Analysis of riboflavin content in a vitamin pill by fluorescence spectroscopy

A       IDENTITAS

          Hari/Tanggal    : Selasa /17Juli 2012

          Waktu               : 09.00-12.00

B       KEGIATAN                                        

         Analysis of riboflavin content in a vitamin pill by fluorescence spectroscopy

C       RINGKASAN KEGIATAN                      

Riboflavin merupakan salah satu koenzim yang berperan dalam berbagai metabolisme energi di dalam tubuh, terutama dalam pemecahan senyawa karbohidrat menjadi gula sederhana. Senyawa kompleks lainnya, seperti lemak dan protein, juga dapat dikonversi menjadi energi. Beberapa metabolisme vitamin lain dan mineral juga membutuhkan peranan vitamin ini. Selain itu, vitamin ini berperan dalam respirasi jaringan tubuh, pertumbuhan badan, dan produksi sel darah merah.

Defisiensi ini dapat terlihat dari warna mata yang cenderung merah, peningkatan sensitifitas terhadap cahaya matahari, peradangan di mulut, dan bibir pecah-pecah. Efek lainnya juga terlihat pada kerusakan jaringan kulit, keriput, dan kuku pecah. Gejala awal defisiensi adalah sakit tenggorokan dan bibir pecah-pecah. Bila telah parah, penderita akan mengalami anemia, gangguan saraf, pembengkakan lidah. Defisiensi vitamin B2 ini sering dialami oleh para pecandu alkohol.

Salah satu cara untuk menganalisis kandungan B2 dalam suatu pill adalah dengan flurescence spectroscopy. Fluoresensi spektroskopi atau fluorometry atau spektrofluorometri, adalah jenis spektroskopi elektromagnetik yang menganalisis fluoresensi dari sampel. Ini melibatkan penggunaan sinar cahaya, biasanya sinar ultraviolet , yang menstimulus elektron dalam molekul senyawa tertentu memancarkan cahaya cahaya tampak namun tidak selalu demikian. Perangkat yang mengukur fluoresensi disebut fluorometers atau fluorimeters. flurescence spectroscopy banyak digunakan dalam analisis biomedikal karena lebih selektive dan lebih sensitive.

Fluorescence. If a molecule is irradiated with light whose energy corresponds to the energy difference between its ground electronic state and one of its excited electronic states, absorption of a photon can occur and a rearrangement of the molecule's electronic distribution occurs as it reaches the excited state. For example, an electron may be excited from a p molecular orbital to a p* molecular orbital. Because light absorption only involves the movement of electrons (no nuclear rearrangement) and electrons are very light (approx. 1/2000^th of the mass of a proton), light absorption is a very fast process. It normally occurs on the time range of femto-seconds (10^-15 s). Once excitation of the molecule has occurred by the absorption of the energy of a photon, the molecule can subsequently return to the ground state by losing its energy again in two possible ways:

a) by transferring heat to the surroundings (radiationless transition), or

b) by the emission of light (fluorescence or phosphorescence).                         

For the majority of molecules radiationless transition is the predominant mechanism of deexcitation, and such molecules are termed non-fluorescent. For certain molecules, however, particularly when they have rigid structures and are unable to undergo any great degree of vibrational motion, fluorescence can become the major form of de-excitation. The fluorescence quantum yield (i.e., no. of photons emitted/no. of photons absorbed) can then approach unity.

The concentration of the analyte of interest is the sum of the concentrations due to the unknown and the standard additions to each sample, and can be calculated as follows:

[analyte ]= Cunk ⋅Vunk    + Cstd ⋅Vstd

                               Vt                          Vt                                                                                                   (1)

where Cunk and Vunk are the unknown analyte concentration and volume, Cstd and Vstd are the standard analyte concentration and volume, and Vt is the total volume of the sample. The standard addition method will allow us to make measurements of standards in a solution having the same matrix composition as the unknown sample. This method works for any analytical signal (i.e., measured quantity or function of a measured quantity) that is linearly proportional to the concentration of the analyte of interest. The fluorescence intensity (F) of an optically dilute (Absorbance<0.05) fluorescent sample is

F = 2.3K ¹¹φ Poεbc = Kc                                                         (2)

where K” is an instrument constant, φ is the fluorescence quantum yield, P0 is the incident excitation power, ε is the molar extinction coefficient, and c is the analyte concentration. Because all of these factors are constant for a particular sample and a particular set of instrumental conditions, they can be condensed into a single constant, K. Thus, the standard addition method can be applied to fluorescence spectrometry. Substituting equation 1 into equation 2.