Measurement Reproducibility using a UV-Visible Spectrophotometer with One Drop Accessory

Download PDF October 8, 2017

Introduction

Measurement Reproducibility using a UV-Visible Spectrophotometer with One Drop Accessory
V-630
UV-Visible Spectrophotometer

The SAH-769 One Drop accessory is used for fast and accurate quantitation of small volumes of proteins and nucleic acids. To make a measurement a single drop of sample is pipetted onto the cell (5μL for 1mm or 0.6μL for 0.2 mm optical pathlengths respectively).

This application note demonstrates both the reproducibility and linearity of the SAH-769 One Drop accessory using calf thymus DNA with both 1 mm and 0.2 mm pathlength cells.

Experimental

Measurement Conditions
Wavelength260 nmBandwidth1.5 nm
ResponseMedium

A drop of sample was dispensed, measured, and repeated 10 times. The sample was wiped from the cell in between measurements. The procedure can be seen in Figure 1.

Figure 1. Measurement procedure using the SAH-769 One Drop accessory.

Keywords

UV-0021, V-630BIO, UV-Visible/NIR, Biochemistry, SAH-769 One Drop, Microsampling

Results

The absorption values obtained using a 1 mm pathlength cell are shown in Table 1 and the calibration curve in Figure 2. Table 2 illustrates the reproducibility of the concentration values calculated from the absorbance data.

Table 1. SAH-769 absorbance measurement reproducibility using a 1 mm pathlength cell.

ng/µL 0 2.4 4.8 9.6 19.3 38.6 77.2 154.4 308.8 617.5
1-0.00080.00470.00840.01790.03210.06760.1330.2590.5160.998
20.00030.00590.00930.01460.03320.06770.1310.2600.5181.003
30.00120.00560.00830.01620.03340.07050.1300.2600.5191.003
40.00150.00630.00720.01810.03450.06790.1300.2620.5121.024
5-0.00020.00670.00710.01660.03290.06760.1320.2640.5170.993
6-0.00130.00530.00880.02040.03310.06720.1300.2590.5220.996
70.00130.00490.00820.01700.03360.06950.1290.2590.5110.996
80.00020.00360.00890.01680.03260.06800.1330.2600.5091.006
90.00270.00580.00890.01770.03150.06760.1340.2600.5091.000
10-0.00040.00430.00690.01530.03530.06920.1320.2670.5090.995
Avg.0.00040.00530.00820.01710.03320.06830.1310.2610.5141.001
S.D.0.00120.00100.00080.00160.00110.00110.00150.00260.00470.0089
C.V. %N/A17.910.39.63.31.61.21.00.90.9

Figure 2. Calibration curve created using data from Table 1. The calibration equation is: Abs = 0.00163 x Conc. + 0.00366 and the correlation coefficient is 0.9998.

Table 2. Concentration reproducibility using a 1 mm pathlength cell.

ng/µL 0 2.4 4.8 9.6 19.3 38.6 77.2 154.4 308.8 617.5
1-2.80.62.98.817.539.379.8157.3315.0611.5
2-2.11.43.46.718.239.478.0157.7316.3614.6
3-1.51.22.97.718.341.178.0157.9316.9614.7
4-1.31.72.28.918.939.577.7158.8312.4627.6
5-2.419.2.18.018.039.378.7160.0316.0608.8
6-3.11.03.110.318.139.177.5157.0318.7610.4
7-1.40.72.88.218.440.577.4157.1312.0610.3
8-2.10.03.28.117.839.679.3157.8311.1616.5
9-0.61.33.28.617.139.380.0157.4310.9612.8
10-2.50.42.07.119.540.378.9162.2311.0609.9
Avg.-2.01.02.88.218.239.878.5158.3314.0613.7
S.D.0.750.590.521.000.680.660.951.632.885.48
C.V. %N/A57.418.712.23.71.71.21.00.90.9
*Detection limit: 50 ng/µL
*Quantitation limit: 100 ng/µL

The absorption values obtained using a 0.2 mm pathlength cell are shown in Table 3 and the calibration curve in Figure 3. Table 4 illustrates the reproducibility of the concentration values calculated from the absorbance data.

Table 3. SAH-769 absorbance measurement reproducibility using a 0.2 mm pathlength cell.

ng/µL 0 38.6 77.2 154.4 308.8 617.5 1235 2470 4940
10.00080.01220.01960.04600.08420.1660.3260.6461.297
2-0.00110.01650.02230.04300.08470.1660.3300.6451.319
3-0.00010.01600.01950.04270.08730.1700.3280.6531.320
4-0.00040.01120.01760.04300.08630.1650.3310.6541.284
50.00690.01520.01800.04100.08600.1680.3270.6471.307
6-0.00050.01390.01840.04240.08450.1700.3260.6601.303
70.00060.01370.02060.04500.08390.1700.3310.6591.312
80.00190.01270.02000.04400.08750.1680.3300.6581.301
90.00110.01300.02120.04350.08510.1690.3380.6631.296
100.00070.01340.01940.04260.08790.1710.3260.6641.308
Avg.0.00100.01380.01970.04330.08570.1680.3290.6551.305
S.D.0.00120.00170.00150.00140.00150.00200.00370.00710.0110
C.V. %N/A12.27.43.31.71.21.11.10.8

The absorption values obtained using a 0.2 mm pathlength cell are shown in Table 3 and the calibration curve in Figure 3. Table 4 illustrates the reproducibility of the concentration values calculated from the absorbance data.

Figure 3. Calibration curve created using data from Table 3. The calibration equation is: Abs = 0.00264 x Conc. + 0.00281 and the correlation coefficient is 0.9999.

Table 4. Concentration reproducibility using a 0.2 mm pathlength cell.

ng/µL 0 38.6 77.2 154.4 308.8 617.5 1235 2470 4940
10.00080.01220.01960.04600.08420.1660.3260.6461.297
2-0.00110.01650.02230.04300.08470.1660.3300.6451.319
3-0.00010.01600.01950.04270.08730.1700.3280.6531.320
4-0.00040.01120.01760.04300.08630.1650.3310.6541.284
50.00690.01520.01800.04100.08600.1680.3270.6471.307
6-0.00050.01390.01840.04240.08450.1700.3260.6601.303
70.00060.01370.02060.04500.08390.1700.3310.6591.312
80.00190.01270.02000.04400.08750.1680.3300.6581.301
90.00110.01300.02120.04350.08510.1690.3380.6631.296
100.00070.01340.01940.04260.08790.1710.3260.6641.308
Avg.0.00100.01380.01970.04330.08570.1680.3290.6551.305
S.D.0.00120.00170.00150.00140.00150.00200.00370.00710.0110
C.V. %N/A12.27.43.31.71.21.11.10.8
*Detection limit: 50 ng/µL
*Quantitation limit: 100 ng/µL

*The detection limit is calculated using 3.3σ. The quantitation limit is calculated using 10σ. σ is the standard deviation in 0 ng/µL of sample concentration.

About the Author

Toshifumi Uchiyama is a member of the electronic spectroscopy team located at the JASCO main laboratory in Tokyo