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Alliance iS HPLC Systems User Guide
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Alliance iS HPLC Systems User Guide
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Wavelength cutoffs for mixed mobile phases

Mobile phase absorbance

Mobile phase absorbance
Topic LCI-USG-0021, Last Updated 11/08/2024

Consult this table to obtain the absorbances at several wavelengths for frequently used mobile phases.

To reduce baseline noise, choose the mobile phase carefully.

The best mobile phase for your application is one that is transparent at the chosen detection wavelengths. With such a mobile phase, ensure that any absorbance is caused only by the sample. Absorbance by the mobile phase also reduces the linear dynamic range of the detector by the amount of absorbance the Autozero function cancels. Wavelength, pH, and concentration of the mobile phase affect its absorbance. Examples of several mobile phases are given in the following table, where the absorbances are based on a 10-mm path length.

Note: When running normal-phase solvents, install the Waters hexane/THF conversion kit applicable to your system (see Waters hexane/THF compatibility kits).
Caution: When operating the Alliance iS Bio HPLC System above pH 10, ensure that you are using the optional high pH kit. For further assistance, contact Waters.
Table 1. Mobile phase absorbance measured against air or water
Mobile phase Absorbance (AU) at specified wavelength (nm)
200 205 210 215 220 230 240 250 260 280
Solvents
ACN 0.05 0.03 0.02 0.01 0.01 <0.01 n/a n/a n/a n/a
IPA 1.80 0.68 0.34 0.24 0.19 0.08 0.04 0.03 0.02 0.02
MeOH (degassed) 1.91 0.76 0.35 0.21 0.15 0.06 0.02 <0.01 n/a n/a
MeOH (not degassed) 2.06 1.00 0.53 0.37 0.24 0.11 0.05 0.02 <0.01 n/a
Unstablized THF (fresh) 2.44 2.57 2.31 1.80 1.54 0.94 0.42 0.21 0.09 0.05
Unstablized THF (old) >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 2.5 1.45
Acids and bases
Acetic acid, 1% 2.61 2.63 2.61 2.43 2.17 0.87 0.14 0.01 <0.01 n/a
Diammonium phosphate, 50 mM 1.85 0.67 0.15 0.02 <0.01 n/a n/a n/a n/a n/a
Disodium EDTA, 1 mM 0.11 0.07 0.06 0.04 0.03 0.03 0.02 0.02 0.02 0.02
Hydrochloric acid, 0.1% 0.11 0.02 <0.01 n/a n/a n/a n/a n/a n/a n/a
Phosphoric acid, 0.1% <0.01 n/a n/a n/a n/a n/a n/a n/a n/a n/a
Triethylamine, 1% 2.33 2.42 2.50 2.45 2.37 1.96 0.50 0.12 0.04 <0.01
Trifluoroacetic acid, 0.1% 1.20 0.78 0.54 0.34 0.22 0.06 <0.02 <0.01 n/a n/a
Buffers and salts
Ammonium acetate, 10 mM 1.88 0.94 0.53 0.29 0.15 0.02 <0.01 n/a n/a n/a
Ammonium bicarbonate, 10 mM 0.41 0.10 0.01 <0.01 n/a n/a n/a n/a n/a n/a
HEPES, 10 mM, pH 7.6 2.45 2.50 2.37 2.08 1.50 0.29 0.03 <0.01 n/a n/a
MES, 10 mM, pH 6.0 2.42 2.38 1.89 0.90 0.45 0.06 <0.01 n/a n/a n/a
Potassium phosphate, dibasic (K2HPO4), 10 mM 0.53 0.16 0.05 0.01 <0.01 n/a n/a n/a n/a n/a
Potassium phosphate, monobasic (KH2PO4), 10 mM 0.03 <0.01 n/a n/a n/a n/a n/a n/a n/a n/a
Sodium acetate, 10 mM 1.85 0.96 0.52 0.30 0.15 0.03 <0.01 n/a n/a n/a
Sodium chloride, 1 M 2.00 1.67 0.40 0.10 <0.01 n/a n/a n/a n/a n/a
Sodium citrate, 10 mM 2.48 2.84 2.31 2.02 1.49 0.54 0.12 0.03 0.02 0.01
Sodium formate, 10 mM 1.00 0.73 0.53 0.33 0.20 0.03 <0.01 n/a n/a n/a
Sodium phosphate, 100 mM, pH 6.8 1.99 0.75 0.19 0.06 0.02 0.01 0.01 0.01 0.01 <0.01
Tris HCl, 20 mM, pH 7.0 1.40 0.77 0.28 0.10 0.04 <0.01 n/a n/a n/a n/a
Tris HCl, 20 mM, pH 8.0 1.80 1.90 1.11 0.43 0.13 <0.01 n/a n/a n/a n/a
Surfactants
Brij 35, 1% 0.06 0.03 0.02 0.02 0.02 0.01 <0.01 n/a n/a n/a
CHAPS, 0.1% 2.40 2.32 1.48 0.80 0.40 0.08 0.04 0.02 0.02 0.01
Polyoxyethylene sorbitan monolaurate (Tween 20), 0.1% 0.21 0.14 0.11 0.10 0.09 0.06 0.05 0.04 0.04 0.03
Sodiumdodecyl sulfate (SDS), 0.1% 0.02 0.01 <0.01 n/a n/a n/a n/a n/a n/a n/a
Triton X-100, 0.1% 2.48 2.50 2.43 2.42 2.37 2.37 0.50 0.25 0.67 1.42
Waters PIC reagents
PIC A, 1 vial/L 0.67 0.29 0.13 0.05 0.03 0.02 0.02 0.02 0.02 <0.01
PIC B6, 1 vial/L 2.46 2.50 2.42 2.25 1.83 0.63 0.07 <0.01 n/a n/a
PIC B6, low UV, 1 vial/L 0.01 <0.01 n/a n/a n/a n/a n/a n/a n/a n/a
PIC D4, 1 vial/L 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.01

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