Modulated differential scanning calorimeter solvent loss calibration apparatus and method

A modulated differential scanning calorimeter that accounts for heat flow due to evaporative solvent loss. The calorimeter modulates the temperature applied to a sample and a reference to determine the amount of heat flow that is due to evaporation. By calculating the amount of heat flow due to evaporation, the user can determine how much of the heat flow of any given well is due to the process of interest as opposed to evaporation.

BACKGROUND

1. Field of the Invention

The present invention relates to thermal analysis instruments and methods of using the same. More particularly, embodiments of the present invention relate to using modulated temperature differential scanning calorimetry to account for solvent loss during analyses conducted using open calorimeters.

2. Background of the Invention

Differential scanning calorimeters (DSCs) are used to determine physical properties of a sample by analyzing the sample's response to an applied temperature program. In operation, a DSC measures a temperature differential between a reference and a sample being analyzed in the presence of a temperature profile applied to both the reference and the sample. Using the temperature differential, certain properties of the sample being analyzed can be determined.

Two general types of temperature profiles can be used during DSC experiments. One type of temperature program is linear. The second type of temperature programs, commonly referred to as a modulate temperature program, adds a modulation to a linear temperature program. DSCs using the second type of temperature program are commonly referred to as modulated temperature DSCs. Modulated temperature DSCs are described in U.S. Pat. Nos. 5,224,775 and 6,561,692, both of which are incorporated by reference herein in their entireties.

DSC experiments can be performed using open or closed calorimeters. Open calorimeters are those that have an open sample cell. An open sample cell allows reagents to be easily added to calorimeter cells using automated pipettes. Generally, open DSCs have multiple calorimeter cells arranged in an array. In this manner, many experiments can be run in parallel. Because of the ease with which reagents can be added to calorimeter cells and the array arrangement, open calorimeters are useful in high-throughput applications.

Open DSC experiments typically involve placing a sample in a sample cell in the array, and then adding one or more reagents to the sample. For example, a number of different types of protein can be placed in an array of calorimeter cells as suspensions in water using an array of automated pipettes under robotic control. A controlled amount of ligand solution is added to each cell using automated pipettes under robotic control. By monitoring power signals from the calorimeter cells in response to an applied temperature profile, the interaction of characteristics of the various protein-ligand pairs can be rapidly assessed. This multi-sample cell architecture allows many experiments to be carried out in a rapid and efficient manner.

There is a serious disadvantage to using an open calorimeter arrangement in DSC experiments. This disadvantage is due to unknown solvent loss, which adds an uncontrolled contribution to the calorimeter measurement signal, and as a result, injects uncertainty in the calorimeter measurement.

One potential solution to this problem is to perform a differential experiment using a sample containing protein-ligand pairs, and a reference containing a same volume of solvent only, and assuming that the solvent loss in both the sample and reference cells is the same. In that way, simple subtraction could remove the unknown contribution due to solvent loss. Unfortunately, this does not solve the problem because the presence of the sample can change the rate of solvent evaporation in the sample relative to the reference. Thus, a differential arrangement with subtraction alone does not solve the problem. Further complicating the issue is that uncontrolled contribution to the heat flow caused by evaporation makes assessing enthalpies difficult because selecting the correct baseline is problematic. Similarly, when examining kinetics, the heat flow contribution from the process of interest cannot be disentangled from the heat flow contribution from solvent loss.

SUMMARY OF THE INVENTION

Embodiments of the present invention account for heat flow due to solvent loss in differential scanning calorimetry experiments. For example, differential scanning calorimeters according to embodiments of the present invention will produce a substantially better baseline from which the true thermal characteristics of the sample well may be deduced.

One embodiment of the present invention is a differential scanning calorimeter (DSC) having a sample DSC cell containing a sample comprising a solvent and a specimen to be analyzed and a reference DSC cell containing a reference solution or containing no sample. The DSC of the first embodiment also includes a first heating element for applying a modulated temperature program to the sample and reference. Further, the DSC of the first embodiment includes a first sensor for measuring a temperature of the sample and a second sensor for measuring a temperature of the reference. In addition, the first embodiment includes a computer for capturing the measured temperatures and calculating a heat capacity of the sample due to the applied modulated temperature; and to determine the amount of heat flow attributable to the evaporation of solvent from the sample DSC cell.

Another embodiment of the present invention is a differential scanning calorimeter (DSC) that includes an insulated enclosed housing unit, an array comprising a multiplicity of cells to hold one or more samples and one or more references, one or more sensors for measuring the base temperature of the sensor and the temperatures in the various cells, a heating device for exposing the samples and references to a modulated temperature program, and a computer to use a signal received from the DSC cells to calculate heat capacity independently of any kinetic process that is occurring, and to determine what portion of the received signal is due to solvent loss.

Another embodiment of the present invention is a method for accounting for solvent loss in a differential scanning calorimeter (DSC), including depositing a sample comprising a solvent and a specimen to be analyzed in a sample DSC cell, depositing solvent in a reference DSC cell, heating the sample well using a first heating element according to a modulated temperature profile, heating the reference cell using a second heating element according to the modulated temperature profile, calculating how heat flow due to solvent loss is related to changes in heat capacity as measured from the modulation from the reference cell, and using this information to calculate a baseline due to solvent evaporation from the sample DSC cell, and adjusting a DSC heat flow measurement due to the sample in accordance with the calculated baseline.

Another embodiment of the present invention is a method for accounting for evaporation loss from a solution in a sample DSC, including calibrating the DSC cell to determine the heat flow caused by solvent loss in a prior experiment using only solvent, and then subtracting from the signal from the experiment with a sample the component of heat flow due to solvent loss, where the rate of solvent loss is determined by measuring the change in heat capacity of the solution from the amplitude of the modulation, and adjusting the measured heat flow in accordance with the determined relationship between the heat flow, the solvent loss and the heat capacity as measured by the modulation.

DETAILED DESCRIPTION

A DSC according to one embodiment of the present invention employs temperature modulation in an open DSC to ascertain what portion of the heat flow in a given cell is due to evaporation or loss of solvent. By calculating what portion of the heat flow is due to uncontrolled solvent evaporation the part of the heat flow that derives from other processes (typically the subject of interest) can be more accurately measured. Once the amount of heat flow due to evaporation of solvent of both the reference and sample cells is accounted for, the user may then gain a more precise understanding of the heat flow due to the thermal event being measured. The terms “temperature modulation DSC,” “temperature modulated DSC,” “modulated DSC,” and “modulation DSC” are used interchangeably herein to refer to a DSC experiment, method or apparatus that involves superimposing a periodically varying temperature perturbation during sample heat treatment, such as a linearly increasing sample temperature. Thus, a temperature modulated DSC curve may be a curve having a sinusoidal variation in temperature whose average temperature is increasing with time.

In a typical DSC, there are one or more cells, or wells, in which reagents or materials of interest can be deposited. The terms “cells” and “wells” are used interchangeably in the present specification. In an open cell DSC device, each cell is uncovered. The cells may be configured as part of a larger tray that contains many such open cells in an array. An exemplary such array is a microplate available from Vivactis of Leuven, Belgium. An open cell design allows for rapid and convenient addition of materials into the cells. For example, computers, robots, or other automated material deposition structures can deposit materials into the cells in a high throughput manner via, for example, automated pipettes. Using such automated techniques, an open DSC design array allows for a relatively high number of experiments to be run in parallel, while reducing the costs associated with multiple experiments, or the human labor required for manual deposition. In addition, the open cell design allows for greater flexibility of experimental design due to the multiplicity of possible cell configurations. Manual deposition may still be used within the spirit and scope of the present invention.

Individual cells in a DSC may be of any appropriate size, including so called “micro” or “nano” cells. In addition, the array may include any number of cells, and a single array may employ cells of varying size. In one embodiment of the present invention, each cell is capable of being heated individually by activation of any number of appropriate heating devices. For example, each cell may have its own individual heating element. These individual heating elements may be part of the array of cells, or may be part of a housing unit that houses the array of cells during the experiment. In another embodiment, there may be fewer than one heating element per cell, as each heating element may be capable of introducing a modulated temperature signal to more than one well. To ensure accurate measurements, the heating elements can be calibrated using calibration standards prior to use for analyses in a well-known manner.

FIG. 1Ais a schematic diagram illustrating a DSC according to an embodiment of the present invention. Typically, a DSC according to one embodiment of the present invention includes a housing unit102. In one embodiment of the present invention, housing unit102is an insulated, enclosed structure, similar to an oven, that is capable of housing a DSC cell array103, while a temperature program is applied to one or more DSC cells. For example, inFIG. 1, DSC cells104a,104b,104c, and104dare shown. Heating can be applied to the DSC cells in a number of ways. For example, in one embodiment of the present invention, each DSC cell has a corresponding heating element that can apply heating according to a modulated temperature profile to the DSC cell. For example, as shown inFIG. 1, heating element106asupplies heat to DSC cell104a, heating element106bsupplies heat to DSC cell104b, heating element106csupplies heat to DSC cell104c, and heating element106dsupplies heat to DSC cell104d.

Generally, in DSC experiments the DSC cells are paired. In each pair of DSC cells, one cell is used for a reference, and the other cell is used for the sample being analyzed. For example, inFIG. 1A, DSC cell104amay be paired with DSC cell104b. DSC cell104acan be used for a sample, and DSC cell104bcan be used for a corresponding reference. Similarly, DSC cell104cmay be paired with DSC cell104d. DSC cell104ccan be used for a sample, and DSC cell104dcan be used for a corresponding reference.

Operation of the DSC is controlled using a computer110. Computer110can be any well-known personal computer, microprocessor, sequencer or other computing device that can be programmed to control heaters, cause data to be captured from sensors and stored, and control automatic filling of DSC cells. Such computers are well-known.

FIG. 1Bis a schematic diagram illustrating automatic filling of DSC cells using pipettes. Pipettes150a,150b,150c, and150dare used to fill respective DSC cells152a,152b,152c, and152dautomatically. Pipettes150a,150b,150c, and150dare controlled by a computer, such as computer110. In operation, the pipettes are manually or automatically (under computer control) filled with a sample or reference as desired. Under computer control, the pipettes are positioned over the DSC cell array. Computer110then causes the pipettes to empty their contents into the DC cells over which they are positioned.

In the example shown inFIG. 1B, the DSC cells are configured as sample reference pairs. Pipette150ais positioned over a DSC cell152aand fills DSC cell152awith a sample153a(containing, for example, solvent and protein-ligand to be studied). A heater154aheats sample153aaccording to a temperature profile under control of computer110. A sensor156asenses the temperature of the sample153aand provides the sensed absolute temperature or a differential temperature (in accordance with sensor156b) to computer110.

A DSC cell152bis used as the reference corresponding to the sample in DSC cell152a. Pipette150bis positioned over DSC cell152band fills DSC cell152bwith a reference153b(containing, for example, solvent only). A heater154bheats reference153baccording to a temperature profile under control of computer110. A sensor156bsenses the temperature of the reference153band provides the sensed absolute temperature or a differential temperature (in accordance with sensor156a) to computer110.

Pipette150cis positioned over a DSC cell152cand fills DSC cell152cwith a sample153c(containing, for example, solvent and protein-ligand to be studied). A heater154cheats sample153caccording to a temperature profile under control of computer110. A sensor156csenses the temperature of the sample153cand provides the sensed absolute temperature or a differential temperature (in accordance with sensor156d) to computer110.

A DSC cell152dis used as the reference corresponding to the sample in DSC cell152c. Pipette150dis positioned over a DSC cell152dand fills DSC cell152dwith a reference153d(containing, for example, solvent only). A heater154dheats reference153daccording to a temperature profile under control of computer110. A sensor156dsenses the temperature of the reference153dand provides the sensed absolute temperature or a differential temperature (in accordance with sensor156c) to computer110.

In another embodiment of the present invention, the housing unit has an oven that can be controlled to heat the inside of housing unit102, for example, heater114, according to a modulated temperature program. Such heating will cause each of the DSC elements to experience substantially the same modulated temperature program.

In another embodiment of the present invention, the housing unit can have a heating element, such as heater114, that can provide overall heating to housing unit102, and each of the DSC cells can have corresponding heating elements to heat their corresponding DSC cells, for example, heaters106a,106b,106c, and106d. In one such embodiment, heating element114is controlled by computer110to heat the housing unit according to a linear temperature program, and each of the DSC heating elements106a,106b,106c, and106dis controlled by computer110to heat their corresponding DSC cells according to an oscillating temperature program. In this manner, each of the DSC cells is effectively heated according to a modulated temperature program.

The heating elements are coupled to a computer, such as computer110, through an input/output (I/O) port, for example I/O port112. Computer110is programmed using a temperature program to control the heaters to expose samples and references to temperature profiles, such as modulated temperature profiles, as required. The heating elements can be those typically used in DSC instruments, including for example, triac heaters or resistive heaters.

The DSCs of embodiments of the present invention can be either heat-flux DSCs or power compensation DSCs. In heat flux DSCs, the temperature of the reference and sample DSC cells is measured and used to determine heat flow. The heat capacity can be derived from the measured heat flow. In power compensation DSCs, the differential power required to maintain a constant temperature of the sample and reference DSC cells is measured. The differential power is a direct measure of the heat capacity of the sample.

Each DSC cell also has a corresponding sensor. For example, as shown inFIG. 1, sensor108acorresponds to DSC cell104a, sensor108bcorresponds to DSC cell104b, sensor108ccorresponds to DSC cell104c, and sensor108dcorresponds to DSC cell104d. The sensors can be any sensor used in typical DSC analyses, including for example, thermocouples. As is well known, some DSC beaters can perform the sensor function as well as heat the DSC according to a temperature program. Each sensor is coupled through an I/O board to computer110. Computer110captures and stores the data sensed by the sensors for further analysis as described herein.

The measurements resulting from applying a modulated temperature program to the sample and reference DSC cells are used to measure heat capacities of each sample and reference independently of any kinetic process that may otherwise be occurring. The rate of change of heat capacity (i.e., the derivative of heat capacity) can be used to measure the rate of solvent loss from a DSC cell. From the enthalpy of evaporation of the solvent, the heat flow due to evaporation can be calculated from the rate of change of heat capacity.

FIG. 2is a graph illustrating an exemplary measurement curve204from a DSC experiment according to an embodiment of the present invention. Using the derivative of the heat capacity measured during a calibration run (described below), a “true baseline” curve201is calculated. Curve201represents an exemplary heat flow due to evaporation of solvent. The desired response is the difference between the measured signal and the calculated baseline (i.e., curve204minus curve201). An additional piece of information, the time of the addition of reagent can also be determined as point206by comparing measurement curve204baseline curve201.

“True baseline” curve201can be determined through a calibration procedure according to an embodiment of the present invention during which the DSC is operated using just solvent.FIG. 3is a graphical representation of several curves used in calculating a calibration constant according to one embodiment of the present invention. Curve304is an exemplary heat capacity measured by applying a modulated temperature profile to a DSC cell containing only solvent. The heat capacity measured in such an experiment is a direct measure of the amount of solvent in the cell. As a result the change in heat capacity with respect to time (i.e., the derivate of the measured heat capacity) represents the rate of solvent loss (e.g., evaporation) from the DC cell. Curve305is the derivative of measured heat capacity curve304according to an embodiment of the present invention. This curve is the same as the power curve306(dotted) due to solvent loss multiplied by some constant. As a result, when multiplied by an appropriate calibration constant, the derivative of heat capacity (i.e., the rate of solvent loss) overlays the power curve due to solvent loss. This product (derivative of heat capacity times a calibration constant) is illustrated by curve308(dashed), which overlays the power curve due to solvent loss, curve306. The power curve due to solvent loss is the total signal in an isothermal experiment or the non-reversing heat flow in a scanning experiment.

FIG. 4is a flow chart for determining the calibration constant according to an embodiment of the present invention. In step402, a DSC cell is filled with solvent only. In step404, a modulated temperature program is applied to the DSC cell containing solvent only and a measurement signal collected. In step406, the heat capacity is determined from the measurement signal. In a modulation DSC experiment, the measurement signal can be deconvoluted to produce reversing heat capacity, among other quantities.

In step408, the derivative of the reversing heat capacity is calculated.

In step409, the curve representing the power due to solvent loss is calculated. The power due to solvent loss is the total signal in an isothermal experiment or the non-reversing heat flow in a scanning experiment. The non-reversing heat flow is also obtainable by measuring the heat flow signal of the solvent in a modulation DSC experiment and deconvoluting the signal.

In step410, the constant required to cause the calculated derivative of heat capacity to overlay the power due to solvent loss curve is calculated. This can be done by any of a number of curve fitting techniques.

In step412, the determined constant is stored as the calibration constant.

In another embodiment of the invention, the steps illustrated inFIG. 4may be carried out using temperature modulation DSC performed on a solvent in the presence of one or more other materials. However, such a system must produce no other processes during the thermal treatment other than evaporation of the solvent. Accordingly, it is preferable that the method ofFIG. 4be carried out as indicated in step402, in which solvent only is used in the DSC cell.

The calibration constant determined according to the method outlined inFIG. 4is used to calculate a “true baseline” as shown inFIG. 2.

FIG. 5is a flow chart that depicts exemplary steps involved in a method for accounting for solvent loss in a DSC experiment, according to an embodiment of the present invention.

In step502, a DSC experiment is performed by applying a modulated temperature to a sample disposed in solvent to obtain a heat flow signal. In step504, the heat flow signal is deconvoluted to calculate a reversing heat capacity, as well as the total heat flow. In step506, the derivative of the reversing heat capacity is calculated. In step508, the calculated derivative of the reversing heat capacity is multiplied by the calibration constant calculated in step410to derive the heat flow due to solvent loss, which can be used as a true baseline for subtraction from experimental DCS curves. In step510, the derived heat flow due to solvent loss is subtracted from the curves representing total or non-reversing heat flow (which can also be determined from the deconvolution of step504), as appropriate.

In summary, in accordance with embodiments of the present invention, a calibration constant obtained during heating of a pure solvent can be used together with a heat capacity measurement from a temperature modulation DSC experiment of a sample disposed in the solvent, in order to calculate the “true baseline” as shown, for example, inFIG. 2. During an analysis run, the “true baseline” is subtracted from the total (for isothermal experiments) or non-reversing (for scanning or non-isothermal experiments) heat flow as appropriate, in order to determine the heat flow due to the thermal event being analyzed.

The modulated temperature caused by the individual heating element can be of any appropriate type. For example, the modulation a square wave, a sine wave, a triangle wave, a sawtooth wave, any combination of these in a periodic or random manner, or any other appropriate modulated signal. The modulation can be periodic or aperiodic.