8. Dry air prevents condensation of the sample windows at temperatures below 5
C. Contributions of water vapor from
ambient air to the spectrum strongly interfere, particularly
with the protein bands.
9. It is recommended to select an automatic CO 2 /H 2 O correction when ATR studies are done. For transmission studies, the
automatic correction is not needed if both background and
sample are recorded after sufficient purging of the sample area.
10. The iris opening can be adjusted if needed.
11. Make sure that both background and sample spectra are
recorded after sufficient purging of the sample area (minimally
5 min). The disappearance of water vapor after purging is
particularly visible in the 1800–1500 cm
À1 region (see Fig. 1).
12. To avoid leakage, the diameter of the tissue sample should be
slightly larger than the opening of the hole in the tube used for
positioning the sample on the ATR crystal. Silicon-free grease
can be used at the outer edges after placing the sample holder
with the tissue on the ATR crystal to avoid leakage. Care
should be taken to prevent contact between the tissue and
the grease.
13. If the first spectrum already contains strong bands of the
compound in the protective solution, the solution has leaked
from the sides and directly reached the ATR crystal. In this
case, mount a new sample. Characteristic bands of the protective agent should progressively increase in intensity over time
(dependent on thickness and sample type, saturation is reached
ranging from several hours to overnight).
14. If the absorbance values exceed 2, this indicates the sample is
too thick, which may result in distortion of the spectral bands.
15. The actual ramp rate of the sample is generally different from
the set value of the ramp rate, due to discrepancies between
the temperature that is indicated on the controller unit and the
actual sample temperature which is measured by the
thermometer.
16. Calibrate the system by measuring phase transition temperatures of pure lipid systems with a known melting temperature
(i.e., DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine,
and DOPC, dioleoyl-sn-glycero-3-phosphocholine). Lipid
phase transition temperatures should be measured during heating, since literature values are typically reported from heating
scans.
348
Willem F. Wolkers and Harrie ¨ tte Oldenhof
C. Contributions of water vapor from
ambient air to the spectrum strongly interfere, particularly
with the protein bands.
9. It is recommended to select an automatic CO 2 /H 2 O correction when ATR studies are done. For transmission studies, the
automatic correction is not needed if both background and
sample are recorded after sufficient purging of the sample area.
10. The iris opening can be adjusted if needed.
11. Make sure that both background and sample spectra are
recorded after sufficient purging of the sample area (minimally
5 min). The disappearance of water vapor after purging is
particularly visible in the 1800–1500 cm
À1 region (see Fig. 1).
12. To avoid leakage, the diameter of the tissue sample should be
slightly larger than the opening of the hole in the tube used for
positioning the sample on the ATR crystal. Silicon-free grease
can be used at the outer edges after placing the sample holder
with the tissue on the ATR crystal to avoid leakage. Care
should be taken to prevent contact between the tissue and
the grease.
13. If the first spectrum already contains strong bands of the
compound in the protective solution, the solution has leaked
from the sides and directly reached the ATR crystal. In this
case, mount a new sample. Characteristic bands of the protective agent should progressively increase in intensity over time
(dependent on thickness and sample type, saturation is reached
ranging from several hours to overnight).
14. If the absorbance values exceed 2, this indicates the sample is
too thick, which may result in distortion of the spectral bands.
15. The actual ramp rate of the sample is generally different from
the set value of the ramp rate, due to discrepancies between
the temperature that is indicated on the controller unit and the
actual sample temperature which is measured by the
thermometer.
16. Calibrate the system by measuring phase transition temperatures of pure lipid systems with a known melting temperature
(i.e., DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine,
and DOPC, dioleoyl-sn-glycero-3-phosphocholine). Lipid
phase transition temperatures should be measured during heating, since literature values are typically reported from heating
scans.
348
Willem F. Wolkers and Harrie ¨ tte Oldenhof