Upload
others
View
10
Download
0
Embed Size (px)
Citation preview
Rochester Institute of TechnologyRIT Scholar Works
Theses Thesis/Dissertation Collections
6-1-1973
The Use of Thin Layer Chromatography (TLC) inQualitative and Quantitative Determinations ofPhotographic Developing Agents (in DeveloperSolution) Using a Scanning DensitometerMichael Gilbert
Follow this and additional works at: http://scholarworks.rit.edu/theses
This Thesis is brought to you for free and open access by the Thesis/Dissertation Collections at RIT Scholar Works. It has been accepted for inclusionin Theses by an authorized administrator of RIT Scholar Works. For more information, please contact [email protected].
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.