Encyclopedia of Biophysics

Living Edition
| Editors: Gordon Roberts, Anthony Watts, European Biophysical Societies

Absorbance Spectroscopy: Spectral Artifacts and Other Sources of Error

  • Paul WormellEmail author
  • Alison Rodger
Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-35943-9_776-1
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Synonyms

Definition

Modern spectrophotometers and data acquisition software are usually reliable and easy to use, but the spectra that they produce must always be regarded critically, as there are many possible sources of error and measurement uncertainty. Some of these relate to sample purity and identity. Others relate to the quality and calibration of the instrument, experimental technique, choice of sample cells and wavelength ranges, correct use of software for data acquisition and analysis, appropriate scan parameters that optimize signal-to-noise ratios yet ensure the fidelity of the recorded spectrum, and uncertainties associated with data analysis (e.g., the use of standard curves). A good understanding of spectroscopic principles, and proper instruction and supervision in the use of spectroscopic instrumentation, is essential for reliable spectroscopic work. Other factors and artifacts may also affect the reliability of absorption, linear dichroism (LD), and circular dichroism (CD) spectra, as discussed below. These may lead to distorted spectral band shapes, deviations from the Beer–Lambert law, and unreliable LD and CD data.

Basic Characteristics

A number of issues that affect absorbance data reliability but may not be apparent upon inspection of a spectrum or calibration of an instrument are discussed below.

Solvent Absorption

In absorption spectrophotometry, most samples are dissolved in a solvent, although some spectra are recorded of pure solids or samples that are incorporated in polymer films. The choice of solvent is often determined by sample solubility and stability. For biomolecules, buffers are often used to manage these factors and to ensure that the correct ionic or neutral form of the sample is being analyzed. Some solvents (e.g., pure water and n-pentane) are effectively transparent at wavelengths down to 200 nm, but others are not. A summary of solvent cutoffs in a 1-cm cuvette is given in Table 1. Cutoffs in practice depend on the purity of the solvent as even small percentages of impurity may be present in high molar concentration. Buffers are a more complicated issue as it depends on the concentration required to ensure buffering capacity. A summary of some components as provided by Kelly et al. (2005) is given in Table 2. Reducing the path length or the concentration of the buffer extends the accessible spectral range.
Table 1

Wavelength at which the pure solvent absorbance in a 1-cm path length cuvette is 1 (arranged in order of increasing absorbance) according to Honeywell Burdick and Jackson. An absorbance of 1 is an effective cutoff for solvents since most instruments become unreliable between absorbances of 2 and 3 and some sample absorbance must be considered

Solvent

Wavelength at which A = 1 in 1-cm path length

Acetonitrile UV

190

Pentane

190

Water

190

Hexane UV

195

Cyclopentane

198

Cyclohexane

200

Heptane

200

Isopropyl alcohol

205

Methanol

205

Ethyl alcohol

210

n-Propyl alcohol

210

Trifluoroacetic acid

210

Tetrahydrofuran UV

212

n-Butyl alcohol

215

1,4-Dioxane

215

Ethyl ether

215

Iso-octane

215

n-Butyl chloride

220

Isobutyl alcohol

220

Ethylene dichloride

228

1,1,2-Trichlorotrifluoroethane

231

Dichloromethane

233

Chloroform

245

Ethyl acetate

256

N, N-dimethylformamide

268

Dimethyl sulfoxide

268

Toluene

284

o-Xylene

288

o-Dichlorobenzene

295

Acetone

330

Table 2

Absorbances of selected buffer components at 1 mM in a 1-cm path length cuvette. Tris = tris(hydroxymethyl)aminomethane. (Note: TBS often denotes Tris-buffered saline, which is a mixture of 50-mM Tris and 50-mM NaCl adjusted to pH 7.6 with HCl; it is a mixture of components all of which must be taken into account.) HEPES = 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid. MES = 2-(N-morpholino) ethanesulfonic acid (Kelly et al. 2005)

Component

180 nm

190 nm

200 nm

210 nm

NaCl

>0.5

>0.5

0.02

0

NaF

0

0

0

0

NaClO4

0

0

0

0

Boric acid

0

0

0

0

Na borate (pH 9.1)

0.3

0.09

0

0

Na2HPO4

>0.5

0.3

0.05

0

NaH2PO4

0.15

0.01

0

0

Na acetate

>0.5

>0.5

0.17

0.03

TrisH2SO4 (pH 8.0)

>0.5

0.24

0.13

0.02

HEPES/Na+(pH 7.5)

>0.5

>0.5

0.5

0.37

MES/Na (pH 6.0)+

>0.5

0.29

0.29

0.07

Although, in principle, a double-beam spectrophotometer can correct for a weakly absorbing solvent if it is placed in the reference beam, spectroscopic accuracy and precision deteriorate when the solvent absorbance increases, and stray light (see below) may also degrade the absorbance readings. In the worst case, both the sample and reference cells are opaque at the analytical wavelength, producing a very noisy and unusable spectrum. It is thus usually better to use air in the reference beam and subtract a buffer baseline manually as then one knows how it is affecting the spectrum.

Light-Scattering Samples

Unless there is no alternative, absorption spectra should not be recorded for turbid samples, as the measured absorbances may be misleading. However, many biomacromolecular species are of comparable size to the wavelength of light, and as a result they scatter light. The situation is worse for linear and circular dichroism as such samples may differentially scatter the two polarizations of light used. The theory of light scattering is complicated, depending not only on the size regime of the particles but also on their shape. Thus, if at all possible, one should avoid it occurring rather than trying to correct for it. In general, scattering can be reduced either by reducing the size of the particles or collecting a high percentage of the scattered photons. Three methods that may enable scattered photons to be collected (and thus to avoid the problem of scattering) are to (1) have a wide-angle photomultiplier tube (PMT), (2) place the sample very close to the photomultiplier tube, or (3) have a collecting lens close to the sample to refocus the scattered light onto the PMT.

However, it is not always possible to remove light-scattering artifacts. In such a case, the measured spectrum is a combination of true absorbance and scattering. The scattering contribution is usually apparent as a sloping baseline outside absorbance bands. Nordh et al. (1986) showed that a simple empirical correction can often be subtracted from an observed linear dichroism spectrum to remove the sloping baseline (Meeten 1981).

$$ {LD}^{\mathrm{Scattering}}=a{\lambda}^{-k} $$
(1)

However, a more complete treatment as outlined by Hulst (1981) may be required and is challenging.

The same approach may also work for other spectroscopies.

Stray Light

In addition to any light scattering from the sample, so-called stray light (or stray radiant energy) from anywhere in the system may cause additional artifacts especially at lower wavelengths where lamps struggle for light intensity. In such a case, a significant percentage of the light that does reach the photomultiplier detector may be stray light rather than light of the correct wavelength that has passed through the sample. However, all photons get counted as unabsorbed photons at the monochromator’s nominal wavelength, causing the detector to give a misleading indication that there is less absorption than there really is. The main problem is due to imperfections of the monochromator: long-wavelength stray light bypasses the monochromator. This limits the maximum absorbance at which an instrument will give reliable readings. If the spectrum is “true,” it will follow the Beer–Lambert Law (A = ϵCℓ). A sharp drop-off in intensity on the low wavelength side of a band is often indicative of this problem.

Absorbance Flattening

The phenomenon of absorption flattening is a suppression of the absorbance signal in regions of high absorbance in nonhomogeneous samples causing the Beer–Lambert law to break down. The same issues apply to linear and circular dichroism. To understand this phenomenon, consider the extreme case where half the light beam passes through pure solvent and half passes through a more dense solution of analytes. Half of the incident photons (Io/2) will thus reach the PMT directly, while the other half will pass through a solution of higher absorbance. The percentage transmittance of the sample is then
$$ T=100\%\times \left(\frac{I_o+I}{2{I}_o}\right) $$
(2)
where A = log10(Io/I) is the absorbance of the absorbing half of the sample and log10(Io/Io) = 0 is the absorbance of the transparent half of the sample. The apparent absorbance of the sample is thus significantly reduced compared to the true absorbance had the sample been homogeneous since
$$ {A}_{\mathrm{apparent}}={\log}_{10}\left(\frac{2{I}_o}{I_o+I}\right)=-{\log}_{10}\left(\frac{1+I/{I}_o}{2}\right) $$
(3)
Thus, for an absorbance in the denser part of the sample of 3 or greater, A apparentis equal to log10(2) = 0.301. For intermediate and small absorbances, the effect of light leakage is significant as illustrated in Table 3.
Table 3

Absorbance flattening effects for samples where the light passes through a sample of which half has no absorbance and half has the indicated absorbance

A of clear part of the solution

A of dense part of solution

A apparent of inhomogeneous solution

A for the analogous homogeneous solution

Error (%)

0

3

0.30

1.5

80

0

2

0.29

1.0

71

0

1

0.26

0.5

48

0

0.5

0.19

0.25

24

An effect analogous to absorption flattening can be caused by any arrangement that allows a fraction of the light to bypass the sample solution or to pass through a shorter path length of sample. Unusual sample holders such as capillaries are also prone to this problem (Waldron et al. 2010). In some cases, the problem of absorption flattening can be reduced to acceptable levels simply by diluting the whole solution as shown by the lower absorbance lines in Table 3. For membrane systems, one somehow has to remove the local high concentrations, and simple dilution is often not sufficient. Mao and Wallace (1984) have outlined how this may be done by reducing membrane particle size. Unfortunately, the problem cannot always be avoided, in which case methods such as those of Gordon and Holzwarth (1971) can be used to correct measured data.

Photodegradation

While not specifically a spectroscopic artifact, photodegradation of biomolecules in a light beam is a very common cause of a measured spectrum not being the true spectrum. Fast preliminary scans are the best way to identify whether this is a problem.

Cross-References

References

  1. Gordon DJ, Holzwarth G (1971) Artifacts in the measured optical activity of membrane suspensions. Arch Biochem Biophys 142:481–488CrossRefGoogle Scholar
  2. van de Hulst HC (1981) Light scattering by small particles. Dover Publications Inc., New YorkGoogle Scholar
  3. Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751:119–139CrossRefGoogle Scholar
  4. Mao D, Wallace BA (1984) Differential light scattering and absorption flattening optical effects are minimal in the circular dichroism spectra of small unilamellar vesicles. Biochemistry 23:2667–2673CrossRefGoogle Scholar
  5. Meeten GH (1981) Conservative dichroism in the Rayleigh-Gans-Debye approximation. J Colloid Interface Sci 84:235–239Google Scholar
  6. Nordh J, Deinum J, Nordén B (1986) Flow orientation of brain microtubules studied by linear dichroism. Eur Biophys J 14:113–122CrossRefGoogle Scholar
  7. Waldron DE, Marrington R, Grant MC, Hicks MR, Rodger A (2010) Capillary circular dichroism. Chirality 22:E136–E141CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association (EBSA) 2018

Authors and Affiliations

  1. 1.School of Science and HealthWestern Sydney UniversityPenrithAustralia
  2. 2.Department of Molecular Sciences, Macquarie UniversitySydneyAustralia

Section editors and affiliations

  • Alison Rodger
    • 1
  1. 1.Department of Molecular Sciences, Macquarie UniversityNWSAustralia