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1、When preparing a sample for CD measurements the absorption of light must be consid­ered. For normal light the optical density (OD of the sample is given by the Beer-Lambert law: OD = *l*c, where is the extinction coefficient (OD/cm*Molar, l the path length (cm and c the sample concentration (Mo

2、lar. CD is based on the difference on the absorption of left and right circularly polarized light, so the Beer-Lambert law still applies. The difference though is that instead of the extinction coefficient ( the difference in the extinction coefficients for left and right circularly polarized light

3、is used ( = L R . Therefore the CD signal linearly tracks the sample. From this relationship one would naturally assume that higher sample concentrations will give better CD signals. Unfortunately this case is not true. The is small compared to the abso­lute levels of light. Increasing the conc

4、entration results in less light for the measurement, and the small difference in absorbance can become lost in the noise. Remember that absorbance is the log scale of transmission, which is the true measure of the light energy. An absorbance of 1 means that 90% of the light is being absorbed, 2 mean

5、s 99%, 3 equals 99.9%, and so on. The noise level is not constant but is also a function of the light level, being higher at low light levels. Therefore the signal-to-noise profile peaks at approximately 0.8 OD before deteriorating. The profile is rela­The researcher must consider what is actua

6、lly absorbing the light. The ideal situation is for the sample molecules and nothing else to be the active chromophores. Unfortunately, in the UV and particularly the deep UV (below 250 nm, many things absorb light. This only reduces the amount of light available for the measurement and adds nothing

7、 to the CD signal. When taken to the extreme there is so little light passing through the sample that CD measurements are impossi­ble. This is frequently the case when taking wavelength scans and it is desirable to penetrate the UV as far as possible. Table 1 shows the wavelengths at which a gi

8、ven solvent has an OD of 1. Values are given for optical path lengths of 1.0 and 0.05 mm. For a given solvent and path length wavelengths longer than the ones in the table will have lower absorbances while shorter wave­lengths result in rapidly increasing absorbances. The problem of solvent abs

9、orbance can be reduced by using short path length cuvettes and scaling up the sample concentration. For exam­ple, a 0.5 mm cuvette with 2X the sample concentration will have the same CD signal as a 1X sample concentration in a 1 mm cuvette, but with 1/2 the solvent absorbance.The same absorbanc

10、e problems occur with buffers and salts added to the sample. Table 2 shows the absorbance values for different compounds over a range of wavelengths. The condi­tions are for a 1.0 mm cuvette containing 10 mM solutions. Changing path length or concentration will scale the absorbance linearly. Th

11、e researcher can adjust concentrations and path length to optimize CD measurements.Table 1: Solvent TransparencyCompound Wavelength (nm for OD = 1.0 1.0-mm PathLength0.05-mm Path LengthH 2O 182 176MeOH 195.5 184F 6iPrOH 174.5 163F 3EtOH 179.5 170EtOH 195 186MeCN 185 175Dioxane 231 202.5Cyclohexane 1

12、80 175-Pentane 172 168Table 2: Absorbance of Various Salt and Buffer Substances in the Far-UV RegionCompound pHNoAbsorbanceAbsorbance of a 10 mM solution in a 1.0 mm Cuvette at:Above 210 nm 200 nm 190 nm 180 nmNaClO 4 170 nm 0 0 0 0 NaF, KF 170 nm 0 0 0 0 Boric Acid 180 nm 0 0 0 0 NaCl 205 nm 0 0.02

13、 >0.5 >0.5 Na 2HPO 4 210 nm 0 0.05 0.3 >0.5 NaH 2PO 4 195 nm 0 0 0.01 0.15 Na Acetate 220 nm 0.03 0.17 >0.5 >0.5 Glycine 220 nm 0.03 0.1 >0.5 >0.5 Diethylamine 240 nm 0.4 >0.5 >0.5 >0.5 NaOH pH 12 230 nm >0.5 >2 >2 >2 Boric Acid, NaOH pH 9.1 200 nm 0 0 0.09

14、0.3Table 2: Absorbance of Various Salt and Buffer Substances in the Far-UV RegionCompound pHNoAbsorbanceAbsorbance of a 10 mM solution in a 1.0 mm Cuvette at:Above 210 nm 200 nm 190 nm 180 nmTricine pH 8.5 230 nm 0.22 0.44 >0.5 >0.5TRIS pH 8.0 220 nm 0.02 0.13 0.24 >0.5HEPES pH 7.5 230 nm 0

15、.37 0.5 >0.5 >0.5PIPES pH 7.0 230 nm 0.2 0.49 0.29 >0.5MOPS pH 7.0 230 nm 0.1 0.34 0.28 >0.5MES pH 6.0 230 nm 0.07 0.29 0.29 >0.5Cacodylate pH 6.0 210 nm 0.01 0.20 0.22Selection of the appropriate cuvette is also important. Cuvettes are available from a wide range of materials, not al

16、l of which are suitable for CD measurements. First, the cell should be capable of transmitting light in the range of interest. Table 3 shows the approximate wavelength range for which the light transmission is better than 80%. For CD measurements in the deep UV the best material is QS quartz. At wav

17、elengths below 200 nm all commonly available cuvettes absorb light, so measurements become difficult regardless of the sample characteristics.Table 3: Selection of CuvetteMaterial Transmission >80%QS Quartz 200 - 2500 nmQH Quartz 230 - 2500 nmQX Quartz 200 - 3500 nmOS “Special Optical Glass” 320

18、- 2500 nmOG “Optical Glass” 360 - 2500 nmPY “Pyrex” 340 - 2500 nmMethyacrylate Plastic 300 - 750 nmPolystyrene Plastic 350 - 750 nmSecond, the cuvette should not be mechanically strained. Strain in the cuvette will depolar­ize the light so that the CD baseline is not flat. When buying cuvettes it is possible to specify “Strain Free” cuvettes from the manufacturer, but this specification is not always sufficient.