Frequently used in biochemistry, biology, and enzymology, Fast Protein Liquid Chromatography (FPLC) is a type of liquid chromatography technique that separates proteins, peptides, nucleic acids and other biomolecules from complex mixtures in a liquid phase. Commonly performed as a part of a bigger lab workflow, FPLC is a form of medium-pressure chromatography, offering advantages such as identification of protein profiles present in body fluids like plasma, urine, and cerebrospinal fluids as well as purification of plasmid DNA, RNA, and synthetic nucleotides to name a few.
A basic FPLC system is typically composed of a pump, injection loop and valve, column, UV detector, conductivity meter, as well as a fraction collector and generally utilizes higher flow rate and lower pressures to pass buffers and samples through a separating column. Good FPLC systems offer several advantages like flexibility to support protein purification techniques for affinity, ion-exchange, hydrophobic-interaction, gel-filtration, reverse-flow, multimodal and size-exclusion chromatography and the ability to accommodate high resolution chromatography media of 5-15 µm beads automation for unattended operation.
Used for many applications spanning a wide range of wavelengths, it is vital for scientific laboratories and academic foundations to acquire an FPLC system that can deliver reliable and reproducible and can minimize the risk of errors. With a plethora of models and types of FPLC equipment in the market, finding the most appropriate one for your application can be quite a daunting and overwhelming task. Don’t fret as LabApp has prepared this simple guide to walk you through factors you should consider in selecting the right FPLC system for your workroom.
Review Application Demands and Needs for your FPLC System
A crucial step in selecting the most suitable FPLC system for your laboratory is reviewing what your application requires of you. You can start by answering these questions:
- Are you doing small-scale protein purification or large preparative and scale-up method development? Answering this can help you decide on the size of equipment to get as well as on the features and functionalities that you should be looking out for. For development, scale-up and production processes, you can look into a complete and customizable FPLC system built with all fluid handling components for smooth delivery and mixing.
- How many and what wavelengths would you want to have access to? Check if your application requires maximum flexibility for simultaneous monitoring of multiple wavelengths.
- How many columns would you want hooked up at once? See if you would need multiple column configurations for faster sample analysis.
- Do you require software and computer controlled components? FPLC systems with pre-programmed application protocols and method templates can minimize sample preparation times.
Depending on your specific application requirements, you can also shop for the following FPLC parts and FPLC accessories:
- Autosampler - aids in quick sample loading on a chromatograph, when getting an autosampler be sure to consider its sample volume range, cycle and load times, precision and accuracy as well as container types
- Fraction collector or valve - used to collect and store in different containers all chromatographically separated materials
- Injection loop and valve - for transferring pre-loaded samples onto columns
Aside from these, ensure you obtain maximum control and optimal sample processing by matching the FPLC system you are planning to buy with the computer and software you are using.
Consider Lab Area Requirements where the FPLC will be Situated
Before you start browsing FPLC systems, review your available lab space first as this can influence the convenience of location and accessibility of your equipment. For a large and open workroom, you can check out modular FPLC systems that can be configured and expanded for access to more detection methods and higher throughputs.
Establish and Decide on Budget Allocations for your FPLC Purchase
Look into your laboratory funding and see how much you are willing to allocate for your FPLC system purchase. If you have sufficient resources on hand, you can always opt for new FPLC systems for your academic and industrial research needs. Consider instrument lifestyle costs as this can also help you decide whether to buy new units for your lab. If you have a limited budget, you can go for one of the popular options these days, purchasing a used, recertified, and refurbished-to-specification FPLC system. As most Original Equipment Manufacturers (OEMs) have tight schedules in supplying new lab instruments, you might want to go for economically priced units from trusted used equipment suppliers. For any urgent unit replacement needs, backups, or to meet your market demands, you can always rely on industry-leading distributors for quality and easily available certified pre-owned lab equipment, saving up to 70% of your budget.
FPLC Pump Type Preference
In charge of moving the mobile phase through the column and FPLC system at a specific flow rate and under a constant pressure, pumps ensure the constant flow of the liquid catalyst, essential in avoiding changes in elution time of sample components and result errors.
Most FPLC systems make use of two cylinder-piston pumps, utilizing one for each buffer and mixing the output of both in a chamber. There are also simpler units that operate with a single peristaltic pump designed to draw both buffers from different reservoirs with the help of a proportioning valve and a mixing chamber. Both types allow the fraction of each buffer entering the column to be constantly varied.
Normally made of resistant materials, these pumps offer two types of operation, delivering constant (isocratic pump) and variable (gradient pump) mobile phase composition. Pumps may also vary in terms of flow range, going from milliliters per minute in benchtop systems to liters per minute for industrial scale purifications.
In terms of flexibility, you can consider the following pump types and identify the best one for your application:
- Binary Pumps - can pool and combine two eluents together at the same time, made of two independent pumps that allow flow for each solvent, high-pressure proportioning
- Quaternary Pumps - can pool and combine four different eluents at the same time, made of one pump, low-pressure proportioning
It is vital to get corrosion-resistant FPLC pumps that are compatible not just with your solvents but also with your buffers and solutes. Acquire a unit that can offer constant flow delivery, independent of back pressure and low dead volume to avoid possible issues when it comes to solvent changeover.
FPLC Column Chemistries and Types
FPLC columns are glass or plastic cylinders packed with beads of resin, also known as the stationary phase, and filled with the buffer solution. These tubes are responsible for carrying out separation of sample components and compared to HPLC ones, FPLC columns have a maximum pressure of roughly 3-4 MPa.
Considered as the heart of a chromatograph and can make or break your analysis, getting the best choice of column for your system is critical. Understanding your compounds of interest, such as the physicochemical characteristics of the molecules to be separated and the interactions between these molecules through chromatography media or resins, can help you select the most fit column component of your FPLC system. Choose the right one for your application by familiarizing yourself with different types of columns based on their composition and methods of separation. You should consider column length and particle size/porosity as these factors can affect the efficiency, sensitivity, and speed of your sample analysis. Some FPLC columns are described below for your reference:
- Normal Phase Columns - separate sample components based on polarity of sample components, widely used in pharmaceutical analysis
- Reversed Phase Columns - separate sample components also based on polarity but happens opposite the normal phase columns, based on hydrophobicity
- Ion Exchange Columns - separate sample components based on the attractive ionic force between molecules and charged stationary phase, surface charges
- Size Exclusion/Gel Filtration Columns - separate sample components based on size
In need of columns and column parts compatible with your FPLC system? Browse convenient online store showcasing a large inventory of liquid chromatography parts and accessories from well-known scientific brands.
FPLC Flow Rate
Besides column dimension, stationary phase particle size, and temperatures, an FPLC system pressure is also affected by the flow rate implemented. Utilizing the most appropriate flow rate is crucial as using a higher than usual one can impact your system’s chromatographic quality, offering insufficient time for your analyte to interact with the stationary phase.
Review your column particle size, length, and internal diameter as these will help you identify the right flow you need. Large diameter columns usually require higher flow rates. Shorter columns and elevated flow rates result to faster analyses. You can refer to the table below for the typical flow rates used based on your column’s internal diameter, injection volume, and loading capacity.
|Internal Diameter (mm)||Typical Injection Volume (µL)||Loading Capacity (mg)||Typical Flow Rates|
FPLC Detector Types Required
FPLC detectors are units used to measure the amount of individual molecules eluted off a chromatography column. Picking out the right detector will depend on the analytes you want to detect, the level of required sensitivity, properties of eluents or liquid solvents used in the separation, and the type of information required by your experiment.
Some popular non-destructive FPLC detectors in the market include:
- pH Detector - monitors and measures pH values
- Conductivity Detector - detects ions in a solution, highly sensitive and susceptible to effects of temperature variations, measures inorganic ions and small organic substances such as organic acids and amines
- UV/Vis Single Wavelength Detector - measures a sample’s absorption of light on a single wavelength
- UV/Vis Multiple Wavelength Detector - measures a sample’s absorption of light on multiple wavelengths and are more sensitive, for routine analysis of a wide range of applications
- UV/Vis Diode Array Detector - presents multiple photodiode arrays to obtain information through a wide range of wavelengths at one time
- Refractive Index Detector - based on changes in refractive index of eluting compounds in the mobile phase, less sensitive compared to a UV-Vis detector
Keep in mind that a good detector offers low noise, low detection limits, low dead volume to eliminate memory effects, high sensitivity towards solute over mobile phase and a large dynamic linear range.