Translator for HPLC HINTS and TIPS for Chromatographers

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)

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: Area / Height & Exponential Modified Gaussian (EMG).

Saturday, April 21, 2018

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

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, we install a "restrictor" in its place. A restrictor 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 provides about 1,000 psi (~ 70 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 and show poor pump performance. An HPLC column, with its densely packed small particles inside adds a great deal of back-pressure to the HPLC system and that back-pressure greatly improves the stability of the baseline and prevents pulsations by acting as a dampener and system buffer. Most HPLC pumps will not operate properly without 20 or more bars of back-pressure on their outlets to prevent cavitation and pulsation. 

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; 
  • Testing the performance of a detector module;
  • 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 a test (P/N, source, 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)".