The Three Most Common HPLC Questions and How To Solve Them

The three most common HPLC related questions I am asked each week can be summarized below. Test your basic chromatography knowledge. Before reading the answers, see if you can answer them correctly on your own.

• "What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?"

• "How Should I Wash or Regenerate My HPLC Column?"

• "How Can I Tell if the Sample is Retained On the HPLC Column? or What Does It Mean When No Chromatography Took Place?"

Let us address each question in order and attempt to provide accurate answers (I have included links after each question to articles with more detailed explanations).

What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?

• Retention times must be reproducible from run to run.The causes of an unstable baseline and/or changing peak retention time(s) are often related. Common reasons include: Column temperature fluctuations, inadequate mobile phase mixing or degassing, leaks, dirty column, sample overload, lack of pH or buffering control (weakly ionizable samples can be very sensitive to changes). *Full Article link with detailed answers, here.

How Should I Wash or Regenerate My HPLC Column?

Note: Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide or booklet which comes with the new column. Additional information can be found on the vendor's website or by contacting them directly.

• Two issues must be addressed to answer these types of questions. (1) Always wash your column with a specific column wash solution which is stronger than your analysis solution. The use of a stronger solution (In this context, "stronger" means better at dissolving the samples and faster at eluting them from the column) as the wash solution requires regular use to maintain the column. Failure to regularly wash your column may result in compounds accumulating on the column over time (fouling the column) resulting in poor reproducibility, higher back-pressures, contamination and/or poor peak shape. (2) Next, always wash your column after each analysis. This should be a separate step, not incorporated into your analysis method. The analysis method should not include the column re-equilibration steps at all. A second, separate wash method should always follow each analysis method which includes the rinsing of the column with a "stronger" solution for an adequate period of time, then adjustment back to initial conditions where re-equilibration can take place to get it ready for the next analysis run. These are fundamental guidelines of good method development and follow well established principles. Developing methods in this way should increase the lifetime of your columns and improve the reproducibility of results obtained (better %RSD run-to-run).

For more information on washing bound proteins off RP HPLC columns, please refer to this linked article found here.

How Can I Tell if the Sample Is Retained On the HPLC Column? or What Does It Mean When the Sample Comes Out At or Near the Column Void Volume?

• Chromatography is a tool which when used properly adds one or more additional dimensions of physical or chemical characterization information to your analysis data. It does so first by using on-column RETENTION. Samples must be run under conditions which allow the material to interact with the chromatography support for a period of time. We define this time as the retention time. A sample which does not interact at all with the column support material will elute off the column early (and not be retained) at the "column void time" (or column dead time). We refer to this void time as the "T zero" time. When a sample elutes at or near the T zero time, no chromatography has taken place and no method has been developed. It is as if the HPLC column was not used. How do you know what the "T zero" time is (it will be different for different methods)? You must first calculate the HPLC column's dead volume. Once you know the column dead volume and flow rate, you can calculate the T zero time. A scientifically valid HPLC method will include conditions which retain the sample on the column for a long enough period of time to insure that it is interacting with the support. This allows for separation from other compounds to take place and is the purpose of chromatographic resolution. Without this retention mechanism, you are just flow-injecting the sample past the column and skipping all chromatography. It would be far simpler to just place the sample in a spectrophotomer cell as no retention or additional data would be obtained using that technique.

• When first learning liquid chromatography, two of the very first calculations you must learn to use in HPLC are: Column Dead Volume (aka: Column Void Volume) and the K prime of a sample (aka: Peak Capacity Factor). Do you know how to calculate these? They are calculated and reported for each method used. You should be able to tell anyone who asks you what the values are for each method. A chromatographer must know and understand them before using an HPLC system or running a method. They are also critical to method specificity and proper validation. Here are links which after reading and practicing, should make you an expert in these two fundamental calculations. 

"Determination of HPLC Column Dead Volume / Dead Time (T zero)";

"K Prime (also known as: Capacity Factor or Retention Factor): One of the Single Most Important HPLC Parameters of All".

So, how did you do answering these basic questions? If you have put in the needed study time and practical experience to learn and use these fundamentals of high-performance liquid chromatography, then you should have been able to easily provide correct answers to all three questions. If not, then it is time to go back and study up on those basic liquid chromatography texts and article links, plus get more supervised hands-on time with the instruments.

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.

HPLC Column PORE SIZE (or Pore Diameter) and Retention Time

Think of your typical porous bare silica support as a big sponge full of holes. All of those holes (pores) are where the sample will migrate through before emerging out the other side. With conventional chromatography supports, most of the interaction takes place inside the particle, not on the surface. The size and number of these openings relate to retention time. Besides particle size (particle diameter), pore size is one of the most important characteristics of silica based chromatography supports.


The pore size or pore diameter is often expressed in Angstroms (i.e. 80 A = 8 nm). The degree of porosity relates to the hydrodynamic volume of your sample and is inversely related to the surface area of the support. The larger the surface area of the support (smaller pore size), the longer the possible retention of the sample. For small drug molecule samples under 1,000 daltons (an estimate only) we often use high surface area supports with small pore sizes between 60 and 150 Angstroms (~ 200 to 500 square meters per gram). These provide high retention characteristics useful in separating apart many small compounds in one analysis run. For larger molecules (i.e. peptides and proteins), we employ supports with larger pore sizes (~300 Angstroms). Particles with small pores have larger surface areas which can provide more interaction with the sample. Note: Pore size is often determined using the BET Nitrogen adsorption/desorption equation. Due to endcapping of the support (e.g. C8 or C18), the actual value obtained is often 20-30% less than the original value.

When comparing bare silica columns or trying to identify similar conventional columns for use in a method, pore size must be considered. Manufacturer's publish the pore size in Angstroms (*sometimes in nm) for their different supports. Choosing columns with similar pore sizes is just one of many parameters needed to provide similar retention characteristics. 

Hydrophilic Interaction Chromatography (HILIC)

Perhaps you have a polar sample which shows poor or no retention under reverse phase conditions. HILIC may provide you with an alternative method for retention and separation. HILIC is a unique mode of chromatography which uses numerous retention mechanisms. The most important mechanisms involve surface layer liquid-liquid partitioning, adsorption and various types of ionic interactions.

Sometimes referred to as "aqueous normal phase chromatography", this hybrid technique utilizes a stationary phase which is very polar (e.g. silica, amino or a diol column) and a mobile phase which is made up mostly of organic phase with some water added. The retention mechanism is based on the idea that adding a low percentage of polar phase (water in this case) to a polar surface will result in a water layer forming. Typically this hydrophilic layer results when as little as 2 or 3% water is added to the mobile phase. The remainder of the mobile phase is an organic solvent (ACN is the most popular, but many others can be used). The polar charged analyte(s) will partition into and out of this adsorbed water layer (often, a cation exchange process takes place, but their may be a purely electrostatic mechanism going on as well). Unlike conventional reverse-phase chromatography, in HILIC increasing the organic content of the mobile phase increases the retention! Put another way, increasing the water content of the mobile phase and decreasing the organic portion (as in an HILIC gradient method) results in retention and then elution of very polar analytes. 

With the HILIC mode, sample elution (retention) decreases as you increase the polarity of the organic solvent. Based on this information, good HILIC column wash solutions usually use alcohols in place of ACN  (IPA, Ethanol and Methanol; with Methanol being a stronger eluter). For best results, consider incorporating an alcohol wash after each analysis. Allow plenty of time for the column to equilibrate too.

As with other modes of chromatography, the use of additives, buffers and pH can all play a role in retention and separation plus improve reproducibility. When developing methods, be sure and evaluate their role. Because of the low water content of most methods, buffers must be chosen carefully to insure full solubility. Ammonium formate and acetate are popular as are acids such as formic acid. Regarding pH, the low aqueous portion will mean that the actual pH of the final solution will be much closer to neutral.

 

• Caution: Sales and marketing people sometimes stick an HILIC label on an existing silica column to create a new product. No "special" HILIC columns are needed to develop an HILIC method. Since HILIC is a mode of chromatography, not a support type, most any high quality, NP silica column can be used.

 

As some HILIC methods may be hard to reproduce (very sensitive to changes in composition and long equil times) they are best used by more experienced cinematographers, only after conventional methods have been unsuccessful.

Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero):

Column Hold-up Volume, Column Dead Time or 'Column Void Volume' (the preferred name) are all different terms we apply to find the internal volume of a packed column  (divided by the flow rate and usually expressed in minutes for the Column Void Time). You must know what this value is BEFORE starting to run an HPLC method or perform liquid chromatography. The value for column void volume changes for different column dimensions and different column support types (e.g. fully porous, superficially porous etc) .

Are you peaks or samples eluting at or near the column void volume? If so, for most modes of chromatography, this implies that no chromatography has taken place and no HPLC method has been developed (SEC/GPC separate based on hydrodynamic volume, so elution at or near the column volume means the sample(s) were excluded from the column). Individuals with little to no chromatography training or experience often make this mistake and create methods which show poor retention. Make sure your methods are designed to retain each sample for a long enough time period on the column (K prime). How do you know how long is long enough? Start by estimating the Column Void Volume (use our table or calculate it for an estimate) then, calculate the K prime value for your sample. The K prime for each peak should be at least 1.5 (>2.0 is the accepted standard for most regulatory authorities) for the method to be useful and selective. *A more accurate value of column void volume will be found by measuring the void volume of your column (please read on).

Knowing the Column Void Volume and the Flow Rate used allows you to calculate the Column Void Time (which is the most useful initial value). Determining  the column void time or T0 ("Tee Zero" as we call it), is necessary to find other important chromatography values such as: the Resolution, Separation Factor and Capacity Factor (K prime aka: "K1") in a chromatography separation. Ideally, it is measured by injecting a sample which is unretained by the column & mobile phase (it passes right through the column support with little to no interaction). It may also be easily estimated for most fully porous, spherical, bare or coated silica supports if you know a few physical specifications of the column and media used. You should first estimate it, then measure it (the two values should be close, +/- 15%). Note: A practical "tip". You can also estimate T0 by noting when the small injector valve pressure peak ('blip') appears on the baseline. It results from the pressure change which occurs from switching the injection valve from the "load" to "inject" positions. Use a low UV wavelength to observe this deflection on the baseline.

Here is short list of typical HPLC column dimensions and their associated estimated void volumes for fully porous silica supports. At a flow rate of 1.000 ml/min these values would also be the same as the void time in minutes.

COLUMN DIMENSIONS (I.D. x Length (mm))                 VOID VOLUME (ml)

                         2.1 x  50                                                                  0.12
                         2.1 x 100                                                                 0.24
                         2.1 x 150                                                                 0.37
                         2.1 x 250                                                                 0.61
                         2.1 x 300                                                                 0.73

                         4.6 x  50                                                                  0.58
                         4.6 x 100                                                                 1.16
                         4.6 x 150                                                                 1.75
                         4.6 x 250                                                                 2.90
                         4.6 x 300                                                                 3.49

                       10.0 x 100                                                                 5.50
                       10.0 x 150                                                                 8.25
                       10.0 x 250                                                               13.75
                       10.0 x 300                                                               16.49

•  Column Void Volume Equation for Std Sized, FULLY Porous Supports:

Column Volume (ul) = (d^2 *Pi * L * 0.7) / 4 ;

•  Column Void Volume Equation for SUPERFICIALLY Porous Supports (e.g. Fused-Core, Core-Shell etc):

Column Volume (ul) = (d^2 *Pi * L * 0.5) / 4 .

   Note: Column Diameter & Length are in mm. Volumes are estimates (always measure to find the actual value).

[Note: All you need is the column's length and ID to estimate it. For most fully porous supports, use a 'Pore Volume' value of 0.70 in the above equation. This is the most commonly measures pore volume found for non-encapped, fully porous spherical bare silica support (please check with the manufacturer for the actual value of your support). For superficially porous supports, use a value of 0.50. Estimating the value will often get you close to the measured value, but due to the unique chemistries used to prepare supports, it is only an approximation.

Always measure the actual void volume of your specific HPLC column with a compound which is unretained by your column. For RP applications which utilize at least 20% organic, Uracil or Thiourea are often used, but some inorganic salts (e.g. sodium nitrite and sodium nitrate) have also been shown to work as well. When determining the "Column Void Volume", you are really measuring the void volume of the column plus any extra-column volume from the injection volume plus all lines connecting the injection to the column and the column to the flow cell. Note: This is very different from the "System Dwell Volume" which includes the volume from the pump (or gradient valve) to the column head.

A more detailed version of this table with other common HPLC Column Sizes and Tubing Volumes for capillary lines are available at the following links (Link #1) or (Link #2).