Chemistry of Glycerol Dichlorohydrin As a Reagent for the Determination of Vitamin a Robert Scott Allen Iowa State College

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1949 Chemistry of glycerol dichlorohydrin as a reagent for the determination of vitamin A Robert Scott Allen Iowa State College Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons Recommended Citation Allen, Robert Scott, "Chemistry of glycerol dichlorohydrin as a reagent for the determination of vitamin A " (1949). Retrospective Theses and Dissertations. 13111. https://lib.dr.iastate.edu/rtd/13111 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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ProQuest Infonnation and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 NOTE TO USERS This reproduction is the best copy available. UMT CBEMISTRY OF GLYCEROL DICHLOROHXDRIN AS A REAGENT FOR THE DETERMINATION OF VITAMIN A by Robert Soott Allen A Dlsaertatlon Submitted to the Graduate Faculty In Partial Ftilflllment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Miajor Subjects Biochemistry Approved: Signature was redacted for privacy. In Charge of^Major Work Signature was redacted for privacy. Head of Major Department Signature was redacted for privacy. Dean of ^Graduate College Iowa State College UMI Number DPI 2329 UMI' UMI MicroformDP12329 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 0 01. \i S A u TABLE OF CONTENTS INTRODDCTION 1 REVIEW OF THE LITERATURE 4 Discovery of Vitamin A •••••••»•••••••••••••••••• 4 Properties of Vitamin A ••••••••••••••••••••••••• 5 Development of Assay Methods 7 Biological Assay 7 The growth method ••••••••••••••••••••• 6 Singlo'^ose growth method 11 Vaginal smear method 11 Liver storage method 11 Physioo-ohemioal Methods 12 The ultraviolet absorption method ••••• 12 Colorimetrio methods 16 The antimony trichloride method •• 20 The glycerol dichlorohydrin method 25 Chemistry of the Vitamin A Determination 34 The Antimony Trichloride Method 34 The Qlycerol Dichlorohydrin Method 40 APPARATUS 43 METHODS 46 Purification of Qlycerol Derivatives 46 Activation of Glycerol Derivatives •••••••••••••• 46 Qualitative Tests 46 Antimony Trichloride Method 46 Acid Methods 47 Preparation of Vitamin A and Carotene Solutions • 48 Absorption Spectra 49 Optical Density Measurements 50 Preparation of GDH Containing Added Impurities •• 51 EXPERIMENTAL RESULTS 52 •r~9:>3S' Ill Esqperlments with Olyoerol Diohlorohydrin (QDH) •• 52 Shell Olyoerol Diohlorohydrin 52 Purif ioation 52 Qualitative activation tests 54 HCl activation 57 Sulfuric acid activation 67 Chlorosulfonic acid activation 69 Activation with antimony trichloride .. 80 Activation by heating 90 Activated Shohan Olyoerol Diohlorohydrin ••• 93 Deactivation 93 Activation with heat 94 Solvent-reagent ratio study 94 Reaction with /3<-carotene •••••••••••••• 105 The effect of added impurities on the ODH-vitamin A color reaction •••••••• 112 Recovery and reactivation of used re agent 113 Eastman Olyoerol (75^ 25^ Di ohlorohydrin ••••••••••••••••••••••••••••• 119 Qualitative activation tests •••••••••• 119 Activation with antimony trichloride •• 122 Paragon Olyoerol «(»V-Dichlorohydrin 125 Qualitative activation tests 125 Activation with antimony trichloride •• 131 Experiments with Two Olosely Related Olyoerol Derivatives 134 Olyoerol Monoohlorohydrin 134 Qualitative activation tests 134 Purification 140 Activation with antimony trichloride •• 141 1,2,3-Trichloropropane 144 Qualitative activation tests 144 Attempted an.timony trichloride acti vation 149 Iv Investigation of the ICeohanlsia of the GDH-Vlta- mln A Reaction 150 Formation of Anhydrovltaoln A 150 Quenching Bxperlaents •«•••••••••••••••••••• 152 TJltravlolet Absorption Spectra as the ODE- Vitamin A Color Reaction Aged 158 1)5 Solvent-reagent ratio 158 5tl Solvent-reagent ratio ••••••••••••• 159 DISCUSSION 162 SUMKAHY AND CONCLUSIONS 191 LITERATURE CITED 196 ACKNOWLEDOMBNTS 218 -1- INTRODUCTION The analysis of vitamin A by oolorlmetrlc methods has been the topic of many investigations In the past twenty five years* Numerous methods have been proposed, tested and fi nally discarded with the conclusion that they lacked speci ficity and/or the required sensitivity to measure small amoimts of vitamin A in biological materials* The method that appears to be the most accepted and most used in biological and investigational work is the so-called Carr-Prlce or antimony trichloride method* Although this method produces an Intense blue color with vitamin A which permits testing of very small quantities of vitamin A, the color builds up to a maximum and then begins to fade within 5 to 10 seconds at room temperature. The color must be de veloped in the absence of moisture to avoid the formation of a suspension of antimony oxychloride which results in a false instrument reading. The color development and fading rate are dependent upon the intensity of light striking the reac tion mixture. Many improvements in technique and apparatus for measuring the blue color have tended to minimize the dis advantages of the antimony trichloride method, but for the average analyst a reliable and sensitive method which can be used without numerous precautions and special techniques would be highly desirable. -2- Wlthln the last foup years a new colorlmetrlc reagent, which has many desirable features for the measurement of vitamin A, has been introduced. The reagent is glycerol diohlorohydrin that is activated by vacuiim distillation with a small amoxint of antimony trichloride. The violet color pro duced with vitamin A is stable for several minutes but has an extinction coefficient only about one-fourth that of the an- tiirony trichloride color. Traces of moisture apparently do not interfere with the color reaction. The color may be measured with practically any type of colorimeter or spectro photometer, and Sobel and Snow (211) reported a method of measurement which approximates the sensitivity of the anti mony trichloride method. Eecause of the advantages of the activated glycerol dichlorohydrin method (214), it is being used in some analytical laboratories. Regarding the use of activated glycerol dichlorohydrin, Dann (43) has stated that "the reagent appears more likely to displace antimony trichloride than any other which has yet appeared, and it deserves to be widely tested". As yet, very little information regarding the chemistry of the ac tivation of glycerol dichlorohydrin or its reaction with vi tamin A has been reported. This investigation was initiated in an attempt (1) to determine the nature of the activation of glycerol dichloro hydrin with certain acids, metallic and nonmetallic halides. 3- acyl halldes and other compounds, (2) to determine the struc tural requirements necessary In glycerol derivatives to pro duce good color when nixed with a vitamin A solution, (3) to determine the effect of various solvent-reagent ratios on the absorption spectra of the products of the reactions of vita min A with activated glycerol dlchlorohydrln, (4) to deter mine the effect of added impurities on the development of color, and (5) to determine the probable mechanism of the re action of vitamin A with activated glycerol dlchlorohydrln. -4- REVIEW OP THE LITERATURE Discovery of Vitamin A Although the medicinal use of cod liver oil was general in Western Europe by 1840 (83), it is only since the recog nition of deficiency diseases that progress regarding the cause of avitaminosis has been made. Detailed accounts of early work dealing with this subject are presented in Sher man and Smithes Monograph (201) and the Medical Research Council's Survey of the Vitamins (125)* In 1913, McCollum and Davis (117) found that growth ceased prematurely in rats fed on a synthetic diet in which the fat supplied was lard* I^on the addition of butterfat or an ether extract of egg yolk to the diet growth was re sumed and it was oonoluded that the effect might be due to the presence of complex organic lipins or substances asso ciated with the latter. Osborne and Mendel (167, 168) ob served that

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