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)".

HPLC PEAK Fronting and Tailing, Common Reasons For It

All users of HPLC need to know and be familiar with the correct terms used to describe non-Gaussian shaped peaks. Two of the most common undesirable peak shapes, peaks that show "Fronting" and peaks that show "Tailing" indicate problems with the HPLC method.  A quick refresher on why you may observe an HPLC peak front or tail on the chromatogram follows. 

Peak FRONTING: First, let us define what peak fronting looks like. The leading edge (front) of the peak is vertical, straight up and non-Gaussian in shape. This sharp increase in signal is easy to spot. 

Common Reasons for Peak FRONTING:

• Poor sample/peak capacity. In other words, too low a K prime (not enough retention on the HPLC column) resulting in no chromatography taking place. To solve this problem you must develop a proper HPLC method which first retains the compound(s) of interest, holds them long enough to obtain an acceptable K prime and resolve them away from other peaks, then elutes them off the column.

• Injection Solution Too Strong:Your sample(s) should be dissolved in the mobile phase and not in a solution that is "stronger" in elution strength than the mobile phase. Example: If you method is 100% aqueous, do not inject the sample in a solution with organic solvent. Follow fundamental good chromatography guidelines.

• Column Fouling / Overloading of sample. When the HPLC column is overloaded with sample, the peak shape will show fronting. Decrease the injection volume and/or concentration, as appropriate, in 10x graduations until the peak shape is normal.

• Saturation of the Detector: Just as with overloading the column the peak shape may change, overloading the detector's measuring range may also result in saturation of the signal and loss of accuracy. Decrease the injection volume and/or concentration, as appropriate, in 10x graduations until the peak shape is normal and back on-scale.

Peak TAILING: First, let us define what peak tailing looks like. The trailing edge (tail) of the peak slowly drops off towards the baseline and  is non-Gaussian in shape. For those with GC experience it appears similar to a peak that "bleeds" and continues to interact with the column for an extended period of time.

Common Reasons for Peak TAILING:

• Flow path Diffusion (from extra-delay volume). Poorly swaged fittings/connectors, a column with a void, incorrectly sized capillary connection lines may all contribute to peak tailing. Optimize the flow path, column and connections.

• pH dependence for ionizable compounds. If the sample is easily ionized and the difference between the pka of the sample and the mobile phase is less than 2 pH unit, tailing may result. Being sure to work within a safe pH range for your column, increase or decrease the mobile phase pH to be > 2 pH units away from the sample's pka to reduce tailing.

• Type 'A' silica or heavy metal contamination of the support. Many older style column supports did not use ultra-pure, heavy metal free packing material. These material often interacted with the sample on the column resulting in changes in retention, The use of more modern type 'B' or 'C' packings has eliminated many of these problems.

• Residual silanol groups present on support. As with the earlier type 'A' supports, non fully end-capped supports with residual silanol groups often resulted in secondary, extended retention effects. Use of more modern, fully end-capped, ultra-high purity packing materials (and/or mobile phases which better address these residual groups) often allow Gaussian peak shapes without the need for many additives.

• Column Fouling / Overloading of sample. When a column is not washed of all retained material after each analysis, it may build up over time and change the surface chemistry of the support. This may lead to changes in retention, especially delays in both binding and elution. Wash, regenerate or replace the column to solve.

You may also be interested in reading a related article; "Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)".

A Case of Changing Solution pH. Formic Acid Stability in Solution (Methanol)

Real life examples help to better illustrate problems that I am called in to troubleshoot for clients. As a professional scientific consultant, many of my clients have spent months (sometimes years) trying to solve an analytical problem on their own before I am brought in to make the diagnosis and propose a solution. Many years of working in a wide range of scientific fields allows me to identify problems quickly and efficiently saving clients the most money and allowing them to resume work on their projects.

This was the case during a recent consult for a major cannabis testing laboratory. They were having a great deal of difficulty obtaining reproducible results for their analytical testing screens (14 compounds in their analysis with a need for repeatable and accurate results). Variations from 25% to 50% were observed run-to-run over the course of seven days. They assured me they were doing everything in the same way. To begin the troubleshooting process, we started by looking at the actual data gathered and the actual method(s) used to acquire the data. These were evaluated to see if they followed good practices and techniques, also to make sure they had SOP's in place which were clear. Good SOP's must include enough detail to allow anyone reviewing them to prepare samples, standards and/or solutions in the exact same way. Additionally, the HPLC instrumentation was checked and tested to verify it was performing as designed.

After reviewing their training and methodologies on-site, a number of areas of concern were quickly identified. One of the most likely reasons for the variation in values over time was found to be caused by a common mistake in the preparation of mobile phase solutions for the HPLC system. To save time, the client's scientists prepared all organic solvent solutions in advance (~ one month or more), then filtered and stored them at room temperature. For example, their solutions of 0.1% formic acid in HPLC grade Methanol were pre-mixed and stored in glass one liter bottles. These bottles were then put aside, for an average of one month before use. This finding proved key as someone with proper HPLC training would be aware of a well known problem when formic acid is left in pure organic solvent, especially methanol, over time (less so with ACN). Briefly, the formic acid content degrades quickly over time and is often found to be only half of what it was initially after just three or four days (If you have not done so already, this is a simple and useful experiment to run in your lab, monitoring the acid level by titration, not with a pH meter, over time at room temperature in methanol)! This degradation continues over time reducing the amount of acid in solution. If the acid is added to the solution to enhance ionization (i.e. LC-MS; LC-MS/MS) or provide acidification to maintain the sample in a fully ionized form, then as the level of acidification decreases, so does the solution's ability to maintain it. In other words, your HPLC method may change over time (resulting in an in-valid method).

•  I have always promoted the importance of making and using freshly prepared mobile phase solutions (daily), especially where any aqueous solutions are used (to prevent degradation of additives and/or bacterial or fungi growth). However, this precaution does not normally apply to many pure organic solvents, but there are a few very important exceptions to this, formic acid and methanol in this example. 

Changes were made to their SOP's to insure that future solutions of formic acid in methanol were not prepared in advance, but instead, fresh on the day needed only. This coupled with a few basic improvements to their column washing, equilibration and overall training resulted in %RSD of only 0.3% for future analysis runs.

 
As a side note, I have been asked why solutions of formic acid in methanol are sold commercially for HPLC use? I have no answer to this, but respectfully remind everyone that just because something is offered for sale, does not mean it should be purchased. Ask yourself if the item is appropriate for your application? It may not be suitable for your use or application. 

BTW: Please be sure to flush your HPLC system of all organic acids (e.g. acetic, formic) after use and do not leave them in the HPLC system overnight. Even 1% levels of organic acids may be corrosive to stainless steel. 

Air Bubbles Exiting the HPLC Vacuum Degasser. Reasons Why

A common question we are asked to solve relates to why air bubbles might be observed exiting out of an HPLC vacuum degasser module  (where the mobile phase leaves the degasser ports to go to the pump heads and/or gradient valve)? Troubleshooting and answering this question is most easily accomplished if you first have a solid understanding of the HPLC flow path, how to make proper connections and are familiar with performing routine maintenance on the HPLC system. 

• Key Point: HPLC systems utilize Teflon low-pressure tubing to transfer the mobile phase (solvents) from the mobile phase bottles to the HPLC pump. The Teflon lines are permeable to gas in the atmosphere. Gas is diffusing through the plastic tubing used to transport your solvents. This is one of the reasons why we purge the entire flow path of the HPLC system before use, each day. Overnight, gas has diffused into the system so we start by flushing (purge) the mobile phase from each bottle, through the degasser, through each channel all the way to the pump head, to waste.

To start the troubleshooting process to find the reason why air bubbles may be observed existing the HPLC vacuum degasser module, we examine the flow path.

Reasons For Air Bubbles Exiting The HPLC Vacuum Degasser:

• Loose Connections: If the low pressure fittings (nuts and ferrules)  which secure the Teflon tubing to the degasser are damaged or loose, air may enter the system resulting in bubbles. Most vacuum degassers 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 (such as found on many of the generic small benchtop degasser which use the micro-chambers or the HP/Agilent model G1379-series). Inspect the tubing and fittings used for proper seating depth, wear and/or damage. Replace parts as needed and re-install using the correct amount of torque.

• Flow Rate Too High 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. 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.

• Choice of Mobile Phase Liquid: 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.

• 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).

• Vacuum Degasser Damage: HPLC Vacuum degasser modules, like most other component parts of your HPLC system break down over time and require professional diagnostic testing, cleaning and repair. Under ideal conditions, most inline electronic vacuum degassers require diagnostic testing and cleaning or repair every 4 to 5 years. *Many show signs of contamination or failure before that time. The internal vacuum tubing becomes contaminated and worn over time. The vacuum pump is an electromechanical part which is exposed to all of the mobile phase additives and solvent vapors during use. Other internal component parts such as vacuum valves or restrictors may also become contaminated or worn over time. The vacuum degassing membranes (or tubing) themselves can stretch from use and wear out over time. The vacuum chambers may be exposed to incompatible chemicals or over-pressured resulting in internal leakage. Certain chemicals may also attack and even dissolve the degassing membranes causing more internal damage and contamination of the mobile phase. These devices do not have any "contamination" detection alarms and the vacuum sensors sometimes become damaged over time leading to false vacuum levels being reported. Never rely on the module's built-in error alarm system as proof of compliance (no more than you would the reported flow rate shown on the computer screen. It must be measured to be known). Regular professional HPLC degasser testing and service are required to maintain the modules and meet compliance requirements.

 Any of the above causes may contribute to air being drawn into the degasser 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 degasser. 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. Replace any damaged fittings with new ones. Check the solvent pickups 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 degasser making any unusual sounds? 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. 

Additional Information:

• "Agilent / HP Brand Vacuum Degasser Troubleshooting Tips, Errors and Repairs"; https://www.hplctools.com/agilent_degasser_error_troubleshooting.htm

• "Waters Alliance and Acquity Brand UPLC Vacuum Degasser Troubleshooting Ti[s and Errors"; https://www.hplctools.com/waters_degasser_error_troubleshooting.htm

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