Set “Signal Stability Conditions” and let the instrument wait until the signal is stable
◆ Set a “Maximum time to wait” if time is critical

▪Collecting enough baseline to determine the zero point

◆ 30 min is the default.
◆ Make sure you have enough time to capture peaks and valleys to get a good average

▪Not disturbing the instrument while the baseline is being collected

◆ Due to the data reduction, it is wise to wait a few minutes after the baseline has
finished to disturb the calorimeter by inserting the samples.

TAINSTRUMENTS.COM
Baseline collection

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Baseline collection

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Baseline problems

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Baseline problems

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Baseline problems

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Baseline problems

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Baseline problems

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Baseline problems

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Baseline problems

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Baseline problems

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 Baseline collection

•When looking at absolute energies or comparing two curves, it is essential that

you have a baseline that is set to zero. There should be no heat being
produced which means an empty calorimeter and empty reference.

•Collecting a good baseline means:

▪Waiting until the baseline is stable before starting collection
◆ Set “Signal Stability Conditions” and let the instrument wait until the signal is stable
◆ Set a “Maximum time to wait” if time is critical

▪Collecting enough baseline to determine the zero point

◆ 30 min is the default.
◆ Make sure you have enough time to capture peaks and valleys to get a good average

▪Not disturbing the instrument while the baseline is being collected

◆ Due to the data reduction, it is wise to wait a few minutes after the baseline has
finished to disturb the calorimeter by inserting the samples.

TAINSTRUMENTS.COM
 TAM Thermostat

An oil based liquid bath system for a

outer
continuously circulated heat sink medium tank

Calorimeter
that prevents any thermal event in a test
sample or from the room environment
from altering the constant temperature
bath inner
tank

Peltier
cooler

fine heater
over flow
system

TAINSTRUMENTS.COM
Calorimeters

TAINSTRUMENTS.COM
Calorimeters

Wait 15-20 min for sample

to equilibrate with the oil bath

TAINSTRUMENTS.COM
Calorimeters

The move into the

measurement area causes
a disturbance.
Instrument waits 45 min for
signal to be correct.

TAINSTRUMENTS.COM
 TAM Applications

Pharmaceuticals
Life Science
Material Science

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TAM Applications

Stability

TAINSTRUMENTS.COM
TAM Stability Testing

Least stable X
exo

X
Heat Flow

X
Most stable

Time
TAINSTRUMENTS.COM
 TAM Stability Testing

40°C 30°C
60
200

40

Normalized heat flow (µW/g)

Normalized heat flow (µW/g)

100

20

-100
-20

-200
-40

-300
-60

8 16 24 32 40 1 2 3
Time (ho ur) Time (da y )

Three different lots of the same material at two different temperatures.

TAINSTRUMENTS.COM
 Active Pharmaceutical Ingredient Stability

(~75% Relative Humidity)

P,µW Pin[1](t) Pin[2](t) Pin[3](t) Pin[4](t)

Conditions:
60 TAM Temperature 50 °C
Side A: API and hygrostat filled w/ saturated NaCl
inside 4 mL SS screw cap ampoule

Lowering ampoule 40
Side B: Empty hygrostat inside 4 mL SS screw cap
into calorimeter ampoule

20

70% Amorphous : 30% Crystalline (w/w)

100% Crystalline
5 10 Time,hour

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Stability with TAM and Compared with HPLC

Oxidation of Meclofenoxate Hydrochloride

Containing dl-a-Tocopherol

50°C

40°C
30°C
30°C
 HPLC data
23°C
Ο TAM data

Otsuka T., Yoshioka S., Aso Y. and Terao T., Chem. Pharm. Bull., 42(1) 1994

TAINSTRUMENTS.COM
 Hydrate Formation in Ethinyl Estradiol

600 Measuring
temperature: 45C
P/m /uW g-1

400
• Blue trace: 100 %RH
200
• Red trace: 95 % RH
0
0 20 40 60 80 100 120
• Green trace: 88 %RH
Time /h
600

400
Rate equation:
P/m /uW g-1

200

0 P = k (T )  f (q / H )
-200
-5 0 5 10 15 20 25 30
-1
q/m /J g

15 Peter Vikegard, J&J – Cilag Ag, Switzerland

8
TAINSTRUMENTS.COM
Avrami’s Model

P = k (T )  f (q / H )
f (q / H ) = 2(1 − q / H )  [− ln(1 − q / H )]1/ 2

600

400
• Blue trace: experimental
P/m /uW g-1

200 data
0
• Black trace: fitting
-200
-5 0 5 10
-1
15 20 25 30 equation (k=0.0005 s-1)
-4 q/m /J g
x 10
Avrami rate constant

5
4
• Rate constant as a
3 function of relative humidity
2
1
0
82 84 86 88 90 92 94 96 98 100
Relative humidity (%)
RHcritical

TAINSTRUMENTS.COM
TAM Applications

Compatibility Testing

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Compatibility Measurements

Basic sample set,

2-component test

A B Standard
mixture
Additional mixture
samples for special
tests

The term compatibility refers to a Gas phase Interaction Intimate

mutual physical or chemical interaction zone exposure
interaction between two or more
components of a mixture, which
leads to a change in the mixture By Lars-Gunnar Svensson, Celsius
Materials CMK, Karlskoga, Sweden
or component properties.

TAINSTRUMENTS.COM
 Evalutation of Compatibility Measurements

50:50 mixture of two components A and B

A+B (measured)

A+B (theoretical)

Time (a.u.)

If the heat flow curve of A+B (measured) differs from A+B (expected), this is
an indication that the materials affect each other or are incompatible.

16
2
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 Compatibility Between Wax and Mineral Wool

16Data provided by Svensson, Bodycote Materials AB, Sweden (2003)

3
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 Compatibility Experiment with TAM

TAM Temperature: 50 °C
Aliquot of water added to ampoule

Ampoule
removed

164
Schmitt, E.A.; Peck, K.; Sun Y.; Geoffroy, J-M. Thermochim ACTA, 380, 175-183 (2001)

TAINSTRUMENTS.COM
 Compatibility Experiment with TAM

TAM Temperature: 50 °C
Relative Humidity: 75%

Phipps, M.A.; Mackin, L.A. Pharm. Sci. Tech. Today, 3(1), 9-17 (2000)
TAINSTRUMENTS.COM
TAM Assistant Analysis (1 of 2)

• Data must be collected with the mass of

individual components entered.
1. Open data file
2. Click Analysis/Compatibility
3. Add Mixture and select Results file (.rslt)
4. Change the time scale (if required)
• Select … button to the right “Mixture
measurement signal” field
5. Generate Report
• Copy plot and information to
alternative program for presentation (if
required)

TAINSTRUMENTS.COM
TAM Assistant Analysis (2 of 2)

Step 3

Step 4

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ASA with Mg-Stearate and Sucrose

TAM III at 40 °C

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Amine-Lactose Interactions

One approach to perform excipient compatibility screening is to add water to the powder
mixture. The graph shows the response of an amine-lactose interaction at different
temperatures with 20% water added.

Schmitt, Peck, Sun & Geoffroy, Thermochim. Acta, 380, 175-183, (2001).
TAINSTRUMENTS.COM
Drug - Excipient Stability

Time in days
Another approach to perform excipient compatibility screening is to compress (make
a tablet) with the ingredients. The figure shows the same compressed mixture at
several different temperatures and clearly the rate of reaction increases as the
temperature increases.

Selzer, Radau & Kreuter, Int. J. Pharm., 171, 227-241, (1998).

TAINSTRUMENTS.COM
Linear and Exponential Fitting

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Kinetic Analysis with TAM Assistant

Effect of Temperature

Isothermal Models

TAINSTRUMENTS.COM
 TAM Applications
Amorphicity and Polymorphic Studies

TAINSTRUMENTS.COM
 Definition of Crystallinity

•The degree of crystallinity is a measure of crystal

imperfection
•Imperfections increase the energy (enthalpy) of the crystal
•The enthalpy increase of a crystal relative to a reference
crystal of high crystallinity can be measured by
calorimetry, either as heat of solution or heat of
crystallization.

17
4
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 Relevance of Crystallinity

•The presence of imperfections (amorphicity) in a crystal

affect relevant properties.
•Properties affected are: chemical stability, solubility,
bioavailability, surface energy.
•To have a material well characterized it is very important to
have a good control over these key properties.

17
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 Characterize Amorphicity with TAM

1. The Microhygrostat Method

2. The Controlled RH Perfusion method
3. The SolCal Method

17
6
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 The Microhygrostat Method

Microhygrostat:
Glass tube with pure
solvent or a solvent
saturated by a salt
Solid (e.g. sat. NaCl (aq))
sample

Developed independently by:

Angberg, Uppsala University and Byström, Astra Zeneca (1992) TAINSTRUMENTS.COM
Moisture Induced Crystallization

The induction time

depends on:
• The vapor activity
• The temperature
• The sample size
• Presence of
crystals or seeds

Bystrom, Thermometric Application Note 22004

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 Recrystallization as a Function of Relative
Humidity

120

• Amorphous lactose
100
57 %RH • Exp temperature:
mass )/mW g-1

100 %RH 25C

80
• Sample mass: ≈ 30
mg
Heat flow / (mW/g

60
• Microhygrostat: Pure
40
water (100 %RH) and
sat. aq. NaBr (57
%RH)
20

0 2 4 6
Time (ho ur)
Time /h

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The Controlled RH Perfusion Ampoule

a
f
e

f
a) inlet of dry nitrogen
b) dry flow line
c) humidifiers and thermal
equilibrators c
d) sample ampoule
e) outlet
f) wet flow line
g) sample powder bed

d
g

TAINSTRUMENTS.COM
 Moisture Induced Crystallization

RH ramp

L.E. Briggner (Astra-Zeneca), Thermometric Application Note 22022

TAINSTRUMENTS.COM
 Moisture Induced Crystallization

Highly crystalline lactose

P/µW 30 → 40 %RH 30 → 40 %RH

80
0.3%
69.04mJ amorphous!
20

- 69.295mJ
-40
89.25mJ 69.04mJ

-100 40 → 30 %RH

0 2 4
Time/hour

L.E. Briggner, AstraZeneca, (2002)

See also: L. Mackin, et. al. Int. J. Pharm. (2002), 231, 227-236 TAINSTRUMENTS.COM
 The Solution Calorimetry Method

SolCal = Precision Solution Calorimeter

Crushing ampoule (1 mL)

in stirrer

Reaction vessel with

solvent (100 or 25 mL)

Sapphire tip
TAINSTRUMENTS.COM
 Dissolution of Lactose

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4
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Heat of Solution for Different Lactose Forms

Heat of solution Amorphicity

Type
(J /300 mg) content (*)

Monohydrate 43.89 0%

Spray dried -49.93 100 %

Mixture 1 32.91 11.7 %

Mixture 2 41.42 2.7 %

(*) degree of crystallinity = 100-% amorphicity content

TAINSTRUMENTS.COM
 Standard Curve <10%

18 Hogan & Buckton, Int. J. Pharm., 207, 57-64, (2000)

6
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 Summary – Amorphicity & Crystallinity

• Measure enthalpy of crystallization (and sorption effects)

• 10 to 500 mg (typically 50-150)
• High sensitivity (towards 0.1%)
• High reproducibility
• Easy to adapt method after substance
• Hydrophobic or hydrophilic substances
• Calibration important (standard curve)
• Timescale 1-5 hours

18
7
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 Benefits of TAM Over Other Techniques

Sensitivity
Detection limits below 1%, towards 0.1% is possible
Results are highly reproducible

Versatility
Adapt method to substance (different solvent vapors, sample
sizes and operating temperatures)

Sample throughput
The sample throughput attainable with TAM IV and TAM 48 can
not be achieved by any other method today.

18
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 Polymorphism Introduction

The study of Polymorphic behaviour of drugs and

excipients is an important part of preformulation work
because it affects:

▪ bioavailability mediated via dissolution

▪ solid state reactions (stability)
▪ hygroscopicity
▪ mechanical stability
▪ compactability
▪ batch and source variation

18
9
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 Main Issues

•Relative stability of polymorphic pairs

•Equilibrium transition temperatures
•Assesment of stability (meta-stable or stable)
•Kinetics of polymorphic transitions

19
0
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→  Transformation of Tripalmitin

Hongisto, Lehto & Laine, Thermochim. Acta, 276, 229-242, (1996).

TAINSTRUMENTS.COM
 →  Transformation of Tripalmitin

Note: the results were obtained below the DSC sensitivity level

The enthalpy does not change with temperature indicating that the same
transition has been observed at all the different temperature.

Hongisto, Lehto & Laine, Thermochim. Acta, 276, 229-242, (1996).

19
2
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 Solution Calorimetry

Temp offset, mK

20

-20
Calibration 1

Calibration 2
Baseline

Baseline

Baseline

Baseline
Break
Pause

End

0 15 30 45 Time, min

T = 45 °C

19
3
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 Heat of Solution

polymorph A solHm,A

tHm Solution

polymorph B solHm,B

tHm,A to B = solHm,A- solHm,B

19
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Which Direction? A → B or B → A?

Answer: most likely is the direction

for which tHm < 0 (exothermic)

⚫ tHm = solHm,A- solHm,B < 0

⚫ A more stable than B

⚫ tHm = solHm,A- solHm,B > 0

⚫ B more stable than A

195

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 Transition Temperature of a Polymorphic Pair
(A and B)
Transition enthalpy from SolCal
+ solubility of form A and B

The transition temperature, Ttrans,

between form A and B
References:
Chong-Hui Gu and David J. W. Grant. (2001) Estimating the relative stability of
polymorphs and hydrates from heats of solution and solubility data. J. Pharm. Sci.
90 (9).
Koji Urakami et al. (2002) A novel method for estimation of transition temperature
for polymorphic pairs in pharmaceuticals using heat of solution and solubility data.
Chem. Pharm. Bull. 50 (2).

TAINSTRUMENTS.COM
 Results with 4 drug compounds

The estimated Ttrans for indomethacine indicates monotropic relationship. This is

also supported by DSC data interpreted with the “Heat of fusion rule” (Burger &
Ramberger, 1982). fusHg > fusH, Tfus,g > Tfus,

Data from : Koji Urakami et al. (2002) A novel method for estimation of transition temperature for
polymorphic pairs in pharmaceuticals using heat of solution and solubility data. Chem. Pharm. Bull. 50 (2).

TAINSTRUMENTS.COM
Polymorphic Transformations

Polymorphic transformations can occur via two distinct

mechanisms:

Directly via molecular rearrangements in the dry state

”slow process”
or

Via a solvent phase like a solvent- mediated polymorphic

transformation (smpt)
”can be made to run fast”

TAINSTRUMENTS.COM
Crystallization from a Super-Saturated Solution

ci
A
Typical solubility

solubility
curves for a
polymorphic pair cA B
c
cB
temperature

Typically the most soluble polymorph is not the most stable form (Form A)

TAINSTRUMENTS.COM
The Solvent Mediated Process

Initial dissolution of A

Nucleation (induction) Crystallization of B

Dissolution of A

A→B

TAINSTRUMENTS.COM
SMPT with Microcalorimetry

Solvent mediated polymorphic transformation = SMPT

dmA dmB
The Heat Flow: P= DHm + C H m
dt dt

The enthalpy change: tHm = DHm+ CHm

(D = dissolution, C=crystallization)

TAINSTRUMENTS.COM
Water Slurries of a Meta-Stable Drug Compound

Solvent mediated polymorphic

transformation = SMPT

Slurries with pure drug were

prepared directly into 3-ml glass
vials serving as vessels for the
calorimeter

Operating temperature: 45oC.

Approximately 70 mg drug and
2 g water

Peter Johansson (2002) J&J-PRI, Switzerland

TAINSTRUMENTS.COM
 Results with Three Meta-Stable Drug Lots

Results from water

slurries with pure drug at
45 °C – variation in
particle size investigated.

tHm = -20.1 J g-1

(std dev = 1.2 J g-1)
n=6
Blue trace: lot A (largest particle size)
Green trace: lot B
Red trace: lot C (micronized)

20
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 Before and After

30

20

P/ W
10

-10
0 100 200 300
Time /h

FormA Form B

TAM is non destructive. After measurement is complete

additional analysis can be made on the same sample.

20
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 TAM Applications

Temperature Scanning

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 Applications of Slow Scanning

•Polymorphism
•Crystal Hydrates
•Heat Capacity

•Melting Behavior
•Glass Transitions

20
6
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 Heat Capacity Determination
Temperature Empty ampoule Sapphire

25.6
Method:
1. Insert empty ampoule in sample side
2. Change the thermostat temperature by
25.4
typically 1°C (blue curve)
200
3. Restore the original temperature
4. Fill ampoule with sample
25.2 5. Repeat the temperature change with the
Temperature (°C)
Heat flow (µW)

same limits (cyan curve)

6. Calculate Cp from the energy difference
0 25.0
between the curves and the sample
weight

• For best accuracy apply calibration

24.8 substance with known Cp

-200
24.6

0 6 12 18 24 30
20
7 Time (hour) TAINSTRUMENTS.COM
 Heat Capacity Determination with TAM III

Blank - 3320 stainless steel ampoule, empty

Sample - 3320 stainless steal ampoule, geothermal water
Temperature
28

10
27
26.83 J

9.579 J
The Geolgically heated water

Tempera ture (°C)

0

Hea t flo w (mW)

26

Temperature (˚C)

Heat flow (mW)

is a strong electrolyte with a -19.43 J
heat capacity almost 20% 25 -10
lower than that of pure water.
24 -20

-53.99 J

23 -30

Results 0 2 4 6 8 10
Time (hour) Time (h)

mass /g T /C corr. factor Cp /J K-1g-1

peak 1 2.2767 2 0.9125 3.457
peak 2 2.2767 4 0.9125 3.463
20mean 3.460
8
TAINSTRUMENTS.COM
 Heat Capacity Using the Step Isothermal

Lehto, Laine, Ylianttila, Hyysalo and, Jokela, J. Therm. Anal., Vol. 53, 685-695 (1998)

20
9
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  →  Transformation of Tripalmitin

On heating
I.  →  transformation
at 40°C (exo)
II. Melting of  form at
III
65°C (endo)
On cooling
III. Crystallisation of  form
I (exo)

The results indicates that the

 →  transformation
II
is irreversible.

Note: Sharps peaks due to

the slow scanning rate.

21 Thermometric Application Laboratory, internal results (2003)

0
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 Hexatriacontane - Function of Scanning Rate
Hexatriacontane
Temperature, TAM III Thermostat #209

78 2.0
•TAM – 2 °C/hr (top)
•TAM – 1 °C/hr (bottom)
1.5
Temperature (°C)

76
Heat flow (mW)

1.0

74
0.5 •QDSC – 0.5, 2.0, and 10 °C/min
0.0

-0.5
72

-1.0
210 240 270 300 330 360
Time (min) 0

Hexatriacontane
Temperature, TAM III Thermostat #209
-5

78 1.0

Heat Flow (W/g)

-10

76 0.5
––––––– Hexatriacontane
Temperature (°C)

Heat flow (mW)

––––––– Hexatriacontane
––––––– Hexatriacontane
-15

74 0.0

-20

72 -0.5 70 72 74 76 78 80 82
Exo Up Temperature (°C) Universal V4.5A TA Instruments

25 26 27 28 29 30

21 Time (ho ur)

1
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Hexatriacontane - Function of Scanning Rate

212

Marti et al (2004) J. Thermal Analysis Calorim 77 TAINSTRUMENTS.COM

TAM Applications
Life Sciences

TAINSTRUMENTS.COM
 Other Methods used as Bioassays

•For the metabolic response:

Glucose, glutamine,
failed to give results for many Endogenous
systems
•The drug effect has to be
specified prior to analysis (e.g.
cell growth)
Lactate CO2, H2O
•One assay has to be used to
assess each biological
response – many methods Both ways give heat
All changes in metabolism
are measured by
microcalorimetry

If something is happening calorimetry will tell you!

TAINSTRUMENTS.COM
Rapid detection of Mycobacteria in culture

•A typical bacteria will produce heat

at a rate of about 2 pW

•Detection is achived by their

replication

•With a calorimetric detections limit of

200 nW, only 100 000 growing
mycobacteria are required for
detection

•Thus the calorimeter will detect a

infection well before either a colony
is visible or all oxygen is consumed
(as in BACTEC 960 MGIT).

TAINSTRUMENTS.COM
Rapid detection of Mycobacteria in culture

•Tuberculosis (TB) is a major concern for public heath

▪TB kills about 1.5 million people worldwide each year
▪An additional 0.2 million people per year die from HIV-associated TB
▪Also, other mycobacterial infections are emerging and their incidence
increasing.

•Culture-based detection techniques is the gold standard.

•In many developing countries, simple solid culture media are used, and
detection is achieved by visual inspection. Detection is very slow, since
one must wait until a visible colony appears.

•Detection of mycobacterial growth using liquid culture is currently

achieved using a radiolabelled substrate (e.g. BACTEC 12) or
fluorescent indicators sensitive to oxygen concentration (e.g. BACTEC
960 MGIT). However, such indicators are expensive additions to culture
media.
From: O. Braissant*, D. Wirz, B. Göpfert, A.U. Daniels
Tuberculosis (2009), doi:10.1016/j.tube.2009.11.001 TAINSTRUMENTS.COM
Different pathogenic mycobacteria

(MOTT – Mycobacteria other than tuberculosis)

From: O. Braissant*, D. Wirz, B. Göpfert, A.U. Daniels
Tuberculosis (2009), doi:10.1016/j.tube.2009.11.001
TAINSTRUMENTS.COM
 M. tuberculosis

From: O. Braissant*, D. Wirz, B. Göpfert, A.U. Daniels

Tuberculosis (2009), doi:10.1016/j.tube.2009.11.001
TAINSTRUMENTS.COM
 Effect of Growth Hormone on Cells

Addition of GH should give

an immediate metabolic response

followed by

anabolism = increase in biomass

21
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 Long-term Studies of GH on Lymphoid Cells
The proliferative response upon addition of human growth hormone, hGH
Heat Flow,µW
Heat flow, µW
50

A negative slope - cells are dying off

The larger positive slope value - the higher proliferation rate
40

0.4 nM GH
30
0.2 nM GH

20

PBS
10

5 10 15
Time,hour

Schön and Walum, 1997 TAINSTRUMENTS.COM

 Initial Metabolic Response - Addition of GH

Heat Flow,µW

20
PBS

0.2 nM GH

18
0.4 nM GH - 0.7 µW

16 5 nM - 2.2 µW

10 20 30
Time,min

22Schön and Walum, 1997

1
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 Results - GH on a Lymphoid Cell Line

•Kinetic information: 10 min for maximum metabolic GH

stimulation; another 20 min before growth starts
•Dose response curves can be constructed based on the
initial metabolic response
•Dose response curves can be constructed from the growth
rate with high sensitivity

22
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 Drug Efficacy

Flow calorimetry: Leukemia (T-lymphoma) cells exposed to the anti-cancer drug methotrexate. The
final drug concentrations were (a) 0, (b) 0.2, (c) 0.5, (d) 1.0, (e) 2.0, (f) 4.0µM (ref 6).

a
b
c

d
e
f

Bermudez, Backman and Schon., Cell. Biophys. 20, 111-123, (1992). TAINSTRUMENTS.COM
 Microorganism Detection

Microcalorimetry - A Novel Method for Detection of Microorganisms in Platelet Concentrates

and Blood Cultures. Andrej Trampuz, Simone Salzmann, Jeanne Antheaume, Reno Frei,
22
A.U. Daniels University of Basel & University Hospital Basel, Switzerland
4
TAINSTRUMENTS.COM
 Background

The diagnosis of implant-associated infections is difficult due to

organisms attached to surfaces forming biofilms.

Diagnosis can be improved by sonicating retrieved implants to

remove attached microorganisms, followed by culture and
calorimetry of sonicates.

1 mL sonicate fluid was injected in ampoules containing 2 mL TSB

and incubated at 37°C in calorimeter. Positivity by calorimetry
defined as an increase in heat flow rate of ≥10 μW above baseline.

22
Trampuz, A. et al. JCM (2006), 44, 628
5
TAINSTRUMENTS.COM
 S. epidermidis in 3 mL TSB

-4
1.75x10 3 104
3106 3 105 CFU
-4
CFU CFU 3000
1.50x10 300 CFU
CFU 3 CFU
30 CFU
-4
1.25x10
Heat flow (W)

-4
1.00x10

-5
7.50x10

-5
5.00x10

-5
2.50x10
0.3
0.00 CFU
0 CFU

0 4 8 12 16 20 24 28 32 36 40 44 48
Time (h) TAINSTRUMENTS.COM
S. aureus & S. epidermidis in TSB

-4
3.0x10

-4
2.5x10

Staphylococcus aureus
Heat flow (W)

-4
2.0x10

-4
1.5x10 Staphylococcus epidermidis

-4
1.0x10

-5
5.0x10

0.0
0 5 10 15 20 25 30
Time (h)

TAINSTRUMENTS.COM
Suspension of Microorganisms in TSB

S. aureus
P.
(ATCC 29213)
aeruginosa

E. coli

S. aureus
(ATCC 43300)

Prop.
acnes

S. epidermidis (clinical
C. albicans
isolate)

S. Epidermidis (ATCC
12228)
M. fortuitum (clinical isolate)

TAINSTRUMENTS.COM
 Results

Characteristic Aseptic cases Implant infection

(n = 646) (n = 475) (n = 171)
Type of prosthesis
Joint prosthesis (n = 167) 31% 44%
Internal osteosynthesis (n = 479) 69% 56%
Diagnosis
Tissue culture (next slide) 9% 77%
Sonicate culture (≥5 cfu/plate) 5% 89%
Sonicate Gram stain 0% 52%
Calorimetry (> 30 µW) (n = 409) 13/306 (4%) 98/103 (95%)

22
9
TAINSTRUMENTS.COM
 Results

Tissue Sonicate
23
0
TAINSTRUMENTS.COM
 Advantages of Calorimetry

• Rapid detection of microbial growth

(within hours)
• High sensitivity and specificity
• Potential rapid antimicrobial
susceptibility testing (using selective
growth media)
• Potential rapid identification

23
1
TAINSTRUMENTS.COM
 Enzymatic Reaction of Tyrosine

• L-tyrosine (or 4-hydroxyphenylalanine) is a para isomer and is the most

common isomer form found in nature. There are two additional isomers,
namely meta- and ortho-tyrosine (3- and 2- hydroxyphenylalanine, respectively),
which rarely occur in nature.
• Tyrosinases are enzymes that oxidize a broad range of phenols into
ortho-quinones.
• When tyrosine is exposed to a tyrosinase in the presence of oxygen the
benzene ring is oxidized at the hydroxyl group and converted to an ortho-
quinone molecule, which changes the color of the solution.

Tianhong Chen, Heather D. Embree, Li-Qun Wu, Gregory F. Payne “In Vitro Protein-Polysaccharide
Conjugation. Tyrosinase-Catalyzed Conjugation of Gelatin and Chitosan” Biopolymers, 64, 292-302
23
(2002)
2
TAINSTRUMENTS.COM
 Oxidation of Tyrosine by Tyrosinase

• The exothermic oxidation of the

ambient air purged with dry N2 Blank
tyrosine is detected by the
calorimeter. The magnitude of the
reaction (and heat flow) is dictated by
800 the oxygen content inside the
ampoule.
• Data shifted on Y-axis for
600
comparison.
Heat flow (µW)

Ambient air • Picture below shows the comparison

of the solution color change with
400
oxygen content. Blank - left, Nitrogen
Blank
head space – center, and ambient air
– right.
200

Nitrogen

0 1 2 3 4
Time (hour)

Experiment performed in a TAM Air with Admix ampoule.

23
3
TAINSTRUMENTS.COM
 Germination of Quinoa Seeds

23
Sigstad and Garcia, Thermochim. Acta 366, 149-155, (2001).
4
TAINSTRUMENTS.COM
Milk Fermentation

•milk + 1% starting culture

•normal, +dextrose, +NaCl, +Na-benzoate
•Closed 20 mL glass ampoules at 19oC

TAINSTRUMENTS.COM
Milk Fermentation

Lactobactilius culture growth

3
heat production rate / mW

2.5
normal
2

1.5 +dextrose

1 +NaCl

0.5 +Na benzoate

0
0 50 100
time / h

TAINSTRUMENTS.COM
Carrot Juice Spoilage

•8 samples heat treated at 37-58oC

•Closed 20 mL glass ampoules at 25oC (accelerated)

TAINSTRUMENTS.COM
Carrot Juice Spoilage Measured at 25°C

0.5

55oC
heat production rate / mW/g

0.4 52oC Heat treatment

temperatures

0.3 58oC
37-49oC

0.2

0.1

0
0 10 20 30 40 50 60 70
time / h
TAINSTRUMENTS.COM
 Microbiological spoilage prevention

• The goal of preventing microbial spoilage can be reached by many methods,

e.g., natural and synthetic chemical preservatives, and thermal treatments.

• Fresh carrot juice is an extremely perishable food-stuff as it has a neutral

pH, high sugar content and also contains many soil microorganisms.

L. Wadsö, F. Gómez Galindo / Food Control 20 (2009) 956–961 TAINSTRUMENTS.COM

 Live Insect with Single Blade of Grass

1
2
0
.
0

A
c
t
i
ve
Mi
l
l
ip
e
de
!

1
0
0
.
0

8
0
.
0
Heat flow (W)

6
0
.
0
N
o
t
i
c
ede
c
r
e
as
e
ds
i
g
na
ld
u
et
o
O2c
o
n
su
m
pt
i
on

4
0
.
0

C
o
n
d
i
ti
o
ns
:
T
A
MT
e
m
pe
r
a
t
ur
e2
5
°C
2
0
.
0
S
i
d
eA:
Mi
l
li
p
ed
es
e
a
le
d
in
s
i
d
e4m
L
S
Ss
c
r
ew
ca
p
am
p
ou
l
e
B
a
s
e
l
in
e
(
W
~
0) S
i
d
eB:
Em
pt
y4
mL
S
Ss
c
r
ew
ca
p
am
p
ou
l
e

0
.
0
1
0
0
00 2
0
0
00 3
0
0
00 4
0
0
00
T
i
me
(s
)

Note: Single red ant gave ~30 W signal (not shown)*

24* No insects were injured during the course of this experiment.
0
TAINSTRUMENTS.COM
 Effect of Sucrose on Yeast Growth – TAM Air

A
m
p
o
ul
e
lo
we
r
e
di
n
to
me
a
s
ur
i
n
gp
os
i
t
i
on

6
0
.
0
C
o
n
d
i
ti
o
ns
:
T
A
MA
i
r
Te
mp
e
r
a
tu
r
e2
5
°C
5
0
.
0 S
i
d
eA:
1
0mL
Y
e
as
t
i
nAd
d
/
Mi
xa
mp
o
u
l
e
S
i
d
eB:
1
0mL
W
a
te
r
i
ng
l
as
s
am
p
ou
l
e

4
0
.
0
Heat flow (mW)
I
n
j
ec
t
io
n
of
0
.1
mL
C
o
k
e

3
0
.
0
I
n
j
ec
t
io
n
of
0
.1
mL
D
i
et
Co
k
e

2
0
.
0
S
t
i
r
ri
n
g

1
0
.
0

0
.
0
2
0
0
0 7
0
0
0 1
2
0
00 1
7
0
00 2
2
0
00
T
i
me
(s
)

TAINSTRUMENTS.COM
Effect of Blanching on Turnip

TAINSTRUMENTS.COM
TAM Applications
Cement Hydration

TAINSTRUMENTS.COM
Sample Ampoules

▪ Static 20 mL disposable
glass ampoules.
▪ Mixing of solid/liquid
outside calorimeter

TAINSTRUMENTS.COM
Portland Cement Basics

• Silicates hydrate to give strength giving gel, “glue”

• Aluminate and ferrite phases necessary to get a molten phase during
production of cement
• Aluminates react rapidly, interact with admixtures, workability, set, early
strength development
• Gypsum added during grinding to slow down aluminate hydration rate
▪ Higher C3A , lower C4AF generally more reactive
▪ Different sulfate forms have different solubility
◆ Dr. Sandberg, Grace Construction Products, US (2002)

• ASTM Methods available in 2009 related to cement isothermal

calorimetry (www.ASTM.org).
▪ C1679-08 “Standard Practice for Measuring Hydration Kinetics of Hydraulic
Cementitious Mixtures Using Isothermal Calorimetry.”
▪ C1702-09 “New Test Method for Standard Test Method for Measurement of
Heat of Hydration of Cement with Heat Conduction Calorimetry.”

TAINSTRUMENTS.COM
 Portland Cement Basics

The hydration process

undergoes a number of
phases (Young, 1985)
• (I) Rapid initial processes
• (II) Dormant period
• (III) Acceleration period
• (IV) Retardation period
• (V) Long term reactions

The phases has been described in more detail

(Sandberg, 2002)
▪ (I) Dissolution of ions and initial hydration
▪ (II) Formation of ettringite
▪ (III) Initiation of silicate hydration
▪ (IV) Depletion of sulphate

Dr. Sandberg, Grace Construction Products, US (2002)

TAINSTRUMENTS.COM
 Setting Time of Cement

•Assessment of setting time and

early stiffening (diagram)

•Influence of concrete
admixtures

•Influence of glass fillers, waste

products, slags etc.

•Influence of contaminants, e.g.

in water

•Assessments of the efficiency of

mixing

24Dr. Sandberg, Grace Construction Products, US (2002)

7
TAINSTRUMENTS.COM
 Effects of Admixtures

•Admixture is any material which Water/cement ratio 0.5, 23 C,

water only
affects the process and properties
of cement

•Only small differences between

cement lots when tested without
admixture Water/cement ratio 0.5,
23 C, 1.0% water
reducing admixture by

•Very large differences between

weight of cement in all
samples

cement lots when tested with

same admixture!!!

24
Dr. Sandberg, Grace Construction Products, US (2002)
8
TAINSTRUMENTS.COM
Cement Hydration

Cement Hydration

Ampoule lowered into measuring position

550.0
Cement
500.0 Cement (1% wt. NaCl)
Cement (1% wt. Sucrose)
450.0
Cement (1% wt. Na Lignosulfonate pasticizer)
400.0
Heat flow (mW)

Conditions:
350.0 TAM Air Temperature 25 C
600 mW scale
300.0
Cement:Water ratio 1:1 (w/w)
250.0 Side A: ~5 g Cement samples in plastic ampoule
Baseline Side B: ~5 mL Water in plastic ampoule
200.0

150.0

100.0

50.0

0.0

0 20000 40000 60000 80000

Time (s)

TAINSTRUMENTS.COM
 Effect of Contamination

Influence on hydration rate

of a mixture of soil and
sawdust (0; 0.9; 2.5 and
5.9% of w/c=0.6 cement
mortar).

25
Dr. L. Wadsö, University of Lund, Sweden (2002)
0
TAINSTRUMENTS.COM
 TAM Air Reproducibility

25
Dr. Moro , Holcim Group Support, Switzerland (2002)
1
TAINSTRUMENTS.COM
 Temperature Dependency of Cement Hydration

Measurements at 20, 25 and 30 C

P (mW)
P reflects the rate of the process

Q (W)
Q reflects the extent of the process

Time (h)

25
Dr.
2
Johansson , Thermometric AB, Sweden (2002) TAINSTRUMENTS.COM
 Admix Ampoule -Two Identical Ampoules

Excellent reproducibility !

25
Dr.
3 Moro , Holcim Group Support, Switzerland (2002)
TAINSTRUMENTS.COM
 Admix Ampoule Experiment

0.25
––––––– Stucco_w/c 0.50_1
–––– Stucco_w/c 0.50_2
––––––– Stucco_w/c 0.50_5% Salt

0.14hr

0.20

DI water vs. salt solution

Normalized Heat Flow (W/g)

0.15

0.04hr

0.39hr
0.10
0.46hr

0.04hr
0.05

0.00
0.0 0.2 0.4 0.6 0.8 1.0
Time (hr) Universal V4.4A TA Instruments

25
4
TAINSTRUMENTS.COM
Admix Ampoule Experiment

0.12
––––––– Stucco w/c 0.35
–––– Stucco w/c 0.50

0.10

Varied w/c ratio

Normalized Heat Flow (W/g)

0.08

0.06

0.04

0.02

0.00
0.0 0.5 1.0 1.5 2.0
Time (hr) Universal V4.4A TA Instruments

TAINSTRUMENTS.COM
 TAM Applications

Material Science and Industrial Applications

TAINSTRUMENTS.COM
Stability with TAM

TAM Temperature: 40 °C
Sodium Percarbonate QC test (AN 22034)

TAINSTRUMENTS.COM
 Stability of Organic Peroxides

Autocatalytic behaviour of 80% wt. cumene hydroperoxide

Hou, Houng-Yi; Shu, Chi-Min; Duh, Yih-Shing; AIChE Journal,

25
(2001), Vol 47, No 8, 1983
8
TAINSTRUMENTS.COM
 Long-term Behaviour from Short-term Data

200
Specific heat flow

150
Heat flow (µW/g)

Blue curve: measured heat flow data

100
Red curve: Fitted curve of a multi-exponential type to
measured data between 0.1-2 week.
50

0
0 0.5 1 1.5 2 2.5 3 3.5 4
Time (weeks)

200
Specific heat flow

150
Heat flow (µW/g)

100
Prediction of the 4 week behaviour from
data obtained during 0.1-2 week using the multi-
50
exponentional function obtained by the fitting procedure.

0
0 0.5 1 1.5 2 2.5 3 3.5 4
Time (weeks)

25
9
TAINSTRUMENTS.COM
 Repeatability

Three different samples

1.4
Relative specific heat flow

1.2

1.0

0.8

0.6

0.4

0.2

0.0
0 0.5 1 1.5 2 2.5 3 3.5 4
Time (weks)

26
0
TAINSTRUMENTS.COM
 Stabilization of an Energetic Plasticizer (NPN)

26
Wingborg and Eldsäter, Propellants, Explosives and Pyrotechnics 27, 314 -319, (2002).
1
TAINSTRUMENTS.COM
NATO Standard

•STANAG 4582 – ratification in

process

•Dedicated for stability testing of

nitrate ester based propellants
by use of heat flow calorimetry
(HFC)

•A method to establish chemical

stability of SB, DB and TB
propellants for a minimum of
10 years when stored at 25 °C

Figure from Bodycote Materials testing

TAINSTRUMENTS.COM
 RH Perfusion – Two Gas Sources

Conditions:
40 TAM at 25 °C
Side A: Activated carbon inside 4 mL SS
RH perfusion ampoule
30 Side B: Empty 4 mL SS cir-clip ampoule
Heat Flow (We-6)

Carbon B
20
Carbon C
Carbon F
10
25 ppm Ethanol Adsorption onto Activated Carbon

0
0 0.5 1 1.5 2 2.5 3

-10
Time (Hours)
Note: Each sample weighed approximately 15 mg. Heat flow signal was not normalized.

TAINSTRUMENTS.COM
Percarbonate – Vacuum/Pressure Ampoule

Ampoule pressure, Ch 4:1 Signal, Ch 4:1

220

1.8

200
Heat flow (µW)

1.6
Pressure (bar)

180
1.4

160
1.2

2 4 6 8
Time (day)

TAINSTRUMENTS.COM
Moisture Sorption - Vacuum/Pressure Ampoule

Dolomite = Calcium magnesium carbonate CaMg(CO3)2

TAINSTRUMENTS.COM
 Oxidation of Polymers

•Radical chain reaction

▪Initiation, Propagation, Termination, and Chain branching

•Important intermediates
▪Hydroperoxides and radicals

•Solid state oxidation may be heterogeneous in nature

▪Localization and spreading

•Stabilizers are usually added to prevent oxidation

•Effects of prolonged oxidation
▪Chain scission and embrittlement

26
6
TAINSTRUMENTS.COM
 Characterization of Oxidation

•The effects of oxidation is typically studied by analyzing

the formation of volatile, non-volatile and polymer bound
oxidation products as a function of time.

•Alternative: Use rate-sensitive techniques to monitor the

in-situ oxidation.

26
7
TAINSTRUMENTS.COM
Polyamide 6

O H

HO C CH2 N H HO
5 n

Polyamide 6
• Polyamide 6 film from Nyltech, Italy
• Thickness: 40 µm
• Crystallinity: 40 %
• Melting point: 210oC
• Glass transition temp: 70oC (dry), -10oC (100 % r.h.)
• Stabilizer: Irganox 1098 (hindered phenol)
• Sensitive to oxidation!

268

TAINSTRUMENTS.COM
 Response to Nitrogen / Oxygen

Heat flow curves for unstabilized polyamide 6 film exposed to nitrogen and
oxygen at 110C. After four days, the gases were switched .

700
Gas switch
600
Heatflow ( W/g)

500
400
oxygen
300
200
100 nitrogen

0
0 2 4 6 8
Ageing time (d)

Forsström, D., Thesis,”Novel techniques for characterisation of the oxidative stability of

polyamides”, KTH, Dept. Polymer Technology (1999) TAINSTRUMENTS.COM
 Efficiency of Stabilizers

Influence of Irganox 1098 on the Heat Flow time curve for PA6. The presence of
the stabiliser suppresses the heat flow signal indicating the preventing action.

o
Unstabilised 120 C air
400

300
100 ppm
Heat flow (W/g)

200 ppm
200

400 ppm

100

0
0 1 2 3
Time (d)

Forsström, D., Thesis,”Novel techniques for characterisation of the oxidative stability of

27
polyamides”, KTH, Dept. Polymer Technology (1999)
0
TAINSTRUMENTS.COM
Polypropylene (PP)

H CH3 H CH3 H CH3

C C C C C C

H H H H H H

• Isotactic PP from DSM Research

• Powder: Individual particle mass: 50 – 100 µg
• Crystallinity: 53 %
• Melting point: 165oC
• Glass transition temp: -10oC
• Sensitive to oxidation at ambient conditions!
271

TAINSTRUMENTS.COM
 Rate-Sensitive Techniques

•Thermal Activity Monitoring (TAM)

◆ Measure the heat flow by the use of thermopiles (J/s)
◆ TAM is a non specific technique, i.e. sensitive to all heat producing

processes (physical and chemical)

◆ TAM is a quantitative technique

•Chemiluminescence Techniques – CL / ICL

◆ Measure the light intensity by the use of a photon multiplier (counts/s)
◆ CL is sensitive to all light-producing reactions

◆ The most referred mechanism to explain the CL emission is the Russel

mechanism i.e. a bimolecular termination reaction of peroxy radicals.

27
2
TAINSTRUMENTS.COM
ICL - Oxidation of Unstabilized PP Powder in Air

1.2 o
120 C

▪ The ”oxidation
o o o o
130 C 110 C 100 C 90 C
1
rate” is not constant but
CL intensity (a.u)

vary in a characteristic
0.8 way.

0.6 ▪ The service life of the

PP sample roughly
0.4 corresponds to the
onset of the
0.2 accelerating part of the
curve.
0
0 0.5 1 1.5 2
Time (d)
273

TAINSTRUMENTS.COM
TAM  ICL 100oC

20000 1.2
o
tmax=0.92 d 100 C
18000
1
16000

CL intensity (a.u)
14000
Heatflow (µW/g)

0.8
12000

10000 0.6
8000
0.4
6000

4000
0.2
2000

0 0
0 0.5 1 1.5 2

Time (d) 274

TAINSTRUMENTS.COM
 TAM: Heat Flow versus Energy

70000 ▪ Heat flow values are

120oC known at different
60000
temperatures for
50000 different Energies.
Heatflow (µW/g)

40000 ▪ Thus, an apparent

activation energy (Ea)
30000
can be calculated as a
o 60oC function of the ’extent of
20000 70 C 100oC
oxidation’
10000
80oC
0
0 2000 4000 6000 8000 10000

Energy (J/g)

TAINSTRUMENTS.COM
 Curing of Epoxy in TAM Air

3
(mW/g)

▪ D.E.R. 331 epoxy resin

from Dow Chemical
1

25 °C ▪ Jeffamine D-230
hardener from Huntsman

0
6 12 18 24 30 36
Time (ho ur)

TAINSTRUMENTS.COM
 Curing of Epoxy in TAM Air

0.04
––––––– TAMAir_25
––––––– TAMAir_30
––––––– TAMAir_35
0.78hr
––––––– TAMAir_40
––––––– TAMAir_50

0.03
Normalized Heat Flow (W/g)

0.02
1.68hr

7.58hr

2.48hr

0.01
3.14hr

0.00
0 5 10 15 20 25 30
Time (hr) Universal V4.4A TA Instruments

▪ D.E.R. 331 epoxy resin from Dow Chemical

and Jeffamine D-230 hardener from Huntsman
TAINSTRUMENTS.COM
 Adhesive: ESP 110

9000 • Heat flow values are in the

range of mW/g at 50oC
and increases with
70oC temperature.
6000 • Possible exothermic
reactions: curing,
60oC oxidation.

3000
• ESP 110 – usually cured
at 150oC
50oC
• Closed 3 ml glass
ampoules

1 2 3

Time (d)

Data by Lars-Gunnar Svensson, Celsius Materials CMK, Karlskoga, Sweden TAINSTRUMENTS.COM

 Epoxy Curing

Heat prod. rate / mW/g

5

0
1 0 10 20 30 40 50
1
log10(Heat prod. rate / mW/g )

Q(t)/Q(max)
Time / h

0.5
0.8

0
0.6

Extent of reaction
-0.5
0.4

-1
0.2

-1.5
P  rate Q  extent
0 0.5 1 1.5 2 0
0 20 40 60
log10(Time / h)
Time / h
TAINSTRUMENTS.COM
Battery Discharge

•Standard AAA battery in calorimeter discharged through

a load or a resistance.

TAINSTRUMENTS.COM
Self-Discharge (No Load) of Alkaline Battery

AAA battery - new AAA battery - damaged

10

8
Heat flow (µW)

▪ TAM III at 25 °C
4

▪ 4 mL SSt screw cap

2
Signals corrrect
ampoules

0
0 2 4 6 8 10 12
Time (ho ur)

TAINSTRUMENTS.COM
Larger Sample Applications

•Environmental science
•Food applications Soil 17 (3-15-10).rslt, Ca(NO3)2 Glu
Soil25 (3-26-10).rslt, 25 FYM Gluc
Soil 16 (3-19-10).rslt, Straw+N Glu
Soil 23 (3-19-10).rslt, Sludge Glu
Soil 45 (3-19-10).rslt, (NH4)2SO4 Glu

•Large samples 200

Normalized heat flow (µW/g)

150

100

50

0
0 8 16 24 32 40 48
Time (ho ur)

TAINSTRUMENTS.COM
 RH Perfusion – Step RH

Lowering ampoule
into calorimeter
Conditions:
A TAM at 40 C
50 PBO mass = 0.00490 g
Forceps were used to tease small fiber
bundles from a ~11 cm piece.
25 N2 flow rate 120 mL/hr
Normalized Heat Flow (mW/g)

0 RH set to 90% - Adsorption

RH set to 0% - Desorption
B
-25

-50

-75

-100
Poly(p-phenylene-2,6-benzobisoxazole) fiber

25 30 35 40 45 50 55 60 65 70 75 80 85 90
Time (h)
TAINSTRUMENTS.COM
 RH Perfusion – Step RH

Conditions:
70
A TAM at 40 C
PBO mass = 0.00490 g
Forceps were used to tease small fiber
60 bundles from a ~11 cm piece.
N2 flow rate 120 mL/hr
Normalized Heat Flow (mW/g)

50

40

30

20 Total signal still positive (~ 4 W) after ~3

days indicating continuing reaction(s).

10 RH set to 90%

30 31 32 33 34
Time (h)
28
4
TAINSTRUMENTS.COM
 RH Perfusion – Step RH

0
B RH set to 0%
Singnal returns to baseline
(0 W) after 19 hr.
Normalized Heat Flow (mW/g)

-25

Note: Multistep desorption

-50

-75
Conditions:
TAM at 40 C
PBO mass = 0.00490 g
Forceps were used to tease small fiber
-100
bundles from a ~11 cm piece.
N2 flow rate 120 mL/hr

70 71 72 73 74
Time (h)
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 Pop Quiz!!!

PV Work

Conditions:
5.0 TAM Air Temperature 25 C ▪ Lowering ampoules will
600 mW scale
4.0 Side A: Empty glass ampoule w/ 1mL syringe and canula
create frictional heat and
Side B: Empty glass ampoule
1.0 mL injection
work (or pressure) on the
3.0
calorimeter due to small air
2.0
0.5 mL injection
Baseline (~0 W) gap between ampoule and
Heat flow (mW)

1.0 calorimeter wall.

0.0
▪ This example shows that
-1.0 heat flow increases as
-2.0 0.5 mL evacuation 1.0 mL evacuation pressure in the ampoule
-3.0
increases.
-4.0

-5.0

5000 10000 15000 20000

Time (s)

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 TAM Applications

Isothermal Titration Calorimetry

TAINSTRUMENTS.COM
ITC in Biochemistry/Biophysics

An important technique for the characterization of

molecular interactions and for the understanding of
binding thermodynamics.

Most commonly utilized for biological molecules

and biological systems.

20

15
Heat flow (µW)

10

0 1 2 3
Time (ho ur)
TAINSTRUMENTS.COM
 ITC in Biochemistry/Biophysics

• A premier tool for the characterization of

biological macromolecules
▪Antigen – Antibody
▪Peptide – Protein
▪Lipid – Protein
▪Nucleic Acid – Protein
▪Carbohydrate –Protein
▪Small Molecule/Drug – Protein
▪Protein – Protein
▪Protein – Receptor (soluble and membrane-bound)

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 Strength of TAM-ITC Relative to Other Methods

•High accuracy and precision

•General applicability
•No chemical modification necessary
•No immobilization necessary, although possible
•Equilibrium conditions
•Not limited by turbid or particulate suspensions
•Removable cell (important for toxic or radiological
samples)

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 Binding Characterization

•Binding Affinity, K
▪Tightest binding measurable approaches nanomolar
Weakest binding for biological macromolecules millimolar

•Binding Stoichiometry, n
•Binding Thermodynamics, H, G, S and Cp
H measure of the heat released or absorbed
Cp measure of the temperature dependence of H (dH/dT)
G measure of the equilibrium constant (G = -RTlnK)
S measure of ”order” in the system (G = H-TS)

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 Determination of Binding Constants

G = -RTlnKa = H-TS

•The more negative G is, the higher the binding affinity

•Negative H favors the reaction
•S is positive for entropically-driven reactions
•Cp determined from performing binding experiments at
different temperatures, plotting T vs. H. Slope is Cp
•Different combinations of H and S can give same G and
thus Ka, but not necessarily the same specificity
•An ideal thermodynamic profile is a balance between
hydrophobicity (solvent repulsion) and hydrogen bonding (target
attraction)
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 The Titration Experiment

•The relation between H and K Ka = 1/Kd

M+L ML
K = [ML] / [L][M]
Q = ([ML] / [M]total) · H
Q = (K · [L] · H) / (1 + K · [L])
kJ/mol

Calculated
dQ/dAmount

30 <Current>

20

10

0
0.0 0.6 1.2 1.8 [L]/[M],
TAINSTRUMENTS.COM
 Choosing a Binding Model

• Do you know Stoichiometry? (the number of ligands bound per macromolecule)

▪ Sources: related protein/ligand complexes, X-ray or NMR structural data, CD or
NMR titration data (including competitive binding), molecular modeling/docking,
sequence alignments, mass spec, DSC
▪ Use simplest model consistent with available information – start with Independent

• Independent? (one or several identical sites bind the same ligand with the same
enthalpy and Ka, independent of each other)

• Multiple? (two or more binding sites, each capable of binding ligands, but with different
enthalpies and Ka).
◆ Possibly fit using several Independent models

• Cooperative? (two or more binding sites. The binding of the first ligand affects the
binding of succeeding ligands)

• Other binding model?

• Try to run an experiment to determine H

▪ Run experiment to saturate all sites and determine enthalpy by titration curve
▪ Run experiment with excess macromolecule and low ligand and calculate enthalpy
per mole of ligand added.
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 ITC Experimental Design
K [AB]
A+B AB K = [A][B]
H
Constant Equilibrium Constant Constant Enthalpy with
with Varying Enthalpy Varying Equilibrium Constant

K=
H = 4x
6
K = 10

Incremental Heat Effect

Incremental Heat Effect

H = 3x
5
K = 10

H = 2x

4
K = 10
H = 1x

Moles Ligand Injected Moles Ligand Injected

▪ Generally want to obtain stoichiometry (n), enthalpy (H) and binding constant (Ka) from one
experiment
▪ Enthalpy is directly measured. Receptor should be saturated with ligand at end of titration
▪ To obtain Ka: 10 < Ka[M]T < 1000
▪ [M]T is typically 10 – 100 mM, Ka is typically 103 to 109 M-1
▪ Reminder: KD = 1/Ka
TAINSTRUMENTS.COM
ITC Experimental Design

•For accurate evaluation the value of C*K has to be

between 1 and 1000

Note: C is the concentration of the titrand (reaction vessel)

•Best precision is obtained if the titration curve has a

sigmoidal shape and many data points in the transition
region (e.g. C*K = 50)

•e.g. If K is estimated to 106 M an optimum concentration of

C is 10-3 M.

296

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 ITC Experimental Design

• According to the ligand binding theory, the heat evolved for each injection depends on two variables, i.e. r and
X where r = Ka·[Mt] and X = [Lt]/[Mt].

• This left side of this equation (Independent model) can be plotted versus X for different r values, see the graph.
It can be observed that the shape of the binding curve is ‘nice’ for an r value of 50-100. For an unknown sample
it is important that the binding curves shows a good shape to obtain highest accuracy in the fitting procedure in
determining the reaction enthalpy and the r value. From the r value the equilibrium constant can be found since
r = Ka·[Mt]. In practice binding curves with a good shape is obtained for r values in the range: 10 < r < 1000.

1000 and 
1
50
5
0.8

1 dq 1  1 + r  ( X − 1) 

 = 1−
(1/VDH)dq/dL

V  H dLt 2  (r − X  r + 1)2 + 4 X  r 
0.6
1

0.4

0.2
0.1

0 0.5 1 1.5 2 2.5 3

Ltot/Mtot

TAINSTRUMENTS.COM
 ITC Experimental Design

▪ Choosing correct ligand and receptor concentrations requires an

estimate of stoichiometry and Ka, often from spectroscopic
measurements. ITC is then used to determine Ka accurately
▪ Weak binding (low Ka) – may be limited by concentration
▪ may use multiple syringes and combine results
▪ Strong binding (high Ka)
▪ minimize concentration or injection volume, etc…
▪ try competitive binding (displacement) experiment

▪ Use Experiment Design module to alter Ka, binding model,

stoichiometry and concentrations, and see the effect on the binding
curve. Requires ‘best guess’ inputs of stoichiometry, Ka and binding
model

▪ No idea of the parameters? Try 5 M receptor (in cell), 50 M ligand

(in syringe), 15 x 15 L injections, 25 oC

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 Hints for ITC Experiments

•Small heat of binding? Change temperature, pH and/or buffer

•Temperature: H is temperature and system dependent, varies with

Cp. Conduct experiment at relevant temperature where binding has
measurable H.

• Different buffers have different enthaplies of ionization, affect H of

binding. If low enthalpies observed, use buffer with high ionization
enthalpy

• Low enthalpy may indicate that a non-optimal pH is being used.

• Determine concentrations by absorbance.

▪Scanning wavelength is better than single wavelength in a situation where
additives may interfere.

• Degas both ligand and macromolecule solution

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 Hints for ITC Experiments

▪ All reactions produce heat. Ensure only the desired reaction is

measured.
– pH, ionic strength, choice of buffer and temperature

▪ Diluting a compound produces or absorbs heat (‘heat of dilution’).

▪ To minimize heat of dilution of buffer ‘contaminants’ in protein and

ligand solution: 1) dialyze protein and ligand 2) use the used
dialysis buffer to dissolve the ligand 3) perform ‘blank’ experiment
(titrate ligand into buffer), then subtract blank from ‘real experiment’
data. Caution: analyze blank data for indications of ligand-buffer
interactions.
– Is the ligand too small to dialyze? Be sure to desalt and use dialysis
buffer to prepare and dissolve ligand.

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Hints for ITC Experiments

▪ Instrument default settings are factory-optimized for aqueous solutions.

User can adjust for organic solvents. Order ITC with Kalrez (not Viton) O-
rings if anticipate using organics.

▪ Non-aqueous solvents have different viscosities and heat capacities.

▪ Match [organic] in syringe and sample cell? Extremely difficult if ligand
first dissolved in organic, then diluted with buffer to match [organic] in
sample cell.
▪ Any mismatch in [organic] can result in
heat of dilution masking heat of binding
▪ Proteins easily unfold in organics. If
organics are present, is the ligand
binding to the native conformation?
▪ Example: 5% DMSO decreases Tm of
RNase A by 1 oC.

TAINSTRUMENTS.COM
 Hints for ITC Analysis

• Unexpectedly low stoichiometry could be due to:

▪[receptor] lower than anticipated
▪[ligand] higher than anticipated
▪Receptor contains contaminating proteins
▪Receptor is partially unfolded
▪Multiple binding sites
▪Wrong binding model
▪Insufficient curvature in data: change concentrations

• Solvent protonation
▪Study different buffers at the same pH
▪Plot observed enthalpy versus ionization enthalpy
◆ Slope gives the number of protons released (if negative)

- Slope equals zero than no effect of buffer selected

◆ Y-intercept is the enthalpy of binding (buffer independent)
- Different pH will result in a different plot

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Proton Linkage

•M + L + B ML + BH+; Hreaction
•M + L ML + H+; Hbinding
•B + H+ BH+; Hproton

200

-200
Hreaction
P/ nW

-400

-600
Hproton
-800
IGF-I to the soluble extracellular
-1.000 IGF-I receptor
0 50 100 150 200
Time/min

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DSC may be Helpful for ITC Results Correction

▪ Characterizing the thermodynamics of a binding reaction requires determining ∆H and

∆G at several temperatures, and obtaining ∆Cp to predict the change in ∆H and ∆G with
temperature
▪ ∆H is directly measured by ITC. ∆G is calculated from the binding constant at T:
G = -RT lnKa
▪ Obtaining ∆Cp by ITC requires ∆H measurements at several temperatures, then taking
the slope to obtain ∆Cp. Better to determine ∆Cp by DSC and correct the temperature
effect on ∆H .

Dragan et al., J. Mol. Biol. 343, 371-393, 2004. TAINSTRUMENTS.COM

 ITC Results by using Slow-scanning DSC

▪ If a ligand binds preferentially

to a folded protein, the Tm of 80

the protein will increase. 70

Generally, the more bound 60
0.3 mM

-1
0.75 mM
ligand there is, or the tighter it 0.15 mM

Excess Cp / kJ mol K
-1
50
binds, the more Tm increases. 1 mM
40
1.25 mM
▪ Can determine binding 30
0.075 mM
1.5 mM
constant at Tm. 20
0.05 mM
0 mM
▪ But, useful if very slow or very 10

tight binding, or organic 0

solvents necessary. -10
35 45 55 65 75 85
▪ Valid if comparing relative Temperature / °C
binding of ligands to same
protein Binding of 2’-CMP to RNase A ± 5% DMSO
Ka = 5900 M-1 (-DMSO); 6900 M-1 (+DMSO) at Tm
▪ DSC is a quick way to
determine if two molecules
interact. Also allows Cp
correction of ITC data
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Dynamic Calibration for ITC

dQ/dtDyn dQ/dtHF Baseline

24

18

12

0 20 40 60 Time,min

PDyn = Praw +  · dPraw/dt

where P = dQ/dt
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 Titration Calorimetry in Rational Drug Design

•Screening of pharmaceutical hits and leads

Non-binder — Heat of dilution Binder

5 5
4 4
3 3

P/µW
P/µW

2 2
1 1
0 0
-1 -1
0 10 20 30 40 0 10 20 30 40
t/min t/min

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 CMC of Sodium Dodecyl Sulfate (37 °C)

• 125.18mM SDS injected into 1 mL of water

• MW = 288.38 g/mol

125mM SDS into 1mL Water (5µL injections)

Accu m u la ted h ea t (m J )
-5
-10
Heat flow (µW)

-20
-10

-30

-15
0 50 100
Vo lu m e (µl)

-20

0 1 2 3 4 5 6
Time (hour)

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Need Assistance?

•Check the online manuals and error help.

•Contact the TA Instruments Helpline

▪Phone: 302-427-4070 M-F 8-4:30 EST
◆Select Microcalorimetry Support
▪Email: [email protected]
•Call your local Technical or Service Representative

•Call TA Instruments
▪Phone: 302-427-4000 M-F 8-4:30 EST
•Check out our Website: www.tainstruments.com

•For instructional videos go to: http://www.youtube.com/tatechtips

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 Thank You

The World Leader in Thermal Analysis,

Rheology, and Microcalorimetry

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