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HPLC in Pharmaceutical Applications

HPLC in Pharmaceutical Applications

HPLC is a powerful tool in the development and production of pharmaceuticals

Aimee O’Driscoll

Aimee O’Driscoll, BSc, MBA, has a decade of experience as a development chemist and is a seasoned science writer. She can be reached at

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High performance liquid chromatography (HPLC) is an incredibly useful analytical technique with a broad range of applications. HPLC involves a column of adsorbent material (such as silica beads) through which a sample is passed under pressure. Each component of the sample mixture will interact differently with material in the column, and as such, have a different flow rate. As a result, the various components will leave the column at different times and can be collected separately. Qualitative and quantitative analysis can determine the identity of each component and the composition of the sample.

HPLC is considered a crucial tool in a variety of pharmaceutical applications, including evaluating formulations, checking purity, and monitoring changes due to process adjustments or during scaleup.

When using HPLC, it’s important to know what factors to consider for each application. For example, there are many different HPLC methods to choose from. Of particular concern with pharmaceuticals are regulatory requirements that may determine which equipment and specifications must be used. These are often specified by the United States Pharmacopeia.

Read on to discover the main use cases for HPLC in pharmaceutical applications and the main factors to consider when determining the optimal setup for your application.

Use of HPLC in pharmaceutical applications

HPLC is one of the most useful analytical methods in the development and manufacture of pharmaceuticals. Its applications are not confined to just one area and it is instrumental in a number of critical steps necessary for robust pharmaceutical analysis.

Jade C. Byrd, a director in the Liquid Phase Separation Division (LPSD) at Agilent Technologies, notes that, “HPLC is a very versatile technique that can help (bio-) pharmaceutical researchers and manufacturing facilities fully characterize potential drug or treatment candidates, and ensure the medicines are manufactured in a safe and consistent way.” According to Byrd, typical research experiments might include understanding the chemical properties of small molecules or potential biotherapeutics, ranging from assessing the hydrophobicity of a particular molecule to the sugar structures on a monoclonal antibody that affect immune response.

One specific use case is ensuring the consistency of active pharmaceutical ingredients (API). HPLC can provide quantitative analysis of select molecules, so you can confirm the correct dosage of active ingredients. “For QA/QC testing, HPLC can be useful in ensuring critical quality attributes such as strength/concentration, content uniformity, the detection and quantification of impurities, and the quality and identity of raw materials,” notes Byrd.

Impurities can pose a serious safety risk to patients, and their detection and identification is often facilitated by the use of HPLC. Standard HPLC techniques may be combined with highly efficient detection methods (such as UV detection) to provide a complete and accurate impurity profile.

In a similar vein, HPLC can be vital in evaluating the stability of pharmaceutical products. The composition of formulations can alter over time due to a variety of environmental factors, such as exposure to humidity, oxygen, heat, and light. HPLC can assist in the identification of degradation products as well as determine the extent of change over time.

Indeed, Byrd notes that HPLC can also be useful in determining shelf life; “for example, some biotherapeutics are sensitive to aggregation over time, or if not stored properly, and HPLC can be used to monitor this aggregation.”

Factors to consider when using HPLC

Although HPLC is extremely useful, the right methods and equipment must be chosen for each specific application. There are several major factors to consider here. In pharmaceuticals in particular, regulatory requirements will determine the right methods, equipment, and specifications to use. For example, the Food and Drug Administration (FDA) provides a guidance document, titled Validation of Chromatographic Methods, which covers various types of HPLC methods. Other regulatory bodies that may dictate how products are tested in their respective regions include Health Canada and the UK’s Medicines and Healthcare products Regulatory Agency.

Byrd explains that “There are many factors that affect the quality of the separation (instrument design, column and mobile phase chemistries, injection parameters, and column temperature),” and these variables need to be well understood.

HPLC is not a one-size-fits-all method and there are several options to choose from. Two common approaches are normal phase and reverse phase; in both, the separation is based on polarity. In normal phase HPLC, the adsorbent material is polar and the solvent non-polar (typically an organic liquid), resulting in fewer polar components of the sample being eluted first. Reverse phase involves the opposite setup, so the more polar components will exit the column quicker.

Other types of HPLC include ion-exchange (based on ionic charges) and chiral (for separating enantiomers). In the latter, the stationary phase material has pores that slow down smaller molecules. There’s also ultra-high performance liquid chromatography (UHPLC), which uses a smaller column and smaller particles than HPLC, ultimately resulting in a more efficient process.

HPLC is incredibly versatile, especially when combined with different detectors, such as UV-Visible spectroscopy (UV-Vis), mass spectrometry (MS), and fluorescence. For example, UV-Vis can help determine the concentration of molecules following elution from the column. A highly specific detection method is MS, which can help measure a molecule’s mass-to-charge-ratio and thus its molecular weight. Fluorescence can be particularly sensitive for the right kinds of analytes with parts per billion (ppb) detection limits.

There’s no doubt that HPLC plays an important role in pharmaceutical analysis. Its accuracy and versatility make it a suitable tool for many stages of the development and production of biotherapeutics. While there are a number of factors to take into consideration when designing an HPLC analysis, the technique’s innate flexibility, including its compatibility with multiple advancing technologies, makes it an excellent choice for a broad range of applications.

For additional resources on HPLC, including useful articles and a list of manufacturers, visit