In my opinion there are two possible reasons for negative absorbance:

1) you have a problem with instrument calibration, which means that your reference sample for baseline correction is contaminated (moisture or other surface-impurities). In this case I would suggest obtaining or creating clean fresh reference sample.

2) your sample shows luminescence triggered through excitation of specific wavelength giving higher total light output. You have to note that though the source in UV-VIS spectrophotometer is monochromatic, the detectors are not, they register net total light output from your sample (whole spectra produced by interaction of source beam with the sample).

Can you elaborate on what kind of setup you use for UV-VIS spectroscopy? Are samples in liquid state (transmission configuration) or powders (reflection setup, mixed with BaSO4 or other matrix)?

In which spectral range is the absorbance lower than baseline?

the wavelength that I used is from 600 to 200 nm. It's start with positive absorbance (higher than baseline) then at certain pick (300 to 200 nm) it started to become negative.

Even, when I'm doing UV-Vis titration, the absorbance getting negative as the concentration increase.

I think that the solution that you are using is unabsorbed in the region 200- 300 nm. It means that it is non absorbing in UV region (200 - 300 or upto 350nm)

After that it starts absorbing in the visible region (400 - 800 nm) based on your functional group of the compound under study.

No matter how high concentration is taken in UV region.

May be it becomes more negative or less negative as concentration increases due to some factors like stray light radiation etc,

can I assume that, my receptor had reached it limitation to bind with the analyte?

Yes. I'm using quartz cuvette. I'm using Deionize water and Methanol as solvent because the solution that I'd prepared compose of ligands (diamide ligand - pyridine diamide) which dissolve in methanol and anion salt (Tetraethylammonium Nitrate, Tetraethylammonium Chloride and Potassium Chromate) which dissolve in deionized water.

I'm using Shimadzu 1601 series (double beam).

My blank composition is ligand, Methanol and deionozed water.

While, my sample compositions are Ligand, Anion salt, Methanol and deionized water.

I attach one of figure of my UV-Vis Spectra.

L6 stand for my ligand.

thanks for the information given. I will try improving my method as suggested.

Have you looked at the absorbance of your blank relative to a water blank (or looked at reference/sample intensity instead of absorbance)? As others have mentioned, it is likely that the components in your blank are strong absorbers and so you are seeing an instrumental artifact (stray light).

Only solvents should be considered in blank composition.

In your blank ligand is free and in the sample ligand is bounded and maube change the absorption spectra, therefore, blank absorbes more light than sample. So, follow John McCaffrey advise.

So, am I need to remove methanol from my blank sample?

Just using ligand and deionized water for blank sample?

what is your blank?

Is your ligand colored?

Is your sample colored?

I agree with everything Farooq wrote, but I also wanted to add that you should make sure that the blank has, as close to possible, the same composition as the sample, except without the chromophore. If you have a single beam instrument, you can also try leaving the cuvette in the spectrometer and replace the blank with the sample solution using a pipet (rinsing in between with a bit of the sample solution). This will ensure that no change in cuvette orientation will have occurred that might alter the light path.

There is no physical meaning of negative absorbance. Please try with different solvents. If the solvents do not give positive absorbance, then use distilled water as solvent. I hope this will work.

In UV-VIS spectroscopy we use combination of Deuterium lamp (for UV region) and Tungsten Halogen lamp (for visible region) for detection of absorbance of light of particular wavelength. In the case of UV region (using Deuterium lamp) and DI water as solvent, doesn't De lamp create free radicals hitting the water, dissociating it and creating radicals at various wavelengths of the UV spectrum ? I am confused why the lamp is used as a reference lamp behind its direct effect on radical generation. Can somebody tell me ?

please tell me that how i can set absorbance range 0-1 during wavelength scan in my UV-S. when i am setting it 0-1 half of the graph disappers and goes down.  I have tried same sample on other uv which gave spectrum btw 0-1 so please tell what is the problem with my UV-S. I have attached both files here.

Was it the same sample? or only equal? 

is it a solvent in a cuvette?

what kind of UV-S do you use? double beam? both?

Has a Baseline correction been used in both cases?

please give us more information about your sample, spectrometer und parameters do you use for your measurments.

Do you use a referrence solution according to your sample? 

try to increase the light path by using 4cm cuvet. otherwise I thing you are working under the LOD

baseline means that the sample solution has lower absorbance than reference solution . make sure from solubility of complex and try to read more than one solution absorbance for the same reading.

I think this link is very useful, 

http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/

i am also facing the same problem, but i solved the it, i measured the the blank and sample with the same cuet but before i was using different cuets for measuring the blank and sample

i am also facing the same problem, but i solved the it, i measured the the blank and sample with the same cuet but before i was using different cuets for measuring the blank and sample

i am also facing the same problem, but i solved the it, i measured the the blank and sample with the same cuet but before i was using different cuets for measuring the blank and sample.

I am also facing the same problem, but i solved the it, i measured the the blank and sample with the same cuet but before i was using different cuets for measuring the blank and sample.

I am also facing same problem.

One of my product have derivative method using copper sulphate solution and I am getting negative result in reference wavelength.

what is the reason behind it?

Anyone can help me?

Based on a quick websearch, a 1 molar solution of of copper sulphate will have an absorbance of ~2.8A at the absorbance peak of 635nm. What is the concentration of copper sulphate (assuming it is the primary absorber), and what wavelength (or wavelength range) are you using?

I am using wavelength 580nm and 410 nm.

I am getting negative result at 410 nm.

Yes copper sulphate is primary absorber and conc. is 0.0125 g/mL.

Sorry I didn't see your response earlier.

I was able to track down the expected UV-VIS absorbance spectrum for copper sulphate. Copper sulphate is a very weak absorber at 410nm. See the paper:

http://www.arcanum-butler.k12.oh.us/userfiles/8/my%20files/arcanum%20writing%20guide%202016/2016%20lab%20report%20spectrophotometry%20analysis%20of%20copper.pdf?id=1749

[Note: don't be confused by that Concentration plot on page 3 - the author apparently swapped the numbers for the labels. X axis (concentration) should be 0 - 0.45M, Y axis (absorbance) should be 0 - 1.5]]

Depending on whether you used anhydrous or pentahydrate, your 0.0125 g/mL corresponds to 0.05 - 0.08M.

When you say 'negative result' - do you mean a 'result close to zero', or do you mean a large negative number? Every measurement includes noise.

thanks for reply.

yes I got large negative number.

Interestingly, I came across a paper on the UV-Visible spectra of boric acid solutions in various solvents, and the authors report negative absorbances:

http://ijpsr.com/bft-article/uv-visible-spectral-analysis-of-boric-acid-in-different-solvents-a-case-study/?view=fulltext

It is possible that at sufficiently high concentrations, some solutes can react with with the solvent, thereby, decreasing its absorbance. In the case of aqueous solutions of boric acid, for example, the lone-pairs of the water can bind to the boron, causing the n -> sigma* electronic transition to shift to higher energy, and shifting the absorption band well into the ultraviolet region.

Thanks Hitoshi,

Can you explain how to calculate absorbance for calculation. Should we add negative result or take ZERO for negative value?

The absorbance, A, at a given wavelength is the logarithm (to the base 10) of the ratio of the intensity of light (having the given wavelength) reaching the detector in the absence of the chromophore/sample, I0, (aka the blank or reference) to the intensity of light (having the given wavelength) reaching the detector in the presence of the chromophore/sample, I.

The equation is: A = log10(I0/I)

Since %Transmittance = I/I0 x 100%, the absorbance is related to, %T, through the equation:

A = -log10(%T/100%)

Negative absorbances have meaning and should not be discarded. A negative absorbance means that the the intensity of light passing through the sample is greater than the intensity of light passing through the reference. If the experiment is performed correctly, a negative absorbance may have an important significance.

- Hitoshi.

I also come across negative result for Olson solution (soil samples extracted with sodium bicarbonate to test available phosphorous in the soil). I get stacked and looking possible answer. My spectrophotometer is the JENWAY model 6300.

I will be happy if anyone can help with this problem

Please attach your sample and blank/reference spectra, and let me know the wavelength (range) of interest. Excel format is easiest if you want me to take a close look.

Have you generated a calibration curve?