ATR PRO Penta

Overview of the ATR PRO PENTA

The ATR method is widely for measuring solutions without the need for sample preparation, but since the current mainstream single-reflection ATR requires a sample concentration on the order of several percent, it was challenging to measure low-concentration samples of less than 0.1% as required by many biological samples.

ATR PRO Penta
Figure 1. ATR PRO Penta

The newly developed ATR “ATR PRO PENTA” (Figure1), a highly sensitive multiple reflection ATR, can measure low concentration samples of 0.1% or less using small amounts of samples several microliters by adopting a uniquely designed 14-reflection Ge prism (Figure 2.).

Time Reflective Ge Prism
Figure 2. Time Reflective Ge Prism

Here, we introduce that this new ATR as a useful tool in the biochemistry field for samples such as lipids, sugars, and proteins.

Quantitation of low concentration solutions

The spectrum of 0.5%, 0.1%, and 0.05% sucrose aqueous solutions and their calibration curve is shown in Figure 3. Each spectrum is the spectrum of the sucrose aqueous solution, the difference spectrum by which the spectrum of water is subtracted. Very high linearity is obtained in concentration (Figure 3b). It suggests that ATR PRO PENTA is sufficiently quantifiable even for low concentration regions that are previously untestable.

Difference spectrum of each sucrose aqueous solution Figure 4.Sucrose content metering (1056 cm-1 peak height)
Figure 3. Difference spectrum of each sucrose aqueous solution

* The quantitation limit of sucrose aqueous solution in this experiment was 0.042%, and the detection limit was 0.014%.

Difference spectrum of each sucrose aqueous solution
Figure 3b. Difference spectrum of each sucrose aqueous solution

Secondary structure analysis of IR proteins using extremely low concentration samples

IR-SSE (IR protein secondary structure analysis) was performed using the spectrum (difference spectrum with water) of 0.01% lysozyme aqueous solution (Figure 5). Even at a low concentration of 0.01%, the peaks of amide I and amide II are clearly separated, and the peak shape is clear, so amide I information can be used for secondary structural analysis (Figure 6). Figure 7 shows the resulting concentration values. The results of the secondary structure analysis of the difference spectra measured using a heavy aqueous solution were reasonably consistent with 1 obtained by Sarver and his team [1].

[1] Sarver, R. W., Krueger, W.C., 1991. Anal. Biochem., 194, 89-100.

* Since proteins adsorb on Ge, the spectral intensity does not change if they are thinner than a certain concentration.

* Strong acids and strong alkalis cannot be measured as they damage the crystal.

Measurement results and difference spectra of 0.01% lysozyme aqueous solution with ATR PRO PENTA
Figure 5. Measurement results and difference spectra of 0.01% lysozyme aqueous solution with ATR PRO PENTA
SSE spectra of 0.01% lysozyme heavy aqueous solution
Figure 7. SSE spectra of 0.01% lysozyme heavy aqueous solution

SSE quantitation

Structure Percentage (%)
α-Helix40
β-Sheet22
β-Turn18
Other20

・For a sucrose aqueous solution, a calibration curve with good linearity was obtained, and the detection limit was found to be 0.014%.
・Detection of lysozyme at a concentration of 0.01% was possible, and an SSE analysis was performed based on the Amide-I band in the difference spectrum.