Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, February 9, 2019

Part 3, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's); Routine Backup of HPLC Data, Methods And Related Data:

As a scientific consultant, I often review clients 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 third and final example of a 'must have' SOP' which should be in place for any laboratory performing HPLC analysis.

Part 3 of 3:
Routine Backup of HPLC Data, Methods And Related Data:

Another area which is often overlooked and can have disastrous consequences when ignored relates to regular software backups. Successful management of analytical instruments requires that all of the software which is required to operate and maintain them on a daily basis is safely backed up. This should include any needed patch files, hot-fixes or service packs too. Separate copies of all key software applications and licenses should be stored off-site, in a safe location where they can be assessed if and when needed by authorized individuals. This location may be another office location or even a home. It might even be located on a separate computer server or on the 'cloud'. However, for it to be a safe location, it must be off-site and protected from loss due to a hard-drive crash, data corruption, flood, fire, theft, earthquake or other disaster.


  • Regular backups (automated are best!) of all acquired data, methods and related information needed to restore the data should be performed on a daily basis. Backups must also be tested and verified on a periodic basis (if you do not verify them, how do you know that they work?). Verification also serves to train you how to restore damaged data or software which is something that you do not want to learn how to do when you actually need it for the first time.
  • You may also wish to address the use of suitable electrical power backup modules (UPS) to protect the computers used to acquire data from and run methods from power outages or surges. An article which addresses this topic can be found at this, "Power and Surge Protection for Computers & Analytical Instruments (e.g. Uninterruptible Power Supply AKA UPS)" LINK.
  • SOP's describing Backup and Restoration of key Applications and Data should be created which detail the types of backups made, the frequency of backups, which applications and/or files are backed up, using which backup software products or applications, the restoration process used and how often it should be tested to ensure that you can still restore your data.

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 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.

Saturday, January 5, 2019

Part 2, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's); HPLC System Preventative Maintenance Frequency & Procedure (PM)

As a scientific consultant, I often review 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 second example of a 'must have' SOP which should be in place for any laboratory performing HPLC analysis. 

Part 2 of 3:
HPLC System Preventative Maintenance Frequency & Procedure (PM):
 
Regular cleaning, inspection and maintenance of the modules which make up the HPLC system should be carried out on a regular schedule. The frequency will depend on how the system(s) are used and what part lifetimes are typical. Preventative maintenance must not be confused with repairs or servicing to restore operation of the system after damage has occurred. Many companies perform PM services every 4, 6 or at most, 12-months. 
  • PM procedures should include the inspection, cleaning and replacement of normal wear and tear parts such as: filters, frits, valve seals, injection seats/seals, pistons, piston seals and lamps. The PM provides an opportunity to inspect the condition of the modules and parts to insure they are operating properly. The goal should be to replace worn parts before they contribute to poor reproducibility or a failure. If the parts are found to be damaged, then that would be considered 'service' not maintenance and indicates that previous data collected on the system may be unreliable. Don't delay performing PM services on your instruments. Regular maintenance is a requirement. Your related SOP's should address which modules require regular maintenance ( A policy SOP), how often it is performed (A frequency SOP), who performs it and what training have they had (Training Requirements SOP. Also document in the specific Instrument's Logbook), which parts and tools are required (The actual PM SOP for the module) and what tests are performed to insure that it was done correctly (Separate SOP's for each Test).

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 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.

Saturday, December 8, 2018

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

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 should 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, mobile phase preparation often include 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 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. This will set the standard from which the other SOP's can be based on.
  • Be sure to prepare FRESH, filtered mobile phase solution for use each day. Prepare only enough solution for one or two days use (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.
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 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.

Saturday, November 3, 2018

HPLC Mobile Phase Composition and LC-MS Electrospray Voltage

I am often asked about the importance of selecting and optimizing the MS Electrospray Interface (ESI) voltage. To better understand why it is necessary to do so and how it effects the results obtained, let us review some key facts about ESI first.

  • While a gas sheathed flow of volatile mobile phase is directed into the MS source, a strong positive or negative electric field (KV) is applied across the MS inlet. The effluent is atomized and evaporated to form ions (voltage polarity determines positive/negative mode).
  • Too high of a capillary voltage may produce electrical arcing resulting in damage to the system (e.g. PEEK needle may melt, burn and/or clog).
  • Too low of a capillary voltage and ion evaporation will not occur.
  • The voltage needed to produce efficient desolvation and ion evaporation are directly related to the sheath gas flow rate, the mobile phase composition and the flow rate.

What Can You Do To Insure Finding A Suitable ESI Capillary Voltage?

  1. High quality HPLC methods which utilize fully volatile mobile phases and first retain, hold, then elute all samples are needed to generate LC-MS or LC/MS-MS methods. Optimize the HPLC column type, dimensions, MS compatible mobile phase composition and flow rate before optimizing the MS settings. If you have enough sample available, use an infusion method (flow injection) to establish the initial MS settings needed to detect the sample before continuing with the LC/MS method development optimization.
  2. The HPLC mobile phase and any dissolved additives or buffers used for LC/MS analysis must be of high purity and fully volatile.
  3. Make sure your sample is fully dissolved in the mobile phase and filtered (0.22 u filter) before injecting into the system.
  4. Basic samples can be protonated to form [M+H]+ clusters in acidic mobile phases.
  5. Acidic samples can be deprotonated to form [M-H]- clusters in basic mobile phases.
  6. The electrospray ionization (ESI) process used in LC/MS or LC/MS-MS analysis is affected by the surface tension of the HPLC mobile phase used. Water has a higher surface tension than most organic solvents (i.e. Methanol, Acetonitrile, Ethanol, IPA). Using conventional flow rates with highly aqueous mobile phases requires a higher initial voltage for ion evaporation to occur. IOW: Mobile phase mixtures high in water content will require a higher capillary voltage.
  7. Higher organic solvent content usually leads to better atomization / droplet formation and require less capillary voltage to maintain.
  8. Lower HPLC flow rates usually lead to to better atomization / droplet formation and require less capillary voltage to maintain.
  9. To optimize the ESI capillary voltage it is necessary to carry out experiments trying different voltages and monitoring the signal (S/N of a standard or sample) to find the best voltage which results in good signal quality and low noise.

Optionally, ESI signal output may be enhanced using: Adducts or changing the solution chemistry with other mobile phase additives.