Nucleotide separation in phosphate free buffer

Phosphate - the problem

Phosphate + Acetonitrile (any concentration between 5-100 mM; (75-100%, maybe less) but small volumes necessary) Phosphate crystals!

Atlas of Canine and Feline Cytology, Saunders, 2001

Phosphate Acetonitrile

Phosphate - the problem

Phosphate Acetonitrile now flow HPLC

HPLC - a solution

Nucleotide separation in phosphate free buffer

HPLC - a solution Reference: 0.1 M phosphate; pH 5.5-6.5; ACE C18 AR, 2.1 mm ×15 cm, GTP/GDP: no separation; AMP/ADP/ATP: weak separation 0.1 M MES; pH 5.5: better than phosphate; still bad 0.1 M MES; pH 5.5 + 2% methanol: better than phosphate; still bad 0.1 M MES; pH 5.5 + 6% methanol: like phosphate 0.1 M HEPES; pH 7.5: like phosphate 0.1 M triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile: good resolution and separation (excellent for GTP/GDP)

Triethylammonium bicarbonate buffer 0.1 M triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile

GTP GDP 0.1 M phosphate, pH 6.0 (ACE C18 AR, 2.1 mm ×25 cm column) 100 µM GTP, 100 µM GDP in 8 mM PIPES pH 6.8

GDP alone GDP GTP GTP alone 0.1 M triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile (ACE C18 AR, 2.1 mm ×15 cm column)

Triethylammonium bicarbonate buffer 0.1 M triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile Expensive (48$ per l) Lower concentration (35 mM)

Triethylammonium bicarbonate buffer 35 mM triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile

100 µM GTP, 100 µM GDP

lower concentration (35 mM)

Triethylammonium bicarbonate buffer A real experiment (GTP and GDP in tubulin from Cytoskeleton.com)

GTP GDP 45% 55% 0.1 M phosphate, pH 6.0 (25 cm column) ?

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35 mM triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile (15 cm column) GDP GTP 19-22% 78-81% Peaks are not really nice, because of the low pH of the injected sample

Triethylammonium bicarbonate buffer It also works with AMP/ADP/ATP

AMP ADP AMP ADP ATP [Acetonitrile] (3.5-4% ?) ATP

Triethylammonium bicarbonate buffer Does not form salts in Good buffers (MES, HEPES, PIPES) or water Can be mixed with acetonitrile, isopropanol Can be mixed with 5 mM Mg 2+ (I have not tried Ca 2+ )

Triethylammonium bicarbonate buffer Limitations HCO 3 CO 2 + OH (pH!)

Triethylammonium bicarbonate buffer Limitations 1) Cool the buffer (4 °C) 2) Make fresh buffer every day

Triethylammonium bicarbonate buffer 35 mM triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile Addition of 80mM HEPES pH 7.4 results in a more stable pH But there is no good nucleotide separation anymore

Triethylammonium bicarbonate buffer A short manual 1) 86-95% water (4 °C) 2) Add 3-10% triethylammonium bicarbonate buffer (1 M) and 2-4% acetonitrile 3) pH to 7.4 with acetate (7.1 when you need more than 6h for your experiments) Always store on ice; the pH of the buffer should be ≤8.0

Keep in mind that substances in your samples might bind to the column; include the one or the other cleaning step between your experiments when you have many samples