Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, April 8, 2017

LC-MS Contamination? Another Possible Cause. Are your Mobile Phase Bottles and Filters Clean ?

One of the more common LC/MS problems I am asked to help solve deals with contaminated LC-MS or LC/MS/MS systems. Over time, many systems will become contaminated with a wide variety of plasticizers, detergents, salts, metals and ion pairing agents that routine source cleaning will not remove. Often, these compounds are introduced to the system through the tools used (e.g. pipettes) chemicals, solvents, mobile phase additives or even the samples themselves. "Dirty" samples sometimes persist inside the system long after the analysis work is complete, leaving material in poorly maintained injection valves but also through the use of poorly washed / contaminated and fouled HPLC columns. Even the modern inline HPLC vacuum degasser has proven to be a source of contamination. 

In addition to the above mentioned sources of contamination, another more obvious source of contamination should always be addressed early in the process of cleaning the system. Specifically, the glass mobile phase bottles and the associated solvent pickup filters used with them. Contamination in these areas directly infuses the system with undesirable material, so good practices must be maintained to reduce this source of potential contamination. 

As a general guideline, we should not place our mobile phase reservoir bottles in any type of dishwasher or wash them using any soaps. These will leave a residue easily detected by even the weakest mass spectrometer. Avoid contamination by purchasing high quality glass bottles with vented caps to keep dust out. If rinsing with organic solvents (and/or freshly prepared and filtered high resistance water) does not clean them, you can try a Nitric Acid rinse (30%) followed by a neutralizing wash in 2M Sodium hydroxide. Follow-up with a few rinses of HPLC Grade water (or LC/MS grade) then re-fill with an appropriate mobile phase. Don't forget to replace those solvent pickup filters too. Most of the sintered glass style filters are designed to be disposed of (not cleaned or put in an ultrasonic cleaner!) so dispose of them and install new filters and fresh mobile phase into those recently cleaned bottles before you start looking for the source of contamination in the more expensive parts of the instrument. - Please don't re-contaminate an expensive HPLC or LC/MS system and your data because you skipped replacing a $10 part. Keep commonly used spare parts around and always maintain a clean system.

Saturday, March 4, 2017

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 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 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. 




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.

Saturday, February 4, 2017

Determine the HPLC System Dwell Volume (Gradient Delay Volume)



Note: The total HPLC 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 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) 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) and runs to 0% ‘A’ and 100% ‘B’ for about 10.0 minutes.
(5) Flush and degas both solutions, ‘B’ first, then ‘A’ through the system until you get a nice clean, flat baseline.
(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 flow rate and 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 is 1.000 ml/min. Your 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) 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 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. There are many other methods which can be used as well. This proposed example serves to illustrate the concept.

Saturday, December 31, 2016

Peak Purity Determination by HPLC Diode Array Chromatography Software: Limitations and Uses



"Peak Purity" Determination by HPLC 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 the 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 second dimension of data, but a scanning, multi-wavelength 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 seen, allowing for a 3D plot of the spectra to be recorded much like a “fingerprint”. Pure compounds which 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).  When a properly developed HPLC separation method or analysis 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 sample's spectral profile to detect the presence of an impurity that has co-eluted with the sample. This additional dimension of analysis is required to improve the confidence level that a peak is in fact correctly identified and does not contain any co-eluting compounds.



Diode-Array 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). It is impure by UV/VIS analysis. Note: It does not indicate that the compound is impure, but rather 'the peak' being measured is.

“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 a measure of 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.

Points to consider when using "Peak Purity" software:


  • The absence of any spectral differences across the peak is not an indication of actual purity;
  • Compounds similar to your sample may have similar absorbance profiles;
  • 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. Inputting poor quality settings or using a poor quality method often leads to misleading purity results.
  • 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 which elute at or near the column void volume (demonstrates lack of a method and specificity, fails validation).


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.

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). The correct detector sample rate, signal wavelength and bandwidths need to have been selected and used (Reference Wavelength OFF). The peaks shown in your chromatogram should have excellent symmetry with good on-column retention (K-prime), baseline separation (> 2.0) and very low baseline noise levels. The two 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. 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 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.



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. * In many major laboratories, due to a lack of training, I see it being used incorrectly by most chromatographers on a regular basis. 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 the 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. 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. 

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.

©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.

Saturday, November 26, 2016

HPLC Detector Optical SLIT WIDTH Selection

A few notes on HPLC Optical Slit Width selection:

   Notes: 
  1. The chosen slit width setting determines the amount of light which is directed to the detector.
  2. For most HPLC methods, a slit width value of 4 nm is suggested. 
  3. Bandwidth should be set at least as wide as the optical slit width.

Characteristics of Narrow Optical Slit Widths:
  • Less light falls on the detector
  • Less signal intensity
  • Increased baseline noise
  • S/N ratio decreases
  • Spectral resolution improves which allows for more accurate spectral identification.
Characteristics of Wide Optical Slit Widths:
  • More light falls on the detector
  • Greater signal intensity
  • Decreased baseline noise 
  • S/N ratio improves
  • Spectral resolution decreases and detail is lost. Less accurate spectral identification and an increase in errors for spectral library matching.

Saturday, October 29, 2016

Notes on Cleaning bound Protein from RP HPLC columns:



First, a few comments:

  • ·         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 which comes with the new column. Their guidelines supersede these!
  • ·         Columns are consumable items. After a suitable amount of use, the time and materials required to regenerate them may cost more than the purchase of a replacement column. Always have a new, spare column on hand.
  • ·         Protect your detector. Before washing or cleaning the column, disconnect the column outlet line and direct the column to waste only.
  •      Column Storage solutions are not the same as column wash solutions. Never store a column in buffer or ion pairing containing solutions.

For RP supports, if buffers have been used, always start by washing the column down with ultra-high purity water and some organic solvent (e.g. Water/MeOH, 95%/5%) to remove all salts. Use about 10 column volumes to flush these off.


Many polymeric resins (e.g. PS-DVB) can be effectively cleaned using 0.1 M Sodium Hydroxide solution or a mobile phase solution containing equal parts of isopropanol (IPA) and 3 M Guanidine hydrochloride at ~ 50 °C. Optionally, some success has been reported using other solutions such as: 5M Urea (pH 7) buffer solution; 1 M NaCl (pH 7) and even mixtures containing some methylene chloride solvent. Check with the manufacturer!


For RP silica based supports, we often use a series of wash solutions. In most cases, pure water or pure organic solvents such as MeOH or ACN will not remove bound protein. An acid, base or ion pairing reagent is often needed. One of the first general wash solutions to start with is a 1% Acetic acid solution in Methanol (50/50). If desired a stronger acid such as Trifluoroacetic acid (TFA) can be swapped for the acetic acid (where possible, start with a weaker acid). This can be followed with a later solution where IPA replaces the MeOH (50/50). In both cases, adjust the pH to ~ 2.5. 5 M Urea solution has also proven effective in removing bound protein from silica supports too. Another salt solution that has shown some promise is 1 M sodium phosphate solution, pH 7.0. Run the salt solutions for about one hour at a moderate flow rate. Follow up with rinses of water and MeOH (80/20), then 90% MeOH/Water.