Dear all, 

I would also thank you very much for all your advice and comments, they was really helpful for me  as well.

Am also got negative result in one of my experiments, its happened when I diluted the suspension 20 times before the test with uv-vis. but with the diluted factor was just  ten times the absorption  was fine (between 0-1).

Please let me know if you can explain more about this phenomena.

Regards 

Salam 

Do you find this recording a full spectrum and if yes, at what wavelength does it happen?

Or do you find this while doing a calibration curve and the curve is intersecting the y-axis below 0?

How much negative?

If significantly then

some error in your analysis.

Instrument is not working properly (lamp Energy),

solvent quality is not good, etc. etc.

(Mrs/Miss or Mr.) Abilash, first are you sure that your quivetts are absolutely clear?! I am apologize for this first note, but it would be also source of such problem.

Which type of UV-VIS spectrometer you have 'single' of 'double beam'?

@Tobias : I was scanning from 400-800nm. Took water as reference. From 800nm onwards the spectrum is below the 0 (i.e. in negative)

@ Vineet: Not much negative, just below the zero line

@Ivanova: Cuvvette may not be so clean. It is a double beam spectrometer.

From the questions posed by all three of you, it appears that it is unusual to see the spectrum in the negative region unless there is a mistake somewhere. Even if the baseline is not done properly, I think this problem may come. is it? Please suggest what all errors can lead to this

What is the solvent in your sample? Is it water just like in the reference? If you draw the baseline using water and your sample is a non-aqueous solution you might observe a negative absorbance as a result, since the solvent in your sample (ethanol, methanol, ether, etc.) could absorb less light than water at certain wavelengths.

(Mrs/Miss or Mr.) Abilash, when your instrument is a double beam, that the problem would be of one of the 'photo diode' (from the 'reference' site). You may change it alone (it is easy) with new one. But after that you should obligatory calibrate the instrument.

The 'beam splitter' would also be a problem, but most probably the problem is in the 'photo diode'.

Yes, check cuvet, solvent match, and make sure you warmed up device long enough prior to measurements.

Hello Abilash,

I do agree with the responses you have received so far but I am suggesting a simple method to isolate the problem - whether it is the photo-sensor or the sample preparation method

Before you start, make sure you have "CLEAN" couvettes for both the sample and the reference.

Step 1: Dry RUN:

Place "empty" couvettes in the sample and reference chambers and run the spectra for the range of your interest.

Step 2: Water (H2O) only test:

Fill both the couvettes with water and run the scan again for the same wavelength range.

Step 3: Solvent only test:

Repeat the scan for the solvent in both places.

The outcome of the above tests should give you fair idea as what is wrong. Still if you are getting the NEGATIVE values, then it has something to do with the sensor electronics (i.e. the photo-diode or photocell).

I would suggest to pay a closer look at the spectral response of a photo-diode (or a photo-cell). The electrical output is not a flat signal across the spectrum. It DOES HAVE a peak, mostly towards the IR range (>800nm). Such peak might be playing a role in your case.

Hope my suggestions are useful to you. I shall be very interested to hear from you.

Thanks and best luck!

-Prasanna Waichal

Are your cuvettes a matched pair? If they are not a matched pair, then you could get slight discrepancy in your resulting spectra (due to each cuvette scattering slightly differently because of slight differences in the glass). Look for a marking (either a number or a letter) on the top corner of the cuvette. If there are no markings on the top of the cuvette then I would assume that they are not matched.

Hope that helps and that you are able to track down the source of the problem.

It could be that in your sample, for some reason (reaction), you have unbalanced with reference and so you have a negative response. Check for the exact balance of blank before and after reading of sample.

Change cuvette reference to test and test to reference, if any change in spectra than it is cuvette problem. otherwise sample like plasma, extract, multiple residue causes such types of error.

If u have found this thing with some particular sample only and not with other samples then I assume..it is not the problem with the cleaning issue or photodiode...etc...I think the problem is with the AUTO ZERO option. I guess, if u can auto zero at a wavelength probably on the red side of the wavelength u initially choose for auto zero, u may get a better spectrum. It usually happens if the wavelength at which u choose to do auto zero has some finite absorbance.

Gangula, be merciful and tell us what was happening. :-) I need to know.

Hello Friends,

There are several suggestions leading to same possible cause/s. That is why, I have suggested him some simple steps so that we can really identify the problem.

As Shahnawaz Rafiq has also mentioned to take care of the RED zone (higher wavelength side), I think this is a combined effect of the sensor spectral response as well as absorbance of some chemical at those specific wavelengths.

Lets wait to hear more from the author - Mr. Gangula.

Thanks!

I agree that there is a mismatch of the matrix. In double beam spectrophotometers the Absorption of the reference channel is subtracted from your sample's Absorption; see attached paper.

I am sure there is a mismatch between the solvents that were used for study. First baseline has to be corrected by keeping the bare solvent in both the Cuvettes if you are using a double beam spectrophotometer. After correcting baseline you have to keep the sample+solvent in the rare side of the cuvette chamber as is designed in the Shimadzu spectrophotmeter. Also the sample that you are taking for analyses should not be taken from a source where sample is already dissolved in some other solvent. I am sure if you keep these things in mind, you will get very good spectrum.

I think that the problem may be in the cuvet, solvent used.

Thank you all for your valuable suggestions. As some of you said, the cuvettes are not so clean. Also I have used proper solvent for taking baseline. Now it is fine. I realized it is very important to take care of small things like cleaning of cuvettes, cuvettes of same type, taking proper baseline etc....Thank you all once again

Dear all, 

I would also thank you very much for all your advice and comments, they was really helpful for me  as well.

Am also got negative result in one of my experiments, its happened when I diluted the suspension 20 times before the test with uv-vis. but with the diluted factor was just  ten times the absorption  was fine (between 0-1).

Please let me know if you can explain more about this phenomena.

Regards 

Salam 

Thanks a lot to all the people who have put forth their views. It was very helpful to know what all the factors that would be affecting the spectra. Thanks again.

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