Thin Layer Chromatography (TLC) is an important and very economical separation method with a wide variety of applications. It is a powerful tool for studying large families of biomolecules, especially when portability, high throughput and low cost are required. TLC is attractive due to its simplicity, low cost and convenience of operation. For polypeptides and steroid hormones previous methods give a few femtomole/sample limits of detection(LOD). TLC/MPD permits sub-attomole per sample sensitivity and excelent linearity.

We performed TLC separation and MPD quantitation of a mixture of sex steroids (testosterone and estradiol) with attached ¹²⁵I. Urine spiked with these radioiodinated steroids was gradually diluted, separated and quantitated using TLC/MPD until the limits of detection were achieved. Good signal to noise ratios can be seen for samples containing a few attomoles/sample. The limit of detection was about 1 attomole/sample. Equally impressive limits of detection have also been shown for a radio-iodinated polypeptide - porcine insulin. A mixture of sex steroids with an attached I¹²⁵ labeled histidine moiety [testosterone-3-(O-carboxymethyl)oximino-(2-[¹²⁵I] iodohistamine and oestradiol-6-(O-carboxymethyl)oximino-(2-[¹²⁵I] iodohistamine] was submitted to TLC separation. We used reverse phase TLC glass plates with silica (Merck, RP-8 F254). The solvent system used was acetonitrile and water (2:1) and separation was performed at room temperature. To measure the sensitivity of TLC/MPD, a mixture of two steroids was dissolved in methanol/water (9:1). A 10 microliter sample of this solution (about 150 fg/sample) was placed on a silica plate and inserted into the TLC tank. After development the TLC plates were dried and measured with the MPD Imager. All peaks were resolved with an excellent signal-to-noise ratio. We also studied 5, 25 and 125-fold dilutions of the above mixture of steroids, i.e. the samples were 30, 6 and 1.2 femtogram/sample, respectively. All were easily detectable (see Figure 1). We documented the excellent linearity (see Figure 2). Quantitation by the MPD Imager is very reliable and precise. In over 100 measurements we proved that the error of quantitation of ¹²⁵I at the 10 pCi level is below 1 percent. In TLC/MPD a potential source of error is during the deposition of the studied material on the TLC plate. We performed a series of measurements, wherein about 40 attomole of spiked steroid was deposited on the TLC plate either manually or using a semi-automatic instrument. A precision of a few percent has been achieved, see Figure 3. Actually, at very low amounts of material, the reproducibility of TLC/MPD is slightly better than that of HPLC/MPD.
For development of a TLC assay for insulin we used porcine insulin pre-labeled at the tyrosine moiety as [3-[¹²⁵I] iodotyrosine insulin. Reverse phase TLC was performed with C8 silica plates (MERCK, RP-8 F254) and mobile phase consisting of acetonitrile : water : TFA (50:25:1.5). Ten microliters of radio-labeled insulin dissolved in the mobile phase was spotted onto TLC plates and separated by performing all chromatography in a temperature controlled oven at 27 C. Following separation, the TLC plates were dried and measured with the MPD Imager. Concentrations of insulin from 700 to 5 attomoles per sample demonstrated excellent linearity (R= 0.99).