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Yüksek basınçlı sıvı kromatografisi (HPLC), yüksek bir analitik çözünürlüğe sahip bir sıvı çözücü içinde çözülmüş bileşenleri ayırır, tanımlar ve miktarını belirler.

HPLC işlemi süreklidir ve kapalı bir devrede çalışır. Pompa, enjeksiyon sistemi, ayırma kolonu ve dedektör olmak üzere dört ana bileşenden oluşur.

Bu teknik aşağıdakilerin kullanımını gerektirir:

• Sabit faz - paslanmaz çelik kolonlara (iç çapı 2-5 mm arasında olan), genellikle silikadan yapılmış küresel katı gözenekli parçacıklar (3-10 um ile).
• Hareketli faz - sıvı çözücü.

HPLC mekanizması aşağıdaki adımlardan geçer:

1. Hareketli faz, partiküllerin ve mikroorganizmaların yokluğunu garanti etmek için filtreli bir kanal aracılığıyla sisteme sokulur. Pompalar içinden pompalanır, sürekli olarak yüksek basınç altında ve kontrollü bir akışla kolondan geçirilir.
2. Enjektör, bir numune hacmini (uL) ekstrakte ederek ve onu sürekli olarak kolondan geçen mobil faza sokarak başlar.
3. Numune (hareketli faz ile) sabit fazın mikroküreleri arasında bulunan gözeneklerden geçer. Bu işlem belirli bir basınçla gerçekleşir. Numunenin bileşenlerini, mobil faz ile sabit faz arasında bölünebilme yeteneklerine göre ayırır.
4. Kolondan ayrıldıktan sonra bileşikler saptanır ve tanımlanır.
5. Bu bilgiyi bir kromatograma aktaracak olan dedektör vasıtasıyla nicelendirilirler.




Bileşenlerin HPLC'deki alıkoyma süresi, sabit faz ve mobil faz ile etkileşimi ile belirlenir. Örnek malzemenin her bir bileşeni farklı bir afiniteye ve moleküler ağırlığa sahiptir, bu da kolonun farklı zamanlarda ayrılmasına ve ayrılmasına neden olur.

Bu yöntem, mikropartiküllerle kolonların kullanılması nedeniyle yüksek basınçlar ve bileşiklerin ayrılmasında yüksek verim kullanarak analizde daha yüksek verimlilik sunar. Bu nedenle, farklı amaçlar için yaygın olarak kullanılmaktadır:
‣ üretim - farmasötik ve biyolojik ürünlerin üretim sürecinde;
‣ yasal konular - idrarda performans arttırıcı ilaçların saptanması;
‣ araştırma - karmaşık bir biyolojik numunenin veya benzer sentetik kimyasalların bileşenlerinin birbirinden ayrılması;
‣ tıbbi sorunlar - kan serumundaki D vitamini seviyelerini tespit etmek.



REFERANSLAR
1. Kamiya biomedical company. Cellular Antioxidant Activity Assay. Rev. 13470931.
2. WATERS- HPLC: High Performance Liquid Chromatography. Available at: https://www.waters.com/waters/en_US/HPLC---High-Performance-Liquid-Chromatography/nav.htm?cid=10048919&locale=en_US. Accessed on: 25/03/2020.
3. Gerber, F.; Krummen, M.; Potgeter, H.; Roth, A.; Siffrin, C.; Spoendlin, C. (2004). "Practical aspects of fast reversed-phase high-performance liquid chromatography using 3μm particle packed columns and monolithic columns in pharmaceutical development and production working under current good manufacturing practice". Journal of Chromatography A. 1036 (2): 127–133.
4. JICKELLS, S.; NEGRUSZ, A. – Clark’s Analytical Forensic Toxicology. Cambridge: Pharmaceutical Press (2008);
5. SKOOG, Douglas A; HOLLER, F. James; Nieman, Timothy A. Principles of instrumental analysis. Brooks Cole; 5th edition (September 3, 1997).
6. David Harvey. Chromatographic and Electrophoretic Methods. Analytical Chemistry 2.0. 2016.







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