Sometimes the counts from the light striking a pixel is not sufficiently different from the counts when the pixel is in the dark to make a reliable absorbance reading. This can happen either when the light source is weak in that spectral region, or when the sample is so strongly absorbing that very little light strikes the detector. When this happens, the software may report a blank cell or a value above 3. There could be several reasons for this. Review the tips below for your specific spectrometer type.
VISIBLE ONLY SPECTROMETERS (SpectroVis Plus, Go Direct SpectroVis Plus, Go Direct Visible, etc)
1. The absorbance values for all of your samples need to be between 0.1 and 1.0 absorbance units for best results at all wavelengths of interest. If your samples are too concentrated, they will cause too much noise and result in empty cells or readings of 3. If this is the case, then start by diluting your samples. Why is the absorbance reading on my device (spectrometer/colorimeter) unstable or nonlinear at values above 1.0?
2. The light source in the spectrometer may have burned out or is otherwise not working properly. To test this, switch the spectrometer to Uncalibrated mode and look at the spectrum across the entire wavelength range. If Uncalibrated mode shows flat areas, this indicates that something may be wrong with the light source. Detailed instructions are available here: How do I check the lamp output of my spectrometer?
UV-VIS SPECTROMETERS (Go Direct UV-VIS, Go Direct UV-VIS/Fluorescence, etc)
1. The absorbance values for all of your samples need to be between 0.1 and 1.0 absorbance units for best results at all wavelengths of interest. If your samples are too concentrated, they will cause too much noise and result in empty cells or readings of 3. If this is the case, then start by diluting your samples.
2. Make sure you are using UV-VIS compatible cuvettes. Standard plastic cuvettes are very effective at cutting off UV wavelengths. If you are using standard plastic cuvettes this may be the problem. You need to use UV-VIS compatible plastic cuvettes or quartz cuvettes to fix this problem. Even some UV-compatible cuvettes contribute a lot of noise to this region of the spectrum. Try retaking the data using only quartz cuvettes. For more information about cuvettes available from Vernier, see What is the difference between the types of cuvettes you sell for Spectrometers and the Colorimeter?
3. Consider the solvent you are using for your background measurement. If you are using a concentrated solvent that absorbs in the UV, like nitric acid, the detector will not receive any light, creating a high noise threshold which eliminates reporting of these values. You can test this by taking a background with water and then taking a spectrum of your solvent, if your sample has high absorbance readings, this could be your problem. You can fix this by diluting your solvent or switching solvents.
4. The light source in the spectrometer may have burned out or is otherwise not working properly. To test this, switch the spectrometer to Uncalibrated mode and look at the spectrum across the entire wavelength range. If Uncalibrated mode shows flat areas, this indicates that something may be wrong with the light source. Detailed instructions are available here: How do I check the lamp output of my spectrometer?
You may also be interested in What are some tips for improving my Fluorescence/UV-VIS Spectrophotometer data?
OCEAN OPTICS Spectrometers
Check the tips above for your spectrometer type first. But if you are using an Ocean Optics spectrometer, here are some additional things to consider:
A. Is the latest version of Logger Pro installed on your computer? If you have a new Ocean Optics spectrometer and are running an old version of Logger Pro, Logger Pro may assume that the device is a different type of Ocean Optics spectrometer. This issue has been seen with Logger Pro 3.4. Installing the latest version of Logger Pro will fix the problem. Logger Pro updates are available at: https://www.vernier.com/downloads/
B. If you are using an Ocean Optics device, there may be a chance that you (or a helpful student) accidentally connected a visible detector to a UV-VIS light source. Unfortunately, there is no way to tell from the outside of the box whether it is a UV detector or not. You have to detach the spectrometer from the light source and then plug the spectrometer into Logger Pro. In Logger Pro, choose Set Up Sensors from the Experiment menu. Select your spectrometer and click Restore Defaults. The wavelength range reported tells you whether or not you are attached to a visible detector or a UV/VIS detector.