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)".

Determine the HPLC System Dwell Volume (Gradient Delay Volume)

Note: The total HPLC gradient system dwell volume is different than the HPLC column’s void volume. Two different terms for two very different measurements.

When we perform gradient HPLC analysis, the mobile phase composition is changed over a period of time. The mobile phase is mixed in real time by the pump(s), mixer and/or valves, then transported to the injector and finally, on to the head of the HPLC column. The total volume of liquid contained between where the mobile phase is mixed and the head of the column helps us determine when the newly mixed solution arrives at the column head (it is not instantaneous). This delay is often referred to as the gradient delay time (or delay volume) and its value will vary for different HPLC systems due mainly to differences in tubing dimensions used, pumping system type and the design of the flow path. 

For example: If the system dwell volume is found to be 1 ml and the flow rate used is 1.000 ml/min, then the gradient delay time is one minute. 

So how do we know what the system dwell volume or gradient delay volume is? Well, we measure it of course!

Measure the ‘System Dwell Volume’ (aka: Gradient Delay Volume)*:

(1) REMOVE any HPLC column(s) and install a Zero Dead Volume Union (*ZDV) or a restriction capillary of know volume in its place.

(2) Prepare Two Different Mobile phase solutions:

Bottle ‘A’: HPLC grade Methanol (MeOH).

Bottle ‘B’: HPLC grade Methanol with 0.1% acetone added (v/v).

(3) Set your UV/VIS detector to 265 nm (8 nm Bandwidth, Reference OFF).

(4) Program a suitable system flow rate and create a simple Gradient Method (linear change) which starts at 0.0 minutes with 100% ‘A’ (HPLC grade Methanol) and 0% B (HPLC grade Methanol with 0.1% acetone added) and runs to 0% ‘A’ and 100% ‘B’ for about 10.0 minutes (actual times used will depend on your selected flow rate).

(5) Flush and degas both solutions, ‘B’ first, then ‘A’ through the system until you get a nice clean, flat baseline. Make sure their is enough backpressure on the pump (>40 bars) to obtain a stable signal (use a restrictor or back-pressure regulator if needed).

(6) No injection should occur during this method.

(7) Start the method (RUN) and observe the 265 nm signal over time. At some point you should observe the signal begin to rise. When you see this signal change occur, the acetone has finally made it from the pump head to the detector’s flow cell. Make note of the time this occurs. 

Using the known flow rate and observed signal change time, you can now estimate the total system dwell volume. 

Example: If you observe the signal start to rise steeply at 2.00 minutes and your flow rate was 1.000 ml/min. Your system dwell volume would be 2.000 mls. 

A more accurate system dwell volume value can be obtained by next running the same method with an injection of acetone (e.g. 1 ul) and noting the time at which the injection peak is first seen. That will give you the time it takes the sample (and therefore the volume needed) to go from the injector to the flow cell. If you subtract this time off the system dwell time you recorded in the last test, you will have the actual measured time from the pump head (or proportioning valve) to the head of the column (vs the flow cell). Normally the volume contained in this tubing and flow cell are very small relative to the volume in the rest of the system, so we can ignore them. However, when using some of the very low volume columns (e.g. 2.1 x 50 mm), the volume contained in these areas can become significant so when appropriate, we need to be aware of them.

Failure to take into account changes in HPLC system dwell volumes can result in methods which no longer work or provide different results. This is because the gradient rate change you program in your method may not allow enough time for the new mobile phase composition to reach and flow all the way through the column in the time that you have programmed. A common mistake we see is when users forget to adjust the gradient profile when changing column dimensions or program changes using too fast a time.

BTW: One common trick we use to improve compatibility between systems which have different dwell volumes is to include an initial (time 0.0)  isocratic hold-time into the start of each method. If all systems used have system delay volumes under 3 mls, then add a 3 minute isocratic hold time at the start of each method (if 1.000 ml/min flow rates are used), before any gradient starts. While not the best way to deal with the issue, this type of “cheat” can make it possible to quickly adapt a method for use on several different system types.

*Note: This is a generic method to determine the system dwell volume or gradient delay volume. Detector signal buffering and flow cell volume also adds to the delay and in some cases, must also be accounted for too. There are many other methods which can be used for this determination as well. This proposed example serves to illustrate the concept only.

Modern HPLC Method Development Tips (PART II):

This is the second of two articles (Part I) which will provide suggestions on how to improve the HPLC (UHPLC) method development process. - PART II

INITIAL METHOD DEVELOPMENT:

1. Before you start, learn what you can about your sample, its hazards, solubility and properties. Conduct a quick literature and/or keyword search on the web using a popular search engine (e.g. GOOGLE). You can often find many journal articles, white papers, application notes and chemical data on the web in just a few minutes.
2. Determine which liquids your compound(s) are soluble in. Use a pipette and several glass vials or tubes with different solutions (pH is important too). This will narrow down the types of mobile phase and chromatography modes that you can use.
3. Column choice is the most important part of method development. The stationary phase that you choose has the single greatest effect on selectivity! Select the right column and mode of chromatography. Most RP methods should start with at least a modern ultra high purity, metal free type B silica column with a C8 or C18 support (*But you must select the column type that best suits your application). For small molecules (<1,000 Daltons) select a standard sized analytical column with a 2.5 to 5.0 micron particle size and pore size between 60 and 120 Å (e.g. 4.6 x 150 mm; 3.0 x 100 mm). *For larger peptide or protein molecules you will need a particle with a large pore size (~ 300 Å). For optimal results, columns with very small internal volumes should be paired with HPLC systems with similar ultra-low internal delay volumes. Your HPLC system should be optimized to balance the needs of good mixing, low delay volume and proper sampling rate for your method. If the method is likely to be transferred to different types of HPLC systems, then you may want to initially stay away from the < 2.1 u particle supports for your method development. At this time, these tiny particles can not be reliably packed into columns as well as the larger sized particles and generally have much poorer %RSD values than larger particles (i.e. 2.5 to 5 u). Method development is initially easier & generally more rugged using conventional particle sizes [Keep things simple when starting out. You can always change later on]. Once you have selected a column and decided on a flow rate, make sure you calculate and measure the actual column void volume to find the 'Tzero value' (column dwell volume). You will need this value to find out if your compound has been retained on the column (K prime) and to also determine most of the needed performance calculations and parameters.
4. Once you have selected an HPLC column (if possible, please start with a brand new column) you will need to test it and establish a baseline to show that it meets both the manufacturer’s specifications and, far more importantly, that it meets your method’s requirements. In most cases, do not use the manufacturer’s QC test solution for this. Those stds are designed to allow the column to easily pass manufacturing QC, not the more critical requirements that you may need to prove. We prefer to use a real sample mixture (~2 compounds) that are similar to the type proposed for the method. They must be well retained on the column (K prime of > 2), easy to prepare, stable and reliable. This std and the specific method you develop for it, will be used to prove the column’s performance (i.e. R, S, K prime, Tzero, Plates). It will be used again, when the column’s performance is called into question.  
5. Notes on Mobile phase and additives: Keep it simple. Avoid the use of any additives such as ion-pairing reagents when first starting out. They are overused in general and can cause problems later on. If required, they can always be added later on. Use only HPLC grade solvents, fresh RO HPLC grade Water and the highest purity acids, bases and/or additives, if required. Use only filtered (0.22u) products. Always dissolve samples in the mobile phase (or a weaker solution) for injection. For many RP methods a low pH mobile phase (~ pH 2.5) provides a good starting place. Samples with a pKa far enough away from this value are likely to be retained, stable and unaffected by small changes in pH. *pH is usually one of the last parameters which is optimized.
6. Use a Gradient Method to find the approximate elution conditions of the mixture AND make sure you are detecting all of the compounds injected onto the column [For dedicated RP isocratic methods, start with a high organic mobile phase % (i.e. 95%) and record the results. Continue to reduce the organic content in steps of 10% and observe where the best compromise exists between retention and elution]. For RP gradient methods, start with a very high aqueous % (e.g. 98%) and run your gradient, slowly, to a very high organic % (e.g. 95% or 98%, not 100%!) to make sure you retain, hold, and then elute everything. Your mobile phase conditions must be strong enough to elute everything off the column during the gradient portion of the run (long enough hold time). Please Do NOT include the gradient reversal portion back to initial conditions (wash and re-equilibration) of the gradient as part of the same analysis method. The method should end after the gradient has reached and been held at its maximum level for a period of time (we refer to this as the "hold time"). It should NOT switch back to the initial conditions to re-equilibrate the column. Don't make this novice mistake. Column flushing and re-equilibration should be a separate method and/or step, separate from your analysis method. If you include your column flushing (washing) and re-equilibration steps as part of your analysis method, you are also forcing the baseline's slope to change radically. This may interfere with integration plus include extra peaks and baseline changes that you are going to have to integrate, identify and explain to others (e.g. auditors). Additionally, including these wash steps as part of the same method wastes time as you must wait for these steps to complete before you can start to process the data obtained in the analysis method. Summary: Develop methods like the professionals do and create separate Analysis and Flush/Re-Equilibration Methods (or steps).
7. If your compounds can be seen with a UV/VIS detector, then make sure you are using a Diode Array Detector (aka, DAD or PDA) set to scan a wide range of wavelengths (e.g. 210 - 410 nm). Method development should not be performed using a single or multi-wavelength detector. This invites errors, limits the utility of the method and does not result in any cost savings. You must have a full scanning detector so you can detect all possible peaks at the same time. If you use a single or dual wavelength detector you may not know if an impurity has been introduced or if you have a co-eluter (because you will have no way to detect it). Generic starting settings for Wavelength Bandwidth should be ~ 8 nm, software based Reference Wavelength OFF and the sampling rate must be set to collect at least 20+ peaks/sample peak of the narrowest sample peak observed and integrated (at ½ height). Run with scanning turned 'on' for all analysis methods and review the spectral data for each run. This provides important qualitative data about the compounds which may be used for purity determination and also to demonstrate how the method is selective for the compound(s) of interest. *If your compounds do not absorb well (weak chromophores), then you may still want to have a UV/VIS detector inline (first) with a secondary detector second. The UV/VIS detector will still be very useful for troubleshooting and detecting other compounds. Select an appropriate type of detector and compatible mobile phase as required for the secondary detector (e.g. RID, EC, FLD, ELSD, CAD, MS...). Note: If available, LC-DAD-MS is one of the most useful instrument setups for LC method development.
8. This needs to be repeated (from Part I)....  Before starting ANY HPLC analysis, the HPLC pump must be running and operating with no problems, achieving a stable baseline, steady flow rate with as little pulsation as possible (~ 1% ripple or less). Accuracy depends on this. Do not begin any HPLC analysis unless the HPLC pumping system is working perfectly.