to form adenosine diphosphate (ADP) and adenosine triphosphate (ATP). It is the product of incomplete
hydrolysis of ATP.
ATP、 ADP、 AMP have an absorption peak at 254 nm, and its content can be determined by high
performance liquid chromatography.
Reagents and Equipment Required but Not Provided：
High-efficiency liquid chromatograph (C18 column (4.6×250 mm), ultraviolet detector (VWD)),
desktop centrifuge, adjustable pipette, mortar/ homogenizer, brown EP tube, 50 syringe filters (water, 0.45
µm), syringe, suction filter, filter membrane (organic, water), 50 brown injection bottle (2 mL), acetonitrile
(chromatographically pure, 500 mL), ultrapure water.
Preparations before the experiment:
1. Use 500 mL of chromatographically pure acetonitrile (mobile phase A) and 1000 mL of configured
mobile phase B to filter with a filter membrane to remove impurities in the solvent to prevent clogging the
chromatographic column. (Acetonitrile uses 0.45 µm organic filter membrane for suction filtration, and the
configured mobile phase B uses 0.22 µm aqueous filter membrane for suction filtration).
2. Ultrasound the prepared mobile phases A and B for 30 minutes to remove the gas in the solvent to
prevent clogging the chromatographic column and affecting the experimental results.
3. Preparation of ATP standard: 1 μmol/mL ATP standard solution is diluted with distilled water to 0.5
μmol/mL, 0.1 μmol/mL, 0.05 μmol/mL, 0.01 μmol/mL, 0.005 μmol/mL ATP standard solution. (The
prepared standard concentration is for reference only and can be adjusted according to the actual sample
concentration). The standard is filtered use an aqueous syringe filter into the brown injection bottle to be
tested (please place it at room temperature before testing, so as not to affect the retention time).
3. Preparation of ADP standard: 1 μmol/mL ADP standard solution is diluted with distilled water to 0.5
μmol/mL, 0.1 μmol/mL, 0.05 μmol/mL, 0.01 μmol/mL, 0.005 μmol/mL ADP standard solution. (The
prepared standard concentration is for reference only and can be adjusted according to the actual sample
concentration). The standard is filtered use an aqueous syringe filter into the brown injection bottle to be
tested (please place it at room temperature before testing, so as not to affect the retention time).
3. Preparation of AMP standard: 1 μmol/mL AMP standard solution is diluted with distilled water to
0.5 μmol/mL, 0.1 μmol/mL, 0.05 μmol/mL, 0.01 μmol/mL, 0.005 μmol/mL AMP standard solution. (The
prepared standard concentration is for reference only and can be adjusted according to the actual sample
concentration). The standard is filtered use an aqueous syringe filter into the brown injection bottle to be
tested (please place it at room temperature before testing, so as not to affect the retention time).
