Project

Bridge Ion Separation Technology - A Revolutionary New Tool for HPLC

Goal: To foster new research applications of Bridge Ion Separation Technology (BIST), a new separation mode for HPLC discovered by SIELC. Potential project members can join this project and get a free BIST column by sending an email to research@sielc.com. You can also send us a request through ResearchGate for the full text of our publication in the Journal of Separation Science introducing BIST!

Date: 11 July 2022

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Brad Widawer
added 6 research items
Toluenesulfonic acid is a rare strong acid in that it is a solid under normal conditions. Using SIELC’s newly introduced BIST™ method, this strong acid can be retained on a negatively-charged, cation-exchange BIST™ A column. There are two keys to this retention method: 1) a multi-charged, positive buffer, such as N,N,N’,N’-Tetramethyl-1,3-propanediamine (TMDAP), which acts as a bridge, linking the negatively-charged tartrazine analytes to the negatively-charged column surface and 2) a mobile phase consisting mostly of organic solvent to minimize the formation of a solvation layer around the charged analytes. Using this new and unique analysis method, Toluenesulfonic acid can be retained and UV detected at 270 nm.
2-Naphthalenesulfonic acid and 1,5-Naphthalenedisulfonic acid, also known as Armstrong’s acid, are two similarly-structured compounds. 2-Naphthalenesulfonic acid is commonly used in dye production and 1,5-Naphthalenedisulfonic acid is used in synthesizing the salts of basic drugs and in electrokinetic chromatography. Using SIELC’s newly introduced BIST™ method, these two acids can be retained on a negatively-charged, cation-exchange BIST™ A column. There are two keys to this retention method: 1) a multi-charged, positive buffer, such as N,N,N’,N’-Tetramethyl-1,3-propanediamine (TMDAP), which acts as a bridge, linking the negatively-charged tartrazine analytes to the negatively-charged column surface and 2) a mobile phase consisting mostly of organic solvent to minimize the formation of a solvation layer around the charged analytes. In this case, however, various alcohols were used as the mobile phase instead of MeCN and water. Methanol (MeOH), which is relatively polar, behaves like a high-aqueous solution in BIST™; as the alcohols become progressively less polar (ethanol and isopropanol), the BIST™ effect increases due to the minimization of a solvation layer. Using this new and unique analysis method, 2-Naphthalenesulfonic acid and 1,5-Naphthalenedisulfonic acid can be retained and UV detected at 270 nm.
Sunset Yellow is a popular orange-yellow synthetic dye used in a wide variety of food products. Using SIELC’s newly introduced BIST™ method, Sunset Yellow, which ionizes in water, can be retained on a negatively-charged, cation-exchange BIST™ A column. There are two keys to this retention method: 1) a multi-charged, positive buffer, such as N,N,N’,N’-Tetramethyl-1,3-propanediamine (TMDAP), which acts as a bridge, linking the negatively-charged Sunset Yellow analytes to the negatively-charged column surface and 2) a mobile phase consisting mostly of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes. The effect of reducing the solvation layer by increasing the organic component concentration in the mobile phase can be clearly seen above. Using this new and unique analysis method, Sunset Yellow can be retained and UV detected at 480 nm.
Brad Widawer
added an update
Our research group, led by Yury Zelechonok, has published our lab's work on BIST in the Journal of Separation Science!
Feel free to contact us and if you would like a copy of the full-text of our paper.
 
Brad Widawer
added a research item
Chlorpheniramine Maleate is a popular antihistamine used to provide relief from the symptoms of allergies, hay fever, and the common cold. Using SIELC’s newly introduced BIST™ method, Chlorpheniramine Maleate, which separates in water, can be retained on a positively-charged anion-exchange BIST™ B column. There are two keys to this retention method: 1) a multi-charged, negative buffer, such as Sulfuric acid (H2SO4), which acts as a bridge, linking the positively-charged Copper and peptide to the positively-charged column surface and 2) a mobile phase consisting mostly of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes. Using this new and unique analysis method, Chlorpheniramine Maleate can be separated, retained, and UV detected at 210 nm and 275 nm.
Brad Widawer
added a research item
Succinic acid has 2 acid groups that ionize in solution. This allows us to generate fairly strong BIST™ retention at even 70/30% Acetonitrile/Water in the mobile phase. This method is compatible with Mass Spectrometry (117 m/z).
Brad Widawer
added an update
We are happy to announce that our lab's paper introducing BIST™ to the scientific community has been published in the Journal of Separation Science! The article can be found at the link below and should be available on the page for our project soon.
 
Brad Widawer
added a research item
Folic acid, the artificial form of Folate (Vitamin B9) makes an excellent candidate for BIST™ retention due to its 2 acid groups. With BIST™, we can generate fairly strong retention at even 70/30% Acetonitrile/Water in the mobile phase. This method is compatible with Mass Spectrometry (440 m/z) and UV detection at 275 nm.
Brad Widawer
added a research item
The production of Bivalirudin can often result in the production of its degradant, Asp9-Bivalirudin. Separating these two is important in order to purify the Bivalirudin and maintain its efficacy as an anticoagulant. These two similar compounds can be easily separated on a BIST™ A+ column, and their retention times can be controlled by adjusting the pH of the TMDAP buffer.
Brad Widawer
added 3 research items
Trifluoroacetate (TA) and Difluoroacetate (DA) make for excellent test cases of BIST™ since they are of like charge but with slightly different chemical structures, which can be exploited for separation by BIST™. These organic anions are able to be separated with a gradient method due to the different electron distribution due to the extra Fluorine atom TA has.
BIST™ is a versatile and powerful chromatographic tool that can be utilized to separate compounds of different charges. Here, we demonstrate the retention and separation of Copper (II) ion, Chromium (III) ion, and Benzenesulfonic Acid.
Phenylephrine, Epinephrine, and Norepinephrine are similarly structured neurotransmitters with slight chemical and structural differences. These differences can be exploited through the BIST™ mechanism to retain and separate these compounds.
Brad Widawer
added 5 research items
Allura Red, with its 2 negative charges and its intense color, makes for a logical candidate for BIST retention. Here we show BIST retention of Allura Red on a BIST A column.
A typical MP for use on a BIST B or BIST B+ column utilizes Sulfuric acid (H2SO4) as the ionic modifier since the sulfate ion deprotonates fairly easily and is therefore readily available in solution to form bridges and retain analytes. However, since the sulfate ion is not the most volatile of compounds, it is not a viable ionic modifier for mass spectrometry-compatible methods (or LC-MS). We discovered that Hexafluoroglutaric acid (HFGA) works as a viable alternative for LC-MS-compatible BIST methods. HFGA deprotonates readily in solution and is volatile enough to be compatible for use with a mass-spec. We successfully tested our theory and were able to generate BIST retention of Diquat and detect it (via positively-charged electrospray ionization, ESI+) on a mass-spec.
As we have discussed previously, BIST works when the water (i.e. polar) component of the mobile phase has a low concentration, minimizing the solvation layer the water forms around the charged analytes. In theory, the polar MP component does not need to be water. Here, we demonstrate BIST retention of a mixture of three different amines: Dopamine, m-Xylylenediamine, and 2,4,6-Tris(dimethylaminomethyl)phenol (tris-DMP). These amines can be separated and retained on a BIST B column with a step-gradient, non-aqueous MP. The gradient shifts the MP from a higher polarity (Isopropanol and Methanol mixture) to a lower polarity (just Isopropanol). This gradient allows enough time for the BIST mechanism to separate the amines while also delivering reasonable retention times.
Brad Widawer
added 5 research items
Separation of Perchlorate, Iodide, Trifluoro acetate, Nitrate, Bromide, Mesylate, and Chloride ions on a BIST A+ column.
Copper peptide GHK-Cu is a copper complex of glycyl-L-histidyl-lysine and can be naturally found in urine, saliva, and plasma. Research has shown that it may promote wound healing, attract immune cells, stimulate collagen, and act as an antioxidant and anti-inflammatory compound. Using SIELC’s newly introduced BIST™ method, Copper peptide GHK-Cu, which separates in water, can be retained on a positively-charged anion-exchange BIST™ B column. There are two keys to this retention method: 1) a multi-charged, negative buffer, such as Sulfuric acid (H2SO4), which acts as a bridge, linking the positively-charged Copper and peptide to the positively-charged column surface and 2) a mobile phase consisting mostly of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes. Using this new and unique analysis method, Copper peptide GHK-Cu can be separated, retained, and UV detected at 210 nm.
Polymers with charged monomeric units are difficult to separate using ion-exchange chromatography due to very strong and often irreversible interactions with the oppositely charged stationary phase of the column. Usually, an extremely high concentration of the ionic modifier, up to several molar, needs to be used to facilitate an ion-exchange process. This high ionic modifier concentration, however, is usually not desirable because of the significantly increased viscosity of the MP and the salt formation in the pump components. With BIST, these polymers can be separated and retained with relatively weak ionic modifiers (in the mM regime). Here, we show a separation of two different Polyglutamic acid polymers of different sizes; the two differently-sized polymers interact differently with the BIST stationary phase due to each polymer's unique size-dependent charge. Gradients with increasing concentrations of water were used to generate BIST separation in a reasonable time frame.
Brad Widawer
added 4 research items
To separate anions with BIST, an cation exchange column with a negatively charged surface is required in addition to an MP with a doubly-charged cation as the ionic modifier. Common examples of such a modifier include magnesium acetate, calcium acetate, and multi-charged organic amines, such as N,N,N’,N’-tetramethyl diaminopropane (TMDAP). This method utilized an isocratic method to separate four organic acids: Maleic acid, Ascorbic acid, Nicotinic acid, Fumaric acid, and Oxalic acid.
To separate anions with BIST, an cation exchange column with a negatively charged surface is required in addition to an MP with a doubly-charged cation as the ionic modifier. Common examples of such a modifier include magnesium acetate, calcium acetate, and multi-charged organic amines, such as N,N,N’,N’-tetramethyl diaminopropane (TMDAP). This method utilized an isocratic method to separate four organic acids: Maleic acid, Nicotinic acid, Aconitic acid, and Fumaric acid.
Using SIELC’s newly introduced BIST™ method, Fluoroacetic acid, Difluoroacetic acids, and Trifluoroacetic acid can be separated on a negatively-charged, cation-exchange BIST™ A+ column, contrary to conventional chromatographic wisdom. There are two keys to this retention method: 1) a multi-charged, positive buffer, such as N,N,N',N'-Tetramethyl-1,3-propanediamine (TMDAP), which acts as a bridge, linking the negatively-charged anion analytes to the negatively-charged column surface and 2) a mobile phase consisting mostly of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes. Other positively-charged buffers that can generate BIST™ include Calcium acetate and Magnesium acetate. Using this new and unique analysis method, these anions can be separated, retained, and detected with a Conductivity Detector.
Brad Widawer
added 13 research items
Here we demonstrate a separation of oppositely charged molecules of varying charges that, in typical reverse phase chromatography settings, are either difficult to retain or produce poor peak shape and efficiency. BIST™ conditions can, instead, produce high efficiency, retention and symmetrical peak shape. Lisinopril, Trimetazidine, m-Xylylenediamine, 2,4,6-Tris(dimethylaminomethyl)phenol, and Desmosine were separated using a gradient with increasing water concentration. This gradient allows for BIST separation to occur in a reasonable time frame.
When an ionic modifier with double-charged ions (H2SO4) replaces an ionic modifier with single-charged ions (Perchloric acid, HClO4), separation of common pesticides Paraquat and Diquat occurs, as shown in blue (near-void elution with a TFA ionic modifier is shown in red). Since the interactions that govern BIST™ occur close to the surface of the SP, small differences in the charge position within the solute molecule significantly influence the analyte’s retention time and enhance the column’s selectivity. It is clear that a single-charged ionic modifier can provide neither retention nor selectivity.
Proteins and peptides are usually positively charged molecules in an environment with an acidic pH. As a result, BIST can be used to retain and separate these molecules. A high selectivity, which correlates strongly with the number of positive charges in the peptide/protein molecules, can be seen with the retention of Cytochrome C in the attached figure.
Brad Widawer
added an update
SIELC’s new separation mode BIST™ (Bridge Ion Separation Technology) has unlocked new applications that were previously difficult to achieve in the field of liquid chromatography. With this project, we are charging the LC community to continue to discover new and unique applications for BIST™. To aid in this discovery, SIELC is offering any research group that wants to join this project a free BIST™ column for discovering new applications. Check out our website (sielc.com/bist-applications) for example applications of BIST. We offer 4 different BIST columns to choose from: 2 Anion Exchange (BIST B and B+) and 2 Cation Exchange columns (BIST A and A+). Once you have decided which column you would like to order, you can contact us at research@sielc.com to request an order form.
Once you have your column, you can begin experimenting with different methods and samples. A reminder: mobile phases should have a relatively high organic component concentration (like MeCN), a double-charged ionic modifier (like H2SO4), and the SP and analyte should have the same charge (and be opposite to that of the ionic modifier). We ask that in return for this free column that you publish your research results to this project so that the entire LC community can continue to push the envelope of our communal understanding of LC. You can submit your results by emailing us or filling out the form at the following link: https://tinyurl.com/BIST-Project. We will post and credit submissions.
 
Brad Widawer
added an update
SIELC has recently discovered a new liquid chromatography separation mode that we have named BIST™ (Bridge Ion Separation Technology). This new mode of separation can be used with any charged organic or inorganic compound, and with both small molecules, such as drug and dye compounds, and big molecules, such as polymers and proteins, alike. Some literature is already available for download (see attached brochure). You can peruse through our brochure or check out our website (sielc.com/bist) to get an introduction and see some application examples for this new technology. Some separations were previously very challenging without BIST™, and others offer alternative selectivity that was previously unavailable with traditional separation modes.
SIELC Technologies has started this collaborative project on ResearchGate to build a shared database of new, transformational HPLC research using BIST™. Our goal is to create a collaborative, communal effort to unlock new applications of BIST™. In order to facilitate this goal, we are offering a free BIST™ column to any research group that wants to join this project. We ask that you publish your research with BIST™ to this project to share with the LC community and to make BIST™ a staple in every lab's HPLC toolkit, just like IEC, RP, and HILIC modes. If you would like to learn more about this opportunity, please contact us at research@sielc.com.
 
Brad Widawer
added 2 research items
BIST™ in action! When an ionic modifier with double-charged ions (H2SO4) replaces an ionic modifier with single-charged ions (Trifluoroacetic acid, TFA), retention of Dopamine occurs, as shown in red (pre-void elution with a TFA buffer is shown in blue). Conventional chromatographic wisdom tells us this shouldn’t be happening, yet positively-charged Dopamine is retained when a H2SO4 ionic modifer and a high MeCN concentration are employed in a positively charged column (BIST™ B).
Brad Widawer
added a project goal
To foster new research applications of Bridge Ion Separation Technology (BIST), a new separation mode for HPLC discovered by SIELC. Potential project members can join this project and get a free BIST column by sending an email to research@sielc.com. You can also send us a request through ResearchGate for the full text of our publication in the Journal of Separation Science introducing BIST!