Liquid Chromatography Systems Buying Guide

Liquid Chromatography Buying Guide image

Liquid chromatography (LC) is essential in many laboratories for the separation, identification, purification and quantification of various compounds in different matrices. This Buying Guide will walk you through the aspects you need consider in choosing the right LC for your lab.


1. Background
2. Types of LC Systems
3. System Performance
4. A Guide to LC Components
5. Considerations For System Configurations
6. Modular and Integrated Systems
7. Service and Support
8. Other Considerations
9. Success in LC
10. Current and Future Trends
11. Summary

BACKGROUND

In liquid chromatography, the sample is carried across a stationary phase (the solid support in LC columns) by a moving liquid (the mobile phase or eluent). The components of the sample are separated based on their affinity with the stationary phase.

“Classical” or “traditional” LC relied on gravity to get the mobile phase and sample through the column. But as smaller particles were used to improve separation power, higher pressures were needed to create the desired flow through the column, high pressure liquid chromatography, or HPLC, was developed. Eventually as advances in instrumentation took place and performance continued to improve, the acronym HPLC came to stand for high performance liquid chromatography.

TYPES OF LC SYSTEMS

LC systems can be general purpose or application specific. Application specific LC systems are used in the separation of specific analytes such as, ion chromatography (IC), used in the separation of ions, and gel permeation chromatography (GPC, also known as size exclusion chromatography, SEC), used in the separation of large biomolecules such as proteins and nucleic acids and synthetic polymers. Note that there are dedicated LC systems for bioseparations, but if you are not routinely doing SEC, you may use special columns for “bioseparations” on an existing HPLC system.

Another application specific LC system is supercritical fluid chromatography (SFC), which is a hybrid of liquid and gas chromatography. It is used in the separation of compounds that are difficult to analyze by either liquid or gas chromatography, such as hydrophobic and chiral molecules, lipids, fuels, natural products, surfactants, and thermally labile analytes. In 2012, a system called UltraPerformance Convergence Chromatography (UPC2) was introduced. It combines SFC with UltraPerformance Liquid Chromatography (UPLC), enabling scientists to tackle routine and complex separation challenges that SFC can solve, while delivering reliability, robustness, sensitivity and throughput that the UPLC technology has demonstrated since its first introduction in 2004. UPLC is a general-purpose LC technology which uses sub-2µm particles, resulting in higher resolution, increased sensitivity, faster run times, and reduced solvent consumption. The smaller particle size generates higher back pressure compared to conventional HPLC columns. Ultra high-performance liquid chromatography (UHPLC) is the general term given to LC systems that can withstand pressures up to 1000 bar. (UPC2 and UPLC are trademarks of Waters Corp.)

General-purpose LC systems (commonly known as HPLC) are used in the separation of different types of molecules, depending on what column is used (e.g. reversed phase, normal phase). There are different HPLC platforms, depending on the goal of the separation, and the corresponding flow rate requirements. Table 1 summarizes the different LC systems in the market, and what each is typically used for.

Table 1. Different kinds of LC systems

Application-Specific LC

LC System

Characteristics

Ion chromatography (IC)

Separation and quantitation of inorganic anions and cations, and low molecular weight water-soluble organic acids and bases

Use in research and development labs, routine analyses

GPC/SEC

Separation and quantitation of large biomolecules and synthetic polymers

Used in research and development labs

Supercritical fluid chromatography (SFC)

Analysis of hydrophobic and chiral molecules, lipids, fuels, natural products, surfactants, and thermally labile analytes

Uses CO2 as primary mobile phase

UltraPerformance convergence chromatography (UPC­­2)*

SFC using 3 to 1.7 µm particles, resulting to higher resolution, increased sensitivity, faster run times, very low solvent consumption

*UPC2 is a trademark of Waters Corp.

General-purpose LC

LC System

Characteristics

Semi-preparative Preparative

Isolation and/or purification of compounds

Sample goes from detector into fraction collector

Analytical

Quantification and or/identification of compounds

Samples goes from detector into waste

·   Standard

·   Flow rates up to 10 mL/min

·   Microflow capillary, nanoflow

·   Used for sample limited applications, like proteomics

Flow rates between 0.01-100 µL/min (microflow) and 0.01-1 µL/min (nanoflow)

UHPLC

(UPLC*)

Uses sub-2µm particles, resulting to higher resolution, increased sensitivity, faster run times, reduced solvent consumption

Employs high-performance pumps that deliver 15,000 psi (1000 bar) or more of pressure

*UPLC is a trademark of Waters Corp

Buying Guide Tip: Consider what you are planning to separate and detect at the moment, and possibly in the future. How much do you have for your analyses? Do you need fast separation?

SYSTEM PERFORMANCE

The overall performance of an LC system, and the quality of data generated, depend on the different components that comprise the system (the different components will be discussed in the next section). Consider the following performance attributes:

Buying Guide Tip: Which performance attributes are most important to you? Not a single instrument can provide all…

Accuracy is the closeness of the values obtained by the LC analysis to the "true" value. The accuracy of an LC system is influenced by the injector, pump, and detector.

Accuracy of the pump is very important. The pump must maintain accurate and consistent flow rate, necessary for stable and repeatable interaction between the analyte and the stationary phase.

Dynamic range is a performance parameter of the detector. There are two “dynamic range” terms that are used: “dynamic range” and “linear dynamic range”. “Dynamic range” is that range of analyte concentration over which the detector continues to respond to changes in analyte concentration. The “linear dynamic range” is the concentration over which the detector output is linearly related to the analyte solute concentration.

Precision is the closeness of the results of multiple LC analyses carried out under the same conditions, with the goal that repetitive analysis of the same sample gives similar results. The precision of an LC system often lies in the ability of the sample injection system to introduce samples onto the column in a very reproducible manner. The injector should be able to draw the same amount of sample in replicate injections. Precision is also influenced in part by the pump, stability of the column oven (if used) and detector.

Precision of retention times: Excellent retention time precision is required, especially if this parameter is used for compound identification.

Resolution is a measure of how well peaks are separated, and is largely dependent on the stationary phase (column) and the mobile phase.

Robustness: The LC system is robust if it is able to perform difficult analyses while still maintaining quality results.

Selectivity is the ability of the LC method to separate two analytes from each other. It is largely dependent on the characteristics/properties of the mobile and stationary phases.

Sensitivity is the ability of the system (method used) to discriminate between small differences in analyte concentration. The sensitivity of an LC system depends a lot on the detector, but is also influenced by the column parameters, the performance of pump, choice of eluent, and electronic noise.

Speed, Throughput: Fast separations can be achieved in several ways: by using fast systems that use sub-2µm particles (ultra-high performance chromatography, UHPLC), high temperature, using other column platforms (core-shell or superficially porous particles, monolith), shorter columns, and increasing flow rate.

Buying Guide Tip: If you need an LC system that offers fast separation, consider the possible ways of doing this. Will investing in a new LC system, such as a UHPLC, be the best solution? Or are you willing to try new column platforms that offer fast separations without having to buy a specialized LC system?

A GUIDE TO LC COMPONENTS

Every LC system has the following key components:

  • pump
  • sample injector
  • column or columns
  • detector(s)
  • data handling system (computer)

Table 2 lists the things you need to consider for each of the different components.

LC systems are usually equipped with degassers to remove dissolved gases from the eluents. For applications that require close control of column temperature, column thermostats/ovens are usually included in the LC system. The other advantage of using a thermostat is that better chromatograms are usually obtained by maintaining column temperature.

Buying Guide Tip: Maintaining column temperature using a column thermostat usually results in more stable and reproducible chromatograms.

There are many detectors for liquid chromatography, some of which are: UV-Vis, photodiode array (PDA), fluorescence, mass spectrometer (MS), refractive index, electrochemical, and evaporative light scattering (ELSD).

Buying Guide Tip: Choosing the right detector depends on the analyte(s) you wish to detect, the level of sensitivity required, properties of the eluents used in the separation, and the type of information you require (for example, a mass spectrometer would be most useful for compound identification).

Table 2. LC system components considerations

Component

Things to consider

Pump

Solvent compatibility, resistance to corrosion

Accurate and precise flow rate

Accurate and precise mixing of solvents

Availability and convenience of replacing worn out parts

Injection, Autosampler

Highly precise even at low volumes

Accurate injection volumes

Range of injection volumes

No/Minimal carry-over

Is there a need to cool/heat the samples?

Detector

Low noise, drift

Low detection limits

High sensitivity

Large dynamic range

Good stability and reproducibility

Sensitivity towards solute over mobile phase

Computer (Software)

Ease of use

Flexibility (if it is something you require)


Buying Guide Tip: It will save time (time in training and learning to use the system) and cost if the system software is compatible with the current software used in your laboratory. Also, if you are planning on using other detectors, consider the software’s compatibility with the other detectors?

CONSIDERATIONS FOR SYSTEM CONFIGURATIONS

HPLC systems can be classified into four basic types, depending on use, as illustrated in the diagram below:

I. Basic Isocratic Systems

Simple, routine analysis (QA/QC)

 

 

 

II. Gradient Systems

Method development, complex analyses/separation, and dial-mix (autoblend) isocratics for routine analysis

IV. Fully automated gradient systems with state-of-the art detectors

Methods development, research

III. Fully automated, dedicated systems (gradient, isocratic); high-throughput

Cost-per-test, round-the-clock analyses (clinical and environmental laboratories)


Buying Guide Tip: When deciding on the type of system, think not only about your immediate needs. Consider what you will be using the system for in the future.

Note that there are LC systems with integrated sample preparation modules.

MODULAR AND INTEGRATED SYSTEMS

A modular LC system is composed of separate modules (the modules are different components of the LC) stacked and connected to function as one unit. In an integrated system, the modules/components are built inside a single housing. Modular systems provide maximum flexibility and upgradability, and are more easily serviceable since internal components are more accessible (downtime is reduced during repair or maintenance service). One can access the latest technology by switching modules. On the other hand, integrated systems tend to be more affordable.

SERVICE AND SUPPORT

It is important that the LC system manufacturer can provide not only excellent hardware, but also a responsive and knowledgeable service.

Buying Guide Tip: Know what kind of servicing interval the system will need and how much this is going to cost.

Make sure the company provides excellent customer support. A company’s support reputation may change over time; check out a company’s customer support reputation from current users. Good local sales and service representatives can help you interface with the company to make sure that problems are solved at a reasonable time. Also, a good support means the company is able and willing to help you should you decide to venture into new uses/applications for your LC system.

OTHER CONSIDERATIONS

Ease of Use A new LC system needs to be fairly easy to use/understand. Some vendors include technical training for lab personnel in the price of the LC system. This will prove beneficial since you can ask questions in person, instead of having to call or find answers in the instrument’s manual.

Maintenance Beyond the simplicity of operation and ease of use, consider the LC system’s ease of maintenance. If special maintenance is required, do you have lab personnel trained to do it? Can you afford the downtime required for the maintenance?

Availability of supplies and accessories Make sure supplies and accessories for the system are easily accessible.

Cost Consider not only the initial investment, but also the long-term cost of ownership. When you think about the return on your investment, consider aspects such as productivity (higher throughput with the same or higher quality results) and savings on solvents.

Space How much space does the system require? Do you have this space in the lab?

SUCCESS IN LC

In the end, your success in LC depends on three things that have been covered in this guide:2

  • The suitability of the instruments to the work you want it to do. Consider your performance requirements, which are influenced by the different components of the LC system, as well system configuration that best suits your current and possibly, future needs.
  • Maintenance. Will your lab be able to keep it running and maintain it? No matter how new/old, or what kind of LC system it is, it will only run as well as you look after it. System maintenance is critical in keeping the system running well enough to produce good results and reducing maintenance call outs.
  • The service and support you receive. The vendor should offer you excellent service, be able to help you in solving problems on a timely manner, and assist you should you start out in new directions/applications for your LC system.

CURRENT AND FUTURE TRENDS

LC systems are almost ubiquitous in any laboratory, since they can be used to analyze a broad range of analytes in various matrices. The use of mass spectrometer as a detector in LC systems is becoming more common. LC-MS has found applications in clinical diagnostics, and is expected to continue to find new applications. Since the introduction of the first system about a decade ago, UHPLC is going mainstream, being adapted in many laboratories across the world. Future LC systems that can withstand even much higher pressures than current UHPLC systems will enable the use of even smaller particles, yielding even faster and better separations.

In ion chromatography, IC-MS is becoming more common. An IC system that operates at higher pressures, allowing for the use of columns packed with smaller particles, was introduced earlier this year. The smaller particles provide better performance than conventional columns.

Liquid chromatography will continue to be an essential analytical instrument. As novel applications for LC emerge, and users require better system performance, we will continue to see improvements and new developments in LC instrumentation.

SUMMARY

Visit the SelectScience Separations product and user product reviews directory for an overview of the latest UHPLC/UHPLC, SEC, IC products from leading manufacturers.

Keep up to date with the latest techniques and advances in Liquid Chromatography by visiting the SelectScience Separations pages for application notes, videos and the latest news.

Additionally, watch on-demand webinars and attend future events.

Recent Liquid Chromatography Webinars:

Advances in Chiral Compound Separations using UltraPerformance Convergence Chromatography (ACQUITY UPC2)

Application of Ultraperformance Nanoscale Liquid Chromatography and High Resolution Accurate Mass Tandem Mass Spectrometry in ‘Omic Biomedical Studies of Infectious Disease and Cancer

REFERENCES

1. Skoog, D.A.; Leary, J.J.. Principles of Instrumental Analysis, 4th Ed. 1992. Orlando: Saunders College Publishing.

2. McMaster, M. HPLC: A Practical User's Guide, 2nd Ed. 2007. New Jersey: John Wiley & Sons.

Editor's picks

Kerry Parker

Editor-in-Chief

Agilent 1200 Infinity Series LC (Agilent Technologies)

Product image

4 out of 5

“This equipment is really amazing. It is very easy to use. Results are reliable."
Cromwell Flores, WAHA





Read more

Nexera UHPLC (Shimadzu)

Product image

4 out of 5

"Easy to use, user friendly software. Very satisfied with the system functions."
Marija Mladic, VU University Amsterdam




Read more

ACQUITY UPC² System (Waters)

Product image

4 out of 5

"This is really an excellent high throughput system. A rugged and robust system. The speed is matchless."
Sunil Dubey, BITS Pilani




Read more

ACQUITY UPLC System (Waters)

Product image

4 out of 5

"Very robust instruments. Great savings in time and money. Support is good."
Rushinkumar Jadawala, Bristol Myers Squibb




Read more

UltiMate® 3000 Basic LC System (Thermo Fisher Scientific)

Product image

4 out of 5

“The system presents exceptional characteristics, and the results and configurations offered by the Chromeleon software are amazing."
Enrique Carrasco Correa, University of Valencia

Read more

Prominence HPLC System (Shimadzu)

Product image

4 out of 5

“Easy to operate. Separation performance is good with any kinds of column, conventional and core-shell...”
Karen Kloke, Mayo Clinic



Read more