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.
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 |
Wavelength cutoffs for mixed mobile phases
