Kapiler elektroforez yöntemi ile PVI kaplı kapilerde inorganik anyonların  hassas analizi: Kemometrik deneysel tasarım ile metod optimizasyonu
High sensitivity  analysis of inorganic anions in PVI coated capillary by capillary electrophoresis with sample stacking: Method optimization using chemometric experimental design
Yazar: SİRUN ÖZÇELİK
Danışman: DOÇ. DR. NEVİN ÖZTEKİN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Kimya Eğitimi Bilim Dalı
Derece: Yüksek Lisans, 2013
Konu: Kimya = Chemistry

Özet:
Kapiler elektroforez (CE) hızı, yüksek çözünürlüğü, yüksek verimliliği, otomasyon, düşük elektrolit ve örnek tüketimi gibi özellikleri ile büyük biolojik moleküllerden küçük inorganik iyonlara kadar birçok bileşiği ayırabilen güçlü bir analitik tekniktir. 
Elektroosmotik akış (EOA) elektroforetik ayrımlarda büyük önem taşımaktadır. EOA?ın kontrolü ayrımların optimizasyonunda oldukça önemlidir. 
Küçük inorganik anyonlar EOA?tan daha hızlı ve ters yönlü elektroforetik mobiliteye sahiptiler ve bu durum aşırı göç sürelerine neden olur. Sonuç olarak bu anyonlar normal moda (dedektör katot ucunda iken) dedekte edilemez. Anyon analizleri için daha iyi bir seçenek olarak elektroosmatik akış genellikle bastırılır veya ters çevrilir. Bu durumda daha hızlı bir ayrım sağlarken  bütün anyonların dedeksiyonu da gerçekleşir.
Bu çalışmanın ilk bölümünde,  bir kapiler kaplama işlemi tanımlanmıştır. Kaplama malzemesi olarak polivinil imidazol (PVI) kullanılmıştır. PVI ile kaplanmış kapilerde EOA büyüklüğü ve stabilitesi, farklı tampon çözeltileri, tampon konsantrasyonları ve pH?larda, iyonik şiddet ve organik çözücü eklemeleri gibi şartlar altında incelenmiştir. Geliştirilen kaplama işlemi daha sonra inorganik anyonların analizinde uygulanmıştır. 
Düşük duyarlılık ve dedeksiyon limitleri eser miktardaki anlaitlerin CE?deki analizlerinde ciddi sorunlar oluşturmaktadır. Bu sebeple, duyarlılık arttırmak için örnek sıkıştırma yöntemleri geliştirilmiştir. Örnek sıkıştırma yöntemleri CE?de düşük dedeksiyon limitlerinin aşılmasında oldukça önemli bir yere sahiptir. 
Çalışmanın ikinci bölümünde ise çeşitli inorganik anyonların ayrımı ve tayini için örnek sıkıştırma metodu geliştirilmiştir. Duyarlılık numunelerin elektrokinetik enjeksiyonu ile geliştirilmiştir. Çalışmada, CE koşullarının optimizasyonu için kemometrik deneysel tasarım (fraksiyonel faktöriyel tasarım ve merkezi kompozit dizaynı) kullanılmıştır. Fraksiyonel faktöriyel tasarım (FFD) deneysel faktörleri taramak  ve  önemli olan faktörleri belirlemek için kullanılmıştır. Ardından merkezi kompozit tasarım (CCD) önemli faktörler arasındaki ilişkiyi hesaplamak ve optimum örnek sıkıştırma koşullarının belirlenmesi için kullanılmıştır. Örnek sıkıştırma yönteminin optimizasyonu için yanıt fanksiyonu olarak pik yükseklikleri kullanılmıştır. Optimum koşullar 20 mM Tris tamponu pH 7,5, 89 mM NaCl  ve enjeksiyon süresi 2 dakika olarak bulunmuştur. 
Kalibrasyon eğrileri çalışma aralığı (50-300 ng / mL) içinde doğrusaldır. Dedeksiyon limtleri (LOD) 5,42 ng/mL?ye kadar belirlenmiştir.

Abstract:
Capillary electrophoresis (CE) is a powerful analytical technique capable of separating compounds ranging from large biological molecules to small inorganic ions due to its beneficial features including fast, high resolution, high efficiency, automation, low electrolyte and sample consumption.
Capillary electrophoresis allows simultaneous separation of cations and anions by utilizing a large EOF in a narrow diameter (50-75 µm id) capillary. In capillary electrophoresis, fused-silica is most commonly used capillary material due to its good thermal conductivity, ultraviolet (UV) transparency and uniform inner diameter. The chemical properties of the capillary surface play an important role in the separation process. The electroosmotic flow (EOF) alter with charge of capillary wall. Above pH 2 the silanol groups dissociates; this results in a negative charge of the capillary wall. The higher the pH, the stronger the EOF. EOF also plays a important role in electrophoretic separations. Control of the EOF is of major importance for the optimization of separations.
Small inorganic anions having high mobilities migrate faster than the EOF and in the opposite direction, resulting in excessive migration times. As a consequence they would not be detected in the normal mode (detector at cathode). A better alternative for anion analyses by CE is to suppress or even reverse the direction of the electroosmotic flow. This allows for more rapid separations and ensures that all anions are detected.
To avoid these problems most approaches for controlling EOF focus on altering the charge at the capillary surface. The charge of wall can be changed by modification of capillary by coating. Modification of capillary is performed by modification of the capillary wall by noncovalent and covalent bonding. Modification by noncovalent bonding, such as physical adsorption, has shown some advantages including good reproducible ability, homogeneous chemical derivatization reactions on the capillary inner wall, as well as simplicity and speed of the coating process. Therefore, investigation of good coating reagents and different coating procedures are of great importance for applications in CE.
The first part of this study, we described a capillary coating process. Poly vinyl imidazole (PVI)  as coating material was be used. The magnitude and stability of EOF in capillaries coated with PVI were investigated under different buffers, buffer
concentration and pH, ionic strength and organic solvent additives. The developed coating process was then applied to analysis of inorganic anions.
Due to the tertiary amine groups, PVI is a weak cationic polyelectrolyte and its positive charge density depends on pH and also on the concentration and the electrolyte type. The average pK for the protonation of the amino group is 5. 
A very simple coating procedure  was used by flushing with PVI solution for more than 5 min and and the PVI solution left in the capillary for 10 min. Finally,  the capillary was rinsed with  water for 5 min, then with running buffer for 5 min. 
Conventional EOF mobility is mainly measured using a neutral marker: A small amount of the marker is injected into the capillary and its migration time on voltage application is measured.
where EOF is the EOF mobility, Ld is the length of the capillary to the detector, Lt is
the total capillary length, t is the migration time, and V is the applied voltage.
However, in the case of low mobilities this method is unsuitable (enormously high run times, background electrolyte depletion, overheating, peak dispersion, etc.). A more useful method for determination of low EOF mobility has been introduced by Williams and Vigh [1]. This method based on the formation of three bands of a neutral marker in set order in the capillary, and measurement of migration times: The first zone of a marker is injected and forced by pressure for a certain time into the capillary; then the second zone is injected and pushed to an equal extent. After that a voltage is applied to move both zones by EOF. In the last step, the third zone of the marker is injected and it acts as a reference for EOF mobility calculation.
ere t1, t2, t3 are the migration times of zones 1, 2, and 3, tinj is the time of zone injection, Ld is the length to the detector, Lt is the total length of the capillary, V is the applied voltage, tmig is the time of voltage application; and trampup and trampdown are the times required for a linear change of voltage from 0 to V.
Coating solution was obtained  by dissolving 0.5 %  in 0.1 M NaCl. It is expected that the magnitude of EOF is dependent on the concentration of PVI in a coating solution. The EOF decreased significantly with an increase in the concentration of PVI. The mobility of the EOF was almost constant at a concentration exceeding 0.02%. The EOF mobility was suppressed to ~10-fold smaller than that in the bare capillary. The result showed that PVI suppresses the EOF effectively by dynamic coating. 

The effect of pH on the EOF was investigated at different pH values in different background electrolytes (acetate, citrate, phosphate and tris).  No significant increase in the EOF was observed in the pH range of 3.0?9.0. 

Influence of different electrolytes (NaCl, NaNO3, NaClO4 and NaSO4) in running buffer was investigated. Any  change of EOF with the increase of running buffer concentration was not observed .
The PVI-coated capillary showed a long term stability up the 40 runs. Coating had good tolerance to o.1 M HCl, 0.1 M NaOH and some organic solvents. The run-to-run and day-to-day RSDs were between 0.5- 1.02%. The  PVI-coated capillary
The second part of this work, an on-line stacking CE method for detection of inorganic anions was  developed. The  PVI-coated capillary was successfully applied to analysis inorganic anions.
The poor sensitivity and detection limits in terms of concentration are among the most serious drawbacks of CE methods for the analysis of trace analytes. In last decade, significant advances have been accomplished for solving this weakness. Thereby, stacking methods have been developed to fulfill the sensitivity requirements. Stacking methods are very important in overcoming the poor detection limits in capillary electrophoresis (CE). In this study, the separation and determination of several inorganic anions by a stacking method was described. The possibility of sensitivity enhancement by using electrokinetic injection of the sample was also evaluated. 
Chemometric experimental design (fractional factorial design and central composite design) was used for optimisation of stacking conditions. Experimental design methods were utilized to make the developing process more efficient and cost-effective due to the many factors involved in the optimization of this analytical method. Fractional factorial design (FFD) was used to screen the experimental factors and for identifying the most significant factors. Then the central composite design (CCD) is used to estimate the relationship between all factors and find the optimal stacking conditions. 
The response function used the peak heights for the optimization of the stacking conditions. The optimum conditions were achieved using 89 mM NaCl at 20 mM TRIS buffer pH 7.5, and  injection time of 2 min. Under these conditions the analysis time of inorganic anions is 6.5 min.  
The repeatability for the inorganic anions with PVI-coated capillary was investigated under the optimal conditions.  The RSD values (n=7) for migration times of inorganic anions was  below 1 %.
The calibration curves were linear between 70?300 ng/mL of inorganic anions. Correlation coefficients were all 0,980 or better. The limits of detection (LODs) were 7.81 nM for NO2-  and 5.42 nM NO3- .