In previous articles we have discussed how the choice of column
particle size directly changes the system backpressure. Smaller particles generate higher back-pressures. We have also
discussed the importance of HPLC
tubing selection to minimize delay volume and diffusion within the HPLC's laminar flow path. Let us now focus on how the tubing's internal diameter and length impacts the total
HPLC back-pressure (or pressure drop)
observed.
Key Points:
- Try to optimize the plumbing of your HPLC system.
- HPLC Tubing lengths between connections (or HPLC modules) should always be as short as possible.
- Pressure drop is dependent on the tubing length and inner diameter. Doubling the inner diameter of the tubing will decrease the pressure by a factor of 16.
Once the HPLC tubing connection lengths have been minimized, the next critical
dimension which affects band broadening, delay volume and peak-width is the internal
diameter (ID) of the tubing. The tubing selected should be narrow enough to
reduce the undesirable spread of the peak(s) inside the tubing, but not be so
narrow or restricted to result in clogs or obstructions (which is why good
chromatography guidelines should be followed insuring that each sample is fully
dissolved and filtered before
injection). Commonly used tubing ID’s for most analytical HPLC systems are:
0.010” (0.25 mm), 0.007” (0.17 mm) or 0.005” (0.12 mm). By far, 0.007” (0.17 mm) is
the most commonly used size for modern analytical HPLC analysis as it offers a compromise between low delay-volume and modest back-pressure (with fewer clogs). However, in addition to the much lower internal
volumes which accompany the narrower ID’s, the pressure drop measured across equivalent
lengths of tubing may change dramatically and this should be noted during set-up, selection and operation. Take the time to learn what "normal" backpressures are under specified conditions.
Understanding how the HPLC system backpressure changes as the internal diameter of the tubing varies is extremely useful in troubleshooting a number of common HPLC problems.
Let us compare the pressure drops measured across three
popular HPLC tubing ID’s of the same length (40 cm) using common HPLC mobile
phase solvents. This table will help illustrate the observed backpressure changes that
the tubing ID and liquid have on the pressure drop.
PRESSURE DROP (in bars):
SS Capillary Tubing, 40 cm length,
flow rate 1.000 mL/min.
Mobile Phase / Tubing ID
|
Water
|
ACN
|
MeOH
|
MeOH/Water (1:1)
|
IPA
|
0.010” (0.25 mm)
|
0.7
|
0.2
|
0.4
|
1.2
|
1.5
|
0.007” (0.17 mm)
|
2.7
|
1.0
|
1.6
|
5.1
|
6.2
|
0.005” (0.12 mm)
|
10.4
|
4.0
|
6.3
|
19.1
|
24
|
Note: Pressure drop is also a
function of tubing length so if we halve (1/2) the length of tubing used, we also
will reduce the pressure drop by one-half.
Note the four-fold change that narrowing the tubing ID has
at each ID reduction. The change is more dramatic when viscous solutions are used
(i.e. MeOH/Water or IPA). If you re-plumb any part of your HPLC system with new
tubing, then awareness of this physical change will assist you in
troubleshooting many types of HPLC problems (to know which types of pressure
changes indicate a real problem and which types of pressure changes are normal).
Changes to the overall length or ID may result in noticeable changes to the
total system backpressure. As an experienced chromatographer knows, when HPLC solvents are mixed together (e.g. gradient analysis) the pressure does NOT always follow a linear progression. In some cases, a reaction occurs between the solutions resulting in an overall change to the final viscosity of the mixture which may not be expected or understood by novice chromatographers (e.g. mixtures of MeOH/Water and ACN/Water are very well know examples which show these properties).
You can download a free, more detailed table of 'HPLC Tubing Backpressure Examples' in PDF Format at this link: