Backpressure Changes, Pressure Drop from HPLC Tubing Selection (0.007, 0.005, 0.010")

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:  

1. Try to optimize the plumbing of your HPLC system.  
2. HPLC Tubing lengths between connections (or HPLC modules) should always be as short as possible. 
3. 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:

HPLC Capillary Tubing Connection Volumes:

The length and internal diameter of the HPLC interconnecting tubing used in your system really does matter. The total volume contained in the tubing can dilute your sample or separated peaks. This can effectively undue the work of separating the peak(s) on a column. Extra volume in the tubing can also have the effect of increasing the gradient delay factor for your method (the greater the volume of the tubing from the pump head to the column inlet, the greater the delay in the solvent mixture arriving at the column). In general, keep the the total delay volume as low as possible. This is accomplished by connecting the various modules together using the shortest lengths of tubing possible. For systems which use standard sized HPLC columns (e.g. I.D.'s of 3.0 to 4.6mm and lengths from 100mm to 300mm) the tubing internal diameter should be 0.17mm (0.007"). For systems which use very short, mini or micro bore sized HPLC columns (e.g. I.D.'s of 1.0 to 2.1 mm and lengths from 50mm to 250mm) the tubing internal diameter should be 0.12mm (0.005"). Looked at another way, if the total column volume is less than 750 ul, consider using the smaller internal diameter tubing (0.17mm) to reduce band broadening. 

Here are some tubing volumes to help you evaluate the effect changing the I.D. or length has on the tubing that you use.

I.D. (mm)	I.D. (inches)		ul / cm	ul / inch
0.12	0.005		0.127	0.323
0.17	0.007		0.249	0.632
0.25	0.010		0.507	1.288
0.51	0.020		2.026	5.146
1.02	0.040		8.103	20.581

Pressure Drop Across an HPLC / UHPLC Column

Many of you prefer tables of data over equations that you must work out. So, instead of providing you with another equation, I have done some basic measurements for you to provide a general overview of how particle size (porous) effects System backpressure.

For simplicity, let us start with a few parameters. Pore Volume = 0.70; Linear Velocity = 1.44 mm/sec; Solvent Viscosity = 0.89 cP at 25C (Water). 

Pore Volume and Flow Resistivity will vary by column type. Obviously the back pressure will be higher with more viscous solvents (e.g. EtOH is 1.20 cP) and lower with less viscous solvents (e.g. ACN is 0.34cP). A Table of HPLC Solvent Viscosity values can be found here [ http://www.hplctools.com/lcsolvent.htm ]. Linear flow rates have been used for all column I.D.'s to better illustrate the relationship between column dimensions and flow rate. If you double the flow rate, then the pressure will approximately double as well. 

Note that when run at traditional linear velocities, most 2.5u particles are within the maximum pressure limits of most HPLC systems (under 400 bars). Only the newer sub 2.0 micron particles used in long columns exceed the 400 bar limit. The higher maximum pressure limits of many UHPLC systems allow the use of higher flow rates with these particles. Naturally, you should optimize both column efficiency and system dwell volume when developing any UHPLC method. Failure to optimize the dwell volume (and minimize all volumes) may result in very poor chromatography separations. Meeting any/all backpressure requirements to run a method does not translate to success in sample analysis. Successful ultra-fast separations require ultra-low system dwell volumes, higher sampling rates and usually smaller flow cell volumes.

HPLC Column I.D. (mm)	Particle Size (u)	Column Length (mm)	Flow Rate (mL/min)	Observed System Back Pressure (Bars)
4.6	5	250	1.000	89
4.6	5	150	1.000	54
4.6	5	100	1.000	36
4.6	5	50	1.000	18
4.6	3.5	250	1.000	182
4.6	3.5	150	1.000	109
4.6	3.5	100	1.000	73
4.6	3.5	50	1.000	36
4.6	2.5	250	1.000	357
4.6	2.5	150	1.000	214
4.6	2.5	100	1.000	143
4.6	2.5	50	1.000	71
4.6	1.9	250	1.000	618
4.6	1.9	150	1.000	371
4.6	1.9	100	1.000	247
4.6	1.9	50	1.000	124
3.0	5	250	0.430	90
3.0	5	150	0.430	54
3.0	5	100	0.430	36
3.0	5	50	0.430	18
3.0	3.5	250	0.430	184
3.0	3.5	150	0.430	110
3.0	3.5	100	0.430	74
3.0	3.5	50	0.430	37
3.0	2.5	250	0.430	361
3.0	2.5	150	0.430	217
3.0	2.5	100	0.430	144
3.0	2.5	50	0.430	72
3.0	1.9	250	0.430	625
3.0	1.9	150	0.430	375
3.0	1.9	100	0.430	250
3.0	1.9	50	0.430	125
2.1	5	250	0.210	90
2.1	5	150	0.210	54
2.1	5	100	0.210	36
2.1	5	50	0.210	18
2.1	3.5	250	0.210	184
2.1	3.5	150	0.210	110
2.1	3.5	100	0.210	73
2.1	3.5	50	0.210	37
2.1	2.5	250	0.210	360
2.1	2.5	150	0.210	216
2.1	2.5	100	0.210	144
2.1	2.5	50	0.210	72
2.1	1.9	250	0.210	623
2.1	1.9	150	0.210	374
2.1	1.9	100	0.210	249
2.1	1.9	50	0.210	125

* The results obtained in this table from are from one of our HPLC systems and reflects the total system backpressure (what the pressure gauge reads), with the column inline. Your results may vary due to differences in HPLC system used, flow path, tubing ID, column choice and mobile phase selected.