absorbs a sufficient quantity of light within a spectrum of wavelengths that can be considered to be fairly specific for that molecule. If the molecule of interest does not absorb a sufficient quantity of light within the range of the spectrophotometer, or the specificity is not sufficient, then the molecule can be reacted with reagents that will produce another molecule that can meet these criteria. The challenge in Clini- cal Chemistry and much of Laboratory Medicine is to design methods that allow the determination of a molecule of interest in a sea of other molecules that have similar physical and chemical characteristics.
2.4 The Chemistry of the Absorbance of Light
The spectral range that one may utilize to determine molecules of interest is some- what limited. The infrared region of the spectrum begins at 700 nm and extends to 5,000 nm. Molecules absorb light in the infrared region as the result of vibrational motions of atoms as well as bending, rotating and twisting [6]. Infrared spectroscopy is useful in identifying fairly pure solutions of a molecule, because the absorbance is specific for functional groups making up the molecule. One needs to examine absorb- ance versus a fairly wide spectrum and relate the absorbance peaks to known func- tional groups. Sometimes the absorbance spectrum may give way to a ‘fingerprint’
pattern compared to known compounds.
At the other end of the spectrum, with wavelengths below 400 nm, begins the ultraviolet region . The human eye cannot detect ultraviolet light. There are many molecules that absorb in this region; however, the use of the ultraviolet region is limited because of absorbance by materials used to hold the sample, such as glass, and by solvents. Most organic solvents absorb significant amounts of light between 100 and 300 nm [6]. For example, acetone absorbs light at 340 nm and ethanol at 210 nm. Water, the most common solvent for Laboratory Medicine purposes, absorbs at 191 nm [6]. For these reasons, most wavelengths usable in determinations fall between 340 and 700 nm.
Most molecules of interest do not absorb enough light between the ranges of 340–700 nm without some chemical reaction. Usually, as in the case of creatinine , the molecule of interest will shift the absorption band of a reagent, allowing for quanti- fication of the analyte. Bilirubin is unique in that it absorbs light without the need of reaction. Lipemia is unique in that it is the major source of turbidity , a form of light scattering.
As mentioned before, light, a form of electromagnetic radiation can interact with matter. If the light is scattered by the matter, no change in the matter occurs. If the matter absorbs the light, then there is an alteration of the molecule in some fashion.
Radiation with short wavelengths, such as X-rays, when they interact with matter can break chemical bonds. Often, this interaction is destructive. At long wavelengths,
wavelength (nm) 160000
140000 120000 100000 80000 60000 40000 20000 0 -20000
300 350 400 450 500 550 600
250
extinction coefficient
carotene
wavelength (nm) 60000
50000 40000 30000 20000 10000 0 -10000
300 350 400 450 500 550 600
250
extinction coefficient
bilirubin
Fig. 2.2: Absorbance spectra for carotene and bilirubin.
O N N N N O
M P M P V M M V
carotene
bilirubin
Fig. 2.3: Polyene structure of carotene and bilirubin.
2.4 The Chemistry of the Absorbance of Light 17
such as infrared light, nearly all the chemical bonds can absorb the light and the energy is given off in some form of heat, because it represents vibrational energy.
In regards to analytical methodology and spectrophotometry , absorption of visible and near ultraviolet radiation is the most interesting and useful form of absorption.
Molecules absorbing light in this region undergo electron excitation [7]. If the surface of a bulky material is very smooth, it may reflect light, for example, a mirror. If the surface is rough, it will reflect light diffusely. The latter effect is the one we experience in everyday life. Diffuse reflection demonstrates the color of the object [7]. Scattered light , as occurs with a powder, does not demonstrate a color, because there is no interaction with the matter itself [7].
For absorption in analytical chemistry, the most important sources of color for objects are transition metals and transitions between molecular orbitals [7]. The absorption by transition metals is an important facet for the chemistry of minerals and inorganic chemistry and has been used to detect and quantify transition metals in clinical chemistry. They are notable for the absorption of a specific wavelength of light, with the transition of an electron in a d-orbital to a specific higher energy level, which is known as a ligand field effect [7]. The resulting absorption band often is very sharp.
Far more important for the purposes of clinical chemistry are absorption and color in organic molecules . Absorption by organic molecules can be described by the molecular orbital theory [7]. Much of the knowledge concerning color in organic molecules is derived from the study of organic dyes. In dyes, it has been noted that conjugated double bonds , as occur in carotene , give rise to a particular color (Fig. 2.2 and Fig. 2.3).
Dyes possess several resonance structures [7]. In molecular orbital theory, there are several types of orbitals involved, including bonding or antibonding orbitals of sigma (σ or σ*) or pi (π or π*) types, and in addition, n-type nonbonding orbitals [7].
In the lowest energy state of the molecule, all of the bonding and nonbonding orbit- als are fully occupied, while the antibonding orbitals are empty [7]. When the organic molecule absorbs light, the energy of the light now becomes part of the molecule, often described as a transition of orbitals from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), i.e., an electron transfers from a nonbonding or bonding orbital to an antibonding orbital, usually n → π* and π → π* [7]. In formaldehyde the π → π* produces a strong absorption band at 185 nm and the n → π* produces a strong absorption band at 290 nm [7]. Neither of these two bands produce a color in the visible region, but addition of another double bonded carbon to formaldehyde produces acrolein (Fig. 2.4), and shifts the n → π* absorption from 290–330 nm, much closer to the visible region in the spectrum [7]. Addition of another double bonded carbon to acrolein (Fig. 2.4) produces a molecule with yellow absorbance.
Continued addition of double bonds in a polyene fashion produces dyes, such as carotene (Fig. 2.2 and Fig. 2.3). Absorbance of this molecule is predominated by the
π → π* transition [7]. In organic molecules, the p-orbitals are unshielded from electro- static interactions; the electrostatic interactions from other electronic orbitals have their effect on the absorption bands, broadening them out, with the result that the absorption band for organic molecules are typically very broad. Also, in organic mol- ecules, the change in energy levels between the LUMO and the HOMO states depend on the length of the π system in the molecule and the bond length; molecules like bilirubin are rather plastic and there can be small variations in the length of the π system, causing a dispersed population of molecules absorbing light [8].
As the number of double bonds increases in a dye, so does the wavelength of light absorbed by the dye [8]. If the wavelength of light absorbed is 420 nm, then the color of the light absorbed is blue, and the solution appears yellow; if the wave- length is 540 nm, the color of the light absorbed is green and the color of the solu- tion is red; if the wavelength of light absorbed is 640 nm, then the color of the light absorbed is orange and the color of the solution is blue; and if the wavelength of the light absorbed is 740 nm, then the color of the light absorbed is red and the color of the solution is bluish green [8]. As the number of π electrons increases in a polyene, so does the wavelength, with 16–30 p-electrons placing its absorbance in the visible region between 400 and 550 nm [8]. Beta-carotene is a typical example, with a strong orange absorption (Fig. 2.2 and Fig. 2.3). Bilirubin also has a polyene structure with nitrogens in the place of some of the carbons (Fig. 2.3). Carotene has 11 double bonds while bilirubin has ten. In both carotene and bilirubin, the double bonds are laid out in adjacent fashion without ring structure formation. Benzene shows aromatic resonance because it has three double bonds arranged in hexagonal ring structure.
Neither carotene nor bilirubin demonstrate aromatic resonance. Because bilirubin does not demonstrate aromatic resonance as in benzene, it acts as a strong chromo- phore in the visible region of light (Fig. 2.2). The absorbance spectra for carotene and bilirubin are almost identical in the visible region. Bilirubin is not quite as strong as carotene in its molar absorptivity, but both molecules represent important natural dyes.
Polyenes do not need to exist only in a straight chain, but may also occur in cyclic form, and they can represent strong absorbers of light if they do not present with benzenoid conjugation and aromatic resonance [7]. The porphyrins are strong rep- resentatives of this class and include chlorophyll and heme . Of course, bilirubin is the breakdown product of heme. Bilirubin has a strong potential for causing interfer- ence because its absorption band covers a broad area of the spectrum. In the past, if
O C
H H H2C
O C H formaldehyde acrolein
Fig. 2.4: Structure of formaldehyde and acrolein.
2.4 The Chemistry of the Absorbance of Light 19
one directly measured absorbance without a chemical reaction, bilirubin has a strong potential for causing interference, because its absorbance might be measured as well.
This interfering potential is an important problem for co-oximetry, where hemoglobin in its various forms is measured without chemical alteration.
One of the most common ways to minimize interferences with methods is using a sample blank. In a manual spectrophotometer, that process is rather easy, because one would use a sample diluted with buffer as the blank in the analyzer. Automated equipment used to present a challenge, because automated analyzers used a single blank channel for multiple samples. More modern automated analyzers solved this problem by taking multiple readings. Thus, an initial reading is taken after the addi- tion of the sample and the buffer or other diluents, prior to adding the reagents neces- sary to start the reaction. This blank reading for the sample is held in the analyzer’s computer and subtracted from the final readout answer. The multiple readings approach works well, but may be confounded if the blank reading is so high that the final total absorbance is very high. If the final total absorbance reading is very high, it may exceed the photometric error allowed for the assay and result in a flag or code, indicating that there is a problem with the sample [9].
An alternate approach to taking a blank reading is to subtract out a baseline of the interferent. This approach is commonly used to deal with the effects of hemo- globin present in the sample and is mentioned here because its use has the potential to cause problems with bilirubin. This method is commonly referred to as the Allen correction . In the classic Allen correction, in addition to the wavelength chosen for the primary chromogen (usually chosen close to its maximum value of the absorption curve after subtracting out the absorbance for the reagent), two other wavelengths are chosen, one to the left of the chromogen wavelength and the other to the right of the chromogen wavelength (Fig. 2.2) [9]. This method works if the spectrum of the inter- ferent differs from that of the chromogen in the reaction. It assumes that the spectrum of the interferent is approximately the same on both the left-hand side (lhs) and the right-hand side (rhs), and that the chromogen has minimal absorbance at these two wavelengths. The formula for calculating the Allen correction is
(2.5)
In many modern analyzers, a modified version of the Allen correction is used, where only one wavelength is used to adjust for the presence of hemoglobin. Use of only one wavelength may incur effects from other potential interferents, such as bilirubin.
Corrected Achromogen =Achromogen−(Alhs+Arhs)
2 .