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

This page describes a simple method to obtain some large spectrum (about 800 nm) with the
Thermotronic Genesis 10 simple beam spectrometer family, with the Datalyse software.

For the connection of a spectrometer to a USB computer, see
this link.

For data acquisition with Datalyse on the Macintosh, see this link.
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1) Introduction:

2) Principle:

3) Notice:

3-1) selection of the device and synchronization:

3-2) Setting the zero OD value for the scan:

Table of zero settings:

3-3) Making the baseline:

3-4) Making the spectrum:

4) Spectrum obtained with UV/Vis and Vis model, on a large range of wavelengths: limitations and advices:

4-1) UV spectrum of viral DNA using Genesis-10 UV/Vis and Genesis-10 UVscanning models:

4-1-1) Quartz cuvettes:

4-1-2) UV transparant plastic cuvette:

4-2) UV spectrum of proteins using Genesis-10 UV/Vis and Genesis-10 UVscanning models; experimental results and computed model values:

4-3) Comparative UV spectrum of a DNA preparation and proteins:

4-4) Examples of large band spectrum made mostly in the visible ligth with Genesis10-Vis and Genesis10-UV/Vis.

4-4-1) CuSO4 solution spectrum:

4-4-2) KMnO4 solution spectrum:

5) Conclusions:

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Note 1: About the firmware:

Note 2: The ExPASy (Expert Protein Analysis System):


1) Introduction:

These spectrometers are not particulary concived to perform spectrum even tough a software theoretically allows to obtain spectrum of 100 nm bands. Here is a procedure to obtain with the Datalyse software, some large band spectrum both for Genesis 10 visible and Genesis UV/visible (included the UVscanning declination) models (see links above). The firmware of the spectrometer must be 2.xx [note 1].

The difficulty to obtain spectrum with these spectrometers is the high energy pics which appear when the device changes of source beam filter. These pics, for certain particular wavelength, give negative OD overtaking the limits of the devices.

Indeed, once these particular wavelength identified, it becomes possible to obtain very good spectrum with respecting only two essential directions :

- The baseline and the spectrum itself, HAVE to be made in the same cuvette.

- The zero of the device HAS to be made at some specific wavelengths, on the air, without any cuvette, even empty. The appropriate wavelength to make the "zero", which depends on the range of the wavelength to scan and the model of spectrometer, are given below in the notice (section 3-2).

For more details about the principle of these kind of simple beam spectrometer, see "The use of the spectrometer Spectronic 301 in spectral mode".


2) Principle:

The wavelength range of these devices, 325 to 1100 nm for "Genesis-10 Vis" and 190 to 1100 nm for "Genesis-10 UV/Vis", are obtained by a monochromator containing a holographic grating. The first order spectrum is used to obtain the desired wavelength. To optimize the spectral purity of selected wavelength, there is a set of 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. This delay, might interfere with the spectral acquisition, as we will see below in the delay setting in Datalyse. Furthermore, the energy changement induced, imposes to find a correct zero value to avoid out of range negative values as explained above.

This link shows onto a similar device, the step by step procedure to follow to obtain a spectrum (including commented baseline and spectrum) with a simple beam spectrometer, with the Datalyse software. The procedure is very similar for the Genesis 10 family.


3) Notice:

3-1) selection of the device and synchronization:

The following screen shots, show the specific commands needed to obtain a spectrum with a genesis-10 as example, piloted with Datalyse.

First, be sure the spectrometer initialisation is ended and choose the correct device in the list: press "OK":

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If the synchronisation is effective, the green message, shown on the below picture, appears:

3-2) Setting the zero OD value for the scan:

! Note: it is strongly not recommended to use the keyboard of the spectrometer when it is connected to a computer. All the comands have to be performed from Datalyse.

To set the zero OD value of the spectrometer, choose the so called "Measure at on Wavelength..." menu, and input the appropriate value. In the present case, for the Genesis-10 Vis, it is 680 nm. Make the zero value from the Datalyse menu. The spectrometer will set "0 OD" at 680 nm. Don't use the spectrometer keyboard to make the zero value:

--

This table gives the value of the wavelength to use for setting the zero of the spectrometers according to the spectral range values:

Genesis 10 UV/Vis, UVscanning:

190 nm -> 660 nm: zero wavelenght: 300 nm

350 -> 1000 nm: zero wavelenght: 484 nm

Genesis 10 vis:

325->1000 nm: zero wavelenght: 680 nm

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3-3) Making the baseline:

For the baseline, choose a range of wavelength values compatible with the zero value previously set, and select a "Delay" of 1 (second):

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3-4) Making the spectrum:

For the spectrum, keep exactly the same values that used for the baseline (Datalyse keep it by default):

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4) Spectrum obtained with UV/Vis and Vis model, on a large range of wavelengths: limitations and advices.

4-1) UV spectrum of viral DNA using Genesis-10 UV/Vis and Genesis-10 UVscanning models:

4-1-1) Quartz cuvettes:

The next graph shows a viral DNA spectra made with a Gensis-10 UV/Vis model (zero value of the device performed at 300 nm). 3 curves are prensented; the baseline of the Tris buffer containing DNA, the spectra of the DNA (scan of the DNA solution corrected from the baseline), and the sum of these two curves, giving the level of OD reached for this analysis (conditions: quartz cuvette, 1.5 ml, 1 cm of optical way).

This graph clearly shows:

- The spectrometer has to be used with a minimum of 205 nm. Lower values give unprecised results, near the limits of the device (3 OD).

- It is adviced to use the spectrometer with wavelength superior to 210 nm.

- For this kind of sample, never exceed 0,1 DO (at 260 nm) of DNA in the cuvette to avoid out of range values.

This picture, shows a full scaled representation of the DNA spectra showed in the precedent graph. The ponctually discontinuous aspect of the spectra from 190 to 230 nm, indicates we are near to the limits of the OD device capacity. The ratio of OD (260/280 nm) is 2.12. It is compatible with the value, generaly considered to be obtained with a protein free DNA extract. Amersham recomends these following values for nucleic acids purification: RNA (260/280 nm) > 1.8; DNA (260/280 nm) > 1.9. DNA sample from courtasy of Dr J. Cebrian.

4-1-2) UV transparant plastic cuvette:

Because of the greater absorbance of UV light from this cuvette [ref1] compared to the quartz, it is interesting to test it on a DNA spectra, to check the capacities of the spectrometer, by a comparative evaluation of the (260/280 nm) ratio. The next picture, shows spectrum and baseline of another DNA preparation:

The upper figure, shows a baseline and a spectra of a DNA preparation, analysed in plastic "UV transparant" and quartz cuvettes. We can see the dramatic absorbance of this kind of plastic cuvette. The limit for the baseline is 250 nm. This wavelength, isn't suitable for a spectrum scan (see the out of range value, traduced by a negative value of the cyan curve, falling to zero at the scale of this graph). Despite of this, the next picture shows that the results are usable from 260 nm.

This graph, presents the two full scaled DNA spectrum, shown in the previous figure. Although the 260 nm wavelength value reaches the limit of the transparancy for the UV light of the platic cuvette, the two spectrum (quartz cuvette and plastic cuvette) are very similar from 260 to 350 nm. The (260/280 nm) OD ratio is 1.93 for the "plastic" spectra and 2.10 for the "quartz" spectra. These values are in the same range.

These results show:

- This plastic cuvettes are usable in this conditions with a minimum wavelenght of 260 nm.

- The spectrometer gives good results even in these limit conditions.


4-2) UV spectrum of proteins using Genesis-10 UV/Vis and Genesis-10 UVscanning models; experimental results and computed model values:

This part shows comparative spectrum of two proteins, trypsin, and bovin serum albumin (BSA). The lambda max, near to 280 nm and the absorbance at this lambda max is compared to some theoritical values calculated from the primary sequence of the aminoacid residues using the tools avaible on the Expasy website. [note 2]

The upper graph shows the spectrum of trypsin and BSA with a large OD scale. Among the inflections in the spectrum curves, we can see the ones mostly due to double bonds at 220 nm and the others, more specificaly due to the peptid bonds, at 230 nm.

This graph presents the same spectrum than the precedent graph, full scaled sor the absorbances around the 280 nm pics.

Here is the theoritical results, obtained from Expasy "ProtParam" tool [note 2], compared to the experimental results.

Theoritical data (ref 2, ref 3) are computed with this physical conditions: 1% protein in 6.0 M guanidium hydrochloride, 0.02 M phosphate buffer pH 6.5.

Experimental results obtained with 0,1% protein, in HCl N/1000 for trypsin and distilled water for BSA.

Lambda max:------- ------- ---------OD:

Theoritical:-------experimental:----Theoritical:-------experimental:-----

Trypsin ----- 278 ---------------278 ----------------1,377 ------- ------0,130*10=1,30

BSA - ----- --276 ------- -------276 ----------------0,673 ------- ------0,060*10=0,60

- These results shown:

- Similar lambda max, which indicate a good precision of the spectrometer, even if the OD measured is very low.

- The theoritical OD is in the same range than the theoritical values (with an error of 6 and12 %). Of course, these calculated OD are indicative, but very pertinant regarding to these two protein spectrum.

If you have any doubts about the values given by your spectrometer in scan mode, you can find a simple and fast method to check its calibration at this link.


4-3) Comparative UV spectrum of a DNA preparation and proteins:

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This picture shows a compilation of the DNA and protein spectrum of the sections 4-1 and 4-2. We can see at 230 nm on the protein spectrum that the inflections of the curves due to peptid bonds, correspond to a local minima in the DNA spectrum, showing the hight putity level of the preparation.


4-4) Examples of large band spectrum made mostly in the visible ligth with Genesis10-Vis and Genesis10-UV/Vis.

4-4-1) CuSO4 solution spectrum:

The following measures, show compared spectrum in large band of wavelength (from 325 to 1000 nm), obtained with the Genesis10-Vis and Genesis10-UV/Vis models.

The upper picture shows a spectra obtained using a standard plastic cuvette, with a Genesis10-UV/Vis spectrometer. The baseline made with a zero setting of the device at 484 nm, shows the energy variations appearing at the filters changements (see section 2 for explanations). This setting, allows to obtain a continuous spectra from 350 to 1000 nm.

This graph shows two spectrum of the same CuSO4 solution. One, is obtained with a Genesis10-Vis with a zero set at 680 nm and scanned from 325 to 1000 nm. The other-one, corresponds to the spectra of the precedent picture, made with a Genesis10-UV/Vis.


4-4-2) KMnO4 solution spectrum:

The following measures, represent compared spectrum in large bands of wavelength from 325 to 1000 nm and from 210 to 1000 nm, obtained with the Genesis10-Vis and Genesis 10 UV/Vis models.

The above graph, shows a spectra of a KMnO4 solution, obtained using a standard plastic cuvette with a Genesis10-Vis spectrometer. The baseline with a zero set at 680 nm, shows the energy variations appearing at the filters changements (see section 2 for more details).

Note the differencies with the baseline of a Genesis10-UV/Vis (section 4-4-1).

This figure shows the spectrum of the same KMnO4 solution. One, corresponds to the spectra obtained using a standard plastic cuvette with the Genesis10-Vis model (details in the precedent picture). The second-one, is obtained using a quartz cuvette with a Genesis10-UV/Vis spectrometer.

The differencies observed between the two spectrum below 510 nm are due to the different optical properties of the used cuvettes (plastic and quartz).


5) Conclusions:

All these examples, show that the genesis 10 spectrometer family, with an appropriate zero setting on the air, allow to obtain usefull large band spectrum, both in UV and visible ligth.

The Datalyse driving of these devices, allows a very convenient and powerfull way for an optimal exploitation of these devices.


Note 1: About the firmware: the spectrometer, needs a 2.xx firmware version to be piloted. You can upgrade, if needed, the firmware of your spectrometer with the GenFlash thermotronic software. The upgrade procedure can be made with a "USB" PC using a USB to RS232 adaptor. Some upgrade with this kind of adaptor, using a Macintosh running a virtual PC was successfully perfomed.

After upgrading, sometimes the initialization of the spectrometer might take about 20 min. You can correct this by using Datalyse to send these following comands to the spectrometer:

UNLOCK 4092

DEBUG

if the answer to this last comand is "DEBUG=2", send:

DEBUG = 0

SAVE

Restart your spectrometer.

However, if your spectrometer is under warranty or if you are not familiar with this kind of manipulation, it is strongly adviced to contact your resailor or the very competant Thermotronic team to make it. An error could make your spectrometer unusable. The firmware upgrade and modification is entirely under your own responsability.


Note 2: The ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB) is dedicated to the analysis of protein sequences and structures as well as 2-D PAGE. The SIB is an academic not-for-profit foundation established on March 30, 1998 whose mission is to promote research, the development of databanks and computer technologies, teaching and service activities in the field of bioinformatics, in Switzerland with international collaborations.

To find the data computed for a particular protein, you can search in the data base by name or protein number. You can have a direct access with the protein number link of this page (ref 2) for Trypsin and BSA. Once you have found the protein you are interrested in, at the bottom of the web page, click on "ProtParam" in the Sequence analysis tools list. Several subsequences are proposed. Just go at the bottom of the page and click on "submit" to have the full protein computation. You can have a direct access to the results for trypsin and BSA with the links of this page (ref 3).


References:

ref 1: Microcuvette dispolab, Kartell, 1941 pmma.

ref 2: trypsin protein number: P00761, BSA protein number P02769.

ref 3: trypsin ProtParam results: P00761 ProtParam . BSA ProtParam results: P02769 ProtParam


Special thanks to Alessandra Albano for the English correction of this page.

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

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