TA Instruments Nano ITC Series, Nano ITC 5301, Nano ITC 5300, Nano ITC 5302 Getting Started Manual

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Nano Isothermal

Titration Calorimeter

(Nano ITC)

Getting Started Guide for

Models 5300, 5301, 5302

Revision F Issued February 2011

Notice

The material contained in this manual, and in the online help for the software used to support this instrument, is believed adequate for the intended use of the instrument. If the instrument or procedures are used for purposes other than those specified herein, confirmation of their suitability must be obtained from TA Instruments. Otherwise, TA Instruments does not guarantee any results and assumes no obligation or liability. TA Instruments also reserves the right to revise this document and to make changes without notice.

TA Instruments may have patents, patent applications, trademarks, copyrights, or other intellectual property covering subject matter in this document. Except as expressly provided in written license agreement from TA Instruments, the furnishing of this document does not give you any license to these patents, trademarks, copyrights, or other intellectual property.

TA Instruments Operating Software, as well as Module, Data Analysis, and Utility Software and their associated manuals and online help, are proprietary and copyrighted by TA Instruments. Purchasers are granted a license to use these software programs on the module and controller with which they were purchased. These programs may not be duplicated by the purchaser without the prior written consent of TA Instruments. Each licensed program shall remain the exclusive property of TA Instruments, and no rights or licenses are granted to the purchaser other than as specified above.

TA Instruments can accept no liability for loss or damage, however caused, arising from the faulty or incorrect use of its products.TA Instruments shall not be liable for any damages caused by interactions between exogenous materials (e.g. chemicals) and parts of the instrument. This includes interactions of gaseous, liquid or solid materials with, for instance, ampoule surfaces and/or parts of the instrument. It also includes gases or vapors leaking from ampoules (e.g. originating from chemical reactions producing gaseous substances), with subsequent cause of damage to the instrument.

Page 2 Nano ITC Getting Started Guide

Introduction

Important: TA Instruments Manual Supplement

Please click the TA Manual Supplement link to access the following important information supplemental to this Getting Started Guide:

• TA Instruments Trademarks

• TA Instruments Patents

• Other Trademarks

• TA Instruments End-User License Agreement

• TA Instruments Offices

Nano ITC Getting Started Guide Page 3

Notes, Cautions, and Warnings

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This manual uses NOTES, CAUTIONS, and WARNINGS to emphasize important and critical instructions. In the body of the manual these may be found in the shaded box on the outside of the page.

NOTE: A NOTE highlights important information about equipment or procedures.

CAUTION: A CAUTION emphasizes a procedure that may damage equipment or cause loss of data if not followed correctly.

A WARNING indicates a procedure that may be hazardous to the operator or to the environment if not followed correctly.

Regulatory Compliance

Safety Standards

EMC Directive

This instrument has been tested to meet the European Electromagnetic Compatibility Directive (EMC Directive, 2004/108/EC). The Declaration of Conformity for your instrument lists the specific standards to which the unit was tested.

The instrument was designed specifically as a test and measuring device. Compliance to the EMC directive is through IEC 61326-1 Electrical equipment for measurement, control and laboratory use - EMC requirements (1998).

As noted in the IEC 61326-1, the instrument can have varying configurations. Emissions may, in non-typical applications, exceed the levels required by the standard. It is not practical to test all configurations, as the manufacturer has no control over the user application of the instrument.

Immunity Testing

The instrument was tested to the requirements for laboratory locations.

Emission Testing

The instrument fulfills the limit requirements for Class A equipment but does not fulfill the limit requirements for Class B equipment. The instrument was not designated to be used in domestic establishments.

Page 4 Nano ITC Getting Started Guide

Low Voltage Directive (Safety)

In order to comply with the European Low Voltage Directive (2006/95/EC), this equipment has been designed to meet IEC 1010-1 (EN 61010-1) standards. To comply with requirements in the USA, this instrument has been tested to the requirements of UL61010a-1.

WARNING: If this instrument is used in a manner not intended or specified in this manual, the protection provided by the instrument may be impaired.

DANGER: High voltages are present in this instrument. Maintenance and repair of internal parts must be performed only by TA Instruments or other qualified service personnel.

Safety

High voltages are present in this instrument. Maintenance and repair of internal parts must be performed only by TA Instruments or other qualified service personnel.

Electrical Safety

You must unplug the instrument before doing any maintenance or repair work; voltages as high as 125/250 VAC are present in this system.

Lifting the Instrument

The Nano ITC is not a portable instrument. In order to avoid injury, particularly to the back, please follow this advice:

WARNING: Use appropriate care when unpacking or moving the instrument. It may be too heavy for some individuals working alone to handle safely.

Nano ITC Getting Started Guide Page 5

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Table of Contents

Introduction ......................................................................................................................................... 3

Important: TA Instruments Manual Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Notes, Cautions, and Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Regulatory Compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Safety Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Lifting the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Chapter 1:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Instrument Models Covered in this Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

The Nano Isothermal Titration Calorimeter (ITC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Measuring Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Reaction Vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Syringe/Stirrer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Buret Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Options and Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Instrument Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

Chapter 2:

Unpacking/Repacking the Nano ITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

Introducing the Nano ITC .............................................................................................. 9

Batch/Incremental Titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

Titration/Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Calculation of Equilibrium Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Installing the Nano ITC ................................................................................................ 17

Installing the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Inspecting the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

Choosing a Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

On . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

Near . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

Away from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

Power Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

Setting Up the Nano ITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

Connecting the Cables and Cords . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Starting the Nano ITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Shutting Down the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Nano ITC Getting Started Guide Page 7

Chapter 3:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

Calibrating Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Heat of Protonation of Tris Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Experiment Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Preparing and Degassing the Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Degassing Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

Preparing the Sample and Reference Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28

Loading the Buret . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

Installing the Buret Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31

Starting the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Cleaning the Nano ITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33

Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36

Maintaining the Nano ITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37

Purging the Nano ITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Use, Maintenance, & Diagnostics ................................................................................ 23

Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Experiment Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Troubleshooting the Nano ITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

Minimizing Blank Corrections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

Operating at Non-Ambient Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Stirring Speeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

Stable Instrument Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

Appendix A:

Index ................................................................................................................................................... 43

Buret Position Functions ........................................................................................... 41

Page 8 Nano ITC Getting Started Guide

Chapter: 1

Introducing the Nano ITC

Overview

There are three ways in which a calorimeter may be designed. Heat measurements may be based on the following:

• A temperature rise measured in a system of known heat capacity, (ΔT)

• The measured change in power (typically resistance heating) required to maintain a system at a constant temperature (power compensation), and

• A direct measure of the heat flowing between the system and large heat sink maintained at a constant temperature (heat flow)

Each method (ΔT, power compensation, and heat flow) has its advantages and disadvantages. The TA Instruments Nano Isothermal Titration Calorimeter (ITC) uses a differential power compensation design for maximum sensitivity and responsiveness.

The Nano ITC is available in three configurations: the Standard Volume model with 1-mL measurement

cells made of either 24K gold or Hastelloy

Standard Volume ITCs shipped before September 2009 were labeled “Nano ITC incorporates second generation technology featuring enhanced baseline stability and increased sensitivity.

alloy, and the Low Volume model with 190-µL gold cells.

”. The Nano ITC

Instrument Models Covered in this Guide

This guide covers Nano ITC instrument models 5300, 5301, and 5302.

Nano ITC Getting Started Guide Page 9

The Nano Isothermal Titration Calorimeter (ITC)

Buret assembly

The Nano ITC (shown in the figure below) consists of the measuring unit (calorimeter block and two nonremovable reaction vessels), the buret assembly, which includes the stirring system, and a cleaning accessory. With the exception of the power on/off switch located on the back of the calorimeter unit, all functions of the Nano ITC are controlled remotely by the computer through the USB connection.

Figure 1 Nano ITC

Applications

Batch/Incremental Titration

In incremental or continuous titration, one of the reactants is placed in a syringe or buret external to the reaction vessel. If individual, repeated injections are made, incremental titration takes place (as seen in the example below); if only one injection is made, it is continuous injection calorimetry.

Figure 2 Incremental titrations.

Page 10 Nano ITC Getting Started Guide

The baseline data, i.e. heat flow in the regions before and after each titrant pulse, shows the power required

to maintain a zero temperature difference between the sample and reference cells.

The baseline in this region is a function of heating by stirring. The baseline is used to calculate the area or the heat from each pulse in the reaction vessel during the titration or batch reaction. The thermogram constructed from the integrated peak areas is then used for data analysis.

Titration/Data Analysis

A single titration calorimetric experiment yields heat data as a function of the ratio of the concentrations of the reactants. Titration data, in the form of heat change versus volume of titrant added, can be examined for both analytical (thermometric titrimetry) and thermodynamic (titration calorimetry) information.

Other corrections must be made to the heat data to account for heat effects associated with titrant dilution and any temperature difference between titrant and titrate solutions. These corrections are most easily accomplished by performing a blank titration experiment and subtracting the blank heat data from the experimental thermogram.

In the case of quantitative reaction of added titrant, the analysis of the thermogram is quite simple. All peak areas will be the same (with the possible exception of the last peak) and ΔH calculated from the incremental heat and the number of moles of titrant added per increment. The titrant concentration is calculated from the total heat divided by the ΔH for the reaction.

Calculation of Equilibrium Constants

The equilibrium constant for a given reaction may be simultaneously determined with the enthalpy change, if the magnitudes of K and ΔH for the overall reaction taking place in the calorimeter are within certain limits. The family of curves presented in the figure below shows that increased overall curvature of the thermogram is generated with decreasing values of the association constant, K

Figure A below shows the effects of varying magnitudes of the enthalpy change ΔH. Figure B shows the effects of varying the equilibrium constant K.

Figure 3 Calculation of equilibrium constants.

Nano ITC Getting Started Guide Page 11

System Components

TED-controlled block

Thermal shield

Reference cell

Sample cell

Control heater

• Nano Isothermal Titration Calorimeter

• Personal computer (optionally available from TA Instruments)

• ITCRun and NanoAnalyze software

• Power cord

• Getting Started Guide (this manual)

• Data Collection and Analysis Software

• 1 each 2.5-mL filling syringe with 16-gauge, 8-inch long needle (Standard Volume ITC)

• 1 each 0.5-mL filling syringe with 16-gauge, 8-inch long needle (Low Volume ITC)

• 1 each 100-µL and 250-µL syringes (with Nano ITC Standard Volume)

• 1 each 50-µL syringe (with Nano ITC Low Volume)

• 1 each buret drive

• USB cable

The components that make up the Nano ITC system are briefly described in the following sections.

Measuring Unit

The measuring unit includes the calorimeter block and two non-removable reaction vessels (sample and reference cells). Access tubes extend downward from inside the buret mounting cavity on the top of the calorimeter. The access tubes serve as conduits for the filling syringe, titrant delivery, and reference needle. They also provide for titrant equilibration and as a thermal barrier to the environment outside the calorimeter.

Figure 4 Nano ITC measuring unit.

The Nano ITC utilizes a differential power compensation design. Semiconducting thermoelectric devices (TED) are used for temperature control and to detect temperature differences between the sample and

Page 12 Nano ITC Getting Started Guide

reference cells. A proportional/integral/derivative (PID) control loop uses a control heater on the sample

Platinum

Access Tube

Teflon Bushings on Needle to Dampen Stirring Noise

24K Gold Reaction Vessel

Titrant Delivery Needle

Stir Paddle

Titrant Exit Point

cell to maintain a zero temperature difference between the sample and reference cells. The power required to maintain this zero difference is used as the calorimeter signal and is monitored as a function of time. If a reaction that produces heat occurs in the sample cell, the heat required to maintain the zero difference decreases by the amount of heat supplied by the reaction, resulting in a peak in the thermogram.

A calibration heater located on the outside of the sample cell is used to provide precisely controlled heat pulses for electrical calibrations, and to verify instrument performance.

The entire measuring unit is encased within an insulated air-tight canister which has been purged on a vacuum pump and filled with dry nitrogen at the factory. This is to prevent possible condensation and evaporation of moisture around the unit which would create excessive baseline noise.

CAUTION: The purge port valve on the back of the Nano ITC should remain in the closed position at all times to maintain the integrity of the nitrogen purge.

NOTE: Purging of the canister is not a routine maintenance operation; contact TA Instruments before proceeding.

Reaction Vessel

The calorimeter uses two matched reaction vessels with options of 1-mL gold, 190-µL gold, or

1-mL Hastelloy match as closely as possible the thermal properties of the sample cell. Accordingly, a reference needle is placed inside the reference cell during operation to correspond to the titrant needle in the sample cell.

. The vessels are accessed through platinum tubes. The reference cell is constructed to

Figure 5 Sample cell assembly.

CAUTION: Extreme care should be taken not to bend the syringe needle, because this would impair proper stirring and possibly damage the reaction vessel.

Nano ITC Getting Started Guide Page 13

Syringe/Stirrer

Plunger position indicator

Buret handle

Syringe plunger

Rotating buret shaft

Graduated syringe barrel

Knurled syringe mounting knob

Reference cell with reference needle

Sample cell with syringe needle/stirrer

Nano ITC Standard Volume systems include two syringes of 100 and 250 µL capacities. Two buret syringes are provided with 100 µL and 250 µL capacities. The only difference in dimension between the two is the inner diameter of the syringe barrel; the needles are identical in order to maintain the thermal and mechanical properties.

The Nano ITC Low Volume system includes one 50-µL syringe. This syringe uses a shorter needle. To avoid possible damage, do not use the larger syringes with the Low Volume ITC. If you feel a stiff resistance or if the buret handle does not readily slip fully into place in the ITC, do not force it. Remove the buret and check to see if the correct size syringe is installed. You can verify the feel of the normal resistance by installing a buret with no syringe in place.

The titrant syringe needle also functions as the stirrer and extends down into the reaction vessel from the top when the buret is mounted. The needle is balanced for optimum stirring efficiency. It has two Teflon bushings to help dampen stirring noise and ensure that the needle spins true within the cell access tube (see the figure below).

Figure 6 Orientation of buret, syringe, needles, and cells during experiments.

CAUTION: To avoid possible damage, do not use the 100 or 250 µL syringes with the Nano ITC Low Volume instrument. Syringes are shipped with warning labels which may, if desired, be affixed to the ITC by the customer to serve as a reminder.

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