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Mobile Phase Preparation BIST™ B and BIST™ B+ Columns

The following procedures will produce a mobile phase (MP) solution containing 80% Acetonitrile (MeCN), 20% water, and 0.2% H2SO4. The resulting concentrations can be modified for your particular needs by adjusting the initial volumes of solution.

Chemicals
HPLC-grade Acetonitrile (CAS #75-05-8)
HPLC-grade Water
HPLC-grade H2SO4 (CAS #7664-93-9)
Equipment
1 L graduated cylinder
100 mL beaker
1 L solvent bottle (qty. 2)
Analytical scale

The first step in making BIST™-compatible Mobile Phases is to make 1 L of 0.2 % w/v H2SO4 in water and 1 L of 0.2% w/v H2SO4 in Acetonitrile. This will make further Mobile Phase preparations faster (and safer). The procedure below will be based on using 98% H2SO4 as the basis for the dilution. The numbers will change accordingly if you use 35% H2SO4 as your basis.

Preparation of 0.2% Sulfuric acid Stock Solution (using 98% H2SO4)

  1. Ensure you are wearing proper PPE: gloves, lab coat, lab goggles and working under a fume hood.
  2. In the 1 L graduated cylinder, pour in about 500 mL of water.
  3. Weigh 2.04 g of 98% H2SO4 reagent
  4. Add the H2SO4 into the 1 L graduated cylinder.
  5. Rinse the beaker a few times with about 50 mL of water, adding each wash to the cylinder.
  6. Fill the graduated cylinder to the 1 L mark with water.
  7. Transfer the contents of the graduated cylinder into the 1 L solvent bottle.

This will be your acidic water stock solution.

  1. In the 1 L graduated cylinder, pour in about 500 mL of acetonitrile.
  2. Weigh 2.04 g of 98% H2SO4 reagent.
  3. Add the H2SO4 into the 1 L graduated cylinder.
  4. Rinse the beaker a few times with about 50 mL of acetonitrile adding washes to the cylinder.
  5. Fill the graduated cylinder to the 1 L mark with acetonitrile.
  6. Transfer the contents of the graduated cylinder into the 1 L solvent bottle.

This will be your acidic Acetonitrile stock solution.

  1. Mix 800 mL of acidic acetonitrile and 200 mL of acidic water solution into the 1 L MP bottle to produce a 80/20/0.2% Acetonitrile/Water/H2SO4 mobile phase.

Notes:

  • This procedure provides a slightly lower concentration of water and Acetonitrile. The actual MP composition is close to 79.9/19.9/0.2, but for most practical purposes it can be written as 80/20/0.2.
  • We observed no significant changes in the Acetonitrile sulfuric acid stock solution for a month of storage at room temperature. However there are some indications that Acetonitrile can react very slowly with concentrated sulfuric acid. Ideally, the Acetonitrile sulfuric acid stock solution should be kept at a lower temperature.

Sulfuric acid as mobile phase ionic modifier in HPLC