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Clinical
and Biological Applications of
Capillary Electrophoresis
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Capillary electrophoresis (CE) is a versatile analytical platform
that can be used to separate and quantify a wide variety of compounds
of clinical and biochemical interest. Analytes can be separated
by mass, charge or isoelectric point using a variety of methods
including gel electrophoresis, zone electrophoresis, or isoelectric
focusing. One major advantage of CE is the small sample and reagent
volumes required. This makes CE ideal for the development of
assays for analytes in mouse blood where sample volumes are limiting.
We use CE routinely for analysis of hemoglobin variants, HbA1c,
and erythrocyte glutathione. An assay for UDP-N-acetylglucosamine
is under development. This is a schematic of the major components of a capillary electrophoresis
instrument. Different capillaries, buffers, and polarities are
used in assays for proteins, carbohydrates, DNA, electrolytes,
and many other compounds of biological interest. Example: Analysis of HbA1c in Human Blood
Electropherograms of hemolysates from normal and diabetic subjects.
Samples were prepared by adding 10 µl of erythrocytes to
200 µl of KCN/EDTA hemolyzing reagent. The capillary was
filled with a solution containing 0.375% methylcellulose and
2% ampholytes (pH 6.7 to 7.7). Samples were pressure injected
for 15 seconds into the capillary inlet, then the inlet and outlet
were immersed in 100 mM phosphoric acid and 80 mM borate (pH
10), respectively. Detection at 415 nm is relatively specific
for heme-containing compounds. HbA2 is a minor variant that can
be quantified and used in the diagnosis of beta thalassemia.
HbA0 is adult hemoglobin while HbA1c is glycated adult hemoglobin.
HbA1c is quantified based on peak area as a percentage of total
peak area (peak area of adult HbA0 + HbA1c).
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