Circular Dichroism Test Chemistry - Steady State Measurements
This test is for calibration only, and will not usually be performed during a service visit. For further details contact techsup@photophysics.com .
5.1 Camphorsulphonic acid (CSA)
Camphorsulphonic acid has been widely used for the calibration of CD spectropolarimeters because it has excellent optical purity and is very stable in solution. However, some care does need to be taken in its preparation in order that it can be used for calibration purposes. The reason for this is that CSA is hygroscopic and this makes it difficult to weigh accurately. However, CSA has been studied in depth and it is known to have a ?e of 2.36 at 290.5nm. From this it has been deduced that a 1mg/mL solution of CSA in water would have an ellipticity of 335 millidegrees when measured in a 10mm path cuvette. As it is known from the literature that the extinction coefficient of anhydrous CSA is 34.5 at its absorption maximum (285nm), this means that the concentration of a 1mg/mL solution can be determined accurately from its absorption characteristics. A solution containing exactly 1mg/mL will have an absorbance of 0.2972 AU in a 20mm pathlength cuvette. Using proportionality, one can then calculate the CD value for a newly prepared CSA solution once its absorbance at 285nm as been accurately determined.
| Test Sample:- | Camphorsulphonic acid (CSA) in water |
| Solute: | (1S)-(+)-10-camphorsulphonic acid |
| Source: | Sigma C-1395 |
| MW: | 232.3 |
| Conc: | 1mg/mL in distilled water |
| Measurement Conditions (PiStar) | ||
| CD Calibration | CD peak Ratio | |
| Wavelength Range: | 260-320nm | 180-320nm |
| Wavelength step: | 0.5nm | 1nm |
| Bandwidth: | 2nm | 2nm |
| Optical Path: | 10mm | 0.5mm (cylindrical) |
| Counts/step: | 5,000 | 25,000 |
| Nitrogen Purge: | no | yes |
Fig 5.1. The frame on the top shows the absorption spectrum of a 1mg/mL solution of CSA measured with a 20mm optical path. The absorption at the 285nm maximum is used to establish the CD of the same solution at 290.5nm. A typical CD spectrum of CSA, obtained using a 10mm optical path and as used for calibration purposes, is shown above.
Fig 5.2 The CD spectrum shown here has been obtained using the same 1mg/ml standard solution but now measured in a 0.5mm cylindrical cuvette. A 5 point Savitzky-Golay smooth has also been applied in order to make the spectrum as clean as possible in the far UV. The ratio of the two CD peaks should be close to 2.08.
5.2 Pantoyllactone
Pantoyllactone is also useful for calibration purposes as it has its CD maximum at a wavelength close to where UV CD measurements are made in protein studies. Its use for calibration purposes is particularly relevant when making stopped-flow CD measurements where the optical characteristics of the stopped-flow cell cause a smaller CD signal in the far UV to be produced relative to the same solution when measured in a regular cuvette. An accurately prepared pantoyllactone solution allows the stopped-flow configuration to be calibrated using the exact operational conditions required for CD kinetic measurements i.e. stopped-flow cell in place, same wavelength region, increased monochromator bandwidth.
| Test Sample:- | Pantoyllactone in water |
| Solute: | Pantoyllactone |
| Source: | Sigma P-2625 |
| MW: | 130.1 |
| Conc: | 0.15mg/mL in distilled water |
| CD value: | -186mdeg at 219nm |
| Measurement Conditions (PiStar) | |
| Wavelength Range: | 190-260nm |
| Wavelength step: | 0.2nm |
| Bandwidth: | 2nm |
| Optical Path: | 10mm |
| Counts/step: | 10,000 |
| Nitrogen purge: | Yes (10L/min) |
Fig 5.3. The CD spectrum of a 0.15mg/mL solution of pantoyllactone as measured in a 10mm optical path. At the wavelength maximum of 219nm, the CD value of a 0.15mg/mL solution should be -186 millidegrees. Calibration using pantoyllactone is recommended prior to stopped-flow CD measurements in the far UV.
5.3 Cyanocobalamin (Vitamin B12) in water
| Test Sample:- | Cyanocobalamin |
| Solute: | Cyanocobalamin or Vitamin B12 |
| Source: | Sigma V-2876 |
| MW: | 1355 |
| Conc: | 0.2mg/mL in distilled water (0.15x10 -3 M) |
| Measurement Conditions (PiStar) | |
| Wavelength Range: | 200-600nm |
| Wavelength step: | 1nm |
| Bandwidth: | 2nm |
| Optical Path: | 0.5mm or 1mm |
| Counts/step: | 35,000 |
| Nitrogen purge: | No |
Fig 5.4 The CD spectrum of a 0.2mg/mL solution of cyanocobalamin (vitamin B12) as measured using a 1mm optical path.
5.4 Protein solutions
| Sample:- | Lysozyme in water |
| Solute: | Lysozyme |
| Source: | Boehringer 1243 004 |
| MW: | 14,400 |
| Conc: | 0.2mg/mL in distilled water |
| Sample:- | Cytochrome c in water |
| Solute: | Cytochrome c |
| Source: | Sigma C-2506 |
| MW: | 12384 |
| Conc: | 0.2mg/mL in distilled water |
| Measurement Conditions (PiStar) | |
| Wavelength Range: | 180-260nm |
| Wavelength step: | 0.2nm |
| Bandwidth: | 2, 3 or 4nm |
| Optical Path: | 0.5mm |
| Counts/step: | 40,000 |
| Nitrogen purge: | Yes (10L/min) |
Fig 5.5. CD spectra for 0.2 mg/mL solutions of lysozyme (top) and cytchrome c (above). These scans were acquired using a 2 nm monochromator bandwidth and a 0.2 nm wavelength increment. The time for each scan was about 8 minutes. No signal processing/smoothing has been applied.
Fig 5.6. CD spectra for 0.2 mg/mL solutions of lysozyme (top) and cytochrome c (above). These scans were acquired using a 4 nm monochromator bandwidth. All other parameters were the same as in Fig 5.5.