Top Five Reasons for Using µPAC HPLC Columns in Your Laboratory

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Top 5 Reasons for Using µPAC HPLC Columns

Team TFS
Team TFS

050622 µPAC Blog.jpg


At Thermo Fisher Scientific™, we have been at the forefront of proteomic research since the start. With our product and workflow innovations, we have provided researchers with unprecedented possibilities to teach us more about the proteome, from identification of peptides to determination of protein quantities, discovery of disease biomarkers and post-translational modifications (PTM), to protein interactions.


Of course, our Thermo Scientific™ Orbitrap™ mass spectrometry technology is most likely the eye-catching innovation in proteomics, but also our front-end solutions such as Thermo Scientific™ Vanquish™ Neo UHPLC system and Thermo Scientific™ EASY-Spray™ columns continue to push the boundaries of research. With our new Thermo Scientific™ µPAC™ HPLC columns, we now offer a unique approach to drive proteomics research to the next level. Taking advantage of the micro-pillar backbone structure in the µPAC separation channel, a significant step in reproducibility can be made. Utilizing photolithographic manufacturing, full control over pillar dimension and positioning is achieved, creating identical flow channels repeatedly. Next to the remarkable reproducibility, this also allows the creation of longer separation paths — currently up to 200 cm — to provide maximum separation power in combination with ultra-high MS detection sensitivity.


You don’t have to take our word for it. Read below to hear what our customers — researchers like yourselves — have to say about µPAC HPLC columns, how they use them in their proteomic workflows.


1. Perfectly ordered structure


µPAC HPLC columns could revolutionize MS-based proteomics. When you look inside the column and see the perfectly ordered structure, you can understand how it increases the separation power, offering more sensitivity. Of course, also the low back pressure is important here, as it allows for longer columns, pushing sensitivity even more.

We use µPAC in shotgun proteomics and see an overall increase in identifications on peptide and protein level. And we will continue to use them in a range of proteomics methods to see what workflows we can now perform that were not possible before.

— Francis I. and Kris G.


2. Column robustness and stability


In our facilities, we perform a wide variety of proteomic projects. On of our main projects is the investigation of biomarkers in preclinical and clinical studies, for both human and model animals with our samples typically biofluids and tissues. One of our biggest challenges in this is to optimize our workflows. For our biomarker research we have implemented a clinical proteomics approach, which requires not only high-speed scanning equipment, but also has extra high demands on the column technology we use.


This is where the µPAC HPLC columns come into play. Not only do they provide outstanding chromatographic performance, but they come with the highest reproducibility and robustness as well. We would be looking at cohort studies of up to 200 patients, which would result in 400 samples, and up to 1,200 injections. This can only be done in a high-throughput, high-reproducibility fashion for label-free quantitation.


In our hands, µPAC HPLC columns are much less prone to clogging than sub-2µm particle columns. This has allowed us to design a method of 60 min total analysis time, with the majority of our peptides eluting in a gradient window of 12-56 minutes. The low carryover of the µPAC also allowed us to reduce the time required for washing and equilibration, increasing our throughput. The robustness simply allows us to run more injection on a single column, minimizing the number of column exchanges within the LC-MS study.

— Allan S.


3. Low back pressure and flow flexibility


I heard about µPAC HPLC columns via some of its earliest users. Particularly the idea of a low back pressure, extra-length column without carryover made µPAC interesting to try in our core facility. We typically have a full range of proteomic samples coming in, from protein SDS-gel band to complex human proteomes and isoforms, running in labeled and unlabeled methods.


Before working with µPAC HPLC columns, our column setup depended on sample complexity, longer columns (50 cm) and gradient for highly complex and shorter column (25 cm) and gradient for less complex samples. So yes, that does take quite a number of column exchanges, but it also allows for method optimization. However, now with µPAC, the 50 cm length version is the one used all the time, for all samples that are coming in. Highly complex samples are run at 300 nL/min, using a shallow gradient, and when less complexity samples come through, you simply increase the flow to 600-1000 nL/min and take a steeper gradient, but with the performance of that 50 cm long column. This is one of the things about the low back pressure of the µPAC that is really great, the low back pressures — despite the long lengths of 50 cm and 200 cm — and the flexibility it provides in method optimization, without column exchanges. The µPACs last for months in our lab, performing thousands of samples — blank and QC samples.

— Duncan S.


4. Extra resolution separations in my existing LC-MS setup


Our group is particularly interested in developing and improving chemical cross-linking mass spectrometry methods, to map protein confirmations and study protein-protein interactions. We already had developed a cleavable linker within the ground, and then we learned about the µPAC HPLC columns, their stability and their push of chromatographic resolution.


We compared the µPAC 200 cm nanocolumn against our self-packed 50 cm long columns. Where before we observed peaks that were not always that well resolved, µPAC really improved the separation on a very complicated sample. We benchmarked the µPAC 200 cm against our C18 packed-bed columns, using 1 µg of cross-linked E coli ribosome, run in a 240 min gradient. µPAC allows us to find 26% more cross-linking sites and 32% more cross-link spectral matches.

— Andrea S.


5. Retention time reproducibility


I used to always pack my own columns. However, during a project in a different lab, I learned about µPAC HPLC columns and it was fascinating to work with them. We used the 200 cm long µPAC, and started optimizing the running conditions, including gradient lengths and gradient types, resulting in the methods that the lab continued to run.


We used the method to benchmark the 200 cm µPAC against state-of-the-art packed 50 cm long columns, and immediately saw an increase in reproducibility, both for peptide retention times and proteins identified. Over a triplicate run, 98% of all proteins identified overlapped with µPAC, and on the packed-bed column this was only 75%. This is a really important result, as we still sometimes struggle with reproducibility. But with µPAC, it becomes much less of an issue.

— Gabor T.


To all proteomics researchers who are using or transferring to µPAC HPLC Columns … thank you for looking to us to help you push your science forward and sharing your stories with us.  Your continued success is our inspiration — so let’s continue to win together.


If you are interested in learning more about the µPAC HPLC columns from Thermo Fisher Scientific, please visit our µPAC pages.