Plotting absorption changes

Plotting changes in the absorption as a function of sample concentration gives a more quantitative way of evaluating dependence than spectral overlay plot.  Such a profile also helps to evaluate more precisely useable limits, or dynamic range, of the spectrometer.

The data matrix that you previously loaded in Igor is a two-dimensional grid of data. For spectral overlay plot you picked data in vertical groups, or columns. Within each of these groups/columns sample concentration was the same and you observed change of intensity with wavelength.

Likewise, you can select data in horizontal groups/rows. Within each row wavelength remains the same, but concentration changes from one point to the next. You can plot rows of a matrix against concentration calibration that you have previously calculated in Excel and copied into CP_ wave.

Typical titration matrix will contain many more rows than columns and it is not practical to plot every row.  Moreover, only a part of the spectrum shows changes in absorption. Therefore, you can safely clip and thin displayed data without loss of information.  To select the region that you want to plot profiles for, first open spectral overlay plot and examine it. Show plot Info from Graph menu or by clicking Ctrl+I. Info window has two cursors – round and square. Drag round cursor (called cursor A) to one of spectra with intermediate intensity – Info window will display the name of the wave that cursor is currently on and position information.  Using keyboard left/right keys move cursor from one limit of spectral changes to another. Take a note of point numbers for both positions – this is your row index range.

Knowing row index range decide how frequently to pick up rows for overlay. Graph with over 10 traces will be excessively busy and difficult to analyze.  If your selected range is from row 20 to row 180, for example, you may want to overlay every 15^th or every 20^th row.

Making an overlay plot of rows in a matrix is almost identical to what was described for overlaying columns in spectra overlay plot. The main difference is following:

Graphical User Interface

 In the New Graph dialog make sure you are in the “More Choices” mode. Select matrix name as a Y Wave and CP_ wave as an X Wave, add it to the list. By default Igor will pre-fill ranges of selected matrix to entire first column. This has to be changed to a fixed index of the first row and column range from 0 to the index of the last column (number of columns minus one). When waves are properly prepared column range of the matrix should be the same as row range of CP_ wave. Click Add button.

Add other profiles you want to overlay by incrementing row index by the value you selected when examined spectra (by 10, 15, or 20…).

The rest is identical to spectral overlay plotting.

Command line

Similarities between row and column overlay are even closer when using command line.

Bring command window forward by selecting it or by pressing Ctrl+J. In the command line type:
Display MyMatrix[20][0,25] vs CP_MyMatrix
and execute command by pressing Enter.  Here [20] is index of the first row to overlay and [0,25] is the entire range of columns in the matrix. Also not that name of calibration wave has changed to CP_MyMatrix.

Return to the command window and start appending profiles using the following command:
AppendToGraph MyMatrix[30][0,25] vs CP_MyMatrix

The row index should be incremented with each trace ([30], [40], [50] etc. ) until you display the entire region that you selected.

What do profiles show?

When you examine overlaid profiles you may notice several significant points. It will help in analysis if you make a duplicate of overlay plot as you have done for spectral overlay and change scales of one of them to the lower range.

• All profiles are linear over some range, but slopes are different. Slope of a profile at each wavelength is the coefficient of molar extinction.
• All profiles come to saturation at more or less the same level. This level corresponds to noisy maximum seen in the spectral overlay.
• At very low concentrations profiles a no longer liner too. Instead, absorption intensity begins to oscillate randomly as you approach zero concentration. This limit is determined by internal noise of the spectrometer.