ANALYTICAL CHEMISTRY I (CHEM-301)

DEPARTMENT OF CHEMISTRY

UNIVERSITY OF CRETE

WINTER SEMESTER 2023-2024

DETERMINING The HbA1c (Hemoglobin A1c) concentration in

blood using HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY(HPLC)

PROFESSOR : Χρυσοβαλάντου Χατζηιωάννου

STUDENT : ΠΕΝΤΑΡΗ ΟΛΥΜΠΙΑ (3195)
• chem3195@edu.chemistry.uoc.gr

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INTRODUCTION
WHAT IS HbA1c
• HbA1c is a form of hemoglobin that is linked to sugar in human blood(glycosylated hemoglobin).It
shows a person’s average blood glucose levels for the last 2-3 months. It is used to diagnose and
monitor diabetes.
WHAT IS HPLC
• Is a modified column chromatography that is used to seperate, identify and quantify each component
in a complex mixture.

PROCEDURE
1. SAMPLE COLLECTION: A blood sample is collected from the patient. This is typically done
through a venipuncture (drawing blood from a vein) and the sample is collected in a special tube that
contains an anticoagulant to prevent clotting.

2. HEMOLYSIS: In the laboratory, the blood sample is often subjected to a process called hemolysis,
which breaks down the red blood cells to release the hemoglobin within them.

3. SAMPLE PREPARATION: The hemosylate(the liquid containing released hemoglobin) is then

prepared for analysis. This may involve dilution and filtration to ensure a clean and homogenous
sample.
4. CALLIBRATION: Preparation of a series of HbA1c reference standards with known
concentrations. Inject these standards into a HPLC instrument to create a callibration curve. This
curve correlates peak areas with HbA1c concentrations.

5. HPLC ANALYSIS: The prepared sample is injected into a HPLC instrument. In the HPLC system
the hemoglobin is seperated into its different components including HbA1c, using a column with a
stationary phase and a mobile phase(solvent). During hemolysis hemoglobin is seperated in
glycosylated hemoglobin and non-glycosylated hemoglobin. By using an ion-exchange column that
its stationary phase is negatively charged, non-glycosylated hemoglobin can not be easily eluted,
because it is positively charged. But, glycosylated hemoglobin can be eluted from column firstly,
because it is little positive charged. HbA1c is seperated because of different retention time1 , which
is based on the attraction between solute ions and charged sites bound to the stationary.

6. DETECTION : As the hemoglobin components elute from the column, they pass through a
detector2, which measures their absorbance at a specific wavelength. HbA1c has a distinct
absorbance peak, making it easily distinguisable from other hemoglobin components.

7. DATA ANALYSIS: The detector is linked to a computer, so chromatography software is going to be

used in order to integrate the peak area of HbA1c. Futhermore, comparison happens between the
peak area and the callibration curve, in order to determine the concentration of HbA1c in the blood
sample.

8. REPORTING: The concentration of HbA1c is being reported as a percentage of total hemoglobin

and then interpreted according to the clinical guidelines3.

1 It determines the identity of the targeted component .Is the internal between the injection of a sample and the
detection of substances in that sample. It’s the time required for the solute to pass through a chromatographic
column.
2 It can be a UV, IR, MASS , FLUORESENCE , ELECTROCHEMICAL SPECTOMETERE
3 For instance: ADA(AMERICA DIABETES ASSOCIATION) guidelines for diabetes managment

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BIBLIOGRAPHY
• Manahan, Stanley E.
Environmental chemistry / Stanley E. Manahan.-6th ed.
ISBN1-56670-088-4
• ΑΝΑΛΥΤΙΚΗ ΧΗΜΕΙΑ
DANIEL C. HARRIS/CHARLES A. LUCY
Νικολαος Α. Χανιωτακης
PUBLICATIONS : BROKEN HILL
• HPLC : A Practical User's Guide [0-471-75401-3; 0-470-07909-6] McMaster, Marvin C.
YEAR: 2007