HPLC Column Cross-Sectional Area and Scaling

Here is a simple formula to use when scaling up or down Internal Column Diameter to maintain retention values (under constant linear velocity). Flow rate must be adjusted to account for any changes made to the column's cross-sectional area. We usually refer to these types of changes as the "Scaling Factor". To determine the scaling factor, we need to know the internal column diameters of the two columns we are scaling from (actually, we need to know the radius, but once we have the diameter, we simply divide the diameter by 2 to obtain the radius). *In this discussion, changes in cross-sectional area are the only parameters we are concerned with as column length does not affect scaling.

• Scaling Factor = (S);
• Column #1 Radius =  (R1);
• Column #2 Radius =  (R2).

     S = R2² / R1²

Example #1: 250 x 4.60 mm column scaled down to a 250 x 2.10 mm column. 

          Answer = 0.208. 

• If the original flow rate was 1.000 mL/min, the the scaled down flow rate would be 0.208 of the original or 0.208 mL/min for the 2.10 mm ID column. *For practical use and application, we often use either 200 ul/min or 210 ul/min to simplify the value.

Example #2: 250 x 4.60 mm column scaled up to a 250 x 10.00 mm ID semi-prep column.

          Answer  = 4.726. 

• If the original flow rate used was 1.000 mL/min with the 4.60 mm ID column, then we would increase the flow rate to 4.726 mL/min on the 10.00 mm ID column to maintain the same relative velocity (and relative retention). *For practical use and application, we often use 5 mL/min to simplify (round off) the value. 

Notes:

1. Flow rate optimization should always be carried out by running a standard at different flow rates and plotting the plate height (N) vs the flow rate. Test flow rates that are slightly below the predicted linear velocity and up to 2 times higher than that rate to find and optimize the flow rate for your sample (it must be determined through experimentation for your specific method). 
    
2. HPLC Columns packed with sub 2 micron supports may have optimum flow rates 2 to 5 times more than the predicted std linear flow rate so actual testing is critical to determining the most efficient flow rate. I recommend optimizing the flow rate used with analysis methods which use any particles which are 2.5 microns or smaller in diameter.
   

Changing from Reverse Phase (RP) to Normal Phase (NP) Mode (or vice versa) in your HPLC System

Two closely related HPLC questions which have the same answer are commonly found in my email folder each week. Both questions deal with concerns about switching mobile phase solutions used in HPLC. Here are the two questions with an explanation regarding what information is needed to answer them:

1. Can I switch-over an HPLC system which has reverse phase (RP) columns and RP solvents installed to one with Normal Phase (NP) columns and solvents (e.g. such as a C18 column with water and acetonitrile switched over to a silica column with Heptane and IPA )?
2. What is the best way to switch or change-over from a reverse phase (RP) mobile phase made up of water with buffer and acetonitrile to one that is all organic for normal phase (NP)?

To answer these questions we first must review the specified materials used in the HPLC system that are in contact with the mobile phase solutions (the 'wet' parts). 

If you have a system rated for use with most RP solutions, then you will want to verify that the same system is also rated for use with the NP liquids you are considering too (Refer to the manufacturer's product manual or specification sheet). Some HPLC systems may require no changes at all, while others may not be compatible for use. Some systems have seals, tubing and/or valve components that may NOT be chemically compatible with the proposed solvents. Use with incompatible solvents (or additives) may result in damage or destruction of the system. The instrument manufacturer will often provide the needed information inside the specific instrument's Operator / User Manual (*always contact the manufacturer if you have any questions regarding the safe use of the system). In some cases, the vacuum degasser, pump piston seals, tubing, injector seals and/or other parts may need to be replaced with chemically compatible parts before use.  The newer seals or parts may also have different operational limits (e.g. pressure) or specifications than the ones they replace. For example, the maximum pressure ratings with the different parts may be different (some RP to NP conversions result in much lower max pressure ratings and reduced part lifetimes). Many of the newer vacuum degasser units may have no chemical compatibility with the proposed HPLC solvents or additives. Proceed with caution. Check and verify compatibility first.

Anytime you switch from one liquid type to another, you must insure that the new solution is fully soluble (miscible) with the old solution being displaced from the system. The solutions used must be miscible and must not result in precipitation of any contents or contamination of the flow path (and/or plugging of lines) may result. 

Basic guidelines: 

• If any buffers or additives have been used, begin by flushing the system with the same solution, but without the buffer or additives dissolved. We want to remove those buffers and salts first. Flush the entire flow path. Flushing out these salts will greatly reduce the chances of system contamination or plugging. In the case of aqueous buffer solutions, initially flushing with ultra pure water will remove them. 
• Next, if the new solvent is not fully miscible with the old solvent (e.g. Water to Hexane...), then flush the system with an intermediate solvent that is fully soluble with both liquids (in the Water to Hexane example, IPA would be an excellent choice). In fact, for many aqueous to normal phase conversions, IPA provides the miscibility needed to change back and forth from a highly polar aqueous mode to a non-polar organic mode. *Consult a table of Solvent Physical Properties for guidance
  

Summary: 

Verify instrument chemical compatibility and the possible need to replace any seals or parts.

With the proper precautions and checks, many research grade analytical HPLC system can be routinely switched between RP and NP modes.

To switch from NP to RP mode (or RP to NP), flush the system of any salts, buffers or additives, verify liquid miscibility of the solvents, then use an intermediate solvent if needed to change over. 
A table of commonly used HPLC Solvent Properties will help you determine which liquids can be used as intermediate solvents for this purpose.

Air Bubbles Exiting the HPLC PUMP, Reasons Why.

Reasons For Air Bubbles Exiting The HPLC Pump:

• Pump Cavitation: When the pump pressure fluctuates wildly up and down, at very low pressures, this is often due to 'pump cavitation'. It is caused by a loss of priming inside the pump (Air, instead of liquid is in the pump's flow path). The HPLC pump should be primed with fresh, degassed mobile phase (following proper procedures) to restore smooth, stable flow. Often, this can be accomplished using the pump, set to a high flow rate, to draw liquid from the bottles. In cases where the pump is not strog enough, manually priming the low pressure lines using a syringe (~ 20 mL) filled with mobile phase and opening (or disconnecting) a fitting at the pump's outlet may aid in priming the system. Note: Depending on the configuration of your HPLC system, to fully prime an HPLC pump, you may need to run 20 or more mLs of solution through EACH channel. Please keep this in mind every time you use the system and every time you prepare or change a mobile phase solution. This article on baseline/pressure fluctuations may assist you in troubleshooting.

• Loose Connections: If one or more of the low-pressure fittings (nuts and ferrules)  which secure the Teflon tubing to the pump (or vacuum degasser) are damaged or loose, air may enter the system resulting in bubbles. Most pumps use plastic finger-tight style fittings 1/4-28 (or 5/16-24). The threads are soft and can be deformed. When access to these fittings is difficult, sometimes the fittings are left loose and will allow small amounts of air to be drawn in. A build up of salts and/or buffers on the exposed fittings can also allow air into the system (and the presence of deposits on the fittings indicates poor maintenance and a LEAK !). Inspect the tubing and fittings used for proper type, seating depth, wear/condition, cleanliness and/or damage. Replace parts as needed and re-install using the correct amount of torque.

• Flow Rate Too High, Too Low or Not Enough Degasser Equilibration Time: Degassing efficiency is directly related to the flow rate. Lower flow rates increase the residence time of the mobile phase in the degassing membrane or tubing, improving the gas removal. Higher flow rates provide less time for gas extraction and result in lower degassing efficiency (which equals bubbles in the outlet line). Check with the manufacturer regarding the optimal flow rate range for your degasser to insure you are working  within an acceptable range. Allow enough time for the degasser to reach its set-point and stabilize before use. If the degasser is not operating properly or is unable to "keep up" with the flow rate, then bubbles may be frequently observed in the outlet lines. 

• Choice of Mobile Phase Liquid: The miscibility of the liquid is also important. If the new mobile phase is not compatible with the previously used mobile phase, pump cavitation may result. Always flush the pump with an intermediate liquid that will dissolve in both the old and new fluids to flush them out before introducing the new mobile phase solution. (such as pure water or IPA, as applicable). The solubility of air (gas) in the specific solution used also affects the efficiency of the vacuum degasser. Aqueous solutions usually hold less gas than popular organic solvents (though air bubbles can be harder to "push" through in water). The amount of dissolved gas inside the liquid relates directly to the time needed to reduce it to acceptable levels for use in HPLC. Be sure to allow enough time to properly degass the new solution.

• Dirty or Obstructed Solvent Pickup Filters (Bottle filters): Bottle filters should be cleaned or replaced at regular intervals, following routine maintenance SOPs. When they become fouled or obstructed, a vacuum may form as the liquid is drawn into the system. This may result in air being sucked into the tubing or through a fitting (remember that the low pressure Teflon tubing used to connect the bottles to the degasser and pump is porous and allows gas to diffuse through it). The pickup filters should not obstruct the normal flow of solvent (typically they are 10-20 u in porosity). * a quick troubleshooting tip to rule out an obstructed solvent pickup filter is to temporarily remove the filter from the bottle. Observe the back-pressure on the pump to see if it increases and priming is restored. If so, the filter may be clogged. Always replace the filter with a fresh, clean filter and never operate the HPLC without the solvent filters installed.

• A Sticking Check Valve: The pump's inlet and outlet check valves must function perfectly, all of the time, to maintain proper flow and pump function. If an inlet check valve is not fully closing properly to seal off the high pressures generated inside the pump, then the pump will not be able to maintain pressure or flow. Inspect the check valve. Remove and clean it, per the manufacturer's guidelines (often this involves placing the check valve assembly in a beaker with solvent such as IPA and sonicating for 20 minutes to remove any residues. If cleaning fails to restore proper valve function, then replace the check valve with a new one.

• Worn Pump Piston Seals (or Pistons): When the piston seals begin to leak, air is allowed into the system. Pump piston seals require regular replacement (they are normal wear items). Scratched or worn pistons may also result in leaks with air getting into the system. Inspect and Test them both for pressure tightness on a scheduled basis or anytime you suspect a problem. Flush the pump with a suitable liquid, then run a high-pressure test to determine if they pass or fail the manufacturer's leak tightness and high pressure tests. Be sure to perform a physical inspection too.

• Contaminated or Obstructed Pump Outlet Filter: Most HPLC pumps have a small disposable outlet filter installed at or near the pump outlet line (Note: In the case of most Agilent brand HPLC pumps, a small PTFE filter may be found at the outlet valve or inside of the prime-purge valve). These filters should be replaced at regular intervals (monthly is strongly recommended), especially if any aqueous buffers or solutions are used (a they contribute to contamination). Contaminated pump outlet filters may result in a number of pressure instability problems. Abnormally high back-pressure during operation OR when vented to waste are indications it is obstructed. Regular scheduled replacement is the best way to prevent lost time and reduce system contamination.

 Any of the above causes may contribute to valves not functioning properly or air being drawn into the HPLC system. Troubleshooting should begin with the easiest and obvious areas first. Check the condition of the low pressure tubing used to make the connections to and from the mobile phase bottles and pump. If it is kinked, twisted or damaged, replace it with new tubing. Check the fittings used (nuts and ferrules) for tightness and to insure they have been installed properly and are not leaking. Repair all leaks. Keep the system clean (it is easier to monitor and troubleshoot problems when it is clean). Replace any damaged fittings with new ones. Check the solvent pickup filters monthly to insure they are clean and not obstructed. Make sure the flow rate you are using is within the acceptable range for your degasser. 

Has your degasser module been professionally cleaned and serviced within the last 5 years? Are any degasser errors being generated? Is the vacuum degasser making any unusual sounds? Is liquid being emitted from the vacuum pump exhaust port? If any of the answers to these questions are 'yes', then have the HPLC vacuum degasser professionally diagnosed for problems so that repairs can be made to restore function.

Do your HPLC Methods Meet Good Chromatography Fundamentals? HPLC Training: RETAIN, SEPARATE and RESOLVE

When an HPLC or LC-MS method is not developed properly, it may not be selective for the sample and may not show any retention on the column. When this happens, everything injected may elute out at the same time and appear to be 100% pure . *These types of errors are easy to spot by anyone with formal training and experience in chromatography concepts (note: "years" on the job are not the same thing as years of practical knowledge and/or formal training in the technique. We routinely provide consulting services to clients with 10 or more years on the job performing chromatography analysis, but whom have not received any formal training during this time and make errors of this type).

Developing HPLC Methods which follow good chromatography guidelines and fundamentals should be key goals of HPLC method development. When developing an HPLC ("UHPLC") method, you must develop an analysis method which is selective for the compound of interest. 'Selectivity' is the most important variable to focus on when developing methods. Your method must demonstrate that it can: (1) Retain; (2) Separate and (3) baseline Resolve all peaks present (and any possible impurities or related substances), in a reliable and repeatable way. Failure to demonstrate that your HPLC method meets these basic requirements AND is selective for the sample being analyzed means your method is invalid.  

*You may be surprised to know that many HPLC methods (including some published papers and "Validated" Methods) do not meet these basic requirements. In this case, knowledge is truly power. If you have the practical knowledge and understanding of this technique, you will be able to easily spot these invalid methods. Make sure you review other methods as part of your training. Never assume because someone else published it or "did it that way", that it is valid. It may not be. An average of 20% of the methods I review do not meet these basic requirements and are invalid.

• Do your HPLC methods meet these requirements? 
• Can you demonstrate to others, who are knowledgeable in the technique, that your method follows good fundamentals? 

You should be able to demonstrate knowledge of these basic principles and have confidence in them.

Proper HPLC method development training must include and stress the following three practical, fundamental concepts of Retain, Separate and Resolve:

• Demonstrate that using your HPLC Method, that the sample is RETAINED on the Column. *Screen many columns to find the best one, early in the process. For most modes of chromatography, you do this by first estimating then measuring the column void volume. How do you know if it is retained long enough? Next, you calculate the K prime (Capacity Factor) of your sample to insure it meets basic chromatography guidelines (or regulations). * K prime > 1.5 (or > 2.0 for most regulated environments). Note: While retention is required, K prime is not applicable to SEC modes of chromatography.

• When satisfactory retention is achieved (K prime), begin optimizing the method to SEPARATE and baseline RESOLVE all compounds. This is achieved using proper HPLC Method Development techniques plus the practical knowledge and experience which comes from good training. Please refer to these two linked articles for more information; "Modern HPLC Method Development Tips (PART I)" and "Modern HPLC Method Development Tips (PART II)".