HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?

HPLC Injection Volume and Solution Tips: For best results, the choice of injection solution and amount must be carefully selected. Successful HPLC & LC-MS methods shall observe good chromatography fundamentals. 

• How much sample can I inject on my column? The HPLC injection volume must be carefully selected to avoid overloading the column and also maintain good quality peak shape (Good peak shapes, Gaussian are ideal, are preferred for accurate integration and quantitation). Too large an injection volume and the peak shape may be broad and result in co-elution, column fouling and/or poor reproducibility. Too low an injection volume may lead to no-detection, poor reproducibility and/or inaccurate integration. Choose an appropriate Injection Volume (and concentration) that is appropriate for the COLUMN and Method used (their is no universal answer as they depend on YOUR column and method). Start, by learning what your HPLC column's "dead" volume is (Determining the HPLC Column Volume Link here).  As a general guideline, keep the volume low and inject no more than ~ 1% of the column's dead volume (maximum for most columns is ~ 1 to 2 %, but if the peak shape is excellent, sometimes up to 3% is possible). The actual capacity will be different for different column support types, dimensions etc, so it is best not to guess. Use a volume that is within the injector's most accurate range (for most auto-injector's, the optimal range may be found away from the extreme limits, often between 20% and 80% of capacity, but please refer to the documentation for your injector for specifics). Once an acceptable volume has been identified, then you can vary the concentration to find the best sample load for your analysis conditions.
  
• NOTE: To find the true and correct answer to "How Much Can I Inject (Load) onto my column" requires that you conduct a 'Loading Study' [To run a loading study you will prepare a batch of samples of increasing concentrations levels which can be individually injected, then evaluated on YOUR column, using YOUR method. This is how we determine the MAXIMUM amount possible which can be loaded and still provide good quality results. All other methods are just estimates.

• What should I dilute my sample in? Dilute samples using the mobile phase solution (in the case of gradient compositions, use the "initial" mixture to avoid precipitation). Your sample(s) should be FULLY dissolved in the mobile phase and not in a solution that is chemically incompatible with the flow path or is "stronger" in elution strength than the initial mobile phase. The diluent should not interfere with the analysis or loading of the sample onto the column. Example: If your method is 100% aqueous, then do not inject the sample(s) in a solution that contains organic solvent (i.e. ACN). *Peak fronting, splitting, precipitation and/or distortion (broad shapes) may result from using a diluent that is stronger than the mobile phase.
  

• My sample solution is cloudy or has "stuff" floating in it. ONLY Inject sample solutions which are 100% fully dissolved, in-solution. Injecting samples which have precipitated out of the solution OR which are not fully dissolved in solution (100%) may result in line obstruction, clogging, column fouling and invalid data collection/results. Take the time to find a mobile phase that your sample fully dissolves in to avoid problems. Troubleshooting and repairing an HPLC system for clogs and/or column contamination is both time consuming and expensive.

• Filter sample solutions to prevent clogs and reduce column fouling. Make sure the sample is first fully dissolved in the solution and do not use a 'filtering' step as a cheat to remove undissolved sample. Filtering is used to protect the system from particles that we can not easily observe which may clog the system. Please refer to the article; "Syringe Filter Selection for HPLC or LC/MS samples"; for more information on filter selection.

• Improve Injector reproducibility: Leave the vial cap slightly loose so it does not make a full seal. *This prevents a vacuum forming inside the vial, resulting in injection volumes which may be lower than the selected volume. "Loose caps" can greatly improve accuracy and reproducibility when larger OR multiple volumes are injected from the same vial. Additionally, if the total sample vial volume is very small (i.e. ~ 200 ul), utilize a vial insert of the correct dimensions and type for improved accuracy. When using vial inserts, check that the needle height is correct for the vial insert used.  Do not use the entire sample volume! Never use more than 90% of the vial volume or air may be aspirated resulting in invalid data collection.
  

• Prevent sample carryover problems by regularly inspecting and servicing your HPLC injector (Manual valve and Autoinjector maintenance tips will be found at this LINK). Replace common wear parts such as rotary valve seals and needle seats on a regular basis (Do not "clean" and re-use seals). Carryover troubleshooting Tips will be found at this LINK.
  

•  Calibration Volumes for Quantitation: When creating a new calibration table for a group of standards, use the SAME VOLUME for each standard and vary the concentration ("calibration level") only with each vial. As we have seen, injection volume is a variable which may change peak shape and integration accuracy. If you inject the same volume of liquid for all standards (and samples too), then you remove this variable. Using the SAME injection volume for all standards and samples helps to reduce problems. *Note: Thought it may not be approved, if you thoroughly test varying the injection volume across the range used for the calibration to demonstrate no undesirable changes to peak shape, loss of resolution/separation, and it is reproducible and accurate for the analysis method, then you can vary injection volume. Link to: HPLC Calibration Article.
   

 

Please note that these are general guidelines only and the mode of chromatography (e.g. NP/RP/HILIC/SEC), scale (prep vs. analytical) and/or specific method used must be optimized for best results. Follow these basic guidelines to prevent analysis problems, prolong column and system lifetimes and increase reproducibility and accuracy.

Cannabis, Cannabinoid (Hemp, THC, CBD, CBN, Marijuana) HPLC Analysis and Testing, Areas for Improvement [*Updated 8/2021]

Over the past few years we have observed an exponential growth in the number of state-level, legal businesses (in the USA) who offer Cannabis Analysis (e.g. Potency Analysis or Profiles) and/or related businesses such as Hemp Oil Extraction. Most related products which incorporate Marijuana, CBD, THC, Cannabinoids, Terpenes and/or other related compounds require formal analytical laboratory testing which should follow good laboratory practices. This article is targeted to help many of the people involved in this new analysis business (or anyone using chromatography as the analytical technique of choice for the same goals).

As a professional chromatography consultant, I have seen a large increase in the number of requests for my services to this new market. Most of these new businesses have questions about obtaining professional training, correct analysis procedures, improving reproducibility, documentation, optimizing method development, how to receive professional training in maintenance of the HPLC system(s) and need hands-on help to optimize the procedures used. Many users are not achieving acceptable results and need help finding out why. They want to know where they can take a class to learn HPLC method development and how to perform the required tests. 

These new businesses would benefit greatly from professional guidance EARLY in their setup and establishment, to improve the internal methods and procedures of analysis used. Time spent on the "front-end" of any process is always time spent wisely (in this context, knowledge and practical experience = confidence). A chromatography professional can quickly identify areas which may need improvement and/or suggest changes that can directly improve your company's accuracy, reproducibility, increase efficiency and of course, impact your bottom-line too. Focus is placed on the exact areas that will benefit you (rather than wasting time with non-targeted approaches, sales biased classes and trial-and-error approaches). 

• Please note that there are NO SCIENTIFICALLY LEGITIMATE ONE DAY, ONE WEEK or ONE MONTH LONG TRAINING CLASSES THAT CAN TEACH YOU HOW TO PERFORM HPLC METHOD DEVELOPMENT or ANALYSIS. NONE AT ALL. Most types of "Certification" offered are completely without scientific merit or value. The training needed takes many years of hands-on experience, in an industrial laboratory (not a school), to acquire just a basic level of proficiency (*Emphasis on 'Basic", not intermediate or expert). Be very cautious of anyone who claims to be able to provide you with all the training you need in a short time period.
  

Generating accurate and reproducible analytical data, esp. with HPLC, SFC or GC requires a great deal of knowledge, formal training and practical hands-on experience (not something which is taught at most university or school programs). These complex techniques require years of bench time and professional hands-on experience to learn). Shipping or selling products which contain unacceptable levels of impurities or which do not meet basic testing or regulatory qualifications could pose a health and/or liability risk. Hire people who have the needed training from industry before setting up the laboratory.

It has been my professional experience that some of the most common training areas that client's would benefit from are: GLP (Good Laboratory Practices/Procedures and SOPs) and additional instrument operational training to demonstrate proficiency in analytical chromatography. Address these areas early on and continuously update them to reduce errors and improve results. Training should continue on a regular basis to gain experience.
 
While each confidential consultation visit I have with a client may show different key issues which need to be addressed, many labs can start to improve their analytical results by addressing and improving how they address:

1. Documentation: Laboratory methods and sample analysis must be conducted using clearly written documentation. This should initially include having Standard Operating Procedures (SOPs) in place for all methods, procedures, qualification, verification and tests used. They should include SOPs, Document Control and Policy documents which also address: Training, Calibration, Maintenance, Frequency of the same, Mobile phase preparation, pH measurement, use of the balance and so on... I find that it is best to create an initial SOP Template to insure document uniformity (i.e. include such fields as: Business Name, SOP #, review/approval date(s)/names, Rev #, materials & tools needed, purpose, procedure steps, pass/fail definitions... plus any needed supporting documents).
2. Sample Preparation Methods: Be sure to document, test, review / standardize specific sample preparation methods, for each sample type. Variations in: temperature, extraction solvent or the solution(s) dissolved in, homogenization or grinding methods, mixing, times used, glass or plastic containers used may result in significant variation of the final reported results.
3. Correct Poor Reproducibility and/or Baseline Instability Issues: In chromatography analysis, if the method(s) used are not stable and reproducible, every time they are run, then little to no scientific value can be obtained from them. Methods used must follow basic good chromatography fundamentals and meet basic guidelines. Baseline noise or instability may directly impact integration results (which directly impacts reported results). Instrumentation must be operated in clean, climate controlled rooms. Failure to reproduce a result within acceptable limits (these will vary per method type) will invalidate the method used. Make sure that SOP's are followed, mobile phase solutions are made fresh each day (do not pre-mix solutions with acids and let them sit for several days before use; do not "top off" bottles), solutions should be degassed, HPLC columns are properly washed and re-equilibrated before each analysis, instruments are maintained (per a SOP) and serviced on a regular basis. 
4. Develop HPLC methods that follow good chromatography fundamentals: Retain, separate and resolve ALL peaks. Insure peak K primes are 2.0 or higher. If you have co-eluted peaks in your method, then method development is not finished. If you have ghost peaks or changing retention times, then you need to stop running samples and find out why. Be careful whose method(s) you use. A method that is "Validated" may not be scientifically valid method to use. Have the method checked by an experienced chromatographer.
   
5. Continuous Training is Required to become Proficient: To be proficient, at a basic level in chromatography, takes most chromatographers several years working in an industrial environment to gain practical hands-on time. That assumes that they have had professional training outside of college, in an industrial lab, and can demonstrate an understanding of the fundamentals of good chromatography. Note, that method development skills require a much higher level of understanding and hands-on training to acquire the needed skill set. Make sure your scientists have the needed level of training to operate, run analysis methods and troubleshoot any issues that come up (and issues will come-up, even under ideal conditions). Please do not make the mistake of thinking they will "figure it out" on their own. Hire people who already have several years of industrial chromatography experience, then provide them with additional training opportunities to advance their skills in the application areas that your business needs.  Get them help NOW, you will save money and time, plus get back on track moving forward with your project.
   

If you want to surpass your competitors and provide clients with the most accurate data, then investing in your employees professional knowledge and hands-on technical training is the fastest route to do so. This is an experience based technique where decades of practical knowledge are needed to improve your skill set. A professional can quickly provide you with practical information and show you techniques that will help you move forward. 1-2 days of on-site training often translates to nearly one years worth of knowledge. What is one-years worth of lost time worth to you?
  
Additional Resources:

• Modern HPLC Method Development Tips (PART I): 
• Modern HPLC Method Development Tips (PART II):
• EXPERT CANNABINOID HPLC Training and CONSULTING SERVICES: 

Changing from Reverse Phase (RP) to Normal Phase (NP) Mode (or vice versa) in your HPLC System

Two closely related HPLC questions which have the same answer are commonly found in my email folder each week. Both questions deal with concerns about switching mobile phase solutions used in HPLC. Here are the two questions with an explanation regarding what information is needed to answer them:

1. Can I switch-over an HPLC system which has reverse phase (RP) columns and RP solvents installed to one with Normal Phase (NP) columns and solvents (e.g. such as a C18 column with water and acetonitrile switched over to a silica column with Heptane and IPA )?
2. What is the best way to switch or change-over from a reverse phase (RP) mobile phase made up of water with buffer and acetonitrile to one that is all organic for normal phase (NP)?

To answer these questions we first must review the specified materials used in the HPLC system that are in contact with the mobile phase solutions (the 'wet' parts). 

If you have a system rated for use with most RP solutions, then you will want to verify that the same system is also rated for use with the NP liquids you are considering too (Refer to the manufacturer's product manual or specification sheet). Some HPLC systems may require no changes at all, while others may not be compatible for use. Some systems have seals, tubing and/or valve components that may NOT be chemically compatible with the proposed solvents. Use with incompatible solvents (or additives) may result in damage or destruction of the system. The instrument manufacturer will often provide the needed information inside the specific instrument's Operator / User Manual (*always contact the manufacturer if you have any questions regarding the safe use of the system). In some cases, the vacuum degasser, pump piston seals, tubing, injector seals and/or other parts may need to be replaced with chemically compatible parts before use.  The newer seals or parts may also have different operational limits (e.g. pressure) or specifications than the ones they replace. For example, the maximum pressure ratings with the different parts may be different (some RP to NP conversions result in much lower max pressure ratings and reduced part lifetimes). Many of the newer vacuum degasser units may have no chemical compatibility with the proposed HPLC solvents or additives. Proceed with caution. Check and verify compatibility first.

Anytime you switch from one liquid type to another, you must insure that the new solution is fully soluble (miscible) with the old solution being displaced from the system. The solutions used must be miscible and must not result in precipitation of any contents or contamination of the flow path (and/or plugging of lines) may result. 

Basic guidelines: 

• If any buffers or additives have been used, begin by flushing the system with the same solution, but without the buffer or additives dissolved. We want to remove those buffers and salts first. Flush the entire flow path. Flushing out these salts will greatly reduce the chances of system contamination or plugging. In the case of aqueous buffer solutions, initially flushing with ultra pure water will remove them. 
• Next, if the new solvent is not fully miscible with the old solvent (e.g. Water to Hexane...), then flush the system with an intermediate solvent that is fully soluble with both liquids (in the Water to Hexane example, IPA would be an excellent choice). In fact, for many aqueous to normal phase conversions, IPA provides the miscibility needed to change back and forth from a highly polar aqueous mode to a non-polar organic mode. *Consult a table of Solvent Physical Properties for guidance
  

Summary: 

Verify instrument chemical compatibility and the possible need to replace any seals or parts.

With the proper precautions and checks, many research grade analytical HPLC system can be routinely switched between RP and NP modes.

To switch from NP to RP mode (or RP to NP), flush the system of any salts, buffers or additives, verify liquid miscibility of the solvents, then use an intermediate solvent if needed to change over. 
A table of commonly used HPLC Solvent Properties will help you determine which liquids can be used as intermediate solvents for this purpose.

Air Bubbles Exiting the HPLC PUMP, Reasons Why.

Reasons For Air Bubbles Exiting The HPLC Pump:

• Pump Cavitation: When the pump pressure fluctuates wildly up and down, at very low pressures, this is often due to 'pump cavitation'. It is caused by a loss of priming inside the pump (Air, instead of liquid is in the pump's flow path). The HPLC pump should be primed with fresh, degassed mobile phase (following proper procedures) to restore smooth, stable flow. Often, this can be accomplished using the pump, set to a high flow rate, to draw liquid from the bottles. In cases where the pump is not strog enough, manually priming the low pressure lines using a syringe (~ 20 mL) filled with mobile phase and opening (or disconnecting) a fitting at the pump's outlet may aid in priming the system. Note: Depending on the configuration of your HPLC system, to fully prime an HPLC pump, you may need to run 20 or more mLs of solution through EACH channel. Please keep this in mind every time you use the system and every time you prepare or change a mobile phase solution. This article on baseline/pressure fluctuations may assist you in troubleshooting.

• Loose Connections: If one or more of the low-pressure fittings (nuts and ferrules)  which secure the Teflon tubing to the pump (or vacuum degasser) are damaged or loose, air may enter the system resulting in bubbles. Most pumps use plastic finger-tight style fittings 1/4-28 (or 5/16-24). The threads are soft and can be deformed. When access to these fittings is difficult, sometimes the fittings are left loose and will allow small amounts of air to be drawn in. A build up of salts and/or buffers on the exposed fittings can also allow air into the system (and the presence of deposits on the fittings indicates poor maintenance and a LEAK !). Inspect the tubing and fittings used for proper type, seating depth, wear/condition, cleanliness and/or damage. Replace parts as needed and re-install using the correct amount of torque.

• Flow Rate Too High, Too Low or Not Enough Degasser Equilibration Time: Degassing efficiency is directly related to the flow rate. Lower flow rates increase the residence time of the mobile phase in the degassing membrane or tubing, improving the gas removal. Higher flow rates provide less time for gas extraction and result in lower degassing efficiency (which equals bubbles in the outlet line). Check with the manufacturer regarding the optimal flow rate range for your degasser to insure you are working  within an acceptable range. Allow enough time for the degasser to reach its set-point and stabilize before use. If the degasser is not operating properly or is unable to "keep up" with the flow rate, then bubbles may be frequently observed in the outlet lines. 

• Choice of Mobile Phase Liquid: The miscibility of the liquid is also important. If the new mobile phase is not compatible with the previously used mobile phase, pump cavitation may result. Always flush the pump with an intermediate liquid that will dissolve in both the old and new fluids to flush them out before introducing the new mobile phase solution. (such as pure water or IPA, as applicable). The solubility of air (gas) in the specific solution used also affects the efficiency of the vacuum degasser. Aqueous solutions usually hold less gas than popular organic solvents (though air bubbles can be harder to "push" through in water). The amount of dissolved gas inside the liquid relates directly to the time needed to reduce it to acceptable levels for use in HPLC. Be sure to allow enough time to properly degass the new solution.

• Dirty or Obstructed Solvent Pickup Filters (Bottle filters): Bottle filters should be cleaned or replaced at regular intervals, following routine maintenance SOPs. When they become fouled or obstructed, a vacuum may form as the liquid is drawn into the system. This may result in air being sucked into the tubing or through a fitting (remember that the low pressure Teflon tubing used to connect the bottles to the degasser and pump is porous and allows gas to diffuse through it). The pickup filters should not obstruct the normal flow of solvent (typically they are 10-20 u in porosity). * a quick troubleshooting tip to rule out an obstructed solvent pickup filter is to temporarily remove the filter from the bottle. Observe the back-pressure on the pump to see if it increases and priming is restored. If so, the filter may be clogged. Always replace the filter with a fresh, clean filter and never operate the HPLC without the solvent filters installed.

• A Sticking Check Valve: The pump's inlet and outlet check valves must function perfectly, all of the time, to maintain proper flow and pump function. If an inlet check valve is not fully closing properly to seal off the high pressures generated inside the pump, then the pump will not be able to maintain pressure or flow. Inspect the check valve. Remove and clean it, per the manufacturer's guidelines (often this involves placing the check valve assembly in a beaker with solvent such as IPA and sonicating for 20 minutes to remove any residues. If cleaning fails to restore proper valve function, then replace the check valve with a new one.

• Worn Pump Piston Seals (or Pistons): When the piston seals begin to leak, air is allowed into the system. Pump piston seals require regular replacement (they are normal wear items). Scratched or worn pistons may also result in leaks with air getting into the system. Inspect and Test them both for pressure tightness on a scheduled basis or anytime you suspect a problem. Flush the pump with a suitable liquid, then run a high-pressure test to determine if they pass or fail the manufacturer's leak tightness and high pressure tests. Be sure to perform a physical inspection too.

• Contaminated or Obstructed Pump Outlet Filter: Most HPLC pumps have a small disposable outlet filter installed at or near the pump outlet line (Note: In the case of most Agilent brand HPLC pumps, a small PTFE filter may be found at the outlet valve or inside of the prime-purge valve). These filters should be replaced at regular intervals (monthly is strongly recommended), especially if any aqueous buffers or solutions are used (a they contribute to contamination). Contaminated pump outlet filters may result in a number of pressure instability problems. Abnormally high back-pressure during operation OR when vented to waste are indications it is obstructed. Regular scheduled replacement is the best way to prevent lost time and reduce system contamination.

 Any of the above causes may contribute to valves not functioning properly or air being drawn into the HPLC system. Troubleshooting should begin with the easiest and obvious areas first. Check the condition of the low pressure tubing used to make the connections to and from the mobile phase bottles and pump. If it is kinked, twisted or damaged, replace it with new tubing. Check the fittings used (nuts and ferrules) for tightness and to insure they have been installed properly and are not leaking. Repair all leaks. Keep the system clean (it is easier to monitor and troubleshoot problems when it is clean). Replace any damaged fittings with new ones. Check the solvent pickup filters monthly to insure they are clean and not obstructed. Make sure the flow rate you are using is within the acceptable range for your degasser. 

Has your degasser module been professionally cleaned and serviced within the last 5 years? Are any degasser errors being generated? Is the vacuum degasser making any unusual sounds? Is liquid being emitted from the vacuum pump exhaust port? If any of the answers to these questions are 'yes', then have the HPLC vacuum degasser professionally diagnosed for problems so that repairs can be made to restore function.

Mobile Phase Preparation; Part 1, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)

As a scientific consultant, I often review company overall laboratory operations and make recommendations regarding documentation and procedures which may improve their accuracy and results. Some of these recommendations come in the form of SOP's.

Here is the first example (of three installment posts) of a 'must have' SOP' which should be in place for any laboratory performing HPLC analysis.

Part 1 :  
Procedure(s) For the Preparation of MOBILE PHASE :

Proper documentation of HPLC methods shall always include all of the information that someone would need to reliably reproduce the method in another laboratory. This includes the instrument brand, model, module numbers, configuration, details of the column type with the dimensions and particle size, flow rate, mobile phase composition, all detection parameters including flow cell dimensions, path length/volume, wavelength(s) and bandwidth (if applicable), sampling rate, injection solution, injection volume, sample concentration and other critical information. 

• An area which is often overlooked is HOW the mobile phase solutions are prepared. In addition to stating the chemical grades used, pH measurement checks/adjustments and if any filtering is required (esp. for Aqueous Solutions), mobile phase preparation often includes weighing, dispensing and mixing steps, each of which needs to be described in detail if they are to be reproduced. Without clear directions, the composition of the mobile phase may be different. For example, do you weigh or measure (volume) out all liquids? What type of glassware are used to measure volumes? Volumetric flasks, beakers, graduated cylinders (and if so, what tolerance grade or class are they?) When mixing two solutions, do you measure and prepare them separately in two containers (if so, which containers?), then mix them (how do you mix them)? Do you fill one container with one liquid, then fill to the desired level with the second one? Do you need to check the pH of the solution (as well as how to adjust the pH of the solution? With what?)? When and how? Do you have a SOP for the pH meter and how/when to calibrate it? Number of standards used (usually 3 are used)? Is the final solution filtered, and if so, by what method (be specific)? I have seen people use different methods each time they prepare a solution. As you can see, each procedure results in a final composition which will be different. Different mobile phase compositions usually lead to different results. The important message here is to use the SAME method to prepare solutions and to document it in a SOP for the method (and for all methods). Additionally, be sure there is formal training to insure that everyone prepares solutions in the same manner. Most labs will need multiple SOP's for mobile phase preparation, but as a general guideline, you should have one master SOP for the preparation of mobile phase solutions. The SOP should also include detailed information regarding STORAGE time limits for each type.This will set the standard from which the other SOP's can be based on.

• Be sure to prepare FRESH, filtered aqueous mobile phase solution for use each day. Prepare only enough solution for one or two days use maximum (refrigerate the solution to get 2 days), then dispose of any remaining solution and start with fresh solution. Do not store aqueous solutions at room temperature or for extended periods.
  

• Some types of ORGANIC solvents may require special SOP notes regarding preparation (e.g. pre-filtering), stabilization additives (e.g. mol sieves, dry Nitrogen gas, etc), types of storage (e.g. amber glass bottles away from light), expiration dates after opening (e.g. Acetic Acid, DEA, TEA, THF, etc). Be sure to include these in your SOPs.
  

Notes on SOP creation and editing: Make sure you have several people review the draft SOP's before approving. Sometimes what appears clear to you may in fact have a different meaning to someone else. Clear laboratory procedures should contain enough detail that people with different backgrounds will each carry out the procedure in the same manner. Often, these types of documents will go through many drafts and even after approved, should also be open to future suggestions to make them even better.

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PART #2 of this three-part series, "HPLC System Preventative Maintenance Frequency & Procedure (PM); Part 2, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)" can be found at this link:

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. Be cautious when reviewing any "sales" brochures or articles on these detectors as a great deal of misinformation may be found.

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 (sales literature is often very biased to make one system fail). 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 very unusual 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. Due to the high level of training needed, difficulty to operate and maintain, high cost of operation and poor reproducibility, IMHO they should be a "last choice".

Detection: NOT “Universal” detectors (sourced to marketing misinformation from vendors and early academic reviews which over simplified their 'operation', not of the actual commercial instruments). While detection is partially based on the analyte’s chemical or physical properties, the actual output observed is in fact also 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. Use high-purity grades of mobile phase and additives. 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. Flatter baselines allows for better quality peak integration.

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. Exhausting these large volumes of solvent vapor and gas into a fume hood is just as important during site prep. Be sure and factor these costs and the required space into any site-prep plan.

Operational Reproducibility and Method Transfer:  Recording the exact detector settings used in the method may not provide any guarantee of being able to duplicate the results obtained. No two instrument models are the same so results may vary (similar to LC/MS). Results obtained for each sample are relative to the specific instrument, the chosen settings & method used (again, much like LC/MS) and the internal condition of the detector used. 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 with some success. High quality reproducible methods are achievable with both types of detectors, but will require calibration tables with many additional standards per order of magnitude.

Linearity and Output Characteristics: Except in the most narrow concentration ranges, neither detector is likely to provide a linear response. Different samples will need their own full calibration table and curve fit, per method. 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, quadratic, sigmoidal, exponential, log…etc). 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 at different retention times. This is most easily observed during a gradient analysis. As the mobile phase composition changes, so does the response for EACH sample (this is NOT a UV/VIS detector).

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 (yes, ever one of them). Optimization of detection conditions usually involves making multiple measurements (Peak and Baseline S/N ratios) to find the best settings to use with each sample type and method. This optimization process is time consuming and changes may need to be made to the method over time as the detector fills up with baked-on sample material (changing the spray pattern via nebulization changes).

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 are often 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 only 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:

•  “Advanced Evaporative Light-Scattering Detector (ELSD) For A Variety of HPLC Applications”;
• “Evaporative Light-Scattering Detector (ELSD) Optimization Procedure. Varex ELSD IIA, Technical Note # 0024”;