Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, December 9, 2017

Evaporative HPLC Detectors; CAD (Charged Aerosol Detector) and ELSD (Evaporative Light-Scattering Detector)

  • If you wish to read about their development and/or operating principles, then please review the early published patents and many articles available through the web.
E.L.S.D. modules for HPLC applications were first developed and commercialized in early 1980. CAD units were first described ~ 2001 (US patent 6,568,24) and commercialized after 2004. Both types of evaporative detectors have undergone many updates over the years. They are complementary and focused on the same application areas where conventional UV/VIS detectors do not provide for or allow detection of specific compounds. While claimed limits of detection vary by manufacturer, both designs are highly sample and method dependent so fair comparisons are rare. Significant differences in cost between the two detectors are noteworthy, with CAD units currently costing several times as much as ELSD units. Let us take a look at some of the characteristics and uses of these niche detectors.

Applications: CAD and ELSD are both used with a wide range of non-volatile sample types. Targeted at compounds which have weak or no UV chromophore (e.g. Carbohydrates, fats, lipids, triglycerides, polymers, surfactants, oils).
Thousands of application notes and journal articles are available for both types of detectors (esp. for ELSD with almost 40 years of use) and a keyword search on the web is the best way to find them. As someone who was involved in the early development and design of these detectors, I have used them successfully to develop several hundred different types of methods. They have proven to be useful for a number of difficult applications, but their higher cost and even higher training and skill requirements still place them outside of most users labs. As with LC/MS detectors, CAD/ELSD modules may require far more maintenance and advanced training to use than most chromatographers have received. As such, it is my opinion that you consider their potential use in your projects only after other more conventional methods have failed to provide results. 

Detection: NOT “Universal” detectors (sourced to marketing misinformation). While detection is not fully based on the analyte’s chemical or physical properties, the actual output observed is in fact partially based on the properties of the mobile phase (volatility and purity), sample volatility/stability and especially the many different custom detection settings chosen by the user (gas flow, heater temperatures, flow rate, specific detector used, level of contamination inside the detector). As such, their output is very subjective since it is based on both the specific chromatography method selected, the condition of the detector, the lab environment used-in, and the detailed operational settings chosen by the operator. They can detect everything from dirt, buffers, undissolved chemicals or particulate matter in your mobile phase. Even pressure changes on the detector's exhaust line can effect the output.

“Destructive” Detectors: As with an LC/MS system, the mobile phase is evaporated away from the sample and sample collection is not possible at the exhaust. They are best used as a secondary detector, with a primary detector sch as a UV/VIS module placed in front of the CAD/ELSD (to increase your chances of detecting something that the CAD or ELSD may miss). ELSD and CAD units will NOT detect all samples. If sample collection is required, a low volume, micrometer valve flow-splitter can be fitted to the evaporative detector’s inlet port. Note: Depending on the flow-splitter's split ratio, the detector’s signal output may be reduced.

Mobile Phase Requirements: Evaporative detectors require a fully volatile mobile phase (similar to LC/MS requirements). The use of non-volatile additives can contaminate or damage them (no phosphate buffers!). Use of non-volatile buffers or additives, low purity materials, contamination of the gas, mobile phase or by samples may result in excessive noise levels limiting detection. Examples of Mobile phases used: "Popular LC/MS and HPLC Volatile Mobile Phase Buffers"

Isocratic and Gradient Capable: Unlike RID or EC, CAD/ELSD allows the use of gradients and the use of UV obscuring solvents. Because the mobile phase is evaporated away, little to no baseline drift occurs during gradient analysis (often improving integration results). Sample types which dissolve best in solvents such as methylene chloride, acetone, chloroform or other strong UV absorbing solvents may find that these detectors assist in developing better quality methods. Reduced gradient baseline drift plus the option of using UV absorbing solvents are two characteristics which make them well suited to application areas such as lipids, polymers and oils.

Gas Requirements: Similar to the requirements of an electrospray LC/MS system, both CAD and ELSD modules use very large volumes of high-purity gas (i.e. Nitrogen) to safely evaporate the mobile phase away. Be sure and factor this cost and the required space into any site-prep.

Operational Reproducibilty and Method Transfer:  Recording the exact detector settings used in the method may not provide any guarantee of being able to duplicate the results some time later. No two models are the same so results may vary (similar to LC/MS). Results obtained are relative to the specific instrument, the chosen settings & method used (again, much like LC/MS). Compare the many critical heat, gas flow and atomization related CAD/ELSD settings to the more common UV/VIS detector where only the wavelength, bandwidth and flow cell dimensions need to be specified to easily duplicate the detector setup. CAD/ELSD internal contamination levels, nebulizer spray patterns, gas flow rates, quality of the mobile phase and operator training may all contribute to variations. *As with all methods and detection systems, proper training and good method design will insure success.

Quantitation: Can be used for quantitative analysis across a wide dynamic range spanning multiple orders of magnitude. High quality reproducible methods are achievable with both types of detectors.

Linearity and Output Characteristics: Except in the most narrow concentration ranges, neither detector is likely to provide a linear response. Quantitation can be improved through the use of larger numbers of calibration levels (more than normal) plus a high quality chromatography data analysis software package which includes many available non-linear curve fit options (polynomial, sigmoidal, exponential, log…). Output often changes across orders of magnitude so be sure to optimize the curve fit for each sample type. Different sample types will often have different response outputs too.

Optimization Process:Unlike a UV/VIS or RID system which simply needs to warm up and stabilize, CAD and ELSD systems may require a methodical optimization process of adjusting the flow rate, gas flow and heating temperatures to optimize the measured S/N peak ratios for each sample and each method used. Optimization of detection conditions may involve making multiple measurements (Peak and Baseline S/N ratios) to find the best settings to use with each sample type and method.

Operational Complexity: Methods which utilize CAD/ELSD systems may be more complicated and time consuming to learn, use and validate then conventional detectors. Specialized detector cleaning procedures may be needed. The detectors may become internally contaminated during use (sample builds up inside the unit). Failure to clean and maintain them may lead to high noise levels and/or inaccurate results. Due to the additional maintenance needs, lack of traditional linearity, and overall complexity, we recommend their use when: (1) Conventional detectors or methods of analysis are not possible or unsatisfactory and (2) where the operator has demonstrated a high level of practical hands-on training through use of the detector and/or has sufficient experience (advanced level) in chromatography.

For more information:

Saturday, November 4, 2017

Repair Missing Or Corrupted Windows System Files Using the System File Checker Tool

It has been awhile since I posted any Microsoft Windows software tools or tips so here is a MS utility program. Most laboratory instruments operate under MS Windows software control and you will be able to keep things running smoothly if you take the time to learn and use many of the available utility programs. 

  • Note: Before using any software utility program, make sure you first have permission and authorization to do so. Back up your system programs and any data files. Create a Restore Point to protect the basic settings too. Take precautions before using any utility programs and do so at your own risk.

In addition to the very useful "Restore Point" feature found in Windows (discussed in an earlier post, which is great for solving a number of issues), Microsoft has another time-saving utility tool built into most versions of Windows (i.e. XP, Vista, 7, 8 and 10). The Windows System File Checker (simply known as, SFC) offers users the option to scan their Windows operating system files for corruptions AND restore any found corrupted files, all automatically! Running the utility is very easy. First, make sure your account has Administrator privileges, then use the Run Command prompt to run the program SFC 'As Administrator' *Type:  sfc /scannow 

More information about this useful utility can be found on Microsoft's website support page.

Saturday, October 7, 2017

Preparation of Phosphate Buffered Saline (PBS)


While not commonly used in liquid chromatography, PBS solution is commonly used in preparing samples. Because chemical forms and grades vary, there are several recipes on the web. By popular request, I am provided a common laboratory recipe for a 1X PBS solution here.

To make Phosphate Buffered Saline (PBS), 1x pH 7.4 solution:


1. To a 1-liter Beaker, add the following four anhydrous salts:

a. 200 mg KCI

b. 8,000 mg NaCI

c. 240 mg KH2PO4

d. 1,400 mg Na2HPO4

2. Add about 850 ml of deionized water and stir to dissolve the salts. Adjust the pH to 7.4 with 10% phosphoric acid (phosphate solutions should be adjusted with phosphoric acid only). Transfer to a 1 L volumetric flask (class-A). Fill to the “line” with more deionized water. Cap and mix a final time to insure a uniformly mixed solution.
3. Filter the final solution through a suitable 0.22 micron filter before use.

Note: pH increases with decreasing temperature.

Saturday, September 2, 2017

HPLC Solvents, Acetonitrile and Methanol, Key Differences and Properties

Widely used in RP HPLC method development, Acetonitrile (ACN) and Methanol (MeOH) are the two most common solvents you will use with water or aqueous buffers to develop methods. So, besides the fact that Acetonitrile is well know to have a higher elution capacity than Methanol, what other properties should chromatographer's be aware of? Let's discuss a few that all chromatographers should know.

First, a few comments about the preparation of mobile phase solutions. 

     There are two common methods of preparing binary mixtures, V/V, of mobile phase solutions.
  • Method #1 is to fill a volumetric flask with a specific volume of the "A" solution, then fill the flask up to the line with the "B" solution.
  • Method #2 is to fill a graduated cylinder (or volumetric flask) with a specified amount of "A" solution; fill a second graduated cylinder (or volumetric flask) with a specified amount of the "B" solution and then mix the contents of both together.
Whichever method you use, please fully document it in your HPLC method so anyone reading it will be able to accurately reproduce it. The two methods described above are both correct in design, but will result in solutions with different properties.

For HPLC grade solvent (*we should always use HPLC or LC-MS grade solutions in HPLC analysis), ACN has the lowest absorbance (~ 190 nm) of the two making it well suited for low UV applications. HPLC grade MeOH has a slightly higher UV cut-off, around 205-210 nm, limiting its use in the very low UV ranges.

There is a significant difference between ACN and MeOH in their ability to dissolve many types of buffer salts AND samples. These differences are critical in method development. 

Solubility of the Mobile Phase:
  • A common reason for gradient runs to show poor reproducibility or to fail may be associated with running high concentrations of buffer combined with high concentrations of organic solvent. Most aqueous / organic solutions containing salt solutions of less than 10 mM concentration are not likely to precipitate under most gradient conditions (running to a max of 98% organic, not 100%). If high percentages of organic solvent are mixed with more concentrated buffer solutions, then the higher salt concentrations may precipitate out of solution during the analysis (resulting in clogs, leaks, plugs and/or inaccurate results). Be cautious when mixing organic solvents and buffers together for gradient analysis. Make sure the solutions used will stay in solution and be stable at all concentrations used. Also verify that the buffering capacity is still present when high organic concentrations are used (as your buffer will be diluted). *Not sure if the salt will stay in solution? Just mix up a sample at the same concentration for a test. Look at it. Is there any turbidity or particulate visible? You should have your answer.
  • Methanol's overall better solubility characteristics (better than ACN) mean that it does a better job of dissolving most salts (esp NH4, K and Na) at higher concentrations resulting in better performance and less precipitation.

Solubility of the Samples (effect on Peak Shape, Selectivity & Retention):
  • A fundamental requirement of liquid chromatography is that the sample fully dissolves in the mobile phase (initial mobile phase). Dissolve the sample in the mobile phase or in a slightly weaker strength solution (not a stronger solution) before analysis. This insures it will be loaded onto the head of the column as a concentrated slug improving peak shape and RSD. If the sample does not fully dissolve in the mobile phase then you are not in fact analyzing the whole sample. Another area where Methanol may be superior to ACN can be found in its ability to fully dissolve more types of samples. This improved solubility may result in better overall peak shape. Methanol also has different selectivity, often better than ACN (not just the elution strength) which may result in peaks eluting at different retention times than expecting. This is another reason why we always try different mobile phase mixtures containing either ACN or MeOH when developing RP methods. Please never assume that one solvent will be better than the other. Too many novice chromatographer's use only ACN as their main organic solvent for method development. Please don't make their mistake as such a strategy indicates a lack of practical experience and knowledge. You must first try them both separately (ACN & MeOH) to evaluate the results with your own sample (best to start with comprehensive gradients at different pH values, as applicable). You will be rewarded for putting in the initial time to test both types of solutions as no simulator has yet been developed which can predict a truly accurate result with your own sample(s). You may be surprised to learn how many samples show better peak shape and performance using MeOH solutions. If no improvement is seen, document it and move forward with more confidence.

  • ACN is less viscous than MeOH ( 0.34 vs. 0.54 respectively) and if used alone will result in lower column and system back-pressures overall. Less gas will dissolve into ACN vs MeOH. Mixtures of ACN and Water will also exhibit an endothermic reaction (cooling the solution) which can trap gas inside the solution. If you pre-mix your mobile phase, let it rest for several minutes after preparation.
  • MeOH is more viscous than ACN alone. It also has an unusual property where a 50/50 mixture of MeOH and Water will result in a much higher system and column back pressure than either MeOH or Water alone will (*ACN has a similar property, but the peak pressure occurs between 60-70%). The effect with methanol is very Gaussian with a peak pressure observed with a 50/50 mixture. An exothermic reaction results from an initial mixture of the two solutions (MeOH and Water) releasing some gas. When preparing solutions it is best to allow the solution to rest for a few minutes to out-gass before topping off or using in the HPLC system.

I hope that this short discussion about some of the differences between these two popular HPLC solvents will aid you in developing better quality HPLC and LC-MS methods.

Reference: Table of HPLC Solvent Properties