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\begin{document}
<<setup, include=FALSE, cache=FALSE>>=
library(knitr)
## set global chunk options
opts_chunk$set(fig.align='center')
opts_chunk$set(tidy=TRUE)
## set code/output width
options(width=85)
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@

\title{Equilibrium in CHNOSZ}

\author{Jeffrey M. Dick}

\maketitle
This document defines the concepts, explains the organization of functions,
and provides examples of calculating equilibrium in CHNOSZ. It also
highlights some applications of the methods (i.e. to reproduce published
diagrams) and includes an Appendix on details of the equilibration
calculations.

\section{Concepts}
\begin{description}
\item [{Species~of~interest}] Chemical species for which you want to
calculate relative stabilities.
\item [{Basis~species}] Species in terms of which you want to write all
formation reactions of species of interest.
\item [{Formation~reactions}] Stoichiometric chemical reactions showing
the mass balance requirements for formation of 1 mole of each species
of interest from the basis species.
\item [{Chemical~affinity}] Negative of the differential of Gibbs energy
of a system with respect to reaction progress. For a given reaction,
chemical affinity is the negative of Gibbs energy of reaction; $\boldsymbol{A}=2.303RT\log(K/Q)$,
where $K$ is the equilibrium constant and $Q$ is the activity quotient
of species in the reaction ($\log$ in this text denotes base-10 logarithms,
i.e. \texttt{log10} in R).
\item [{(1)~Reference~activity}] User-defined (usually equal) activities
of species of interest.
\item [{(1)~Reference~affinity}] ($\boldsymbol{A}_{{\it ref}}$) Chemical
affinity of formation reaction with a reference activity of the species
of interest.
\item [{(1)~Maximum~affinity~method}] Comparison of reference affinities
for given balance coefficients in order to calculate stability regions
on a predominance diagram.
\item [{Balance~coefficients}] ($n_{balance}$) The number of moles of
a basis species present in the formation reaction of each of the species
of interest. Reactions between any two species of interest then are
``balanced'' on this basis species. Can be a quantity other than
basis species (e.g., balance = 1, or length of amino acid sequence
of protein).
\item [{Predominance~diagram}] Diagram showing fields of maximal stability
(i.e. greatest activity at equilibrium) for species of interest as
a function of two variables (aka equal activity diagram).
\item [{(2)~Starred~affinity}] ($\boldsymbol{A}^{*}$) Chemical affinity
of formation reaction with unit activity of the species of interest
(aka ``starved'' affinity because the activity of the species of
interest drops out of $Q$).
\item [{(2)~Total~balance~activity}] The sum of activities of this basis
species contributed by each of the species of interest. (In Appendix:
activity of the immobile or conserved component; $a_{\mathrm{ic}}$.)
\item [{(2)~Equilibration~method}] Comparison of starred affinities in
order to calculate activities of species of interest for given balance
coefficients and total balance activity.
\item [{Speciation~diagram}] Diagram showing the activities of species
of interest, usually as a function of 1 variable (aka activity diagram).
\item [{Boltzmann~distribution}] Algorithm used for the equilibration
method when the balance coefficients are 1.
\item [{Reaction~matrix}] Algorithm used for the equilibration method
when the balance coefficients are not all 1.
\item [{Normalization}] Algorithm used for large molecules such as proteins;
chemical formulas and affinities are scaled to a similar molecular
size (e.g. a single residue; ``residue equivalent'' in Appendix),
activities are calculated using balance = 1, and formulas and activities
are rescaled to the original size of the molecule.
\item [{Mosaic}] Calculations of chemical affinities for making diagrams
where the speciation of basis species depends on the variables.
\end{description}
The numbered groups above are connected with two distinct approaches
to generating diagrams:
\begin{enumerate}
\item With the \textbf{maximum affinity method} for creating predominance
diagrams, the user sets the reference activities of the species of
interest; the program compares the reference affinities at these conditions
to determine the most stable species (highest activity, i.e. predominant
at equilibrium).
\item With the \textbf{equilibration method} for creating predominance or
activity diagrams, the user explicitly sets the total balance activity
or the program takes it from the reference activities of the species.
The starred affinities are used to calculate equilibrium activities
using one of two techniques (Boltzmann distribution for balance =
1, reaction matrix for balance $\ne$ 1).
\end{enumerate}
The affinities used in these calculations can be calculated using
\texttt{affinity()}, which works with a single basis set, or with
\texttt{mosaic()}, which uses multiple basis sets to account for basis
species that themselves may change as a function of the variables
of interest (e.g. ionization of carbonic acid as a function of pH).
This document focuses primarily on the \texttt{affinity()} function;
for more information on mosaic diagrams see the help page (type \texttt{?mosaic}
at the R command line).

Step-by-step examples of some of the calculations, particularly the
reaction matrix algorithm, are provided in the Appendix. For further
description of the equilibration method applied to proteins see \citet{DS13}
(also with a derivation of energetic distance from equilibrium using
the \textbf{starred affinity}).

\section{Organization}

The function sequences below assume you have already defined the basis
species and species of interest using \texttt{basis(...)} and \texttt{species(...)}
(ellipses here and below indicate system-specific input).

Note that if \texttt{equilibrate()} or \texttt{diagram()} is called
without an explicit \texttt{balance} argument, the balance coefficients
will be taken from the first basis species (in the current basis definition)
that is present in all of the species. Depending on the system, this
may coincide either with balance = 1 or with balance $\ne$ 1. In
the case of \texttt{normalize = TRUE} or \texttt{as.residue = TRUE},
the balance coefficients (for the purposes of the equilibration step)
are temporarily set to 1.
\begin{enumerate}
\item Maximum affinity method, balance = 1

\begin{enumerate}
\item Typical use: simple mineral/aqueous species stability comparisons
\item Function sequence:\\
\texttt{a <- affinity(...)}\\
\texttt{diagram(a, balance = 1)}
\item Algorithm: $\max\left\{ \boldsymbol{A}_{{\it ref}}\right\} $
\end{enumerate}
\item Equilibration method, balance = 1

\begin{enumerate}
\item Typical use: simple aqueous species activity comparisons
\item Function sequence:\\
\texttt{a <- affinity(...)}\\
\texttt{e <- equilibrate(a, balance = 1)}\\
\texttt{diagram(e)}
\item Algorithm: Boltzmann distribution
\end{enumerate}
\item Maximum affinity method, balance $\ne$ 1

\begin{enumerate}
\item Typical use: mineral/aqueous species stability comparisons
\item Function sequence:\\
\texttt{a <- affinity(...)}~\\
\texttt{diagram(a, balance = ...)}
\item Algorithm: $\max\left\{ \boldsymbol{A}_{{\it ref}}/n_{balance}\right\} $
\end{enumerate}
\item Equilibration method, balance $\ne$ 1

\begin{enumerate}
\item Typical use: aqueous species activity comparisons
\item Function sequence:\\
\texttt{a <- affinity(...)}~\\
\texttt{e <- equilibrate(a, balance = ...)}~\\
\texttt{diagram(e)}
\item Algorithm: Reaction matrix
\end{enumerate}
\item Maximum affinity method, normalize = TRUE

\begin{enumerate}
\item Typical use: protein/polymer stability comparisons
\item Function sequence:\\
\texttt{a <- affinity(...)}~\\
\texttt{diagram(a, normalize = TRUE)}
\item Algorithm: $\max\left\{ \boldsymbol{A}^{*}/n_{balance}-\log n_{balance}\right\} $
\end{enumerate}
\item Equilibration method, normalize = TRUE

\begin{enumerate}
\item Typical use: protein/polymer activity comparisons
\item Function sequence:\\
\texttt{a <- affinity(...)}~\\
\texttt{e <- equilibrate(a, normalize = TRUE)}~\\
\texttt{diagram(e)}
\item Algorithm: Scale formulas and affinities to residues; Boltzmann distribution
(balance = 1); Scale activities to proteins
\end{enumerate}
\end{enumerate}

\section{Examples}

\subsection{Amino acids}

Basis species: $\mathrm{CO_{2}}$, $\mathrm{H_{2}O}$, $\mathrm{NH_{3}}$,
$\mathrm{H_{2}S}$, $\mathrm{O_{2}}$. Species of interest: 20 amino
acids. (Only the first few lines of the data frame of amino acid species
are shown.)

<<AAsetup>>=
library(CHNOSZ)
data(thermo)
basis("CHNOS")
species(aminoacids(""))[1:5, ]
@

Code for making the diagrams. Function names refer to the subfigure
labels.

<<AAfigures>>=
res <- 200
aa <- aminoacids()

aaA <- function() {
  a <- affinity(O2=c(-90, -70, res), H2O=c(-20, 10, res))
  diagram(a, balance=1, names=aa)
}

aaB <- function() {
  a <- affinity(O2=c(-90, -70, 80), H2O=c(-20, 10, 80))
  e <- equilibrate(a, balance=1)
  diagram(e, names=aa)
}

aaC <- function() {
  a <- affinity(O2=c(-71, -66, res), H2O=c(-8, 4, res))
  diagram(a, balance="CO2", names=aa)
}

aaD <- function() {
  a <- affinity(O2=c(-71, -66, 80), H2O=c(-8, 4, 80))
  e <- equilibrate(a, balance="CO2")
  diagram(e, names=aa)
}

aaE <- function() {
  basis("O2", -66)
  a <- affinity(H2O=c(-8, 4))
  e <- equilibrate(a, balance="CO2")
  diagram(e, ylim=c(-5, -1), names=aa)
}

aaF <- function() {
  species(1:20, -4)
  a <- affinity(H2O=c(-8, 4))
  e <- equilibrate(a, balance="CO2")
  diagram(e, ylim=c(-5, -1), names=aa)
}
@

Note that for the plot we use the 1-letter abbreviations of the amino
acids, unlike the full species names (\texttt{aminoacids()} is a function
in CHNOSZ that returns their names or abbreviations).

<<AAnames>>=
AA <- aminoacids("")
names(AA) <- aa
AA
@

The annotated figure is shown next. The actual code used to set up
the plots, add labels, etc. is in the source of this vignette (not
shown in the PDF).

<<showtime, include=FALSE>>=
showtime <- function(st) {
  # plot time in lower-right of figure region
  f <- usrfig()
  par(xpd=TRUE)
  if(st[3] > 2) col <- "red" else col <- "black"
  text(f$x[2], f$y[1], paste(round(st[3], 1), "s\n"), adj=1, col=col)
  par(xpd=FALSE)
}
@

<<AAplot, echo=FALSE, message=FALSE, results="hide", fig.width=13/2, fig.height=9/2, cache=TRUE>>=
layout(t(matrix(c(1:7, 11, 8:10, 12), nrow=4)), widths=c(1, 4, 4, 4), heights=c(1, 4, 4))

## row 0 (column titles)
opar <- par(mar=c(0, 0, 0, 0))
plot.new()
plot.new()
text(0.5, 0.5, "maximum affinity", cex=1.4)
plot.new()
text(0.5, 0.5, "equilibration", cex=1.4)
plot.new()
text(0.5, 0.5, "equilibration", cex=1.4)
par(opar)

## row 1 (balance = 1)
opar <- par(mar=c(0, 0, 0, 0))
plot.new()
text(0.5, 0.5, "balance = 1", srt=90, cex=1.4)
par(opar)
# figure A
st <- system.time(dA <- aaA())
showtime(st)
title(main="loga(species) = -3", cex.main=1)
label.figure("A", col="blue", yfrac=0.9, xfrac=0.1)
# figure B
st <- system.time(dB <- aaB())
showtime(st)
title(main=paste("loga(total species) =", round(dB$loga.balance[1], 2)), cex.main=1)
label.figure("B", col="blue", yfrac=0.9, xfrac=0.1)

## row 2 (balance = nCO2)
opar <- par(mar=c(0, 0, 0, 0))
plot.new()
text(0.5, 0.5, 'balance = "CO2"', srt=90, cex=1.4)
par(opar)
# figure C
st <- system.time(dC <- aaC())
showtime(st)
title(main="loga(species) = -3", cex.main=1)
label.figure("C", col="blue", yfrac=0.9, xfrac=0.1)
# figure D
st <- system.time(dD <- aaD())
showtime(st)
title(main=paste("loga(total CO2) =", round(dD$loga.balance[1], 2)), cex.main=1)
label.figure("D", col="blue", yfrac=0.9, xfrac=0.1)

## right (speciation at different total activity of CO2)
par(xpd=NA)
lines(c(-66, -64.5), c(4, 9), lty=2)
lines(c(-66, -64.5), c(-8, -8.5), lty=2)
par(xpd=FALSE)
# figure E
st <- system.time(dE <- aaE())
showtime(st)
title(main=paste("loga(total CO2) =", round(dE$loga.balance[1], 2)), cex.main=1)
label.figure("E", col="blue", yfrac=0.9, xfrac=0.1)
# figure F
st <- system.time(dF <- aaF())
showtime(st)
title(main=paste("loga(total CO2) =", round(dF$loga.balance[1], 2)), cex.main=1)
label.figure("F", col="blue", yfrac=0.9, xfrac=0.1)

@

Comments on the plots:
\begin{itemize}
\item The equal-activity lines in Figures \textcolor{blue}{A} and \textcolor{blue}{B}
are \emph{identical}. For balance = 1, the maximum affinity method
and the equilibration method should produce the same predominance
diagrams. (More precisely, because balance = 1, the conditions of
equal activity of any species of interest \textbf{are independent
of} the actual value of that activity.)
\item Figures \textcolor{blue}{C} and \textcolor{blue}{D} are \emph{different}.
For balance $\ne$ 1, the maximum affinity method and the equilibration
method will generally produce difference predominance diagrams. (Because
balance $\ne$ 1, the conditions of equal activity of any species
of interest \textbf{depend on} the actual value of that activity.)
\item Both Figures \textcolor{blue}{E} and \textcolor{blue}{F} are constructed
using the equilibration method, to calculate activities of species
as a function of $\log a_{\mathrm{H_{2}O}}$ at $\log f_{\mathrm{O_{2}}}=-66$.
Figure \textcolor{blue}{E} shows the results for the default settings
($a_{\mathrm{CO_{2}}}$ is the sum of activities present in all species,
taken from initial species activity of $10^{-3}$) and the crossing
lines indicating equal activities \emph{are identical to the positions
in Figure }\textcolor{blue}{\emph{D}} at $\log f_{\mathrm{O_{2}}}=-66$.
\item Figure \textcolor{blue}{F} shows the results for a lower total activity
of $\mathrm{CO_{2}}$. Consequently, the activities of the predominant
species decrease from ca. $10^{-2}$ in Figure \textcolor{blue}{E}
to ca. $10^{-3}$ in Figure \textcolor{blue}{F}. Also, the stability
region of the smaller glycine has grown at the expense of the neighboring
bigger amino acids, so that the crossing lines indicating equal activities
in Figure \textcolor{blue}{F} \emph{are closer to those shown in Figure
}\textcolor{blue}{\emph{C}} at $\log f_{\mathrm{O_{2}}}=-66$.
\item In other words, a lower equal-activity value causes the stability
region of the species with the smaller balance coefficient to invade
that of the species with the larger balance coefficient.
\item Figures \textcolor{blue}{A}, \textcolor{blue}{B}, \textcolor{blue}{C},
and \textcolor{blue}{D} are all equal activity diagrams, but have
different constraints on the activities:

\begin{itemize}
\item Maximum affinity method (Figures \textcolor{blue}{A}, \textcolor{blue}{C}):
Equal activities of species set to a constant value.
\item Equilibration method (Figures \textcolor{blue}{B}, \textcolor{blue}{D}):
Equal activities of species determined by overall speciation of the
system.
\end{itemize}
\end{itemize}

\subsection{Proteins}

Basis species: $\mathrm{CO_{2}}$, $\mathrm{H_{2}O}$, $\mathrm{NH_{3}}$,
$\mathrm{H_{2}S}$, $\mathrm{O_{2}}$, $\mathrm{H^{+}}$. Species
of interest: 6 proteins from the set of archaeal and bacterial surface
layer proteins considered by \citet{Dic08}. 

<<PRsetup, results="hide">>=
basis("CHNOS+")
organisms <- c("METJA", "HALJP", "METVO", "ACEKI", "GEOSE", "BACLI")
proteins <- c(rep("CSG", 3), rep("SLAP", 3))
species(proteins, organisms)
@

Code for the figures.

<<PRfigures>>=
prA <- function() {
  a <- affinity(O2=c(-90, -70, 80), H2O=c(-20, 0, 80))
  e <- equilibrate(a, balance="length", loga.balance=0)
  diagram(e, names=organisms)
}

prB <- function() {
  a <- affinity(O2=c(-90, -70))
  e <- equilibrate(a, balance="length", loga.balance=0)
  diagram(e, names=organisms, ylim=c(-5, -1))
}

prC <- function() {
  a <- affinity(O2=c(-90, -70, res), H2O=c(-20, 0, res))
  e <- equilibrate(a, normalize=TRUE, loga.balance=0)
  diagram(e, names=organisms)
}

prD <- function() {
  a <- affinity(O2=c(-90, -70))
  e <- equilibrate(a, normalize=TRUE, loga.balance=0)
  diagram(e, names=organisms, ylim=c(-5, -1))
}

prE <- function() {
  a <- affinity(O2=c(-90, -70, res), H2O=c(-20, 0, res))
  e <- equilibrate(a, as.residue=TRUE, loga.balance=0)
  diagram(e, names=organisms)
}

prF <- function() {
  a <- affinity(O2=c(-90, -70))
  e <- equilibrate(a, as.residue=TRUE, loga.balance=0)
  diagram(e, names=organisms, ylim=c(-3, 1))
}
@

The plots follow. As before, the code used to layout the figure and
label the plots is not shown in the PDF.

<<PRplot, message=FALSE, echo=FALSE, results="hide", fig.width=13/2, fig.height=9/2, cache=TRUE>>=
layout(t(matrix(1:12, nrow=4)), widths=c(1, 4, 4, 4), heights=c(1, 4, 4))

## row 0 (column titles)
opar <- par(mar=c(0, 0, 0, 0))
plot.new()
plot.new()
text(0.5, 0.5, 'balance = "length"', cex=1.4)
plot.new()
text(0.5, 0.5, "normalize = TRUE\n(balance = 1)", cex=1.4)
plot.new()
text(0.5, 0.5, "as.residue = TRUE\n(balance = 1)", cex=1.4)
par(opar)

## row 1 (equilibrate 2D)
opar <- par(mar=c(0, 0, 0, 0))
plot.new()
text(0.5, 0.5, "equilibration", srt=90, cex=1.4)
par(opar)
# figure A (balance = "length")
st <- system.time(dA <- prA())
showtime(st)
label.figure("A", col="blue", yfrac=0.9, xfrac=0.1)
# figure C (normalize = TRUE)
st <- system.time(dC <- prC())
showtime(st)
label.figure("C", col="blue", yfrac=0.9, xfrac=0.1)
# figure E (as.residue = TRUE)
st <- system.time(dE <- prE())
showtime(st)
label.figure("E", col="blue", yfrac=0.9, xfrac=0.1)

## row 2 (equilibrate 1D)
opar <- par(mar=c(0, 0, 0, 0))
plot.new()
text(0.5, 0.5, "equilibration", srt=90, cex=1.4)
par(opar)
# figure B (balance = "length")
st <- system.time(prB())
showtime(st)
label.figure("B", col="blue", yfrac=0.9, xfrac=0.1)
# figure D (normalize = TRUE)
st <- system.time(prD())
showtime(st)
label.figure("D", col="blue", yfrac=0.9, xfrac=0.1)
# figure F (as.residue = TRUE)
st <- system.time(prF())
showtime(st)
label.figure("F", col="blue", yfrac=0.9, xfrac=0.1)
@

Comments on the plots:
\begin{itemize}
\item All of the plots shown are calculated using the equilibration method.
The balanced species is amino acid residues (specified by balance
= ``length'' in Figures \textcolor{blue}{A} and \textcolor{blue}{B}.
In the other figures, \texttt{normalize = TRUE} and \texttt{as.residue
= TRUE} internally reset the balance to 1 after scaling the protein
formulas to single amino acid residue equivalents). Activity of the
balanced species (amino acid residues) is set to 1 (log activity =
0).
\item Figure \textcolor{blue}{B} shows that balancing on length produces
drastic transitions between activities of the proteins. This either/or
type behavior is a consequence of the large sizes of the balancing
coefficients, which become exponents on the activities in the expression
for $Q$ (or coefficients on the logarithms of activities in $\log Q$).
\item Figure \textcolor{blue}{D} shows that coexistence of proteins with
comparable activities can be predicted using \texttt{normalize = TRUE}.
Here, the protein formulas and affinities are scaled down to their
``residue equivalents'', then the equilibrium among the residue
equivalents is calculated (with balance = 1), and the activities are
rescaled to the original proteins. For example, a residue activity
of 0 corresponds to $10^{-2}$ for a 100-residue protein and to $10^{-3}$
for a 1000-residue protein. 
\item Figures \textcolor{blue}{E} and \textcolor{blue}{F} are like \texttt{normalize
= TRUE}, except that the rescaling to original protein size is not
performed. Note the higher activities of the residue equivalents (Figure
\textcolor{blue}{F}) compared to the proteins (Figure \textcolor{blue}{D}).
\item Compare the equilibration plots above with the maximum affinity plots
below. Here the equal activities of the proteins are intentionally
set to a very low value: this causes a difference in the plot using
balance = ``length'', but the second and third diagrams remain equivalent
to those in Figures \textcolor{blue}{C} and \textcolor{blue}{E} above
(verified by the \texttt{stopifnot} statements; \texttt{dA}, \texttt{dC}
and \texttt{dE} refer to the diagrams above). This behavior is consistent
with that seen in the amino acid example, where the maximum affinity
and equilibration methods give equivalent results for balance = 1
but different results for balance $\ne$ 1.
\end{itemize}
<<PRplot2, message=FALSE, results="hide", fig.width=6, fig.height=2, cache=TRUE>>=
layout(t(matrix(1:3)))
species(1:6, -111)
a <- affinity(O2=c(-90, -70, res), H2O=c(-20, 0, res))
d1 <- diagram(a, balance="length", names=organisms, main='balance = "length"')
d2 <- diagram(a, normalize=TRUE, names=organisms, main="normalize = TRUE")
d3 <- diagram(a, as.residue=TRUE, names=organisms, main="as.residue = TRUE")
stopifnot(!identical(d1$predominant, dA$predominant))
stopifnot(identical(d2$predominant, dC$predominant))
stopifnot(identical(d3$predominant, dE$predominant))
@

With balance = ``length'', changing the equal activities to \emph{lower
values} increases the relative stabilities of the \emph{smaller proteins},
which is why the stability field of the larger protein from BACLI
disappears while that of the smaller protein from METJA grows. Because
of the drastic activity changes at the stability transitions (see
Figure \textcolor{blue}{B} above), a large change in equal activities
(to a minuscule activity = $10^{-111}$) is used here to demonstrate
this effect, and even then the visual impact on the predominance diagram
is subtle. Therefore, naturally occurring relative abundances of proteins
are better modeled using the \texttt{normalize} or \texttt{as.residue}
approaches.

\clearpage

\section{Applications}

Many of the help-page examples and demos in CHNOSZ use these methods
to reproduce (or closely emulate) published figures. Below is not
a comprehensive list, but just some highlights.

\subsection{Maximum affinity method}
\begin{itemize}
\item The ``Aqueous Aluminum'' example in \texttt{?diagram} shows predominance
fields for aqueous species (balance = 1, after \citealp{TS01})
\item The ``Fe-S-O'' example in \texttt{?diagram} shows stability fields
for minerals (balance $\ne$ 1, after \citealp{Hel70c}).
\item Next is an example of using \emph{unequal activities} of species (mineral
activity = 1; variable aqueous species activity indicated by the contours
(logarithm of activity)) to plot aqueous species \textendash{} mineral
stability boundaries (balance = 1, after Figure 14 of \citealp{Pou49}).
\end{itemize}
<<Pourbaix, message=FALSE, results="hide", fig.width=4, fig.height=3, cache=TRUE>>=
basis(c("Cu+2", "H2O", "H+", "e-"))
species(c("Cu+2", "copper", "cuprite", "tenorite"))
for(loga in c(-1, 0, -2, -3)) {
  species("Cu+2", loga) 
  a <- affinity(pH=c(1.6, 7.6, 400), Eh=c(-0.2, 1, 400))
  if(loga==-1) d <- diagram(a)
  else d <- diagram(a, add=TRUE, names=NULL)
  iCu <- which(d$predominant == 1, arr.ind=TRUE)
  text(a$vals[[1]][max(iCu[, 1])] - 0.03, a$vals[[2]][min(iCu[, 2])] + 0.2, adj=1, loga)
}
water.lines(d)

@

\subsection{Equilibration method}
\begin{itemize}
\item Speciation of reduced and oxidized glutathione, after \citet{SB01}.
Two moles of reduced glutathione (GSH) are oxidized to produce one
mole of oxidized glutathione containing a disulfide bond (GSSG), according
to
\[
2\mathrm{GSH}\rightleftharpoons\mathrm{GSSG}+\mathrm{H_{2}}
\]
First, we define a basis set that includes GSH; this becomes the balanced
basis species, so we can set its total activity in the call to \texttt{equilibrate()}.
This total activity is the initial concentration of GSH that will
be speciated among GSH and GSSG. If the aqueous species have equal
concentrations (or activities), the fraction of GSH that has been
oxidized is actually 2/3, because the formation of one mole of GSSG
consumes two moles of GSH. That is why the blue lines (fraction of
starting GSH that is oxidized) are higher than the black lines (aqueous
species distribution). Although the caption to Fig. 3 of \citet{SB01}
reads ``percent GSH that has been oxidized to GSSG'', the lines
in their figure are closer to the black lines in the figure below.
\end{itemize}
<<glutathione, results="hide", message=FALSE, fig.width=5, fig.height=4>>=
basis(c("GSH", "NH3", "H2S", "H2O", "H+", "e-"))
basis("pH", 7)
species(c("GSH", "GSSG"))
a <- affinity(Eh=c(-0.3, -0.1))
# initial millimoles of GSH
mM <- c(10, 3, 1)
M <- mM * 1e-3
for(i in 1:3) {
  e <- equilibrate(a, loga.balance=log10(M[i]))
  diagram(e, alpha=TRUE, lty=c(0, i), add = i!=1, legend.x=NULL, ylim=c(0, 1), yline=1.6, lwd=2, ylab="aqueous species distribution")
  fGSH <- 1 - (10^e$loga.equil[[1]] / M[i])
  lines(e$vals[[1]], fGSH, col="blue", lty=i)
}
mtext(side=2, "fraction of GSH oxidized to GSSG", las=0, line=2.6, col="blue", cex=0.8)
mtext(side=2, "- - - - - - - - - - - - - - - - - - -", las=0, line=2.1, cex=0.8)
legend("topleft", lty=1:3, legend=paste(mM, "mM GSH"))
@

\subsection{Mosaic diagrams}

The examples using \texttt{mosaic()} shown below are based on the
maximum affinity method, but equilibration calculations are also possible
(if a suitable example from the literature is found it will be added
here; see also the \texttt{blend} argument of \texttt{mosaic()} to
equilibrate the basis species rather than compose the diagram using
the predominant basis species).
\begin{itemize}
\item See the examples in \texttt{?mosaic} and \texttt{demo(\textquotedbl{}mosaic\textquotedbl{})}
for calculations of mineral stabilities in the Fe-S-O-$\mathrm{H_{2}O}$
system (after \citealp{GC65}).
\item A calculation of copper solubility limits and speciation with aqueous
glycine based on Fig. 2b of \citet{AD01}. We use \texttt{mosaic()}
to speciate the basis species glycine (activity $10^{-1}$) as a function
of pH. The stability fields are shown for \emph{unequal activities}
of the minerals (unit activity) and aqueous species ($10^{-4}$).
This is essentially a composite of three diagrams, with glycinium,
glycine and glycinate in the basis at low, mid and high pH. This demo
also modifies the thermodynamic database (with \texttt{mod.obigt()})
to use Gibbs energies taken from \citet{AD01}, and bypasses the default
plotting of labels by \texttt{diagram()} in order to customize their
format and placement.
\end{itemize}
<<Aksu_Doyle, message=FALSE, results="hide", fig.width=6, fig.height=4.8, cache=TRUE>>=
demo("copper", ask=FALSE)
@

\appendix

\section*{Appendix}

Two different methods of calculating the equilibrium activities of
species in a system are described below. These are referred to as
the \emph{reaction-matrix approach} and the \emph{Boltzmann distribution}.
Each method is demonstrated using a specific example that has been
described previously \citep{Dic08,DS11} (the ``CSG'' example).
The results shows that two approaches are equivalent when the molar
formulas are normalized.

\section{Standard states, the ideal approximation and sources of data}

By chemical activity we mean the quantity $a_{i}$ that appears in
the expression 
\begin{equation}
\mu_{i}=\mu_{i}^{\circ}+RT\ln a_{i}\,,\label{eq:mu}
\end{equation}
where $\mu_{i}$ and $\mu_{i}^{\circ}$ stand for the chemical potential
and the standard chemical potential of the $i$th species, and $R$
and $T$ represent the gas constant and the temperature in Kelvin
($\ln$here stands for the natural logarithm). Chemical activity is
related to molality ($m_{i}$) by
\begin{equation}
a_{i}=\gamma_{i}m_{i}\,,\label{eq:activity}
\end{equation}
where $\gamma_{i}$ stands for the activity coefficient of the $i$th
species. For this discussion, we take $\gamma_{i}=1$ for all species,
so chemical activity is assumed to be numerically equivalent to molality.
Since molality is a measure of concentration, calculating the equilibrium
chemical activities can be a theoretical tool to help understand the
relative abundances of species, including proteins.

For the CSG examples below, we would like to reproduce exactly the
values appearing in publications. Because recent versions of CHNOSZ
incorporate data updates for the methionine sidechain group, we should
therefore revert to the previous values \citep{DLH06} before proceeding.

<<AddObigt>>=
data(thermo)
mod.obigt("[Met]", G=-35245, H=-59310)
@

\section{Reaction-matrix approach}

\subsection{CSG Example: Whole formulas}

Let us calculate the equilibrium activities of two proteins in metastable
equilibrium. To do this we start by writing the formation reactions
of each protein as
\begin{equation}
{\it stuff}_{3}\rightleftharpoons\mathrm{CSG\_METVO}\,\label{react:CSG_METVO}
\end{equation}
and
\begin{equation}
{\it stuff}_{4}\rightleftharpoons\mathrm{CSG\_METJA}\,.\label{react:CSG_METJA}
\end{equation}
The basis species in the reactions are collectively symbolized by
${\it stuff}$; the subscripts simply refer to the reaction number
in this document. In these examples, ${\it stuff}$ consists of $\mathrm{CO_{2}}$,
$\mathrm{H_{2}O}$, $\mathrm{NH_{3}}$, $\mathrm{O_{2}}$, $\mathrm{H_{2}S}$
and $\mathrm{H^{+}}$ in different molar proportions. To see what
${\it stuff}$ is, try out these commands in CHNOSZ:

<<ProteinFormation>>=
basis("CHNOS+")
species("CSG",c("METVO", "METJA"))
@

Although the basis species are defined, the temperature is not yet
specified, so it is not immediately possible to calculate the ionization
states of the proteins. That is why the coefficient on $\mathrm{H^{+}}$
is zero in the output above. Let us now calculate the chemical affinities
of formation of the ionized proteins at 25 $^{\circ}$C, 1 bar, and
pH = 7 (specified by the logarithm of activity of $\mathrm{H^{+}}$
in the basis species):

<<ProteinAffinity>>=
a <- affinity()
a$values
@

Since \texttt{affinity()} returns a list with a lot of information
(such as the basis species and species definitions) the last command
was written to only print the \texttt{values} part of that list. The
values are actually dimensionless, i.e. $\boldsymbol{A}/2.303RT$.

The affinities of the formation reactions above were calculated for
a \emph{reference value of activity of the proteins, which is not
the equilibrium value}. Those non-equilibrium activities were $10^{-3}$.
How do we calculate the equilibrium values? Let us write specific
statements of the expression for chemical affinity (2.303 is used
here to stand for the natural logarithm of 10), 
\begin{equation}
\boldsymbol{A}=2.303RT\log(K/Q)\,,\label{eq:affinity}
\end{equation}
for Reactions \ref{react:CSG_METVO} and \ref{react:CSG_METJA} as
\begin{equation}
\begin{array}{rl}
\boldsymbol{A}_{3}/2.303RT & =\log K_{3}-\log Q_{3}\\
 & =\log K_{3}+\log a_{{\it stuff},3}-\log a_{\mathrm{CSG\_METVO}}\\
 & =\boldsymbol{A}_{3}^{*}/2.303RT-\log a_{\mathrm{CSG\_METVO}}
\end{array}\label{eq:A3_METVO}
\end{equation}
and

\begin{equation}
\begin{array}{rl}
\boldsymbol{A}_{4}/2.303RT & =\log K_{4}-\log Q_{4}\\
 & =\log K_{4}+\log a_{{\it stuff},4}-\log a_{\mathrm{CSG\_METJA}}\\
 & =\boldsymbol{A}_{4}^{*}/2.303RT-\log a_{\mathrm{CSG\_METJA}}\,.
\end{array}\label{eq:A4_METJA}
\end{equation}
The $\boldsymbol{A}^{*}$ denote the affinities of the formation reactions
when the activities of the proteins are unity. I like to call these
the ``starved'' affinities. From the output above it follows that
$\boldsymbol{A}_{3}^{*}/2.303RT=104.6774$ and $\boldsymbol{A}_{4}^{*}/2.303RT=314.1877$.

Next we must specify how reactions are balanced in this system: what
is conserved during transformations between species (let us call it
the immobile component)? For proteins, one possibility is to use the
repeating protein backbone group. Let us use $n_{i}$ to designate
the number of residues in the $i$th protein, which is equal to the
number of backbone groups, which is equal to the length of the sequence.
If $\gamma_{i}=1$ in Eq. (\ref{eq:activity}), the relationship between
the activity of the $i$th protein ($a_{i}$) and the activity of
the residue equivalent of the $i$th protein ($a_{residue,i}$) is
\begin{equation}
a_{residue,i}=n_{i}\times a_{i}\,.\label{eq:a_residue_i}
\end{equation}
 We can use this to write a statement of mass balance: 
\begin{equation}
553\times a_{\mathrm{CSG\_METVO}}+530\times a_{\mathrm{CSG\_METJA}}=1.083\,.\label{eq:a_residue}
\end{equation}

At equilibrium, the affinities of the formation reactions, per conserved
quantity (in this case protein backbone groups) are equal. Therefore
$\boldsymbol{A}=\boldsymbol{A}_{3}/553=\boldsymbol{A}_{4}/530$ is
a condition for equilibrium. Combining this with Eqs. (\ref{eq:A3_METVO})
and (\ref{eq:A4_METJA}) gives
\begin{equation}
\boldsymbol{A}/2.303RT=\left(104.6774-\log a_{\mathrm{CSG\_METVO}}\right)/553\label{eq:A_METVO}
\end{equation}
and
\begin{equation}
\boldsymbol{A}/2.303RT=\left(314.1877-\log a_{\mathrm{CSG\_METJA}}\right)/530\,.\label{eq:A_METJA}
\end{equation}
Now we have three equations (\ref{eq:a_residue}\textendash \ref{eq:A_METJA})
with three unknowns. The solution can be displayed in CHNOSZ as follows.
Because the balancing coefficients differ from unity, the function
called by \texttt{equilibrate()} in this case is \texttt{equil.reaction()},
which implements the equation-solving strategy described in the next
section.

<<ProteinActivities>>=
e <- equilibrate(a)
e$loga.equil
@

Those are the logarithms of the equilibrium activities of the proteins.
Combining these values with either Eq. (\ref{eq:A_METVO}) or (\ref{eq:A_METJA})
gives us the same value for affinity of the formation reactions per
residue (or per protein backbone group), $\boldsymbol{A}/2.303RT=0.5978817$.
Equilibrium activities that differ by such great magnitudes make it
appear that the proteins are very unlikely to coexist in metastable
equilibrium. Later we explain the concept of using residue equivalents
of the proteins to achieve a different result.

\subsection{Implementing the reaction-matrix approach}

CHNOSZ implements a method for solving the system of equations that
relies on a difference function for the activity of the immobile component.
The steps to obtain this difference function are:
\begin{enumerate}
\item Set the total activity of the immobile (conserved) component (aka
total balance activity) as $a_{\mathrm{ic}}$ (e.g., the 1.083 in
Eqn. \ref{eq:a_residue}).
\item Write a function for the logarithm of activity of each of the species
of interest: $\boldsymbol{A}=\left(\boldsymbol{A}_{i}^{*}-2.303RT\log a_{i}\right)/n_{\mathrm{ic},i}$,
where $n_{\mathrm{ic},i}$ stands for the number of moles of the immobile
component that react in the formation of one mole of the $i$th species.
(e.g., for systems of proteins where the backbone group is conserved,
$n_{\mathrm{ic},i}$ is the same as $n_{i}$ in Eq. \ref{eq:a_residue_i}).
Calculate values for each of the $\boldsymbol{A}_{i}^{*}$. Metastable
equilibrium is implied by the equality of $\boldsymbol{A}$ in all
of the equations.
\item Write a function for the total activity of the immobile component:
$a_{\mathrm{ic}}^{'}=\sum n_{\mathrm{ic},i}a_{i}$.
\item The difference function is now $\delta a_{\mathrm{ic}}=a_{\mathrm{ic}}^{'}-a_{\mathrm{ic}}$.
\end{enumerate}
Now all we have to do is find the value of $\boldsymbol{A}$ where
$\delta a_{\mathrm{ic}}=0$. This is achieved in the code by first
looking for a range of values of $\boldsymbol{A}$ where at one end
$\delta a_{\mathrm{ic}}<0$ and at the other end $\delta a_{\mathrm{ic}}>0$,
then using the \texttt{uniroot()} function that is part of R to find
the solution. 

Even if values of $\delta a_{\mathrm{ic}}$ on either side of zero
can be located, the algorithm does not guarantee an accurate solution
and may give a warning about poor convergence if a certain tolerance
is not reached.

\subsection{CSG Example: normalized formulas (residue equivalents)}

Let us consider the formation reactions of the normalized formulas
(residue equivalents) of proteins, for example
\begin{equation}
{\it stuff}_{12}\rightleftharpoons\mathrm{CSG\_METVO(residue)}\,\label{react:CSG_METVO_residue}
\end{equation}
and
\begin{equation}
{\it stuff}_{13}\rightleftharpoons\mathrm{CSG\_METJA(residue)}\,.\label{react:CSG_METJA_residue}
\end{equation}
The formulas of the residue equivalents are those of the proteins
divided by the number of residues in each protein. The \texttt{protein.basis()}
function shows the coefficients on the basis species in these reactions:

<<ProteinBasis>>=
protein.basis(species()$name, normalize=TRUE)
@

Let us denote by $\boldsymbol{A}_{12}$ and $\boldsymbol{A}_{13}$
the chemical affinities of Reactions \ref{react:CSG_METVO_residue}
and \ref{react:CSG_METJA_residue}. We can write 
\begin{equation}
\boldsymbol{A}_{12}/2.303RT=\log K_{12}+\log a_{{\it stuff},12}-\log a_{\mathrm{CSG\_METVO(residue)}}\label{eq:A3_METVO_residue}
\end{equation}
and

\begin{equation}
\boldsymbol{A}_{13}/2.303RT=\log K_{13}+\log a_{{\it stuff},13}-\log a_{\mathrm{CSG\_METJA(residue)}}\,.\label{eq:A4_METJA_residue}
\end{equation}
For metastable equilibrium we have $\boldsymbol{A}_{12}/1=\boldsymbol{A}_{13}/1$.
The $1$'s in the denominators are there as a reminder that we are
still conserving residues, and that each reaction now is written for
the formation of a single residue equivalent. So, let us write $\boldsymbol{A}$
for $\boldsymbol{A}_{12}=\boldsymbol{A}_{13}$ and also define $\boldsymbol{A}_{12}^{*}=\boldsymbol{A}_{12}+2.303RT\log a_{\mathrm{CSG\_METVO(residue)}}$
and $\boldsymbol{A}_{13}^{*}=\boldsymbol{A}_{13}+2.303RT\log a_{\mathrm{CSG\_METJA(residue)}}$.
At the same temperature, pressure and activities of basis species
and proteins as shown in the previous section, we can write $\boldsymbol{A}_{12}^{*}=\boldsymbol{A}_{3}^{*}/553=2.303RT\times0.1892901$
and $\boldsymbol{A}_{13}^{*}=\boldsymbol{A}_{4}^{*}/530=2.303RT\times0.5928069$
to give 
\begin{equation}
\boldsymbol{A}/2.303RT=0.1892901-\log a_{\mathrm{CSG\_METVO(residue)}}\label{eq:A_METVO_residue}
\end{equation}
and
\begin{equation}
\boldsymbol{A}/2.303RT=0.5928069-\log a_{\mathrm{CSG\_METJA(residue)}}\,,\label{eq:A_METJA_residue}
\end{equation}
which are equivalent to Equations 12 and 13 in the paper \citep{Dic08}
but with more decimal places shown. A third equation arises from the
conservation of amino acid residues:
\begin{equation}
a_{\mathrm{CSG\_METVO(residue)}}+a_{\mathrm{CSG\_METJA(residue)}}=1.083\,.\label{eq:a_residue_2}
\end{equation}
The solution to these equations is $a_{\mathrm{CSG\_METVO(residue)}}=0.3065982$,
$a_{\mathrm{CSG\_METJA(residue)}}=0.7764018$ and $\boldsymbol{A}/2.303RT=0.7027204$.

The corresponding logarithms of activities of the proteins are $\log\left(0.307/553\right)=-3.256$
and $\log\left(0.776/530\right)=-2.834$. These activities of the
proteins are much closer to each other than those calculated using
formation reactions for whole protein formulas, so this result seems
more compatible with the actual coexistence of proteins in nature.

The approach just described is not actually used by \texttt{equilibrate(...,
normalize = TRUE)}. Instead, because balance = 1, the Boltzmann distribution,
which is faster, can be used.

\section{Boltzmann distribution}

\subsection{CSG Example: Normalized formulas}

An expression for Boltzmann distribution, relating equilibrium activities
of species to the affinities of their formation reactions, can be
written as (using the same definitions of the symbols above)
\begin{equation}
\frac{a_{i}}{\sum a_{i}}=\frac{e^{\boldsymbol{A}_{i}^{*}/RT}}{\sum e^{\boldsymbol{A}_{i}^{*}/RT}}\,.\label{eq:Boltzmann}
\end{equation}
Using this equation, we can very quickly (without setting up a system
of equations) calculate the equilibrium activities of proteins using
their residue equivalents. Above, we saw $\boldsymbol{A}_{12}^{*}/2.303RT=0.1892901$
and $\boldsymbol{A}_{13}^{*}/2.303RT=0.5928069$. Multiplying by $\ln10=2.302585$
gives $\boldsymbol{A}_{12}^{*}/RT=0.4358565$ and $\boldsymbol{A}_{13}^{*}/RT=1.364988$.
We then have $e^{\boldsymbol{A}_{12}^{*}/RT}=1.546287$ and $e^{\boldsymbol{A}_{13}/RT}=3.915678$.
This gives us $\sum e^{A_{i}^{*}/RT}=5.461965$, $a_{12}/\sum a_{i}=0.2831009$
and $a_{13}/\sum a_{i}=0.7168991$. Since $\sum a_{i}=1.083$, we
arrive at $a_{12}=0.3065982$ and $a_{13}=0.7764018$, the same result
as above.

\section{Notes on implementation}

\subsection{CSG example: another look}

All the tedium of writing reactions, calculating affinities, etc.,
above does help to understand the application of the reaction-matrix
and Boltzmann distribution methods to protein equilibrium calculations.
But can we automate the step-by-step calculation for any system, including
more than two proteins? And can we be sure that higher-level functions
in CHNOSZ, particularly \texttt{equilibrate()}, match the output of
the step-by-step calculations? Now we can, with the \texttt{protein.equil()}
function introduced in version 0.9-9. Below is its output when configured
for CSG example we have been discussing.

\begin{small}

<<ProteinEquil, results="hide">>=
protein <- pinfo(c("CSG_METVO", "CSG_METJA"))
basis("CHNOS+")
swap.basis("O2", "H2")
protein.equil(protein, loga.protein=-3)
@

\end{small}

The function checks (``check it!'') against the step-by-step calculations
the values of $\boldsymbol{A}^{*}$ calculated using \texttt{affinity()},
and the equilibrium activities of the proteins calculated using \texttt{equilibrate()}.
(Note that Astar/RT in the second line after the first ``check it!''
can be multiplied by $\ln10$ to get the values shown above in Eqs.
\ref{eq:A_METVO_residue} and \ref{eq:A_METJA_residue}.)

\subsection{Visualizing the effects of normalization}

A comparison of equilibrium calculations that do and do not use normalized
formulas for proteins was presented by \citet{Dic08}. A diagram like
Figure 5 in that paper is shown below.

<<ProteinSpeciation, results="hide", message=FALSE, fig.height=5.5, cache=TRUE>>=
organisms <- c("METSC", "METJA", "METFE", "HALJP",  "METVO", "METBU", "ACEKI", "GEOSE", "BACLI", "AERSA")
proteins <- c(rep("CSG", 6), rep("SLAP", 4))
basis("CHNOS+")
species(proteins, organisms)
a <- affinity(O2=c(-100, -65))
layout(matrix(1:2), heights=c(1, 2))
e <- equilibrate(a)
diagram(e, ylim=c(-2.8, -1.6), names=organisms)
water.lines(e)
title(main="Equilibrium activities of proteins, normalize = FALSE")
e <- equilibrate(a, normalize=TRUE)
diagram(e, ylim=c(-4, -2), names=organisms)
water.lines(e)
title(main="Equilibrium activities of proteins, normalize = TRUE")
@

Although it is well below the stability limit of $\mathrm{H_{2}O}$
(vertical dashed line), there is an interesting convergence of the
activities of some proteins at low $\log f_{\mathrm{O_{2}}}$, due
most likely to compositional similarity of the amino acid sequences.

The reaction-matrix approach can also be applied to systems having
conservation coefficients that differ from unity, such as many mineral
and inorganic systems, where the immobile component has different
molar coefficients in the formulas. For example, consider a system
like one described by \citet{See96}:

<<SulfurSpeciation, results="hide", message=FALSE, fig.width=6, fig.height=5.5>>=
basis("CHNOS+")
basis("pH", 5)
species(c("H2S", "S2-2", "S3-2", "S2O3-2", "S2O4-2",  "S3O6-2", "S5O6-2", "S2O6-2", "HSO3-", "SO2", "HSO4-"))
a <- affinity(O2=c(-50, -15), T=325, P=350)
layout(matrix(c(1, 2, 3, 3), nrow=2), widths=c(4, 1))
col <- rep(c("blue", "black", "red"), each=4)
lty <- 1:4
for(normalize in c(FALSE, TRUE)) {
  e <- equilibrate(a, loga.balance=-2, normalize=normalize)
  diagram(e, ylim=c(-30, 0), legend.x=NULL, col=col, lty=lty)
  water.lines(e)
  title(main=paste("Aqueous sulfur speciation, normalize =", normalize))
}
par(mar=c(0, 0, 0, 0))
plot.new()
leg <- lapply(species()$name, expr.species)
legend("center", lty=lty, col=col, lwd=1.5, bty="n", legend=as.expression(leg), y.intersp=1.3)
@

The first diagram is quantitatively similar to the one shown by \citet{See96},
with the addition of color and the water stability line. If we use
the normalized formulas (divide whole formulas by moles of $\mathrm{H_{2}S}$
in their formation reactions), the range of activities of species
is smaller at any given $\log f_{\mathrm{O_{2}}_{\left(g\right)}}$,
as shown in the second diagram. Although \texttt{normalize = TRUE}
was implemented to study the relative stabilities of proteins, it
might also be relevant to other systems where the molecules are in
various stages of polymerization.

\section*{Document history}
\begin{itemize}
\item 2009-11-29 Initial version containing CSG example (title: Calculating
relative abundances of proteins)
\item 2012-09-30 Renamed to ``Equilibrium in CHNOSZ''. Remove activity
comparisons, add maximum affinity method.
\item 2015-11-08 Add sections on concepts, organization, examples and applications;
move most material from the previous version of the document to the
Appendix; now uses the \textbf{knitr} vignette engine.
\end{itemize}
\bibliographystyle{plainnat}
\bibliography{vig}

\end{document}
