HPLC-Definition, principle uses in clinical diagnostics

High-performance liquid chromatography (HPLC) is a powerful analytical technique used to separate and identify compounds in a liquid mixture. It is a versatile technique that can be used in a wide variety of applications, including the pharmaceutical industry, environmental analysis, and clinical diagnostics.

Principle of HPLC

The basic principle of HPLC is the partitioning of compounds between a mobile phase and a stationary phase. The mobile phase is a liquid that flows through the column, while the stationary phase is a solid that is packed into the column. The compounds in the sample are attracted to the stationary phase to varying degrees, depending on their chemical properties. The compounds that are more attracted to the stationary phase will spend more time in the column, while the compounds that are less attracted to the stationary phase will spend less time in the column. This difference in retention time allows the compounds to be separated from each other.

Parts of an HPLC system

An HPLC system consists of the following components:

  • A pump: The pump provides the high pressure that is needed to force the mobile phase through the column.
  • A sample injector: The sample injector injects a small sample of the mixture into the mobile phase.
  • A column: The column is packed with the stationary phase and is where the separation of the compounds takes place.
  • A detector: The detector measures the amount of each compound as it elutes from the column.
  • A data system: The data system collects the detector signal and displays it in a graphical format.

HPLC Applications in Clinical Diagnostics

a. HbA1c– Glycated hemoglobin estimation is done by HPLC method and considered gold standard

b. Identification and Quantification of Hemoglobin variants like HbA2, HbS

c.HPLC is used for quantification analytes like Adrenaline (epinephrine) , Noradrenaline in urine or serum for diagnosis of tumors such as Phaeochromocytoma, neuroblastoma.

e. Analysis of vitamins like vitamin A vitamin B1, amino acids, and other small molecules

d.HPLC-MS -HPLC with mass spectrometry is considered current reference method for measuring serum cortisol and urinary free cortisol. It has diagnostic sensitivity of 100% and specificity of 98℅ for diagnosing cushing syndrome. Unlike RIA and enzyme linked immunoassay HPLC-MS is not affected by cross reactivity with steroid metabolites or synthetic glucocorticoids.

HPLC Chromatogram

A HPLC chromatogram is a graphical representation of the separation of compounds in a liquid mixture by HPLC. It is a plot of the detector signal (typically absorbance) versus the retention time of the compounds. The retention time is the time it takes for a compound to elute from the column.

The chromatogram is divided into two axes: the x-axis is the retention time and the y-axis is the detector signal. The peaks in the chromatogram represent the individual compounds in the mixture. The height of the peak indicates the amount of the compound in the mixture, and the width of the peak indicates the resolution of the separation.

The concentration of a compound can be determined by measuring the height of the peak in the chromatogram.

Here are some of the key features of an HPLC chromatogram:

  • Retention time: The retention time is the time it takes for a compound to elute from the column. The retention time is affected by the properties of the compound, the properties of the stationary phase, and the composition of the mobile phase.
  • Peak height: The peak height indicates the amount of the compound in the mixture. The peak height is proportional to the concentration of the compound in the mixture.
  • Peak width: The peak width indicates the resolution of the separation. The peak width is narrower for better separation.
  • Baseline: The baseline is the level of the detector signal when no compound is present in the mobile phase. The baseline should be flat and free of noise.

Interpreting an HPLC chromatogram

The interpretation of an HPLC chromatogram is a two-step process:

  1. Identify the peaks: The first step is to identify the peaks in the chromatogram. This can be done by comparing the retention times of the peaks to the retention times of known compounds.
  2. Quantify the peaks: The second step is to quantify the peaks in the chromatogram. This can be done by measuring the height or area of the peaks.
sample graph

Advantages of HPLC

HPLC has a number of advantages over other analytical techniques, including:

  • High resolution: HPLC can separate very closely related compounds.
  • High sensitivity: HPLC can detect very small amounts of compounds.
  • Wide range of applications: HPLC can be used to analyze a wide variety of compounds.

Disadvantages of HPLC

HPLC also has some disadvantages, including:

  • Expensive: HPLC systems are relatively expensive.

further reading on HPLC visit-https://www.ssi.shimadzu.com/service-support/faq/liquid-chromatography/knowledge-base/hplc-basics/index.html

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