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)".

Common Causes of Baseline Noise in HPLC, UHPLC.

Achieving a flat baseline which does not exhibit spikes, ghost peaks, drift or wander in an unpredictable manner should be a primary goal when performing HPLC analysis or developing methods. Methods which result in flat baselines and have well defined, sharp peaks allow for accurate sample area integration. Integration algorithms perform poorly in quantifying peaks on sloped, drifting or noisy baselines. Excessive baseline noise contributes to many problems, including poor quantitation, high %RSD errors, peak identification errors, retention time variation and many other critical problems. Properly developed HPLC methods are reproducible methods which apply and utilize good chromatography fundamentals. Note: "Noise" is a relative term, often w/o meaning. You should always describe it scientifically, measure and compare the signal to noise ration (S/N) of the baseline vs the peak plus note any cyclical patterns (useful in troubleshooting).

Note: A lack of proper training in the operation of the HPLC system, improper start-up or poor quality maintenance of the chromatograph (Examples: failure to degas and purge the system lines before use; poor mixing; an air bubble stuck in a check valve, a bad detector lamp or a leak will often result in baseline noise) are the main causes of noise. Your HPLC system must be optimized for your specific application. Be sure and allow time for the mobile phase to reach full equilibration with the system before starting any analysis. Do not start an analysis until the baseline is stable.

In this article, we will discuss how temperature fluctuations, inadequate mixing, inadequate degassing and flow cell contamination can result in excessive baseline noise. We will provide suggestions on how to reduce or eliminate these problems. Troubleshooting should be done on-site, not over the web or telephone.

TEMPERATURE FLUCTUATIONS:

To obtain reproducible results, the temperature of the HPLC column must be kept constant during each analysis. Laboratory room temperatures often vary by several degrees during the course of one day and these changes will often change the retention characteristics of the sample(s). The 'On' and 'Off' cycling of power from an air conditioner or heating unit will often cause the baseline to drift in a cyclical manner, up and down, during the day (this can often be seen as a clear sine wave pattern when you zoom-in to study the baseline trace over time). Temperature also changes the refractive index of the mobile phase. Light based detectors (UV/VIS, RI...) will show this change as drift up or down). In some cases, a temperature change of plus or minus one degree C from run-to-run can cause changes in retention times which effect reliability of the method. 

To reduce temperature fluctuations, you must control the temperature of the column and mobile phase (if applicable) during the analysis. This is most commonly done by: (a) using equilibrated mobile phase at the start of the day or analysis, (b) keeping the interconnecting lines as short as possible (esp. any which exit the column and go to detectors/flow cells), (c) insulating any stainless steel lines with plastic tubing to reduce heat loss and (d) using a thermostatted column compartment to maintain the column at a single set temperature throughout the day. Control of the column temperature will remove 'temperature' as a variable from your analysis. Temperature should be a constant run to run, not a variable. Be sure and document the temperature selected as part of your method.

INADEQUATE MOBILE PHASE MIXING:

The associated noise and ripple of incomplete mixing can reduce the limit of detection (LOD) and increase integration error. Both high pressure (with separate pumps) and low pressure pumping (one pump with a multi-channel proportioning valve) systems depend on efficient mixing to reduce noise. For gradient analysis, failure to completely mix the mobile phase solution before it enters the HPLC column often results in excessive baseline noise, spikes and poor reproducibility. "Mixing" is often initially accomplished by combining the flow paths of more than one solvent channel together, using a multi-channel gradient valve or tubing. Mixing also performed directly in a mixer installed in the flow path of an HPLC pump. This mixer is often a static mixer (a simple 'Tee', a tube filled with baffles, a frit or beads, valve orifice or microfluidic device) of low volume design for chromatography use, but allows adequate mixing of the liquids within a prescribed flow rate range. The best mixers incorporate longitudinal and radial mixing in-line. A mixer with too low a volume or of insufficient design can result in poor mixing of the mobile phase (note: incorrect solvent compressibility settings can also cause mixing and noise problems too). To reduce mixing problems, first insure that the mobile phases used are fully soluble with each other. Next, make sure that any mixer used is appropriate for the flow rates and volumes you will be using. If needed, run a gradient valve test to insure that each valve channel is working properly, not leaking or introducing any cross-flow leakage to another channel. Monitor the baseline for pressure stability (% ripple), drift and artifacts  (e.g. spikes) in real time to spot problems and make adjustments to correct them. 

INADEQUATE MOBILE PHASE DEGASSING:

For the best results, continuously degas your mobile phase. Reducing the amount of gas will also improve signal to noise levels of detection, reduce drift and reduce pump cavitation. If you are using an electronic vacuum degassing module, make sure it is maintained and working 100%. A faulty degasser may cause more damage (contamination) to your system and methods. Maintain and Repair them just as you do for your other instrument modules. Gas bubbles may cause check valves to malfunction (get stuck), baseline noise spikes to appear randomly, flow rates and/or pressures to become irregular, detector outputs to show high levels of noise (from air in the flow cell) and also cause the loss of prime or cavitation in pumps. To achieve the best balance of low noise levels and high reliability, both aqueous and organic mobile phases should be fully degassed before and during use. This can be accomplished through stand-alone inline vacuum degassing modules or through gentle continuous helium gas sparging (*Helium makes an excellent choice of gas as it is not soluble in the mobile phase. Never use Nitrogen or Argon gas, they are soluble in the liquid!). In all cases, degassing must be continuous (not just done one time). Continuous degassing reduces cyclical noise and signal variations. For this reason, I do not recommend using ultrasonic baths to degas mobile phase solutions as these are not used in a continuous mode. The mobile phase solution starts to re-absorb gas as soon as you stop sonicating the solution. This results in continuous baseline drift (up and down).

Removal of gasses is critical to the function of a modern HPLC pumping system. The liquids used are compressed to very high levels which forces out solubilized gas from the solutions. This is best accomplished before the liquid is transferred into the pump. These gas bubbles must be minimized to achieve desirable baselines. *Even if you use a high pressure pumping system, an inline degassing system reduces the amount of noise and baseline drift. Properly maintain and service your degasser to insure compliant operation. IOW: Whichever method you use, always degas your mobile phase solutions.

FLOW CELLS:

One other less common cause of baseline spikes and random noise is due to either a dirty flow cell (i.e. the windows) or an air bubble trapped inside the flow cell. If the flow cell is suspected of having one of these problems, then it should be carefully rinsed or flushed out with an appropriate mixture of suitable solutions to expel the air bubble or remove the contamination. If possible, keep a spare, 'known good' flow cell on hand to swap out for troubleshooting purposes. This can help to quickly determine where the problem is. This flow cell must be the exact same size and type (volume and path length) for this purpose. If the cell's windows are contaminated and flushing does not restore them, then many manufacturer's offer kits which allow you to replace the windows and gaskets used. Warning: When attempting to clean or repair any flow cell, be sure and work within the manufacturer's operational specifications for the specific flow cell. Some flow cells are not designed to withstand even very low back pressure and damage can result if you exceed their maximum pressure or chemical rating.

Many other types of problems not mentioned in this short article can also cause baseline noise. For example, a sticking inlet or outlet valve on the pump, worn piston seals, worn out detector lamp(s) or detector electrode (EC) can induce noise. In all cases, the cause must be investigated in a logical, step-wise manner. Demonstrate what is working and rule out items one-by-one.

Reference: http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html

External (ESTD) vs. Internal Standard (ISTD) Calibration in HPLC

Reliable quantitation of sample analytes using HPLC analysis requires accurate and reliable quantitation of a standard(s). For chromatography applications, we commonly use either an External Standard or an Internal Standard, as applicable, to insure reliable quantification of the sample.

• NOTE: A quick comment about calibration methods. Before you begin to create any calibration tables or analyze any standards/samples, please make sure that your current chromatography method follows good chromatography fundamentals. It must be selective for the sample type, retain the compound(s) with good K prime values, be reproducible and resolve apart all of the samples and possible impurities with near to perfectly symmetrical peak shapes. Your calibration results will only be as good as your original method. A poor quality method may not provide reliable results so be sure and spend as much time as possible developing the initial HPLC method to be as rugged and reliable as possible before starting any quantitation or calibration. *Poor quality method development is the number one reason for problems with quantitation.

Methods of Quantitation, Peak-height vs. Peak-area: Both types of response provide a measurement of the detector signal output. Proper and reproducible integration of the signal output is critical. Peak area is the most popular choice in chromatography, but peak height measurements can also be used if the peaks have near perfect symmetry (very rare, so peak area is far more reliable for integration). Whichever method you chose, you must use it consistently and document it well.

Definitions, External & Internal Standards: For most samples, there are two commonly used types of standards used. When known standards are run separately from the actual samples (in their own chromatogram) and their response is compared to that of the sample in another chromatogram, then we refer to this as an External Standard (ESTD). When the standard is added to the sample and analyzed at the same time we refer to this as an Internal Standard (ISTD). With an Internal Standard we are comparing the instrument's response to the sample to a reference standard with similar response characteristics, both run together.

External Standard (ESTD) Calibration Notes: The sample must fall within a range bracketed by the calibration solution. I suggest that you include a range which covers concentration values which are ~ 50% or more outside of the expected range. Dissolve the final calibration standards into the mobile phase (or a weaker solution) when preparing the injection vials from the stock solution. At least five (5) different concentration values should be used per order-of-magnitude (larger range = more stds). *Inject the same volume of solution (different concentration) for each calibration standard point ("level") onto the column. Do Not inject different volumes of solution from one std vial to create different concentrations. Plot peak response vs concentration. Ideally, you should have a linear response and the line will go through the origin (true zero intercept, ideally, though matrix effects/or the use on non std detectors such as the ELSD or CAD may require complex curve fits/formulas to describe the response). Once you have injected all of the standards, repeat the process again at least three more times (or use multiple injections) to determine overall reproducibility before constructing the final calibration table.

Internal Standard (ISTD) Calibration Notes: Internal standards are commonly used when many sample preparation steps are required before the sample can be injected onto the column. The internal standard may compensate for any losses during filtration or extraction. Selection of the Internal Standard is critical. Some of the characteristics of a good ISTD should include: It must be different than the sample, well resolved and must not elute where any sample peaks could be expected; It should not elute where any interfering matrix or other compounds could appear; It should have a similar linear response as the sample (Inject a fixed volume/concentration); Available in a high purity form from one or more commercial sources (certified method); Must be stable and not react with the sample or mobile phase solution. 

Add it to the samples before any extraction procedures. Base the amount of ISTD concentration such that it is between 1/3 and 1/2 of the expected concentration of the sample(s). The sample's target concentration range is a good value to use. *Because of these and other strict conditions, finding a suitable Internal Standard can take some time and testing.

Common HPLC Calculations:

Capacity Factor / Retention Factor / Capacity Ratio:  k1 (K Prime)

k1 = T(R) - T(0) / T(0)
where T(R) equals the retention time of the peak in minutes and T(0) is
the retention time of an unretained peak. *For chromatography to take place, K Prime must be > 1.00 and for most modes of chromatography, should be greater than 1.5 or 2.0 for all samples !


Tailing Factor: USP: 't'

t = W(5.0)/tw/2

where tw equals the distance between peak front and T(R) at 5% of peak height units. W(5.0) equals peak width at 5% height, in minutes.


Theoretical Plates: USP and ASTM, 'N'

N = 5.54 x (T(R)/W(50))2          

Assumes width at peak half height (50)
* More info can be found at this link.


Resolution: USP, ASTM, 'R'

R = (T(R)(b)-T(R)(a)) x 2.35/(W(50)(b) + W(50)(a))/2

Assumes width at half height (50%) with peaks (a) and (b).

*Notes: Visually, "Baseline" resolution is R = 1.5. Your goal should be R = or > 2.0. ** R of 1.5 provides 99.8% separation which means you cannot accurately quantify a 0.1% impurity so develop the method to have a resolution value of at least 2.0.


Note: The appropriate formula(s) for use with your samples may depend on which of the many pharmaceutical guidelines and regulations apply in your country. Always consult the appropriate guidelines.