Sampling, Separation, and Resolution of Amino Acids in Tears by Phenol Red Thread and CE-LIF.

Collection, sample handling, and analysis of amino acids in nano-to-microliter volume tears is highly important for prospective examination of eye health condition. This thesis presents a new method for tear sampling by phenol red thread (PRT) and an optimized method developed for amino acids separation and analysis by capillary electrophoresis (CE). Capillary electrophoresis is a fast and efficient method for separation of low volume samples, which makes it ideal for tear studies. PRT is a fast, minimally invasive, and a low volume collection tool for subsequent chemical analysis. Volume scale of tear collection with PRT can be as low as 100 nL, and as fast as 15 seconds. Thread is flexible and does not cause any irritation nor discomfort. Derivatization of amino acids can be performed directly on the thread making it not only a low volume collection tool, but also a support for chemical reactions. Capillary electrophoresis with laser induced fluorescence (CE-LIF), is a simple separation and analysis technique that provides efficient separation with low detection limits. The use of various chromatographic conditions and buffer additives allows CE to separate a broad spectrum of analytes. With dimethyl sulfoxide (DMSO) as an additive to a CE buffer and dilution of a chemically complex matrix, more amino acids were resolved in a tear sample for quantitation analysis. The ability to customize fused-silica CE using polydimethylsiloxane (PDMS) connections can result in a multidimensional separation technique to enhance separation and resolution. Transfer from one dimension to another was performed by an online heart-cut method, which allows for the selection of a region of interest for separation in a second dimension. A micro-cross junction interdimensional device, with a 50 µm i.d. channel, creates an efficient heart-cut without resulting in prolonged migration times or peak broadening. These projects were designed to enhance the collection of tears and for subsequent analysis of amino acids by CE-LIF system. The PRT collection method collects <1 microliter in volume, requires fifteen seconds to perform, and is minimally invasive. Thus, it is promising for tear chemical analysis in order to create a database of tear chemical content. Separation optimization developments with DMSO addition to CE-LIF buffer can allow resolution of co-migrating analytes in various complex biological samples. PDMS micro-cross junction device can create an efficient online heart-cut for 2D CE separation of complex samples.