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.
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|
|Buffer 2 (%)||0||100||0||0|
|Buffer 3 (%)
|HbA1c (binary gradient): Flow: 1.3 ml/min|
|Buffer 3 (%)
|Injection volume:||10 to 20 µl|
|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.
- 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.
- 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.
|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|
|15007/B||Reaction vials, heavy metal free, 1000 pcs.|
|55033||PEEK prefilter, 5 µm, 5 pcs.|
|15010||PEEK prefilter housing, 1 pc.|
These products are not available in the United States and Canada.