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)".

PEAK PURITY Determination by HPLC Diode Array Chromatography Software (UV/VIS): Limitations and Uses

"Peak Purity" software determination by HPLC UV/VIS detection is one of the most abused and easily misunderstood features found in advanced liquid chromatography systems (e.g. HPLC, UHPLC and CE).

For HPLC, one or more inline detectors can be used which provide additional data about a fully resolved peak’s physical or chemical properties. The data obtained can be compared to that of a pure standard, or known impurity. For compounds which absorb light in the region of most UV/VIS detectors (~ 200 to 900 nm), a single wavelength detector (e.g. UV/VIS) provides a very limited second dimension of data (retention time is the first dimension), but a scanning, multi-wavelength UV/VIS detector can add a second and third dimension of data to the retention time. Scanning detectors, commonly known as Diode-Array Detectors (aka: DAD or PDA) are commonly used in HPLC and CE analysis (they are required for routine method development). A scanning DAD can provide detailed sample UV/VIS spectra across a range of wavelengths for each peak, at any retention time recorded, allowing for a 3D plot of the spectra to be recorded much like a “fingerprint”. "Pure" compounds which do absorb light across a pre-defined wavelength range should show identical spectral profiles (“slices”) across the upslope, apex and down slope of the resolved peak. "Impure" peaks may show dissimilar spectra across the width of the peak revealing the presence of a co-eluting peak or impurity. Impure peaks may also NOT show any dissimilar spectra at all (because some compounds may not be detected). When a properly developed HPLC analysis method is used to evaluate the purity of a sample, the single dimension of “retention time” is evaluated with additional dimensions of analysis such as the UV/VIS peak spectra. "Peak Purity" relies on the detection of a sample's spectral profile to detect the presence of an "impurity" (that may have co-eluted with the sample). This additional dimension of analysis (full Spectra) is required to improve the confidence level that a peak may in fact be correctly identified (qualitatively) and does not contain any co-eluting compounds. IOW: "Peak Purity" does not actually test for purity.

Diode-Array 'Software' based Peak purity determination by HPLC is a qualitative assessment of the impurity profile of the sample. It is designed to reveal impurities, NOT prove peak purity. BTW: We really should rename it “Peak Spectral Impurity Assessment" because that is in fact what we are measuring. The algorithm used for Peak Purity determination is designed to confirm the presence of one or more impurities by comparing spectral data slices (multiple slices taken at the apex and both the upslope and down slope sections of the peak).  A mismatch would indicate the peak has not been fully resolved (one or more co-eluting peaks are present). In other words, it is impure by UV/VIS analysis. Note: It does not indicate that the compound is impure, but rather 'the peak' being measured is. As you can see, the concept makes sense, but the how it is used in many laboratories is flawed leading to invalid reports and data.

• “Peak Purity” does not in fact indicate the actual purity of the compound, but instead indicates when a peak may be found to contain impurities. It is an estimated measure of PEAK Impurity.

In simple terms, IF the spectral slices obtained from one peak are not identical, than the peak may contain one or more impurities. Co-elution is the most likely reason for this.

Points to consider when using "Peak Purity" software:

• The absence of any spectral differences across the sample peak are not an indication of actual purity;
• Compounds similar to your sample may have similar absorbance profiles (fooling the system);
• The relative concentration of actual impurities may not be high enough to detect;
• The compounds / impurities may not absorb light at the wavelengths scanned;
• The HPLC method used, the software settings and the parameters that you chose in the ‘Peak Purity’ software menu have a huge effect on the results obtained. Different people often get different results for the same sample. Inputting poor quality settings or using a poor quality method often leads to misleading purity results. This is an advanced software feature requiring many years of training to use. Again, it does NOT test for purity.
  
• The peak of interest must be retained on the column (K prime > 2) and resolved apart from any observed peaks. Don't use peak purity to analyze peak(s) which elute at or near the column void volume (Low K prime values may demonstrate that good chromatography fundamentals were ignored. Poor quality methods fail validation). Poor quality HPLC method and poorly selected DAD "Purity" settings result in invalid results (audits, recalls etc may result from reliance on a subjective "software" feature).

We prefer to think of HPLC 'Peak Purity Assessment' as a null test. If the recorded peak spectral data slices are different, than you probably have co-elution and/or impurities present (so try and develop a better method to resolve the peaks apart). If no differences in the spectra are seen (they are similar), then the peak may be pure or may contain compounds with similar spectra as are commonly seen with related reaction synthesis products or compounds. So only when you detect differences in the acquired spectra can you be confident that there IS a qualitative difference or impurity present. You will not know what percentage of impurity level is (since you do not know what it is).

When configuring the Peak Purity parameters for your sample, you must start with a very high quality HPLC method (A "validated method" is not necessarily a high quality method. "Validation" does not in fact insure that the method follows good chromatography fundamantals). The correct detector sample rate, threshold, slope, signal wavelength and bandwidths need to have been properly selected and used (Reference Wavelength always OFF). The peaks shown in your chromatogram should have excellent symmetry with good on-column retention (K-prime, as applicable to mode), baseline separation (> 2.0 for non-SEC modes) and very low baseline noise levels. The two Peak Purity spectral reference points should be manually selected and placed at times before and after the peak of interest in clear baseline areas where no other peaks or spectra are seen (never use the instrument default settings for reference points!). Select at least 7 spectra from the sample peak for comparison (more detail can be provided with more spectra, but be careful not to select spectra near the baseline or the noise limits). If your method and chromatogram are not of the highest quality, then please do not use the automated "peak purity" analysis feature, instead spend time improving your method.

SUMMARY: 

The HPLC UV/VIS Peak Purity Analysis (“Peak Spectral Purity”) feature is very complex and has many software settings which must be set up correctly to obtain any scientifically useful data regarding possible peak impurity levels. 

• Do NOT use the system default settings / values for 'Peak Purity' ! They are just place holders for actual values (which you must calculate and fill in the correct values for your method).
  

 

* Due to a general lack of formal training, I often see this software feature being used incorrectly by most chromatographers. This is worth repeating... the HPLC method used to obtain the original data must be of the highest quality and the training of the operator must also be at the highest level. To use this advanced software feature successfully, an advanced understanding of the fundamentals of chromatography are required as are a detailed understanding of all of the peak purity software features (how to set the correct threshold, obtain reference baselines, Set sampling rate, noise levels, signal extraction, normalization settings…). Routine HPLC training classes do not cover these types of tasks. Years of specialized training and practical experience are required to use these tools. Never use the “automated” versions or the manufacturer’s default values to find “Peak Purity”. The only correct way to use these features is to manually tune the method and settings to your specific sample. Failure to customize the method and settings used may result in invalid data and incorrect "purity" determinations. 

Due to very complex software setup needed for "Peak Purity" determination by UV/VIS spectra, the requirement for a high quality HPLC method and a high quality data-set,it is our opinion that few should ever use it. In general, the recommendation for most chromatographers is to not use this feature unless first having demonstrated the required skills and advanced understanding of the fundamentals of chromatography. Most of the methods that we professionally review where "Peak Purity" data have been used as part of the method have been found to be based on invalid methods, resulting in any "purity statements" issued as unscientific and invalid. Please proceed cautiously and request professional review of any methods which employ it BEFORE committing to relying on it.

©Copyright, March 1, 1996 by William Letter of Chiralizer Services (Plainsboro, NJ) from a portion of material presented in an HPLC Diode Array Method Development Class.

HPLC Peak Splitting. Common Reasons For It

True "Split" HPLC peaks, not resulting from co-elution of another peak, can be caused by a number of chromatography problems. Here are a few examples and their solutions:

1. Sample overload. Sample overloading is one of the most common reasons for observing peak "splitting". Reduce the sample concentration by factors of ten to see if the peak shape improves. 
2.  A poor quality HPLC method. Poor quality methods which do not use mobile phase solutions which are at an appropriate pH (*If the pH of the mobile phase is close to the pKa of the sample, then split peaks may result); which does not dissolve the sample in (should be fully soluble) or are unstable, show sample or mobile phase precipitation can cause this effect. Always check solubility before starting.
3. A partially plugged or fouled column. A dirty or fouled column (from not washing down properly with a solution which is STRONGER than the mobile phase). Analysis methods should be followed by separate wash methods to remove all bound material and any late eluters,
4. Wrong injection solution. Peak splitting may be the result of dissolving and injecting your sample in a solution that is stronger than your mobile phase. Dissolve and inject samples in the mobile phase or in a solution which is a slightly weaker solution (not stronger).
5. A poorly packed column, void at column inlet, a dirty frit or poor mechanical connection (i.e. improperly swaged fitting). These types of structural or mechanical defects can each result in peak "splitting" (all of these are less common today than in the past using modern HPLC columns). When present, a dirty inlet frit can be replaced with a new one, or the column can sometimes be backflushed to remove any accumulated material. Connections should always be double checked.
6. Detector data rate set too low. Too few peaks collected over time may result in integration errors and inaccurate peak symmetry problems. Read more about how to determine the best data collection rate at this link.

HPLC K Prime. Also known as: Retention Factor, Capacity Factor): One of the Single Most Important HPLC Parameters of All

The role of Capacity Factor / Ratio (K prime) in liquid chromatography is to provide a calculation or ratio which defines how much interaction the solute (sample peak) has with the stationary phase material relative to the mobile phase (IOW: the relative time the sample spends interacting with the support vs. the mobile phase). If this interaction is too short (i.e. K prime less than 1.0), then no chromatography has taken place and you have just developed a "flow-injection" method (the same as if no column was used) instead of a chromatography method. The method fails all validation and is NOT fit for purpose. Sample Retention must be long enough to demonstrate that the method developed is specific to the sample and shows good selectivity (retention) for the sample analyzed. This is true for most, but not all modes of liquid chromatography (3). 

• New and ever 'experienced' users lacking training in HPLC often make this error, developing methods where the sample has little to no retention on the column. We routinely review methods where the actual K prime of the key sample is measured to be at or near 0, failing to show any chromatography took place in the method (*The Journals are filled with thousands of examples of invalid HPLC methods of analysis). This mistake is made because the author(s) have no formal training in liquid chromatography and do not understand the basics of the technique. 
• Note: Slowing down the flow rate to "show" a later elution time DOES NOT increase K prime (a very common novice mistake) !!! 
  

Observance of the fundamentals of chromatography are key to developing high quality HPLC methods. For most modes of HPLC, highest on this list of basic fundamentals is that the sample(s) be retained on the HPLC column used and not eluted out at or near the column's void volume (we often refer to this time in minutes as, "T-zero" or "t0"). Many chromatography methods fail this simple test of retention and are invalid as written. Knowing what a compound's retention or capacity factor is allows us to be confident that it has been retained and eluted past this critical point, but to first calculate K prime, we first need to know the HPLC column's void volume. 

Calculation and/or measurement of the Column Void Volume should be one of the very first chromatography method development tasks you learn to perform. Knowing the column void volume allows you to determine the retention time of an unretained sample and the resulting retention factor (K prime) of each sample eluted after it. To do this, you must calculate the column void volume AND inject a sample which will not be retained by the column to determine what time an unretained sample will be eluted off the column. This establishes the 'T' zero time, or T(0). The time it takes an unretained compound to elute off the column is critical to know. If your HPLC method does not retain the sample on the column long enough past this time, then you are not allowing any chromatography to occur. Once you have this T(0) value, you can then determine the retention factor (the "K Prime") of your actual sample(s) using the simple ratio formula below. Your final method should baseline separate all compounds apart and, if properly developed, each sample peak will often have K Prime values between 2.0 and 10.0. K prime values of greater than 10 are acceptable, but often show minimal improvements to resolution. Try and insure that the earliest eluting peak in your sample has a K Prime of  >1.5. Do not develop methods which only result in K Primes of less than 1.5 (an indication of poor quality chromatography). 

Note 1: Many regulatory agencies (e.g. The USA FDA) requires that K prime values for HPLC separations be equal to or greater than 2.0 to meet Specificity acceptance criteria (System Suitability/Method Validation). After all, if it elutes at or near the void volume, then your method is not specific for anything. Besides being unscientific in design, your method will fail System Suitability and fail validation. IOW: It does not meet this basic requirement.

• K Prime, K1 (Capacity Factor or Retention Factor) Formula:

       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). 
• *The 'K Prime' of your sample must be > 1.00. A value greater than 1.5 should be your goal.

Example #1: 
 T(0) found to be 2.90 minutes and the sample elutes at 5.80 minutes. k1 = 5.80 - 2.90 / 2.90. k1 = 1.00.

Example #2:
 T(0) found to be 2.90 minutes and the sample elutes at 9.10 minutes. k1 = 9.10 - 2.90 / 2.90. k1 = 2.13.

Example #3:
 T(0) found to be 1.75 minutes and the sample elutes at 1.74 minutes. k1 = 0. No retention and no chromatography have taken place at all. The method is invalid.


Note 2: I see and read published HPLC methods (including "Validated Methods" !) every week which ignore this fundamental requirement and present data showing little to no retention of the primary sample on the column. Most are RP methods run on popular C18 columns and show the main peak of interest eluting out as a nice sharp peak right at the void volume.  These methods often describe the sample analyzed as "100% pure" and are fully validated (because the person doing the work may not have had any HPLC experience or training)! A mixture will always look like a single peak by HPLC when no 'chromatography' is employed to separate out all of the possible components. The sample must be retained on the column for a period of time before we can conclude anything about its purity by the method employed.

Note 3: In some cases, when other modes of chromatography are utilized (e.g. ion exchange, size exclusion chromatography (SEC / GPC), K prime is not as relevant. The mode of chromatography can affect the interpretation. For example: This is because size exclusion chromatography relies on the sample's interaction with well defined pores inside the support (inclusion/exclusion) to separate based on molar size. A variety of pore sizes can be used to "filter" the sample. So a large K prime value might be normal for a molecule that is low in molecular weight and spends a lot of time working its way through the column. A high molecular weight sample might just "shoot" through the column due to little or no interaction with the pores. You still need to have retention on the column, but now it is determined by how long it takes the sample to find its way out of the column. SEC columns are bracketed by Pore Size (e.g Mw. Excluding all samples that do not "fit"). With size exclusion columns, determine the Retention and Exclusion times, not the K prime). This article is specific to thr more common cases where traditional HPLC NP or RP modes are used. In these cases, low K prime values indicate no retention took place and the method fails all claims of specificity for the sample (selectivity is absent or poor). HPLC methods with little to no selectivity fail scientifically as no chromatography has taken place.

HPLC Peak Tailing - Some of the Most Common Reasons For it

Three easy ways to minimize chromatography peak tailing:

(1) Tailing often results from using “Type – A” HPLC silica. Type-A silica often contains more acidic silanol groups and metal impurities than Type-B. To improve peak shape, use modern “Type – B” silicas which are of higher overall purity, have less metal contamination and feature minimal silanol ionization under higher pH conditions.

(2) Minimize ionic interactions and utilize a buffer or ion pairing agent (e.g. TFA 0.02%) in your mobile phase. Select a buffer that is at least 2.0 pH units away from your sample's pKa and use the smallest concentration or amount that gets the job done. For LC/MS or MS/MS applications, remember to only use volatile buffers and avoid the use of ion pairing agents unless absolutely necessary (and if used, use at the lowest possible concentration to avoid source contamination).

(3) Always use a freshly washed and equilibrated column. Is the column fouled or the inlet frit dirty? If the head of the column is fouled from sample overloading or from a failure to wash off strongly retained compounds from many runs (much more common problem), then your peak shape and reproducibility will suffer. Incorporate a washing step in between your analysis methods which utilizes a solvent which is stronger (in concentration) than your mobile phase to wash off any strongly retained material after each run. For example, if you normally end a method with an 80% concentration of ACN, utilize a separate wash method which has 95% ACN in it. Allow enough wash time for this work.

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.

Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)

   Let's take a quick look at two different problems which you may encounter when operating an HPLC system. We start with the basic observation and then look at the most likely causes so we can begin the troubleshooting process and repair the problem. An automated HPLC system's flow path typically consists of: The Solvent Pickup Filters (in the mobile phase reservoirs); The Pump(s); AutoSampler; AutoInjector; Column and one or more Detectors.*You should have a good understanding of this flow path before you proceed to diagnose the problem(s).

 *A gradual increase of pressure for the same method over time is often due to column fouling or a dirty inlet frit (e.g. PTFE frit). This article specifically focuses on the causes of a sudden change, not a slow change over time.

   Sudden System Back Pressure Changes: We will assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the back pressure has changed from what is normally seen. The problem must lie within the flow path of the system.

   Excessive High Pressure: Typical reasons for this are:

1.      A fouled or plugged column;
2.      Wrong flow rate (higher than normal);
3.      Inlet frit/filter plugged or restricted;
4.      Plugged line;
5.      Wrong mobile phase composition.

   Large Drop in Pressure: Typical reasons for this are:

1.      A leak at a fitting, column or line (Number one reason);
2.      Wrong flow rate (lower than normal);
3.      Wrong mobile phase composition. 

• Start by checking the method parameters to insure that they have not changed (i.e. flow rate, mobile phase composition). Check for leaks or plugs. If the column is suspect, replace it with a zero dead volume union (ZDU) and restrictor and flush the system. Replace the column with a new one or wash the current column according to the column manufacturer's guidelines.

   Sudden Peak Shape Changes: We will again assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the peak shape of one or all of the peaks has changed from what is normally seen. *The key thing to keep in mind is that the change occurs all of a sudden, not because of poor initial method development.

   Typical reasons for this are:

1.      Tailing or Split Peaks: Sample overload, change in flow rate, mobile phase composition (e.g. composition or pH), void formation, dirty frit, injection solvent too strong or a fouled column.
2.      Fronting: Commonly seen when overloading sample on column.
3.      Ghost Peaks: Usually due to a contaminated mobile phase, contaminated sample vial or contaminated injector (e.g. rotor seal).
4.      Broad Peaks: Large sample injection volumes or extra column volume (bad connections with the system or tubing) are usually to blame. Try reducing the injection volume by a factor of 10 and see if the problem goes away. You may also want to wash the column as it may be fouled with sample.

   These are just two common problems we see when using HPLC systems. Note that a dirty or fouled column can cause many of these problems so take care of your columns and wash and test them regularly to insure they are in compliance. There are many other commonly seen problems besides these. If you would like to see a specific problem featured on this blog, then please send me a request.



  

Proper Wavelength Selection for HPLC Method Development (or Purity Determination)

Selecting the best HPLC wavelength(s) to monitor during an analysis method for use in quantitation and/or purity determination requires both knowledge and careful attention. Here is the basic procedure to use:

Step 1. Create the Method. To determine which UV/VIS detector wavelength(s) should be chosen for the analysis of your sample, you will first need to create a general HPLC Method which retains and resolves the compound(s) of interest on the column (goal is a K prime of >2.0, less than 10.0). Be sure and utilize a scanning diode array detector in full scan mode (often referred to as a photo-diode array detector, PDA or DAD) to scan all relevant wavelengths of your samples (e.g. 210 to 450nm). Note: Your choice of mobile phase and detector settings will effect the S/N values.

Step 2. Determine the lambda max of the sample's spectra using the Data analysis software. Once you have completed the analysis, review the spectral data to determine which prominent peak wavelengths have the maximum signal to noise (S/N) ratio. These “peaks” can be used as the individual wavelengths for integration and purity determination. By sure and double check that any detector options which use a “reference wavelength" are turned ‘OFF’ when running these methods (more info on “reference wavelengths” can be found on this blog in another post). With the wavelength selected, chose an appropriate bandwidth for use (narrow).

Step 3. Edit the HPLC method to use the discreet wavelengths found in step 2. Whenever you run a real sample, continue to use the full scanning mode of the detector so you will know about any other components which absorb at wavelengths far away from and/or near the peak wavelengths. These compounds can add or subtract signal from the main peak making it appear to be more or less concentrated (or more or less pure) than it actually is. If you only monitored the sample with a single wavelength detector, then you would miss this vital information and make errors in your purity or concentration determinations.

Conclusion. (1) Using a multi-wavelength, scanning HPLC detector such as a DAD is one of the most important tools you can use to create accurate and reliable chromatography methods. Always use a scanning DAD for method development to prevent errors. (2) Learning how to correctly use and set up the detector's settings, parameters, special features and options may prevent false or misleading results. Only after you have developed a reliable and repeatable method with good sample retention and peak shape can you begin to report accurate integration and concentration values (and/or make UV/VIS "purity" determinations).