UV-Vis Spectrometry – Troubleshooting & DIY

UV-Vis Spectrometry

UV-Vis spectrometry is a simple, but sensitive technique that’s most often used to quantify, and less commonly used to identify. The concept is straightforward: light goes in, and less light comes out. Spectrophotometry is so attractive because of the technique’s sensitivity: its detection is linear across three orders of magnitude. Nearly any sample concentration can be determined by choosing an appropriate dilution. By applying the Beer-Lambert law, analysts can quantify the amount of a chemical present in a sample with very high precision.

Beer Lambert Law © VerdantThe Beer-Lambert Law

By measuring the amount of light that didn’t make it through (absorbance, A). Every chemical has a unique physical property called an extinction coefficient, which is a measure of how much light it absorbs (ε = color intensity in solution). This value is specific to a given wavelength, so this is sometimes written with a subscript, such as ε690. When you combine that information with the amount of sample the light passed through (l – the path length), you can determine an exact concentration (c).

Important! Wavelengths are usually chosen by the absorbance maximum (highest peak), but in advanced cases, different wavelengths can be used to bring the sample’s absorption into the linear range without dilution.

What’s the difference between spectrometry and spectrophotometry?

Not much – the difference is what you do with the information. Spectrometry is evaluating a profile of electromagnetic radiation for a compound across an entire spectrum. That can be an entire range, such as all UV & visible light, or it can be a limited subset like the near infrared. A UV-Vis spectrum can be used to identify compounds in a sample, or tell inorganic chemists about the nature of the bonds in a complex. Spectrometry applies to any part of the entire electromagnetic spectrum.

Spectrophotometry deals with measuring the amount of light being transmitted or absorbed at any point along that spectrum. That enables an analyst to to determine the concentration within a sample, and provides researchers with important information about a sample’s physical properties.

Important! Light is measured in moles of photons. Absorbance is a unitless measurement because the moles cancel during calculation – another name for ϵ is the molar absorption coefficient.

Proper Maintenance

 


Troubleshooting UV-Vis Spectrometry

I don’t see any peaks! (Flat Spectrum)

First, ensure that your lamps are turned on, warmed up, emitting light, and that the light path to the detector is not blocked.

If your UV-Vis uses a monochromator instead of a polychromator & photodiode array, it’s possible that the motor has failed or become stuck. If this is the case you will have a flat graph as it reads every data point to be the same, but it will be offset from the baseline to some extent when running a sample compared to a blank. To confirm, compare a blank to a sample covered in electrical tape, which be read as a sample with a saturated absorbance signal. Motors fail often are not too hard to replace, but this will be a precision servo. Replacing it in the proper alignment can be challenging.

If you can visibly confirm that your lamps are turned on and working, then you may have a problem with the detector. If you have a working monochromator, it’s reasonable to expect that a single photodiode may have failed. But in the case of a polychromator and photodiode array, it’s very unlikely that an entire array failed at once. Trace the wiring inside the instrument and to the computer, checking for loose cables, unseated connectors, or broken/sharply bent wires.

I don’t see any peaks! (Noisy Spectrum)

If

My spectrum is flat on the left or right side! (Spectrum Trails Off Flatly)

If your spectrum begins to flatten toward the left or right side, it means you have a dying lamp. If it’s toward the left side, in the UV range (~200 nm), then it’s your deuterium lamp. If it’s to the right side, in the red light range (~700 nm), then it’s your tungsten lamp. Both lamps emit overlapping wavelengths of light in the center of the spectrum, so that’s why you’ll only see dead zones in the far ends are specific to each lamp.

My spectrum is flat on the left side! (Noisy Left Side)

You’re most likely using the wrong type of cuvette. Plastic cuvettes won’t work because most polymers are linked by a carbon-carbon single bond, which absorbs strongly in the UV range. Fused quartz cuvettes are used because it’s one of the few substances which allows ultraviolet light to pass absorbs ultraviolet light.

 

 

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