General Guidelines for Additives

  • Use only high-purity volatile additives for the mobile phase, such as pH modifiers, buffer salts and ion pairing reagents.

  • Choose the grade of additives that has the lowest concentration of non-volatile impurities. Common impurities are iron, copper, sodium and potassium.

  • Avoid using non-volatile additives, such as citrate, phosphate, potassium, sodium, sulfonic acids and Tris.

  • Use the lowest concentration of non-volatile additive required to achieve the desired chromatographic performance.

  • Make sure that the mobile phase pH is within the allowed range of the detector: 2-12. More importantly, make sure that the mobile phase pH is well within the operating range of the LC column and the system.

TIP

Many columns will exhibit bleed, which refers to the release of non-volatile impurities into the eluent, if operated near the extremes of their pH range. For most silica-based columns, the pH range is 2 to 8.

Overview of typical additives and buffers for use with the detector

Additive/Buffer

pKa

Buffer range

Recommended max. concentration

pH at max. concentration in water

Acetic acid

4.76

3.8 – 5.8

0.3% v/v

3

Ammonium acetate

4.76

3.8 – 5.8

10 mM

 

Ammonium bicarbonate

7.8, 9.8, 10.3

7 – 11

10 mM

 

Ammonium formate

3.75

2.8 – 4.8

10 mM

 

Formic acid

3.75

2.8 – 4.8

0.3% v/v

2.5

Trifluoroacetic acid (TFA)

0.3

--

0.1% v/v

1.9

  • Acetic acid (AA) and especially formic acid (FA) are recommended as primary choices of acidic modifier for use with the detector. They are relatively volatile and the pH at typical concentrations is well within the compatibility range of most columns.

  • Trifluoroacetic acid (TFA) is frequently used as acidic modifier with the detector. However, as indicated in the table above, the pH of 0.1% v/v TFA is beyond the range of many columns. Use a lower concentration, if possible, to minimize column bleed.

  • TFA and other perfluorocarboxylic acids, such as heptafluorobutyric acid (HFBA), can function both as acidic modifiers and anionic ion pairing reagents. These additives are less volatile than AA and FA and will more readily form non-volatile salts with other additives or sample components. If you use these additives, do not use basic additives, such as ammonium salts or amines.

  • TFA is known to be unstable after exposure to air. Single-use ampoules of TFA are therefore recommended if choosing this additive.

  • If using cationic ion pairing reagents, such as TEA, practice the same precautions as with anionic pairing reagents to avoid formation of non-volatile salts with other additives and to stay within the range of column and detector pH compatibility.

  • AA and FA and their corresponding ammonium salts are recommended as primary choices to control pH within the above indicated buffer range.

  • Ammonium bicarbonate may be used to control pH at higher levels but requires use of an elevated evaporation temperature, such as 50 °C. Although the buffer range extends to pH 11, make sure to keep the pH below the detector maximum of 12. More importantly, make sure to keep the pH well below the compatibility range of your column.

  • If using a pH electrode to adjust buffer pH, only do so in aqueous solutions without organic modifiers. Always make pH measurements in separate aliquots of the aqueous solution to avoid cross-contaminating the mobile phase buffers with non-volatile salts from the pH electrode calibrations.