Most people cringe when they hear a word like â€œspectrometerâ€ (or the closely related spectrophotometer, colorimeter, or spectroscope). Â But if youâ€™re involved in science, you will probably run across one.
A spectrometer is an instrument that breaks light into its component parts.Â By saying â€œlight,â€ I mean almost any kind of electromagnetic radiation.Â It could be radio waves (i.e. nuclear magnetic resonance or electron spin resonance), infrared (FTIR), near infrared (usually called NIR), ultraviolet or visible (UV/Vis or Atomic absorption), or x-rays (EDXRF, Â as well as a plethora of other instruments).Â This includes some of the older medical blood analyzers.Â Here, we will concentrate on ultraviolet/ visible spectrometers, also called UV/Vis.
Letâ€™s talk about how a UV/Vis spectrometer works.Â Â Most science books show the experiment where a ray of light is split into individual wavelengths by passing the beam through a prism.Â A beam of white light is sent into one side of the prism.Â After going through the prism, a rainbow of colors appears on the other side. Â A â€œspectrometerâ€ does the same thing. Â Â It mayÂ use a diffraction grating instead of a prism, a digital camera to identify the wavelengths, and a computer to calculate the results.Â But the goal is the same:Â To identify the intensity and wavelength of each ray of light. A graph showing wavelength vs intensity is called a â€œspectrum.â€ Â A normal UV/Vis spectrometer will plot wavelengths from 800 nanometers (nm) to 200 nm.
To get a better understanding of a basic spectrometer, please read the September 6 article in CSL -â€œKickstarter Project Makes Spectrometers for Affordable for Citizen Scientistsâ€.
For chemical work, a light is put in front of the spectrometer and fed through a chemical substance, usually a water solution.
Very few light sources give a constant output over a wide range.Â For visible light (the Vis part of UV/Vis), tungsten halogen or tungsten lamp works. Â While ultraviolet (UV) work normally uses a deuterium lamp. A tungsten lampâ€™s output varies dramatically with wavelength.Â Often, a filter that absorbs at the peakÂ wavelength makes theÂ output more uniform over the entire visible range.Â Light emitting diodes (LEDs) donâ€™t produce light over the entire spectrum.Â For this reason, they arenâ€™t normally used.Â A typical visible spectrometer covers 800 nm to 400 nm.Â A UV instrument goes from 400 nm to 200 nm.Â Some tungsten lamps provide usable light into the UV (say down to 300 nm).Â Wavelengths below 200 nm are considered part of the â€œfar UVâ€ and are not normally used.
A safety note.Â If you are aligning a UV/Vis instrument, do not look directly into the lamp, especially with deuterium lamps.Â Align the instrument using a screen or by peaking the controls.Â Looking directly into the beams can damage the eyes. Â Â In addition, the deuterium lamps use high voltages and produce hazardous ultraviolet (UV) radiation.
Letâ€™s do a mental experiment.Â Take a piece of glass two inches thick and shine a beam of light through it.Â Do the same thing with a piece of glass half an inch thick.Â Hereâ€™s the question- do you think the same amount of light gets through the two pieces of glass?
This concept is called â€œpath lengthâ€.Â More light will get through the half inch thick glass than the two inch thick glass.Â The thicker the glass, the less light gets through.
For spectrometers, the standard path length is 1 centimeter (cm).
How do you keep water solutions 1 cm thick? By using glass or plastic containers, called cuvettes.Â These cuvettes commonly are made in quartz and plastic.Â The quartz are best, working down to 200 nm (that also means expensive).Â The plastics cut off at about 300 nm, depending on the specific material.Â Each cuvette holds about 3 mls of liquid and are reasonably priced from lab suppliers. Â For medical blood analyzers, the cuvettes are custom made with many sample cells per part.
Two scales are commonly used in UV/Vis work.Â These scales are absorbance (also called optical density or OD) and transmittance.Â Transmittance often optimizes spectral comparison work.Â Thatâ€™s answering the question-are the two spectra the same?Â On the other hand, if you want to know how much is in the liquid.Â This is concentration work and normally uses Â absorbance.
Regardless of the scale used, you need a blank.Â Different instruments generate the blank differently.Â Some take the beam of light and split it into two separate beams (double beam instruments).Â One beam goes through the blank, the other beam goes through the sample.Â Many instrumentsÂ â€œmemorizeâ€Â blanks.Â And many medical analyzers ratio the active portion of the spectrum to an unused, â€œreferenceâ€ (or blank) portion of the spectrum.Â But the result is the same.Â If the blank is messed up, your sample will be, too. Take blanks often and check the blank results.
Fun and Games at work
One part we made was a custom multicell cuvette for a blood analyzer. We used an actual blood analyzer for the testing.Â Â One customer requirement was for the plastic to absorb very little light.Â But they also wanted all of the cells to absorb a similar amount of light (â€œmatchedâ€ cells).Â We started getting complaints on parts that had tested as â€œgoodâ€.
It was a quality inspector who found the problem.Â They noticed that occasionally a cuvette would test good initially, then test â€œbadâ€ when removed from the shipping bag and retested.Â Could something on the polyethylene bag transfer to the cuvette?
I got the job of answering that question.Â I went through many bags, and eventually found one that had a small amount of a waxy material on it.Â The waxy material was analyzed by microscope fourier transform infra-red (FTIR) and spectral library search came up with a low quality hit for erucyl amide (also called erucamide).Â More work was done.Â We sorted out the waxy bags, washed them with a solvent, and dried the solvent on FTIR salt plates.
The salt plates were run.Â This time, we got a high quality spectral hit for erucamide.
The plastic bag manufacturer was asked why they used erucamide to make their polyethylene bags.Â Erucamide is a â€œslip agentâ€.Â The erucamide is applied during blow molding and stops the two sides from sticking.Â While erucamide is needed for molding, the amount was not controlled.Â We happened to get a lot of bags with a large amount.
Only a few of cells of the cuvette touched the bag.Â This caused a large variation from cell to cell, so the cuvette was no longer â€œmatchedâ€.
Who would have thought a packaging bag would have caused this problem?Â Lesson learned- even very minor parts of a process can have a major effect on product quality.