Complete Solution for Organic Acid Analysis

Robert Puryeara, Piotr Macecha and Itaru Yazawab

aImtakt USA, Portland,OR, bImtakt Corp., Kyoto, Japan


Abstract: Organic acids come in a wide variety and are difficult to analyze on a single stationary phase. Our new Intrada Organic Acid column was designed for this purpose.

Introduction: Whether you are looking into the metabolomics of a disease through changes in TCA cycle carboxylic acids, or trying to analyze phosphoric or even sulfuric acids, attaining good chromatography for organic acids (OA), can be quite a challenge. This is true for several reasons. First, OAs are polar making them difficult to retain on traditional reversed phase columns without ion-pairing or derivatization, which further complicates analysis and data interpretation. Second, the presence of multiple pKa values can lead to variations in the charge states of your analytes, which may cause poor peak shapes including tailing, shouldering and even split peaks. Additionally, OAs are typically poor chromophores, making them difficult to analyze by UV-Vis detection, and CAD does not work well with gradient elutions.

Recognizing these challenges, we developed them most complete solution for OA analysis, with a novel stationary phase called Intrada Organic Acid. This proprietary multi-mode column optimizes the use of multiple stationary phase structures specifically targeting OAs, delivering the most complete LC-MS-based solution on the market today.

Experimental Conditions: Running conditions are shown within the figures. MS detection was used in all cases.

Result and Discussion: Figure 1 shows simultaneous analysis of the entire suite of OAs that comprise the TCA cycle. We achieved very good retention of these polar compounds with times ranging from 2.3 minutes to 5.4 minutes, without using ion-pairing or derivatization. We accomplished these results with excellent peak shapes for all analytes. Oxaloacetate does show some minor tailing with these conditions, but this is most likely due to the highly unstable nature of this compound, which spontaneously decarboxylates. Despite this challenge, we were able to achieve nearly perfect peak shape for oxaloacetate.

Figures 2 and 3 highlight the ability of Intrada Organic Acid to expand its capabilities into strong as well as inorganic acids,

Figure 1: Improved analysis for all organic acids in the TCA cycle

as shown for phosphoric and sulfonic acids, respectively, and their organic derivatives. Figure 2 shows excellent retention and peak shape for not only the highly  

Figure 2: Analysis of a variety of phosphoric acids

polar phosphoric acid, but also AMP, ADP and ATP, which are also part of many metabolomic analyses. We accomplished these results due to the special design of this column’s stationary phase and because these columns only use metal-free column housings. We have previously shown phosphorylated compounds will readily interact with the stainless-steel column housings in traditional columns, resulting in the formation of metal (Fe, Ni, or Cr) salts, which can cause ion-suppression when using LC-MS. In figure 2, there is no evidence of metal salt formation due to the lack of ion-suppression, which could interfere with accurate data interpretation.


Figure 3: Analysis of a variety of sulfonic acids.

Figure 3 shows the analysis of several different sulfonic acid compounds. Conditions in this method were modified for ion-exchange mode and resulted in excellent retention and peak shape for all compounds, except for ammonium thiosulfate. This is likely due to the presence of the additional sulfur on this molecule, but conditions could be optimized if this was your primary analyte of interest. The ability to optimize elution through the use of a wide variety of mobile phases, presents a further advantage of our column design over current ion-exchange columns, providing end-users with multiple strategies to accomplish their separation objectives.


Figure 4: Improved peak shape for polycarboxylic acids

In figure 4 we show a strategy for using mobile phase optimization to overcome the challenges when analyzing OAs with multiple pKa values, in the analysis of polycarboxylic acids. For this specific challenge, we were able to achieve single sharp peaks for these multivalent compounds, utilizing a combination of normal phase mode in neutral conditions and ion-exchange. By using this strategy, we did not observe any peak splitting, shouldering or tailing, which are a common problem for these complex compounds. Therefore, under certain circumstances normal phase conditions might be preferred.

Conclusion: Organic acids comprise a staggeringly broad group, ranging from the simple single carboxylic acid-containing compounds, to strong phosphoric or sulfuric acids. They can be complex, as in the case of polycarboxylic acids, where multiple acidic residues and pKA values present significant chromatographic woes. As a group, they can be as challenging in their analysis as the varieties that are analyzed today. Equally varied are the solutions that chroma-tographers employ to analyze them. From the use of ion-chromatography, complex derivatizations, or even the use of ion-pairing reagents, these analyses used to require convoluted strategies, in terms of the use of a many different column types, analytical techniques and/or sample preparation methods to achieve analytical goals.

Here we show that our newly developed Intrada Organic Acid column can analyze a vast array of very different OAs, on a single column. The specially developed multi-mode stationary phases utilized in this column’s design provide the broadest capability for the analysis of OAs under MS-friendly conditions, on the market today. We have shown that its versatility, combined with its ability to be used with a wide variety of mobile phase conditions, enables optimization to meet a wide range of needs. Never before has such a solution been available to chromatographers. We have shown that whether you are analyzing simple carboxylic acids, like those in the TCA cycle, strong acids like phosphoric or sulfuric acids, or complex multivalent species with multiple pKa values, Intrada Organic Acid could be a single column solution for all your OA analysis needs. Please feel free to reach out to one of our column experts to learn how this revolutionary column could be used in your laboratory.

Detection of Selenocyanate in Biological Samples by HPLC with Fluorescence Detection Using König Reaction

Ryu Mochizuki,a Kyohei Higashi,b Yusuke Okamoto,a Hiroko Abe,c Hirotaro Iwase,c and To s h i h i ko Toi d a

a Graduate School of Pharmaceutical Sciences, Chiba University; 1–8–1 Inohana, Chuo-ku, Chiba 260–8675, Japan

b Faculty of Pharmaceutical Sciences, Tokyo University of Science; 2641 Yamazaki, Noda, Chiba 278–8510, Japan

c Graduate School of Medicine, Chiba University; 1–8–1 Inohana, Chuo-ku, Chiba 260–8670, Japan.


Chem. Pharm. Bull.67, 884–887 (2019). Received March 28, 2019; accepted May 22, 2019


Abstract: We developed a simple and sensitive HPLC method for the determination of selenocyanate (SeCN­). The König reaction, which is generally used for the determination of cyanide and thiocyanate, was applied for the post-column detection, and using barbituric acid as a fluorogenic reagent made it possible to detect SeCN­ with high sensitivity. The limits of detection (LOD) and quantification (LOQ) were 73.5 fmol and 245.1 fmol, respectively. Subsequently, the amounts of SeCN­ in human blood and in cultured cell samples were analyzed, and no SeCN­ was detected in human whole blood. Interestingly, we have found that some of the spiked SeCN­ decomposed to cyanide in human whole blood. Ascorbic acid suppressed the decomposition of SeCN­ to cyanide by reducing the ferric ion, which is typically involved in SeCN­ decomposition. Then, SeCN­ was detected in cultured HEK293 cells exposed to selenite. The established HPLC method with fluo-rescence detection of SeCN­ is useful for investigating small amounts of SeCN­ in biological samples.


Scherzo SS-C18

Conversion of N-acetyl-d-glucosamine to nitrogen-containing chemicals in high-temperature water

Mitsumasa Osadaa Shinya Shojia Shin Suenagaa Makoto Ogatab (2019)

aDepartment of Chemistry and Materials, Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano 386-8567, Japan

bDepartment of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College, Nagao 30, Iwaki, Fukushima 970-8034, Japan

Fuel Processing Technology Volume 195, 1 December 2019, 106154Received 31 May 2019, Revised 6 July 2019, Accepted 14 July 2019, Available online 19 July 2019.

Abstract: To demonstrate the conversion of renewable biomass to platform chemicals, we previously reported the noncatalytic conversion of N-acetyl-d-glucosamine (GlcNAc), which is obtained from chitin, to nitrogen-containing chemicals; however, various aspects of this process were not clarified. Herein, we reported updated and expanded results for the synthesis of nitrogen-containing chemicals from GlcNAc in high-temperature water at 180–280 °C and 25 MPa with a reaction time of 5–34 s. The main products were 2-acetamido-2,3-dideoxy-d-erythro-hex-2-enofuranose (Chromogen I) and 3-acetamido-5-(1′,2′-dihydroxyethyl)furan (Chromogen III) with the maximum yields of 37.0% and 34.5%, respectively. Although 3-acetamido-5-acetylfuran was expected to form by the dehydration of Chromogen III, a yield of only <1% was obtained, likely because the dehydration of Chromogen III is difficult in the absence of a catalyst. The evaluation of the effects of acid and base catalysts on the dehydration of GlcNAc revealed that the acid catalyst suppressed the transformation of GlcNAc to Chromogen I and promoted the transformation of Chromogen I to Chromogen III, whereas the base catalyst had the opposite effects on these processes. The synthesis of nitrogen-containing chemicals from GlcNAc in high-temperature water is an environmentally benign method for utilizing renewable chitin biomass.

Unison UK-Amino

Unison US-C18

ASMS 2016

ASMS was such a great show this year. We saw a lot of familiar faces and had such a great time visiting San Antonio. Especially the River Walk. Yazawa-san our lead column creator came all the way from Kyoto to ASMS to reveal his latest creation, Dacapo DX-C18. This is a wide pH range column with a revolutionary polymer coated silica particle, giving it a stable functionality range from 1 to 12 pH. Ask us to know more.


MSACL 2016

IMG_1091 IMG_1096


We had such a great time in Palm Springs at MSACL this year. Here are a couple of photos of our new robot statue. He certainly got a lot of attention and let people know that we have HPLC columns and that we are quite a bit different from other column companies out there. This is the kind of thing that makes working at Imtakt so much fun.

Welcome to Our New Website

Columns at the Fields Park


Here at Imtakt USA we are excited to unveil our brand new website. Besides a fresh new style, we have added quite a bit of new content to enhance your web-browsing experience… well, at least while you’re on our site. We have made it even easier and faster to find the application notes, poster presentation or peer-reviewed journal article that you are looking for, all in one search. Searching will be more powerful and fun since you can simultaneously search by keyword, category or column stationary phase. This should really get your inquisitive searching juices flowing. It will also be much easier to find the column that you are looking for since we added more than 150 new part numbers. You will now be able to seamlessly go from application note to purchasing the correct column in just a few clicks.

This is all part of our continuing effort to give our customers what they want without any nonsense. Just straight-forward good science. Just like our columns. We hope that you will have fun on the new site and we are looking forward to hearing your feedback.

Pittcon 2015

Pittcon Booth 2015  Horyuji

We hope that everyone had a fun time at Pittcon 2015 in New Orleans. I know that we sure did. Thank you to all the customers and vendors that stopped by our booth and we are looking forward to continuing to work with you throughout the year. If you came by our booth you probably noticed our new column display piece which started life as a model of the famous Horyuji 法隆寺 temple in Japan. Our new display has five levels with the five major brands lines illuminated with side-lit LEDs and a large Imtakt USA sign on top. What a great way to represent the Japanese heritage of the Imtakt brand while highlighting the columns that we provide. If you see us at the next trade show stop by and take a look, it’s pretty cool.