Thin-Layer Chromatography

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28 2 Physical Methods of Detection The emission of thermal electrons is subject to statistical fluctuations (lead shot effect). It is influenced by the current strength, the number of electric charges liberated and the frequency of the radiation [55] (see [40] for further details). Range of application and spectral sensitivity: The photomultipliers most frequently employed in scanners possess antimony-caesium cathodes. These alloy cathodes are primarily sensitive to the short-wavelength part of visible light (Tab. 4). The long-wavelength limit which depends on the cathode material is ca. k = 650 nm in the red region of the spectrum. If this does not suffice for the determination an antimony-alkaline metal alloy is employed as the cathode material [56—58]. The range of application into the short-wavelength region of the spectrum depends on the window material employed in the photomultipher. Borosilicate glass (KOVAR glass), for example, only transmits radiation down to about 280 nm, Suprasil down to 185 nm and fused quartz down to 160 nm. Hence fluorimeters which primarily detect long-wavelength radiation (fluorescent radiation) are often equipped with type S-4 detectors (Tab. 4), whose windows absorb a part of the short-wavelength radiations. Table 4. Characteristics of the most important photomultipliers with Sb-Cs cathodes and 9 amplification steps [50]. Type S-4 S-5 S-19 RCA 1P21 931B 1P28 1P28/VI 1P28A 1P28A/VI 4837 Origin HAMAMATSU EMI 1P21 R105 R105UH 1P28 R212 R212UH R454 R282 R106 R106UH 9781A 9661B 9781B 978 IB 9781R 9665A 9783B Examples of scanners employing S-4 detectors: Spectral — response [nm] 300.. 650 3OO...65O 300.. 650 185...650 185...650 185...650 185...650 185...650 160...650 160. .650 Wavelength maximum sensitivity 400 400 400 340 340 340 450 450 340 340 Window material borosilicate borosilicate borosilicate UV glass UV glass UV glass UV glass UV glass fused silica fused silica • UV/VIS chromatogram analyzer for fluorescence measurements (FARRAND) • Spectrofluorimeter SPF (AMERICAN INSTRUMENT CO.) • TLD-100 scanner (VITATRON) /./ Detection oj Aosoroing zuosiances The majority of detectors, which are employed for the measurement of absorption, employ UV glass (e.g. Suprasil). All type S-5 photomultipliers possess sheaths of this material, so that they ought to be usable in the far UV region if N2 purging is employed (to remove O2) (Tab. 4). Scanners with S-5 detectors • KM 3 chromatogram spectrophotometer (C. ZEISS) • SD 3000 spectrodensitometer (KRATOS/SCHOEFFEL) • CD-60 densitometer (DESAGA) Photomultipliers of type S-19 employ fused quartz instead of UV glass; this transmits down to A = 160nm although this far UV range is not normally employed in scanners. Scanners with S-19 detectors • CS 920 scanner (SHIMADZU) • CS 930 scanner (SHIMADZU) Photoelements andphotodiodes: Both photoelements and photodiodes are photoelectric components depending on internal photoelectric effects. In the case of photoelements incident quanta of light produce free charge earners in the semiconductor layer; previously bound electrons become free. Thus, the nonconducting layer becomes conducting. In addition, the migrating electrons produce "holes" which increase the conductivity. The radiation energy is directly converted into electrical energy. The construction of a photoelement is illustrated in Figure 21. Fig. 21: Principle of construction of a photoelement [1]. - 1 light-transmitting metal layer, 2 semiconductor layer, 3 metal plate. hv
3U z rnysicai meinous oj veiecuun Photodiodes produce an electric field as a result of pn transitions. On illumination a photocurrent flows that is strictly proportional to the radiation intensity. Photodiodes are sensitive and free from inertia. They are, thus, suitable for rapid measurement [1, 59]; they have, therefore, been employed for the construction of diode array detectors. 2.2.3.2 Principles of Measurement The scanners commercially available today operate on the basis of the optical train illustrated in Figure 22. They can be employed to • detect absorbing substances against a nonfluorescent plate background (Fig. 22A: recording a scanning curve, absorption spectra, quantitative analysis of absorbing substances); • detect absorptions indirectly because of luminescence diminishing (Sec. 2.2.2). [Here, however, it is necessary to introduce a cut-off filter before the detector to absorb the shortwave excitation radiation (Fig. 22B)]; E ) Pr A M o Fig. 22: Optical trains of the commercially available scanners. (A) absorption, (B) fluorescence quenching and true fluorescence. R = recorder, I = integrator, E = detector, Pr = sample (TLC plate), S = mirror, M = monochromator, L = lamps (incandescent lamp ®, deuterium lamp 0 and mercury lamp ©), Fl = cutoff filter. • detect and analyze quantitatively fluorescent substances against a nonfluorescent background without spectral analysis of the fluorescent light (Fig. 22B). [An additional filter is necessary here too (Sec. 2.3.3).] On emitting phases it is not possible to determine directly {in situ) the fluorescence and absorption spectra of compounds that absorb in the excitation range of luminescence indicators without distorting the measurement signal. Direct Determination of Absorbance Determination of absorption spectra in reflectance: If there is no luminescence radiation absorption spectra can be determined using the light path sketched in Figure 22A. If absorbing substances, such as, for example, dyestuffs, caffein or PHB esters, are determined spectrophotometrically after chromatography, then, depending on the wavelength, these components absorb a proportion (/abs) of the light irradiating them (70). The chromatographic zone remits a lower light intensity (7ref) than the environment around it. A) ~ Albs = Aef Therefore it is possible to determine absorption spectra directly on the TLC plate by comparison with a substance-free portion of the layer. The wavelengths usually correspond to the spectra of the same substances in solution. However, adsorbents (silanols, amino and polyamide groups) and solvent traces (pH differences) can cause either bathochromic (ketones, aldehydes [60, 61], dyestuffs [62]) or hypsochromic (phenols, aniline derivatives [63]) shifts (Fig. 23). However, these absorption spectra can be employed as an aid to characterization, particularly when authentic reference substances are chromatographed on a neighboring track. The use of differential spectrometry yields additional information [64]. Quantitative analysis is usually performed by scanning at the wavelength of greatest absorbance (Amax). However, determinations at other wavelengths can sometimes be advantageous, e.g. when the result is a better baseline. An example is the determination of scopolamine at X = 220 nm instead of at Amax = 205 nm or of the fungicide vinclozolin at X = 245 nm instead of at AmaI = 220 nm. Absorbance (reflectance) scanning: The positions of the chromatographically separated substances are generally determined at AmaI. As the chromatogram is scanned the voltage differences produced at the detector are plotted as a function of position of measurement to yield an absorption scan (Fig. 24). Conclusions concerning the amount of substance chromatographed can be drawn from the areas or heights of the peaks [5].
Page 1 and 2: Hellmut Jork, Werner Funk, Walter F
Page 3 and 4: VI Foreword in many cases, an elega
Page 5 and 6: Contents Parti Methods of Detection
Page 7 and 8: A1V L,omenis Hydrochloric Acid Vapo
Page 9 and 10: 1 Introduction The separation metho
Page 11 and 12: Number 600 too 200 1 Introduction 1
Page 13 and 14: Colorless substances absorb at wave
Page 15 and 16: 14 rnysicat Methods oj Detection B
Page 17 and 18: io z rnysicai Meinous oj ueieciwn F
Page 19 and 20: 22 2 Physical Methods of Detection
Page 21: 26 2 Physical Methods of Detection
Page 35 and 36: 3 Chemical Methods of Detection Eve
Page 37 and 38: Chemical Meinoas oj ueiecuun Fig. 3
Page 39 and 40: 62 3 Chemical Methods of Detection
Page 47 and 48: uj • improving the detection sens
Page 49 and 50: 82 3 Chemical Methods oj Detection
Page 59 and 60: 30-- 20 4- 10 •• P-7 P-8 —I 1
Page 65 and 66: lit J ^ncniiLui lvicinuus uj [129]
Page 69 and 70: 122 4 Documentation and Hints for C
Page 71 and 72: T LsvLumcniuuuri unu liiruijur \^nr
Page 73 and 74:
130 4 Documentation and Hints for C
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zone size and distribution of subst
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138 4 Documentation and Hints for C
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Reagent for: Cations [1-7] Preparat
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146 Alizarin Reagent Detection and
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150 Aluminium Chloride Reagent UV l
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4-Aminobenzoic Acid Reagent Reagent
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158 2-Aminodiphenyl — Sulfuric Ac
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162 4-Aminohippuric Acid Reagent 1
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Reagent for: Ammonia Vapor Reagent
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Ammonium Thiocyanate — Iron(III)
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174 Amy lose — Potassium lodate/I
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1/0 Anuine — Aiaose neageni Mobil
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15 L Anmne — uipnenyiamine — rn
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186 Aniline — Phosphoric Acid Rea
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i s\j Jiituuuz — -I ninuiH. S1LIU
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li»4 X-Anilinonaphthalene-1-sulfon
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198 Anisaldehyde — Sulfuric Acid
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Reagent for: • Ketoses [1] • Gl
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Antimony(III) Chloride Reagent (Car
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Antimony(V) Chloride Reagent Reagen
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214 Berberine Reagent Under UV ligh
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218 2,2'-Bipyridine - Iron(III) Chl
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222 Blue Tetrazolium Reagent Note:
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224 Bratton-Marshall Reagent Substa
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Bromocresol Green — Bromophenol B
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232 Bromocresol Purple Reagent Meth
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236 tert-Butyl Hypochlorite Reagent
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240 7-Chloro-4-nitrobenzo-2-oxa-l,3
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244 Copper (II) acetate — Phospho
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Copper(II) Sulfate Reagent Reagent
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2,6-Dibromoquinone-4-chloroimide Re
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2,6-Dichlorophenolindophenol Reagen
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296-Dichloroquinone-4-chloroimide R
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204 Zp-Uichloroqumone-4-cMorovniOe
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268 2,5-Dimethoxytetrahydrofuran
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Lii. t-uimeinyiummocmnumaiaenyae
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276 ^,4-Uimtrophenylhydrazine Reage
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280 Diphenylboric acid-2-aminoethyl
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Diphenylboric Anhydride — Salicyl
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Reagent for: Fast Blue Salt B Reage
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tasi mue salt a Keagent 1 5 10 15 2
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ZVb tluorescamine Keagent Sulfonami
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300 Formaldehyde - Sulfuric Acid Re
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304 Hydrochloric Acid Vapor Reagent
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308 Hydrogen Peroxide Reagent radia
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312 8-Hydroxyquinoline Reagent Proc
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310 iron(iii) L^moriae — fercnion
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5Z\) isonicotinic Acid Hydrazide Re
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JZt Ljeaa{ii) Aceiaie Basic neagem
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In situ quantitation: The fluorimet
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332 Lead(IV) Acetate — Fuchsin Re
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JJO manganeseinj ^nionae — zuijun
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Mercury(II) Salt - Diphenylcarbazon
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2-Methoxy-2,4-diphenyl-3(2H) furano
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jis 3-Meinyi-z-Denzotniazoiinone-ti
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jji i,*-ivupninoquinone-4-sulJonic
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JDO mnnyarui — ^.oiuatne iteagent
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360 4-(4-Nitrobenzyl)pyridine Reage
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Reagent for: • Steroids [1-8] •
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Peroxide Reagent (1-Naphthol - N 4
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1,2-Phenylenediamine — Trichloroa
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Phosphomolybdic Acid Reagent Reagen
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Reagent for: o-Phthalaldehyde Reage
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384 o-fhthaiaiaenyae neageni [16] G
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Pinacryptol Yellow Reagent Reagent
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Potassium Hexacyanoferrate(III) —
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396 Potassium Hexacyanoferrate(III)
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4UU fyrocatechol Violet Reagent 1 2
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Reagent for: Rhodamine 6G Reagent
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Silver Nitrate — Sodium Hydroxide
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Preparation of Reagent Dipping solu
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Tetracyanoethylene Reagent (TCNE Re
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Trichloroacetic Acid Reagent Reagen
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tz.t i rinuruoenzenesuijonw ACIU ne
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4/5 vanadium{ v) — auijunc Acid R
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432 Vanillin — Phosphoric Acid Re
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t JO vamuin — roiassium tiyaroxid
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Layer Mobile phase Migration distan
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Index ABP = 2-amino-5-bromophenyl(p
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Benztriazole -, derivatives 281 ff
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Disaccharides 154,161,163,179,181,
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430 maex Isoniazide 318 Isonicotini
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40U maex -, side on 27 -, window ma
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464 Index Triphenodioxazines 411 Tr
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Prof. Dr. H. Jork UniversitSt des S
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VIII Preface to Volume lb Particula
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Contents Foreword Preface to Volume
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XVI Contents Potassium Dichromate -
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4 Introduction For the same reason
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8 Introduction [36] Wilson, I. D.,
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12 / Activation Reactions The inorg
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16 1 Activation Reactions The excit
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20 1 Activation Reactions These few
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24 1 Activation Reactions Migration
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28 / Activation Reactions of malona
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32 / Activation Reactions Table 2.2
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36 / Activation Reactions In situ q
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40 / Activation Reactions [4] Egg,
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2 Reagents for the Recognition of F
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48 Table 4: (continued) Functional
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52 2 Reagents for the Recognition o
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3 Reagent Sequences Thin-layer chro
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60 5 Reagent Sequences A B Fig. 23:
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t>4 5 Reagent Sequences Here the al
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68 3 Reagent Sequences tert-Butyl H
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72 3 Reagent Sequences tert-Bnty\ H
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76 3 Reagent Sequences Cerium(IV) S
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80 3 Reagent Sequences Sodium Hydro
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84 3 Reagent Sequences Sodium Hydro
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OO J xvcugcric uc Tin(II) Chloride-
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92 3 Reagent Sequences Tkanium(III)
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96 3 Reagent Sequences Reagent 4 Co
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100 3 Reagent Sequences Nitric Acid
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104 3 Reagent Sequences Reagent 2 R
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108 3 Reagent Sequences Tin(Il) Chl
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112 3 Reagent Sequences 1 2 3 4 M 5
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116 3 Reagent Sequences air. It was
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120 3 Reagent Sequences Hydroxylami
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124 3 Reagent Sequences Borate Buff
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128 3 Reagent Sequences Diphenylami
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132 3 Reagent Sequences Tible 1: Co
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136 3 Reagent Sequences Reaction Th
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140 3 Reagent Sequences [36] Bomtra
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144 Acridine Orange Reagent Reactio
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148 Ammonium Monovanadate-p-Anisidi
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152 Ammonium Thiocyanate Reagent Me
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156 4,4'-Bis(dimethylamino)thiobenz
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160 N-Bromosuccinimide Reagent Proc
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164 N-Bromosuccinimide-Robinetin Re
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168 Cacotheline Reagent Method The
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172 Chloramine T Reagents Table 1:
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176 Chloramine T-Mineral Acid Reage
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180 Chloramine T-Sodium Hydroxide R
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184 Chloramine T- Trichloroacetic A
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Reagent for: /7-Chloranil Reagent
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192 p-Chloranil Reagent Procedure T
Page 347 and 348:
196 Chlorine-Potassium Iodide-Starc
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200 Chlorine-4,4'-Tetramethyldiamin
Page 351 and 352:
Reagent for: Chlorine - o-Tolidine
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208 Chlorine-o-Tolidine-Potassium I
Page 355 and 356:
Cyanazin (hR{ 5-10) appeared as gra
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216 Copper(II) Sulfate-Sodium Citra
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220 Dansyl Chloride Reagent Reactio
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224 Dimedone-Phosphoric Acid Reagen
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228 N,N-Dimethyl-l,4-phenylenediami
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4-(Dimethylamino)benzaldehyde - Ace
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4-(Dimethylamino)benzaldehyde-Acid
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240 4-(Uimethyiammo)-oenzaiaenyae-A
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244 4-(Dimethylamino)-benzaldehyde-
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4-(Dimet hy lamino)-benzaldehyde -
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260 Dimethylglyoxime Reagent Reacti
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264 3,5-Dinitrobenzoic Acid-Potassi
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Fast Black Salt K- Sodium Hydroxide
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272 Fast Black Salt K-Sodium Hydrox
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276 Fast Blue Salt BB Reagent Refer
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280 Iodine Reagents Table 1: (conti
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284 Iodine Vapor Reagent Reaction I
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288 Iodine Vapor Reagent [4] Andree
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292 Iodine Solution, Neutral Reagen
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Iodine-Potassium Iodide Solution, A
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300 Iodine-Potassium Iodide Solutio
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304 Iodine-Potassium Iodide Solutio
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3U8 lron(iii) cnionae Reagent Prepa
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Iron(III) Chloride-Potassium Hexacy
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316 Iron(III) Chloride-Potassium He
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320 8-Mercaptoquinoline Reagent Ref
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324 l,2-Naphthoquinone-4-sulfonic A
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328 Nitric Acid Vapor Reagent Prepa
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Reagent for: 4-Nitrobenzenediazoniu
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References [1] Fuhremann, T. W., Li
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340 Palladiumfll) Chloride Reagent
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344 Phosphoric Acid Reagent Storage
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Reagent for: 0-Phthalaldehyde- Sulf
Page 425 and 426:
Potassium Dichromate-Perchloric Aci
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1 3 5 6 7 8 9 10 11 12 Fig 2: Chrom
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Reaction The mechanism of the react
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364 Potassium Hexaiodoplatinate Rea
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joo tvtassium Hydroxide Reagent Lay
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Preparation of the Reagent Dipping
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Potassium Nitrate-Sulfuric Acid Rea
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380 Potassium Peroxodisulfate-Silve
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Reaction The reaction mechanism has
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388 Sodium Hydroxide Reagent Prepar
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References ouuium tiyaroxiae Reagen
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In situ quantitation: The absorptio
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c o Fig. I: Reflectance scans of a
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Procedure Tested Nitrophenols [28]
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408 Sulfanilic Acid-N-(l-Naphthyl)-
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412 Tetrabromophenolphthalein Ethyl
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,j\-ietramethyl-l,4-phenylenediamin
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4-nitroaniline) and 25 ng (2-amino-
Page 461 and 462:
424 Thymol-Sulfuric Acid Reagent Fi
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4/8 i m[ii) ^nionae-nyarocnionc Aci
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432 Tin(IV) Chloride Reagent Migrat
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436 Titaniumflll) Chloride-Hydrochl
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Vanillin Reagents The "aldehyde aci
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Procedure Tested Indole Derivatives
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IVICUIUU The chromatograms are drie
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452 Vanillin-Sulfuric Acid Reagent
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456 Named Reagents and Reagent Acro
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460 Index Aminoglycoside antibiotic
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464 Index Cactus alkaloids lb 229 C
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468 Index -, phenols la 73 -, prech
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472 Index Ergocornine lb 243 Ergocr
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476 Index Halogen lamp la 22 Hexoba
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480 Index Lipids la 44-46, 89,191,2
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484 Index Orelline lb 307 Orellinin
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488 Index PRimine lb 244,246,247 Pr
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492 Index Stimulents lb 268,270 Str
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496 Index -, bioautoradiographic de
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Thin-Layer Chromatography

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