Translator for HPLC HINTS and TIPS for Chromatographers

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.

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), Installtion 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, large temperature charges occur, a lamp is changed, a flow cell is changed, the optics become dirty or contaminated or due to normal wear and age. 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 have complete documentation of these wavelength checks.

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 manufacturer's 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. The instrument's 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 most detectors today for their built-in verification. However, you can still prepare your own test solution.

Notes: A reminder that the solution that you prepare the wavelength check standard(s) in will directly effect the results obtained. If you prepare it in a solution which has strong absorbance in or near the region you test, the results 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). 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) will result in different signal outputs and different background solutions will also result in different results which can not be directly compared. For each test, you must use 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.