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[Chromatography Q & A] Which buffer solution is used for the HPLC mobile phase?

A mixture of water and organic solvents is often used for the mobile phase of HPLC. However, when the dissociation state of the target component changes depending on the pH, the pH should be kept constant with a buffer solution.

The selection points of the buffer solution to be used for the mobile phase are shown below.

  1. The pH can be kept constant.
  2. The buffer solution is stable when mixed with organic solvents.
  3. No unnecessarily reaction occurs with sample compounds.
  4. Degradation of the solid phase (e.g., cleavage of chemical modification group, carrier dissolution) or equipment does not occur.

However, even if these points are met, a case is assumed where, for example, if a buffer solution has a high absorbance when using an absorption detector, it adversely affects the stability or sensitivity of the detector. It is desirable to use a reagent appropriately assured by the detection method.

Table 1 shows the reagents commonly used for preparation of buffer solutions. The choice of buffer solution to be used is not particularly determined, and the optimal conditions are examined for each analysis, but phosphate buffer solution seems to be commonly used. The reason is not sure, but some are presumed.

  1. Easy to prepare.
  2. Many different types of phosphates are readily available (commercially available).
  3. It has a buffer capacity in a wide pH range (available in wide pH range).
  4. Absorption in the low-wavelength UV region is lower than other acid salt buffers.
  5. Many application examples were used in the past.

Particularly when selecting from past analysis examples, the abundance of applications seems to be a factor of being used frequently.

Recently, however, LC/MS has been increasingly used, and instead of nonvolatile phosphate, a buffer solution combining volatile salts such as acetic acid and formic acid and volatile bases such as pyridine, trimethylamine and ammonia has been used. Table 2 shows the relationship between the pH and composition of the volatile buffer, and representative examples.

  • Table 1. Reagents used for preparation of buffer solutions
    Reagent Chemical formula Molecular weight
    1. Acids
    Citric acid HO2CC(OH)
    (CH2COOH)2・H2O
    210.14
    Acetic acid CH3COOH 60.05
    Boric acid H3BO3 61.83
    Phosphoric acid H3PO4 98.00
    2. Bases
    Potassium hydroxide KOH 56.11
    Sodium hydroxide NaOH 40.00
    Tris [tris(hydroxymethyl)
    aminomethane]
    H2NC(CH2OH)3 121.14
    3. Salts
    Sodium citrate C6H5O7Na3・2H2O 294.10
    Sodium acetate CH3COONa・3H2O 136.08
    Sodium carbonate Na2CO3・10H2O 286.14
    Borax (sodium tetraborate) Na2B4O7・10H2O 381.37
    Dipotassium hydrogen phosphate K2HPO4 174.18
    Disodium hydrogen phosphate Na2HPO4 141.96
    Trisodium phosphate Na3PO4・12H2O 380.12
    Potassium dihydrogen phosphate KH2PO4 136.09
    Sodium dihydrogen phosphate NaH2PO4・H2O 156.01
  • Table 2. Relationship between pH and composition of volatile buffer, and representative examples
    a. Relationship between pH and composition
    pH Range Composition of buffer
    Around 2 Acetic acid-formic acid
    2.3~3.5 Pyridine-formic acid
    3.5~6.0 Trimethylamine*-formic acid or acetic acid
    5.5~7.0 Collidine-acetic acid
    7.0~12.0 Trimethylamine*-carbon dioxide
    6.0~10.0 Ammonia-formic acid or acetic acid
    6.5~11.0 mono- (or tri-) ethanolamine-hydrochloric acid
    8.0~9.5 Ammonium carbonate-ammonia

    * Triethylamine may be used.

    b. Representative examples
    pH Ingredients and contents in 1 L of water
    1.9 Glacial acetic acid: 87 mL, 88% Formic acid: 25 mL
    2.1 88% Formic acid: 25 mL
    3.1 Pyridine: 5 mL, Glacial acetic acid: 100 mL
    3.5 Pyridine: 5 mL, Glacial acetic acid: 50 mL
    4.7 Pyridine: 25 mL, Glacial acetic acid: 25 mL
    6.5 Pyridine: 100 mL, Glacial acetic acid: 4 mL
    7.9 Ammonium Hydrogencarbonate: 2.37 g
    8.9 Ammonium carbonate: 20 g

1) Kanto Branch of The Japan Society for Analytical Chemistry, "High Performance Liquid Chromatography Handbook, revised 2nd edition", Maruzen (2000)

In addition, when fractionation is performed by HPLC, operation to remove the buffering agent is necessary. To remove nonvolatile buffering agents, complicated operations are required, and thus also in this case, volatile buffers are effective. In any case, it is essential to use appropriate buffer solution according to the intended use.

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