Gel Permeation Chromatography (GPC) is a form of size exclusion chromatography which can be used to 'clean' complex mixtures of large, interfering molecules from samples further used for analytical methods like HPLC, GC or MS. Cleaning of such samples results in clearer peaks, meaning low concentration analytes are not drowned out by noise. Previously, cleanups were ran manually which is time consuming and introduces human error. However, KNAUER have now released an automated GPC sample cleanup system which makes cleanup operations fast, simple and effective.
AZoM spoke to Maximilian von Delbrück, Product Manager at KNAUER, about the theory behind GPC cleanups, KNAUERS new system and the benefits automated sample prep can bring to your lab.
Why is Gel Permeation Chromatography used for sample cleanup? How does it work?
Prior to many analysis procedures of complex mixtures interfering components in samples must be removed. Mainly these are high-molecular substances such as lipids and proteins.
Sample preparation based on Gel Permeation Chromatography (GPC) separates molecules according to their size and shape. The column material (solid phase) features heterogeneous pore sizes and works as a molecular sieve. Large molecules are too big to enter the small pores of the column material and elute first. Small molecules, which are the targets of analysis, do enter the pores and travel a longer distance until they exit the column. Their retention time in the column is longer.
The principle behind GPC
When is GPC Cleanup applied?
GPC cleanup is applied for samples which are hard to analyse with GC- or LC-MS (liquid- or gas chromatography-mass spectrometry). In the case of complex samples like oil, feed, and soils a cleaning step is an efficient way of reducing the high content of interfering substances. As a result, the signal/noise ratio of the MS analysis is significantly increased.
For the multiresidual analysis of several different target molecules GPC is the right choice because the separation is based on size meaning all chemically diverse target molecules are collected. In contrast, other cleaning techniques select only a group of molecules based on their specific chemical properties.
GPC Cleanup is an established method according to several guidelines:
- Method 984.21 - AOAC international
- SW-846 Method 3640A - US Environmental Protection Agency
- AEN 12393 and EN 1528 - European Standard
- L 00.00-34 Method in accordance with §64 LFGB (formerly §35 LMBG)
What industries do you think GPC sample preparation benefits most?
The general cleaning approach can be employed on a wide range of sample matrices such as foodstuffs, tissues, plants and environmental samples. The most successful applications address olive oil, nuts & seeds, feed and sludge & waste samples.
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What are the benefits of an automated sample preparation compared to manual cleaning?
Manual sample preparation based on GPC is work-intensive and time consuming. Through automated sample preperation the workload is limited to the initial step of sample loading. After sample loading, up to 15 samples are fully processed without manual intervention.
Human resources and time can be saved by the automation of the GPC cleanup method. Further, the reproducibility and quality of the cleanup are improved and kept at a constant level.
How does the daily handling of the AZURA GPC Cleanup system look like? How much time do you save?
In the first step the column is conditioned with eluent. During this equilibration period the 15 sample loops can be filled with sample. After loading is completed, the samples are sequentially processed. Each sample is automatically injected and passed over the column. The first fraction which includes the high-molecular weight substances is sent to the waste. The second fraction yielding the target molecules is collected for each sample. The collection window is determined by a reference run with standard components in advance.
Loading of the 15 sample loops takes approximately 30 min. After this step the system takes over. Including the initial equilibration of the column and the regeneration after each run, the total time for cleaning 15 samples is about 16 hours. Typically, the users of the AZURA GPC cleanup system would load their samples in the afternoon and by the next morning all of the samples are prepared.
What exactly does the AZURA GPC Cleanup system offer?
The system features 15 sample loops controlled by two selection valves. Stored in a tray with drainage system the wound-up sample loops are easily accessible for inspection and replacement. The GPC tubing guide sorts outlet tubings coming from the fraction collection valve. Besides the waste, 15 fractions can be collected. The elution of separated reference standard components is monitored by a variable single wavelength UV detector.
A compact pump based on dual-piston technology ensures constant flow rates, crucial for high-resolution GPC. A pressure sensor at the pump monitors the system pressure during the runs. In addition to the injection valve, a second valve is included to bypass the GPC column or select between two columns.
The AZURA GPC Cleanup System
What features of the AZURA GPC Cleanup System make sample preparation as simple as possible?
The user´s main point of contact is the software. Common HPLC software with all its functions, is unnecessarily complicated for GPC cleanups. Instead we use Mobile Control, a specially designed control software with a touch-optimized user interface and with clearly structured menus to guarantee intuitive handling.
The software is run on a tablet, allowing the remote control of the whole system with a few fingertips. This means, for example, loading sample loops can be facilitated by the push of a button.
How is the risk of sample contamination minimized when using a GPC cleanup system?
The system provides 15 sample loops for 15 samples. The volume of each loop is typically 1 ml. Each sample is stored in its own sample loop waiting for injection. Hereby, the risk of cross-contamination is reduced to a minimum. In contrast, an Autosampler uses the same sample loop, tubing and syringe to inject all different samples. On top there are many screw joints increasing the risk of a carryover.
Do KNAUER have any examples of GPC cleanup separations that they have ran?
There are two application notes available on our website. One is describing the GPC cleanup of olive oil for subsequent pesticide analysis. The second note is comparing the GPC cleanup technique and Online SPE (solid phase extraction) processing sludge samples.
Where can our readers find out more about the AZURA GPC Cleanup System and KNAUER?
Visit our website to find out more about our GPC Cleanup system. If you're interested in trying the control software you can download a demo version on our website too.
If you would like to find out the specifications of the AZURA GPC Cleanup system please download our GPC cleanup white paper using the link below.
Download the White Paper for More Information
About Maximilian von Delbrück
Between 2004-2009 Max studied biochemistry at the University of Bayreuth. 2016 he finished his PhD in biochemistry at the Max Delbrück Center for Molecular Medicine in Berlin. Max joined KNAUER in 2016 as Product Manager for FPLC.
Disclaimer: The views expressed here are those of the interviewee and do not necessarily represent the views of AZoM.com Limited (T/A) AZoNetwork, the owner and operator of this website. This disclaimer forms part of the Terms and Conditions of use of this website.