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ADInstruments

Application Note

AEM42b 1 August 2001

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.

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 UnitsConversion... is sufficient to calibrate the channel. If

such an output is not provided, the Absorbance extension

provides a means to make the conversion.

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)

Installing the extension

To use Absorbance, place the Absorbance(4) file 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).

Concentration

Absorbance

Figure 1

. Absorbance

versus sample

concentration

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.

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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ADInstruments

Application Note

AEM42b 3 August 2001

Document Number: AEM42b

Copyright ADInstruments Pty Ltd, 2001.All rights reserved.

MacLab, PowerLab, and PowerChrom areregistered trademarks, and Chart and Scopeare trademarks, of ADInstruments. Other

trademarks are the properties of theirrespective owners.

Contacts

International (Australia)

Tel: +61 (2) 9899 5455Fax: +61 (2) 9899 5847E-mail: [email protected]: www.adinstruments.com

North America

Tel: +1 (888) 965 6040Fax: +1 (866) 965 9293E-mail: [email protected]

Europe


Japan

Tel: +81 (3) 5820 7556Fax: +81 (3) 3861 7022

E-mail: [email protected]

Asia


www.ADInstruments.com

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 first order (or approximately

first order) chemical reaction, then the absorbance versus

time graph may be fitted 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.

Figure 7

. Recorded Transmittance data

Figure 8

. Calculated Absorbance values

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

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