What is HPLC?

High Performance Liquid Chromatography (HPLC) is a type of purification technique, which with the help of high pressure separates different analytes and detects them.

 

Image HPLCs used in our laboratory. Image Source: Shimadzu, Waters

The major components:

• Stationary phase (also called as Column)
• Mobile Phase
• Pump
• Detector (containing a light source)
• Injector

Pictographic representation of functioning of HPLC. Image Source: Laboratory Info

1.Stationary Phase (Column):The columns are tightly packed with gel-like material through which the solution of interest (samples) shall pass. This gel-like material is usually silica to which long carbon chains are attached and makes the column non-polar. In such columns, polar substances can be separated and this is called Reverse Phase HPLC. When the columns used are polar, then non-polar substances can be separated and this is called Normal Phase HPLC.

From the samples, based on the affinity to the packed material, different molecules will elute at different times and can be measured with the help of a detector.

Image: Different types of columns      Image: Pictorial representation of elution

2.Mobile Phase: Based on the sample of interest and column, a suitable polar or non-polar solution will be chosen as mobile phase. Mostly, acetonitrile, methanol or water is used in different ratios as mobile phase, along with a buffer of suitable pH.

These solutions are degassed, to avoid air bubbles from getting trapped in the column and interfering with the elution of samples.

 

3.Pump: This feature helps pump the mobile phase into the column. Usually there are four pumps in the HPLC and is called Quaternary pump. There are two types of pumps:

a. Isocratic Pump: This feature helps pump the mobile phase into the column. Usually there are four pumps in the HPLC and is called Quaternary pump. There are two types of pumps:

b .Gradient Pump: In these pumps, different ratios of mobile phase solvents can be programmed to be mixed at different time intervals according to the required analytical method

 

 

Image:  Quaternary Pumps Image Source: Ardent Scientific

4.Detectors: The separation of the analytes in the solution can be detected only in the presence of a detector, even though the separation actually happens in the column. The separated components send out electrical signals and this is detected in the detector as a chromatogram. Different types of detectors are used to detect different types of analytes and there is no universal detector to detect all types of samples. The commonly used detectors are Ultra Violet (UV), Visible (Visible), Photo Diode Array (PDA) and Refractive Index (RI) detectors.

a.UV Detectors: These are used to detect samples which can absorb and emit UV  light.

b. PDA Detectors: These help in scanning the samples through the entire spectrum i.e. from 198nm to 700nm.

c. RID Detectors: These help in detecting samples (such as sugars and alcohols) which do not absorb either UV or Visible light.

Image: Pictorial representation of flow of light through UV and PDA detector. Image Source: Hitachi .

Image: Pictorial representation of flow of light through RI detector.

Image: Example of Chromatogram

What is calibration in HPLC?

Calibration is the comparison of measurement values obtained by a HPLC with those of a standard value.

Why is Calibration of HPLC necessary?

HPLCs, tend to deviate from their standard values based on their usage, temperature at which the HPLC is placed and other factors. Calibration helps in getting the best measurements out of them.

Majorly, calibration of HPLC consists two components: 1) Injector performance 2) Detector Performance

In both of them, different parameters like linearity, accuracy, repeatability are performed.

• Linearity is when increasing concentrations of solutions are used as a measurement. If the HPLC is able to detect the increase linearly, it is said to be calibrated.
• Repeatability is the process of measuring the same solution multiple times and still obtaining the same result.
• Accuracy is the process of analyzing different concentration of samples and the HPLC able to detect the change in concentration accordingly.
• The flow rate of the pump is also calibrated to check with the pump is able to pump out the mobile phase at the mentioned flow rate.

These parameters are essential in determining the proper functioning of instruments/equipments. Apart from these, other parameters like carry over check, noise and drift, temperature check of column oven/auto-sampler, gradient proportion check etc. can be performed based on the instrument/equipment.

For calibration, a solution of Caffeine in water at different concentrations is used. Caffeine has two useful lambda maximums that can be used for wavelength accuracy checks in the ultraviolet region, 205 nm and 273 nm and linearity testing of UV/VIS detectors.

        

–Nikhila Balajepalli, MSc