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Denaturing HPLC (DHPLC)

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There are columns on the market for performing fragment analysis, they include the DNASep Column, the Helix DVB column and the Zorbax Eclipse dsDNA Analysis Column.

The ion-pairing agent used with either column is triethylammonium acetate (TEAA), which mediates binding of DNA to the stationary phase. Acetonitrile is used as an organic agent to achieve subsequent separation of the DNA from the column.


Typical Method:

Using the system described above, the following reagents and column can be used
Standards of ds DNA
• Deonised water
• HPLC grade acetonitrile
• Buffer A, comprising of 100 mM TEAA to pH 7.0, 0.1 mM EDTA,
• Buffer B, comprising of 100 mM TEAA to pH 7.0, 0.1mM EDTA and 25 %v/v acetonitrile
• Helix DVB column, 3mm ID x 50 mm length


Set the detection wavelength to 260 nm.

A typical binary gradient cycle is:
A flow rate of 0.45 ml/minute
Percentage of buffer A, when time is 0.00 minute – 55%
Percentage of buffer A, when time is 0.30 minutes – 50%
Percentage of buffer A, when time is 6.00 minutes – 32%
Percentage of buffer A, when time is 7.00 minutes – 32%
Percentage of buffer A, when time is 7.05 minutes – 55%

Samples and standards may need to be diluted with deionised water.

Run the standard to ensure that the required peaks are well resolved. The column may need to be washed, or a wait may be required for the column to equilibrate to the required temperature.

Between changes in temperature, always ensure that the column has had sufficient time to equilibrate to the required temperature.

Optimisation of the denaturing temperature can be performed by trial or error. Here injection
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Source:


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