Physico-chemical methods for the identification of narcotics (cont.). Part Va - Paper chromatography


The Bulletin on Narcotics presents in this issue the continuation of the Canadian series of articles on the "Physico-chemical Methods for the Identifi-cation of Narcotics ". Parts I to IV appeared in the Bulletin, Volumes V (1953) to VII (1955).


Author: Klaus Genest, Charles G. Farmilo
Pages: 20 to 37
Creation Date: 1959/01/01

Physico-chemical methods for the identification of narcotics (cont.). Part Va - Paper chromatography

Klaus Genest
Charles G. Farmilo
Organic Chemistry and Narcotic Section, Food and Drug Directorate Department of National Health and Welfare, Ottawa (Ontario) Canada

The Bulletin on Narcotics presents in this issue the continuation of the Canadian series of articles on the "Physico-chemical Methods for the Identifi-cation of Narcotics ". Parts I to IV appeared in the Bulletin, Volumes V (1953) to VII (1955).






A. Compounds to be reviewed
B. Chromatographic apparatus and techniques
C. Choice and classification of solvents
D. Solvents used for narcotics
E. Detection of solutes on paper chromatograms
i. Chromogenic reagents for narcotics
ii. Physical methods for detection
iii. Biological methods for detection
F. Sensitivity of narcotic identification by paper chromatography
G. R f values for narcotic identification
I. Problems from narcotic control
II. Detection of narcotic addiction and toxicological problems
III. General approaches to analysis
1. Fischer's method for narcotics
2. Macek's method for alkaloids


List of narcotics and related compounds
A. Solvents with acetic acid
B. Solvents with acetic acid and acetate ester
C. Solvents with formic, citric, propionic & hydrochloric acids
D Alkaline solvents
E. Neutral solvents
Sensitivity of detection of narcotics by paper chromatography
R f papaverine / R f morphine for various systems
Number of convictions under Opium and Narcotic Drug Act (1949-1954)
Systems for separating and identifying codeine and hydrocodone
Survey of narcotics used by sixty-two addicts during 1953-1955 in Munich, Germany
R f values for narcotics and basic drugs detected in the urine of addicts
Classification of alkaloids, narcotics and related drugs

* For ease of handling, these tables have been placed in a pocket at the end of the Bulletin


Apparatus for ascending paper chromatography
Apparatus for circular paper chromatography
Diagram showing Fischer's elution technique
The group separation analysis of alkaloids by paper chromatography according to Macek

1. Introduction

Paper chromatography provides a means of separating and identifying narcotics in one operation. It is complementary to other methods of identification such as colour and crystal tests, ultra-violet, infra-red, and X-ray diffraction methods, and can be used in conjunction with the above tests. Paper chromatography simplifies the separation phase of analytical problems, and replaces, in some cases, the more laborious "shake-out" procedures for quantitative and qualitative determinations.

More than eighty publications dealing with paper chromatography of narcotics have appeared up to the present. ** These methods are summarized in the following text by detailed tabulations. A really critical analysis of the methods described in the literature cannot be presented in this review. Such a comparison would require a special study in which the techniques could be tested using uniform material and under constant conditions. Therefore the object of this review is to correlate the paper chromatographic results available in the literature; to obtain a systematic approach to the problem of narcotic identification, and to illustrate some typical examples of difficult problems in narcotics analysis which were solved by paper chromatography.

Received for publication 3 December 1958.

2. Outline of the general method

In modern paper chromatography introduced by Martin and his co-workers, 1944, a small spot of a solution containing the mixture to be examined is placed near one end of a filter-paper strip or sheet. After this has dried, the end of the paper nearest the spot is placed in a trough containing a developing solution, usually an organic solvent saturated with water, and the whole is enclosed in a suitable chamber, the atmosphere of which is saturated with both water and solvent vapours. In "reversed phase" chromatography, water cannot be used as the stationary phase, and the paper is then saturated with rubber or silicone, vaseline or oil, or formamide, etc. In either system, the developing solvent advances by capillary action past the spot along the paper. After some time has elapsed the solute mixture will be found to have moved from its point of application, and to have separated wholly or partially into its components. These usually appear as well-defined circular areas, providing a careful control of conditions is kept. Otherwise, tailing of the separated components occurs. The position taken up by a substance on a paper chromatogram operated under a particular set of controlled conditions is specified by a term, the R f value, which is the ratio of the distance travelled by the solute to that travelled by the advancing solvent front, both distances being measured from the point of application of the mixture. The mechanism involved in the resolution of mixtures on the filter paper may not be a single one. Adsorption and ion exchange are believed to play some part in effecting fractionation. The predominant factor, however, is usually that of partition between two immiscible phases, a mobile solvent phase, and a stationary phase composed of a cellulose-water complex. The R f value of a solute is related to the partition coefficient.

Owing to its wide application, combined with the simplicity of the equipment and techniques involved in its operation, paper chromatography has assumed great importance as a method for qualitative and often quantitative analysis of micro quantities of materials. The subject of paper chromatography is still rapidly expanding, and for the information of those interested in delving more deeply into the history, theory and other specific applications, some textbook references * are given in a section immediately following the narcotic bibliography.

Systematic identification of narcotics by paper chromatography

A. Compounds to be reviewed

* The authors wish to acknowledge the fact that in this brief review of the general aspects of paper chromatography material was drawn freely from the texts, and in particular from the review in the Extra Pharmacopoeia (87)

According to the 1957 list of substances falling under international conventions, there are about sixty-eight narcotics. These include natural products - e.g., raw opium, coca leaves and cannabis; manufactured drugs - e.g., derivatives of morphine, codeine and thebaine - and synthetic narcotics - e.g., methadone, pethidine, moramides, thiambutenes, morphinans, etc. There are a number of chemically related compounds which may be found along with the narcotics in their natural sources - e.g., papaverine and narcotine in opium, or which are homologues of synthetic compounds. These homologues may not be legally classed as narcotics, but have been included in our lists of compounds because they may interfere with the identification of narcotics. The compounds discussed in this paper are shown in table I, preceded by the roman numerals used to designate them throughout the tabulations.


List of narcotics and related compounds


List of narcotics and related compounds

Racemorphan Levorphanol
d, l or dl-Methorphan
Cotarnin. e

B. Chromatographic Apparatus and Techniques

Many forms of apparatus have been devised for paper chromatography. For ascending chromatography the equipment is the simplest (see, for example, figure 1) and consists of a chamber (F), such as a tube, tank, jar, or cylinder, fitted with a cover (B), and a vessel for the solvent (D), placed at the bottom of the chamber. The paper may be formed into a cylinder (C). Various authors (2, 3, 17, 18, 22, 35, 36, 51, 55, 67) have used ascending paper chromatography for narcotic analysis because of technical advantages. For descending chromatography, the essential requirement is a reasonably airtight chamber or cabinet, preferably of glass, in which hangs a strip or sheet of paper. The upper end of the paper dips into a supported trough containing the solvent, and is retained in position in the trough by a weight such as a glass rod. A reservoir of solvent, usually the water-rich layer, after separation of the mobile phase, placed at the base of the chamber, ensures saturation of the atmosphere in the chamber with vapour. The results obtained with descending chromatography have been found to suit special purposes (11, 12, 21, 55, 62, 68, 73). For quantitative purposes, the descending method appears to be preferable. For slow-moving substances, better resolution is achieved by permitting the solvent descending the paper strip to drip off the serrated lower edge of the paper. This method has been employed in chromatography of complex mixtures of alkaloids (34), and is often referred to as "Durchlauf" chromatography.

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FIGURE 1. Apparatus for ascending paper chromatography

A : 10" x 8"
D: 10" Dia. 2"
B : 12" x 12" x ?"
E: 2 cm.
C : 5" Dia. x 20"
F: 111/2" Dia. x 24"

Miram & Pfeifer (39) used a combination of the descending and ascending techniques to achieve the separation of seventeen opium alkaloids from raw opium extracts, nineteen alkaloids from poppy capsule extracts, and fourteen alkaloids from tinctures. One paper strip was subdivided into three buffered zones using two solvent systems consecutively.

Two-dimensional chromatographic techniques were used by Krogerus (30, 32) and Nadeau (50, 50 a). This technique is useful for separation of pairs of alkaloids such as papaverine and narcotine with similar R f values. In this process, a spot of the mixture is placed near one corner of a large square sheet of filter paper and one edge dipped into a long trough containing the solvent. When the solvent front has almost reached the opposite edge, the sheet is removed, dried, and turned through a right-angle, replaced in the trough, now containing a second solvent, and the development repeated. Instead of using a long trough, the sheet may be rolled into a cylinder or even into a spiral provided that the whorls of the spiral are kept from touching by some convenient means and stood in the developing solvent. When using the second solvent, the cylinder or spiral is formed by folding the paper in a direction at right angles to the original direction.

Particularly rapid separations of mixtures of narcotics have been obtained on the circular paper chromatogram, a method which provides for the resolution of solutes into concentric bands instead of spots. The paper is prepared by inserting a wick into a hole in the centre of a circular filter paper. Several drops of the mixture and reference substances to be examined are applied to a circle around the hole and air dried. The paper disc is then placed horizontally over one section of a Petri dish of smaller diameter so that the wick dips into the solvent contained therein. The upper surface of the paper disc is covered by a plate or second dish. The solvent rises up the tail by capillary action and radiates from the centre of the paper disc. Development is often complete within fifteen minutes. Abbaffy & Kveder (1) have applied circular paper chromatography to the separation of methadone from hyoscine and ephedrine. An example of a circular paper chromatographic apparatus which is used in this laboratory is shown in figure 2. A seed culture dish is used (A and E, figure 2) as a chamber with a circular filter paper (B), and a dentist's cotton roll as a wick (C).

C. Choice and Classification of Solvents

The number of solvents suitable for paper chromatography is large, and the choice is based on empirical rules. Block, Durrum & Zweig (81) state certain simple rules which can be used as a practical guide. If the factors of adsorption and ion exchange are neglected, the movement of the solvent and solute is a function of its solubility in the developing solvent. The solvent should be one in which the compounds to be separated have a small but definite solubility. If the substances are too soluble, they will appear at or near the solvent front of the chromatogram. If they are too insoluble in the solvent, they will remain at the point of application. Thus, solvents for water-soluble substances are usually water-containing organic liquids, whereas solvents for substances soluble in organic solvents but insoluble in water are often aqueous solutions of organic solvents.

Munier & Machebœuf et al. (1 a, 14, 40 to 49) investigated a wide variety of solvent systems for chromatography of a number of classes of organic compounds. They also tried to establish rules for selection of solvents based on the dissociation constant (K) of the substance. In case of basic substances, such as alkaloids and synthetic narcotic amines, the classification is as follows:

pK of base

Solvent type

>2 and <3
Neutral or acid
>3 and <7
>7 and <12
Acid or alkaline

The systems containing an alcohol and water with or without the addition of an organic or mineral acid were considered by Munier & Macheboeuf as most satisfactory. Neutral solvents were generally thought to be unsatisfactory for base separations, but in combination with a buffered stationary phase they were found to be quite useful (47, 48). Many subsequent investigators have confirmed this and have often used butanol (BuOH *) in combination with other polar and non-polar solvents.

D. Solvents used for Narcotics

Since the first publication in 1949 on paper chromatography of alkaloids by Munier & Macheboeuf (40), approximately eighty papers on narcotics have appeared. In this literature, about seventy-two different acid, neutral and alkaline solvent systems are recommended for use in separating forty narcotics and related compounds. These solvent systems have been classified along with the R f values and references in tables II A, II B, II C, IID and IIE. ** About forty-five of these solvent systems contain an organic-solvent-phase, acid and water in various proportions, while another twenty-five are neutral. They contain non-polar or polar organic-solvents either alone or in combination with varying amounts of water. Fewer alkaline solvent systems have been recommended for narcotic analysis. In the following classification, the three major solvent types have been subdivided into three main groups - acid, neutral or alkaline.

* n-BuOH or iso-BuOH, Chemical Abstracts abbreviations for solvents, are used throughout the text.

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FIGURE 2. Apparatus for circular paper chromatography

A: 9 3/4 " Dia. x 3"
D: 4" Dia. x 2 1/8"
B : 10 1/4" "
E : 9 3/8" Dia. x 3 1/4"
C : 3/8" Dia. x 1 3/8"

Acid Solvents


  1. Acetic acid + BuOH * (19) and other alcohols (5)

  2. Acetic acid + acetate + BuOH (11) and other alcohols (3)

  3. Formic, citric, propionic acids + BuOH (6) and other alcohols (3)

  4. Hydrochloric acid + BuOH (8)

Neutral Solvents

  1. Alcohol + aqueous phase (10)

  2. Ethers, ester, etc. + aqueous phase (2)

Alkaline Solvents

  1. Ammonia + alcohol + water (3)

  2. Aqueous amine solution (5)

  3. Ammonia + ether organic solvents (1)

The most popular acidic solvent is BuOH: AcOH : H 2O. Nineteen different combinations of these constituents have been tried on about a dozen opiates. It is thought that esterification occurs in the preparation of this system, and some authors (19, 71, 72 a) have controlled this process by adding acetate to the solvent, and sought thereby to obtain better separation of the narcotics. About fourteen such acetate-containing systems have been studied. Thies & Reuther (71, 72 a) claimed that improved separation of papaverine and narcotine can be demonstrated and that demixion can be avoided by the addition of acetate. Storage of acetate solvents for a period of four months without a sign of turbidity has been reported.

E. Detection of Solutes on Paper Chromatograms

Several methods are available for revealing the position of colourless substances on a paper chromatogram. The more important of these are:

  1. Spraying or exposing to the vapours of a chromogenic reagent to yield coloured spots or zones;

  2. Exposing to ultra-violet light to reveal substances which fluoresce, or quench the faint paper fluorescence;

  3. Locating radioactive centres or atoms by means of a Geiger-Mueller counter or by autoradiography;

  4. Hydrolysing the physiologically "bound" narcotic by an enzyme to release the narcotic.

By far the largest use has been made of a sensitive chemical reagent for detection of the narcotics. Details of these reagents and other methods of detection of narcotics are given in the following paragraphs.

For ease of handling, these tables have been placed in a pocket at the end of the Bulletin.

Numbers in parentheses are examples reported, not references.

i. Chromogenic reagents for narcotics

  1. Dragendorff's reagent has been widely used as a chromogenic agent. There are a number of preparations known by this name, all of which seem to be modifications of the original formula which were adapted for purposes of paper chromatography by Munier and Macheboeuf (40-49) and listed by Block, Durrum & Zweig (81). This reagent has found the most widespread use in narcotic identification (6, 7, 11, 12, 14, 17, 17 a; 25-27, 32, 35, 36, 39, 40, 52, 55-57, 62, 63 a, 71-75). Basic substances usually show orange spots on a yellow background with this reagent. Fischer & Otterbeck (19) recommend washing the chromatogram with well water or with slightly alkaline water (1 to 2 drops 40% NaOH per litre), to produce orange spots on a white background.

  2. Potassium iodoplatinate is equally reliable for narcotic identification and produces blue to violet colours. The following authors (5 a, 9, 18, 21, 29, 40, 50, 64) used a reagent prepared according to Munier (40). Another formula for this reagent was given by Zaffroni (80) and referred to by Buchi (12) for use in narcotic identification.

  3. Iodinevapour (40) and iodine dissolved in petroleum ether (22, 51) yield a brown, unstable deposit on a brown background. The reagent has not been widely used for narcotic identification.

  4. Iodine in KI, known as Wagner's, Bouchardat's or Lugol's reagent and under many other names, has been employed (6, 7, 12, 32, 35, 39, 52) in paper chromatographic detection of narcotics. Bettschart & Flück (7) describe the different shades, which range from yellow-brown to dark brown and blue obtained with opiates. These authors state that the sensitivity of this reagent is less than that of Dragendorff's reagent.

  5. Thallium in KI has been recommended by Munier (40) and employed by Mannering (35)for the detection of narcotics on paper chromatograms. Munier states that this reagent gives yellow spots with a number of alkaloids. Mannering obtained dark yellow spots against a light yellow background with opiates and synthetic narcotics. Narceine gives a dark blue permanent spot. The other spots fade rapidly.

  6. Mercuric iodide in KI, known as Mayer's or Valser's reagent, has been used by Csobán & Hegedüs (16) for detection of opium alkaloids. The spots, after spraying, were washed and mercury precipitated as mercuric sulfide. Kosir & Kosir (29) used modified methods of treatment of the paper after spraying with this reagent.

  7. Potassium iodate in HCI solution called Mohr's reagent and listed in Merck's Handbook (88) has been used by Pfeifer (55), and Svendsen (69) and Miram & Pfeifer (39). Miram & Pfeifer list many of the shades of yellow produced by phenolic opiates with Mohr's reagent.

  8. Potassium ferrocyanide and ferrichloride solutions, known as Prussian or Berlin Blue, or Kiefer's reagent have been found (12, 28, 39, 45, 63 a, 69, 70) to give blue colours with the phenolic alkaloids-e.g., morphine, narcotoline, laudanine.

  9. Nitric acid has been used by Pfeifer (55) and Miram & Pfeifer (39) and gives yellow colours with cryptopine, laudanine, laudanosine, morphine, narcotoline and thebaine.

  10. Potassium permanganate recommended by Pfeifer (55), Miram & Pfeifer (39) and Svendsen & Paulsen (63 a), gives yellow colours with some opiates and narcotics.

  11. Ammonia yields a yellow spot with narcotoline after drying at 100°C according to Pfeifer (55). No other alkaloids give a test.

  12. Ammoniacal silver nitrate has been used for a general chromatographic spray reagent, and gives good tests with phenolics and a less pronounced reaction with some other opium alkaloids (39).

  13. 4- aminoantipyrine with potassium ferricyanate in phosphate solution reacts with alkaloids having phenolic hydroxyls unsubstituted in the para-position - e.g. laudanine, narcotoline - or labile carbon linkages - e.g., coramoline (39).

  14. Diazo-reagents - for example, sulfanilic acid and nitrite -have been employed (39, 55, 77) for detection of opiates and synthetic narcotics, in particular those containing phenolic hydroxyl groups.

  15. Phosphomolybdic acid with morphine on the chromategram in an ammonia atmosphere produces blue colours (69). A similar phenolic reagent for morphine called the Folin-Ciocalteu reagent is used for morphine (70).

  16. Sulphuric acid containing reagents, the classical colour tests - e.g., Marquis', FeCl 3/H 2SO 4, Froehde's, Mandelin's, Wasicky's, Erdmann's reagents and concentrated H 2SO 4 itself (39) - have been adapted to the detection of opiates in paper chromatography.

  17. Hydrochloric acid vapours have been used to give a pink or red colour with porphyroxine-meconidine (5, 38, 39, 78).

ii. Physical methods of detection

Ultra-violet light inspection of chromatograms, alone (2, 3, 4, 24), or prior to spraying with chromogenic reagents, has been widely recommended for detection of narcotics (6, 7, 8, 11, 12, 39, 55, 78). Only a few of the narcotics - e.g., papaverine, narcotine and laudanine, which have isoquinoline moeities in their molecules - fluoresce, while morphine, codeine and thebaine, which have a phenanthrofuran nucleus with varying degrees of saturation, may be located at 2537 ? by their ultra-violet-quenching effect.

The sensitivity of ultra-violet detection depends upon the wavelength of light causing the emission, quenching, or other spectral effects. It would be most helpful to all analysts if authors recording ultra-violet results would specify the maximum wavelength emitted by their source. There is a deficiency of this kind of data in the. literature. The ultraviolet technique for narcotic identification has been extended by spraying the chromatograms with fluorogenic reagents which enhance or modify the ultra-violet effect (5 a, 12, 24). Heating the chromatograms (6, 7, 39) may also change the fluorescence properties of the compound, and may be used in conjunction with ultra-violet inspection.

There are no known examples of the use of radioactive centres in narcotic molecules for their detection in paper chromatography, although undoubtedly this technique is generally applicable.

iii. Biological methods for detection

Seibert et al. (67) describe a method using β -glucuronidase to hydrolyse the morphine mono-glucuronide dihydrate isolated by means of paper chromatography from dog urine concentrate prior to detection of the sugar and alkaloid moeities. The eluate of the chromatogram spot was hydrolysed in vitro with β-glucuronidase (24 hr. 37°C pH 5.0), and the enzyme solution chromatographed. The glucuronolactone was identified by an aniline phthalate sugar spray, and the morphine moeity detected by Dragendorff's reagent. A spray of the enzyme may also be used. In an alternative method, the enzyme may be applied to the spot containing the conjugate morphine glucuronide and the paper incubated and then developed. The glucuronic acid and morphine parts are detected as separate entities as mentioned.

F. Sensitivity of Identification by Paper Chromatography

Sensitivity may be defined as the minimum amount, expressed in gamma, which is detectable by a method. The micro-analyst, for the purposes of paper chromatography, can use the following classification to describe sensitivities - e.g., a trace is an amount less than 1 gamma; a small amount from 1 to 50 γ a large amount, 50 to 100 γ a very large amount, 1 mg or over. The sensitivity of the paper chromatographic method is shown in table IIl. These data represent the collection of the rather limited number of reagents and ultraviolet methods of detection of narcotics which have been studied from the point of view of their sensitivity.

A number of factors influence the sensitivity of detection by use of colour test reagents, as follows.

  1. Composition of the chromogenic reagent;

  2. Technique of application - e.g., spray, dip solution or exposure to vapours;

  3. Chemical treatment of the stationary phase - e.g., electrolytes, pastes and other coatings;

  4. he type of paper, including make and thickness, etc.;

  5. The time of observation of the colour;

  6. The influence of daylight on the stability of the colours.

Sensitivity of detection of narcotics by means of fluorescence and absorbance, which are important physical properties connected with identification by ultra-violet light, are influenced by the wavelength of the source of irradiation - e.g., shade of colour and absorbance - and the technique of observation - e.g., transmitted or reflected light. A more detailed discussion of sensitivity is given by Genest & Farmilo. *

G. R f Values for Narcotic Identification

The ratio of the distance travelled by the solvent front to the distance to the centre of the spot from the origin is the R f.

This discussion is to be found in part Vb of this series; it will be published in a later issue of the Bulletin on Narcotics

Distance travelled by the centre of the spot
R f =
Distance travelled by the solvent front

This simple ratio is a complex function of both the chemical structure of the compound being separated and the chromatographic system. Besides the theoretical relationship between the partition coefficient and R f, there are many experimental conditions which influence its reproducibility. Many workers in the field of paper chromatography have observed the influence of the following experimental conditions on the R f: the temperature; chamber size; volume of solvent; the type of paper, size and chemical treatment; the length of travel of solvent front; the plane of the paper (i.e., vertical in ascending and descending techniques; horizontal in circular techniques); the shape of the chamber; the equilibration time for saturating the paper with the mobile and the inert solvent phases; the form of the compound, salt or free base. General studies on parameters influencing R f were made by Saifer & Oreskes on circular paper chromatography (63) and by Clayton (15).

The effect of equilibration time on R f was observed (5, 6, 7) by measuring,the reproducibility of the R f, or by weighing the paper before and after various intervals of time in the saturated atmosphere of the chamber. The relative volumes of the solvent and the chamber affect the reproducibility of the R f Clayton (15) defined the "critical volume" as the volume of solvent needed to saturate a specific chamber under conditions which would produce a constant R f. He found that 175 ml of BuOH: AcOH: H 2O were just above the critical volume for a 14-litre chamber for chromatographing amino acids. A lesser amount altered the R f value but did not change the order of the amino acids. Although the absolute R f value will vary with experimental conditions, the relative order of compounds on a chromatogram is the same for a given system. For example, the opiates are separated in order of increasing R f value, as follows: morphine, codeine, thebaine, papaverine and narcotine, which order remains unchanged with the variations of the conventional BuOH: AcOH: H 2O system. Gore & Adshead (22) found that the narcotic salts have lower R f values than the free bases, while Resplandy (60) and Buchi & Schumacher (11) reported them to have identical R f values.

In spite of the many factors influencing the R f values, the compilation of these values in tabular form is very useful for identification, provided that reference standards are included on the chromatogram. It is not recommended that R f values be used as a constitutive property of the narcotics unless the conditions of chromatography are rigorously controlled. It is simpler to run the standards, as mentioned above, if available, on the same chromatogram, so that experimental variations may cancel out. Especially when dealing with a large number of compounds, it is important to list R f values for correlation and identification. This has been done in Tables II A, B, C, D and E. *

The R x value is sometimes more conveniently reproducible and useful than R f.

Distance traveled by substance
R x =
Distance traveled by reference compound X

See footnote p. 20.


Reference number




















0.5 2 2 4 2 10 3 1             3   3 35
  5 5 5 5 5                         6.7
10 a
10 a
30-40 a
5 2.5                   5 2.5 2.5   2.5 1.0 29
  15.20                                 62
  5                                 61
  5 5 5 5 5
{20 d}
      10               12
{50 c}
  60 30     15                   15     30
Prussian blue
1                                 28
  1                                 12
  5           10       10     10      
5 a
2 2 2 5 3 35 1 1           2   2   35
  10 10 10 10 10         5               12
13 a
Thallium iodide
10 3 3 7 4 10 3 2           3   5   35
I 2-KI
13 a
  20 15 15 15 15 15       20               12
  20 20   20   20       20               6.7
  200 80 8 5 2 10 40 3           80   50   35
KIO 3 + HC1
50                                 69
Phosphomolybdic acid + NH 3
NaNO 2 + NH 3
510                                 69
1                                 70
Eosine b
0.5 0.5                   0.5 0.5 0.5   0.5 0.5 29
Mayer's + 1% HgCl 2 b
2.5 5.0                   5.0 2.5 2.5   2.5 1.0 29
Mayer's b e
25 20                   15 25 20   10 15 29
Valser's b f
1.0 2.5                   2.5 2.5 5.0   7.5 25 29
Valser's b c
0.5 2.5                   2.5 2.5 2.5   5.0 1.0 29
CNBr + benzidine
5 a
UV (325 mμ)
50 25 15 5 1 20
0.5           50   200   35
UV (? mμ)
50 50  
UV (? mμ)
50   100 20 0.5
UV (? mμ)
30 200 100 15 3.0
      50               12
UV (?: mμ)
13 a
UV (? mμ)
UV (Wood's) g
5 a
UV (Wood's) h
5 a

a Per 10 ml urine.

b On paper str s, not chromatograms.

c pH < 5.8.

d pH > 5.8

e After Water wash.

f HgI 2 + KI + H 2O (+0.5% HgCl 2).

g Before CNBr treatment.

h After CNBr treatment.

In Durchlauf chromatography it is not possible to calculate R f values, since the solvent front has run off the paper, and R x values must be used. Macek et al. (34) used scopolamine as the reference alkaloid, and calculated R Sc (scopolamine) values for a Durchlauf chromatograrm. Goldbaum (21) reported R Cod (codeine) values, Thomas & Roland (73) published R Mor, (morphine) values using ascending and descending chromatographic systems. In this review, for purposes of evaluation of the efficiency of a number of solvent systems, the ratios of R f values for the fastest and the slowest compounds was calculated. These data have been tabulated in table IV, and the R f papaverine : R f morphine ratios given.

The results in table IV show that the opiate R P/M ratios vary from 1.2 to 28.3 for a wide range of chromatographic systems. This ratio is one measure of the effectiveness of separation achieved by a given system. Within certain limitations, the higher the R P/M value, the better the separation. A high ratio does not always mean that a good separation of all major alkaloids has been achieved. It does mean that a good separation of the pair has been achieved. R f values for ether solvents have not been included, since their R P/M = ∞. Bettschart (6) showed the R f values of papaverine and morphine to be 0.68 and 0.0, respectively, while codeine and thebaine, which cannot be separated from morphine in this system, still show infinite R P/M ratios.

It is necessary to establish the objective of the given chromatographic system before condemning it on the basis of a low R P/M ratio. An example of a useful system which has a low R P/M ratio was reported by Asahina (2, 3). His objective was the quantation of morphine alone. All the other major opiates with Asahina's solvent remain at approximately one location on the paper. Morphine is separated. The opposite situation obtains in the Zaffaroni system (79, 80). Reichelt (59 a, 59 b), using the paper impregnated with formamide containing ammonium formate (8%) and CHCl 3 as a solvent, found the R f values of 0.00, 0.21, 0.70 and 0.88 for morphine, codeine, thebaine and papaverine respectively. The R P/M value would be infinite in this example, and represents a very good separation of these four opiates. Many other examples of the usefulness of R f, R x ratios in paper chromatography can be found in the literature.


R f papaverine/R f morphine for various systems


Rf V


Rf I

Pre-treatment of paper

BuOH : AcOH : H 2O
1.3 ,,
1.3 [ *]
BuOAc : BuOH : iso-BuOH : AcOH : H 2O
1.4 ,,
BuOH : HC1
1.45 [ *]
1.45 ,,
Iso-AmOH : NH 4OH
1.5 ,,
BuOH : AcOH : H 2O
BuOH : AcOH : H 2O
1.7 [ *]
BuOH : AcOH : H 2O
DOBRO (18)
1.8 ,,
BuOH : AcOH : H 2O
Buffer pH 3.4
BELLES (5 a)
BuOH : HCl
SUN (68)
1.8 ,,
iso-PrOH : H 2O
BuOH : AcOH : H 2O
BELLES (5 a)
BuOH : HCl
PrOH : H 2O
DOBRO (18)
2.0 ,,
Buffer pH 3.0
2.1 ,,
iso-BuOH : AcOH : H 20
2.2 ,,
PrOH : H 2O
2.3 ,,
BuOH : H 2O
McIIvaine pH 6.8
BELLES (5 a)
3.1 ,,
BuOH : phosphate pH 6.5
s-BuOH : H 2O
Iso-AmOH : AcOH : H 2O
CURRY (17)
BuOH : H 2O : citric acid
4.3 ,,
iso-BuOH : AcOH : H 2O
4.7 ,,
dioxane : HCOOH : H 2O
4.9 ,,
s-BuOH : H 2O
BuOH : H,O
BuOH : H 2O
5.6 ,,
BuOH : H 2O
McIIvaine pH 5.0
5.6 ,,
BuOH : H 2O
Citrate pH 5.5
6.7 ,,
7.4 ,,
CHCl 3: BuOH : H 2O
Citrate pH 4.0
SUN (68)
BuOH : H 2O
12.0 [ *]
BuOAc : BuOH : AcOH : H 2O
BCHI (11, 12)
Iso-BuOH : Toluene : H 2O
Kolthoff pH 3.5
BCHI (11, 12)
28.7 [ *]
Iso-BuOH : Toluene : H 2O
Kolthoff pH 3.5

* Corresponds to
  1. Specific problems in the analysis of narcotics

The literature mentioned in this review contains many examples of the application of paper chromatography to practical problems of narcotic identification, encountered in different parts of the world. Some examples to illustrate this international aspect of the analytical problems and the wide use of paper chromatography in their solution have been selected for detailed discussion. These examples also illustrate the use to be made of the tables II A, B, C, D and E for selecting the appropriate solvent system most suitable for the separations which are inherent in the problems. First the papers are grouped under four headings: pure compounds; analysis of pharmaceuticals; natural products; forensic, toxicological and drug metabolism studies.

A number of publications (1, 1 a, 5, 6, 7, 13 b, 13 c, 16, 17, 17 a, 18, 19, 22, 23, 26, 27, 29, 30, 31, 34, 37, 38, 40-49, 50, 51, 52, 54, 55, 59, 60, 62, 63, 64, 64 a, 65, 66, 67, 71, 72 a, 73, 77) provide the necessary data on pure compounds to establish a variety of paper chromatographic procedures. In most of these studies of pure drugs, the narcotics were investigated in combination with other pharmaceuticals-e.g., alkaloids, analgesics, local anaesthetics, etc. Problems in the pharmaceutical field which require analysis of tablets, injections, etc., containing pure materials, are probably the simplest ones. Paper chromatography of medicinal narcotic preparations have been reported (5 a, 8, 12, 14, 32, 33, 36, 57, 58, 59, 59 a, 59 b, 62, 63 a, 65, 74). The related problems of analysis of papaver somniferum and its preparations (2, 4, 5, 8, 11, 12, 13, 25, 33, 39, 57, 60 a, 69, 70), papaver setigerum (4, 28 a), erythroxylon coca (13 a), cannabis sativa L. (4 a) and papaver rhoeas (5, 78) have received attention. Applications of paper chromatography have been made in problems of analysis in forensic science laboratories where narcotics and other substances of toxicological importance isolated from tissues have been determined (5 a, 17, 18, 19, 20, 28, 54, 61, 75). Still other problems in the related biochemical fields of drug metabolism and addiction have been investigated (5 a, 9, 20, 26, 27, 35, 53, 67, 75).

In the next sections four examples are given, with some discussion of the kind of drugs involved; the special problems of separation; the way in which the problem is solved and those which remain to be solved.

EXAMPLE I. - Problems from Narcotic Control

It is obvious that no single solvent system will be able to separate all the narcotics in the International and Canadian Narcotic Schedules, and this will not be necessary since only a limited number are of practical importance in legal cases. For example, in the laboratories of the Food and Drug Direc- torate in Canada, the narcotics most frequently encountered in police cases 1949-1954 leading to convictions for illegal possession of drugs are listed in Table V.

These narcotics are presented to the Food and Drug Laboratories for analysis in various forms. A similar heterogeneity of samples has been reported by Dobro & Kusafuka (18) in the Far East Criminal Investigation Laboratory, Tokyo Japan. The type of samples encountered by them were "decks" (paquets) of narcotics, needles, syringes, cotton, pipes, cigarettes, etc., containing small amounts of the suspected narcotics.


Number of convictions under Opium and Narcotic Drug Act (1949-1954)


Number of convictions under Opium and Narcotic Drug Act (1949-1954)


Number of cases

Poppy capsules
Derivative of morphine
Hydrocodone (Dicodide ®)
Levorphanol (Dromoran ®)
Alleged drugs
Methadone *

During 1954-1956, six convictions for illegal possession of methadone were made.

The narcotics listed in table V can be chromatographed by the standard iso-BuOH: AcOH: H 2O (10: I : 2.4) solvent system. [ *] In this example, two pairs of narcotics, codeine-hydrocodone and pethidine-levorphanol, have similar R f values and are not readily separated.

The use of table II is demonstrated by the example codeinehydrocodone.

Thirteen solvent systems have been listed in table II for the separation of this pair. The best of these have been selected, and are shown in table VI, which illustrates the solution of this problem of separation.

Another example of a pair of alkaloids which often occur together is papaverine-narcotine. The attention of several workers has been drawn to these alkaloids because of the difficulty of separating them in the solvent systems employing alcohols and water with or without acid (see table II A). If butyl acetate is added to a BuOH: AcOH: H 2O system. a good separation results (71). (R f narcotine, 0.63 and R f papaverine, 0.18.) Bettschart (6, 7) separated opiates morphine, codeine, thebaine, papaverine, narcotine and narceine on buffered paper (pH 3-10) using Et 2O as the mobile phase. Krogerus (32) recommended several solvent systems by which papaverine and narcotine can be separated: Et 2O: AcOH (R f narcotine, 0.85; papaverine 0.53) and dioxane: H 2O : HCOOH (R f narcotine 0.89; papaverine 0.77). Pfeifer (55) separated narcotine, papaverine and narcotoline in Et 2O: NH 4OH. In a later work (39) he used water saturated Et 2O and obtained the following R f values at pH 6.5 and 4.0 (phosphate-citrate buffer) using paper strips: R f narcotine 0.94; 0.73 and R f papaverine 0.87; 0.28. Häussermann (23) suggested 25% (NH 4) 2SO 4 in 0.5 N HC1 as an inorganic solvent to separate narcotine (R f 0.45 and papaverine R f 0.3). A study of the quantitative determination of codeine, thebaine, and narcotine by Holubek et al. (25) in poppy capsules showed that codeine could be separated from narcotine and papaverine on formamide impregnated paper with a benzin: benzene solvent containing 8% NH 4 formate (R f narcotine 0.68: papaverine 0.32).


Systems for separating and identifying codeine and hydrocodone

Solvent system

Rf value





BuOAc :
BuOH :
AcOH :
H 2O
0.37 0.27
Fischer (19)
85 15 40 22      
C 2H 4Cl 2:
AcOH :
H 2O
Vidic (75)
20 8 2        
BuOH :
AcOH :
H 2O
0.62 0.49
Salvesen (63 a)
100 10 50 50      
CHCl 3:
    0.21 0.47
Reichelt (59 b)
BuOH :
CHCl 3:
H 2O
40 10 50   0.34 0.53
Wagner (77)
30 20 50   0.32 0.41  

EXAMPLE 2. - Detection of Narcotic Addiction and Toxicological Problems

Problems of detection of the narcotics employed by addicts and of drugs in tissues in toxicological cases are closely related in degree of difficulty and in the methods used for their solution. In narcotic addiction the problem is to determine the drug employed by the addict. Paper chromatography has been used successfully by Jatzkewitz & Lenz (27) in surveying the urines of out-patients of Munich University Hospital in Germany to detect the drugs used by them. A study of 1,000 urine samples of 462 patients examined for the presence of basic drugs during 1953-1955 detected 62 addicts. The results in table VII show the names of the narcotics and the number of patients using each of the drugs.

The problem of detection of narcotics in the urine of addicts is complicated by the presence of other basic drugs which can interfere in the analysis. These drugs may be used in the course of treatment or may be taken by the addict when the supply of the drug of addiction is not available. Because this study is a typical example of the problems encountered in practical applications some elaboration of the methods employed for separation and detection by paper chromatography will be of interest. These problems are: first, the diffi- culty of separating narcotics from other basic drugs; second, other use of alternative solvent systems to isolate difficultly separable pairs of drugs; third, the use of alternative chromogenic reagents to detect group specific drugs.


Survey of narcotics used by 62 addicts * during 1953-1955 in Munich, Germany


Survey of narcotics used by 62 addicts * during 1953-1955 in Munich, Germany

Drug name

Number of patients per year


Int. pharm.




Morphine (or Dilaudid)
Morphine or Hydromorphone)
7 4
1 4
7 2 2
1 6
1 1
9 10 7
5 1
19 35 14
68 *

some addicts were found to be using more than one drug at a time.

In the following tabulation (table VIII) the opiates of interest and some of the interfering non-narcotic bases are shown with their R f values in two systems I Jand/or I V - BuOH: CHOOH: H 2O (12 : 1 : 7) (S.S. 2043 B paper) and II V, C 2H 2Cl 2AcOH : H 2O (20:8:2) (S.S. 2043 B paper).

In the first two columns of table VIII the R f values for the BuOH : CHOOH : H 2O system as found in two different laboratories; the Clinical Institute of the German Research Institute for Psychiatry in Munich (Max Planck Institute) and the Institute for Legal and Social Medicine of the Free University of Berlin, are shown. The difficulty of obtaining exact reproducibility of R f values between laboratories is obvious in this comparison and was discussed in detail by Vidic (75). The pairs of narcotic substances which are not resolved in this solvent system are morphine-Dilaudid,® Dicodid®-codeine and Dolantin®-Polamidon® The groups of non-narcotic bases and narcotics which are not separable are Cliradon®-Preludin®-Merotonin® and Dolantin®-Ritalin®-Dromoran.® In order to distinguish these substances on the chromatograms, Jatzkewitz recommended the use of a diazotized sulphanilic acid to detect the phenolic group in morphine, Dilaudid, Dromoran and Cliradon. Vidic (75) suggested the solvent system C 2H 2Cl 2: AcOH: H 2O (II V) for resolution of these groups. Reference to table VIII shows that morphine-Dilaudid, Dicodid-codeine and Dolantin-Polamidon were resolved, while Eucodal-codeine were separable according to both authors. Nicotine, which can be readily detected in either the I or II V system, was found occurring by itself in 421 of the 1,000 urine samples.


Rf values for narcotics and basic drugs detected in the urine of addicts


IJ *

IV *


Spray reagent - diazotized sulfanilic acid

Dilaudid ®
Eukodal ®
Weakly brown
Acedicon ®
Weak brown → colourless
Dicodid ®
Very weakly yellow brown
Colourless→ rose
Colourless → yellow brown
Bright brown
Cliradon ®
Luminous orange
Colourless → white on a weakly coloured background
Colourless → weak ochre yellow
Colourless → white on a weakly coloured background
Dolantin ®
Rose → colourless
Dromoran ®
Ticarda ®
Pale carmine → citron yellow
0.71-0.77 **
0.68-0.72 **
Polamidon ®
Pale carmine → citron yellow

"J" refers to the values obtained by Jatzkewitz & Lenz (27), and "V" to those obtained by Vidic (75).

Probably R f for demethylated Ticarda found in urine. Vidic, E., Artzneimittelforschung, 1957, 7, 314-319.

Metrotonin® (N-dimethylphenylisopropylamine HCl), Ritalin® (methyl-phenidylacetate HCl), Preludin,® (2-phenyl-3-methyl morpholine), Pervitin® (1-phenyl-2-methylamino- propane HCl) were found to have R f values close to Dolantin and were not readily identified by R f value in the Jatzkewitz system, I J. This author recommended using the diazotized sulphanilic acid colour reagent, which gives no colour with pethidine and a detectable effect with the others. Dolantin® can be separated from the Preludin®-Pervitin® and Ritalin®-Metrotonin® pairs of non-narcotic drugs by Vidic system, II V.

In this study, ten narcotics were detected in the presence of eight interfering bases by use of two solvent systems. The resolution of pairs of narcotics from interfering bases was achieved by solvent I J, while the individual pairs were separated by solvent II V. In combination with group-specific colour reagents, paper chromatographic techniques were successfully applied to detect narcotics used by addicts.

More than one narcotic substance was detected in the urine of addicts - for example, in two cases, two narcotics and in one case, three narcotics (Eukodal,® Cliradon,® Polamidon®) were found. In the remaining 59 cases only one narcotic was detected. The presence of non-narcotic medicinal bases in the urine of patients in 699 of the 1,000 samples was detected; 301 had one or more bases present, 228 had only one basic drug present. In 88 cases of the 1,000 a narcotic substance was detected; 19 cases of this number were indefinite - i.e., not clearly identified as positively containing narcotics. The findings expressed in part in table VII indicate that methadone and Cliradon® were the most frequently used during 1953, while methadone, morphine and pethidine were popular in 1954. In 1955, methadone and morphine were again most widely used, with no cases of pethidine and only two cases of use of Cliradon.® These results would seem to indicate a more frequent use of synthetic narcotics in the Munich area than in Canada (see table V).

A problem of screening urine samples from soldiers for narcotics was studied by Mannering et al. (35). In this study, 1,103 urine specimens were examined and 412 were found to contain morphine, believed to be present as such, or as deacetylated heroin. Mannering et al. (35) believed that opium alkaloids other than heroin and morphine are of little more than academic interest in forensic medicine. In spite of this remarkable statement, they demonstrated that their chromatographic systems were capable of separating at least seven natural and two synthetic opiates. However, in the application to biological materials, they search for morphine only.

The samples showed additional spots in 42 % of the morphine negative cases and in 54 % of the morphine positive cases. In some of these cases codeine was identified. It is interesting to note that, of 691 morphine negative samples, 36 gave spots with the same R f and colour as the morphine spot; but failed to give positive reactions with Froehde's, Marquis' and Mecke's reagents. These cases were indefinite.

In addition to the narcotic addict survey, the tissues from fifteen autopsy cases were analysed for presence of narcotics. Morphine was identified in extracts from post-mortem bladder urine, livers, kidneys, brain and stomach contents. Blood samples (120) were examined, and none showed sufficient morphine to permit identification beyond a reasonable doubt.

Another toxicological problem involving the detection of antihistamines, narcotics and other poisons has been studied by Belles & Sievert (5 a). Antihistamines are detectable with difficulty in the presence of narcotics by ordinary colour tests. These substances yield similar colour reactions with Marquis', Froehde's and Buckingham's (concentrated Froehde's) reagents. An interesting account was given of cases in which antihistamines taken intravenously were mistaken for heroin. These substances were later correctly identified by paper chromatography.

Goldbaum & Kazyak (21) mention the difficult toxicological problem of identifying microgramme quantities of basic drugs in extracts isolated from organs. Their approach to the problem is based on cross comparison of R f values obtained from four chromatograms, each at a different pH value. No practical application to case work was cited to illustrate the method.

EXAMPLE III. - General Approaches to Analysis

  1. Fischer's Method for Narcotics

Another example of the use of several solvent systems to separate a large number of compounds in one unified proce- dure was given by Fischer (19). He reported four solvent systems (see tables II A, II B, II C and II E) for four-hour separations of nineteen analgesics using a test-tube chromatographic technique.

The most difficult problem encountered by Fischer was the separation of Eukodal® and codeine. A micro test for codeine using FeCl 3 in H 2SO 4 was used to distinguish them since paper chromatographic separation was not possible with these systems. The test was applied to the residue of a CHCl 3 eluate of the spot. The elution step was carried out as shown in figure 3. Fischer's technique again illustrates the common procedure of combining micro tests and chromatography for identification of pairs of drugs which are difficult to resolve. The property of fluorescence of the substances can sometimes be utilized in this way. In routine investigations for hospitals, police officers and physicians, the question of time is an important one, and the Fischer method is short and fits these requirements.

  1. Macek's Method for Alkaloids

A still more general method of analyses is required when a wider variety of bases are to be separated. Macek et al. (34) reported a systematic analysis of alkaloids by means of paper chromatography. They chose sixty-six alkaloids shown in table IX which are representative of the major groups used in pharmacy. The flow sheet shown in figure 4 illustrates the chromatographic group separations into which the alkaloids are subdivided. A sample containing alkaloids is spotted three times on a chromatogram alongside of which are the reference compounds codeine, oxycodone, and heroin. The formamide-impregnated chromatogram is then developed with solvent A, chloroform. The references codeine (R f 0.2), oxycodone (R f 0.35) and heroin (R f 0.8) act as group division markers as shown in the second row of figure 4. Two strips, one containing the standards and the other the unknown mixture, are sprayed with Dragendorff's reagent to position the groups. A further unsprayed strip is cut into sections using the following ranges, group I, R f 0.0-0.2; II, R f 0.2-0.35; III, 0.35-0.80; IV, R f 0.8 as cut-off guides. Ethanol eluates were prepared and group I was chromatographed using the system B Durchlauf chromatography-chloroform-formamide with scopolamine as the reference compound. The remaining two strips of the original chromatogram are reserved for the identification of ephedrine (group V) and ergot alkaloids (group VI).

FIGURE 3. Diagram showing Fischer's elution technique

Full size image: 27 kB, FIGURE 3. Diagram showing Fischer's elution technique


Classification of alkaloids, narcotics and related drugs

Pyridine and Piperidines
Lysergic acid
Isolysergic acid

The R-scopolamine values on the system B Durchlauf chromatogram were measured, and group I alkaloids subdivided into three groups - 1 A, R Sc 0.0-0.1, I B, R Sc 0.1-0.4 and I C, R Sc 0.4. The alkaloids in group I C are identified by R Sc values, examination under ultra-violet light, with spectrophotometric examination and four spray reagents. Again, after elution, groups I A and I B are chromatographed with solvent F, MeOH : NH 4OH (5 %) : C 6H 6 (1 : 1: 2). Groups I A and I B yield further subgroups I AA, I AB, I AC and I BA, I BB, containing the alkaloids as shown in figure 4. If it is necessary, the sub-groups I AA, etc., after elution, are chromatographed using solvent G, BuOH : AcOH : H 2O (4: 1 : 5). The nine alkaloids in group I A and the six in group I B are identified by ultra-violet methods and four different spray reagents.

The remaining alkaloids in the groups II and III from chromatogram No. 1 were further subdivided using solvent G. There are eight alkaloids in group II and eleven alkaloids in group III. Solvent C, formamide/C 6H 6: CHCl 3(1:1), solvent D, formamide/C 6H 6 and solvent E, formamide/C 6H 6 : benzin (1: 1) were used for further separation of group IV of chromatogram No. 1. Solvents B, F, or G, were used to identify ephedrine, group V. Solvents B or E were used to resolve seventeen ergot components in group VI.

For identification of the chromatographed compounds the authors recommend the following reagents: Dragendorff's, sodium nitroprusside, p-dimethylaminobenzaldehyde (PDN-AB), concentrated sulfuric acid; potassium permanganate, cerous sulfate, Konig's reagent, Pauli's reagent, 2,4-dinitrophenylhyrazine, as general and group specific chromogenic agents. The physical methods employed were ultra-violet inspection, fluorescence; direct ultra-violet spectrophotometry of the spots to obtain absorption maxima and minima.

The total amount of each alkaloid required for the complete group separation and analysis of the sixty-six substances was estimated to be 0.5-1.0 mg per compound. *

  1. Appraisal of the paper chromatographic method

Paper chromatography is a rapid, simple, economical and effective means of separating compounds that are difficult to determine in naturally occurring mixtures and complex pharmaceutical products. It also provides small quantities of the desired compounds in a pure state, or with so few impurities that the amount present may be determined by any of the simple instrumental and chemical methods, ultra-violet and infra-red spectrophotometry, colorimetry, non-aqueous titrimetry, polarography, optical rotation, fluorimetry and radiometry.

The main difficulty in routine quantitative determination has been the small quantities separated; time-consuming techniques were required to complete the determination, and the precision was not usually of the order required for control analysis. These disadvantages have been eliminated in two ways - by systems which separate milligramme quantities, and by instrumental methods and micro techniques.

Two other systematic approaches to the analysis of alkaloids and related bases have appeared since this paper was submitted: Curry, A. S., Methods of Biochemical analysis, 1, 39-76 (1959). Interscience publishers Inc., N.Y.; and Waldi D., Archiv der Pharmazie, 292/64, 206-220 (1959).

Full size image: 96 kB

The following statements summarize the main factors concerning paper chromatography.

  1. The R f value is dependent on a multiplicity of factors, and may be difficult to reproduce so that reference compounds are chromatographed simultaneously.

  2. The R f value is a constitutive property of a substance which is a constant like the melting point when it is determined under carefully controlled conditions.

  3. The precision of quantitative chromatography has seldom been discussed. A study by Pazdera et al. (53 a) of a quantitative elution procedure shows variance ±0.5 to ±2.4% depending on the method of quantation of the eluate coupled with chromatography. Block, Durrum & Zweig (81) list qualitative estimates of accuracy which vary from ± 3% to ±15 % and include direct and indirect methods of measuring the compounds.

  4. For qualitative work using an ascending technique (18 a) only one man-hour and 22 hours of elapsed time are required for any determination. For quantitative work the time required for analysis depends on the technique (e.g., descending, ascending, etc.). Using one elution/strip/descending technique, 3.25 man-hours and 8.5 hours elapsed time were required (52 a). In another method where twenty strips carrying replicates of two opium samples in which three substances were determined, 3 man-hours and 25 hours of elapsed time were required (20 a). The elapsed time in each of these cases is mainly taken up by the equilibration and developing stages of the chromatographic process, while the man-hours include the actual working time of the analyst.

  1. The number of compounds which can be separated from a complex mixture depends upon the techniques which are used. An unidimensional chromatogram is limited to about twelve compounds. Macek et al. (34) considered the minimum ΔR f = 0.08 for separation of two compounds. By the use of multibuffered strips of special shapes, as many as nineteen compounds have been separated (39). There is a possibility according to Cramer (83) that some substances which travel with an R f value > 0.8 may not be separated, since the spot may grow in size with increasing R f. This phenomenon depends upon the solvent solubility of the substances. In the conventional BuOH system, there are several narcotics which travel as lines or tight spots and have R f > 0.8 which are readily distinguished.

  1. Summary

A review of the literature on paper chromatography has shown that it can be used in conjunction with many other methods of analysis. These methods have been applied to a large number of practical problems in identifying complex pharmaceuticals, natural products, biological materials, etc. The wide variety of approaches which are possible has been illustrated by practical examples to demonstrate the power, simplicity and economy of paper chromatography.

7. References

  1. ABAFFY, F. & KVEDER, S. (Methadone HCl, Ephedrine HCl, Hyoscine HCl, separation by paper chromatography). Acta Pharmaceutica Jugoslavica, 1956, 6, 209, abstracted in Journal of Pharmacy and Pharmacology, 1957, 9, 630.

1 a. ALLOUF, R. & MACHEBOEUF, M. Chromatographie sur papier par deux phases solvantes agissant simultanement. Bulletin de la Société de Chimie Biologique, 1952, 34, pages 215-227.

  1. ASAHINA, H. & ONO, M. Determination of morphine in opium tincture by paper chromatography. United Nations document, ST/SOA/SER.K/40 (1955).

  2. Ibid. The paper chromatographic determination of morphine in opium. Bulletin of the National Hygienic Laboratory, Tokyo, 1955, No. 73, 59.

  3. Ibid. Quantitative determination of morphine in opium by paper chromatography and spectrophotometry. Bulletin on Narcotics, 1956, 8, No. 4, 39-44.

4 a. ASAHINA, H. Studies on cannabis obtained from hemp plants grown in Japan. Bulletin on Narcotics, 1957, 9, No. 4, 17-20.

  1. AWE, W. & WINKLER, W. Ueber die Alkaloide des Klatschmohns (The alkaloids of Papaver Rhoeas). Archiv der Pharmazie, 1957, 290/62, 367-376.

5 a. BELLES, Q. C. & SIEVERT, H. W. Descending chromatographic behaviour and differentiation of some antihistaminics and alkaloids. The Journal of Laboratory and Clinical Medicine, 1955, 46, 628-640.

  1. BETTSCHART, A. Ueber die Eignung chromatographischer Verfahren für die Trennung von Alkaloidgemischen unter besonderer Berücksichtigung der Tropin und der Opiumalkaloide (The suitability of chromatographic methods for the separation of mixtures of alkaloids in particular tropine and opium alkaloids). Thesis, Zürich, 1954.

  2. BETTSCHART, A. & FLUECK, H. Verteilungschromatographie von Alkaloiden, besonders von Opiumalkaloiden, in gepufferten Systemen (Partition chromatography of alkaloids in buffered systems, in particular alkaloids of opium). Pharmaceutica Acta Helvetiae, 1956, 31, 260-283.

  3. BORKE, M. L. & KIRCH, E. R. Separation of some of the opium alkaloids by surface chromatography. Journal of the American Pharmaceutical Association, scientific edition, 1953, 42, 627-629.

  4. BROSSI, A., HAEFLIGER O. & SCHNIDER, O. Oxy-morphinane, 6. Mitteilung. Die papierchromatographische Bestimmung von Morphinanderivaten und die Verfolgung ihrer Ausscheidung beim Hund (Hydroxymorphinanes, 6th communication. Paper chromatographic estimation of morphinane derivatives and their excretion from dogs). Arzneimittelforschung, 1955, 5, 62-66.

  5. 10 BÜCHI, J. & SOLIVA, M. Die Anwendung der Papierchromatographic in der qualitativen Arzneimittelanalyse (Application of paper chromatography to qualitative analysis of drugs). Pharmaceutica Acta Helvetiae, 1955, 30, 170.

  6. BÜCHI, J. & SCHUMACHER, H. Die papierchromatographische Trennung von Alkaloiden (Paper chromatographic separation of alkaloids). Pharmaceutica Acta Helvetiae, 1956, 31, 417-420.

  7. Ibid. Die Reinheitsprüfung der Alkaloide mit Hilfe der Papierchromatographie 3. Mitteilung: Opium-Alkaloide (Examination of purity of alkaloids by means of paper chromatography, 3rd communication, opium alkaloids). Pharmaceutica Acta Helvetiae, 1957, 32, 273-288.

  1. BÜCHI, J. Die Anwendung der Papierchromatographie in der Arzneimittelprüfung (Application of paper chromatography to the analysis of pharmaceuticals). Mitteilungen der deutschen pharmazeutischen Gesellschaft, 1957, 27, 181-194; in Archiv der Pharmazie, 1957, 290/62 Heft 11.

13 a. BÜCHI, J. & SCHUMACHER, H. Die Reinheitsprüfung der Alkaloide mit Hilfe der Papierchromatographie. 2. Mitteilung: Coca-Alkaloide (Examination of purity of alkaloids by means of paper chromatography. 2nd communication: coca alkaloids). Pharmaceutica Acta Helvetiae, 1957, 32, 194-204.

13 b. CARONNA, G. & BRUNO, S. Saggi cromatografici su carta di alcaloidi (Paper chromatographic assay of some alkaloids). Il Farmaco, 1955, 10, 497-502.

c. CARONNA, G. & SCIALPI, E. Ricerca cromatografica di alcuni alcaloidi (Chromatographic research on some alkaloids). Istituto di chimica farmaceutica e tossicologica dell' Universita di Bari, 1956, 14, parte II, 29-35.

  1. CHEFLET, R. I., MUNIER, R. & MACHEBOEUF, M. Microchromatographie de partage sur papier des acides aliphatiques hydrosolubles et non volatils. Bulletin de la Société de Chimie Biologique, 1951, 33, 840-845.

  2. CLAYTON, R. A. Chamber size and R f values of amino acids. Analytical Chemistry, 1956, 28, 904.

  3. CSOBÁN, G. & HEGEDÜS, I Detection of opium alkaloids on paper chromatograms. Magyar Kém. Folyóirat; 1954, 60, 121-122, Analytical Abstracts, 2, No. 2525 (1955).

  4. CURRY, A. S. & POWELL, H. Paper chromatographic examination of the alkaloid extract in toxicology. Nature, 1954, 173, 1143-1145.

17 a. DEFFNER, M. & ISSIDORIDES-DEFFNER, A Effect of the pH of buffered paper on the R f of alkaloids. Journal of the American Pharmaceutical Association, scientific edition, 1958, 47, 343-346.

  1. DOBRO, M. S. & KUSAFUKA, S. The application of paper chromatography to the analysis of narcotics. Journal of Criminal Law, Criminology and Police Science, 1953, 44, 247-257.

18 a. FARMILO, C. G., GENEST, K., CLAIR, E.G., NADEAU, G., SOBOLEWSKI, G. & FISET, L. United Nations document, ST/ SOA/SER.K58, 1957.

  1. FISCHER, R. & OTTERBECK, N. Zum Nachweis von Analgeticis (Opium-Alkaloiden, deren Derivaten und synthetischen Substanzen) mittels " Test-Tube "-Chromatographie. (Detection of analgetics (opium-alkaloids, their derivatives and synthetic substances) by means of "test-tube "-chromatography). Scientia Pharmaceutica, 1957, 25, 242-248.

  2. FLOOD, H. Über die Herstellung und anorganisch-chromatographische Anwendung von ionenaustauschfähigem Papier (Preparation and inorganic-chromatographic application of paper capable of ion exchange). Zeitschrift für analytische Chemie, 1940, 120, 327.

20 a. GENEST, K. & FARMILO, C. G. Microestimation of opium alkaloids in pharmaceuticals by paper chromatography, Journal of the American Pharmaceutical Association, scientific edition, 1959, 48 (In press).

  1. GOLDBAUM, L. R. & KAZYAK, L. Identification of alkaloids and other basic drugs by paper partition chromatography, Analytical Chemistry, 1956, 28, 1289.

  2. GORE, D. N. & ADSHEAD, J. M. Observations on the paper partition chromatogram as applied to the detection of alkaloids, Journal of Pharmacy and Pharmacology, 1952, 4, 803.

  3. HAUSSERMANN, H. Zur papierchromatographischen Trennung von Narkotin und Papaverin (The paper chromatographic separation of narcotine and papaverine). Archiv der Pharmazie, 1956, 289/61, 303-308.

  4. HORHAMMER, L. & LEUE, K. W. Die papierchromatographische Beurteilung der Tinkturen und Fluidextrakte des DAB 6 (Paper chromatographic examination of tinctures and extracts in the German Pharmacopoeia). Archiv der Pharmazie, 1955, 288/60, 377-388.

  5. HOLUBEK, J., KUDRNÁC, S. & NOVÁK, M. Die Bestimmung von Kodein, Narcotin und Thebain in Mohnkapseln (The estimation of codeine, narcotine and thebaine in poppy capsules). Die Pharmazie, 1958, 13, 95-99.

  6. JATZKEWITZ, H. Ein klinisches Verfahren zur Bestimmung von basischen Suchtmitteln im Harn (A clinical procedure for the determination of basic drugs of addiction in urine). Hoppe-Seyler's Zeitschrift für physiologische Chemie, 1953, 292, 94.

  7. JATZKEWITZ, H. & LENZ, U. Zur Leistungsfähigkeit einer papierchromatographischen Methode beim klinischen Suchtmittelnachweis an Hand von 1000 laufenden Untersuchungen (The efficiency of a paper chromatographic procedure on 1000 clinical routine examinatious on narcotics). Hoppe-Seyler's Zeitschrift für physiologische Chemie, 1956, 305, 53-60.

  8. KAISER, H. & JORI, H. Beiträge zum toxikologischen Nachweis von Dromoran "Roche ", Morphin, Dilaudid, Cardiazol, Coramin und Atropin mit Hilfe der Papierchromatographie (Toxicologic identification of dromoran "Roche ", morphine, dilaudid, cardiazole, coramine and atropine by means of paper chromatography). Archiv der Pharmazie, 1954, 287/59; 224-242; 253-258.

28 a. KLEINSCHMIDT, G. Nachweis des Morphins in Papaver Setigerum, D.C. (The identification of morphin in Papaver setigerum, D.C.). Archiv der Pharmazie, 1958, 291/63, 109-111.

  1. KOŠIR, B. & KOŠIR, J. Dolocanje obcutljivosti novih kromatografskih reakcij na alkaloide (Determination of sensitivity of new chromatographic reactions of alkaloids). Acta pharmaceutica Jugoslavica, 1956, 6, 11-15.

  2. KROGERUS, V. E. & TUDERMAN, L. Eräiden alkaloidisuolojen paperikromatografisesta erottamisesta (The paper chromatographic separation of some alkaloidal salts). Suomen Apteekkariyhdistyksen Aikakauslehti, 1954, 42, 245-258.

  3. KROGERUS, V. E. Eine Methode zur papierchromatographischen Trennung von Papaverin und Narkotin (A method for the paper chromatographic separation of paparerine and narcotine). Suomen Kemistilehti, 1955, 28 B, 117.

  4. KROGERUS, V. E., RAUTIAINEN, I. & WESTERLUND, B. Die papierchromatographischen Trennung der Alkaloide im Tetrapon-Gemisch (The paper chromatographic separation of the alkaloids of Tetrapon). Meddelelser fra Norsk Farmaceutisk Selskap, 1955, 17, 198-206.

  5. KROGERUS, V. E. & TUOVINEN, J. Papierchromatographische Analysierung der Opium-alkaloide einiger galenischer Präparate (Paper chromatographic analysis of some galenic preparations containing alkaloids of opium). Farmaseuttinen Aikakauslehti, 1957, 66, 139-145.

  6. MACEK, K., HACAPERKOVÁ, J. & KAKAC, B. Systematische Analyse von Alkaloiden mittels Papierchromatographie (Systematic analysis of alkaloids by means of paper chromatography). Die Pharmazie, 1956, 11, 533-538.

  7. MANNERING, G. J., DIXON, A. C., CARROLL, N. V. & COPE, O. B. Paper chromatography applied to the detection of opium alkaloids in urine and tissues. The Journal of Laboratory and Clinical Medicine, 1954, 44, 292-300.

  8. MARGASINSKI, Z., SZYMANSKA, A. & WASILEWSKA, I. Chromatography of Alkaloids on filter paper. Acta poloniae Pharmaceutica, 1955, 12, 65-84; Chemical Abstracts, 49, 16349.

  9. MESNARD, P. & BOUSSEMART, E. Separation of alkaloids by partition paper chromatography. Bulletin des travaux de la Société de pharmacie de Bordeaux, 1951, 88, 175-177; Chemical Abstracts, 45, 7298.

  10. MIRAM, R. & PFEIFER, S. Quantitative papierchromatographische Bestimmung von Alkaloiden mittels einer photographischen Methode (Quantitative paper chromatography of alkaloids by means of a photographic method). Pharmaceutische Zentralhalle, 1957, 96, 457.

  11. MIRAM, R. & PFEIFER, S. Zur Papierchromatographie der Mohnalkaloide. 2. Mitteilung: Untersuchung von Auszugen aus Mohn, Opium und deren Zubereitungen (Paper chromatography of poppy alkaloids. 2nd communication Examination of poppy, opium and medicinal preparations of them). Scientia Pharmaceutica, 1958, 26, 22-40.

  12. MUNIER, R. & MACHEBOEUF, M. Microchromatographie de partage des alcaloides et de diverses bases azotées biologiques. Bulletin de la Société de Chimie biologique, 1949, 3l, 1144-1162.

  13. Ibid. II. Utilisation de phases solvantes acides. Considération sur l'importance chromatographique de la constante de dissociation des alcaloides. Bulletin de la Société de Chimie biologique, 1950, 32, 192-212.

  14. Ibid. Essais préliminaires de microchromatographie sur papier de divers alcaloides par des phases miscibles à l'eau. Bulletin de la Société de Chimie biologique, 1950, 32, 904-907.

  15. Ibid. Microchromatographie de partage des bases azotées dans des phases solvantes acides. Comptes rendus hebdomadaires des séances de l'Académie des Sciences, 1950, 230, 1177.

  16. Ibid. Microchromatographie de partage sur papier des alcaloides et de diverses bases azotées biologiques. III. Exemples de séparations de divers alcaloides par la technique en phase solvante acide. (Familles de l'atropine, de la cocaïne, de la nicotine, de la spartéine, de la strychnine et de la corynanthine). Bulletin de la Société de Chimie biologique, 1951, 33, 846-856.

  17. MUNIER, R. IV. Séparation de quelques dérivés nicotiniques et du tryptophane. Bulletin de la Société de Chimie biologique, 1951, 33, 857-861.

  18. Ibid. V. Séparation du constituant azoté des phosphoaminolipides (Choline, Colamine, Sérine). Bulletin de la Société de Chimie biologique, 1951, 33, 862-867.


  1. MUNIER, R., MACHEBOEUF, M. & CHERRIER, N. Microchromatographie des alcaloides et des bases azotées biologiques sur papier traité préalablement par des sels. I. Essais préliminaires sur les "papiers phosphatés ". Bulletin de la Société de Chimie biologique, 1951, 33, 1919-1929.

  2. Ibid. Microchromatographie des alcaloides et des bases azotées biologiques sur papier traité préalablement par des sels. II. Amélioration de la technique des phases solvantes acides. Utilisation de papier imprégné par un sel, dont l'anion est celui de l'acide qui acidifie la phase solvante. Bulletin de la Société de Chimie biologique, 1952, 34, 204-214.

  3. MUNIER, R. Bases azotées (Alcaloides, amines, vitamines) et acides organiques hydrosolubles. Bulletin de la Société chimique de France, 1952, 852-873.

  4. NADEAU, G. Note on the identification of alkaloids by paper chromatography. Clinical Chemistry, 1956, 2, 347-352.

50 a. NADEAU, G., SOBOLEWSKI, G., FISET, L.& FARMILO, C. G Separation and identification of major alkaloids from opium by partition chromatography on paper. Journal of Chromatography, 1958, 1, 327-337.

  1. NAGASAWA, K., KOSHIMURA, E. & OKAZAKI, S. Paper chromatographic estimation of opium alkaloids. I. The determination of morphine. HCl. Bulletin of the National Hygienic Laboratory, Tokyo, 1955, No. 73, 53; Chemical Abstracts, 50, 7393.

  2. NAIMKENT, F. N. & MATEUAMENGUAL, B. Identification and separation of minute quantities of alkaloids by microchromatography on paper. Archivos de farmacia γ bioquímica del Tucumín, 1950, 4, 333-343; Chemical Abstracts, 45, 1724.

  3. PAERREGAARD, P. A new method for quantitative determination of small amounts of morphine in human urine. Acta pharmacologica et toxicologica, 1957-1958, 14, 38.

53 a. PAZDERA, H. J., McMULLEN, W. H., CIACCIO, L. L., MISSAN, S. R. & GRENFELL, T. C. Quality control of pharmaceuticals. Analytical Chemistry, 1957, 29, 1649-1654.

  1. PEDLEY, E. The microchemical differentiation of morphine and nalorphine. Journal of Pharmacy and Pharmacology, 1955, 7, 527-532.

  2. PFEIFER, S. Zur Papierchromatographie der wichtigsten Mohnalkaloide (Paper chromatography of the major alkaloids of poppy). Scientia Pharmaceutica, 1956, 24, 84-92.

  3. RAHMAN, A. A. A. Anwendung der Papierchromatographie auf einige Opium-alkaloide und einige Morphinderivate (Application of paper chromatography to some opium alkaloids and some derivatives of morphine). Archiv der Pharmazie, 1957, 290/62, 321-325.

  4. RAHMAN, A. A. A. Trennung der Inhaltsstoffe der Opiumtinktur Pantopon- und Spasmalginampullen durch Papierchromatographie (Separation of components of opium tincture, pantopon and spasmalgin ampoules by means of paper chromatography). Archiv der Pharmazie, 1957, 290/62, 326-329.

  5. REICHELT, J. Papierchromatographie der Opium-Alkaloide (Paper chromatography of opium alkaloids). Die Pharmazie, 1955, 10, 234-235.

  6. REICHELT, J. Determination of scopolamine and atropine in combined injection preparations. Ceskoslovenska Farmacie, 1954, 3, 330-333.

59 a. REICHELT, J. & SARŠÚNOVÁ, M. Papierchromatographie der Alkaloide IV (Paper chromatography of alkaloids IV). Die Pharmazie, 1958, 13, 21-24.

59 b. REICHELT, J. Papierchromatographie der Alkaloide V (Paper chromatography of Alkaloids V). Die Pharmazie, 1958, 13, 24-27.

  1. RESPLANDY, A. Chromatographie sur papier d'alcaloides par des solutions d'électrolytes. Comptes rendus hebdomadaires des séances de l'Académie des sciences, 1954, 238, 2527-2529.

  2. ROMANO, C. Qualitative toxicological analysis of some alkaloids by paper chromatography. Minerva medicolegale, 1950, 70, 172-174; Chemical Abstracts, 46, 4741.

  3. SABON, F. & MONNET, R. Demonstration of morpholylethylmorphine. Bulletin de la Société de Pharmacie de Bordeaux, 1955, 94, 41. Chemical Abstracts, 50, 17324.

  4. SAIFER, A. & ORESKES, I. Circular paper chromatography. Studies of physical factors that may influence R f values. Analytical Chemistry, 1953, 25, 1539.

63 a. SALVESEN, B. & PAULSEN, A. Papirkromatografisk atskillese og identifisering av noen analgetika beslektet med morfin (The separation and identification of some analgesic compounds related to morphine by paper chromatography). Meddelelser fra Norsk Farmaceutisk Selskap, 1953, 15, 33-43.

  1. SCHMALL, M., WOLLISH, E.G. & SHAFER, E.G. E. Chromatography of organic bases on multibuffered paper. Analytical Chemistry, 1956, 28, 1373-1375.

64 a. SCHUBERT, E. Personal communication (E. Merck & Co.)

  1. SCHULTZ, O. E. & STRAUSS, D. Quantitative papierchromatographische Alkaloidbestimmungen durch Ausmessen der Fleckenfläche (Quantitative paper chromatographic estimation of alkaloids by planimetry of spot area). Deusche Apotheker-Zeitung, 1955, 95, 642-644.

  2. Ibid. Paper chromatographic determination of alkaloids. Arzneimittel-Forschung, 1955, 5, 342; Chemical Abstracts, 49, 12781.

  3. SEIBERT, R. A., WILLIAMS, C. E. & HUGGINS, R. A. The isolation and identification of "bound" morphine. Science, 1954, 120, 222-223.

  4. SUN, Y. T. The paper chromatographic separation of some of the opium alkaloids. Journal of the Taiwan Pharmaceutical Association, 1955, 7, 14; Chemical Abstracts, 50, 17339.

  5. SVENDSEN, A. B. Papierchromatographie und quantitative Bestimmung des Morphins im Opium (Paper chromatography and determination of morphine in opium). Pharmaceutica Acta Helvetiae, 1951, 26, 323-333.

  6. SVENDSEN, A. B., AARNES, E. D. & PAULSEN, A. Zur quantitativen papierchromatographischer Bestimmung des Morphins in Opium (Paper chromatographic determination of morphine in opium). Meddelelser fra Norsk Farmaceutisk Selskap, 1955, 17, 116-123.

  7. THIES, H. & REUTHER, F. W. Verbesserte Lösungsmittelgemische für papierchromatographische Trennungen von Alkaloiden (Improved solvent-mixtures for paper chromatographic separations of alkaloids). Naturwissenschaften, 1955, 42, 462.

  8. Ibid. Reagenz für Alkaloide auf Papierchromatogrammen (Reagent for alkaloids on paper chromatograms). Naturwissenschaften, 1954, 41, 230.

72 a. Ibid. Das Verhalten von Alkaloiden gegen verschiedene Lösungsmittel-Gemische in Papierchromatogrammen (Behaviour of alkaloids towards various solvent mixtures on paper chromatograms). Arzneimittelforschung, 1957, 7, 63-65.

  1. THOMAS, G. & ROLAND, P. Chromatographie sur papier: essai d'orientation pour I'identification des alcaloides en mélange. Annales Pharmaceutiques françaises, 1954, 12, 318-320.

  2. TSUNEMATSU, F. & SAKURAI, H. Determination of atropine in atropine-opium alkaloid injections. Japanese Journal of Pharmacy and Chemistry, 1951, 23, 248-250; Chemical Abstracts, 45, 8203.

  3. VIDIC, E. Die Anwendung papierchromatographischer Methoden beim forensischen Suchtmittelnachweis (Application of paper chromatographic methods for forensic detection of narcotics). Arzneimittelforschung, 1955, 5, 291-295.

  4. VITTE, G. & BOUSSEMART, E. Séparation par chromatographie de partage des constituants alcaloidiques d'un sirop. Bulletin de la Société de Pharmacie de Bordeaux, 1951, 89, 83-85.

  5. WAGNER, G. Zur Papierchromatographie und Papierionophorese einiger dem Morphin verwandter Analgetica (Paper chromatography and paper electrophoresis of some analgetics related to morphine). Die Pharmazie, 1955, 10, 470-477.

  6. WINKLER, W. Über die Alkaloide des Klatschmohns (On alkaloids of Papaver Rhoeas). Thesis, Braunschweig 1957.

  7. ZAFFARONI, A., BURTON, R. B. & KEUTMANN, E. H. Adrenal cortical hormones: analysis by paper partition chromatography and occurrence in the urine of normal persons, Science, 1950, 111, 6-8; Journal of Biological Chemistry, 1951, 188, 763-771; ibid., 1951, 193, 749-767.

  8. Ibid. The application of paper partition chromatography to steroid analysis. I. Ketosteroids. Journal of Biological Chemistry, 1949, 177, 109-116.

  1. Textbook references

  2. BLOCK, R. J., DURRUM, E. L. & ZWEIG, G. A Manual of Paper Chromatography and Paper Electrophoresis, 2nd Ed. (1958), Academic Press, New York.

  3. CASSIDY, H. G. Fundamentals of Chromatography (1957), Inter-science, New York.

  4. CRAMER, F. Papierchromatographie, 4. Auflage (1958), Verlag Chemie, Weinheim.

  5. HAIS, I. & MACEK, K. Papirova Chromatografie (1954), Prague. German edition: Handbuch der Papier-chromatographie, Gustav Fischer, Jena, Vol. 1, Grundlagen und Technik (1958).

  6. Journal of Chromatography (1958 ff), Elsevier, Amsterdam.

  7. LEDERER, E. & LEDERER, M. Chromatography, 2nd ed. (1957), Elsevier, Amsterdam.

  8. MARTINDALE, W. H. The Extra Pharmacopoeia, twenty-third edition (Volume 2). The Pharmaceutical Press, London, 1955. Paper Chromatography, p. 506-508.

  9. The Merck Index, fifth edition (1940), Merck & Co. Inc., Rahway, New Jersey.

  10. OPIENSKA-BLAUTH, J., WAKSMUNDZKI, A. & KANSKI, M. Chromatographie (1957), Panstwowe Wydawnictwe Nankowe, Warsaw.

  11. SMITH, I. Chromatographic Techniques, Clinical and Biochemical Application (1958), Heinemann, London.


See part V b.

Systems for separating and identifying codeine and hydrocodone