1. Регулирование рН элюэнта.
Помимо вышеописанных буферов для урегулирования pH элюента применяют такие кислоты, как муравьиная или уксусная, а так же гидрат аммиака и пиридин. То, какие вы выберете в дальнейшем кислоты, основания и тип буфера, напрямую зависит от конкретного случая. В таблице No3 представлены некоторые из них:
*Примечание. рН элюента должен соответствовать используемой неподвижной фазе, чтобы избежать повреждения или изменения свойств (например, для сорбентов на основе оксида кремния рН элюента должен находиться в пределах 2-7,5).
Систему высокоэффективного жидкостного хроматографа очень важно прочищать органическим элементом. Это делается для того, чтобы не допустить микробиологического загрязнения. Рекомендуется проводить очистку изопропанолом или метанолом. Частота таких дезинфицирующих мероприятий в среднем составляет 2-4 недель.
Даже самые небольшие частички собираются на фильтре и на входе из насоса, тем самым загрязняя его. Эти мельчайшие соединения как будто бы невидимы при UV-детектировании, однако с большей вероятностью могут быть обнаружены благодаря масс-спектрометрии. Именно поэтому рекомендуется менять фильтр раз в 1-2 месяца или после того, как был произведен переход от ACN к метанолу (или наоборот). Различные поставщики и компании поставляют на рынок разнообразные фильтрующие фритты, основной задачей которых является защита LC системы от частиц, с помощью присоединения к жидкостным магистралям. Специалисты рекомендуют использовать фритты из нержавеющей стали или PTFE, главное не брать стеклянные, так как со стекла сложно очистить остатки буферов.
2. Промывка колонки.
Заметим, что в LC разделение проходит в нормальнофазовом режиме, это приводит к тому, что неподвижная фаза каждой HPLC колонки включает в себя ковалентно связанные органические группы, которые придают ей конкретные физические свойства. То, как привитые группы будут раскрываться во время хромотографического разделения и смогут ли они стать причиной как мелких, так и более серьезных мешающих сигналов, зависит от качества модификации неподвижной фазы и процедур промывки. Такое явление может привести к потере чувствительности в MS, однако этого можно избежать, если перед анализом обработать колонки изопропанолом с 0.1 % муравьиной кислоты, а скорость потока должна составлять половину от предписанной для данной колонки в ходе одного часа. Такая процедура помогает исключить несвязанные или плохо связанные группы, свести к минимуму шумы базовой линии и увеличить чувствительность (Рисунок 3).
3. Качество подвижной фазы.
Стоит обратить внимание на рисунки 4-5, которые показывают на то, как воздействует степень очистки ацетонитрила LiChrosolv на насыщенность шумов базовой линии в масс-спектрах. Одни из зарекомендовавших себя растворителей марки Мерк Миллипор “hypergrade for LC-MS LiChrosolv”, необходимы для использования ссистемами масс-спектрометрии, они минимизируют пики примесей, удерживают процесс ионизации, создают аддуты и шумы базовой линии, тем самым увеличивают чувствительность. Кроме этого, растворители с градиентными свойствами, такие как “gradient grade for liquid chromatography LiChrosolv” используются для векторного разделения в LC-UV, а растворители изократического характера подходят для изократического режима.
Для того, чтобы провести процедуру сравнения свойств растворителей, рекомендуется добавить определенный стандарт в органические растворители и вводить их в MS с помощью шприцевого насоса. Как показывает практика, резерпин – одно из практичных веществ в роли определенного стандарта.
После завершения процесса сравнения, сигнал, полученный от резерпина, сравнивают с уровнем фонового шума (отношение сигнал-шум). И при отрицательном, и при положительном исходе электрораспылительной ионизации, получается высокий показатель сигнал-шум (= низкое подавление сигнала) при использовании качественного ACN hypergrade . (Рисунок 6).
Вода для применения в LC-MS, должна быть в первую очередь очищенная, т.е. можно использовать или бутилированную, или отфильтрованную жидкость. Специалисты не рекомендуют использовать обычную проточную воду, так как это часто приводит к загрязнению всей системы.
Свойства LiChrosolv®, специальной воды для хроматографии, и наилучшей, чистой воды Милли-Q®, неизменно высокая и не обуславливается регулярностью ее использования. Немаловажно и то, как тщательно и аккуратно хранится жидкость, так как необходимо исключить возможное засорение и распространение числа микроорганизмов.
На качество воды отрицательно влияет множество факторов, например грязь в лаборатории, асихронные рабочие процедуры, халатность по отношению к оборудованию и неверное использование систем очистки воды (СОВ), все это приводит к росту фоновых шумов MS. На рисунке 7 представлено MS спектры и полный ионный ток (TIC) для воды очищенной системой Milli-Q
Образец №1 был взят из СОВ в начале недели, после того, как приостановили саму систему в течение выходных. Образец №2 был отобран в тот же день, как и первый, но после слива нескольких литров до того, как набрали сверхчистой воды. Обратите внимание, что базовая линия масс-спектрометра с шумами принадлежит для первого образца, притом, что ощутимое уменьшение фонового шума MS было получено после очистки системы. Советуем чистить систему ежедневно по утрам, так это поможет избежать микрозагрязнений на выходе из СОВ.
Если происходит загрязнение в некоторых видах добавок и растворителях, то это приводит к тому, что грязь начинает скапливаться на сорбенте в зависимости от хроматографических условий.
На рисунке 8 изображено скопления пластификаторов, которые были растворены в элюенте, на колонке с обращенной фазой после уравновешивания в течение 0, 15 и 60 минут.
- Хранение растворителей.
- Очистка оборудования.
- Буферы.
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