High performance liquid chromatography mass spectrometry

High performance liquid chromatography mass spectrometry (HPLC-MS) is an effective analytical technique for determining the composition and purity of chemicals.

The combination of high performance liquid chromatography (HPLC) and mass spectrometry (MS) offers great capabilities in physical separation and mass analysis providing accurate data of sample composition.

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High Performance Liquid Chromatography Mass Spectrometry (HPLC-MS) is an ideal tool for identification, quantitation and mass analysis and it is used to determine the chemical composition and purity of chemicals. HPLC-MS has high sensitivity and it is optimal for performing precise reproducible quantitative analyses. High performance liquid chromatography techniques are ideal for heat-labile compounds, such as proteins and vitamins in food products. 

In HPLC-MS, the two techniques (HPLC and MS) are connected by an interface that transfers the separated components from the column of the liquid chromatograph into the mass spectrometer ion source. An interface is needed since HPLC operates in high pressure and the MS system has a high vacuum.

HPLC includes both a mobile phase and a stationary phase of which both can be modified to suit the sample matrix as well as the desired properties to be determined. Usually the mobile phase is adjusted to suit the sample, and then the stationary phase is adjusted to work well with the mobile phase solution. The degree of compound separation is based on the compound’s affinity for the mobile phase. The HPLC methods can be divided into two main categories based on the properties of the stationary and mobile phases. A combination of polar stationary phase and non-polar mobile phase is called "normal phase chromatography" and the opposite of that, non-polar stationary and polar mobile phase is called "reverse phase chromatography".

In the normal phase HPLC, the column is usually filled with silica particles. The silica is polar and will bind to polar molecules in the mobile phase. This means that the least polar compounds elute first and the most polar compounds last. Normal phase HPLC is suitable for highly hydrophobic or hydrophilic compounds, compounds that are not soluble in water or compounds that may decompose in water. An ideal use of normal phase HPLC is separation of isomers. In the reverse phase HPLC, the stationary phase usually consists of C8 or C18 silica (silica derivatized with alkyl chains). Opposite to normal phase HPLC, in reverse phase HPLC the most polar compounds elute first and the least polar compounds last. The stationary phase can be composed of different solids to meet different needs.

Once the HPLC has separated the compounds, they are identified and their contents determined by mass spectrometry , which creates a mass spectrum that is unique for every compound. In mass spectrometry, the compounds and their fragments are ionized using either electron or chemical ionization. The sample is then accelerated through a mass analyzer, which includes either a quadrupole or an ion trap, and the ions are identified based on their mass-to-charge (m/z) ratios. 

HPLC-MS is suitable for samples in liquid form. For solid sample matrices, sample preparation will be necessary. Compounds that are being determined must be extracted to a solvent and filtered before analysis. 

Suitable sample matrices

  • Pharmaceutical products
  • Cosmetics
  • Environmental samples
  • Biological samples
  • Packaging materials
  • Chemical products
  • Food and food products

Ideal uses of HPLC-MS

  • Quality assurance and quality control
  • Quantitative and purity analysis
  • Proteomics analysis
  • Pharmaceutical analysis
  • Non-volatile and complex molecule analysis
  • Isomer separation and identification

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Frequently asked questions

What is HPLC-MS commonly used for?

High performance liquid chromatography mass spectrometry is commonly used for analyzing organic and inorganic compounds. Especially complex samples, often found in the environment, are suitable for HPLC-MS. 

What are the limitations of HPLC-MS?

Compounds analyzed with HPLC-MS need to be soluble in common solvents, like water, different alcohol and i.e. acetonitrile. The instruments usually have limited pump pressure and are often not compatible with aggressive non-polar solvents. 

What kinds of samples can be analyzed using HPLC-MS?

Samples must be in liquid form, solid samples must be extracted to a solvent and filtered. 

What is Measurlabs?

Measurlabs offers a variety of laboratory analyses for product developers and quality managers. We perform some of the analyses in our own lab, but mostly we outsource them to carefully selected partner laboratories. This way we can send each sample to the lab that is best suited for the purpose, and offer high-quality analyses with more than a thousand different methods to our clients.

How does the service work?

When you contact us through our contact form or by email, one of our specialists will take ownership of your case and answer your query. You get an offer with all the necessary details about the analysis, and can send your samples to the indicated address. We will then take care of sending your samples to the correct laboratories and write a clear report on the results for you.

How do I send my samples?

Samples are usually delivered to our laboratory via courier. Contact us for further details before sending samples.