Do your HPLC Methods Meet Good Chromatography Fundamentals? HPLC Training: RETAIN, SEPARATE and RESOLVE

When an HPLC or LC-MS method is not developed properly, it may not be selective for the sample and may not show any retention on the column. When this happens, everything injected may elute out at the same time and appear to be 100% pure . *These types of errors are easy to spot by anyone with formal training and experience in chromatography concepts (note: "years" on the job are not the same thing as years of practical knowledge and/or formal training in the technique. We routinely provide consulting services to clients with 10 or more years on the job performing chromatography analysis, but whom have not received any formal training during this time and make errors of this type).

Developing HPLC Methods which follow good chromatography guidelines and fundamentals should be key goals of HPLC method development. When developing an HPLC ("UHPLC") method, you must develop an analysis method which is selective for the compound of interest. 'Selectivity' is the most important variable to focus on when developing methods. Your method must demonstrate that it can: (1) Retain; (2) Separate and (3) baseline Resolve all peaks present (and any possible impurities or related substances), in a reliable and repeatable way. Failure to demonstrate that your HPLC method meets these basic requirements AND is selective for the sample being analyzed means your method is invalid.  

*You may be surprised to know that many HPLC methods (including some published papers and "Validated" Methods) do not meet these basic requirements. In this case, knowledge is truly power. If you have the practical knowledge and understanding of this technique, you will be able to easily spot these invalid methods. Make sure you review other methods as part of your training. Never assume because someone else published it or "did it that way", that it is valid. It may not be. An average of 20% of the methods I review do not meet these basic requirements and are invalid.

• Do your HPLC methods meet these requirements? 
• Can you demonstrate to others, who are knowledgeable in the technique, that your method follows good fundamentals? 

You should be able to demonstrate knowledge of these basic principles and have confidence in them.

Proper HPLC method development training must include and stress the following three practical, fundamental concepts of Retain, Separate and Resolve:

• Demonstrate that using your HPLC Method, that the sample is RETAINED on the Column. *Screen many columns to find the best one, early in the process. For most modes of chromatography, you do this by first estimating then measuring the column void volume. How do you know if it is retained long enough? Next, you calculate the K prime (Capacity Factor) of your sample to insure it meets basic chromatography guidelines (or regulations). * K prime > 1.5 (or > 2.0 for most regulated environments). Note: While retention is required, K prime is not applicable to SEC modes of chromatography.

• When satisfactory retention is achieved (K prime), begin optimizing the method to SEPARATE and baseline RESOLVE all compounds. This is achieved using proper HPLC Method Development techniques plus the practical knowledge and experience which comes from good training. Please refer to these two linked articles for more information; "Modern HPLC Method Development Tips (PART I)" and "Modern HPLC Method Development Tips (PART II)".