IgorCD Procedures. Data -> Load CD Data

Transcription

1 IgorCD Procedures IgorCD is a set of Igor Pro procedures for processing CD spectra and CD thermal melts. It correctly implements spectral smoothing functions and facilitates subtracting baselines (or constant offsets), correcting baseline shifts, truncting data to a useful wavelength rage, automatically processing multiple spectra, performing principal component analysis (using SVD), and extracting traces at specific wavelengths from multiple spectra. Data can be converted to either delta epsilon (De) or theta brackets [q]. The routines can be used with any CD data that can be exported into a delimited text or Excel format. The procedures can be used for spectra and melts. Utility procedures Data -> Load CD Data Data files should be in the same format as an excel spreadsheet that has been exported as an ASCII text file (tab delimited). The 1st column should contain the wavelength, and the subsequent columns should contain the CD values of any number of spectra collected with the same wavelength range and step size. If the 1st row contains a zero value in the 1st column, or contains arbitrary text, that entire row will be deleted. If a row of data at the beginning of the file contains a blank entry in one of the columns, that entire row will also be skipped. In these cases, or if the first row contains only numeric data, the wave names will be derived from the file name. The CD waves will be named "cd_filename_n" where N in incremented from 0 to n. The data will be deposited in the current Igor data folder (the one with the red arrow). The CD waves will be scaled according to the wavelength column. If the first row contains text data delimited by tabs, the wave names will be obtained from this row instead of from the filename as described above. This allows you to import data with descriptive names. In this case, blank entries in the data file are result in a blank (NaN) entry in the wave for that particular CD spectrum. (Check and make sure the data was imported correctly, especially the first data points.) Macros -> Plot CD Spectra CD data are plotted by selecting the spectra in the data browser (shift click for multiple spectra) and doing "Plot CD Spectra" from the Macro menu. Macros -> Process CD Control Panel This is the main control panel for processing CD data. The capabilities/options are: subtract baseline (or constant), smooth data, correct for spectral offsets, truncate wavelength range, convert to delta-epsilon using either mg/ml or mm concentrations, and apply identical processing to a series of spectra automatically. The processed data are put into a new folder called Processed with the same name as the original data. sample popup box - select CD spectrum of the sample you want to operate on. CD units scroll box - click on arrow to cycle between mdeg->de, mdeg-> [q], DAbs->De, and DAbs - >[q] baseline popup box - select baseline wave. Or set to none and apply a constant baseline offset by subtracting the number in the text field from the spectrum. smooth checkbox and popup - activates n-point Savitzky-Golay smoothing. zero baseline - if checked, corrects spectra for any baseline offset shift by zeroing the long wavelength (10 nm) end of the spectrum. process list of spectra checkbox - if checked, select one sample (e.g., named cd_spec_1) as described above and all spectra with same root name (cd_spec_n) will be processed identically. wavelength range - specify starting and ending wavelengths. Default values are obtained from x-

2 scaling of the slected sample wave. concentration - enter value in mg/ml (proteins) or mm (nucleic acids). Use popup box to select units. subunit mol. wt. - used when concentration is entered in mg/ml to convert into molar quantities (delta epsilon). For proteins, this is the average amino acid mol. wt. pathlength - enter value in centimeters additional multiplication factor - for additional scaling factor Do It - does it Additional routines are available as part of the generic spectral analysis procedures. These addition features are described more fully below.

3 Details The smooth data option allows you to apply the Savitsky-Golay smoothing function with window sizes of 5, 7, 9, 11, or 13 points. Both the sample and baseline data are smoothed before subtracting the baseline. The window size of the smooth is decreased as you approach the ends of the spectra. Two data points at each end of the spectrum are left unchanged since the minimum smooth is 5 points. The wavelength range parameters are initially set to those of the sample data. Note that if the spectrum was stopped before getting to the last wavelength, for example by exceeding the dynode voltage limit, then the wavelength range may be less than the size of the wave. You need to watch out for this because the smoothing function may not be applied properly to the ends of the spectrum if your wave extends out past the useful region of your data. You can truncate the data by entering values into the displayed wavelength range. After subtracting the baseline, the data can be corrected for a baseline offset by checking the zero baseline option. This will average the data over 10 nm at the long-wavelength end of the spectrum and subtract this value from the entire spectrum. The next step executed by the macro is to convert the CD data from the input values into molar delta epsilon units, De = el - er (or theta brackets). The concentration can be given in terms of milli-molar (mm) or gram (mg/ml) quantities by selecting the proper units in the pop-up box. If the concentraion is in mg/ml, then the subunuit molecular weight must be given. This is most convenient for protein solutions. Nucleic acids lend themselves better to mm quantities. It is also possible to select "none" from the concentration pop-up menu. This will result in the spectrum not being scaled by concentration or pathlength or being converted to delta epsilon. The additional multiplication factor is still available The macro can also be used for thermal melts or titrations. In these cases, however, there may not be a baseline spectrum to subtract. By setting the baseline to "none" a constant offset can be applied to the data. Since the routine can operate on any arbitrary wave, be careful that you only attempt to process raw CD data and not data that has already been processed. Processed data are saved in a (new) data folder called "processed" within the current data folder. The name of the newly created wave is taken from the name of the original sample data. If that wave already exists, it will be overwritten. Processing multiple spectra in the same experiment will put all the processed data in the same folder making it easy to graph or export the data. Note: if you open a previously saved experiment that was saved with an older version of the IgorCD, you may get an error message. Go to the command window and type in "initigorcd()" IgorSpec Package This gives several new menu items in the Data and Macros menus. Data -> Load Spectrum X Y1 Y2 Imports spectral data similar to that for the Load CD Spectrum Data -> Load Cary Spectra X1,Y1,X2,Y2 Imports data consisting of alternating columns of wavelength and signal. Data can be delimited with

4 either tabs or commas. This is the ASCII format exported from the Cary spectrophotometer. Can also import data that contains only one column; in this case you are prompted for starting and ending wavelengths. There are also several procedures under Macros for manipulating spectra. Macros -> Graph Utilities -> Alternate Trace Styles Change the styles of all traces in a graph to have different colors/styles. -> Cycle Thru Traces (slow, medium, fast) Step thru each trace (slowly, etc) and highlight it while printing the trace number in setvariable control. You can stop the procedure with Cmd-<period> on Mac. You can manually set which trace to highlight with the setvariable control. To delete the setvariable control, mouse Graph->ShowTools, then select control and delete. Macros -> Operate on Spectral Data -> Extract Row from Multiple Spectra This is useful for extracting data at one wavelength from a series of spectra, such as from a melt or titration. The newly created wave will not have any X scaling and you will have to add it manually. First select (by shift clicking) the spectra in the Data Browser window and then select this option. -> Correct Spectrum for Linear Drift/Scatter Intended to estimate a correction for subtraction of scattering from an absorbance spectrum. This routine uses a linear correction and is therefore not strictly correct. Use the cursors to select the region of the spectrum to use as the basis for the correction. -> Offset Spectrum to Zero Selected Region Use the cursors to select the region of the spectrum to use as the basis for the offset correction. -> Decompose Spectrum into 2-Components Use to estimate the fractions of 2 component spectra. Use cursors to select. -> Convert Spectra into Matrix Select multiple spectra in DataBrowser and then this menu item will make a 2D data matrix named data_matrix -> Prinicpal Component Analysis

5 Creates a control panel for doing PCA. First select multiple spectra in DataBrowser and then press RunPCA button. The maximum number of significant vectors will be inserted into the setvariable box of the control panel and the top 5 Eigen values will be printed to the command line. Set the number of EigenVectors you want to use and then plot the basis spectra (will make waves named basis1, etc.), plot Eigenvectors (will make waves named vector1, etc.), calculate spectra (will make a 2D matrix of calculated spectra using the basis spectra and vectors. Cleanup is not yet functional. Installation This package contains this Word document and 2 folders, which should be stored in a convienent location on your hard drive. One folder is named put alias INTO user procedures and contains the procedure files IgorCD and IgorSpec. You should put an alias (Mac) or shortcut (Windows) of these files into the IgorPro/UserProcedures folder. The other folder named put alias INTO igor procedures contains zbootmyproc file. Put an alias of this into the IgorPro/IgorProcedures folder. zbootmyproc will be loaded every time you startup Igor and it will make a new menu item under Analysis that will allow you to load the IgorCD/IgorSpec packages. If you have also installed IgorDenat or other Igor procedures from me, they will also have a zbootmyproc file which will contain the code to load those packages. You will have to edit zbootmyproc file so that all of my packages are available. To do this, remove comments (double slashes //) from the lines you want to be available. These lines are in the Menu and Submenu section at the top of the file.