Translator for HPLC HINTS and TIPS for Chromatographers

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 LC-MS Electrospray Ionization 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 (continuous flow injection) to establish the initial MS settings needed to detect the sample before continuing with the LC/MS method development optimization. Infusion (with a syringe pump) provides the needed time to makes changes, observe how they change the signal for fastest 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 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. This process requires experience to know which settings are likely to enhance the signal and a great deal of skill operating the Mass Spectrometer.

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

Saturday, October 6, 2018

HPLC UV - VIS Wavelength Accuracy Check (" Calibration ") Notes

To verify correct detector wavelength accuracy of your HPLC UV / VIS module it is periodically necessary to measure the wavelength accuracy against know standards using an appropriate SOP ("fit for purpose"). This may be required as part of a Performance Verification (PV), Installation Qualification (IQ) or Operational Qualification (OQ). 

Wavelength accuracy may be adversely affected (or change) when an UV/VIS detector is serviced/repaired, moved, suffers a physical shock (bumped), large temperature changes occur, a lamp or other optical component is changed, a flow cell is changed, the optics become dirty or contaminated, or due to normal wear and age. The wavelength accuracy of any applicable detectors (e.g. UV, VIS, UV/VIS, DAD, PDA) should be measured on a regular basis as part of "Good Laboratory Practices" (GLP). Depending on the regulations or guidelines applied, most authorities require accuracy to be within 2 to 3 nm of a certified standard within the range used. In practice, we generally achieve accuracy of equal to or better than 0.5 nm across a range of UV / VIS wavelengths. Following good laboratory practice (GLP) requires that we establish the frequency and conditions which determine when they should be verified. Complete documentation of these wavelength checks which describe their purpose, specificity, application and detailed procedures (SOP) should be reviewed.

We present a few suggestions in how to measure the detector wavelength accuracy of your HPLC UV / VIS module. 

  • Built-In Test Methods: Most instrument manufacturers incorporate one or more wavelength accuracy checks directly built into their detectors. This allows quick and accurate measurement of the detector's wavelength accuracy for one or more wavelengths in an automated fashion. Most instruments utilize built-in filters (e.g. holmium oxide) which have been treated with chemicals to provide repeatable wavelength spectra which can be used to determine the accuracy of the detector (and adjust it to within specification in most cases, too). If your instrument has one or more of these built-in test filters, then follow the manufacturer's instructions for using them to measure the wavelength accuracy of your detector. 
  • Using a solution of high purity ANTHRACENE: Dissolved in an HPLC grade alcohol (i.e. Methanol ) or Acetonitrile (for low UV checks), anthracene has a lambda max of 251 nm. A solution concentration of ~ 1 ug / mL for HPLC use can be injected using a standardized method (SOP) and the area% evaluated, one-at-a-time, at several different wavelengths (for VWD or single wavelength detectors) as follows: 249, 250, 251, 252, 253 nm. Relative to the baseline, the areas should show a peak at 251 nm. If you have a scanning UV/VIS detector (aka: DAD or PDA), then you can scan all wavelengths around the 251 nm region and plot the results using just one run to obtain the same type of data.

  • Using a solution of high purity CAFFEINE in HPLC grade water: Caffeine has two useful lambda maximums that we can use for wavelength accuracy checks in the ultraviolet region, 205 nm and 273 nm. We often prepare a range of solutions from 5 ug / mL to 500 ug / mL for linearity testing of UV/VIS detectors, but any of those same solutions could be used for wavelength accuracy checking (similar method as described above for anthracene).

  • One of the most widely used methods requires a solution of HOLMIUM PERCHLORATE  solution (NIST). Available for purchase from many chemical suppliers, this acidic solution provides excellent signals for calibration at well documented transmittance bands (i.e. 241.1, 287.1, 361.5 nm and many others out to ~ 640 nm, depending on the solution it is dissolved in). The detector's flow cell can be filled with the solution and measurements made. The solution is also available coated onto quartz slides and is in fact what is found and used in many detectors today as part of their built-in verification. However, you can still prepare your own test solution.

Notes: A reminder that the solution used to prepare the wavelength check standard(s) in will directly affect the results obtained. If you prepare it in a solution which has strong absorbance at or near the region you test, the results obtained may be inaccurate (e.g. a test std dissolved in MeOH used to measure wavelength accuracy at 205 nm would not be an appropriate choice. A standard dissolved in ethyl acetate would obscure the UV wavelengths below its cutoff of ~ 256 nm). Make sure your SOPs state exactly which solutions are used, how they are prepared and which flow cell are used to make the measurements! Flow cells with different dimensions (i.e. path lengths, volumes) will result in different signal outputs. Different background solutions will also result in different results which can not be directly compared (invalid test). For each test, you must use scientifically appropriate methods and the same conditions to make all measurements.

Saturday, August 11, 2018

Cooling Solutions & Mixtures

An alternative to using an ice bath, peltier cooler or recirculating chiller to cool a solution is to prepare a solution whose freezing point is far below that of water. Here are a few salt mixtures which may be used in the laboratory as "cooling - bath" solutions. 

  • Sodium chloride and crushed ice. * ~ 1 part salt + 3 parts crushed iced.  Good for cooling down to -21C.
  • Potassium chloride and water. * ~ 1 part salt + 4 parts water. Good for cooling down to -11C.
  • Calcium chloride (CaCl2*6H20) and water. *  ~ 2 parts salt + 1.4 parts crushed ice. Good for cooling down to ~ -55C.


Saturday, July 7, 2018

HPLC Tubing and Fittings; An introduction to Nuts, Ferrules and Tubing Choices


Setting up a high pressure liquid chromatography (HPLC) system to run trouble-free takes  patience and a strong set of troubleshooting skills. The patience aspect has usually worn out with most of us, but the troubleshooting skills often come from years of tinkering and practical experience. As a consultant who works with chromatographers on a daily basis, I have found that most chromatographers share many of the same basic HPLC hardware problems. Some of these problems the result of a failure to logically troubleshoot a problem from scratch or by overlooking seemingly minor changes that have been made to the system over time. One common area that is often overlooked in the area of HPLC is that of connection fittings (nuts and ferrules) and tubing selection. Selection and installation of the correct HPLC fittings and tubing can help you avoid future problems while allowing your system to run at peak performance. Common types of high pressure chromatography fittings and tubing found in the laboratory will be discussed in this article.

Please click on this link to download the entire article in PDF format.

Saturday, June 2, 2018

Number of theoretical plates (N), Calculation Formulas

Number of theoretical plates (N):

Often used to quantify the efficiency (performance) of a column (HPLC or GC). "Plates" are expressed per meter of column length and should be calculated based on a retained peak with ideal peak shape or symmetry. 

   N = Plates; tr = Retention Time of Peak; w = Peak width;  w0.5 = Peak width measured at half height.

Two popular formulas are:

Tangent: USP (United States Pharmacopeia / ASTM)

Best for Gaussian peaks. Peak width is often determined at 13.4% of the peak height (w). Inaccurate for peaks which are non-Gaussian, poorly resolved or tail.

   N = 16 (tr / w)2

Half Peak Height: (European Pharmacopeia)

For peaks which are less Gaussian in appearance, using a slightly different formula with the peak width measurement made at the half-height (W0.5). Less accurate for peaks which are poorly resolved or tail.

   N = 5.54 (tr / w0.5)2

  • Other formulas, not included, for calculating Plate numbers include: Half Width, Variance Method, Area / Height & Exponential Modified Gaussian (EMG).
  • Caution. HPLC column "Plate" values should not be used for a final determination of efficiency unless you are comparing all results on the same exact HPLC system, which is setup and run under identical conditions each time. Since the result is based on many possible variables, including how your HPLC system is plumbed (dwell volume & tubing ID), the peak's Kprime and symmetry, the detector used, sampling rate, integration quality, flow cell volume, flow rate, actual column used (to name a few), it can easily be manipulated to be very large or small.

Saturday, April 21, 2018

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.

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...).

Saturday, March 10, 2018

Vespel, Tefzel or PEEK Valve Rotor Seals ?

One of the most common HPLC preventative maintenance parts is the injector valve rotor seal. Worldwide, the majority of these chromatography parts are produced by Rheodyne (IDEX) or Valco Instruments (VICI) and used by the major instrument manufacturers in their products. These valve seals are critical to maintaining a leak free, high pressure seal inside the injector. They are subjected to a lot of wear and chemical exposure with use. They have a finite lifetime which may be very short, in some applications (a few months) or last for many years in others. *The most common reason for rotor seal damage is a lack of flushing when buffers are used. Regular flushing down of the flow path (without the buffer) is required to maintain a clean flow path. Buffer deposits and crystals scratch and damage the rotor surfaces. Depending on the specific use and application, rotor seals are often replaced as a preventative measure once every 6 or 12 months. They should also be replaced whenever they are scratched, heavily worn, no longer sealing well, leak or become contaminated. Failure to maintain your injector's parts may lead to HPLC carry-over contamination problems.

The choice of rotor seal material should be based on: (1) the types of chemicals it will come into contact with; (2) the working pH range; (3) the temperature range.

  • Note: When choosing a valve rotor seal material, please refer to the valve manufacturer's information, compatibility and advice. Blends and properties may vary between vendors so always verify compatibility with them before use.
Common HPLC Rotor Seal Material Types:

 Vespel ®: Chemically, this is a polyimide blend (DuPont). One of the most widely used materials for HPLC valve rotor seals. It has excellent chemical compatibility with most HPLC mobile phases, excellent temperature stability and a pH limit of 10. An excellent choice for most applications.

Tefzel ®: Chemical name, ethylene-tetrafluoroethylene (aka, "ETFE"). It has excellent chemical compatibility with most HPLC mobile phases and a higher pH limit of 14. Tefzel's preferred applications are where very high (>9) or very low (<3) pH solutions or mobile phases are used. Not compatible with some chlorinated solvents and in most forms it has a temperature limit of 50°C.

PEEK: Chemical name, polyether-ether ketone. Known for applications where biocompatibility and / or high temperatures are of concern. Like Tefzel, it has excellent chemical compatibility at room temperature with most HPLC mobile phases. Most vendors report a working pH range between 1 and 14. Unlike Tefzel, it has a much higher temperature limit (i.e. 100°C or higher), but its resistance to some chemicals appear to degrade with increasing temperature. Contraindicated where it will come into contact with solutions of THF, methylene chloride, DMSO or concentrated acids (i.e. nitric or sulfuric). Some sources have observed problems with chloroform as well, especially with PEEK tubing, so it may not be recommended for those applications.

For more information on troubleshooting HPLC injector valves, please refer to this linked article, "Troubleshooting HPLC Injectors (Manual and Automated)".