There is a workbook on Uv Vis spectroscopy, published by Aligent, soft copy of this book in pdf form is available online, the book contains a series of experiments to check performance characteristics of the instrument and different properties of absorbing systems , you can try running these experiments on your instrument, and trying out different combinations of sample and blank introduction schemes see if you get the same results as stated in the workbook .

Here is a link to Fundamentals of UV-visible Spectroscopy - Workbook

https://www.agilent.com/cs/library/primers/Public/5980-1398E.pdf

and another similar

https://www.agilent.com/cs/library/primers/Public/59801397_020660.pdf

Your instrument is a double beam instrument, not a diode array so some parts may not be completely relevant.

This situation is so typical with instrumentation in general, and especially problematic with older instruments. The true measurement basics are kept from the user, the documentation is modest, and the vendor would rather you buy a new instrument. (In our lab, we revive instruments by replacing the electronics and controllers with a system integration tool (DAQ2GO) that runs everything directly from MS Excel(R).

In order to get the proper values, true blanks are needed for each beam involved in the measurement, and at each and every wavelength involved in the measurement. It is worthwhile to measure the light intensity (vs no light) which reaches the detector in the measurement beam(s). When there is little intensity, the signal to noise is much poorer, and the linearity can also suffer. ( Most instruments keep this information from users.)

Scot Abbott, as an employee of Agilent I would like to defend the company in this instance. The manual is publicly available on the website here:

https://www.agilent.com/cs/library/usermanuals/public/1972_7000.pdf.

The Cary 100 Bio was on the market until late 2018 so it is quite recent.

I think Daryl will find this a very capable instrument. I should know, I was on the design team! :)

Hugh, my comments were not aimed at you personally or Agilent.

I too have been involved designing commercial instruments and developing applications. Some of the goals from instrument vendors do not match the long term needs of the field.

As a developer of instrumentation, the common drive is to make the unit (1) easy to use and simple to operate (2) work well (3) look as good as possible (4) where ever possible, incorporate a consumable element. (5) Prevent the user from seeing all the details. These goals make sense to the vendor but the result is often keeping the true raw signals from the user, and sometimes making compromises which optimize appearance, sometimes at the expense of analytical integrity. (I have not seen this in HP/Agilent stuff, but I have seen this in many other instrument offerings.).

There are some negative consequences of these well intended goals: The impression from the appearance of simplicity is that an instrument is an 'answer machine' , which is a falsehood. (2) the intentional lack of information about many items of which an instrument is constructed makes long term repair a very costly reality for customers. (3) Inability to gain fundamental control of the unit makes it difficult to adapt for other uses (e.g. adding custom automation, etc.).(4) The predatory lock on software, security choices, etc. make the unit less friendly as time goes on. (A lot of good instruments get scrapped because the software is lost, out of date, etc. Good for vendors. Bad for customers).

thx all for the input. But tbh Scot has a point. What confused me a lot is that different software applications (like scan or simple reads) differered in terms of the howto-descriptions as mentioned above.

And to make it worse - as nice as the primer and the workbook are to read - i found a very good answer to my question elsewhere.

In the Shimadzu UV Vis Talk Letter Vol 9.

As i am not using the most current Software Version - this may have been updated by Agilent - if not: in my opinion it would be an improvement to add information like this somewhere prominent. At least in my opinion it is not 100% clear - and if you check the Yale video i linked - i am not alone with that.

Quote from the mentioned letter:

Measuring a Solution Using a Single-Beam Spectrophotometer

1: Place the solvent in the cell and place the cell in the cell holder.

2: Perform blank correction.

3: Remove the cell and dispose of the solvent. Then add the measurement solution and place the cell in the cell holder. (Rinse the cell with the sample solution two times, then use the third solution for measurement.)

4: Measure the solution.

Measuring a Solution Using a Double-Beam Spectrophotometer

1: Place the solvent in two cells and place the cells in the sample beam and reference beam cell holders.

2. Perform blank correction

3. Remove the cell from the sample beam holder and dispose of the solvent. Then add the measurement solution and place the cell in the sample beam cell holder. Leave the reference side the same as in step 1. (Rinse the cell with the sample solution two times, then use the third solution for measurement.)

4. Measure the solution.

I am following this simple instructions now.