ADInstruments

Application Note

The Absorbance extension for Chart 4

This application note explains how to use Absorbance, a Chart 4 for Macintosh extension that converts a spectrophotometer transmittance signal to an absorbance signal. Written by staff of ADInstruments.

Introduction
Absorbance is a calculation extension, which converts a linear transmittance signal from a spectrophotometer to a signal that is linear in absorbance (optical density). This document describes Absorbance v1.0 (or later versions of 1.0 such as 1.0.1, 1.0.2, and so on), which is compatible with Chart v4.1 for Macintosh or later. The description given here may not apply in full to other versions of Absorbance.

Theory
The relationship between absorbance (A) and transmittance (T) is given by: A = log(1/T) or A = log(T) Transmittance values are also often reported as percentage transmittance (%T), %T=100T. Thus absorbance can also be expressed as: A = log(100/%T) or A = 2 log(%T)

Transmittance and Absorbance

Many spectrophotometers provide an analog output that is proportional to transmittance (or percentage transmittance). Transmittance is proportional to the intensity of light passing through the sample. It is often more useful though, to have a signal which is proportional to the concentration of a sample solution. Since the absorbance of a sample (also known as optical density) is proportional to its concentration (Figure 1), this can be achieved by applying a conversion from transmittance to absorbance. This conversion is already done in certain spectrophotometers, which provide an analog output that is linear with respect to absorbance. In this case Units Conversion... is sufcient to calibrate the channel. If such an output is not provided, the Absorbance extension provides a means to make the conversion.
Absorbance

Installing the extension

To use Absorbance, place the Absorbance(4) le in the Chart Extensions folder in the Chart 4 folder. Quit Chart before installing or removing Chart extensions. The extension will be loaded automatically when you start Chart. When loaded it adds the Absorbance... item to any channel function pop-up menu (Figure 2).

Figure 2. A channel function pop-up menu showing that the Absorbance channel calculation is active

Using the extension

Calibration
In order to perform the conversion, Absorbance needs to know the recorded values corresponding to zero and 100% transmittance. These are obtained from calibration data recorded by Chart.

Figure 1. Absorbance versus sample concentration

Concentration

Record calibration data from the spectrophotometer by setting it to dark current, standby, or zero, and using PowerLab with Chart software to record a few seconds
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Application Note

Figure 3. Recording Absorbance calibration data Figure 5. Calibrating in Absorbance units

of data. Then record some data at 100% transmittance by using water (or some other blank solution) in the cuvette (Figure 3). In the main Chart window select the recorded calibration data and choose Absorbance... from the channel pop-up menu. The Absorbance dialog box (Figure 4) appears. It resembles the Units Conversion dialog box, and works similarly.

If the %T item is selected from the Units pop-up menu, the results will be shown in the range 0%T to 100%T. If Absorbance is selected (Figure 5) data will be converted to absorbance units. If the Calculated radio button is clicked, the recorded data are transformed into calculated values and displayed in the dialog boxs preview window. An absorbance of 3 means that 99.9% of the light has been absorbed. However, most information is usually contained within the 01 absorbance range, which corresponds to 100%10% transmittance.

Applying the calibration

After the calibration information has been entered, clicking OK will apply the chosen transformation to all the data in the channel.

Figure 4. The Absorbance dialog box

Select a suitable portion of the 0% transmittance data in the left hand pane of the Absorbance dialog box. The gray value arrows become black (enabled); click the top one. An average 0% transmittance voltage value is then calculated and displayed in the text box to the right of the arrow. Repeat the process for the 100% transmittance data in the right hand pane. Note that is possible to enter the 0% and 100% transmittance values directly in the text boxes, but normally they are unknown and calibration data must be used.

Figure 6. The Absorbance Demo Data le

The Absorbance demo data le (Figure 6), supplied with the Absorbance extension, contains calibration information in the rst two blocks. These readings correspond to 0% and 100% transmittance. The third block contains recorded data for a typical experiment.

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After calibration, Absorbance applies the transformation to the recorded data (Figure 7), to give the result shown in Figure 8. Since absorbance is the logarithm of a ratio and

Application Note

Figure 7. Recorded Transmittance data

Figure 8. Calculated Absorbance values

thus a dimensionless quantity, no units are shown on the vertical axis in Figure 8. If the spectrophotometric data being collected is expected to follow the kinetics of a rst order (or approximately rst order) chemical reaction, then the absorbance versus time graph may be tted with an exponential function. This is possible using the Curve Fit extension for Chart 4, which is available from the ADInstruments website, www.ADInstruments.com.

Document Number: AEM42b Copyright ADInstruments Pty Ltd, 2001. All rights reserved. MacLab, PowerLab, and PowerChrom are registered trademarks, and Chart and Scope are trademarks, of ADInstruments. Other trademarks are the properties of their respective owners.

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