Troubleshooting guide for Isothermal Titration Calorimetry
Contents
1. O e 6f AH 10000 cal mol S o 8 O n 1 00 ja 40 Pri ioe k 5 uM 12 L Figure 6 Simulated titration curve of macromolecule M with ligand L Parameters used in the simulation are indicated in the figure The dilution heat is set to 0 The non zero heat at the end of experiment is due to the decreasing concentration of macromolecule in the cell Page 7 Created 14 07 2011 Updated 11 08 20112. or the major equilibration baseline is shifted both ITC and DSC The former is easily resolved by decreasing concentrations of the reactants while the latter is often caused by dirty cells Clean the calorimeter once again and if the problem persists check the reference cell Sometimes when the heat capacities of the experimental and the reference chambers differ considerably it causes a shift in the equilibration baseline level and thus affects the available dynamic range This happens often when the content of the reference cell dries out gets contaminated or when you re working with concentrated solutions of denaturants Arbitrary value uJ s or ucal s 0 5 10 15 20 25 Time min Figure 2 The signal is out of dynamic range Page d Created 14 07 2011 Updated 11 08 2011 Experiment won t start even though the baseline is flat This problem occurs only on MicroCal machines with an automatic experiment initiation where the instrument evaluates the baseline quality and starts an experiment by itself without intervention from a user encountered this problem very often when working at higher temperatures above 37 C When tired of waiting check the baseline quality remember to zoom in and if you think that the baseline is flat enough go to VPViewer window Find the button named DP and click twice on the red number displayed beneath the DP This will force the program to the next stage in its schedu
3. walls of the reaction cell upon rotation Often when you are under time pressure it is easy to overlook both the t provided that it is an equilibrium reaction and that the next injection is not initiated prematurely Page 2 Created 14 07 2011 Updated 11 08 2011 elevated noise level and the unsteady baseline If in spite of your suspicion you decide to ignore these indications and start the experiment anyway you will observe some weird looking injections Sometimes the signal will oscillate up and down only slowly settling back to the pre injection level Other times the baseline will drift strongly up or down behaving unpredictably To check if the syringe is straight put it on a flat surface e g at the edge of a table Rotate it slowly along the surface watching the distance between the needle and the table If syringe is bent you will clearly see it Then contact your ITC company Large shift of baseline after initiation of stirring If the baseline is perfect before the stirring is on and when you turn it on the baseline goes out of range or never returns to the usual level you definitely got a bent syringe Instrument specific problems The baseline is completely flat no noise The signal is out of dynamic range VP ITC Flat tops of peaks This problem is illustrated in Fig 2 It is specific to MicroCal Instruments both VP ITC and VP DSC and it indicates that either the injection heat is too large for ITC
4. K It s always a good idea to make a reference titration at the beginning of the binding experiment or a batch of experiments Make sure to repeat it again if you change temperature or solution conditions However often the heat produced in the reference titration is not the same as the one you obtain at the very end of your binding experiment In other words at the end of the titration when the reaction is complete and only the dilution heat is expected the obtained heats are different from those measured in the reference titration This phenomenon has two explanations a Heat of dilution of a ligand in buffer solution is not the same as the dilution of this ligand in a solution of the macromolecule b Upon each injection some of the cell content is being pushed out thus decreasing the concentration of the macromolecule originally loaded in the cell Therefore at the end of the titration one has a decreasing concentration of macromolecule although the concentration of the ligand continues to increase This leads to a shift in equilibrium between the components with the consequent dissociation of the ligand Fig 6 shows a simulated titration where the reference heat is set to zero Nevertheless the heat at the end of the experiment is not zero and has a sign opposite to the binding heat Fig 6 2 00000000 Q pt ee PO M cell 0 01 mM 5 5 10 9 15 20 25 ifevindes sa aa N Injection number Vcell 1 0 mL go 4 Vinj 10 yL
5. Troubleshooting guide for Isothermal Titration Calorimetry M R Kasimova This is a very short summary of common problems encountered with ITC equipment and experiments It is based on my personal experience of working with Omega and VP ITC from MicroCal as well as Nano ITC from TA Instruments The first one although a very reliable machine is probably extinct so the following troubleshooting guide is intended mostly for the users of VP ITC and Nano ITC This guide is neither complete nor intended as a substitution for the manuals provided with the ITC machines It to help those students who after wasting a whole day on trying to make an experiment work still cannot figure out what s going on with the baseline and why Also often encountered insufficiency of the assistance available through the producers of calorimetry equipment because the technical personnel is not particularly trained to help with practical problems simply because these people have never done a real life ITC experiment themselves So instead of wasting your time discussing bubbles and the importance of degassing your sample with the producer look through the subtitles below and see if you can find the one describing your specific case Experimental setup equilibration time stirring rate etc Most thermodynamically controlled reactions under typical experimental conditions occur rather fast This means that allowing 5 minutes between injections should be su
6. al binding reactions and if this is the problem the post injectional baseline will have a long sloping tail which can go on for tens of minutes without leveling out Fig 3 In that case check for degradation products or measure the size distribution of your sample DLS HPLC Spectrophotometry before and after the titration Aggregation of the sample can sometimes be avoided by changing the solution conditions pH or ionic strength or by decreasing concentrations of the reactants If you re working with liposomes the tail can be also due to liposome fusion or leakage Why do I not get anything that looks remotely like a titration curve Arbitrary value J s or ucal s 20 40 60 80 100 Time min Figure 4 Comparison of a perfect titration A to an unfortunate one B If instead of a classic titration Fig 4A you get something like the drawing on the right hand side Fig 4B you should re examine your experimental design or check the concentrations In this particular example all of the cell content is saturated in the first injection Fig 4B which means that you either have to increase the concentration in the cell or decrease the one in the syringe It is useful to analyze the heat produced by the first injection to evaluate the enthalpy of binding This will help you to decide which way the concentrations should be adjusted to receive a reasonable heat effect The same explanation is also valid for the case when all in
7. es is often induced upon the contact of a cold sample just from the fridge and the warm cell injector walls Also if experiments are done at the temperatures much higher or lower than 25 C it is beneficial to equilibrate your sample at the experimental temperature before loading it into the cell or the injector Overshoot to the opposite side Two simultaneous reactions with the opposite heat effect General advice X You can learn quite a lot from reproducing your experiment in a glass test tube while observing the progress of the titration through the transparent wall Let s say you want to titrate a peptide solution with liposomes The experiment must be performed without stirring because the peptide fibrillates upon agitation In the real ITC experiment the V 1 mL and Vinj 10 uL In your simulated experiment you can use the same volumes or you can scale Page 6 Created 14 07 2011 Updated 11 08 2011 them up according to your test tube size for example Vee test 5 ML and Vinj test 50 UL Typically a liposome suspension is opaque thus allowing you to observe the progress of its diffusion through the test tube upon each addition Remember that in the real ITC cell the tip of the injection syringe is located close to the bottom of the cell Now you can answer at least two questions a how fast is the suspension mixed with the rest of the solution b does addition of liposomes induce aggregation of the peptide
8. fficient in most cases and an output of your experiment should look like the one presented in Fig 1A It is very important that the level of the baseline after each injection is the same as before If this condition is not fulfilled and the onset of the following injection occurs before the previous one is over Fig 1B you should check the following Arbitrary value uJ s or ucal s 0 5 10 15 20 25 0 5 10 15 20 25 Time min Time min Figure 1 Illustration of sufficient equilibration time A and insufficient one B Page 1 Created 14 07 2011 Updated 11 08 2011 Is the stirring on The stirring of the injection cell is not the most crucial parameter of the experimental setup However it defines how fast the titrant diffuses through the volume of the cell and reaches the titrate consequently enabling the reaction The stirring rate will not affect the total heat measured over the course of an injection but it can affect the injection shape as slower diffusion will prolong the reaction period and thus make the injection profile wider Therefore this parameter is more important when the titrant is a bulky macromolecule or particles for which the diffusion rate is slower A typical stirring rate for protein peptide solutions is between 200 and 300 rpm Sometimes it is desirable to avoid the stirring all together In that case try to increase the interval between the injections to 10 15 min If it still does not help impro
9. jections are of the same size but not the same as the dilution heat Shifts of baseline When the post injectional baseline is flat and horizontal but does not reach the same level as before see Fig 5 there can be several reasons for that Most probably the binding reaction is associated with the dramatic changes in the heat capacity of the system For Page 9 Created 14 07 2011 Updated 11 08 2011 example the baseline shift can be due to the occurrence of an enzymatic reaction see for example Todd amp Gomez Analytical Biochemistry 2001 296 179 187 Another example describe by Myers et al Biochemistry 1987 26 4309 4315 is the baseline shift associated with liposome fusion Figure 5 Illustration of the baseline shift upon injection Bubbles These look like sharp spikes which can appear anywhere in the titration profile If the bubbles are injected with the titrant from the syringe the injection itself will be affected and the height of the neighboring injections can be strikingly different However this is easy to check by taking the syringe out for close inspection On the contrary a visual inspection of the bubbles formed on the walls of the experimental cell is rather difficult In this case their release can be either initiated by injection or happen spontaneously at any time during the experiment Assuming that you do degas your sample remember to pay attention to its temperature The formation of the bubbl
10. le Experiment is running but no heat is visible not even the dilution heat A problem specific to some Nano ITC instruments is a mechanical glitch associated with the automatic injection system Sometimes the plunger of the injector gets stuck and although it appears that the experiment is running no heat is evolving the baseline stays completely flat If so remove the syringe and check if the plunger is moving when you click up or down buttons in the operation menu Also see a section of the same title in Experimental setup above Sample related problems There is an ocean of things that can go wrong if you re working with a system which has not been described before In addition since you do not know what to expect from your titration it s hard to decide what is causing a problem are the solutions too diluted Is there binding at all And so on Although it is hard to describe each individual case there are some problems that occur rather often and these are described below Slow non equilibrium processes Figure 3 An example of injection profiles affected by slow aggregation processes Page 4 Created 14 07 2011 Updated 11 08 2011 Experimental heat measured by a calorimeter is not necessarily produced by the reaction under study Other processes such as degradation of reactants aggregation fibrillation and so on can contribute as well These processes are rather slow on the time scale of typic
11. ving the baseline between two consecutive injections your problem is most likely due to something else see Sample related problems Is the heat produced by an injection too large It is possible that the concentrations of the reactants are too high and it takes longer than 5 minutes for the system to return to equilibrium This is not so easy to check because most of the manuals do not specify the upper limit of the injection heat However usually avoid having the injection heat above 500 uJ This will also save you some of your precious reactant material Sometimes this problem can also be identified by the cut off tops of the reaction peaks see Instrument specific problems Experiment is running but no heat is visible not even the dilution heat Check if the syringe is clogged or stopped It can happen if you waited too long between loading it and inserting it into the injection chamber Sometimes the liquid on the tip dries out forming a hard to break film Clogging can also happen when the syringe is loaded with aggregating protein or synthetic particles which increase the friction between the plunger and the inner wall of the syringe Try to push the plunger by hand to make sure it s moving freely Another thing to check is whether the injector mechanism operates properly see Instrument specific problems Large baseline noise unsteady baseline These problems can be caused by a bent syringe which scraps the
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