Uv Visible Spectroscopy Assignment

B Spectral Properties of Recombinant Cone Pigments

Human trichromatic color vision, at the level of the photoreceptor cell, requires the presence of three cone pigments with broad overlapping spectral absorption. Three genomic and cDNA clones encoding the opsin apoproteins of these pigments were cloned and characterized (76). The amino acid sequences of these opsins are about 41% identical to that of human rhodopsin. The green and red opsins are about 96% identical to each other and about 43% identical to the blue opsin. Previously, the spectral properties of human cone pigments had been studied by a variety of techniques ranging from psychophysical color matching to microspectrophotometry (7). Recently, however, the human cone pigment genes were expressed in tissue culture cells, reconstituted with 11-cis-retinal, and studied by UV–visible spectroscopy (77, 78). The λmax values reported in the two studies were as follows: blue, 426 nm; green, 530 nm; red, 552 and 557 nm for polymorphic variants (78); and blue, 424 nm, green, 530 nm; and red, 560 nm (77). These studies confirmed the assignments based on genetic analysis of the cloned pigment genes.

Analysis of the arrangements of the cone opsin genes on the X chromosome has led to a detailed understanding of the molecular genetics of inherited variations in color vision (79). In males with normal color vision it was proposed that a single red opsin gene resides with one or more green opsin genes in a head-to-tail tandem array. Thus, in one type of color vision defect, anomalous trichromacy, unequal intragenic recombination can theoretically result in an opsin gene that is a hybrid between green and red opsin genes. It was proposed that these hybrids would have anomalous spectral properties (79, 80). This hypothesis was confirmed experimentally at the level of the photoreceptor pigment by obtaining absorption spectra for heterologously expressed hybrid pigments responsible for anomalous trichromacy (81).

Quantitative polymerase chain reaction (PCR) methods have been applied to evaluate in more detail the numbers and ratios of middle- and long-wavelength-absorbing opsin genes in males with normal color vision. It was found that many subjects had more numerous opsin genes than previously suggested, and that in many cases more than one long-wavelength opsin gene was present on the X chromosome (82, 83). The molecular genetics of blue cone monocromacy has also been investigated (84). A genetic model to account for the absence of the green and red genes has been tested in transgenic mice (85). The results suggest that a conserved 5' region interacts with the green or red gene promoter to determine which gene is expressed in a given cone cell.

Sequence information has been obtained from PCR to allow predictions about the identity of specific amino acids involved in human red–green color vision (86, 87). New World monkeys, such as marmosets (Callithrix jacchus), tamarins (e.g., Saguinus fuscicollis), and squirrel monkeys (e.g., Saimiri sciureus), have dichromatic vision with a single long-wavelength pigment. However, there is a striking polymorphism in the pigment, such that females with two X-linked opsin genes are effectively trichromatic. Using pairwise comparisons of opsin gene sequences from a number of individual male monkeys with a range of pigment λmax values, a model was proposed in which three amino acid residues account for the spectral variation between human green and red cone pigments: positions 180, 277, and 285 (86). This model was tested by introducing mutations at these sites in bovine rhodopsin (88) and by expression of mutant and hybrid human pigment genes (81, 89). Thus, of the 15 amino acid differences between human green and red pigments, three hydroxyl-bearing amino acid residues were suggested to be predominantly responsible for the spectral shift: Ser180, Tyr277, and Thr285(81, 86, 88). A more complete analysis of the molecular determinants of red/green color vision was carried out using site-directed mutant pigments and chimeric pigments (90, 91). It was found that of the 15 differences between the 364-amino acid human green and red pigments, only seven amino acid changes were responsible for the observed 31-nm red shift in going from the green to the red pigment: Tyr116 to Ser, Ala180 to Ser, Thr230 to He, Ser233 to Ala, Phe277 to Tyr, Ala285 to Thr, and Phe309 to Tyr.

Red and green visual pigments have also been shown to bind chloride ions, resulting in a large red shift in their absorption maxima. An extensive mutagenesis study of 18 different positively charged amino acids identified His197 and Lys200, in the extracellular loop linking TM helices 4 and 5, as the chloride ion binding site in these pigments (91). These residues are conserved in long-wavelength-absorbing opsins but not in short-wavelength-absorbing opsins or rhodopsins, suggesting that the ability to bind chloride was a key event in the evolution of color pigment genes.

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3.8.7 UV-visible spectroscopy

General principles

Absorption of incident radiation by bonding/non-bonding electrons represents a high energy (~100 kCal/ mole) transition. This corresponds to a high frequency, i.e. low wavelength, absorption band which is observed at 200 ~ 800 nm in the UV and visible range of detection. In solution, electronic absorption spectra are found with broad, generally unresolved bands. These contrast with the vibration fine structure in the vapour phase and with a series of sharp peaks within a continuum in non-polar solvents.

For a solution of an absorbing substance, an absorptivity ratio at a monochromatic wavelength is defined as: {(incident light, Io)/(transmitted, I)} and this is logarithmically related to concentration and optical path-length by the Beer Lambert law: Absorbance (A) = log10(Io/I) = k.c.l., where c mg/ml is the concentration of solute and 1 cm is the distance travelled between parallel optical faces of a suitable cell, and k is a proportionality constant. It is frequently convenient to normalise to a concentration c = 10 mg/ml [i.e. 1] and l = 1 cm, which is expressed as the specific absorbance [A11cm]. Molar absorptivity is defined by the coefficient ε = Mr.(A/cl), and is related to the relative molecular mass, Mr. This coefficient is computed for each wavelength maximum, and also at minima if this is of diagnostic value. It may be useful in showing relationships within an homologous series.

For light source emissivity, the common radiation source is a deuterium lamp covering the operating range 180~ 350 nm and supplemented by a tungsten filament lamp in the near UV, through the visible, into the near-IR, i.e. over the range 320~1000 nm. Standardisation of equipment and monochromators is necessary to ensure the acceptability of data. The wavelength scale is calibrated with a Holmium perchlorate solution, within a tolerance of ±1 nm below 400 nm and ± 3 nm in the 400~600 range (see British Pharmacopoeia, 1993). The absorbance may be checked with NPL calibrated neutral density filters; or should agree within defined corresponding ‘windows’ with absorbances obtained with a potassium dichromate solution of specified strength, at wavelengths 235, 257, 313 and 350 nm. Stray light is usually checked with a 1.2 potassium chloride solution, where the absorbance for 1 cm path length should exceed 2.0 at 200 nm against a water reference. This solution can be replaced by 1 NaBr or NaI at the more accessible wavelengths of 215 or 240 nm respectively. Glass optics absorb UV light below about 300 nm and quartz systems are used to extend the working range down to 200 nm, and even to 185 nm if there are high quality optics and stray light control. At lower wavelengths, absorption of UV-radiation by air requires the use of vacuum systems in research instruments. For practical UV-vis spectrophotometry, the effective working range is 200~800 nm.

Operating conditions

Selection of a suitable solvent is influenced by the wavelength expected to be studied. Water and the lower (polar) alcohols, through diethyl ether and dioxan to nonpolar cyclohexane and light petroleum (‘aromatic free’ in a spectroscopic grade) can be used above 190 nm, whereas chloroform absorbs below ~245 nm. The table below provides a list of cut-off wavelengths. Measured absorbances should be less than 0.4 relative to air using prism monochromators; but higher absorptivity ratios are favoured with modern instruments.

The choice of cells depends on the target range. Silica is essential for measurements at UV wavelengths but glass is acceptable in the visible region; air must be evacuated below ~200 nm. The matched pair required for test solution and solvent reference path should demonstrate effectively identical absorbance when filled with the same solvent. The optical faces of the cells should be parallel; the absorbance of a matched pair of cells containing the same solvent should not differ by more than 0.005 units. In quantitative work all solutions should be at the same temperature; conveniently, they are transferred from a waterbath at, say, 20° and the absorbance measured immediately. Sensitivity of the solution to laboratory and natural lighting should be established in a pilot experiment. One test (British Pharmacopoeia, 1993) of Resolution Power is the discrimination of adjacent maximum (269 nm) and minimum (266 nm) light absorption of toluene with a ratio not less than 1.5.


Quantitative procedures

UV photometry is a frequently used assay technique. Provided that proper calibration checks are maintained, the UV-vis technique is particularly useful for assay of formulations after extraction or separation of the active substance by suitable chromatography. In the assay calibration, there may be some deviation from Beer’s Law. This may be attributable to association in solution or an effect of slit width. The latter should be large enough to gain a reasonable I-value but remain small compared with the (half-) bandwidth for the absorption measured. If in doubt, reduce the slit width slightly and check if the apparent absorbance increases. UV photometric data can also be of value in determining the kinetics of a process, or in following a reaction sequence, such as the disappearance of an absorption peak representing starting material.

Light absorption’ measurements also provide a semi-quantitative test of identity. This relies on the specific absorbance (defined above as the A1cm1 value), or sometimes absorbance at a nominated concentration and path length, either exactly at a specified wavelength, or at the absorption maximum close to a named wavelength. If this test is used as the principal assay of a substance in a formulation, it is advisable to use an authenticated reference substance rather than rely on a published A11. The absorption spectrum may be sensitive to control of pH. Chromophores involving an acidic or basic group will be affected by pH, e.g. the bathochromic shift (to longer wavelength) and hyperchromic peak (greater intensity) of phenates compared with their parent phenol. This is a useful test for a phenolic system.

In subtractive spectrophotometry, the difference between two (or more) spectra measures multicomponent mixtures and is especially useful in formulated product assays. This should be distinguished from the use of second derivative spectroscopy, in which there is computer differentiation of the algebraic function equivalent to the change of slope (i.e. second differential) of the digitalised spectrum (British Pharmacopoeia, 1993). This display sharpens separation of individual UV bands and thereby facilitates lower levels of control. In other applications of computer-aided spectroscopy, modern equipment will provide ‘smoothing’, deconvolution and regression (least squares) analysis.

General chromophores

Absorption bands are particularly evident for conjugated -bond systems. Most single bond transitions are inaccessible, being derived from higher energy σ-orbitals, with wavelengths below 185 nm, i.e. in the ‘vacuum ultraviolet’. Many isolated triple bonds also absorb below 185 nm. The CC (~185) and CN (~190) double bonds exhibit strong –* interactions but unless there is very good control of stray light, measurement is still unreliable in this region. At longer wavelengths there are rather weak n−* interactions, such as NO and keto CO in the range 280~300 nm. Simple benzene compounds show medium intensity multiplets around 254 nm for non-conjugated derivatives, and shifted to longer wavelengths when substituents are conjugated to the aromatic system. In the table at the end of this section there are examples of commonly encountered chromophores, including conjugated alkene, carbonyl and aromatic systems which exhibit bathochromic (longer wavelength) and hyperchromic (enhanced absorptivity) changes. For very extensive catalogues of individual UV spectra, refer to DMS (1960–1971) (1160 substances), Hirayama (1967) (8500 selected values) and Sadtler (1979) (2000 spectra for 1600 compounds). Older spectra of specifically aromatic compounds were collated by Friedel and Orchin (1951). Schemes such as Woodward’s ‘Rules’ (1941, 1942), as further modified by L. and M. Fieser, for conjugated polyenes and en-ones, have considerable predictive power. (For a fuller discussion, see Scott (1964).)


References

British Pharmacopoeia (1993) Appendix IIB, A88-A89, HMSO, London.
DMS (1960–1971) Documentation of Molecular Spectroscopy: UV Atlas of organic compounds, Butterworth,
   London.
R. A. Friedel and M. Orchin (1951) Ultraviolet spectra of aromatic compounds, Wiley, New York.
K. Hirayama (1967) Handbook of UV & visible absorption spectra of organic compounds, Plenum Press.
Sadtler (1979) Handbook of UV spectra, Heyden, London.
A. J. Scott (1964) Interpretation of UV spectra of natural products, Pergamon Press, London.
R. B. Woodward (1941, 1942) J. Amer. Chem. Soc., 63, 1123 and 64, 72.


Solvent cut-off wavelengths

In this table, approximate wavelengths (nm) are specified below which the solvent absorbance may be unacceptable. For quantitative work, the cut-off may be set at a wavelength (L0) where the absorbance for 10 mm pathlength of the solvent exceeds 0.05 absorbance unit (relative to water), i.e. A1 cm > 0.05. For qualitative work, it may still be feasible to work at significantly lower wavelengths and most analysts accept a cut-off based on the wavelength (L1) for A1 cm > 1.0. However, if the UV absorption curve rises steeply, the accessible wavelength range may not be greatly extended.

 

L0

L1

 

L0

  L1

 

 

 

   

 

 

Alcohols

 

 

  Halocarbons (contd)

 

 

methanol

240

205

  1,2-dichloroethane

250

230

ethanol

240

205

  tetrachloroethylene

320

290

n-propanol

250

210

  trichloroethylene

  >400    

 

2-propanol

240

205

   

 

 

n-butanol

245

215

  Miscellaneous

 

 

s-butanol

285

260

  acetonitrile

200

190

isobutanol

250

200

  NN-dimethylformamide

300

270

 

 

 

  dimethylsulphoxide

330

285

Esters

 

 

  nitromethane

  >400    

380

ethyl acetate

280

260

  pyridine

345

325

n-butyl acetate

275

255

  water

190

185

   

   

  

Ethers

 

 

  Alkanes

 

 

diethyl ether

255

220

  pentane

230

200

p-dioxane

290

220

  hexane

225

195

tetrahydrofuran

280

220

  heptane

230

200

2-methoxyethanol

270

200

  cyclopentane

220

195

2-ethoxyethanol

280

210

  cyclohexane

235

200

1,2-dimethoxyethane

300

220

  2,2,4-trimethylpentane

 

 

 

 

 

         [‘isooctane’]

 230

210

Ketones

 

 

  decalin

 250

230

acetone

340

330

   

 

 

butan-2-one [MEK]

345

330

  Aromatic hydrocarbons

 

 

4-methylpentanone [MIBK]

375

335

  benzene

 295

280

5-methylhexanone [MIAK]

350

330

  toluene

 315

285

 

 

 

  chlorobenzene

 310

285

Halcocarbons

 

 

  1,2-dichlorobenzene

 350

295

chloroform

260

240

  o-xylene

 325

290

dichloromethane

245

230

  1,2,4-trichlorobenzene

 350

?

 

 

 

   

 

 


UV absorption bands for typical chromophores

For nonconjugated -systems, the bands may be inaccessible for conventional spectrometers. Conjugated alkene, carbonyl and aromatic systems are at longer wavelengths and more intense.

Chromophore

Formula

Typical wavelength nm (ε at maximum)

 

 

185

200

215

230

245

260

275

290

320

350

 

 

 

 

 

 

 

 

 

 

 

 

Nitrile

—CN

< 180

 

 

 

 

 

 

 

 

 

Ethyne

—CC—

< 180

 

 

 

 

 

 

 

 

 

Sulphone

—SO2

180 (v.)

 

 

 

 

 

 

 

 

 

Ether

—O—

185 (1000)

 

 

 

 

 

 

 

 

 

Oxime

—NOH

190 (5000)

 

 

 

 

 

 

 

 

 

Ethene

—CC—

190 (8000)

 

 

 

 

 

 

 

 

 

Thiol

–SH

 

195 (1500)

 

 

 

 

 

 

 

 

Amine

—NH2

 

195 (3000)

 

 

 

 

 

 

 

 

Bromide

—Br

 

 

210 (300)

 

 

 

 

 

 

 

Iodide

—I

 

 

 

 

 

260 (400)

 

 

 

 

Ketone

> CO

 

195 (1000)

 

 

 

 

275 (25)

 

 

 

Ester

—CO . OR

 

205 (50)

 

 

 

 

 

 

 

 

Carboxyl

—CO . OH

 

205 (60)

 

 

 

 

 

 

 

 

Aldehyde

—CH . O

 

 

210 (1000+)

 

 

 

 

290 (20)

 

 

Sulphoxide

—SO—

 

 

210 (1500)

 

 

 

 

 

 

 

Nitro

—NO2

 

 

210 (s)

 

 

 

 

 

 

 

Nitrite

—O—NO

 

 

 

225 (1500)

 

 

 

 

 

> 300 (w)

Nitrate

—O—NO2

 

 

 

 

 

270 (10)

 

 

 

 

Azo

—NN—

 

 

 

 

 

 

 

 

> 290 (w)

 

Nitroso

—NO

 

 

 

 

 

 

 

 

300 (100)

 

        
Conjugation [n is no. of substituents on conjugated system]

 

 

 

 

 

 

 

 

Nitroethene

CC—NO2

 

 

 

230 (9000)

 

 

 

 

 

 

acyclic Diene

CC—CC

 

 

[217 + 5n]

 

 

 

 

 

 

 

 

 

 

 

  (20 000)

 

 

 

 

 

 

 

heteroannular Diene

 

 

 

 

[224 + 5n]

 

 

 

 

 

 

 

 

 

 

 

 (s)

 

 

 

 

 

 

homoannular Diene

 

 

 

 

 

[253 + 5n] (s)

 

 

 

 

 

Enimine

CC—CN

 

 

 

220 (22 000)

 

 

 

 

 

 

, β-Enone

CC—CO

 

 

 

 

[215 + 12n]

 

 

 

 

> 300 (w)

 

 

 

 

 

 

  (15 000)

 

 

 

 

 

β,' β'-Dienone

(CC—)2 > CO

 

 

 

 

240 (30 000)

 

 

 

 

 

Triene

CC—CC—CC

 

 

 

 

 

260 (35 000)

 

 

 

 

βδ -Dienone

CC—CC—CO

 

 

 

 

 

 

 

 

 

 

Tetraene

(CC—)4

 

 

 

 

 

 

 

 

300 (50 000)

 

            

Aromatics

 

 

 

 

 

 

 

 

 

 

 

Benzene

Ph-H

184 (46 000)

 

200 (7000)

 

 

256 (180)

 

 

 

 

Carboxyl

ArCOOR

 

 

 

230 (?)

 

 

 

 

 

 

Phenol

ArOH

 

 

 

 

 

 

280 (?)

 

 

 

Naphthalene

 

 

 

 

220 (110 000)

 

 

275 (5500)

 

310 (180)

 

            

G.F. Phillips

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