HPLC HINTS & TIPS for
Chromatographers
Tip# 103: Flow Cell Volume
& Path Length (METHOD TRANSFER ISSUES):
Modern UV/VIS detectors offer several different flow cell options.
The option(s) you select can make a big difference in the level of signal sensitivity,
sample dispersion and response you obtain. If you fail to note which type of HPLC flow
cell you use in a particular system, then you may discover some problems when transferring
a method to a different instrument. Always record the flow cell volume and path length
used as part of your method description.
Flow cells usually differ in three ways:
(1) Maximum Rated Backpressure;
(2) Flow Cell Volume and
(3) Flow Cell Path Length. Let’s take a look at these.
Maximum Rated Backpressure: Unless the detector is in series
with another detector, column or has a backpressure regulator on it, the expected
backpressure on a typical flow cell’s outlet is just about one bar as it usually is
directed to an open waste line. *This topic will be discussed in more detail in the future
as part of another “hint and tip” topic. Today we are more concerned about the
remaining two options:
- Flow Cell Volume: Analytical flow cells are commonly offered
in nl to ul sizes. Depending on your instrument setup, column and sample(s), one flow cell
volume may make more sense than another. After you have spent time separating and
concentrating the peak of interest into a tiny volume you do not want to elute it off the
column and mix it with another peak because the cell volume is too large. Ideal cell
volume is a compromise between sample dispersion and sensitivity. The best choice will be
determined mostly by the actual peak volume of your separated sample. The general rule is
that your flow cell volume should be no larger than 10% of your peak volume and ideally ~
2.5% (a 1:40 ratio), but there are some exceptions to this rule. When in doubt, experiment
with different cells and do not forget to consider the total volume of all the connecting
tubing and valves in your system as these contribute to many issues when the column volume
decreases (such as when using mini or narrow bore columns are used). Some common
analytical cell volumes offered by various manufacturers are 2 ul, 6 ul and 13 ul. For
narrow bore columns (~ 2.1mm ID) a smaller cell volume (~ 2 ul) will result in less sample
dispersion, while a larger cell volume may increase overall sensitivity (esp. when used
with a longer path length). Mid-bore or Mid-Size columns (2.1 to 4.6mm ID) often are best
suited to cell volumes around 6 ul to minimize dispersion and still provide good
sensitivity. Larger flow cells such as the common 13ul size often have longer path lengths
which can be used to enhance sensitivity. Standard 4.6mm ID columns often benefit from a
13ul volume cell to provide maximum sensitivity with less concern for dispersion effects
when larger columns are used (e.g. 4.6 x 250mm). Keep in mind that these are general
guidelines only. Most samples contain many peaks of varying width & volume, so you
will need to select the cell volume that is optimized to most of the peaks found in your
sample.
· Flow
Cell Path Length:
The flow cell’s path length affects the intensity of light reaching the detector
(Beer-Lambert law). For the same volume of sample, the apparent concentration of the
sample will appear to be higher if the path length is longer. There is no established
standard for ‘path length’ so it is important that you always known what the
path length of each flow cell is in your detector (10 mm is rather common).
Manufacturer’s offer different flow cells with varying path lengths. Even identical
detectors can use flow cells with identical volumes, but have different path lengths. When
comparing the analysis results obtained from two instruments, always make note of the flow
cell used in each instrument. If the method is to be accurately reproduced on a second
system, then the flow cells used should have the same geometry (volume and path length).
One way that the difference in path length can be used to increase the sensitivity of an
existing method is to use a flow cell with a longer optical path length. For example, if
your current flow cell has a path length of 6 mm you could replace it with one having a
path length of 10 mm. This would increase the sample peak response (as more light would be
absorbed) in your method. *This fact can be useful to squeeze out additional sensitivity
in a method and often does not require any change to the column or conditions.> Bill
Letter, 06/12/10.
HINT's & TIPS
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