Basic Principles of LC, HPLC, MS, & MS

What is (LC) Liquid Chromatography?

Liquid chromatography (LC) is a separation process used to isolate the individual components of a mixture. This process involves mass transfer of a sample through a polar mobile phase and non-polar stationary phase.

How (LC) Liquid Chromatography Works

The device is a column packed with the porous medium made of a granular solid material (i.e., stationary phase), such as polymers and silica, where the sample is injected and the solvent (i.e., mobile phase) passes to transport the sample.

When a sample is injected, it is adsorbed on the stationary phase, and the solvent passes through the column to separate the compounds one by one, based on their relative affinity to the packing materials and the solvent. The component with the most affinity to the stationary phase is the last to separate. This is because high affinity corresponds to more time to travel to the end of the column.

The Differences between LC and HPLC

High-performance liquid chromatography (HPLC), also known as high-pressure liquid chromatography, is an advanced type of LC. HPLC is amenable to a wide range of applications, such as pharmaceuticals and food analysis. It is especially useful for low or non-volatile organic compounds, which cannot be handled with gas chromatography.

The difference between traditional LC and HPLC is that the solvent in LC travels by the force of gravity. In the application of HPLC, the solvent travels under high pressure obtained by means of a pump to overcome the pressure drop in the packed column, which reduces the time of separation. As will be discussed, a continuous flow syringe pump is very useful in HPLC.

Solvent Pumping and Sample Injection in HPLC

HPLC requires a pump to inject the solvent. The common types of pumping are:

1. Direct gas-pressure system, which is inexpensive and reliable; however, changing solvent is difficult.

2. Syringe pumps, which can provide a pulseless continuous flow rate. Syringe pumps are reliable, very accurate, precise, and can have a large capacity. Here is an example of a syringe pump:

Learn more about syringe pumps here: “What Is A Syringe Pump”

3. Pneumatic intensifier, which operates under constant pressure, i.e., any blockage can cause a pressure drop and consequently pulses.

4. Reciprocating pumps, which are an economical solution that provides a constant flow and high pressure, but can cause pulsing.

The sample injector should work within very small volumes and withstand the high pressure of the solvent. Most devices use sample injection valves instead of direct injection because the former have superior characteristics. It is possible to inject the samples into the valve’s loop automatically with an auto-sampler or manually using a micro-syringe.

Sample Detection and Identification in HPLC

Compounds are determined based on their retention time in the column using a graph called a “chromatogram.” Retention time usually represents the x-axis of the chromatogram; however, the y-axis depends on the method used for detection, which is usually a UV detector and measures the intensity of absorbance.

Other types of detectors can be of use, e.g., mass spectrometry, especially with applications that require higher sensitivity than that provided by UV detectors.

What is (MS) Mass Spectrometry?

Mass spectrometry (MS) ionizes atoms or molecules to facilitate their separation and detection in accordance with their molecular masses and charges (mass to charge ratio). MS is used in various applications, e.g., biochemicals and atomic physics.

How (MS) Mass Spectrometry Works

The main processes that take place in a mass spectrometer are:

  1. Sample Introduction: sample is converted into a gaseous phase (except with gaseous samples or samples that are thermally unstable) and is introduced through the inlet to the ionization chamber
  2. Ionization: gaseous sample is ionized to generate cations (in most cases but a few types of MS work with anions)
  3. Separation: ions separate according to their mass/charge ratio by a mass analyzer
  4. Detection: a detector is used to determine the species and quantity of each ion.

Direct Infusion

Sometimes, especially with thermally labile compounds, it is possible to introduce samples directly to the spectrometer in the liquid phase. This method is called direct infusion. In this case, ionization takes place in the condensed phase, and a syringe pump is necessary to continuously deliver the sample into the spectrometer ion source. Syringe pumps are the most common and reliable method for direct infusions. Syringe pumps are also commonly used for delivery calibration solution and matrix addition in MS.

Limitations of MS

MS is a very accurate and highly sensitive technique for both separation and detection. Nevertheless, when the desired component is present in a highly complex mixture, MS alone cannot perform the separation process. This is because several compounds can have a similar molar mass and fragmentation pattern. Therefore, combining MS with another separation process, such as HPLC is ideal.

What is LC-MS?

The combined technique between MS and HPLC is commonly known as LC-MS. Combining the two analytical methods reduces experimental error and improves accuracy. The application of LC-MS is very useful in situations that involve a huge number of compounds, such as environmental effluents.

How LC-MS Works

LC-MS involves separating mixtures in accordance with their physical and chemical properties, then identifying the components within each peak and detecting based on their mass spectrum. The flow rates used in LC-MS should be less than those used for HPLC. This is to ensure complete ionization and to maintain the detection sensitivity of the MS, which starts to decrease beyond 200 µL/min. Therefore, the column in LC-MS is much smaller to accommodate the smaller solvent flow rates and sample volumes.

This makes syringe pumps very convenient for LC-MS because they are very accurate and can deliver very low flow rates. In addition, it is possible to use syringe pumps for sample injection into the system as they can deliver very precise sample dosing.

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