Units of CD Measurement

CD is reported in units of absorbance or ellipticity. Each of these can be normalized for molar concentration of the sample. The most direct data from the Olis CD instrument is absorbance (Abs(L)-Abs(R)). This value is typically reported in milliabsorbance units (mA), which are a thousandth of an absorbance unit.

CD data are also reported as ellipticity (), which is related to absorbance by a factor of 32.98 = 33.98 Abs). Ellipticity is usually reported in millidegrees (mdeg or m°), which are a thousandth of a degree.

Molar ellipticity ([]) is CD corrected for concentration. The units of molar ellipticity are historical (deg×cm²/dmol). Conversion from molar extinction (absorbance corrected for concentration) to molar ellipticity uses a factor of 3298 ([] = 3298). To calculate molar ellipticity, the sample concentration (g/L), cell pathlength (cm), and the molecular weight (g/mol) must be known. If the sample is a protein, the mean residual weight (average molecular weight of the amino acids it contains) is used in place of the molecular weight, essentially treating the protein as a solution of amino acids.

Nitrogen Purging

The function of purging the CD instrument with nitrogen is to remove oxygen from the lamp housing, monochromator, and the sample chamber. The presence of oxygen is detrimental for two reasons. When deep ultraviolet light strikes oxygen, ozone is produced. Ozone causes degradation of optics and can cause respiratory problems. The second reason for removing oxygen is that oxygen absorbs deep UV light, thus reducing the light available for the measurement. To prevent ozone production, the lamp and monochromator are purged with at least 8 L/min of nitrogen (each) when the lamp is on. When measuring CD below 200 nm, increase the flow to the lamp housing, monochromator, and sample chamber¹.

Sample Concentration Effects

Fig. 3
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In a dual beam CD, no concentration calibration is required because both LCPL and RCPL are acquired simultaneously, allowing the direct acquisition of Abs(L)-Abs(R). CD signals obey Beer's law, CD intensity is proportional to the concentration of the active species, so it is tempting to improve the concentration of °sample to increase the signal to noise. This strategy is not always useful, as the signal to noise is a function of the signal strength and the overall light intensity passing through the sample to the detectors. Since absorbance must occur at the CD active wavelengths, increasing the concentration also increases the overall absorbance, thus reducing the amount of light reaching the detectors. This necessitates the need for higher PMT High Volts, which, in turn, increases the noise. The relationship between absorbance and signal to noise ratio is illustrated in Figure 3². Note that there is an optimum absorbance to use (Abs = 0.89). For a 1 mm pathlength cell, this absorbance is achieved with a protein concentration of about 0.1-0.3 mg/mL.

Fig. 4
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The optimal protein concentration is a function of the pathlength of the cuvette. Figure 4 shows a plot of the protein concentration required to produce an absorbance of 0.5³. This is lower than the optimal 0.9 to account for absorbing buffer components. This plot indicates an optimal protein concentration of approximately 0.1 mg/mL for a protein solution, although slightly higher concentrations may be used if buffer absorbance is minimized.

In addition to the sample, buffers and oxygen also absorb UV light. Oxygen can be removed by purging with nitrogen. Buffer components (especially those that contain carboxylates) that have a high absorbance below 200 nm must be avoided (e.g. Tris, dithiothreitol, imidazole, histidine, choloride, etc.). Table 1 describes the cutoff wavelength (wavelength in which Abs=1 for a 1 mm cuvette) for common solvents and buffer components⁴. Cuvettes with short pathlengths are better, since the amount of buffer the light travels through is minimized. The disadvantage of short pathlength cuvettes is the higher concentration of protein which must be used (increased 10-fold relative to a 1 cm cell). Ideally, the buffer concentration is low (< 10 mM).

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