Orcinol reaction with

Purification of the conjugates may be done by reverse phase HPLC separation. Dry the reaction solution under a nitrogen stream and reconstitute in a minimum volume of acetonitrile/water (1 1, v/v). Apply the sample to a 5 pm Cig-silica HPLC column (250 X 4.6 mm, Nucleosil). Elute with a gradient of water to acetonitrile at a flow rate of 1 ml/minute over a time course of 30 minutes. Free BNAH and BNAH-glycan derivatives can be monitored by absorbance at 275 nm. The conjugate peak also will be positive for carbohydrate by reaction with orcinol, which can be detected by spray after spotting a small eluted sample on a TLC plate. 

In 1964, Netrawali et al.66 described fluorescence measurements of calcium leucovorin in NaHC03 solution after paper chromatography and reaction with acidic orcinol. The gray-blue fluorescent species formed on the developed paper-gram was very sensitive for leucovorin, showing a detection limit of 0.1 yg. In the eluting solution, however, the required concentration increased to 0.5 pg. Recovery by this method was 100 15%, and results obtained from fluorescence were lower by 10 to 20% than those obtained by microbiological methods. 

Figure 2-18. Absorbances observed with increasing amounts of RNA assayed by the orcinol reaction.

The column is washed with borate-saline until all the 2-ME is removed, with 0.2 M galactose in borate-saline buffer until dgRTB/dgRT impurities are removed, and with borate-saline buffer until all galactose is removed (determined using the orcinol reaction). 

The mechanically fragmented brain or spinal cord was dehydrated with acetone, as in the isolation of cerebrosides.7 Gangliosides are present in the water-soluble fraction they were recognized by color reactions (for example, Bial s orcinol reaction) based on the presence of sialic acid.159 The ganglioside mixture may be purified by partition... 

Co- ph is a basic -watersoluble substance, resistant to heat in nUralin solution and to oxidation with HjOi or alkaline permanganate, but fairly unstable in acid solutions HCl inactivates in 5-10 min. at 100° C. and in 1 hr. at room temperature). Activity is markedly impaired by treatment with NaOI, NaOBr, KiFe(CN)6 or Na2SOt. The concentrates are still heavily contaminated with creatine and pve a positive orcinol reaction for pentose. (Do-aph could not be replaced by ashed concentrate or by any known enzyme activator or muscle extractive. Further investigap tion of as-aph and its coenzyme had been suspended in view of war-time conditions. 

Measuring the rate of disappearance of ribose-5-phosphate in the first reaction with orcinol reagent (i.e. Bial s test, q.v.). 

Nucleic acid synthesis. A striking observation first made by Cohen (52,54) is that in cells infected with T2r+ only DNA, the nucleic acid found in the virus, is synthesized in contrast to normal bacteria which make about 3 times as much RNA as DNA. RNA when measured as pentose by the orcinol reaction remains constant. This phenomenon is clearly of paramount importance. It has been confirmed for T6 bacteriophage by Kozloff (private communication). However, Herriott s data (129) on E. coli infected with T2 give indication of a fall in RNA. Although protein synthesis is apparent from the moment of infection, DNA synthesis begins only after a lag of 7 to 10 minutes (54). This holds for T4 and T6, as well as T2 (62). As shown in Fig. 6, DNA increases linearly with time after a lag of about 10 minutes and finally levels off at about 100 minutes. Doermann and Cohen (unpublished experiments... 

Sugar phosphates and nucleotides, photosynthetic intermediates extracted from leaves, have been determined by h.p.l.c. on a strong base anion-exchange resin eluted with a borate-phosphate buffer and colourimetrlc detection after post-column reaction with orcinol-... 

General color reactions of this type include the Molisch test with a-naphtfiol and concentrated sulfuric acid and the anthrone reaction with anthrone and sulfuric acid. The latter is used for quantitive colorimetric determinations. Reaction conditions can be chosen so that only ketoses react (HCl - - resorcinol, Seliwanoff s test) or only pentoses (HCl -b orcinol. Rial s test). These reactions help to distinguish the various sugars. 

Synthesis of the remaining half of the molecule starts with the formation of the monomethyl ether (9) from orcinol (8). The carbon atom that is to serve as the bridge is introduced as an aldehyde by formylation with zinc cyanide and hydrochloric acid (10). The phenol is then protected as the acetate. Successive oxidation and treatment with thionyl chloride affords the protected acid chloride (11). Acylation of the free phenol group in 7 by means of 11 affords the ester, 12. The ester is then rearranged by an ortho-Fries reaction (catalyzed by either titanium... 

Discussion. Minute amounts of beryllium may be readily determined spectrophotometrically by reaction under alkaline conditions with 4-nitrobenzeneazo-orcinol. The reagent is yellow in a basic medium in the presence of beryllium the colour changes to reddish-brown. The zone of optimum alkalinity is rather critical and narrow buffering with boric acid increases the reproducibility. Aluminium, up to about 240 mg per 25 mL, has little influence provided an excess of 1 mole of sodium hydroxide is added for each mole of aluminium present. Other elements which might interfere are removed by preliminary treatment with sodium hydroxide solution, but the possible co-precipitation of beryllium must be considered. Zinc interferes very slightly but can be removed by precipitation as sulphide. Copper interferes seriously, even in such small amounts as are soluble in sodium hydroxide solution. The interference of small amounts of copper, nickel, iron and calcium can be prevented by complexing with EDTA and triethanolamine. 

By careful choice of both the reaction conditions and the phenolic compound used, it may be possible to produce a colour that is characteristic of a particular carbohydrate or related group, so giving some degree of specificity to the method. Thus, Seliwanoff s test uses hydrochloric acid and either resorcinol or 3-indolylacetic acid to measure fructose with minimal interference from glucose. The colour produced by pentoses with orcinol (Bial s reagent) or p-bromoaniline is sufficiently different from that produced by hexoses to permit their quantitation in the presence of hexoses. However, none of the methods based on the formation of furfural or its derivatives can be considered to be entirely specific. 

In contrast, the diphenol reactions leading to a blue-purple color are in wide use for sialic acid determinations. Sialic acids are heated either with orcinol and Fe3+ (the Bial reagent23), or with resorcinol and Cu2+ (Refs. 30 and 120) in concentrated hydrochloric acid.107 In 1903,... 

Minimum amounts of 2-3 /ig of sialic acids can be determined in, for example, the orcinol-Fe3+ assay, if the volumes of the Bial reagents23 are one-fifth of those originally described.107 By using this method, sialic acids can be accurately determined only if they have been prepurified, as free or glycosidically bound pentoses, hexoses, or alduronic acids interfere with the reaction by giving green chromo-phores.107 In our experience, an approximate determination of the sialic acid in a complex carbohydrate is only possible if its sialic acid content is >5%. 

The total content of RNA + DNA in tissues may be estimated from the phosphorus content or by color reactions of the sugars.37 545 These reactions depend upon dehydration to furfural or deoxyfurfural by concentrated sulfuric acid or HC1 (Eq. 4-4). Furfural formed from RNA reacts with orcinol (3,5-dihydroxy-toluene) and ferric chloride to produce a green color useful in colorimetric estimation of RNA. A similar reaction of DNA with diphenylamine yields a blue color. 

An amorphous preparation was made similarly from 2,3,4,6-tetra-O-acetyl-(2-hydroxy-D-galactal).9 It was noted that this preparation did not give the reactions reported by Helferich for the 5,6-anhydro derivative with pyrogallol, orcinol, aniline, and m-nitroaniline. However, it did give a voluminous, red-brown precipitate with phloroglucinol. 

The reaction of hemicellulose to heated, concentrated (12%) HC1 is a test of the former s presence. Detection is by the violet or red color developed by one or more of the reaction products (chiefly furfural) with orcinol or phloroglucinol. The coloration is either blue, greenish, or nonexistent with KI-I2 (Whistler and Smart, 1953). The quantity of furfural produced relates directly to the quantity of pentosan present (Smith and Montgomery, 1959). Under the same reaction conditions, hexoses produce hydroxyme-thylfurfural. Furfural is steam-distillable hydroxymethylfurfural is not. 

---