- Temperature Control
- Automated HTCD
- Automated Titration
- Magnetic CD
- Linear Dichroism
- Optical rotary dispersion
- CD of Solid Samples
- Extended Wavelength
Three single-position Peltier thermostatted cell holders (PTC-510, 514, 517) are available to enable temperature control using the three Spectra Manager II software programs. All feature a temperature setting range of -30 to 130°C with a measurement probe that can be placed inside or adjacent to the cell, as well as a magnetic stirrer to eliminate thermal gradients. These cell holders can accommodate cells of 10, 5, 2, 1 and sub-1 mm pathlengths.
The Multiple-position Peltier cell changer accessories are designed to enable high sample throughput and productivity. The six-position holder for rectangular cells allows for automated spectral scans and parallel thermal ramps on up to six samples at a time. Thermal ramping at single or multiple wavelengths as well as thermal ramping with spectral scans at preset temperatures are also possible.
The MPTC-513 system is compatible with optional fluorescence modes including fluorescence (TFA-555), scanning excitation/emission fluorescence (FMO-522) accessory and fluorescence polarization/anisotropy (FPA-580).
Automated High-throughput CD
Automated high-throughput CD can obtain measurements on up to 192 samples without user intervention, removing the possibility of human error
and saving both time and money.
Recent trends in Combinatorial Chemistry and automated synthesis have lead to various new high-throughput measurement techniques. JASCO developed a high-throughput CD measurement system combining an autosampler, syringe pump and flow cell unit for use with the J-1500 / J-1700.
This system can accommodate two 96-well plates (up to 192 samples) and maintain a constant temperature prior to measurement. The system allows automated scanning measurements at predetermined parameters and/or temperature ramping measurements by using a Peltier thermostatted flow cell. In addition to CD, LD and absorbance data, fluorescence can be simultaneously measured as an option. Software control allows samples to be recovered after measurement for further analysis. The analysis program allows batch processing of data including determination of Tm
and secondary structure analysis.
In the case of samples where only very small volumes are available, two microsampling accessories are now available. The microsampling disk (MDS-462) is designed for sample measurements as small as 2 µL with a 0.2 mm spacer and 10 µL with a 1.0 mm spacer.
For thermal ramping studies of microvolume samples, JASCO also offers a capillary cell jacket (CAP-500), which allows for samples with volumes as small at 5 µL to be placed in a Peltier cell holder. Melting curves are now easily measured, including Tm
, ΔS and ΔH values.
Stopped-flow measurements involve the rapid mixing of two or more solutions to trigger a chemical reaction. The reaction kinetics can then be followed by CD, absorbance and fluorescence. All measurement techniques can be acquired on the same instrument when the stopped-flow system is paired with a J-1500 or J-1700 spectrophotometer.
The SFS-600 series is an innovative stopped-flow measurement accessory with a modular design that allows the flow cell unit to be easily installed and removed from the sample compartment without alignment. Two-, three- and four-syringe models are available, offering flexible mixing as well as upgradeability for quench-flow and T-jump experiments. For temperature-dependent kinetic measurements, the options include Peltier temperature-controlled syringes. Stepper-motor-driven syringes allow variable mixing ratios and a mechanical mixer efficiently mixes solutions commonly used in protein folding experiments.
- Standard 2 mm cell (optional 0.5, 1 and 10 mm cells)
- Standard 10 mL syringe (optional 1, 2.5 and 5 mL syringes)
- 5 mL/sec flow rate with 10 mL syringe
- Exact control of flow rate
- Mixing ratio from 1:1 to 1:20
- Dead time of 2.1 ms with a 0.5 mm cell
- Peltier temperature control ranges from 5 to 80°C and 5 to 60°C for the cell and syringe, respectively
Intrinsic fluorescence can be measured on the J-1500 or J-1700 CD spectrophotometers using the scanning emission monochromator (FMO-522) and emission detector (FDT-538) to acquire fluorescence emission spectra. Alternatively, high-pass filters can be used to select specific excitation and emission wavelengths. This low-cost total fluorescence (TFA-555) approach allows for the detection of fluorescence changes during titration or thermal ramp experiments. However, removing the filter set also allows the user to measure the sample’s 90° light scattering, simultaneously with CD and absorbance data collection.
- With the MPTC-513, CD and fluorescence data can be collected either simultaneously or separately on up to six samples
- Fluorescence scanning can be coupled with the titration and temperature control accessories
Fluorescence-detected circular dichroism (FDCD) is used to measure the difference in fluorescence intensities when an optically active sample has been excited with circularly polarized light. This method takes advantage of the chiral specificities and the structural sensitivities of CD and fluorescence and is more specific than standard CD measurements.
Since FDCD selectivity measures the circular dichroism of a specific fluorescent chromophore in a group of non-fluorescent, chiral molecules, it is particularly useful for the study of proteins, which have multiple chromophores. FDCD can be measured with the standard CD detector when paired with the PTC-510, PTC-517 or MPTC-513 cell holder. When samples have no fluorescence anisotropy, this method is effective because the photoselection artifacts are small. However, when the sample has a larger fluorescence anisotropy, the photoselection artifact will distort the FDCD spectrum. The FDCD-551 attachment is specifically designed to reduce or eliminate these artifacts while greatly enhancing sensitivity due to much more efficient light collection.
Fluorescence polarization anisotropy
Fluorescence anisotropy occurs when polarized light interacts with a fluorescent molecule and the resulting fluorescence emission has different intensities along different polarization axes. The J-1500 and J-1700 CD instruments use circularly polarized light that is generated by phase modulation. By controlling the amplitude of the phase modulation, it is also possible to measure linear dichroism (LD), which is the differential absorption of light polarized parallel and perpendicular to an orientation direction. Using this same principle, fluorescence anisotropy can be measured. Adding the FPA-580 polarizer to the emission optics and utilizing the alternating horizontal and vertical polarization allows for the measurement of fluorescence polarization anisotropy.
The ATS-530 is designed to automatically monitor changes in CD, absorbance and fluorescence as a function of solution pH, chemical denaturant, or exogenous ligands for studies involving protein denaturation or ligand binding. Dual syringes are employed and are each equipped with a valve for automated refilling and flushing during extended runs and for maintaining a constant cell volume. Additionally, the titration measurement program automatically corrects for concentration.
Placing a sample in a magnetic field allows for magnetic circular dichroism (MCD) measurements to be obtained. In protein molecules, MCD can be used as a probe to monitor a chromophore’s local environment. Chromophores with large magnetic moments arising from either rotational symmetries (aromatics, porphyrins), unpaired spins (metal complexes), or both (hemes) are sensitive to electronic perturbations and therefore provide information regarding the molecule’s electronic state. The MCD signal intensity is proportional to the magnetic field strength, which can be applied using either permanent magnets, electromagnets or super-conducting magnets.
- Permanent magnets with field strengths up to 1.6 tesla at ambient temperature
- Electromagnets with field strengths up to 1.5 tesla
- Superconducting magnets with field strengths of 8 tesla
The most versatile approach to orienting macromolecules for LD measurements is the couette flow system. This system subjects the sample to a constant gradient over the annular gap between an inner quartz cylinder, which is rotating at high speed, and a fixed outer quartz cylinder. The CFC-573 Couette flow cell unit includes a built-in beam condenser that focuses the beam onto the small LD flow cell to maximize light throughput. The cell unit is easy to install, remove and clean as needed.
- Small (100 µL) sample volume requirements and 0.5 mm pathlength cell
- Continuous variable spinning speeds up to 7,000 RPM
- Temperature control using an external circulator
Optical rotary dispersion
Optical rotatory dispersion (ORD) provides information on chiral molecules even without chromophores, such as saccharides. It can be used to measure the chirality of non-absorbing samples and the determination of absolute configuration.
JASCO offers two methods of ORD detection: optical null and intensity measurement systems. The intensity measurement method, using a fixed analyzer, is simpler and more economical, while the optical-null approach, with its rotating analyzer, is intrinsically more accurate because the measurement is absolute. Since ORD is very sensitive to strain in the sample cell window, cylindrical cells are strongly recommended for use ORD measurement.
CD of Solid Samples
Samples that are insoluble or may change conformation when in solution have traditionally been difficult to characterize by CD. Diffuse reflectance uses an integrating sphere to effectively analyze these samples. In addition, diffuse transmission measurements of pellets or films is easily accomplished with the same integrating sphere, substantially increasing the collection efficiency for scattering samples and providing a suitable alternative to conventional transmission measurements.
Near Infrared (NIR)-ECD spectra of metallo-protein and metal complexes are known to be sensitive to conformations of the metal’s ligands as well as the configuration around the central metal atom, which is used for structural analysis of these molecules.
The J-1500 CD spectrometer can be used for spectral measurements into the NIR region using an extended wavelength range light source and detector. In this configuration, the InGaAs detector kit includes a halogen lamp unit and allows for measurement up to 1600 nm.