Basic Principles of LC, HPLC, MS, & MS

IN THIS ARTICLE:

• High-Performance Liquid Chromatography (HPLC)
  • What is HPLC?
  • HPLC Main Components
  • How Does HPLC Work?
  • Four Steps in HPLC
• Traditional Liquid Chromatography (LC). Differences between LC and HPLC
• Mass spectrometry (MS)
  • What is HPLC?
  • MS Main Components
  • How Does LC-MS Work?
• Advantages of Using Syringe Pumps in HPLC, MS, and LC-MS

HPLC, MS, and LC-MS IN SCIENTIFIC RESEARCH:

• HPLC (High-Performance Liquid Chromatography) is used to separate, identify, and quantify compounds. It is an invaluable component at research science labs in chemistry, pharmaceutical, and biochemistry fields.
• HPLC is an improved type of liquid chromatography (LC) that has largely replaced the traditional LC methods.
• MS (Mass spectrometry) is a highly utilized analytical tool in science labs for determining the masses of different compounds in a sample.
• LC–MS (Liquid chromatography–mass spectrometry) is a technique that merges the physical separation of HPLC with the remarkable mass analysis capabilities of MS.
• Accurate and reliable syringe pumps are available for MS, HPLC, and LC-MS.

High-Performance Liquid Chromatography (HPLC)

• What is HPLC?

HPLC (High-Performance Liquid Chromatography) is an advanced type of liquid chromatography (LC) and an invaluable component in research science labs in fields such as chemistry, pharmaceutical and biochemistry. HPLC offers several advantages over other analytical techniques thanks to its high sensitivity, high selectivity, versatility, and automation. It is used to separate, identify, and quantify compounds in liquid samples. One of the main advantages of high-performance liquid chromatography is its enhanced speed in contrast to other alternative chromatography techniques.

HPLC is a crucial tool used for pharmaceutical applications such as evaluating formulations, checking purity, and monitoring changes due to process adjustments or during scaleup.

HPLC is mostly used for the separation of low-volatility or non-volatile organic compounds.

• HPLC Main Components

HPLC systems have four main components:

• • Pump: Also known as a solvent delivery system. It maintains a constant flow of the mobile phase (solvent that runs continuously to the system such as acetonitrile, methanol, phosphate buffer, etc..) through the HPLC.
  • Injection Valve: It allows for the introduction of the sample solution in the HPLC column. The sample can be injected manually or with an automated injection valve called autosamplers. Autosamplers such as syringe pumps inject the samples automatically with precision and higher accuracy as compared to manual sample injection.
  • Column: It contains a specific stationary phase to separate individual compounds based on a particular physiochemical property. The majority of HPLC columns are made of stainless steel and filled with porous silica particles. Nevertheless, there is a wide range of HPLC column hardware types and packing materials available.
  • Detector: It analyzes the components of the eluted mixture that is collected after being run through the column. Among the commonly used detectors are ultraviolet/visible (UV/Vis), photodiode array (PDA), fluorescence (FL), and refractive index (RI) detectors.

Many factors can influence HPLC separations such as the mobile phase composition, the stationary phase chemistry, or even the temperature.

• How Does HPLC Work?

The components of the sample separate from one another via a process of differential migration as they flow through the stationary phase column. Each component of the mixture travels at different speeds through the column, and the speed will depend on the interaction between the column (stationary phase) and the chemical composition of the sample. The components elute at different times, thus allowing for the separation.

After the separation, a detector gauges the concentration of the analytes and transforms them into electrical signals. The concentration of each component is directly related to the quantity that was eluted from the column.

• Four Steps in HPLC

1. Dissolve the sample in the mobile phase or solvent.
2. Inject the sample. It can be delivered manually or using an autosampler such as a syringe pump that allows a continuous flow of mobile phase. The sample is delivered to the column by the pump.
3. As the sample travels through the column, its various components interact differently with the mobile and stationary phases, causing them to separate from each other at distinct speeds.
4. Once the components exit the column, they are directed towards the detector, where a physical property of the compounds is measured, such as the absorption of light for UV detection.

Traditional Liquid Chromatography (LC). Differences between LC and HPLC

The difference between traditional LC and HPLC is that the solvent in LC travels by the force of gravity, resulting in a slow flow rate and largely limiting the size of particles being used in the column. As mentioned previously in this article, in HPLC, a pump allows the solvent to travel under high pressure, reducing the time of separation and thus increasing efficiency. HPLC columns are filled with smaller stationary particles than the ordinary LC, allowing HPLC to have superior resolving power when separating mixtures.

HPLC is an advanced type of liquid chromatography (LC) and has improved upon and largely replaced the traditional LC methods.

Mass Spectrometry (MS)

• What is MS?

MS (Mass Spectrometry) is an analytical tool highly used in science labs to determine the masses of different compounds in a sample. This technique allows the researchers to identify and quantify the compounds in a mixture as well as detect impurities in a sample.

Mass spectrometry uses an instrument called a mass spectrometer and it works by employing various ionization methods, determining the mass of a molecule by measuring the mass-to-charge ratio (m/z) of its ion. Mass spectrometers function under conditions of significantly low pressure, creating a high vacuum environment. This measure is taken to minimize the likelihood of ions colliding with other molecules within the mass analyzer.

Every ionization method requires careful consideration of specific factors to ensure its effectiveness. These factors include volume, concentration, sample phase, and composition of the analyte solution.

While various mass spectrometers are available in the market, and they may exhibit certain differences, the sample molecules will undergo identical processes regardless of the instrument used.

• MS Main Components

The primary components within a mass spectrometer are the following:

• • Inlet system: The function of an inlet system is to introduce a small amount of sample into the ion source with minimal loss of vacuum. There are a variety of inlets available, with gas chromatography being the most common technique for introducing samples into a mass spectrometer.
  • Ion source: It is the heart of the mass spectrometer. It is where the sample is ionized before it continues to the mass analyzer and detector. Various ionization techniques have been devised to efficiently ionize molecules with distinct characteristics, including EI, CI, ESI, APCI, MALDI, and others.
  • Mass analyzer: It is responsible for taking the ionized masses and sorting them according to their mass-to-charge ratio (m/z). There are various types of mass analyzers, including time-of-flight (ToF), quadrupole, magnetic sector, ion trap, and orbitrap mass analyzers, as well as combination systems like tandem mass spectrometry.
  • Detector: Mass spectrometers offer several types of detectors, with the electron multiplier being the most commonly utilized for routine experiments.

• How does MS Work?

The sample is introduced through the inlet system. Within the instrument, the molecules undergo ionization in the ionization source, transforming them into ions. These ions are then electromagnetically driven into the mass analyzer, where they are separated based on their mass-to-charge ratio (m/z). Once separated, the detector converts the ions’ energy into electrical signals, which are subsequently transmitted to a computer for further processing.

Liquid Chromatography–Mass Spectrometry:

• What is LC-MS?

Liquid Chromatography–Mass Spectrometry (LC–MS) is a technique that merges the physical separation of HPLC with the remarkable mass analysis capabilities of MS. LC-MS instrument is basically an HPLC unit with a mass spectrometry detector attached to it. These coupled systems are widely favored in chemical analysis as they mutually enhance the distinct strengths of each technique. As we mentioned previously, liquid chromatography effectively separates mixtures with multiple components, while mass spectrometry furnishes valuable spectral information crucial for identifying or confirming the suspected identity of each isolated component.

This technique provides a unique capability for rapid, cost-effective, and quantitative measurements of organic molecules for an enormous variety of applications. HPLC differentiates compounds by their physico-chemical properties while MS differentiates compounds by mass (specifically their mass-to-charge ratio). It is this dual selectivity that makes LC-MS such a powerful analytical tool.

LC/MS is suitable for the analysis of large, polar, ionic, thermally unstable, and nonvolatile compounds.

• LC-MS Main Components

LC-MS consists of an HPLC unit with a mass spectrometry detector attached. While HPLC allows the physical separation of the components, MS measures the mass-to-charge ratio of ionic species related to the analyte under the investigation.

• How Does LC-MS Work?

LC-MS operates through a two-step process:

• • In Liquid Chromatography or HPLC, sample components are separated based on their interactions with the mobile and stationary phases, as mentioned in the HPCL section in this article.
  • Once the chromatographic separation is completed, the compounds elute from the column and are ionized at an ionization source. Subsequently, the ionized compounds are introduced into the mass spectrometer for precise mass analysis.

Advantages of Using Syringe Pumps in HPLC, MS and LC-MS.

• Syringe pumps are highly used in HPLC in science labs, in order to minimize background noise during electrochemical detection.
• Syringe pumps provide speed and reliability to small-volume and large-volume, repetitive liquid transfers in HPLC. These small high-precision devices are very useful to ensure stable transportation of the mobile phase.
• When using syringe pumps for HPLC purposes, the sample is placed in the syringe and pumped at a defined flow rate by a syringe pump. If you want to know more about what a syringe pump is, read our article Syringe Pumps in Research Labs. Applications and More.
• Syringe pumps are also used to inject samples in MS to reduce background noise during electrochemical detection. Syringe pumps offer a smooth, pulse-less flow at low flow rates with the high accuracy and precision needed to handle the varied needs of Mass Spectrometry.
• Not only can syringe pumps deliver samples automatically, but they can also deliver a calibration solution automatically.