Hemoglobin detection by HPLC

Protocol: Reverse phase HPLC for detecting globin tetramers

Materials required

1.    Equipment:
o     Shimadzu UFLC system:
    Binary gradient pumps (LC-20A)
    Auto sampler (SIL-HTC)
    Column oven (CTO-20AC)
    UV detector
o    Computer with LC Solutions™ software
o    Column:
    Aeris Widepore 3.6 µm XB-C18 25 cm × 4.6 mm
    SecurityGuard UHPLC Widepore C18 4.6 mm guard column (Phenomenex™)

2.    Reagents:
o    Solvent A: 0.1% Trifluoroacetic acid (TFA), pH 3.0
o    Solvent B: 0.1% TFA in acetonitrile
o    Globin sample for analysis

3.    Consumables:
o    Vials for sample preparation
o    Micropipettes and tips

4.    Safety Equipment:
o    Laboratory coat and gloves
o    Safety goggles

Procedure

1.    Preparation:
o    Prepare Solvent A (0.1% TFA, pH 3.0) and Solvent B (0.1% TFA in acetonitrile).
o    Filter and degas the solvents before use.
o    Prepare the globin samples for injection.

2.    Instrument setup:
o    Turn on the Shimadzu UFLC system and initialize it using the LC Solutions™ software.
o    Install the Aeris Widepore C18 column and the SecurityGuard guard column into the HPLC system.
o    Set the column oven temperature to 70°C.

3.    Chromatographic conditions:
o    Mobile phase:
    Solvent A: 0.1% TFA, pH 3.0
    Solvent B: 0.1% TFA in acetonitrile
o    Gradient elution:
    Begin with 40% Solvent B and adjust as needed for specific analyte resolution.
o    Flow rate: 1.0 mL/min
o    UV detection wavelength: 190 nm
o    Total run time: 8 minutes

4.    Sample injection:
o    Load the prepared globin sample into the auto sampler.
o    Inject the sample into the system.

5.    Data acquisition and analysis:
o    Monitor the chromatographic run using the LC Solutions™ software.
o    Record the chromatograms and analyze the globin peaks for tetramer detection.

6.    Shutdown and cleanup:
o    Flush the system with deionized water to remove residual solvents.
o    Turn off the HPLC system and clean the column as per manufacturer recommendations.

7.    Data reporting:
o    Export and process the data using LC Solutions™ software for peak identification and quantification.

Note: Before every run (i.e. test sample injection), the assay is checked using a Hemoglobin A2 Reference standard (https://www.sigmaaldrich.com/IN/en/product/sigma/h0266), and control samples for peak shape and retention times to ensure the quality of the run.