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

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.

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To View the two previous articles in this series, please click on the links below. 

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

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

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

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.

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 PART #3 of this three-part series can be found at this link: "Routine Backup of HPLC Data, Methods And Related Data:; Part 3, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)";

If you missed Part #1, the article can be found at this link: "Mobile Phase Preparation; Part 1, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)".

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:

The HPLC Restriction Capillary; Troubleshooting, Qualification and Running Without A Column:

Most types of HPLC pumps will not operate properly without 30 or more bars of back-pressure on their outlets to prevent cavitation and excessive pulsation. Columns play a vital role in stabilizing the baseline during an analysis. In this application, they not only aid retention, but act as a cushion or buffer.

When we want to closely replicate the operation of an HPLC system under "normal" conditions and do not want to use an HPLC column in-line (because a column adds variability), we install a "restrictor" such as a restriction capillary in its place. A restriction capillary is often a very narrow ID section of long tubing (capillary) which will restrict the flow of mobile phase through it. For most HPLC systems, a restrictor which is sized to provide about 1,000 to 2,000 psi (~ 70 to 140 Bars) of back-pressure will closely replicate normal operating conditions. The restrictor can be chosen based on length, ID, volume and your flow rate to create this level of back-pressure. You could place a high pressure rated, zero-dead-volume union its place, but in doing so, the system back-pressure may be extremely low ( a few bars) and show poor pump performance. We need to replicate actual analysis conditions during testing or the results obtained may be invalid and unscientific. An HPLC column, with its densely packed small particles inside acts as a pressure pulse buffer and adds a great deal of back-pressure to the HPLC system. That back-pressure greatly improves the stability of the pump operation and overall baseline. HPLC Columns prevents pulsations by acting as a dampener and/or system buffer.

There will be times when you need to operate the HPLC system without an HPLC column installed.

For Example: 

• Troubleshooting sources of contamination, carryover or artifact peaks on a column;
• Measuring the HPLC system delay volume (gradient delay);
• Testing the performance of the injector;
• Testing the performance of the pump (measure % ripple); 
• Testing the performance of a detector module (measure S/N);
• Running HPLC Operational Qualification Tests (OQ);
• Running HPLC Installation Qualification Tests (IQ);
• Running Performance Verification Tests on a Module (PV);
• Running many of the ASTM Tests (e.g. "Baseline Noise & Drift Test").

Example of a commercially available Restriction Capillary (Agilent P/N G1312-67500). You will want to include any needed details of the restriction capillary chosen for your work in the SOP's that you write which utilize it as part of any test (P/N, source, dimensions, volume...).