What you need to know about Spectrophotometry
Spectrophotometry is an important technique that is widely used to:
- monitor bacterial growth
- determine purity and concentration of RNA, DNA and oligonucleotide samples
- quantitate proteins
- measure enzyme activity
- obtain information about cDNA/fluorophore adjuncts prior to microarray analysis
Spectrophotometry consists in passing through the sample (analyte) a beam of light in the UV/visible wavelength range (190 - 770nm). Typically, the analyte is a solution contained in a transparent container called "cell" or "cuvette".
Light is made up of a stream of photons that can be absorbed once they encounter an analyte molecule. This absorption reduces the number of photons in the original beam of light, thus reducing the intensity of the light beam reaching the detector in the spectrophotometer. This detector measures the intensity of transmitted light, which decreases logarithmically with sample concentration.
Because the extent to which an analyte absorbs photons depends on the wavelength of light, spectrophotometry is performed using monochromatic light where all the photons have the same wavelength.
The unit for spectrophotometric analysis is Absorbance (A), which is defined as:
A = log10 1/T
where T = transmittance, i.e. the proportion of light transmitted from the original beam).
The absorbance varies linearly with both the cell pathlength and the analyte concentration. The pathlength is the distance the light beam travels through the analyte solution and it is normally measured in centimeters. These two relationships are combined to yield a general equation called the Beer-Lambert law:
A = E c l
where c = concentration, l = pathlength and E = Extinction coefficient for that particular molecule at the measurement wavelength. The extinction coefficient changes according to the wavelength of light used in the measurement. The optimum range for absorbance measurements is between 0.1 and 1.0 A.
The first step in the spectrophotometric analysis of a new sample is the determination of its absorbance spectrum. This depicts the relationship between absorbance and various wavelengths of light for that particular sample. The spectrum is a plot of absorbance versus wavelength and it is characterized by the wavelength at which the absorbance is the greatest (lmax).
The value of lmax is important for several reasons;
| 1. | lmax is unique to each analyte and it provides information on its electronic structure. |
| 2. | Analytical measurements made at lmax provide with the highest sensitivity and minimize deviations from the Beer-Lambert law. |
After the determination of lmax for a particular sample, a blank should be measured.
The blank is a control solution that is identical to the sample but devoid of analyte. This measurement is necessary, because both the blank and the cuvette scatter some of the light. The intensity of light passing through the sample is then measured and the reading is adjusted to correct for the blank reading.
Finally, the experimental data for transmittance (T) and the absorbance (A) are used to determine the concentration of the analyte.
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