Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, January 6, 2018

UHPLC TIP: Reducing the Column Temperature to Offset Frictional Heating Effects (Causing Poor Resolution)

Column temperature is a critical variable that we adjust and set when developing methods. We use it as a variable during method development to improve peak shape and increase resolution. Once established, it must be carefully controlled during the method to provide reliable and reproducible analysis results. Change the column temperature and you may change the results obtained. This is fundamental knowledge and must not be forgotten.

If you are developing a new UHPLC method OR perhaps scaling an HPLC method to utilize 2.5 micron or smaller support particles, then you may observe a loss of resolution or poor peak shape in the new method. There are many reasons why this may occur, and the most common ones relate to not optimizing all of the method parameters correctly when scaling the method (e.g. dwell volume too large, flow cell volume too large, injection volume too large, sample rate too slow, flow rate not optimized, mobile phase composition changes not in scale with the gradient...). But there is another reason...

Resolution may be reduced or lost when all of the initial scaling and instrument set-up parameters are optimized. What is the most likely reason for this? In many cases the use of substantially higher flow rates (relative to linear flow rates) and the use of smaller diameter particles results in much higher backpressures (you may recall that if you halve the particle size, the backpressure increases 4x). The resulting backpressures might be 2, 3 or even 4 times higher than observed in the original method. While these higher backpressures were well within the operating parameters of the HPLC system used, the results obtained were poor. The Cause? The higher backpressure increased the amount of frictional heating inside the column, raising the actual analysis method temperature and changing the separation conditions. 
Pushing mobile phase through a chromatography column generates heat and pressure. The heat generated increases the actual temperature of the column. In conventional columns (i.e. 4.6 x 150 mm, 5u) at 1.00 ml/min, this heating effect is minimal, but at much greater column pressures, > 400 bars, the frictional effects may be substantial. These types of very high pressures may be seen with methods which utilize columns containing the smallest particles (1.9 to 2.5 micron). Enough to change the temperature in the column by several degrees (e.g. >5 degrees C) and result in different method conditions. So, what can you do about this? The most direct way to address the problem is to run the same method at a lower temperature (perhaps decrease by 5 C to start with). This will slightly raise the backpressure (lower temperature equals higher viscosity), but it should cool the column and restore the original temperature conditions used. You may need to try several temperatures and this may be easiest to do if your HPLC has a column compartment with heating and COOLING capabilities.

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. By popular request, I am provided a common laboratory recipe for the solution here.

To make Phosphate Buffered Saline (PBS) solution:

Method #1:

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

a. 200 mg KCI

b. 8,000 mg NaCI

c. 200 mg KH2PO4

d. 150 mg Na2HPO4

2. Add about 850 ml of deionized water and stir to dissolve the salts. When fully dissolved, fill to the “line” with more deionized water. Stir a final time to insure a uniformly mixed solution.

3. Pour the contents into a laboratory beaker and adjust the pH to 7.0 with 10% phosphoric acid (phosphate solutions should be adjusted with phosphoric acid only).
4. Filter the final solution through a suitable 0.22 micron filter before use.

Method #2:

Optionally, use the same ingredients as specified above, but premix in a beaker with stir bar to make the job easier. 

Place a 1 L laboratory glass beaker on a hotplate stir with stir bar. Fill the beaker with 850 ml of deionized water. Stir at a moderate rate with some heating (~ 35C). Add the dry ingredients to the solution and allow time for them to dissolve. When fully dissolved, remove the stir bar, remove from the heat and carefully pour the contents into a 1 L volumetric flask. Allow the solution to cool to ~ 20C. Fill to the line with deionized water, stopper and mix the final solution (inversion). Pour the contents back into a laboratory beaker and adjust the pH to 7.0 with 10% phosphoric acid (phosphate solutions should be adjusted with phosphoric acid only). 

Filter the final solution through a suitable 0.22 micron filter before use.