This work was supported by UWS and ThermoFisher Scientific. One of the authors (DK) acknowledges the receipt of an Australian Postgraduate Award.

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The authors have nothing to disclose.

RF-PCD يسمح لخلط الفعال للكاشف اشتقاق مع النفايات السائلة بعد عمود HPLC دون استخدام لفائف رد فعل، والتقليل من آثار توسيع نطاق وتحسين أداء الانفصال. وقد أظهرت أساليب RF-PCD أيضا التحسينات في الاستجابة إشارة فيما يتعلق طريقة الكشف. كان Camenzuli وآخرون. 28 أول من أشار إلى...

عالية الأداء اللوني السائل (HPLC) إلى جانب آخر عمود اشتقاق (PCD) هو أداة قوية يمكن أن يكون مفيدا في حل عدد من القضايا في المختبر التحليلي. ويمكن استخدامه للكشف عن المركبات التي لا يمكن اكتشافها إلا مع مجموعة من أجهزة كشف المتاحة 1،2، وزيادة إشارة الحليلة الهدف، والذي يسمح الحدود الدنيا للكشف وتحديد الكميات 3-5 أو انتقائي derivatize على الحليلة المستهدفة من أجل تجنب آثار مصفوفة 6. وتشمل يشيع استخدامها ردود الفعل PCD رد فعل الأمينات، مثل الأحماض الأمينية، مع أورثو-phthaladehyde 7-9، النينهيدرين 9،10 أو fluorescamine 11،12، واشتقاق من أنواع الاكسجين التفاعلية (ROS) لدى ديفينيل 2،2 1-picrylhydrazil المتطرفة (DPPH •) 13،14 أو 2،2&#39;-azino مكرر (حمض 3-ethylbenzothiazoline-6-السلفونيك (ABTS) 15،16، واستخدام كاشف يوديد-أزيد إلى derivatize الكبريت جمركبات ontaining 17،18. هناك، ومع ذلك، العديد من المآخذ على استخدام ردود الفعل PCD مع أنظمة HPLC 6. أساسا بين هذه هو استخدام لفائف رد فعل بين نقطة إضافة كاشف اشتقاق (ق) وكاشف، مما يتيح الوقت لخلط ورد فعل لتحدث 8. هذه رد فعل الحلقات غالبا ما يكون حجم 500 ميكرولتر أو أكثر، وهو أمر مهم مقارنة مع حجم ما تبقى من نظام HPLC 19. استخدام هذه ارتفاع حجم رد الفعل حلقات النتائج في زيادة الذروة توسيع بالمقارنة مع ما سيتم الاحتفال به بدون وجود حلقة رد فعل. هذه النتائج في أقصر، قمم الأوسع نطاقا التي لها حدود أعلى من الكميات والكشف وسلبيا يؤثر قرار الكروماتوغرافي. أرقام 1 و 2 تسليط الضوء على تدهور ذروة الشكل الذي ينتج من إضافة المختلفة بعد العمود كميات رد فعل حلقة. هذا التحليلوقد أجريت مع تكوين مرحلة المحمول من 94٪ الميثانول و 6٪ من المياه الملة-Q. وكان معدل تدفق الطور المتحرك 1 مل / دقيقة، وكان حجم حقن 20 ميكرولتر وكان الطول الموجي تحليل 265 نانومتر. أدرجت لفائف من مختلف أحجام الميتة من 20 ميكرولتر إلى 1000 ميكرولتر بين العمود وكاشف لمحاكاة آثار رد فعل حلقة حجم القتلى في أساليب PCD. تم إعداد هذه الحلقات من أنابيب الفولاذ المقاوم للصدأ قطرها الداخلي 0.5 ملم. تم إجراء تجربة على نظام HPLC تتكون من وحدة تحكم (SCL-10AVP)، وانخفاض ضغط متدرجة صمام (FCL-10ALVP)، مضخة (LC-20AD)، والحاقن (SIL-10ADVP)، وكاشف المساعد الشخصي الرقمي ( SPD-M10ADVP). تم ضخ الطور المتحرك من خلال الغاز الراحل قبل إدخالها في نظام HPLC. تم إجراء فصل باستخدام س 4.6 ملم معرف 5 ميكرون العمود 250 ملم. وقد تم اختيار الظروف التجريبية لتكون نموذجية من ردود الفعل PCD أن تم مؤخرا نشرت في الأدب. الأبسط، ويطلق آخر الأكثر شيوعا الإعداد مفاعل العمود مفاعل أنبوبي غير مجزأة والتي هي بالفعل طويلة، أنبوب رفيع من خلالها السائل يمكن أن تتدفق ورد فعل يمكن أن يحدث. في هذه الذروة نظام توسيع يعتمد على ليس فقط حجم القتلى إضافتها إلى النظام، ولكن أيضا القطر الداخلي للأنبوب نحو ما أبرزته إيجيما وآخرون. 8. وعلاوة على ذلك، لفائف الهندسة تلعب دورا في توسيع العلامة التجارية لوحظ. ستيوارت 20 ذكر أن اللف المفاعل يغير ملامح تدفق الثانوية، مما أدى إلى أفضل خلط، وهذا يعني أن حجم القتلى قد يكون الحد الأدنى. وقد ذكر أن ذروة توسيع ليس كبيرا عند استخدام أنبوبي مفتوحة محبوك لفائف 21. عندما ذروة توسيع هو كبير جدا، ويمكن أيضا اعتبار أنواع أخرى من المفاعلات 20،22. ويمكن أن تشمل هذه المفاعلات السرير أو مفاعلات تدفق مجزأة. هذه المفاعلات هي مفيدة بشكل خاص لردود الفعل البطيئة التي من شأنها أن اشتراطات غير ذلكحلقات رد فعل ه كبير. كما المفاعلات الأنبوبية غير مجزأة هي الأنواع الأكثر شيوعا من المفاعلات المستخدمة في التطبيقات PCD، والباقي من هذه الصفقات المادة مع هذا النوع من الإعداد المفاعل. تصميم العمود تدفق رد فعل (RF) يشتمل على نهاية المناسب متعددة المنفذ الذي يسمح الطور المتحرك للخروج (أو دخول) العمود إما من خلال منفذ واحد يقع في المنطقة المركزية الشعاعية للعمود أو ثلاثة الموانئ الواقعة على الخارجي منطقة جدار العمود (انظر الشكل 3). يتم فصل هذين التيارين باستخدام المناسب نهاية تحتوي على فريت التي يسهل اختراقها المركزي الذي تحيط به حلقة كتيمة التي هي بدورها محاطة فريت التي يسهل اختراقها الخارجي الذي يمتد إلى جدار العمود. بسبب تدفق حلقة عبر المركزي كتيمة غير ممكن بين المناطق التي يسهل اختراقها اثنين. خلال رد فعل اللوني التدفق، وضخ كاشف اشتقاق (ق) ضد اتجاه تدفق مرحلة المحمول إلى واحد أو TWس من الموانئ الخارجية للعمود تدفق رد فعل. يتم خلط شاطف عمود مع كاشف اشتقاق (ق) في فريت الخارجي وتمريرها إلى كشف من خلال منفذ خارجي خال. يمكن استخدام تدفق رد فعل إما لاشتقاق كاشف واحد (1 منفذ للكاشف اشتقاق، 1 منفذ لتمرير شاطف العمود إلى كشف و 1 منفذ سدت) أو نظام كاشف المزدوج (2 الموانئ لالكواشف اشتقاق و 1 منفذ ل تمرير شاطف العمود إلى كاشف). إما أن تدفق من التيار المركزي أن تستخدم للكشف عن شاطف العمود underivatized وفعالية الكشف عن الإرسال المتعدد 23، أو تمريرها إلى النفايات. أسلوب واحد ضبط الرئيسية المتوفرة عند تشغيل RF-PCD اللوني هو نسبة التدفقات المركزية والطرفية. النسبة المثلى لكل اشتقاق يعتمد على عدد من العوامل مثل ما إذا كان سيتم الكشف عن تدفق المركزي أو تمريرها إلى النفايات. لذلك مرة واحدة وقد تم تحديد النسبة المثلىيجب التأكد من أن نسبة تدفق الصحيحة يتحقق قبل كل تشغيل التي يتم تنفيذها. وقد وجد أن استخدام فريت لخلط تيار العمود شاطف وكاشف اشتقاق في نتائج RF-PCD في خلط أكثر كفاءة مقارنة مع تقنيات خلط التقليدية التي عادة ما تستخدم صفر حجم القتلى T-قطعة أو حجم القتلى منخفض W- قطعة لمزيج من التيارين. وقد سمح ذلك لاستخدام الحلقات رد فعل صغيرة نسبيا، أو حتى القضاء على حلقة رد فعل تماما. الحد من نتائج رد الفعل حجم حلقة في قمم أكثر وضوحا بالمقارنة مع الطرق التقليدية اشتقاق آخر عمود. وهذا يعني أنه على الرغم من حقيقة أن ليس كل من شاطف عمود وderivatized، لوحظ أكبر إشارة إلى نسب الضوضاء وحدود وبالتالي أقل الكشف والكميات لا يمكن أن يتحقق. وقد وضعت تدفق رد فعل اللوني للتغلب على الصعوبات مع التكيف من رد فعل PCD<html> الصورة الأعمدة الحديثة HPLC والأنظمة، لا سيما الخسارة في الكفاءة الناجمة عن الفرقة توسيع بسبب آخر عمود كميات كبيرة ميتة الناجمة عن الحاجة إلى توظيف رد الفعل حجم كبير حلقات. عمليات خلط أكثر كفاءة في RF-PCD مقارنة PCD التقليدي يعني أن أحجام التداول حلقة رد فعل أصغر قد تكون عاملة مما يؤدي إلى زيادة في كفاءة الفصل المرصودة. وعلاوة على ذلك يظهر RF-PCD اللوني على حد سواء زيادة إشارة وانخفاض الضوضاء بالمقارنة مع التقنيات التقليدية PCD مما أدى إلى الحدود الدنيا من الكشف والكميات بالمقارنة مع الطرق التقليدية PCD. ميزة إضافية لRF-PCD بالمقارنة مع الطرق التقليدية PCD هي القدرة على رصد تيار underivatized أن elutes من الميناء الرئيسي للعمود RF فضلا عن تيار derivatized أن elutes من المنطقة الطرفية من العمود. RF-PCD هي تقنية جديدة نسبيا ولكنها واعدة أن يعرض مزايا عديدة أكثر من الطرق التقليدية PCD. &gt; ويتحقق اتصال العمود RF في تقريبا بنفس طريقة عمود HPLC التقليدية مع فارق كبير يجري عدد من التجهيزات نهاية على عمود RF. التجهيزات المستخدمة لربط عمود HPLC القياسية لنظام HPLC هي قادرة على أن تستخدم لربط عمود الترددات اللاسلكية لنظام HPLC.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HPLC instrument</td>
			<td>Agilent</td>
			<td>1290 Series HPLC</td>
			<td></td>
		</tr>
		<tr>
			<td>Additional Pump(s) for derivatisation system</td>
			<td>Shimadzu</td>
			<td>LC-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>RF colum</td>
			<td></td>
			<td></td>
			<td>Non-commercial</td>
		</tr>
		<tr>
			<td>PEEK tubing</td>
			<td>Sigma Aldrich</td>
			<td>Z227307</td>
			<td></td>
		</tr>
		<tr>
			<td>Column stoppers</td>
			<td></td>
			<td></td>
			<td>Provided with column</td>
		</tr>
		<tr>
			<td>PEEK tube cutter</td>
			<td>Sigma Aldrich</td>
			<td>Z290882</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Scale Balance</td>
			<td></td>
			<td></td>
			<td>4-point analytical balance</td>
		</tr>
		<tr>
			<td>Stop watch</td>
			<td></td>
			<td></td>
			<td>Non-Scientific equiptment</td>
		</tr>
		<tr>
			<td>Eluent collection vials</td>
			<td></td>
			<td></td>
			<td>Any Small vial with a flat bottom will do e.g. HPLC vials</td>
		</tr>
		<tr>
			<td>HPLC Vials</td>
			<td></td>
			<td></td>
			<td>Will depend on instrument used</td>
		</tr>
		<tr>
			<td>Vessels for mobile phase and derivatisation solution(s)</td>
			<td>Sigma Aldrich</td>
			<td>Z232211</td>
			<td></td>
		</tr>
		<tr>
			<td>General Laboratory glassware</td>
			<td></td>
			<td></td>
			<td>Volumetric Flasks, pippettes, etc. Quantity and volumes will depend on sample preparation method</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>DPPH</td>
			<td>Sigma Aldrich</td>
			<td>D9132</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma Aldrich</td>
			<td>17836</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia</td>
			<td>Sigma Aldrich</td>
			<td>320145</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescamine</td>
			<td>Sigma Aldrich</td>
			<td>F9015</td>
			<td></td>
		</tr>
		<tr>
			<td>4-aminoantipyrene&#160;</td>
			<td>Acros Organics BVBA</td>
			<td>AC103151000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferricyanide&#160;</td>
			<td>AnalaR</td>
			<td>B10204-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

post column derivatization using reaction flow high performance liquid chromatography columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

كانت الطريقة PCD الأولى التي تم تكييفها للاستخدام من قبل RF-PCD في اشتقاق من المواد المضادة للاكسدة باستخدام 2،2-ثنائي-1-picrylhydrazil الراديكالي (DPPH •) 24. وقدم هذا رد فعل من جانب Koleva وآخرون. 25 واستخدمت على نطاق واسع منذ ذلك الحين. يعتمد الكشف على إزالة اللو?...

Watch this Scientific Journal Video about Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns at JoVE.com

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

آخر العمود اشتقاق عن طريق رد الفعل أعمدة تدفق عالية الأداء اللوني السائل

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

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14.9K Views.  University of Western Sydney. A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

Video: Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

نطاق واسع غير المستهدفة التنميط Metabolomic من المصل بواسطة الترا الطيف الأداء اللوني الكتلة السائل (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

الايض غير مستهدفة من مصادر بيولوجية عن طريق Ultraperformance اللوني السائل عالية الدقة الطيف الكتلي (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

عزل وإعداد جدران الخلايا البكتيرية للتحليل التركيبي بواسطة الترا الأداء الكروماتوغرافيا السائلة

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection.

ضبط عمود مقطع التدفق الموازي وتمكين الكشف المضاعفة

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms ...

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially purpose built multiport end-fitting(s). Curtain Flow (CF) columns belong to the AFT suite of columns, specifically the CF column is designed so that the sample is injected into the radial central region of the bed and a curtain flow of mobile phase surrounding the injection of solute prevents the radial dispersion of the sample to the wall. The column functions as an 'infinite diameter' column. The purpose of the design is to overcome the radial heterogeneity of the column bed, and at the same time maximize the sample load into the radial central region of the column bed, which serves to increase detection sensitivity. The protocol described herein outlines the system and CF column set up and the tuning process for an optimized infinite diameter 'virtual' column.

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

الستار العمود تدفق: تحسين الكفاءة والحساسية

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Radiolabeling والكمي لمستويات الخلايا من Phosphoinositides التي كتبها التلألؤ تدفق عالية الأداء اللوني السائل الديناميكي

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.

A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming.

توليف للبرمجة الرئيسية سلسلة اللدائن السائل البلوري باستخدام مرحلتين ثيول-اكريليت رد الفعل

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline ...

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

ومن الطرق المباشرة لGlucosinolate استخراج وتحليل مع الضغط العالي اللوني السائل (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

طريقة مريحة لاستخراج وتحليل مع اللوني السائل عالي الضغط الكاتيكولامينات العصبية وعلى نواتج الأيض

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

كيمياء تدفق مستمر: رد فعل ديفينيلديازوميثاني مع حمض-نيتروبينزويك ف

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...