Relaxation analysis

Introduction

We will determine relaxation rates in the next part of this tutorial, accordingly we will be working with another experiment series, but this time it will be a T1 relaxation series, which you may have spotted earlier. Here we will be following changes in peak intensity rather than changes in peak position. For such an analysis, where the resonance positions do not change for the various points in the series, so we can use a 3-dimensional experiment (with a single shift list) where all of the planes that correspond to different delay times are combined into one spectrum data set. Within CCPN this type of data set will be referred to as a pseudo-3D experiment; the third dimension is not in PPM, it is delay time and corresponds to the "z" axis - i.e. orthogonal to the X-Y plane that is the screen. We could have used separate 2D experiments in a similar manner to the temperature series if we had that sort of data, although we would probably use a single shift list for all experiments.

Following intensity changes

Firstly look at the T1 rates experiment by opening window5. Switch off all spectra except for the T1 experiment. Note at the bottom of this window is a different kind of scrollbar, made up of buttons. These buttons control which planes of the pseudo-3D experiment are visible on screen. With the left mouse button you can shift the visible planes through the series, and with the middle mouse button (try both left and right buttons together if you don't have a middle button) you can extend the number of visible planes. Try this and switch on all of the planes of the experiment:

Then pick some peaks in these planes by using 'Shift' + 'Ctrl' + Left mouse. Note that you will be picking peak maxima in all planes that are visible. If you didn't want pick in a specific plane, simply ensure that it is switched off.

For fine user control we could pick all the peaks in the T1 series, however, if you toggle on the [HSQC:115] spectrum (use the [Spectra] option at the top of the window), you will see that this assigned HSQC aligns well with the T1 series:

We will use this fact to spread the HSQC assignments to all of the planes of the T1 experiment when we do the relaxation analysis.

Before we do the relaxation analysis go to M: Experiment: NMR Series. Clicking on the "T1" row, you will note that the time values for the series are already entered. This is because the spectrum was loaded from a parameter file (AZARA format in this instance) where the time points of the series are already specified. Anyhow, in the top table set |Condition Type| to "delay time". Before you close the popup make sure that 'Unit' is set to seconds ('s'):

Now select M: Data Analysis: Follow Intensity Changes. You will note that the Experiment series is already correctly selected, by virtue of our selection of delay time as the parameter type. Ensure that the "Reference Peak List" is set to "HSQC:115:1" - the one we have assignments for, and that the Fitting Function is set to "A exp(-Bx)"; i.e. an exponential decay. Set the 1H tolerance to 0.05 and the 15N tolerance to 0.1 - this indicates the size of the region with which analysis will try to pick peaks in the pseudo-3D experiment, based on the locations in the assigned HSQC. Finally, click [Group Peaks]. If some fits fail, just click 'OK'.

As with the temperature series you will see that the assignment has propagated across the whole series and that the selected function/graph has been fitted to the peaks. Although this time is is an exponential function that has been fitted to the peak intensity. Again, clicking on [Show Fit Graph] and clicking on [Next Set] in the Fit Graph popup allows you to quickly investigate the quality of fit for all peaks in each group:

Note that the time constant for each group is recorded in the |TC| column. This is calculated from the inverse of the exponential decay rate; i.e. the "B" in "A exp(-Bx)". You may draw a graph for the T1 values or export the data as text in the same manner as the temperature series, and additionally you can choose to create a dedicated T1 list by pressing [Make T1 List]. This simply records the T1 values (as indicated by the TC column) in a list of NMR measurements that are saved when your CCPN project is saved. Accordingly this data may be looked at at any time in the future (and even deleted) by selecting M: Data Analysis: Measurement Lists and selecting the T1 row.

Previous: Following chemical shifts (2)

Next: Scalar couplings