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:

Useful Windows Command Line Programs and Shortcuts

• Warning: These commands and shortcuts should only be used by experienced users who both accept and understand the risks involved. Please backup all systems, programs, applications, data and files before using any utility program or command line.

Command Names:

Command Line Shortcut (to exit from the command line, type exit):

            cmd

View Network Address (shows your local IP address)

ipconfig

View IP address Routes (shows Interface list with IPv4 and IPv6 Route Tables)

            netstat –r

Ping an Address or Host (From the command prompt, type "ping" followed by the IP or name)
           
           ping hostname     ( e.g. ping 192.168.254.01 )
           ping IP address    ( e.g. ping chiralizer.com )

Find Devices on Network (shows device IP and MAC address. *Useful when you know the MAC address but not the IP it was assigned to)

            arp -a

System Config:

            msconfig

Windows Version:

            winver

Add Hardware Wizard:

            hdwwiz

Control Panel Shortcut:

            control

Device Manager Shortcut:

            devmgmt

Disk Cleanup:

            cleanmgr

Display:

            dpiscaling

Print Manager Shortcut:

            printmanagement

Windows Explorer Shortcut:

            explorer

HPLC to UHPLC Conversion Notes (Gradient Time Program Adjustment)

In an earlier article we discussed how to adjust the flow rate, injection volume and column dimensions when scaling an HPLC method UP or Down. The formula's needed to do this are fairly simple. If we adjust for changes in the column dimensions or flow rate, what types of changes are needed to adjust the gradient time? The formula to make this adjustment is also very simple. Here is the information you need.

Terms Used in Formula:

Time in minutes, Gradient (Initial): Tg1

Time in minutes, Gradient (New):   Tg2

Flow Rate in mL/min, Column (Initial): Fc1

Flow Rate in mL/min, Column (New): Fc2

Column Diameter, mm (Initial): Dc1

Column Diameter, mm (New): Dc2

Column Length, mm (Initial): Lc1

Column Length, mm (New): Lc2

• Tg2 = Tg1 x (Fc1/Fc2) x ((Dc2²) / (Dc1²)) x (Lc2 x Lc1)

Here is an example problem to solve for. 

If we start with a flow rate of 1.000 mL/min (Fc1) on a 4.6 x 250 mm column with 5 micron support (Dc1 & Lc1) and have an initial Gradient Time of 10 minutes (Tg1), then what would the new gradient time be if we switched to a sub 2 micron support in a 2.1 x 50 mm column (Dc2 & Lc2) at 0.200 mL/min (Fc2)? 

To solve the equation we will plug-in the values for each part of the equation separately, then multiply them to obtain the result.

  (Fc1/Fc2):    1.000/0.200 = 5

  (Dc2²) / (Dc1²)  4.41/21.16 = 0.21

   (Lc2 x Lc1) = 50/250 = 0.20

  Tg2 = 10 x 5 x 0.21 x 0.20 

  Tg2 = 2.10 (or 2.10 minutes)

If a 2.1 x 50 mm column was substituted for the 4.6 x 250 mm AND the flow rate was changed from 1.000 mL/min to 0.200 mL/min, then the initial programmed gradient time of 10 minutes would be changed to 2.1 minutes

Routine Backup of HPLC Data, Methods And Related Data:; Part 3, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)

As a scientific consultant, I often review clients overall laboratory operations and make recommendations regarding documentation and procedures which may improve their accuracy and results. Some of these recommendations come in the form of SOP's.

Here is the third and final example of a 'must have' SOP' which should be in place for any laboratory performing HPLC analysis.

Part 3 of 3:
Routine Backup of HPLC Data, Methods And Related Data:

Another area which is often overlooked and can have disastrous consequences when ignored relates to regular software backups. Successful management of analytical instruments requires that all of the software which is required to operate and maintain them on a daily basis is safely backed up. This should include any needed patch files, hot-fixes or service packs too. Separate copies of all key software applications and licenses should be stored off-site, in a safe location where they can be assessed if and when needed by authorized individuals. This location may be another office location or even a home. It might even be located on a separate computer server or on the 'cloud'. However, for it to be a safe location, it must be off-site and protected from loss due to a hard-drive crash, data corruption, flood, fire, theft, earthquake or other disaster.

• Regular backups (automated are best!) of all acquired data, methods and related information needed to restore the data should be performed on a daily basis. Backups must also be tested and verified on a periodic basis (if you do not verify them, how do you know that they work?). Verification also serves to train you how to restore damaged data or software which is something that you do not want to learn how to do when you actually need it for the first time.

• You may also wish to address the use of suitable electrical power backup modules (UPS) to protect the computers used to acquire data from and run methods from power outages or surges. An article which addresses this topic can be found at this, "Power and Surge Protection for Computers & Analytical Instruments (e.g. Uninterruptible Power Supply AKA UPS)" LINK.

• SOP's describing Backup and Restoration of key Applications and Data should be created which detail the types of backups made, the frequency of backups, which applications and/or files are backed up, using which backup software products or applications, the restoration process used and how often it should be tested to ensure that you can still restore your data.

Make sure you have several people review the draft SOP's before approving. Sometimes what appears clear to you may in fact have a different meaning to someone else. Clear procedures should contain enough detail that people with different backgrounds will each carry out the procedure in the same manner. Often, these types of documents will go through many drafts and even after approved, should also be open to future suggestions to make them even better.

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To View the two previous articles in this series, please click on the links below. 

"Mobile Phase Preparation; Part 1, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)";

 "HPLC System Preventative Maintenance Frequency & Procedure (PM); Part 2, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)";