Vitamins, absorption analysis

Many pharmaceutical compounds contain chromophores that make them suitable for analysis by UV/Vis absorption. Products that have been analyzed in this fashion include antibiotics, hormones, vitamins, and analgesics. One example of the use of UV absorption is in determining the purity of aspirin tablets, for which the active ingredient is acetylsalicylic acid. Salicylic acid, which is produced by the hydrolysis of acetylsalicylic acid, is an undesirable impurity in aspirin tablets, and should not be present at more than 0.01% w/w. Samples can be screened for unacceptable levels of salicylic acid by monitoring the absorbance at a wavelength of... 

Fluorimetric methods of analysis make use of the natural fluorescence of the analyte, the formation of a fluorescent derivative or the quenching of the fluorescence of a suitable compound by the analyte. Fluorescence cannot occur unless there is light absorption, so that all fluorescent molecules absorb, but the reverse is not true only a small fraction of all absorbing compounds exhibits fluorescence. The types of molecule most likely to show useful fluorescence are those with delocalised ji-orbital systems. Often, the more rigid the molecule the stronger the fluorescence intensity. Naturally fluorescent compounds include Vitamin A, E (tocopherol). 

Vitamin D2 and D3 exhibit identical UV absorption spectra and they do not possess fluorescence. Electrochemical detection is limited and only few methods are applied in food analysis [530,533], MS detection has been applied achieving satisfactory detection limit (10 mol/mL) [534,535],... 

The technique of absorption-luminescence spectroscopy has been recently applied for the analysis of vitamin Bg and related compounds (137—... 

Figure 1 is the ultraviolet spectrum of a 10 mcg/ml solution of vitamin D3 in methanol. The spectrum was obtained using a Cary Model 219 recording spectrophotometer (Varian Instrument Co., Palo Alto, CA). Vitamin D3 and related compounds have a characteristic UV absorption maximum at 265 nm and a minimum at 228 nm. The extinction coefficient at 265 nm is about 17,500 and 15,000 at 254 nm. An index of purity of vitamin D3 is a value of 1.8 for the ratio of the absorbance at 265 to that at 228 nm. The high absorbance at 254 nm enables one to use the most common and sensitive spectrophotometric detector used in high performance liquid chromatography (HPLC) for the analysis of vitamin D3 in multivitamin preparations, fortified milk, other food products, animal feed additives etc. 

In a previous study we have found that, at low temperature, PS-I electron transfer is largely blocked away from A, and that the state (P-700+, A, ) decays with a half-time of 130us. Analysis of the absorption spectrum of that state showed that A, is presumably a quinone radical anion (Brettel et al, 1986). Chemical analysis, following separation by HPLC, has shown that phylloquinone (a naphthoquinone also named vitamin Kj) is the only quinone present in PS-I. We have found 2 moles of phylloquinone per PS-I. Extraction with dry hexane does not change the electron transfer reactions this treatment only extracts only one phylloquinone per PS-I (Biggins and Mathis, 1987). 

We have used stable Isotopes in several other zinc absorption studies. Sample analysis Is In progress from a study In which Zn was used to determine the effects of phtate and cellulose on zinc absorption In man. Studies were also conducted to determine zinc absorption from vegetarian diets, the effect of vitamin B-6 status on zinc absorption, and the effect of level of dietary zinc on zinc and copper absorption. 

A knowledge of molar absorptivities is particularly important in biochemistrs, where UV spectroscopy can provide an extremely sensitive method of analysis. For example, imagine that you wanted to determine the concentration of vitamin A in a sample. If pure vitamin A has A ax (e - 50,100), what is the vitamin A concentration in a sample whose absorbance at 325 nm is A = 0.735 in a cell with a pathlength of 1.00 cm ... 

The absorption spectrum of interferants is commonly linear, but nonlinear interferant absorption has been reported. A number of mathematical techniques have been developed to correct for nonlinear interfering absorption. Most of the correction techniques are based on assuming that the interferents have an absorption profile that can be represented by some mathematical function. The simpler correction techniques, such as the geometric correction technique(s), assume a linear interferant absorption profile. A basic approach to the technique can be seen from the three-point geometric correction technique, a modification of which has found applicability to the analysis of vitamin A in fish oils. Higher-order functions have also been used to describe the interfering absorption, and in these cases more involved formulas have been developed. 

J.A. Sweileh, On-line flow injection solid sample introduction digestion and analysis spectrophotometric and atomic absorption determination of iron, copper and zinc in multi-vitamin tablets, Microchem. J. 65 (2000) 87. 

The problem of detection of the separated species in HPLC experiments requires a physical property specific to vitamin C (preferably both for L-ascorbic acid and its oxidation products). At the time of writing the most commonly used detectors operate by the measurement of the absorption of u.v. or visible radiation. These allow the detection of nanogram quantities of L-ascorbic acid, but they are much less sensitive to dehydroascorbic acid because of its much lower molar absorbtivity (see above). It remains to be seen whether other detectors such as those which are mass sensitive will produce more effective methods of analysis. 

UV/VIS spectrophotometry is a widely used spectroscopic technique. It has found use everywhere in the world for research, clinical analysis, industrial analysis, environmental analysis, and many other applications. Some typical applications of UV absorption spectroscopy include the determination of (1) the concentrations of phenol, nonionic surfactants, sulfate, sulfide, phosphates, fluoride, nitrate, a variety of metal ions, and other chemicals in drinking water in environmental testing (2) natural products, such as steroids or chlorophyll (3) dyesmff materials and (4) vitamins, proteins, DNA, and enzymes in biochemistry. 

Morton, R. A., Stubbs A.L., (1946). Photoelectric Spectrophotometry Applied to the Analysis of Mixtures and Vitamin A Oils, Vol.Tl, pp. 348-350 Morton, R.A., Stubbs, A.L. (1947). A Re-examination of Halibut-liver Oil. Relation Between Biological Potency and Ultraviolet Absorption Due to Vitamin A, Biochem. /., Vol.41, pp. 525-529... 

Stubbs et al. (1962) have devised a simple infrared method for the analysis of Dicumarol, 3,3 -methylene bis (4-hydroxycoumarin), in blood. The method is sensitive to concentrations as low as 0.1 mg % (1 ppm). The Dicumarol in acidified blood serum is extracted with carbon tetrachloride, and the solvent layer, containing the Dicumarol, is scanned in a 10-mm infrared microcell. The absorption at 1667 cm is measured and the concentration of the drug calculated. Compounds known to interfere with the determination of the drug in blood by other methods (e.g., vitamin K, salicylic acid, aspirin, warfarin, and others) do not interfere with the infrared method. 

Three levels of calcium in the diet were next used —low, medium, and high—the diet being free of phosphate. A very considerable difference in Ca absorbed was observed. The vitamin D rats absorbed distinctly more Ca than the vitamin D-free rats at all three levels. It was calculated that deficient absorption accounted for 80 to 90%of the Ca now found in the feces from vitamin D-deficient rats and that only 10 to 20% was due to the extra loss of endogenous Ca. These data were not submitted to statistical analysis, however. 

In order to eliminate irrelevant absorption, Lambertsen and Braekkan (1959) used a mathematical procedure such as is customary in analysis of vitamin A (Morton-Stubbs correction). In this procedure, extinction is measured at three different wavelengths and it is assumed that irrelevant absorption is linear in this region. The authors used 280, 292 (absorption maximum), and. 301 mix as fixation points and obtained as corrected extinction-. Ecorr. = 2.778 Em (l- 352 Em -t- 1.626 Em)-... 

Electrochemical methods of analysis are extremely sensitive and have been exploited to permit the detection of a wide range of analytical targets down to concentrations of the order 10 M in favorable conditions. The relative low cost of these electroanalytical techniques when compared with conventional techniques such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and Atomic Absorption Spectroscopy (AAS) has led to the use of electrochemical stripping voltammetry (Chapter 2.3) and linear sweep voltammetry (Chapter 2.1) for the detection of both inorganic and organic species [1-6]. Target analytes that have been documented include heavy metals (Bi, Cu, Cd, Ga, Mn, Pb, Sb, Sn, V, Zn), cardiac and anticancer drugs, vitamins, and pesticides. However, the limits of applicability for these silent classical electrochemical techniques have been compromised by four main drawbacks ... 

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