Time-course and Kinetics Measurement

The simplest method to monitor a reaction (or in general any sample) over a time course is to set up an array experiment. Each of the array elements is an identical repeat of the same experiment. The interval between the array elements can be adjusted and the entire time-course of the reaction or time-dependent event can be tracked by monitoring the peak height, volume or other observables (such as peak positions).

Read details below, but briefly the steps are:

  1. Test a regular, single element experiment to set proper nt and a fixed gain
  2. array pad to set up the series of experiments to build the time course
  3. Type go to start experiment.

Test single experiment: set nt and gain

Set nt

Set gain

Gain value ranges from 0 to 60 on most instruments. It is important to set the proper gain value. If the receiver or ADC overflows anytime duriing the experiment, the data are likely ruined. Too low gain causes signal loss and artifacts. How much gain is needed to fill up ~50% of the dynamic range of the digitizer and ADC board depends on how strong the signals are.

Test gain value with autogain setting:

Alternative to the autogain method, you can also manually adjust gain according to the following two methods. Note that you need to take into account that signal may grow over time, depending on the experiment.

Set up an array of parameter pad

The proper array parameter in this measurement in most cases is pad (pre-acquisition delay). If pad=600 d1=3 nt=64, the experiment waits 600 secs, followed by 64 scans with 3 secs recycle delay between the scans. To check how long the experiment takes, type time.

If pad is arrayed, pad=600,600,600,600, ..... the experiment waits 600 secs (10 mins) before collecting a 64-scan spectrum, then wait another 10 mins, collect another 64-scan spectrum, .... until the last element of pad is done. All spectra are saved into the same experiment (as in a 2D experiment), with multiple elements. See note below on how to accurately calculate the delay time between the data points.

First set up a regular non-array experiment, and test, estimate and set a proper nt for good signal/noise ratio. To set an array, for the example above:

This sets up an array experiment with 50 element repeat of the same experiment with nt scans with 600 secs waiting in between. Type da and click Process->Text output panel to see the arrayed values.

Type go to start experiment.

Check total experiment time

Type time to check total experiment time.

Other parameters to array if non-stop data collection is desired

If waiting between each experiment is not desired, you can array pad but give it the same value as d1. Alternatively, you can simply array d1 with identical values and leave pad with a small value (normally 0 or 1).

This example sets up 50 experiments in an array each with nt scans with no waiting in between the experiment except the recycle delay d1. Each experiment is saved in a separate spectrum in a single FID data file, just like the array for pad.

Type go to start experiment.

Array data processing and display

As the experiment goes, type wft f full aph to transform and auto-phase all. The 1st element is displayed. Make proper phase adjustment, expand region if necessary. Click Process->autoprocess to apply automatic processing (into a vertical stack mode with offset), but sometimes the macro doesn't work well. Alternatively, refer to the following commands for manual processing and array display:

Printing of arrayed spectra

After processing and proper display is made on screen, modify and enter the following commands:

Integrals can be either on or off just as in single spectrum display/printing.

Data analysis

Total delay between data points (or array elements):

In most cases, you want to plot the observables (peak height or volume) as a function of time and perform fitting and further analysis. Except for simple exponentials, this should be done with your own fitting program with the text files generated below.

Peak height is simply the maximum peak height (relative or absolute) for a peak. Peak intensity is an term where there is widespread confusion about what it means and it is misused to refer to peak height in Varian manuals. It should mean peak volume per unit area. I suggest you stay with the term peak height and peak volume (or integral).

To use peak height as an observable (Y-axis) and time course as the variable (X-axis), follow these steps after processing the array:

To use peak volume (to be continued)


H. Zhou updated Aug 2011