Time resolution

<p>We start to use UPLC a few months ago. Previouly we used HPLC. We are using a BEH C18 column to detect amino acids and biogenic amines by precolumn derivatization. Our gradient is</p><p></p><table border="1" cellpadding="0" cellspacing="0" style="border-collapse: collapse; border: medium none;"><tbody><tr><td valign="top" width="99"><p style="margin-bottom: 0.0001pt; line-height: normal;">Time</p></td><td valign="top" width="98"><p style="margin-bottom: 0.0001pt; line-height: normal;">Flow</p></td><td valign="top" width="96"><p style="margin-bottom: 0.0001pt; line-height: normal;">%A</p></td><td valign="top" width="97"><p style="margin-bottom: 0.0001pt; line-height: normal;">%B</p></td><td valign="top" width="92"><p style="margin-bottom: 0.0001pt; line-height: normal;">%C</p></td><td valign="top" width="100"><p style="margin-bottom: 0.0001pt; line-height: normal;">Curve</p></td></tr><tr><td valign="top" width="99"><p style="margin-bottom: 0.0001pt; line-height: normal;">Initial</p></td><td valign="top" width="98"><p style="margin-bottom: 0.0001pt; line-height: normal;">0.45</p></td><td valign="top" width="96"><p style="margin-bottom: 0.0001pt; line-height: normal;">90</p></td><td valign="top" width="97"><p style="margin-bottom: 0.0001pt; line-height: normal;">8</p></td><td valign="top" width="92"><p style="margin-bottom: 0.0001pt; line-height: normal;">2</p></td><td valign="top" width="100"><p style="margin-bottom: 0.0001pt; line-height: normal;">6</p></td></tr><tr><td valign="top" width="99"><p style="margin-bottom: 0.0001pt; line-height: normal;">3.02</p></td><td valign="top" width="98"><p style="margin-bottom: 0.0001pt; line-height: normal;">0.45</p></td><td valign="top" width="96"><p style="margin-bottom: 0.0001pt; line-height: normal;">90</p></td><td valign="top" width="97"><p style="margin-bottom: 0.0001pt; line-height: normal;">8</p></td><td valign="top" width="92"><p style="margin-bottom: 0.0001pt; line-height: normal;">2</p></td><td valign="top" width="100"><p style="margin-bottom: 0.0001pt; line-height: normal;">6</p></td></tr><tr><td valign="top" width="99"><p style="margin-bottom: 0.0001pt; line-height: normal;">4.03</p></td><td valign="top" width="98"><p style="margin-bottom: 0.0001pt; line-height: normal;">0.45</p></td><td valign="top" width="96"><p style="margin-bottom: 0.0001pt; line-height: normal;">83</p></td><td valign="top" width="97"><p style="margin-bottom: 0.0001pt; line-height: normal;">13.6</p></td><td valign="top" width="92"><p style="margin-bottom: 0.0001pt; line-height: normal;">3.4</p></td><td valign="top" width="100"><p style="margin-bottom: 0.0001pt; line-height: normal;">6</p></td></tr><tr><td valign="top" width="99"><p style="margin-bottom: 0.0001pt; line-height: normal;">4.32</p></td><td valign="top" width="98"><p style="margin-bottom: 0.0001pt; line-height: normal;">0.45</p></td><td valign="top" width="96"><p style="margin-bottom: 0.0001pt; line-height: normal;">83</p></td><td valign="top" width="97"><p style="margin-bottom: 0.0001pt; line-height: normal;">13.6</p></td><td valign="top" width="92"><p style="margin-bottom: 0.0001pt; line-height: normal;">3.4</p></td><td valign="top" width="100"><p style="margin-bottom: 0.0001pt; line-height: normal;">6</p></td></tr><tr><td valign="top" width="99"><p style="margin-bottom: 0.0001pt; line-height: normal;">7.34</p></td><td valign="top" width="98"><p style="margin-bottom: 0.0001pt; line-height: normal;">0.45</p></td><td valign="top" width="96"><p style="margin-bottom: 0.0001pt; line-height: normal;">60</p></td><td valign="top" width="97"><p style="margin-bottom: 0.0001pt; line-height: normal;">32</p></td><td valign="top" width="92"><p style="margin-bottom: 0.0001pt; line-height: normal;">8</p></td><td valign="top" width="100"><p style="margin-bottom: 0.0001pt; line-height: normal;">6</p></td></tr><tr><td valign="top" width="99"><p style="margin-bottom: 0.0001pt; line-height: normal;">8.11</p></td><td valign="top" width="98"><p style="margin...

Comments

  • For this kind of separation, it is very important that the run to run cycle time is absolutely consistent. The peaks which elute up to 6 minutes are eluting based on the initial strength of the mobile phase.  The strength of the mobile phase in the column will vary if the time between injections varies.  The peaks eluting after that time are well retained on the column until the gradient really kicks in.

    This is how the UPC Amino Acid Solution method is run to acheive run to run RT consistency.

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