LOD for scopolamine [August 26, 2004]
Posted: Sun Aug 29, 2004 8:47 pm
By Lily Tan on Thursday, August 26, 2004 - 02:22 am:
The mobile phase that I am using now consists of : 20.9 ACN, 27.9 MeOH, 51.2 0.05M ammonium acetate. Isocatic condition. Flowrate 1ml/min. Column: Symmetryshield RP18, 5um. Using this method, I am able to get the LOD for scopolamine at 25 ng.
The published method is also using the same mobile phase, isocratic condition but the column: Lichrosorb RP18 10 um, flowrate 1.3 ml/min.At this condition, they are able to get the LOD for scopolamine at 12.05 ng.
I have also tried another column: Lichrosorb 5 um but the LOD is 100 ng.
Does the column material, length, flowrate, poresize etc. affect the elution amount? Any suggestions how I can improve my LOD?
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By Uwe Neue on Thursday, August 26, 2004 - 02:30 pm:
There are several things that affect sensitivity. The first one is the amount of retention that you are getting. If you reduce the retention factor by a factor of 2, you will get a two-fold improvement in LOD. The next question is the column volume - column length and column diameter. A smaller volume will result in a better LOD.
Of course, all of this assumes that the baseline is identical, and that no strange things are happening.
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By Mike on Thursday, August 26, 2004 - 03:58 pm:
Reducing k by a factor of x leads to a factor of x improvement in sensitivity?? That would imply a proportional relationship between k and peak height. But I know of no mathematical relationship between k and peak height. Can you tell me the equation.
Also, smaller column diameter only results in better sensitivity when a concentration sensitive detector is used (which is pretty common) and when the volume of sample is limited (which is pretty rare). Of course, this assumes that one is working at the maximum injection volume.
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By Uwe Neue on Thursday, August 26, 2004 - 09:02 pm:
In isocratic chromatography, the peak becomes wider with larger retention. With a concentration sensitive detector, the peak area is proportional to the mass injected. If the peak becomes wider, it has to become shorter. At equal noise, the shorter peak gives a lower signal-to-noise ratio.
The underlying equation is the one that you use to calculate the plate count.
The mobile phase that I am using now consists of : 20.9 ACN, 27.9 MeOH, 51.2 0.05M ammonium acetate. Isocatic condition. Flowrate 1ml/min. Column: Symmetryshield RP18, 5um. Using this method, I am able to get the LOD for scopolamine at 25 ng.
The published method is also using the same mobile phase, isocratic condition but the column: Lichrosorb RP18 10 um, flowrate 1.3 ml/min.At this condition, they are able to get the LOD for scopolamine at 12.05 ng.
I have also tried another column: Lichrosorb 5 um but the LOD is 100 ng.
Does the column material, length, flowrate, poresize etc. affect the elution amount? Any suggestions how I can improve my LOD?
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, August 26, 2004 - 02:30 pm:
There are several things that affect sensitivity. The first one is the amount of retention that you are getting. If you reduce the retention factor by a factor of 2, you will get a two-fold improvement in LOD. The next question is the column volume - column length and column diameter. A smaller volume will result in a better LOD.
Of course, all of this assumes that the baseline is identical, and that no strange things are happening.
-------------------------------------------------------------------------------------------------------
By Mike on Thursday, August 26, 2004 - 03:58 pm:
Reducing k by a factor of x leads to a factor of x improvement in sensitivity?? That would imply a proportional relationship between k and peak height. But I know of no mathematical relationship between k and peak height. Can you tell me the equation.
Also, smaller column diameter only results in better sensitivity when a concentration sensitive detector is used (which is pretty common) and when the volume of sample is limited (which is pretty rare). Of course, this assumes that one is working at the maximum injection volume.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, August 26, 2004 - 09:02 pm:
In isocratic chromatography, the peak becomes wider with larger retention. With a concentration sensitive detector, the peak area is proportional to the mass injected. If the peak becomes wider, it has to become shorter. At equal noise, the shorter peak gives a lower signal-to-noise ratio.
The underlying equation is the one that you use to calculate the plate count.