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Hemoglobin A1c - HPLC


  • High analytic accuracy

  • Economic refill option

  • Components available separately, lot independend

The short time required for analysis has led to HPLC methods becoming generally accepted for the analysis of HbA1c. The high specificity of HPLC techniques means that the measurements obtained are exceptionally reproducible, this in turn meaning a clearly more reliable adjustment of the patient's plasma glucose concentration.

Ion exchange chromatography is used to separate the hemoglobin fractions in the analysis system. A cation exchange column separates the various fractions by virtue of their different charges. These are then eluted using a three-buffer system with an (increasing) ionic strength gradient.

Using the Chromsystems fluid media, buffer 1 elutes only HbA1a and HbA1b. Foetal hemoglobin HbF is eluted together with HbA1c in the second phase with buffer 2. With an appropriate standard, HbF can be quantitated, allowing assessment of thalassemia. Buffer 3 (highest ionic strength and elution capability) elutes HbA0 and also serves as a wash step for the analytical column. The role of the wash solution is simply to transport the sample to the injector and to rinse the injector unit after each injection. A complete analysis requires maximum 5 minutes.


Hämoglobin A1c Chromatogramm



HPLC parameters (HPLC gradient system)

Because of the different void volumes of individual HPLC systems, slight batch to batch differences of the buffers and columns, the following gradient profiles may have to be adapted. Further information is given on the certificate of the column. Start concentration of the gradient is always 100 % Buffer 1.

HbA1c (ternary gradient): Flow: 1.2 ml/min
Time (min) 0 0.6 1.8 2.3
Buffer 2 (%) 0 100 0 0
Buffer 3 (%)
0 0 100 0


HbA1c (binary gradient): Flow: 1.3 ml/min
Time (min) 0 0.1 0.5 2.2 2.21 2.7 2.71
Buffer 3 (%)
0 0 8 8 100 100 0


Injection volume: 10 to 20 µl
Wavelength: 415 nm
Column temperature: ambient (ca. 25 °C)
Run time: approx. 5 minutes


HPLC parameters (automated HbA1c analyzer)

HbA1c analysis with the Chromsystems HbA1c kit does not require any substantial modifications to the components or settings of the HbA1c automated analyzer. The initial start-up which should be done by a Chromsystems employee consists of the following steps:

  • Exchanging the original holders for the buffer bottles by the holders for the Chromsystems buffer bottles (the holders can be changed back again very easily).
  • Mounting the extension for the column heater (this extension can also be unmounted at any time without problem).
  • Installing a prefilter of PEEK (this prefilter can also be used with conventional reagents).
  • Entering the integration parameters.

Pre-analytic treatment


  • Whole blood

For sample drawing a common anticoagulant (e.g. EDTA) should be used.

Stability of specimens

  • At ambient temperature ca. 3 days
  • At +2 to +8 °C up to 7 days
  • Below -18 °C up to 6 months

Once thawed, a sample should not be refrozen.

Sample preparation

  • Add 5 to 10 µl whole blood to 1 ml Hemolysis Reagent, mix well.
  • Incubate 20 min at 37 °C, cool down immediately.
  • Inject 10 to 20 µl of the hemolysis mixture into the HPLC system.

Ordering information

Ordering information

Order no.Product
15100 Analytical column for HbA1c analysers or HPLC gradient systems (equilibrated, with test chromatogram), 1 pc.
Our refill service allows users of Chromsystems reagents routine exchange of the analytical column in order to prevent chromatographic problems.
Warranty: After about 1000 runs you send your analytical column (15100) to Chromsystems. You then will receive a refill column for another 1000 analyses. Each refill column is fully tested and delivered with a test chromatogram.
15100/R Refill-Service, 1 pc.
15001 Puffer 1, 2.5 l
15002 Puffer 2, 2.5 l
15003 Puffer 3, 2.5 l
Save costs and reduce waste with 5l cubitainers:
15001/C Puffer 1 (Cubitainer), 5.0 l
15002/C Puffer 2 (Cubitainer), 5.0 l
15003/C Puffer 3 (Cubitainer), 5.0 l
15004 Hemolysis Reagent, 1000 ml
15005 Wash Solution, 2.5 l
Startup accessories:
15007/B Reaction vials, heavy metal free, 1000 pcs.
55033 PEEK prefilter, 5 µm, 5 pcs.
15010 PEEK prefilter housing, 1 pc.

Please note:
These products are not available in the United States and Canada.

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