ORGANIC AND INORGANIC COMPONENTS OF MANUSCRIPT INKS
I. ESPADALER1, M. C SISTACH2, M. CORTINA1, E. ELJARRAT1, R. ALCARAZ', J.
CABAÑAS1 and J. RIVERA1.
1. Laboratori d'Espectrometria de Masses. Dept. Micr & Proc. CID-CSIC. Jordi Girona, 18. 08034
Barcelona.
2. Arxiu de la Corona d'Aragó. Almbgavers, 77. 08018 Barcelona.
RESUMEN.- En la composición de tintas de manuscritos antiguos intervienen sustancias de
naturaleza inorgánica (FeSO4) y otros de naturaleza orgánica, provenientes de extractos vegetales
(taninos) y del aglutinante utilizado (goma arábiga). Muchos de estos taninos poseen en mayor o
menor grado Ac. Gálico (ac. trihidroxibenzoico), el cual ínteracciona con el hierro para formar el
complejo estable y coloreado que constituye la tinta. El objetivo del presente trabajo se centra en
comprobar la permanencia o no de restos de componentes orgánicos en tintas con diferente
comportamiento y características. En el presente trabajo se describe el protocolo del tratamiento de
las muestras (extracción, derivatización), así como el análisis de los compuestos mayoritános
presentes mediante crromatografía de gases y acoplamiento de cromatografía de
gases/espectrometría de masas (GC/MS).
SUMMARY.- One of the most important causes of degradation in ancient manuscripts is the ink
acidity. The relationship between the important corrosion that some inks provoke in the support and
the fact that a strong black colouring remains in the ink has often been demonstrated. By contrast,
no acidity is shown in light coloured or slightly dark inks. Manuscript samples with iron gall inks
were analysed by Gas Cromatography - Mass Spectrometry (GC/MS) and Scanning Electron
Microscopy - Energy Dispersive X-Ray Microanalysis (SEM-EDX). The samples, on paper and
parchment bases, showed different colour and acidic behaviour. Inorganic elements analysed by
SEM-EDX showed different sulphur and iron distributions on the base. The different ratio between
the sulphur and the calcium was noted also. Gallic acid and sugars in the ink were tested by
GC/MS. These compounds were identified by mass spectral analysis and co-injection of standard
products. The results were different for light inks and dark or acidic inks. The gallic acid remains
only in the dark inks, but in the light and very acidic inks, gallic acid could not be found. Sugars
that come from arable gum were present in the light inks and in those dark inks that were not very
acidic. The carbonized samples retains very small amounts of sugars.
KEYWORDS: manuscripts, iron gall inks, scanning electron microscopy-energy dispersive X-ray
microanalysis (SEM-EDX), gas chromatography (GC), gas chromatography-mass spectrometry
(GC/MS).
INTRODUCTION
For several centuries, iron gall ink has been used to write manuscript documents. We have many
bibliographic reports about ink formulae where we can find not only their essential components but
also how they are prepared (ref. 1-18); the addition of others substances to improve the ink quality
is noted as well. In iron gall inks, the most important components are the organic extracts of tannins
and the arabic gum. The inorganic components are the iron and copper sulphates. Several recipes
from the 15th to 19th centuries have been published in Catalonia (19), Balear Islands and Valencia,
Spain, and in all of the recipes we find the components noted above. Their preparation is based on
tannin extraction, with which the iron that yields the iron sulphate forms a black suspension that is
retained and thickened by the gum arabic. One of the most
important cause of degradation in ancient manuscripts is the acidity ink. The relationship between
the important corrosion that some inks provoke in the support and the fact that a strong black
colouring remains in the ink has often been demonstrated. By contrast, no acidity is shown in light
coloured or slightly dark inks. We tried to explain the acidic behaviour and permanence of black
colour in some inks. In order to do it, we used GC/MS (20-25) to analyse their organic components,
and the same samples were used in SEM-EDX to check the inorganic components.
LABORATORY INKS AND THEIR BEHAVIOUR
Inks were prepared in two groups. For the first set, we prepared different iron gall inks with
different amounts of gallic acid while keeping the amount of iron sulphate the same. The second set
was prepared so that the sulphate changed and the gallic acid were always the same. The amounts of
and the ratio between the iron sulphate and Gallic Acid used to prepare laboratory inks were
selected based on the ink recipes. Most of these recipes delineate specific components and amounts,
and explain the process of extracting tannins, so that it is possible to deduce that the amount of
gallic acid depends on the extraction procedures. In some of them, we added some quantity of
arabic gum. The factors studied in this first step of our v/ork were the pH variation for the inks and
their appearance and colouring at the time of preparation and then three months later. The first
group of inks was prepared with invariable quantities of iron sulphate and arabic gum (16 mg/ml of
each), while the gallic acid changed (See table I). All of these inks showed a dark colouring which
remained after three months.
Table I. pH changes in adding gallic acid when [Fe SO4] = 16 mg/mL.
The following step was to check pH changes with the increase of iron sulphate, whereas gallic acid
concentration remains constant and low (0.04 mg/ml, see table II). At the same time another group
of inks were prepared, and in this case, iron sulphate was the substance kept constant (equal to 0.4
mg/ml) while the gallic acid changed. (See table II).
Table II. pH changes in adding iron sulphate and gallic acid.
Fifteen days later, all of the inks related to the table II were the colour of iron oxides similar to the
lights inks. At a concentrations of 16 mg Fe SCVmL, an important sediment of iron sulphate and
iron oxides was noticed. Only the first sample with 0'4 mg/mL of iron sulphate concentration and
0'04 mg/mL of gallic acid kept the relatively dark colouring, however, it was very clear. The inks in
table III remained dark and they were darker when the amount of gallic acid was greater.
Another group of inks was prepared (see table III). In this case, 5 mg/mL of Gallic Acid solution
was used prior to different amounts of iron sulphate. These inks had 125 times more gallic acid than
those shown in table II. All of them were dark and dense and their acidity increased together with
the amount of iron sulphate added.
Table III. pH changes in adding differents amounts of iron sulphate.
All of these tests led us to the following conclusions: first, the pH of the ink is always acidic and the
acidity increases with the amount of gallic acid if it is enough; as iron sulphate is added, it reacts
with a greater amount of gallic acid, so the ink becomes darker, more compact and more acidic.
Secondly, the degree of acidity of the inks and the variation of the concentration of their
components suggests that the acidic group takes part in the ink formation. This is implied because it
is unlikely that the complex only has stability with the hydroxyl groups when the pH remains below
3. Finally, when arabic gum is added to the ink, pH becomes less acidic.
We divided our work in two parts. First we analysed the inorganic components by SEM-EDX and
then we used GC/MS to identify gallic acid and sugars. All of the eleven samples tested were
obtained from the "Arxiu de la Corona d'Aragó", they were not very valuable, but they were chosen
for their different appearances and acidity levels. Two of the samples were on parchment, a 16 th
century sample with light ink and a 17 th century sample ith dark ink. Another seven samples were
on paper Of the three samples with light ink ,two were from the mid-16th century and the third from
1757. The other four group samples were with dark ink dated from the end of the 16 th century to the
beginning of the 17th century. Three of the four latter samples had slight acidity problems. Another
two samples had a carbonized paper base.
MATERIALS AND METHODS
This study was performed by:
a) Scanning electron microscopy and
b) Analysis by GC/MS.
X-ray microanalysis.
a) Conditions in microanalysis: Beam current 0'8xl0"9 A; accelerating voltage, 20 Kv; 100.000
magnification; working distance 33 mm. Used model JSM 840 (Jeol Ltd.) with scanner AN 10000
(LINK SYSTEMS) with Be window.
b) Reagents employed for GC/MS analyses were: acetonitrile Merck; Bis (Trimethylsilyl
trifluoracetamid) (BSTFA) Merck; triethylamin Merck; pyridine Merck; trimethylchlorosiiane RPE
Carlo Erba; ethyl acetate Merck; sodium sulphate anhydrous RPE Carlo Erba; Fe S04.7H2O
Panreac; gallic acid Merck; arabic gum Panreac; methaiiol Merck; water Millipore; hydrochloric
acid Merck.
The working conditions for GC/MS were as follows: A KONIK KNK- 3000 HRGC (FID detector)
equipped whith a DB-5 column (J & W Scientific, Folsom, CA, USA) 25 m x 0,22 mm , 0,25 µm
film. The carrier gas was H2 (12 psi) and nitrogen was used as make up gas (30 ml/min). The mode
injection was split 1/20. The temperature was initially 60°C for 3 min, then programed at 6°C/min
to 285°C. The working conditions for GC/MS were as follows: A KONIK 3000 gas Chromatograph
was used, coupled with a magnetic Mass Spectrometer "VG TS-250" (Fisons Instruments,
Manchester, U.K.). and VG 11/250 data system. The GC conditions were the same noted above and
the column was coupled to the ion source . Helium was the carrier gas. The mode of lonization was
electron impact and the conditions were as follows: ionization energy 70 eV, mass range 45-500
amu, scan time 2sec/dcc, resolution 500.
a) Scanning electron microscopy and X-ray microanalysis.
The first step in this work was to analyse inorganic components. A previous study was published by
Sistach (19). In our study, two kind of samples were prepared for each kind of ink; one was
imbedded in Spurr resin and the other one was prepared without resin (Sec Fig. 1). We can check
the inorganic elements and their distribution on the base. Specific areas with ink, bordering or
around the ink can be analysed on the surface as well as into the paper base. The following inks
were analysed: light ink, the dark but not black ink, the black but not acidic ink and the very acidic
ink. We checked the penetration of the ink into the paper and the parchment. It is higher when the
base is paper. Similarly, when we looked at the paper surface we noticed particles between fibres
and found then to be precipitated CaSO4.
Fig. 1 SEM Image for a bordering area with and without ink in a paper base. We can see particles
of Ca SO4 on and between the fibres.
SEM-EDX analysis of light and dark-black inks without acidic behaviour. The areas with ink often
show calcium, iron and sulphur, and sometimes aluminium and potasium. The acidity depends on
the ratio between the first three elements noted above (Ca, S, and Fe), when analysing areas that
contain ink or are next to it. compared with other areas far away .The inks with light colour have the
ratio S/Fe < 1 in superficial areas with ink. When (he ink is dark but no acidic the ratio S/Fc can be
greater than or to 1. Another important factor determining ink behaviour is the presence of calcium,
because in light and dark non-acidic inks, this element keeps their ratio Ca/S greater or equal to that
of areas on the surface and into the support, although the iron is not present. For very dark inks on a
parchment support, the ratio is S/Fe >1 and S/Ca >1 in inner areas with ink. This one should be
acidic if the support was the paper.
SEM-EDX Inks with corrosive behaviour. Most of the areas with ink analysed showed a ratio
S/Fe < 1. The reason for finding less sulphur than iron in these particular ink areas for acidic
samples, is probably because the sulphur that comes from the sulphuric acid migrates to the surface
and to the inner zones far away from the ink when paper carbonization is important. This is
supposed to happen because in areas next to the ink we found significant quantities of sulphur
together with calcium, but in this case the ratio S/Ca can be greater than one. So we can compare
the sulphur excess in contrast with the alkaline reserve of the paper. Similarly, we have noted a
great amount of potasium in some samples with acidic ink. The presence of important amounts of
iron in acidic inks can contribute to cellulose degradation also, but if we quantify the acidity that
comes from the sulphuric acid with that which comes from iron, we should infer that it is mostly
due to the sulphuric acid.
b) Analysis by GC/MS.
The second part of our work was directed at the analysis of organic components. Our main aim was
to check whether or not gallic acid remains in the ink. In order to investigate this important point,
we worked in three steps: a) We prepared several iron gall inks with different quantity of gallic
acid. These are represented in Table I. b) We investigated the technical effectiveness in order to
determine whether or not the gallic acid remains in inks previously prepared at the laboratory. The
relationship between the quantity found was confirmed, c) Finally, we tried these established
conditions on original samples which come from the "Arxiu de la Corona d'Aragó". They were the
same samples tested with SEM-EDX.
In order to ensure good conditions for the identification of gallic acid in the laboratory inks, we took
several steps: a) Hidrolysis of the ink (25 uL of ink + 100 uL HC1 3%) for 2 hours at 105°C; b)
extraction of the organic components with ethyl acetate and passing the extract through a Na2SO4
anhydrous column; c) drying of the extract with a rotary evaporator in order to ensure that no water
remains in the sample; d) silylation of the dried samples using two different reagents: (acetonitrile
50 µL: BSTFA 50 µL: Triethylamin 25 µL and pyridine 100 µL : BSTFA 50 µL:
Trimethylchlorosilane 2µL). In order to analyse original ancient inks, we had to remove them from
the support, the process was made mechanically and some parchment and paper fibres can be
removed together with the ink. The ink samples (2-5 mg) were hydrolysed with 100-200 pL of HC1
3% at 105°C for 2 hours. We worked also with paper and parchment samples soaked in
hydrochloric acid to directly yield the hydrolysed components. The same four different kind of
samples were analysed. For each ink test, another sample spiked with standard gallic Acid solution
was prepared and compared with this standard solution. The results varied according to the
characteristics of the ink. The light inks and the very acidic inks did not have any gallic acid
remaining. However, the other inks retain small quantities of acid. We can see in this figure 2 how
light and non-acidic inks contain some sugars (galactose, rhamnose and arabinose) due to
parchment and arabic gum.
Fig. 2 GC/MS Chromatogram of light ink.
However, the very acidic inks retain few organic substances. These compounds could have
disappeared because of acidity. The Chromatograph provides information about gallic acid in
relation to the standard solution. Mass spectrometry allowed us to identify this substance in a
specific m/z: 281 and 458. (See fig 3).
Fig. 3. El Mass Spectrum of gallic acid TMS derivative.
CONCLUSIONS
The pH variations obtained from different inks made with iron sulphate and gallic acid in variable
ratios seem to indicate that the acid group of gallic acid contributes to the complex formation of the
ink. As far as saturated solution of iron sulphate is concerned, the ink will be darker and more dense
as gallic acid increases. Similarly, we determined the pH of ink increases as arabic gum is added.
SEM-EDX tests of ancient inks indicate the following conclusions. The Ca ratio in paper related to
S and Fe in ink. is very important in order to counteract acidity coming from sulphuric acid.
Distribution and ratio of these three elements in the support changes according to ink acidity;
therefore, in very acidic inks, the S from sulphuric is present in great amounts if we compare this
element with Ca in areas around the ink. However, we do not find significant amounts of S in ink
zones because S moves toward to the areas that border the ink, although we do find some iron in ink
zones.
Analysis of the organic compounds analysis in the ink by GC/MS indicate the following
conclusions: It seems that the fact that light inks are found is due to the scarce quantity of gallic
acid used in ink preparation, as this acid could disappear with the passage of time. The dark colour
of ink that bears no trace of acidity is due to gallic acid remaining in ink. The fact that black colour
is found in very acidic inks that carbonized the base is not because ink retains more gallic acid but
because when the organic compounds and the base carbonize, black colour appears.
It is the particular ratio of the three compounds gallic acid, iron sulphate and arabic gum that
determines ink quality and justifies its later acidic behaviour.
ACKNOWLEDGEMENTS.
The analysis of inorganic components of the ink was performed at the Electron Microscopy Service
of Barcelona University. We acknowledge Dr. R. Fontamau and coworkers for their contribution.
GC/MS analysis were performed at the Mass Spectrometry Laboratory of C.S.I.C. (Barcelona). We
want to thank Maria Guerra and Xavier Huguet in obtaining mass spectra. We want to thank to J.
Ll. Beltrán, Departament de Química Analítica Universität de Barcelona, for his valuable
discussions.
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