Do's and Don'ts : HPLC Analysis

HPLC is an abbreviation for High-Performance Liquid Chromatography. "Chromatography" is a technique for separation, "chromatogram" is the result of chromatography, and "chromatograph" is the instrument used to conduct chromatography.

Among the various technologies developed for chromatography, devices dedicated to molecular separation called columns and high-performance pumps for delivering solvent at a stable flow rate are some of the key components of chromatographs. As related technologies become more sophisticated, the system commonly referred to as High-Performance Liquid Chromatography simply became referred to as "LC". Nowadays, Ultra-High-Performance Liquid Chromatography (UHPLC), capable of high-speed analysis, has also become more widespread.

Only compounds dissolved in solvents can be analyzed with HPLC. HPLC separates compounds dissolved in a liquid sample and allows qualitative and quantitative analysis of what components and how much of each component is contained in the sample.

The solvent used to separate components in a liquid sample for HPLC analysis is called the mobile phase. The mobile phase is delivered to a separation column, otherwise known as the stationary phase, and then to the detector at a stable flow rate controlled by the solvent delivery pump. 

A certain amount of sample is injected into the column and the compounds contained in the sample are separated. The compounds separated in the column are detected by a detector downstream of the column and each compound is identified and quantified.

While operating HPLC, take care of the following points as Do's and Don'ts


1.  Grade of Chemicals and Water
  • Always use HPLC grade chemicals and Water need to be used for the mobile phase and sample preparation. Low-grade chemicals and water lead to precipitation or hazy mobile phase or diluent. Use clear mobile phase bottles for preparation.

2.  Mobile phase Filtration
  • Filter the mobile phase through a 0.45 μm (use 0.22μm if UPLC) membrane filter using filtration assembly.

3.  Column & Instrument Washing
  • Wash the column for every end of analysis with a suitable solvent for at least 30 column volumes. Instruments also need to be washed with organic solvent before and after analysis to eliminate buffer deposition in the lines.

4.  Column Storage
  • A column must be stored in a suitable solvent after washing. Columns should be properly stored with the end cap.

5.  Suction Filter
  • Use a Suction filter and make sure that suction filters are always completely dipped in the mobile phase. Remove and clean the suction filters weekly once to avoid microbial contamination.

6.  Sample Filtration & Vials
  • The sample solution must be filtered through a 0.45μm (use 0.22μm if UPLC) syringe filter before injecting it into the column. Always use new vials and caps with septa.

PEOPLE ALSO READ: General HPLC Column Care

7.  Purging of System
  • Purge the system to remove the air bubble in the tubing before starting the analysis.

8.  Degassing the Mobile phase
  • Remove dissolved gasses in the mobile phase by online degasser or sonicate the mobile phase to remove.

  1. Don't run the empty Pump
  2. Don't use a dirty mobile phase bottle
  3. Don't use the pH of the column outside the range of the column that is being used for the analysis.
  4. Don't use high concentration buffer and ion pair reagents in the mobile phase.
  5. Don't store the column and instrument in water for a long time, will lead to microbial growth.
  6. Don't leave the column without end-capped.
  7. Don't leave the buffer in the column or instrument for a long time.
  8. Don't use low-grade chemicals or water for mobile phase preparation
  9. Don't use the mobile phase without filtration and degassing.
  10. Don't use used vials and caps without septa.
  11. Don't inject the sample without filtration.

Do's and Don'ts: HPLC Autosampler Vials
  • The HPLC vial is a seemingly minor component of an HPLC system but it has the potential to create major problems. This is particularly true for quantitative analysis. The following list of do's and don’ts will help you to avoid some of the more common pitfalls.

Choose your vial carefully
  • There are many different suppliers of HPLC vials, and they each offer different vial options so it is important to choose the vial that is most suitable for your intended use.
  • For example, certified vials may be essential for sensitive trace analysis so that you can be sure that the peaks detected are definitely from your sample and not from the vial.
  • For more routine analysis, certified vials will probably be an unnecessary expense. You may require amber vials for light-sensitive samples to maximize lifetime. Vial inserts may help to inject very small volumes of samples.
  • However, many labs like to use the same vial for all HPLC use and do not have to choose a vial for each analysis. This is usually a good strategy but during method development (or in the initial method implementation phase if the method is already developed) it is a good idea to consider whether the vial may present any potential problems. The vial cap should also be considered for compatibility with both the sample and the HPLC injector.

Label each vial
  • You may think that you will remember what is in each of your vials but it is all too easy to forget or have a mix-up. Thus it makes sense to label vials. In a controlled environment (such as a QC lab) it is not just good sense but essential to label vials, to ensure that all analysis performed is fully traceable. There isn’t a lot of writing space on a vial so a clear and easy-to-decipher naming convention is advisable.

Equilibrate vials to the desired temperature
  • The amount of sample injected needs to be carefully controlled for quantitative analysis, and the temperature of the sample for injection will influence the amount injected. Therefore, it is important to make sure that sample vials are equilibrated to the desired temperature prior to injection.
  • The ‘desired’ temperature may be the ambient lab temperature, or if a temperature-controlled sample compartment in the autosampler is employed, it may be different from the ambient laboratory conditions (usually cooler to prevent solution degradation). In both cases, sample vials should be equilibrated prior to injection.

Do's and Don'ts: Cool On-Column Injections
  • Make sure that the needle size is appropriate for the ID of the column. This will limit the number of “Z”d needles that occur.
  • Have backup needles (if removable) or syringes available.
  • Depending upon the system, some have different guides depending on the ID of the column. Each column ID has an appropriate guide.
  • Turn the pressure down or off before opening the inlet. Failure to do so will launch the insert and spring into the air. The spring may be hard to find.
  • The septum tends to leak when replaced, more than any other inlet. Make sure to leak check the system if not already in the habit of doing so.
  • If using an Agilent autosampler, note it will have a different guide for an on-column needle vs. a normal needle. The guide adds extra support to the fragile needle and needs to be used with the on-column inlet.
  • Remember to remove the syringe cleaning wire from the needle before using (if applicable).
  • The ‘track oven’ option seems to work well.
  • You may be able to get away with larger injection volumes than with a split/splitless inlet because the solvent stays in a liquid for longer.
  • Phase mismatch may be more of an issue than split/splitless.
  • The column is installed as far into the inlet as it can go and held there while the column is secured.
  • If doing manual injections, be VERY careful of the needle because it is very fragile.
  • Remember that everything gets onto the column, so reduce injection size for dirty or contaminated samples.
  • Because everything goes onto the column, higher temperature or extended final hold time may be required in order to remove semi-volatile contaminants from the column that normally wouldn’t have vaporized in a split/splitless inlet.
  • Conduct leak checks using an electronic leak detector. If this is not available, submerge the union in acetone or methanol (a couple of mm in a Petri dish works well as long as the solvent is deep enough to see bubbles).
  • Avoid using snoop (soap and water for capillary unions.)
  • If using EPC, ignore the guard column when programming the column dimensions into the software. The 0.53mm ID guard will offer very little resistance when compared to the 0.32mm ID column, depending on length.
  • Try to use the same ID guard and column if the method allows. This will give you optimal results.

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