Gilles Carpentier's Web Site:
Computer Data Acquisition for Biochemistry Practice Works
Methods and Examples

The use of the spectrometer Spectronic 301 in spectral mode:

1) Characteristics of Spectronic 301:

First, a few comments on the features of this device. It is important to remember some of this information to understand the acquisition procedure for spectral data in Datalyse.

The Spectronic 301 is not designed specifically for spectral analysis yet as it allows you to automatically change the wavelength via a computer connection, it is possible. The characteristics of this device (wavelength range of 325 to 900 nm obtained by a monochromator containing a holographic grating with a  groove frequency of 1200 lines/mm) allows you to obtain a good spectrum of a dye.

Here is some of the technical data that determines the maximum precision of the results we obtain:

Light source: halogen tungsten lamp

Wavelength resolution: 1 nm

Wavelength precision: +/- 1 nm

Wavelength accuracy: +/- 2 nm

Spectral bandwidth (bandpass): 8 nm *

The first order spectrum is used to obtain the desired wavelength. To optimize the spectral purity of selected wavelength, there is a set of 6 filters in the monochromator unit. These filters, automatically positioned according to the current wavelength, will reduce the stray light from neighboring wavelength bands and from the second order spectrum.

It is important to remember the presence of these filters, because the spectrometer needs a little time to move them and that can interfere with the spectral acquisition as we will see below in the delay setting in Datalyse.

These features will be able to give you a good spectrum, especially for large absorbance peaks.

* Definition of the bandwidth:

Although you specify one specific wavelength, in practice it is not possible to pass a "single wavelength", only a band of "wavelengths". If we assume that the energy distribution of this light is triangular, the bandwidth is the wavelength range corresponding to half the peak height. With that assumption the spectral bandwidth correspond to the 3/4 of the energy passed trough the sample. The maximum of the peak correspond to the selected wavelength. The smaller the bandwidth, the greater the energy passed at the selected wavelength, and the better the resolution of the spectra.

2) How to proceed to make a spectrum:

This device is single beam, which means that we first have to obtain a baseline using a cuvette containing only the solvent. This data will then be subtracted from the spectra we want. For more details about this operation, see Spectronic 301 .

- Setting the "zero" OD of the spectrometer:

First we need to set the zero OD value of the spectrometer. The light source does not emit the same amount of energy at all wavelengths, and the cuvette and/or solvent do not absorb the same amount of energy, and when the filters are changed the light energy will change suddenly.

If we make a scan of the absorbance of light with a cuvette containing water (or even air) for instance from 350 to 700 nm, we will obtain a complex spectrum having a very large range of OD (about 2). That means that if you do not set the zero OD value, the OD could exceed the maximum possible value during the scan.

To set the zero, we have to search for the wavelengths region in which the OD is the lowest. First make a quick spectrum with a step of for example 5 nm in baseline or spectrum mode, as shown below (in practice 700 nm is a good value).


An example of parameters for determining a wavelength that is suitable for setting the zero OD of the device

A quick spectrum of a cuvette containing water between 350 and 700 nm. We see that the minimum value is at 700 nm. So, before making a spectrum of the substance, the spectrometer must be set manually to 700 nm and the zero must be set at this wavelength.

3) Setting of delay in Datalyse:

- The spectral scan analysis and how it works:

When a wavelength is chosen a motor will move the grating to the right position. For a step of 1 nm this will take about half a second. When the lambda settings require a changing of filter, this time increases to about 1,5 second. Once the lambda has been set the detector needs a response time of about 2 seconds (3 seconds maximum according to the manufacturer's notice).

For all these reasons a delay function was introduced in Datalyse as you can see in the picture above (the parameters of the spectrum).

For a good spectral acquisition you will need a delay in order to get reliable and stable values. The picture below shows a spectrum of a cuvette containing water (baseline acquisition).

On this picture the delay is 2 seconds and the wavelength step is 1 nm. We clearly see sudden changes of light energy when the filter is changed (green arrows).

- Evidence of the benefit of using delay to get a fine spectrum:

The next picture shows a spectrum of a dye (see legend). Two scans were made, one without delay (red) and one with a delay of 2 seconds (blue). We clearly see that the delay suppressed the artifacts of changing filter (around 360 nm and 450 nm). Further more, the spectrum is smoother.

Example: absorbance spectrum of the basic form of p-nitrophenol (PNP)

Gilles Carpentier's Web Site: "Computer Data Acquisition for Biochemistry Practice Works: Methods and Examples"

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