Authentic reference spectra

National and international compendial [pharmacopoeial] authorities publish authenticated reference spectra. Authentic infrared IR reference spectra are required for proof of identity of official substances by direct comparison with observed IR spectra obtained with the test substance examined in a prescribed manner. 

The advantages of issuing authentic Reference Spectra include ... 

The availability of these data collections is of considerable value, permitting the identification of organic acid spectra by comparison with reference spectra in the collection. As variations in ion intensities can arise when spectra are acquired on different instruments with differing ion source conditions and inlet systems, many laboratories prefer to obtain authentic reference spectra on their own instruments for matching purposes. Such personalized collections are usually limited and do not have the benefit of input of less commonly encountered acid spectra from contributors working in related but different areas. 

Authentic IR reference spectra covering the range 2000-500 cm are published in conjunction with monographs for substances of the British Pharmacopoeia (BPC, London). They are also supplied by the European Pharmacopoeia EDQM, Strasbourg) for a more limited number of official substances, where the cost, or toxicity, of a suitable chemical Reference Substance, or restrictions on its supply by post, favour identification by comparison with an official Reference Spectrum. The International Pharmacopoeia (WHO Collaborating Centre for International CRS, Stockholm) will supply on request paper or electronic copies of an authentic Reference Spectrum covering the full mid-IR range, i.e. 4000-600 cm . ... 

Obtain the IR spectrum of caffeine (use four scans with FT instruments). Compare your data with an authentic spectrum of caffeine and with the data given in Experiment [1 IB]. Your sample may be saved by taping it to a file card with your name, and stored by your instructor in a desiccator. If, later in the year, you isolate caffeine from its natural source (Experiment [IIB]), you will be able to compare the material you have extracted and purified from the plant with your own authentic reference spectrum. 

These NMR spectra are used like a fingerprint to identify a chemical by comparison with its reference spectrum recorded from the authentic chemical under comparable conditions (6). In proficiency tests, NMR is a very useful complementary technique to MS and IR. The limitation is that NMR data is only available in the OCAD as hard copy (PDF). This means that a searchable electronic database that can be used as a library in an instrument is not available. 

Other reference substances are available and can be used as well. Sometimes the resonance of the actual solvent can serve as a reference. Preferably, the referencing method used for the sample should be the same as that used for the authentic reference sample (or library spectrum), or at least the scale... 

NMR spectral parameters, that is, chemical shift (8) and coupling constant (J), may be considerably affected by the sample condition, that is, solvent, pH, sample temperature, concentration, and choice of internal and/or external chemical shift references. Solvent and pH (in water/D20 samples) have the greatest effect. The sample condition should therefore be the same as or comparable to that used for the authentic reference chemical (or library spectrum) and the blank sample. 

Another example of pigment identification by IR microspectroscopy is shown in Figure 10. The bottom spectrum was obtained from a blue pigment from MS 972 (Archaic Mark) the top spectrum is a reference spectrum of Prussian blue. The band corresponding to the C=N of ferric ferrocyanide is common to both spectra. Replicate spectra of blue pigments removed from different locations in MS 972 indicate that the average frequency of this band is 2083 6 cm"1. The ubiquitousness of an iron blue in this manuscript raises doubts about the authenticity of this manuscript. 

Mass spectrum and chromatographic retention index match those of an authentic reference compound. 

Analysis of the authentic reference sample of MDB aldehyde gave a single peak at approximately the same retention time as component D (Fig. 6.9) and an identical mass spectrum (Fig. 6.10), confirming this assignment. 

Analysis of the authentic reference sample of MDB alcohol gave a single chromatographic peak (Fig. 6.15) with a characteristic spectrum (Fig. G. 16). Inspection of the data from the photo ytically degraded PBO showed a minor peak at similar retention time with an identical spectrum (Fig. 6.16), confirming the presence of MDB alcohol in the photolytically degraded PBO. Inspection of the data from the doubly derivatized sample showed a compound of relative molecular mass 266, the spectrum of which is consistent with the TMS (trimethyl-silyi) ether of MDB alcohol. This component (MDB alcohol) will be referred to as component I its structure is shown below ... 

Analysis of a partially derivatized sample of the authentic reference sample of MDB acid gave a peak doublet (Fig, 6.17). The second, sharper, peak is the TMS ester of the acid, The spectrum of the authentic MDB acid is shown in Fig. 6 18 Inspection of the data from the photolytically degraded PBO showed... 

Identification most often relies upon comparison of the GC or HPLC retention time together with the mass spectrum, or of the NMR spectrum with those of authentic reference compounds. Use of GC or LC retention times alone is not adequate, although for GC this may be an acceptable compromise procedure if several derivatives are available. Use of only the mass spectrum is clearly unacceptable since isomers generally provide essentially identical spectra. Naturally, compounds whose structures are unknown may be encountered this presents a much greater challenge since comparison is not then possible. Determination of the structure of such compounds must therefore rely upon determination of the molecular mass by high-resolution... 

The identity of naphazoline hydrochloride may be determined by comparison of its infirated spectrum (KBr) (see Hguie 2) to an authentic reference standard . ... 

When a UV detector works at a fixed wavelength, the obtained information is still very limited. In this case, to identify a compound using different experimental conditions, cochromatography is necessary with authentic reference compounds. DADs are much more useful, because they yield the full record of the UV-Vis spectrum of each compound. Because each class of phenolic compounds has a characteristic spectrum, identification is facilitated, although a safe characterization still requires a standard to compare retention time and UV spectra (see Fig. 2 for an example). 

MS, identification by comparison with mass spectrum stored in the NIST library RT, identification by comparison with retention time of authentic reference compounds KI, agreement with literature data. 

Figure 3.30. (a) UV-vis absorption spectra of the HPAA product (solid line) and the HPDP substrate (dash line) in a H20/MeCN (1 1) mixed solvent, (b) Picosecond time-resolved resonance Raman (ps-TR ) spectra of HPDP obtained with a 267 nm pump and 200 nm prohe wavelengths in a HjO/MeCN (1 1) mixed solvent. Resonance Raman spectrum of an authentic sample of HPAA recorded with 200 nm excitation is displayed at the top. (Reprinted with permission from reference [49]. Copyright (2006) American Chemical Society.)... 

Final identification may be possible by comparison with an authentic spectrum of cyclopropanecarboxylic acid, if it is available in one of the several standard compendia of infrared spectra. A total of about 150,000 infrared spectra are available for comparison purposes. You should check with the reference section of your library to see what atlases of spectral data are available to you. 

FIGURE 21. Mss spectrum of reduction derivation of authentic arsenic and arsenic obtained from TLC development of urine and blood extract left, corresponds to compound C (DMAA) on the TLC plate right, corresponds to compound B (DSMA) on the TLC plate upper, authentic compounds middle, urine of the second day lower, blood of the fifth day. Reprinted with permission from Reference 178. Copyright (1978) American Chemical Society... 

Figu re 12.15 Noninvasive Raman spectra of paracetamol tablets measured through a white, diffusely scattering 1.7 mm-thick plastic container in drug authentication. Conventional Raman and SORS raw data are shown together with the tablets reference Raman spectrum. The acquisition time was 10s and the laser beam power 50mW. Reprinted in part with permission from Ref [51] American Chemical Society. 

PAL activity is assayed spectrophotometricaUy based on the methods of Koukol and Conn (1961) and Kataoka et al. (1983) by measurement of the rate of formation of t-cinnamic acid as the increase in absorbance at 268 nm. The reaction mixture consists of 2 ml of the enzyme preparation and 1ml of 25 pm l-phenylalanine. In the reference mixture, the L-phenylalanine solution is replaced by distilled water. The mixture is incubated at 37 °C for 3h with shaking. The reaction is stopped by the addition of 0.1 ml of 6 N HCl. After 5 ml of peroxide-free ethyl ether is added, the mixture is shaken again vigorously for extraction of the acidified fraction and then centrifuged at 3000 g for 5 min. The ether phase is obtained with a pipette, and the ether is evaporated to dryness under reduced pressure. The residue is dissolved in 4 ml of 0.05 N NaOH and the absorbance of the solution at 268 nm in a 1-cm quartz cell is measured. The enzyme activity is expressed as micromoles of t-cinnamic acid formed per g FW or per mg protein in fresh tissue/h. The reaction product is scanned with a spectrophotometer for comparison of its optical characteristics with those of authentic t-cinnamic acid. The absorption spectrum of the reaction product was identical with that of authentic f-cinnamic acid (Fig. 3). 

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