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6-1-1973

The Use of Thin Layer Chromatography (TLC) inQualitative and Quantitative Determinations ofPhotographic Developing Agents (in DeveloperSolution) Using a Scanning DensitometerMichael Gilbert

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Recommended CitationGilbert, Michael, "The Use of Thin Layer Chromatography (TLC) in Qualitative and Quantitative Determinations of PhotographicDeveloping Agents (in Developer Solution) Using a Scanning Densitometer" (1973). Thesis. Rochester Institute of Technology.Accessed from

THE USE OP THIN LAYER. CHROMATOGRAPHY (TLC)

IN QUALITATIVE AND QUANTITATIVE DETERMINATIONS

04 PHOTOORlPIiIC DEVELOPING AGENTS

(IN DEVELOPER SOLUTION)

USING A SCANNING DENSITOMETER

by

Michael B. Gilbert

A thesis submitted, in partial fulfillment

of the requirements for the degree of Bachelor of Science

in the School of Photograehy

in the College of Graphic Arts and Photography

of the Rochester Institute of Technology

June, 1?73

mvjpg i g Ad_vi st- 'Dr> Ron.a l r"i p"ovic^ s

ACKNOWLEDGEMENTS

The author would like to thank Dr. Schumann, Dr.

Francis, Professor Carroll and Professor Hill of the

College of Graphic Arts and Photography, School of

Photography at the Rochester Institute of Technology;

Dr. Craven Dr. Sowir.ski and Dr. Burns of the College

of Science at the Rochester Institute of Technology;

Elizabeth.Solow,

Assistant Processor, division of

Neurological Surgery, Ind.iana University School <X Medicine

at Indianapolis; Dr. H. Baden oC the Eastman Kodak Company,

Photographic Technology Division at Rochester, New York;

Richard Bilhartz and Barbara Roberts, my Xancae, for their

assistance with the research and preparation of this thesis

Special acknowled.genent is d.ue to the C.I. A. for

financial si'pport for the research of this thesis.

No'ibart Of this thesis may be reproduced in any form

wit-iout permission from the auchoj.-.

TABLE OF CONTENTS

List of Tables iy

List of Figures v

List of Graphs vii

Introduction 1

State of the Art 1

Theorv 1 c-

Objective of Thesis 2k-

Objective I 2^

Procedure 25Observations 27Data and Calculations 30

Conclusion 32

Objective II 86

Procedure 3^

Observations 37Data and Calculations 4oConclusion 80

Obi active III li-2

Procedure -i-2

Observations 57Data and Calculations 9S

Conclusion 6o

Bibliography 70

Aprenyl ices 73

Appendix A 7L

Aonendix 8 79

LIST OF TABLES

Table I Volume of stock solutions I and II used to form

ulate developer solutions 1-4-1-8 ^3

Table II Factorial Experiment for runs 1-X8 k-k

Table III Conversion of Absorption units to Density on.

520 TLC Platen 56

Table IV Density Maxima Dm(l), Dm(2) and Dm(3) for runs

1X-8

'

58

IV

LIST OF FIGURES

Figure 1 Devices used for depositing samples from

prepared solution k

Figure 2 Preparation of Chramatoolates 5

Figure 3 Dimensions of Precoated. Chromaufcogra 6

Figure ^- Application of Samples to Thin L^yer Chroma -

togram 7

Figure 5 Elution Apparatus 8

Figure 6 Application of Visualization Reagent with

; atomizer 9

VltrilriO 1 P. ov,^ O irnlunr. 1^

i'

v~ -"-

Figure 8 Two Dimensional Chromatography 12

Figure 9 Adsorption isotherm of a subntan.ee under

real and ideal conditions 18

Figure 10 'Dens i tome trie Traces 19

Figure 11 Adsorption isothermof two different sub

stances 21

Figure 12 TLC of photographic developing age^t^ 18

prepared in aqueous solution X_th inactive

silica gel used, with n-butanol . aceti c acid:

water solvent svstem 2Q

Figure 13 Step-wise IonizatX"0 of Hyd.roqniuone to a

Qu inone 32

Figure Ik Oxidation of Hydroouinone to a pra benzo

.quinone 33

Figure 15 Formation of hydraauinone monosulfonate 33

j? i rr-, -, ro i_ 8 T-.ro Dimen z i ^1"1 a I T LC 9

v

Figure 17 Use of streaker twice for quantitative ap-

plicati ons of sample to the chro^atomra"! kb

Figure 18 Application of streaker sample to the Chro

ma, togram k7

Figure 19 Uneven and Even Solvent Front ^8

Figure 20 Photovolt TLC Densitometer 520 50

Figure 21 Difference in visual appearance of chroma -

togram after visualization 52

Figure 22 Illustration of how, density maxima Dm(l),Dm(2) and. DX( 3 ) we"9 obtained for species

Rf(l), Rf(?J ^Xl Rf(3) respectively 55

Figure 23 Visualization reagent a.policator for Thin

Layer Chromatography 61

Figure 2k 5D-2000 Chromatogram Analyzer 75

Figure 25 Schematic diagram of a modern split beam

TLC scanner 76

vi

LIST OF GRAPHS

Graph I Log density vs. concentration of hydroquinone

for runs 1-8 6k

Graph II Log density vs. concentration of hydroquinone

for runs 8-16. 65

Graph III Log density vs. concentration of hydroquinone

for runs 17-28-

"

66

Grph IV Log density vs. log concentrationo^ hydro-

quinoue for runs 25-32 67

Graph V Log density vs. concentration of hydroauinone

for runs 33-X)

" "

68

Graph VI Log density vs. concentration of hydroquinone

for runs Ll-48"

69

VI

THE USE OF THIN LAYER CHROMATOGRAPHY (TLC)

IN QUALITATIVE AND QUANTITATIVE DETERMINATIONS

OF PHOTOGRAPHIC DEVELOPING AGENTS

(IN DEVELOPER SOLUTION)

USING A SCANNING DENSITOMETER

by

rticnaei a. Gilbert

An Abstract

TLC was used, for qualitative and quantitative

analysis of photographic d/eveloXag agents hydroquinone,

metol phenidone para, phenvlsned.iam.ine, para aminopheuol,

ascorbic acid and. the oxid.ized. species. Experimental r-on-

ditions and procedures for reporting Rf rallies of the

aqueous solutions of the developing agent were standardized..

Quantitative TLC w-ap done fc" hydrocuinone . The den-

cifv nnca -v i mo0^+-

a T v) ar. -pr>^m-f-

Via "nannpTitr r.onc;T_+-r\'T1^itrir> hrq/.o

of the e luted and visualized chromatogram was correlated, to

the concentration of the hydroauinone. Aoiieous solutions

of.hydroquinone were prepared at four levels of concentre

tion, three levels of pH and two levels of sodium sulfite

concentration for quantitative analysis using TLC.

INTRODUCTION

Recently, in the past twenty years, thin layer

chromatography has played '^n important roLe in au.alitative

and quantitative analysis of many tyoes of substances.

Substances to be analyzed, were complex organic oomoound.s

occurring in nature or synthesized by man. LLC's role in

biological research has been immense and. is an indespensi-

ble tool in laboratories in maPor hospitals.

The chief advantage of TLC, is that it requlresvvery

'little sample, usual ]v miaraarams . to perform the oualita-

tive analysis. In the past ten years, attention in research

has been drawn to quantitative analysis of compounds using

TLC, but much more success has been gained in the field of

gas chromatography. Gas chromatographic instrumentation is

complex and much more costly than TLC instrnmerits

State of the Art

In the 105C s, published papers appeared describing

qualitative determinations of photographic developing

agents 'jsinm paper chrom-atc^a o^'v,~ Mariari 's and

[8arti.nel.liLs a.rticle rave the best description op

experimental

la. Russel p/ip-tiMq 1 Mi a l vol c; i y\ pv. nt ,-m'",i ni"-" ahap4C"'' 8

conditions they used for paper chromatography. The

LuValle and. Pannel paper did. not describe experimental

conditions.^ Conditions ofLuValle'

s and.Pannel'

s ex

perimentation were not explicitly defined and. made it

difficult to repeat their results. The author of this

paper found two references to published papers involved

with qualitative determinations of photo chemicals using

pacer chromatography.

After WoX d War II, agon Stahl did Intense, research

in establishing a near type of chromatography. It was

similar to the older paper chromatography but had. distinct

advantages. Thin layer chromatography was coming of age

but did. not achieve recognition untilStahl'

s textbook was

^ km iVi 1

.cViqH -.v. 1 OKI

"This boo't systematizes its voluminous

literature and applicable method.3 so that the

text can be used as alaboratory

haud.book.

TLC can ba used for organic a,rd inorganic

mixtures ranging from 10~^ to10--'

gram and

has been applied to a large group of complex

compounds: d_rums, amino acids, natural and

SE;rnthetic products, vitamins, steroids, dyes,S 1 1 ">-o -pq "8

o

'3. Mari.ani qnd p # Martinelli, Sci. Ind, Photon-. (Fr. ) ,

p. 276.

-* J . A. Pannel and.

.T.S,LuValle Analytical Chamistry,

Vol. 2 5, p. 1566.

^Thin Layen Chromatography--A Laboratory Ha.nd.boo1',Edited bv Egon Stahl

,

5"Thiu Layer Chromatography ,

"Researoh/Develop^-ant

,

pp. 36, X

Reference was made in Stahl Ls textbook to qualitative

analysis of photochemicals In a published paper by J.

Eggers, separation and. detection .conditionswere not re

ported for mixtures of o, m, and. para aminophenols and

N-methyl-p-aminophenol.7Stahl suggests that TLC of

phenols and their sulfonic acids should be selected with

acidic solvents, of high polarity. Solvent systems sug

gested by Dr. Baden of the Eastman Kodak research labora

tories in Rochester, New York, will be listed in Appendix

B.

In qualitative determinations of photographic develop

ing agents using TLC, the Rf value of the developing: agent

is dependent upon the quality of the solvent system,

chroma i, ogram and. activity, Otherdeveloping*

agents

should not have an identical Rf value. There are compli

cations; : Foremost, the sample of -developing agent pre

pared, in aqueous solution requires a preservative or anti

oxidant to enable it to remain unoxidized and potent.

For this reason, sodium sulfite is used in developer form

ulations. ,Photographic developer solutions that develop

silver halide emulsion require adjustment in a pH range

where the developing agent. can ionize. It is good, sense

"Stahl, oo. cit.

7j. Eggers, Photo, u. v/iss., 10, kO (1961).

k

("> J ..1.,!v,ri/O prepare soiUoions or. develop! at and specify con

ditions of preparation. Once prepared, a small sample

size of the Photographic developer can be taken by a micro

pi pet, syringe or streaker, and. d.eoosited on the chroma-

togram. Preliminarily, the sample size taken should con

tain 1 to 5ug'

of the developing agent for qualitative

analysis .

L-

'4T*--

V

1

A- .- -_-_i-___i*i ......... ......

......

Figure 1

Devices used, for deoositinm samples from prepared solutions

A chroma t ora,m is a coating of silica rel alumina

d iatom i.ceous earth, Xies-Xgur or ox pies of metals that are

absorbents coated, on a support. This support usual l.v is

p;lass ,

Tray to holdg/osi platts

Slurry being poured into the applicator

(When handle it turnvd at itatwi, slurry itO'Kftoffed ftom itauom of eppticctor, whichit pulled alonf plo-.tt produc-ng a film, theUiitkneil tteinf gowned by tht flam**)

(a) Preparation of larger chromatoplatej

(I) Applying a small amount ofa slurryto o clean slide

(ii) Spreading by means ofanother slide

Figure2"

Preparation of Chromatoplates

It-

the past, coating was done in the laboratory to a spe

cified, quality of absorbent and coating thickness. This

is a teddous procedure hi.it recent lv the advent of ore

coated substrates of silica ^.Ip-iina aud pal layers

have eliminated, this burden.

26.

David Abbott An Intro iuar.i on t.o ijhv,n'rj.r/r),'rT'-'i,.^v r>

Figure 3

Dimensions of Precoated Chromatogram

Prior to sample deposition, it may be necessary to acti

vate the chromatogram by heating it in an oven. Water

from, the atmosphere can be absorbed, by the chromatogram

substrate and. form an immobile phase. It will also af

fect adsorption properties of the substrate to the depos

ited sample. Once the sample is deposited on the chromato

gram substrate, it is dried. Forced, (heated) air drying

may oxld.ize the developing agent.

7

uwwm(a) Taking a sample from a

clearly labelled tube(b) Applying the sample to paper

(c) Streaking a mixture for preparative

chromatography(d) Drying the spots on the paper

9Figure k

Application of Samples to Thin Layer Chromatogram

Elution in the appropriate solvent system is the next

step. The chromatogram is placed in the confine of a ves

sel that has the solvent. This will ensure an atmosphere

saturated, with solvent vapors. Otherwise there will not

be an even front travel.

Ibid. ,

p . 10

8

Solvent

Plate in the course ofdevelopment

a)Tank10

b) Chamber PMtes

Figure 5

For precoated.flexiblesupports, chamber plates have bet

ter properties over, other closed, vessels. During the elu-

tlon, the apparatus should be isolated from any temperature

or: humidity changes since this will affect the rate and

evenness of travel of the solvent system and the ad.sorption

of the developing agent to the chromatogram substrate.

The elution-of the solvent system in the chamber plate is

ascending. After a period of time, when the solvent front

has migrated up the chromatogram -a 'distance, the elution

is stopped by removing the chromatogram from the confines

of the apparatus. The position of the solvent front is

noted.

Visualization of a compound is necessary if it is not-

visible'. Photographic developing agents can be made vis

ible bv spraying the chromatogram with ammoniacal ,IN

lIbid. , p. 28.

silver nitrate. Spraying with an atomizer will make de

veloping agents visible so that measurements for ?_f values

can be made. Application of the visualization reagent for

qualitative purpose should, be sufficient to make the sub

stances visible. If too much visualization reagent is ap

plied, the sharpness will decrease. For quantitative pur-

#

poses, little is found, in the literature about the amount

of visualizing reagent needed.

Spots

which have

been located

Position; ofspots which

have yet

to be located

(b)

Weight to keep paper f.._

4

Compressed oir

or hand bellows

44

4JJSolution of

locating reagent

JITip -r-n ro 8

'

Application of Visualization Reagent with Atomizer

Before spraying with, the visualization reagent and.

taking measurements, the ohromatogram should be air d.ried.

Cqye *'.ras taken when mixing and spravihT the silver nitrate

Spravi.ng was done under a blower hood.. When finished, the

ovcess oilirer nitrate wan disposed of immeai.ate.lv.

1 1Ibid

. ,p . 1 6

10

n {> vaivioo i w i cac! 'I.OVGJ.UMJ.Ui a^OHU O'. I--.1 k-/^ P.. ^ iy ^ 1 iA-1

bymeasuring the ratio of distance the developing agent

traveled after eluti on and the distance the solvent front

traveled.

The Rf value

Point ofapplication

. . ..

"

< i

,

.

'. ).<_ Centre/ ofspot

So.vent ' .

front

<v=i

The Rx value

Points of application

ap* ;.

*-Spots of-

standard substance X

R*=*-

1 2Figure 7

"

Rf. and R Values

1 a

A variety of visualization reagents are available.

Two dimensional TLC can be used to improve separation

of developing agents prepared in. one solution. Some deveL

oping agents may have similar R valuesfor

a particular

chromatogram substrate and solvent system. Two dimension

al TLC is done by allowing the chromatogram (with sample

12 rbid., p. 17.

-3 S.K. Bibliography on. visualization reagents

11

spotted in the lower left hand corner) to elute in the

ascending direction for a period, of time. Afterwards,

the chromatogram is removed, dried and. rotated

90'

counter.

clockwise and placed in another solvent system. Elution

in the second solvent system is continued for a period of

time and then stooped. Two Rf. values, for the first and

second elutlons, are used, to specify the v. and y coordi

nates on the chromatogram where the d.evelooing agents sep

arated to.

Steps In Two Dimensional Chromatography:

1. sample preparation and. deposition.

2. activation

3. first elution

k. drv

5. rotate90'

counter-clockwise

6. second elution

7. dry

8. visual! act ion

STTPS 3+51

IP

SOLVEWT SYSTEM X SOLVENT STfttfT^X

ro 1 at

visua

taini

e rabl

ardi z

el iX4

ar^ s

curve

penen

soot

m a a ^ 1 :

togra

howe"

the m

usingprnnan

trica

Figure 8

Two Dimensional Chromatography

"While TLC qualitative analysis are

T vo 1 V QimTilr^ to ri ryiio-ino*

T-l-o-nHo mri o

.. .-----__'

/

lization reagents and Rf Tralues,ob~

n^ quantitative information is cons id. -

v niore d.jfficult. .,8a completely stand

ing conditions of sample size and TLC

on, conditions and calibration stand-

can be used to provide calibration

s of spot area versus anoint of corv

t. The amount of the ma.teri.al in a

can also be determined an. th a rl3i"

ityreman t obtained, by photometric or aho-

phi cmc' a

,n a The most accurate results

er are those obtained by extrncti on of

ateria.l in the spot from th absorbent

as solvent an d then date r^ i rp r g the

t colorimetrica,lly or speetroohotome-

1 1nlV

TLC densitometry has been advanced, by introd.u.c.ti on of

Jj-i i.f

Development, k.

"Phiai Laver Chromatography "

Research f

13

'the split reference beam. This has overcome many of the

variables leading to inaccuracies. Determinations can be

made with relative standard deviations of two to three per

cent. Quantitative evaluation of thin layer chromatograms

by conventional methods without extraction of separated

substances are discussed inStahl'

s textbook, Thin Layer

Chromatography- -A Laboratory Handbook.

Theory of Thin Layer Chromatography

In'^TLC, separation of compounds is obtained as it

travels with a migrating solvent through a thin layer of

adsorbent material coated on a glass or flexible support.

Separation of compounds is due to adsorption, partition

or reverse phase mechanisms.

The most common mechanism Is adsorption which Is use

ful for separation of lipophilic organic compounds of medi

um or low polarity. Silica gel and alumina oxide are used

for adsorption chromatography. The material being separa

ted is proportioned, between the migrating solvent system

and the active adsorption sites in the adsorbent. Highly

polar compounds will be strongly adsorbed. . The polarity

of organic compounds, such as photographic developing a-

gents, will be established by the type and number of func

tional groups that have the ability of forming bond.s with

hydrogen.

Partition TLC is useful for polar, water soluble,

Ik

inorganic compounds,, Unactiva4ed silica gel, carrying an

adsobed phase of water, can be used, In partition TLC,

a proportion is established, between the migrating solvent

system and the adsorbed stationary mobile phase. Separa-

tion of substances is similar to factors governing a

separatory funnel extraction, where relative solubilities

..,-. MP*

'

(hence, thepartition'

coefficient ) of the substances de

termine this proportion.

. Reverse phase TLC. is efficient in separating homol

ogous lipophilic compounds. This is a less frequently

used,mechanism.

15

Theory

Consideration of these meahanisms is necessary in

selecting the right migrating solvent (solvent system),

chromatogram substrate, pretreatment (activation) and.

elution conditions J 5

The Rf value of a substance is a number derived for

a compound.

Rf distaaae of centero"

snot from origin

"distance of solvent front from origin

Rf values in adsorption TLC depend, on factors which must

be controlled..

1. Quality of adsorbent- Silica gel, alumina and

1 5kieselgur are of many substrtes to choose

from,"

2, Thickness of adsorbent layer- controlled by the

coating operation.

3, Degree of activity of adsorbent- pretreatment of

chromatogram by placing In dry heated oven or soak

ing the chromatogram in a. treatment solution to

ad. iust pH or chemically impregnating the chromato

gram car, be done.

k. Quality and nature of solvents used- a. variety of

solvent systems with one or more solvent components

15"Thin Layer Chromatography with Eastman Chromagrarn

Sheet and. Developing Apparatus,

"Kodak Publications, reprint

v 1 Q7?-

$-

s

^Reiner Lapp, "Brinkman 3ibliograohy forTLC,"

I965.

16

with many combinations exist.

5. Degree of chamber saturation- There are different

vessels of varying size characteristics. Ideally

the atmosphere surrounding the chromatogram should

be saturated with solvent vapor.

6. Temperature- This will affect the adsorption of a

compound as well as rate of travel of the solvent

system.

7. Running distance- The distance the solvent system

travels should be -kept-, constant by recording the

time of elution and travel distance.

8. Technique of elution- There is a choice of devel

opment apparatus for ascending, descending or hor

izontal development.

9. Amount of sample- 1 to 5 ug samples are recommend

ed as. a starting point. Tailing is observed, with

samples of greater concentration,

10. Impurities present in sample mixture- Water is an

impurity that will affect the degree of activity

of adsorption, sites.

11, Visualization- This is done to render an invisible

compound visible. Attention is drawn to the prob

lem of .tailing. At higher concentrations, tailing

-'Eastman Organic Chemical Catalogue frk6, pp. 391-

392, 1971, published by the Eastman Kodak Company,Rochester, New York.

17

is a visual effect which was observed to be an

unsharp separation of the compound after elution.

Some compounds are not visible after elution and

need to be made visible. There are a variety of

visualization reagents available for invisible

compounds. They react and. form a colored pro-

1 8duct. As mentioned in the "State of the

Art,"

application of the visualization reagent will

have significant contribution to the sharpness

of the detected compound, after elution.

In TLC a substance is separated on the chromatogram

as it is being transported by a mobile phase of a solvent

system. TLC can bring about, a separation, by an absorption

Av -v-. <-% -t-,4- ^ 4-4 y>-wi m^aUi-ivit T" ITIVi,-j 4-

o "1 1 -5 v\ ^, 8f*

.^ a* <... _, .71 _. _ . _ . J_

-*** ---i- -

.--/'>- - - --1' * - - - . -L. _

^._ , _, .

j_

O'

1 <_**.au v>x Ow x vu

TLC can be explained in terms of a Langmuir amp) Freund.lich

3dsorpti on isotherm. These adsorption isotherms dictate

the condition of the amount of a substance adsorbed by an

adsorbent .

l""Eastman TLC Visualization Reagents and Chromatographic Solvents " KodaV Publication?*

.T.T_e; ,Tnlv 1Q71

p. 8.

18

Adsorption isotherm of a substance under

ideal and real conditions.

Concentration' C

on the adsorbent

i deal I

cd Z real II

*p 4 rri 1V>^ QJ. __. . ^.. -.

y

Adsorption isotherm of a substance at

- different levels of concentration

Freuud.i ich and. L?.aTniuir isotherms:

1) M= aP (Freundlich)

2) IX dK?/(l+KP) (Langmnir)

M-is mass of ad.gorba.te/ mass of ad.sorpent

P-eoui libriumpreosur-

of ad.sorbate

a b, and k are constants characteristic of particular

adsorbents which are particularly temperature depen

dent.19

l^Blae^el anH ^el ocha 81 " m .

I'm iimis secondedition, a. XX,

in("'"-"r Oijr>Viti t t'

19

If a substance were deposited on a chromatogram,

eluted, and visualized and the image of the visible sub

stance was scanned with a densitometer, a trace would be

obtained. Traces 5, 6, 7, and 8 (in figure 9) are traces

of a chromatogram with samples of hydroquinoneat

.5, 1.0,

5?0 and 10.0 g/1 concentration after elutionand visuali

zation with .INammoniacal si'ver nitrate /{see figure 10).

Figure 10

Dens 1 tometrie Traces

The non-symmetrical distribution of the separated.

substance at higherconcentration on the chromatogram was

20

seen on the densitometer trace as a skewed gausian dis

tribution. Also, it was noticed, that the maximum density

of the trace was increasing with greater concentration of

hydroquinone.

The reason for this, as explained in terms of the ad

sorption isotherm, is that at higher concentrations there

are not enough active adsorption centers to separate out

the compound. Und.er ideal conditions where there are

enough adsorption centers, the compound will be separated

out sharoly, However, under real conditions, there is a

decreasing linearity. In otherwords, there is a maxi

mum concentration of substance which the adsorption cen

ters can adsorb. Beyond this concentration, tailing will

-

-r

"

-. J-

8Alu >./ 9

In the partition mechanism of separation, much the

same is true for the tailing effect. A. partition mechan

ism involves two phages; one mobile and the other Immobile,

If the substance is not saturated ir the mobile phase,

there will be a sharp separation. The saturated, state of

a solute in a solvent is dictated by the partition co

efficient. When there is a saturated state for the sub

stance in the mobile phase, the substance will separate

from the rest of the migrating substance until equilib

rium of the partition coefficient in the mobile chase is

restored.

Inorganic salts, such as sodium sulfite act as

21

up i.rop.11 1 1 :. c or water etttra,cting particles. This will

affect the equilibrium, of water in the different phases.

Rf values obtained under these conditions will be with

error due to difficulty of measurement xvith tailing ef

fects.

Twod.ifferea.'

-t-.-

-aes will separate out to dif-

ferent positions on-4 '44X.hromatogram during elution if

there is difference in the adsorption properties or parti

tion coefficient with a particular solvent system and

chromatomram substrate.

AWofcfTM \5oTHteft

Figure 11

Adsorption isotherm of two different substances

A sv.atom of separation of claac of compounds can be

22

devised by choosing the appropriate mechanisms which will

bring about the separation.

In TLC, a variety of mechanisms forReparation can be

controlledby*

the choice solvent system, quality of adsorb

ent and degree of activation. Activation is the process

of heating the chromatogram in a dry oven for a period of

*

time to dry off the water from the atmosphere which can be

absorbed by the silica gel. Water will betadsorbed at the

activation sites. This means the degree of the activity

will be determined by the amount of water present in the

atmosphere or in the solvent system. For photographic de

veloping agents and. other non-polar, organic "compounds a

variety of non-polar solvents are used for a solvent sys

tem. If the mechanism of separation in TLC is primarily

adsorption, the chromatogram will be activated and the

solvent system will contain little or no water. There are

other substrates besides silica but 'when activated, do not

exhibit as much adsorptive properties. By not activating

silica and. other chromatograms and poisoning the solvent

system with more polar solvents such as water, the sep

aration can. be shifted to a partition mechanism.

"Generally, hydrocarbons are adsorbed

slightly, if at all, and thus, migrate

fastest. Unsa.tur.ated hydrocarbons are more

strongly adsorbed the more double bonds

they contain. Hyd.rocarbons containing con

jugated double bonds are strongly adsorbed

than those having a system of isolated, dou-

blefbonds. If functional groups are intro

duced into a hydrocarbon, the adsorption

at amity

quence

cnoH "20

is increased, in the following se-

~CH3, O-Alkyl, >C=0, -NH2, OH,

20stahl, op.1 o c

2k

OBJECTIVE OF THESIS

TLC was used as a qualitative and quantitative

analysis of photographic developing agents prepared in

aqueous solution with specified levels of sodium sulfite

concentration and pH. There were three specific objectives

I. R:^ values of developing agents.- B-f values were tab

ulated for aqueous solution of developing agents

metol, hydroquinone, hydroquinone monosulfonate,

phenidone, ascorbic acid, CD-3, para aminophenol,

para phenylenediamine HCL and amidol. Two different,

solvent systems were used.

IT. Two dimensional TLC- Hyd.roquin.one, phenidone, metol

and CD-3 developing agents were mixed into one solu

tion 'and two dimensional TLC was used, to separate the

developing agents from one another.

III. Quantitative TLC- TLC was used for quantitative aual-

vsis of hyd.roquinone prepared, at four levels of con-

.c-entration,three levels of oH and. two levels of so

dium sulfite concentration. The density maxima Dm(#)

obtained after visualization and d.ensitometric scan

ning was correlated to the concentration of hyd.ro-

quinone prepared in developer solutions.

25

OBJECTIVE I

Procedure

1. Two per centsolutions'

of metol, hydroquinonemono-

sulfonate, hydroquinone J phenidone, ascorbic acid,

CD-3, para aminophenol HCL, para phenylenedi amine

HCL and amidol were prepared by adding .2 g of each

developing agent to a 100 ml volumetric flask which

contained 5,g of sodium sulfite and. approximately 80 ml

of water. The CD-3 solution contained,2g

o.f sodium

sulfite. A phenidone/ ascorbic acid solution was

prepared.

2. The pH of the solution was ad-justed to 10.0 with 6N

sodium hyd/roxide. The amidol solution was adjusted

to a pH. of 7.0.

3. The volumetric flasks were brought up to volume by

adding a solution of water and sodium sulfite at lg/l.

Afterwards the solutions were stoppered.

k. 5 ul samples of each solution was drawn up bya'

streaker

and. deposited, on an unactivated silica gel chromato

gram. The chromatogramMwas precoated and. manufactured.

by Eastman Kodak. The samples were placed 1.5 mm from

the bottom edge and 2.5 cm apart from one another.

26

This accomodated eight samples deposited on the 20 cm

wide thin layer chromatogram.

5. The sample was placed in the chamber plate and in

serted into a fish tank that contained a reservoir of

the solvent system. The solvent system contained n-'

butanol; acetic acid and water in relationship of

80:5:15 parts by volume (v/v). 150 ml of the solvent

system was present in the reservoir.

6. After elution of the chromatogram commenced, it was

stopped five and a half hours later. Temperature dur

ing the elution and replications was between70'

and

78*F. with a relative humidity between 30 and 50 %,

7. The chromatogram was withdrawn from the chamber plate

apparatus and dried, inan"

ov^zn at room temperature.

8. Visualization of the invisible developing agents that

were separated during the elution was done by spray

ing with a .IN ammoniacal silver nitrate solution.

This visualizing reagent was prepared by adding ,75ml

of concentrated ammonium hydroxide to 30ml of .IN

silver nitrate. The chromatogram was sprayed under a

blower hood in a repeating pattern with an atomizer

hooked up to a five pound pressure line. Afterwards,

the visualizing reagent was diluted, and dumped because

of the danger of instability of ammoniacal silver ni

trate,

9. Measurements for 'determination of Rf value were taken

27

with a metric rule after the chromatogram had dried.

and before turning an over-all brown. This browning

occured about twenty-four hours after visualization.

10. Steps four through nine were repeated with solutions

one through eight to obtain an estimate of experimen

tal error.

11. Solutions one 'through eight were saved and 50ml of

the photographic developing agent solutions were

placed in an open 100ml beaker for over twenty-four

hours. This was done to encourage aerial oxidation

of the photographic developing agent.

12. TLC of the photographic developing agents one through

eight and their oxidized species was done by repeating

13. A benzene :methanol solvent system (90:10) v/v was

prepared to replace the n-butanbl: acetic acid.:waten

solvent system (80:5:15). Prior to deposition of

fresh samples of photographic developing agents one

through eight, the silica gel chromatogram was acti

vated. Activation, was achieved by placing the silica

gel chromatogram in a d.ry oven at100"

C. for fif

teen minutes.

Observations

1. The phenidone, amidol and sodium hydroquinone mouo-

sulfonate solutions changed color after being

28

deliberately oxidized. Also an additional specie

was formed and. an additional R*. value was calculated

for each specie formed.

2. The benzene:methanol solvent system took one hour to

-lute 10 cm up the chromatogram. The Rf valueswere

generally lower than the*-n-buta-nol:acetic acid:water

solvent system and were 'much sharper visually.

3. The pH of n-butanol'.acetic'acid -.water solvent system

was 2.32 and the pH of the benzene method solvent sys

tem was 6.6

PO

0^, 3 4 5 6 ~7 9

[** som* J^S CD"5 f*"t!0L ^ h*M-

X'%8fe|i

'SKgP

.4 fail

-*$

':%4

<:C 1.1. r__

. -. o -

' t> i - a o%1

e pa. r- i.

Figure 12

TLC Of ngot pgrn php C ^-iyal";a ... .- -.

aqueous solution fo-with inactive silica gel

used. >4th the n-butarol :acat tc -aid: water solvent s-sten

30

Data and. Calculations

1. Rf values of photographic developing agents 1-8 (see

figure 12) .

substrate- silica gel, inactive

sample deposition- 5 ul streaker

solvent system- n-butanol:acetic '-acid:water (80:5:15)

elution time- five and 'a half hours

solution// developing agent'

Rf value replicate average

1 phenidone (2g/l) .83 *.83 .83

2 phenidone (2g/l) .83.8k .8k

and ascorbic.

acid (lOg/l) ,0k

3 para phenyl ene-

diamine HCL (2

g/1) >2 .kk .kj>

k para amin.oohe.nol

5

6

7

8 sodium hydroqui

none monosulfon-

ate .32 .31 .32

20. Rf values of photographic developing agents deliber

ately oxidized by exposure to atmosphere.

HCL (2g/l) .62

CD-3 (2g/l) .ko

amidol (2,4-

diaminophenol ).

.30

metol .58

.61 .62

.ko .ko

.29 .30

.57 .58

31

solutg on ff deyel ooi eg a-ont R-p values oxidized a ceo i eg~ '

i " ' - _-__-,. ,.-..

1 phenidone (2^4) .87 ,27

2 phenidone (2g/l) .84

and ascorbic

acid .08

3 para phenylene-

diamine HCL-43

k para aminoohenol

HCL .71

5 CD-3 M

6 amidol

7 metol.63

8 sodium >ivdrooui-

.21

.04

.34

none monculfon-

ate ,34 .01

3. Rf values of photographic developing agents 1-8

s u i j s Xt r -. 1/ a - :-> I j. .. a.,

.-i ,. i, i v h l, . >

sample deposition- 5 ul streaker

solvent system- benzene :methanol (904 0) \Xv

e .1 ut i on time- one hour

solution developing.^gent R^ value replicate a,rora.ge

1 phenidone (2g/l)

2 -ng-enldcre (2sr/l)and. ascorbic

acid.

3 para nhenvieue

diamine HCL

k para, a.minophenol

HCL

5 amidol

6 CD-3

7 metol

-45 .46 .46

.iik.

.46

.45

.00 .01 .01

.34

.35 .35

.32

.33Q-7

.07 .07 .07

.81. .42 .42

T7

J 1 .38 X8

32

Conclusion

1. The n-butanol :acetic acid:water solvent system

(80:5X5), took five and a half hours to migrat *>>p

the chromatogram. This was four and a half hours

longer than the benzene :methanol (90:10) solvent sys

tem. The advantage of the butanol solvent svstem was

that Rf values were obtained for oxidized species of

hydroquinone, metol, amidol, CD-3 an(l phenidone when

prepared, in aqueous solution form in the presence of

sulfite.

The oxidation of a photographic developing agent in

aoueous solution with sodium sulfite probably re

sulted in the formation of a sulfonic acid of the d.e-

2.

gen will form a para benzo qu inane. However, sod.ium

sulfite will react with, it to form a colorless weak

developing a^ent.

H"

Figure 13

Step wise ionization of hydroquinone to a qu.ino1ic

+ Ofc

33

Figure 14

Oxidation of hydroquinone to a para benzo qui none

^

Figure 15

Formation of hydroquinone monosulfonate

3. A similar mechanism is possible for phenidone, amidol

and CD-3. The phenld.one/ascorhi c acid developer did.

not for;! any additional species as compared, to the

aheuid one/sad i u.m sulfite solution. This indicates

that ascorbic aci.d is a more effective anti-oxidant

than sodium sulfite.

34

4. The sodium, hydroquinone monosulfonate formed, another

specie*, when oxidized. The author has not bee.n able to

attribute the quality of the specie^ of the hyd^-#uinone

monosulfonate. It is possible that it is hydroquinone

di sulfonate.

5. The benzene :methanol solvent system gave a different

set of R^ values for developing agents 1-8. Indepen

dent testing of oxidized species of those developing

agents did not reveal the oxidized species by separat

ing them from the unoxidized species of developing

agents. This was probably clue to the fact that the

benzena :methanol solvent system was at a. higher pH

than the other butanol:acetic acid:water solvent sys-

6. This confirmsStahl'

s statement that more acidic polar

solvent svstemg could, be more successful in separating

sulfonic acids of developing agents.1It is for this

reason that the n-butanol solvent system was superior

to the less nolar and less acidic benzene : methanol

sol u t i on. .

7. The Eastman Kodak Company prepared ? publication on

"Separation of Photographic Developing Agents Using

the Eastman ChromagramSystem."

They reported ap^rcx-

iimate R^ values for the following developing agents

using an activated silica gel chromatogram and a

21Ibid. , p. 369,

35

benzene/methanol (90:10 v/v) solvent system.

developing agent

a. 2,4-diaminopheuol sulfate

b. hydroquinone

c. N-methyl-p-aminophenol sulfate (metol)

d . l-Phenyl-3pyrazolid.one (phenidone)

e.4,4'

-d.imethyl-1 -phenyl _3_pyrazolid

one*

The elution time was thirty minutes which probably

accounted for the difference in Rf values. Also, th.

silica, sheet used, in Obiective 1 was not activated.

Rf value

.08 *

.15

.25

.30

.40

36

OBJECTIVE II

Procedure

1. Two. per cent solutions of metol, hydroquinone, pheni-

done and CD-3 were prepared in a manner similar to

the proceeding procedure in part 1, stction 1.

2. 2 5 ml of each developer solution was placed into a

100 ml volumetric flask and mixed together.

3. 5 ul of the solution, was drawn by a micro-pipet and

the spot was placed on an activated silica chromato

gram 20 mm from the bottom edge and 60 mm from the

left edge. The 5 ul sample was deposited on the chro

matogram twenty minutes after the chromatogram had

been activated.

4. A benzeue/meth.anol (90:10) v/v solvent system was pre

pared and 1.50 ml of the solvent system was Placed in

the reservoir.

5. The spotted- chromatogram was placed in the chamber

plate and. e luted, for one hour in the first solvent

system.

6, After ejlution, the distance the even solvent front

.4

traveled, was measured.

7. The chromatogram was air dried for fifteen minutes.

37

8. Following the90*

counter-clockwise rotation of the

chromatogram, the chromatogram was placed in the sec

ond solvent system (n-butanol:acetic acid:water; and

eluted for one and a half hours. The distance sol

vent system two traveled was measured.

9. The chromatogram was dried for twenty minutes and

visualized with the ammoniacal silver nitrate vis-

ualization reagent.

10, After the chromatogram was dry, two dimensional Rf

values were measured.

Observations

1, The separations of the developing agent are sharp

py-ir! saparcite frpm pne another^mvionc. mcici little prob

lem in recognizing them since each was separated from

one another.

2. There was a slight uneven solvent front travel during

the second, elution in both trials.

3. Before performing the two dimensional TLC, it was

helpful in spotting -u additional sample of substances

on a margin of the chromatogram.. This was not to be

run with the other sample during the second, elution.

By removing the margin sample from the rest of the

chromatogram. after first elution and visualizing it

before the second elution of the other half of the

chromatogram, it was possible to monitor the progress

38

of the separation of substances during the first e-

lution.

4. Figure 1'6 shows the chromatogram in two parts. Part

a is the chromatogram that had the solution of four

developing agents spotted in the lower left hand, cor

ner, eluted in the verticle direction with the ben-

zene/methanol solvent system, rotated90'

counter

clockwise andeluted"

in the second sodvent system

with the n-butanol:acetic acid:watersolven.t system,

and then visualized. Part b was the chromatogram

that had four separate samples of each developing

agent deposited. 20 mm from the bottom edge and elut

ed vertically with the benzene /methanol solvent sys

tem only. Then it was visualized.

39

a) b)

Figure 16

Two Dimensional TLC

40

Data ar-1 Calculations

Rf coordinates for developing agents:

(l) replicate average. (1) replicate average

Rf(x) Rf(x) Rf(x) Hf(y) Hf(y) Rf(y)

1. .28.29 .29 .74 .76

.75

2. .38.39 .39 .50 .52 .51

3. -43 .44 .44.69

*

.73.71

4. .36 .40 .33 .34.35 .35

1. hyd.roqu i non e, 2. metol 3. phenidone 4. CD-'5,

Conclusion

1, Two dimensional TLC can improve the separation of

photographic developing agents prepared in a solution

and. deposited on a chromatogram.

2. Elution time with the t'^o solvent systems required

only three hours.

3. Reversing or changing the order of elution will also

change the Rf coordinates. Prior to the first elu

tion, the chromatogram was .activated. After the

first elution. the chromatogram was not cmtiimt4. but

air dried, in preparation for the second, elution. It

v/qg possible that there was a stationary methanol

phage during the second, elution. It appeared, that

benzene or methanol left over from the first elution

did not effect the separation of the developing

agents during the second, elution to any grat degree.

41

In other words, if the second elution was continued.

to five and a half hours with the n-butanol :a.aotic

acid:water solvent system, the Rf values would *o

approximately the same (as Objective I).

4. Providing there is good even solvent front travel

during both elutions, experimental error should, be

small.

42

OBJECTIVE III

Procedure

1, A silica gel chromatogram, 20X20 cm, *was used for de-

positing eight samples of thehydroquinoTre' d:eve4oper

solution, 2.5 cm apart from one another and 2.0 cm

from the bottom, edge of the chromatogram.

2. Solutions of hydroquinone for quantitative TLC were

prepared, from stock solutions of hydroquinone and so

dium sulfite. By add.ing proper volume of each

stock solution the concentration of yivd^oouincro ,-*c

set at one of the four levels.

3. The four levels of hydEroquinone were set at.5, 1.0,

5.0 and 10.0 g/1. The sodium sulfite concentration

was set at 10 g/1 and 100 g/1. pH was ad.iu.sted. to 9,

11, or 13 with 6N sodium hydroxide after the solution

was brought up to 90% of the final volume. A 1% a_

queous solution of sodium sulfite was used, to bring

the developer solution up to final volume.

4, The hyd.roauinone stock solution was prepared, in the

following manner:

a 500 ml distilled water at125*

F.

b 10 gsodium sulfite (dessioated )

43

c 20 g bydrnnuiuone

d 10 g sodium sulfite

e bring level to 11 volume with,1%

aque- &

sodium sulfite solution

f stopper and. record date of preparation

5. The sodium sulfite stock solution was prepared in the

fol lowi.p. g manner:*

a 750 ml distilled water at75'

F.

b 200 g of sodium sulfite

c bring level to 11 volume

6. Table I was constructed to indicate the proper volume

of stock solutions of hydroquinone (I) and sodium sul

fite (II) to be used, to formulate developer solutions

-*

Table I

run stock

2.50

pit i op I

ml

S t OC'.'s 0

'

4.75

j \^ t, i on. II

ml

ad ius t pH to

1,5 0

2,6 5.00 ml 4. =,0 mi 6

3,7 25.00 ml 2.50 mlQ

k,8 50.00 ml 0.00 ml 9

13 2. $ ml ko,7 4 ml 0

10 ) 14 5!o ml kq. --.o n. 9

11,15 25x0 m.l 47.50 r-i 1 9

1 2,16 50.00 ml 45.00 ml. 9

17,21 2.5 ml ^-75 ml 11

13,22 5.0 ml 4.50 ml 11

19,2-3 254) ml 2.50 ml 11

20,24 50.O0 ml. 0.OQ ml 11

25 ,?9 2.5 ml 49.75 ml 11

26,30 5.0 mlLiO

tqp ml 11

op 01^ i , ^

-1-

25.00 ml 87.40 ml. 11

90 ^ 50.00 ml 45 .00 m 1. 11

44

run

33,3734,38

35,3936,4o

4l,44

42,4543,46

44, 47

Table I (cont'd).

stock solution I stock solution II

2.5 ml

5.0 ml

25.0 ml

50..00ml

2.5 ml

5.0 ml

25.0 ml

50.0 ml

4.75 ml

4.5 ml

2.5 ml

0.0 ml

49.75 "1

49.5 ml

47.5 ml

45.0 ml

ad iust pH to

13

13

13

13

13

13

13

13

The volume of stock solutions I and. II added by buret

for runs 1-48.

7. Table II was the planned factorial experiment which

listed, eachfactor

and level op

hydroquinone, sodium

sulfite and pH. In. addition, it alsolisted what order

the experimental runs were to be made.

H

Y

D

R

0

Qu

I

N

0

N

E

Factorial Experiment

Table II

10.0 100. 00

Q 1 1 n a 1 1 5~

.5T 1 '; 0 23 ^'*-L

4a A< a 7 1 a 2 9 45

1.0e 1 8 34 10 2 6 42

_22 oQ 14 op ''-6

5.0 a 1 0 74 11 27

'

g

790 co 45

"3 1 4?

10.0 u 80a 8 12 28 '!4

a 24 4.0 1.6 n 't.C.

run 1 7 1 2 ?

'odium sulfite

45

8, For each day, sixteen runs were done. There were two

chamber plate apparatuses. The first chamber plate

was confined to do runs that had 10 g/l concentration

of sodium sulfite. The second chamber plate was con

fined to do runs that had 100 g/1 of sodium sulfite

concentration. A chromatogram placed in each chamber

plateaccomodated'

eight runs.. There were four runs

that had their replicates in addition to being on the

same chromatogram. This was the state of the art in

quantitative TLC.

9- Eight solutions were prepared twenty-four hours in ad

vance to sample deposition onto the chromatogram.

From each solution bottle containing 100 ml of the

hydroquinone at the specified level with sodium sul

fite and. -pH, a few drops were transferred to the

streaker trough.

10. The streaker was the d.evice used to deposit a 5 nl

sample on the chromatogram. Deposition was achieved

by placing the streaker in the trough which contained

the solution. It was lifted out by tilting the streak

er out of the solution. A chem wipe towel was used to

wipe off the excess solution from the teeth of the

streaker.

46

Figure 17

Use of the q '/nr, r. _ t' -\ f.pr quant i tat \ ve application!

of sa-ipie to the 'chroma togram

47

11. The streaker was placed on the chromatogram 20 mm

from the bottom edge in one of the eight columns

reserved, for the run and its replicate. This was

repeated for the other concentrations of hydroqui

none .

--rwyjwfr -j

-

Figure 18

Application of streaker sample to the chromatogram

12. The silica gel chromatogram, being inactive with the

eight spotted, samples, was placed in one of the oham-

ber plates. Another chromatogram with eight other

spotted, samples at the higher concentration of sodium

sulfite but the same pH level, was placed in the other

chamber plate apparatus. Samol.es spotted on the chro-

matogr^m .r^-r^ a! lowed to dry pefore elution.

48

13- An n-bntenol : aceti c a?Pd:water (80:5:15 parts by

volume) solvent system was used with the silica gel

chromatogram and was eluted. for five and a half hours

with 150 ml of the solvent system placed in the reser

voir. During the course of elution, the temperature

was between 72 and

7.5*

F. and the relative humidity

was between 30 and 4o4. -At times an* uneven solvent

front was observed. It was theorized that temoera-

ture and. humidity graddents were responsible for the

uneven solvent front.

-

__PE ------

1 '--f^rm ~ZZ~ZZ. '-, P

i

:z, pa

a

\ZZ

X

m -r-,

. .....

a) v.neven solvent front b) even solvent front

Figure 19

To remedy this, the chromatogram wag activated, for

49

fifteen minutes in a100*

C. dry oven and -allowed to

stay oyer night on an open shelf to rejoin equilib

rium with the atmosphere uniformly over the chromato

gram ..

14, After elution, the chromatogram was dried in a low

temperature oven.

15. Visualization of the hydroquinone and species formed

in developer solution was done by spraying the chro

matogram with ,1N ammoniacal silver nitrate with an

atomizer In a reoeatable manner. The amount of sil

ver nitrate used, was enough to saturate the chroma

togram,

16. After visualization, the chromatogram was d.ried and

the visualization reagent was disposed, of by flush

ing it down a sink drain. Visualization, was done one

hour before the d.ensitometric scanning,

17, Quantitization of the hydroquinone eluted. on the

chromatogram was achieved by measuring the maximum

density of the hyd.roquinone reduced by the ammonia.oal

silver nitrate.

18. This 'maximum d.ensity was obtained by using a Photovolt

TLC densitometer, model #520.

50

SCAWN'iKJC-STAG-f

?HYT TVBC

fmvx s,,T _,

mVI|4> SYAG.

CNl|lVT),CfV CPT1C

iwcRoe srfKT V??)TVnJffS-rtf, $v.3 M;

-]T* /V\CTCR. UNtj

a)

Pj 0-1 ] ~na 2 0

Photovoi t 'PTC 4 3-" -. i tQmetC" mod.-^l '52with

a)saa/'^nin"

sta^s b) metric unit a"d c)rpPToripr-

oen

pv>=_ -nr>oc-^anr a for use of this d eraM-o-no t.^n

.-jcr. pal-

lowed, according to the manufacturer'soperatic,- in_

strnctions. The following steps in addition were

"oil owed:

q cjiafor'(= tracing the chromatogram the scanning

stage,meter

and recording -pen units were warmed

no^or twenty minutes.

51

b. Lights in the room were turned off, since they

affected density readings.

c. A Kodak #2 step tablet was traced to observe how

the unit was performing.

d. On a point midway between the sample deposition

line and the bottom edge of the chromatogram, the

densitometer, meter unit and recording pen were

adjusted to zero by controlling ^he intensity of

the tungsten lamp and. line voltage controls to

the metering and recording pen unit.

e. .The densitometer meter unit and recording pen

unit were adjusted to infinite density after cov

ering the photo tube in the scanning stage with

the opaque cover silt.

f , This was done before tracing every one of the

sixteen runs done per day. Usually the runs

were done the day before the scanning and the

visualization was d.one one hour before scanning.

This was done due to the problem of the browning

effect.

52

p*w*w9W(j

' ~l

, s

i R -

act-~ j

j VZZZZZ 'if ^(Pf**1^'**?****

| lft_f*fc'^.j*tf**^

j 1 .

[ 1\'

1| '

s c.~v_i

J Urtmnwi' "* y^T^.^. . _

1j

, 5

"

J i 3 *

i ;

I H j

I' 3

;,

'i'

1 i i !

j ii

i ;

1.

'

i;

^4

j

1'

i j

-;

i l'

|1 !i

J j.

j If11

.. i j

! i !i jt 5

9

jl

_*__J

a)

Figure 21

Difference in visual appearance of chromatogram

after visualization

a) 1 hour after visualization, b) 24 hours after visualization

53

4

PP44.

*fi*-V_-i-

.^'gr.i<W-

t. ?a, .

rr,""*i

1'

;

mm

HEXa

v..a^,

f^ST^ES

^b^wt^

j

... . _w.j#

i C. -:>;%'& f

p4>'

|

^icUi2_5__;a:;-

, I

.

b)

Figure 21

54

19. The paper used for the recording pen was made for the

percent absorption mode of operation of the TLC den

sitometer unit. Table III was constructed to convert

absorption units to density units. This was done by

lining up the density pattern on the linear graph pa

per and reading off what density corresponded to the

percent absorption,

20. For "each specie or Rf value of hydroquinone, a deu-

maxima, Dm was obtained, from the d.ensitometric trace

by referring through Table III for the correct den

sity value. This was best illustrated by Figure 17

which showed a series of samples 1-8 that were de

posited, eluted and visualized in the manner de-

gr> ri Vied in tV-jo opOO or! i ir>e spr>t;i "TI EQch V",m WO S pp . .

sitometrically scanned and. where there was a peak,

a density maxima was noted, by.drawing

aa bar over that

peak.

55

p. .wi ; *ww>rw.mT,r^MTm'~wrJw?rrm

Z\

'

I <%By v

DENSITOi-iETRIC SCAI.M..G OF

THIl, LAYER CHRO..iATOGRA..,S

1

'-"!.. j

| :

\^

run #

Rf(3)

(2)

Hf(l)

Pi c-ur,,-. 09

Illustration of how density maxima D(l)f Dm (2) ^dD"' (a) arore obtained fo"

sn^^i^^ j?,

ti. vo l.v,

RJl)> ^(2) ^ Hf(3)

5^

Table III

Conversion of Absorption to Density

A D

.00 .00

1 !.)

D

.01 .012 .21 .262

.02.025 .22

.275

30

.11 .140

J- 5 .188

.16 .2X

J-79i n-> -L J

.18.22 5

.19.240

A

,41

.42

.03 .040.23

.288 .43

.04 .050 .24

.300 J(4

.05-062

.25.312

.4.5

.06 .076 .26

.325.46

.07.088 .27

.338 .47

.03 .10 .28 .350 .48

.00 J.l .29.362

.37.5.50

53

54

.54

31. .383 .51

.12 .150 .32.395

.52

.13.140

.33

.14 .175.34

^5

36

37 .460

.57

48 .470 .58

.so

.485

Pa

410

42 0

43 ^

844 .56

D A

oa oca ^qq

59

.510 .61

.525.62

.540 .63

.550 .64

.565 .65

.575.66

.583 .67

.600 .68

.612 .69

.628.?0

.633 .71

.6.50 ;72

.660 .73

.675.74

.638 .75

.700.76

.712

.77

.72 5 .78

.738 v79

a^D 80

on 520 TLC Platen

DAD

.762

.778

.790

.800

.312

.32.5

.838

X62

.87 5

.888

.395

.910

.920

,3.5

. 945

.960

.970

.935

1. ,00

.31

.82

.83

.84

.85

.86

-37

.33

.80

.J;

X1

09

.93

.94

.95

X6

.97

.08

00

1.00

1.01

1.02

1.04

1.0.5

1.07

1.08

1.10

1.12

1 .14

- -**

L J_ '4

1.18

1 .20

1.25

1.35

1.50

1X5

2 J0

Oo

A= Absorption; D= Density,

57

21. The log density of Dm(l ) . Dm(2) and Dm(3) xvere tab

ulated for runs 1-48 and plotted as a function of log

concentration of the hydroquinone developing a >nt.

prepared in solution at a fixed sodium sulfite con

centration and pH level. This required six graphs to

do for the 48 runs.

Observations

1. For each corresponding specie* of hydrobuinone there

was an. Rf value.

2. At lower pH levels, the Rf(i) specie did not appear.

3. The appearance of the chromatogram changed with time

after the visualization. At higher concentrations of

"hydroquinone deposited on the chromatogram, insuffi

cient application of the visuiallzation reagent result

ed in d.e.nsities that were not significantly darker

(visually) than the lower concentrations of hydro

quinone .

58

Data and Conclusions

Xhle IV

Density maxima Dm(l), Dm (2) and Dm(3)

run Dm ( 1 ) Jm (J_) 44_jj X ( 2 ) D-_2) _X ( 2 )

for runs 1-48

D~(3) 4D~(

1

5

.03

.05.04

.02

.1.1

.13

.15 -07

.13

.21 .20

.03

2

6

.04

.06 .05 .02

.25

.30 .28.05

.38 .36 .05

3

7

.02

.03 . 03 .01

.33

.44

-.37 .07

*

.68

.65

.67 -03

4

8.03

.05.04 .02

.33

.30 .32.03

.78

.73 .73 .00

9

13

X6

.09 .08 .03

o4*

0? .06.03

.25

.28 .27 .03

10

14

.08

J2 .10 .04

.05

.18 .12

1 a-

>

.3^

.36 .38.03

11

15

.03

,06.05 -03

.10

.09.10 .01

.55

.50

.53 .05

12

16

.05

.09

.07.04

.25

.23 .27 .03

.76

.81,7Q

.05

1721

.04

.04

.05.01

.29

.24 .27

-23

.21 .22 .02

18

32

.06

.03 .05op

.39

.34

.37

.04

.36

45 a 8.01

19

23

X5

.04-0.5

.01

.66

.63 .65 .03

.58

.53 .56

.05

20

24

1.50

.571.04

.93

1 .02

.80 .91 .22

.63

X7 .60 .06

252Q

.34

.31 X3 .03

.14

.13.14 .01

.31

JI 01 OP-- -

26

30

.36

.36 .36 .00

.15

.16 .16 ,01

.40

.41 .41 .00

27 .57

.53 .55.04

.30

.30 .30 .00

.78

.59

.67 J9

59

Table IV (cont'd)

^un Dm ( i. ) DmJ ) ADjl ) ]>__)___2)

ADm(2) Dm ( 3 ) Dm(3) *Qm(3)

28

32

.66

-53 .60.13

.55

.35 .45 .20

.92

.57 .7* .35

33

37

.68

1.35 1.02.67

.51

.81 .66 .30

.10

.63.36

.53

34

38

.66

.86 .76 .20

.79

.83 .81 .04

.20

.15 .18 .05

35

39

1.50

1.20 1.35 .30

1.04

.97 1.01 .07

.76

.57 .65.11

36

40

1.60

1.05 1.33 .55

1.14

.73.94 .41

.65

.63 . 64 .02

41

45

.36

.50 .42 .14

,20

.10 .30 .10

.24

.08 .16 .16

42

46

.74

.46 .7.5.32

,53

.35.83 .18

JO

.35 .23 ,25

4347

.81

.59 .70 .22

1.16

1 ,14 1.15 e02

.80

.68.74

.12

44

48

.86

.88 .37.02

1.75

1.35 1.55 .40

.86

.78

82.03

ii Dm is referred, to as the range, '.'hen plotted, on grabs 1-6,

6o

Conclusion

1. A regressional analysis should be done for Dm(3) vs.

the log concentration of hydroquinone at fixed levels

of sodium sulfite concentration and pH but time did

not permit this to be done.

2. In runs 1-48, the log Dm(3) is an increasing function

of log hydroquinone concentration over all sodium

sulfite concentrations and pH levels. It appears

that with the experimental estimate of error,ADm(3),

that it is a linear relationship.

3. At higher concentrations of sodium sulfite and. pH,

Dm(2) and Dm(3) are increasing with increasing hydro

quinone concentrations. This should, indicate that

the hyd.roquinone monosulfonate formed at higher con

centrations of sodium sulfite and pH. The author

itfould think that the concentration of hydroquirone

viould decrease. The hydroquinone Concentration as

indicated by Dm(3) should then decrease, but this has

not been proven.

4. It xvas observed, that the amount of visualization re

agent sprayed on. a chromatogram with an atomizer, was

a very inaccurate, non-uniforn manner of'

application.

A: device was built to uniformly deposit the vis-

iualization reagent. It was similar to a rotating

postage stamp moistener. The rotating pyrex cylin

der was sand-blasted, to eliminate thellpophl.llic

61

properties of the glass. Th ammoniacal silver ni

trate was picked up by the rotating cylinder and. a

uniform amount of the reagent was applied to the

traveling chromatogram by a meniscus transfer.

However, due to engineering parameters, the quantity

of silver nitrate transferred, was not sufficient to

produce higher densities at higher concentrations of

hydroquinone.

Pi :ni r"e2

By estimation of the amount of silver (farmed byth"

reduction of the IN ammoniacal silvernitr4-.

-elu

tionb"

the hyd -.-, -,,-,.-

.t-i o> 'c ) through trauma.

;s

~ *on

62

densitometry, it should have been possible to estimate

the amount of hydroquinone that was eluted on the chro

matogram.

6. Because of the following problems, which were not

standardized or controlled, non-random experimental

error was introduced.

a. Repeatability of densitometric results using the

Photovolt TLC 520 densitometer.

b. Application of visualization reagent.

c. Instability of the chromatogram after visualiza

tion (see figure 19).

7. Because of those non-random factors that could affect

the experimental error,.the procedure describing

llnQVlf t t"of . Tro clVT-il-,rrir, ^f + V. f* V,r^*,^,.i v.^vir / --

-n>-

-r'T.

-

agent was at best a semi-quantitative analysis. The.

author will propose methods of reducing the experi

mental error to make TLC a more a.ccurate quantitative

analysis in Appendix A.

8. One advantage of using TLC for quantitative analysis

of a photographic developing agent hyd.roquinone wag

that it could determine the quantity of oxidized.

species of hydroquinone formed. There are disadvan

tages of quantitative TLC when compared, to the con

ventional chemical analysis of photographic develop-

22

ing agent. These conventional procedures require

^-Chemical Control Procedures for Black and. White Film

Processing, Motion Picture Film Department, Nov., 1062,Eastman Kodak Co., Rochester, New York,

63

minutes to perform the quantitative analysis.

However, procedures are restricted in the sense that

they can. be applied, only to the specific purpose they

were designed for. Often, they can not be applied to

other developer solutions containing different devel

oping agents. This procedure has been modified to

analysis of other developing agents.* However, these

procedures are of a proprietary nature and are not

readily available on request.

9. Additional. solvent systems and chromatographic sub

strates will be proposed in Appendix B for TLC with

faster elution conditions and better separation of

developing agents.

^ Log density vs. concentration of H

R. 64

run 1-8: pH = 9.0, sodium sulfite^ 10,0 g/1.

Graph I

9 1- -r- 1 4-

1.0

RANGr

2

.01. .02*

2

.07 4

.1

-L.4-4-L-L

.03 .05.

.01

3 43 4

.o/ 01.

.47 -03

D ( 1 ) .n j ,60

.5 1 .0,5.0

10.0

run 9-16:.

pH= 9.0, sodium sulf ite= 100.0 g/1

r~

.

j i_._ . ...i._ .

s I

e i -4

, i .

4-

-. - . _ .

l-ra oh II

1 o0

raids'

\

i z

.03 .0f

D (3)m

3 *j

<03

.Of

/D (2)

_r m

.^ -qj

3 4 56789 10

5 1.0 5.0 10.0

Log concentration of hydroquinone

r"ll Logarithmic, 3*3 Qclcs

7X12081 Log density vs. concentration of H Q

66

run 17-24: pH=

11.0, sodium sulfite^ 10.0 g/1

10r~

aP-

94

i-

Graph III

. | _,_

RftN<r^ /

L j.07l. 2.

L.4

: -1

.01 .0! pV.x

D (1) 3 </m

-0/ .

D (3)m

D (2)m

.0$ .G&

,03 -j a.

1 .0 5.0. 10.0

5 6 7ft

Log concentration of hydroqu i.none

_e_-w

inrg crensity vs. concentration of H Q67

run 25-32: pH=11.0, sodium sulfi.te= 100.0 g/1

T~, 4

I :

-3

Graph IV

"l 8

i TXXTX

,...-(. i i j-

i_J_44-P-P_.

-a

-Tf"

X P'

4-84-

-X-fM_^4

M ' 14--:1-

:Jg'gPj'

D

F

S

1 0.00

.o|

.Oj*0C

.00

-of

3 *f

D (3)m

D (1)m

n ( o\

m

If -35

.Of-13

A A J>r if

.1

T

Y

.01

I P'4

._!___.

I R 7

.5 1 .0

,lpsai

._L

4

4 5 6 7 3

5.0 10.0

Log concentration of hydroquinone

#

Log density vs. concentration of H Q

63

run 33-40: pH=13o0, sodium sulfite^ 10.0 g/1

Graph V

P. r

*

4

: ._._..(......

, , ,

r. . J

"

,

-

X-

Exi

1:48_.E4 ... '.' ' '

'

:i'

r--.

tar-

r .

i ._.

i n

i....

"

LE'R/^-r. 4 1

J.

MEr... /*? Ifl

aZZlEZ"-

-.;- __4^~j_

.._,_-,--

| .,

.4

? A

D (1 ) -3<> 5$m

D (2) -7

.m

D (3) "'.//_m..__--',

7/

*Z.

x .01

.5 1 ,0

h ; -

.

5.0 10.0

Log concentration of hydroquinone

*

12-081

Log density vs0 concentration of H Q 6q

run 41-48: pH=

13.0, sodium sulfite^ 100.0 g/1

_i__UL_..__L-p- ! ii Ml

j

-4-

X-Xrapft

i 4--_

+.

.-.-J....

tc

__j i ..

xx

XXX--Xu..r_a_-fxX4

3i~

RAN6-r

1 2.

I 2

-I- -

L

.-.:p

Jn -J

"Si4

..44

--.-

! .!..

'4

RAw<5-<T

m .ox .*{&

Dm(1);2z^

m

'/4-L *^

01Ii J:

:M'l4J-XXt4XfE i3 4 5 6 7 3 9

.5 1 ,0 5.0 10,0

Loq concentration of hydroquinone

BIBLIOGRAPHY

71

BIBLIOGRAPHY

Abbott, D. and Andrews, R.S., An Introduction to Chromo-

tograohy. Boston, Houghton Mifflin Company, 1965.

Babbit, James, Chromatographic. Analysis . toew York, Reinhold

Book Corporation, 1963.

Blaedel and. Meloche, Elementary Duantltati^/e Analysis, 2nd

edition, Mew York, Harper and Row, 1853.

Distillation Products Industries, "A File of Analytical

Procedures for use. vfith the Eastman ChromagramSystem,"

publication z7-5, Rochester, Mew York, November, 1965.

Eastman Kodak Company,*'Chemical Control Procedures for

Black and. White Fil"1 Processing,"Motion Picture Film

Department, Rochester, New York, November, 1962.

5ao L.-_a.a. a. ._.'._<_'.-, k l, om ixi*

a/ , ,j-xo -apm̂agamo aai.aHi.oc-t.-t. w.

#46, pp. 391-392, Rochester, New York, 1971.

Eastman Kodak Company, "Eastman TLC Visualization Reagents

and ChromatographicSolvents,"

Publication #JJ-5, p.

8, Rochester, New York, July, 1971.

Eggers, J., "Photo, u.Wiss.,"

10, 40, 1961.

James, T.H., "Analysis of Developing Agents by Paper

Chromatography,"Photographic Science and Engineering,

Vol. 3,

Karnsk, F'.W.,"Thin. Layer

Chromatography,."

Research/Devel

opment, 4, 1973-

Laop, Reiner, "Brinkman Bibliography forTLC,"

Westbury,New York, Brinkman Instruments, Inc.

Mariani,B. and Martinelll

, P., "Sci. InJPhotog.,"

p. 276,Series II, No. I, 1956.

Mees, C.E.K. and. James, T.H., The Theory of the Photographic

Process, J rd. edition, Macm i 1 Ian C om pany ,1 06 9 .

72

Pannel, J. A. and LuValle, JX., "Anal.Chem.,"

Vol. 25,p. IjoO. 195 \.

Ra.nderath}Kurt

,Thin L-'ger Chromat ^-raphy 2nd. ed i. t ;

on,

Russel, G., Chemical Analysis in Phot-o^ra p.h.y_, Chapter 8,

New York, Focal Press, Ltd., fQoXT

Stahl, Egon, Thin Layer Chromatography, 2nd edition, New

York, Springer Verlog, Inc., 1969.

Todd, H.N. and. Zakia, R.D., Ph otogra ah i c ?. ens i t oma t vy .

New York, Morgan andI1organ Inc., 1969.

APPENDICES

74

APPENDIX A

Propose Methods for Improving Quantitative TLC

A variety of methods have been, used to quanticize

the amount of substance on a thin layer chromatogram. In

the past, quantitative TLC had limited accuracy (10 to 20

per cent) and was time consuming with hand. operated me-

thods.J

Recently, spectrodensitometric instrumentation

has been made that makes rapid and accurate determinations

on the chromatogram. this later d.ay sophisticated

instrumentation, many of the variables that lead to in

accurate results were eliminated compared to the earlier

Photovolt TLC 520 densitometer. The. Photovolt TLC 520

densitometer did not contain any of the automated features

of the new breed of thin layer chromatogram scanners.

23Kurt Randerath, "Thin LayerChromatography,"

pp.

70-79-

75

Figure242 *

*

5D-2000 Chromatogram Analyzer (Schoeffel Instrument Corp.)

The 5D-2000 chromatogram unit can measure transmission,

reflectance, flouresence:and adsorption. This unit uses a

double beam ratio technique that eliminates many of the

troubles encountered with the Photovolt TLC 520 unit.

Figure 24 shows the schematic diagram of the 5D-2000 unit.

Thin Layer Chromatography, Research/Development,

op, cit. , p. 33.

76

e/Xe-Hg lamp

WRpgR.

Pi o-n x>p 44''

Schematic diagram of a, modern split/reference beam TLC scanner

95lbid.

77

Photographic developing agents may not show any short

wavelength or visible absorption. In this case, the devel

oping agent may have to be visualized. The application of

the visualization reagent would be critical if absorption

of the visualized substance was to be used for quantitiza-

tion. For this reason, a device was constructed to uni

formly apply an accurate amount of visualization reagent

(see figure 2.3). Dr. Carroll was responsible for the sug-

gestion and the author of this thesis believed that such

a device has considerable merit over hand application of

the visualization reagent by a sprayer bottle. Unfortunate

ly, some engineering parameters were not resolved to in

corporate it into the factorial experiment.

Dp . Ca v,T,o1 1 n~\ <^r\ oii-rn-aqf or. +-V.O v_r>cs.r fTr.nrccon^>e>

technique Kodak used for their quantitative determinations

of silver. However, such an apparatus is costly and dif

ficult to obtain.

Dr. Francis suggested two add.it ional alternatives.

The first one was to do a potentiometric titration of the

silver formed, when the hydroquinone was reduced by the .IN

ammoniacal silver nitrate by scraping the blackened areas

off of the chromatogram. The other method, suggested by

Dr. Francis, would be to scrape off the area of the chro

matogram, which was known to contain the hydroquinone, and

perform a sulfate cerrate titration to an indicated end

78

Point with a Ferroin indicator.26

sal Control Procedures for Black and White

Film-Processing, Procedure 407B.

2"Ghemic-

79

APPENDIX B

Solvent systems and substrates may exist that would

have shorter elution times and promote sharper separation

of photographic developing agents. Dr. Harry Baden, of

the Eastman Kodak Company PhotoTechnology*

Division in

Rochester, New York, suggested the following, solvent sys

tems to be used with activated silica gel chromatograms.

solvent system:

1. benzene '.methanol .acetic,acid (90:36:36 v/v)

2. ethyl, acetate :acetoneaacetic acid:water (100:60:

20:20 v/v)

3. chloroform: ethyl acetate racetic acid (l40;40:20

v/v)

4. benzene :methanol: acetic acid (180: 32: v/v)

Stahl listed solvent systems and chromatogram sub

strates used to separate 'phenol, pyrogallol and resarcinol,

1. dioxane: benzene:.acetic acid (2.5:90:4)

2. methanol : benzene : acetic acid (S'.k^'.k)

To what extent the solvent systems would separate the

developing sjgents and the time required for adequate elu

tion is not known. Work remains to be done in this area.