I am developing a UHPLC method for content and purity for a peptide based on 100mM KPF6 and 100mM NH4PF6 pH 3.5. TFA as an ion pair reagent does not give the required separation performance. My question is less about method development HPLC for peptides, but more about the negative effects of KPF6/NH4PF6 in the UHPLC systems used (Waters IClass binary system and Thermo Vanquish Horizon binary system).

The negative effects on 3 instruments were already noticeable after 1 to 2 sequences (extreme pressure fluctuations):

The repairs carried out showed more or less the same damage:

- strongly porous seals in the pump heads

- damage to the pistons

- defective inlet/outlet check valves

One system was used over 2-3 months and showed the most damage.

I will ask my questions right at the beginning:

- What experience do you have in using KPF6, NH4PF6 as additive/buffer in mobile phases for UHPLC analysis or for method development for peptides?

- How can these reagents be used "safely" and robustly without causing damage to the pumps?

These chaotropic reagents seem to be highly corrosive. I would be glad to get some useful advice. Many thanks in advance for your support and advice.

Kind regards

Ronald

Mobile phases are:

Mobile phase A 0.1M KPF6 pH 3.5 / ACN 8:2 (V/V)

Mobile phase B 0.1M KPF6 pH3.5 / ACN 35:65 (V/V)

and

Mobile phase A 0.1M NH4PF6 pH 3.5 / ACN 8:2 (V/V)

Mobile Phase B 0.1M NH4PF6 pH 3.5 / ACN 35:65 (V/V)

Preparation:

0.3M Buffer KPF6 pH 3.5

• 55.2 g KPF6, were weighed into a 2000 mL beaker. 1000 mL water was added and stirred until complete dissolution for 15 min. Sonification for approx. 2 min. It was adjusted to pH 3.5 with Orthophosphoric acid 85% (+/- 255 µL) and stirred well. The buffer was filtered with a Steritop-GP 1000 mL Express Plus PES 0.22 µm into a 1000 mL bottle.

Mobile phase A1: 0.1M KPF6 pH 3.5 / ACN 8:2 (V/V)

• 330 mL 0.3M_ KPF6 buffer pH 3.5, 470 mL water and 200 mL acetonitrile was transferred into a 1000 mL bottle and mixed well. Sonnification for approx.5. min.

Mobile phase B1: 0.1M KPF6 pH 3.5 / ACN 35:65 (V/V)

• 330 mL 0.3M_ KPF6 buffer pH 3.5, 20 mL water and 650 mL acetonitrile was transferred into a 1000 mL bottle and mixed well. Sonnification for approx.5. min.

0.3M Buffer NH4PF6 pH 3.5

• 48.9 g NH4PF6, were weighed into a 2000 mL beaker. 1000 mL water were added and stirred until complete dissolution for 5 min. It was adjusted to pH 3.5 with 25 % NH4OH (+/- 340 µL) and stirred well. The buffer was filtered with a Steritop-GP 1000 mL Express Plus PES 0.22 µm into a 1000 mL bottle.

Mobile phase A1: 0.1M NH4PF6 pH 3.5 / ACN 8:2 (V/V)

• 330 mL 0.3M_ NH4PF6 buffer pH 3.5, 470 mL water and 200 mL acetonitrile was transferred into a 1000 mL bottle and mixed well. Sonnification for approx.5. min.

Mobile phase B1: 0.1M NH4PF6 pH 3.5 / ACN 35:65 (V/V)

• 330 mL 0.3M_ NH4PF6 buffer pH 3.5, 20 mL water and 650 mL acetonitrile was transferred into a 1000 mL bottle and mixed well. Sonnification for approx.5. min.

After sequence, the systems were flushed with

• 85min with ACN/H2O 15/85; 0.4 mL/min
• 15 min with ACN/H2O 75/25; 0.4 mL/min
• 15 min with ACN/H2O 50/50; 0.4 mL/min
• following a low flow ACN/H2O 50/50; 0.1 mL/min