Translator for HPLC HINTS and TIPS for Chromatographers

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).

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 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 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. HPLC Columns prevents pulsations by acting as a dampener and/or system buffer. Most types of HPLC pumps will not operate properly without 20 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 analylsis.

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...).