Using the Cary Spectrophotometer

This document describes the principles of using the Cary Spectrophotometer at the Emory Vaccine Research Center and in the Emory Tetramer Core.

Principles of spectrophotometry

Spectrophotometry is most often used in the biological research laboratory to measure the concentration of some particular substance. There is only one equation that most of you will have to remember, and it is called Beer's Law:

Where Ai is the absorbance (aka OD, or optical density) at wavelength "i", is the "molar extinction coefficient" at wavelength "i", and "l" is the pathlength of the "cell" or cuvette. Since absorbance is a unitless quantity and concentration is usually measured in "moles/liter" and most cuvettes are usually 1 cm in length, the units of are (liters / (mole*cm)).

The importance of absorbance maxima.

Although a substance will have an extinction coefficient at every wavelength, concentrations are typically measured at maxima in the absorbance spectra because this is where the absorbance changes least with changes in wavelength (i.e. the slope of the absorbance spectra is zero).

Spectra versus individual wavelengths

In general, you can use simple wavelength reads in cases such as determining where the peak of a protein coming off of a chromatography column. If you want more information about the quality of your substance, I recommend that you take a full spectrum. When you do take a spectrum, be sure to instruct the software on the spectrophotometer to pick peaks and report the data at the peaks. I also recommend that you save these data files on the spectrophotometer's computer.

The figure below shows a typical protein spectrum. The commonly-found features include: (1) little-to-no absorbance between 310-340 nm; (2) a shoulder at approximately 290 nm; (3) a peak at approximately 280 nm; (4) a trough at approximately 245-250 nm; and (5) a rise between 245-240 nm. For a pure protein, the 260/280 ratio should be between 0.5-0.7.

Simple reads at specified wavelengths

If you wish to determine the concentration of a substance in a number of fractions, you will probably not collect full spectra for each fraction, but instead you will collect data at a few wavelengths, including the approximate absorption maximum.

Never, ever, attempt to measure the concentration of a substance by collecting data at only one wavelength (e.g. at 280 nm for protein solutions). You should always collect data at multiple wavelengths, including one where you expect the absorbance to be zero, and one that will allow you to gain some idea about contaminating substances. For proteins, you should collect data at the following wavelengths: 1) 320 nm, where the absorbance of the protein should be close to zero; 2) 280 nm, near the absorption maximum for most proteins; and 3) 260 nm, which should give you some idea of the level of contaminating nucleic acids or other materials. Note that these are the same wavelengths that should be used to measure the concentrations of DNA or RNA.Use appropriate blanks

In most cases, you'll want to "zero" the spectrophotometer using a cuvette filled with the same buffer as was used for you sample solution. This is particularly necessary if your buffer contains some light absorbing compounds such as DTT or Triton X-100. However, many buffers (PBS, Tris, HEPES) do not contain substances that absorb light in the near UV range that we use most often, and some leeway is permitted here. If you are attempting to measure the concentration of a protein dissolved in a high concentration of a denaturant, your blank should also contain the same concentration of the denaturant.

Dynamic Range and Reliability of Spectrophotometric Data

Although the spectrophotometer will never fail to produce a number, it is essential to remember that those numbers are not always reliable. In particular, the spectrophotometer will not give accurate and meaningful results if the absorbances are either too high or too low.

• Numbers below 0.020. These should be regarded with suspicion, and must be supported with sensible data at additional wavelengths. In some cases, all you want is qualitative information, so it won't matter too much if you get accurate quantitative results. However, where you require accurate quantitative data, you should attempt to either concentrate your sample or dilute it less.
• Numbers above 1.5. The spectrophotometer cannot accurately measure absorbances much above 1.5. If the absorbance of your sample is above 1.5, you should further dilute you sample and measure the absorbance on it again.

Common artifacts

The following are the most common causes of artifacts in spectrophotometric measurements:

1. Air bubbles in the cuvette. Microcuvettes are particularly susceptible to collection of air bubbles in the sample beam. You should always make sure that there are no air bubbles in the sample beam.
2. Particulates in the sample. Particulate matter in the sample will scatter light and result in absorbance readings that are significantly higher than the true reading. Note that particulates are particularly likely to arise if you attempt to dilute denaturant (urea, guanidinium chloride) solubilized proteins into solutions not containing denaturants; this should be avoided, as noted below.
3. Contaminating substances in diluents. Be very careful about the presence of light absorbing substances in the solutions you use to dilute the substances that you wish to measures. One substance that is often forgotten is DTT, which will absorbe UV light when oxidized to the disulfide form.
4. Incomplete mixing of samples can yield improper results. Although this seems like a no-brainer, it can be a matter of serious concern when you are preparing sample dilutions with small volumes (e.g. 2 µl into 98µl).
5. Dirty cuvettes. The cuvettes should be clean on both the outside and the inside. The laboratory should have a cuvette washer, and cuvettes should be washed and then rinsed with clean, high quality solvents.
6. Residual liquid in the cuvette before addition of the sample. This will cause either a) dilution of the sample, resulting in an artifactually low reading; or b) contamination of the sample with a solution containing a "high absorbing" compound, resulting in an artifactually high reading.

Measuring the concentration of denaturant solubilized proteins

Dilute denaturant solubilized proteins only into denaturants. Otherwise, your samples will likely precipitate and you'll end up measuring light scatter rather than the absorbance of your sample. Urea solutions, in particular, should be freshly prepared.

Measuring absorbance of fluorescent compounds, including fluorophore-labeled antibodies

Know the absorbance of you fluorophore, which can be found somewhere in the literature or on the web. The Molecular Probes web site (http://www.probes.com) contains fluorescence spectra for a very large number of compounds).

Protocol: Using the Cary Spectrophotometer

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