ChemoSpec: An R Package for Chemometric

Analysis of Spectroscopic Data

(Package Version 4.4.85)

Bryan A. Hanson∗
e-mail: [email protected]

with contributions from Matt J. Keinsley

DePauw University
Department of Chemistry & Biochemistry
Greencastle Indiana USA

github.com/bryanhanson/ChemoSpec
CRAN.R-project.org/package=ChemoSpec

July 27, 2017

Abstract
ChemoSpec[1] is a collection of functions for top-down exploratory data analysis of spectral data obtained via nuclear
magnetic resonance (NMR), infrared (IR) or Raman spectroscopy. Includes functions for plotting and inspecting
spectra, peak alignment, hierarchical cluster analysis (HCA), principal components analysis (PCA) and model-based
clustering. Robust methods appropriate for this type of high-dimensional data are available. ChemoSpec is designed
with metabolomics data sets in mind, where the samples fall into groups such as treatment and control. Graphical
output is formatted consistently for publication quality plots. ChemoSpec is intended to be very user friendly and to
help you get usable results quickly.

∗ The initial development of ChemoSpec was generously supported by DePauw University in the form of sabbatical funding and a Fisher

Fellowship. Thanks!
1 Introduction Contents

Contents
1 Introduction 2

2 A Sample Workflow 3
2.1 Getting Data into ChemoSpec . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 Preliminary Inspection of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2.1 Plotting the Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 Data Pre-Processing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.1 Normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.2 Bucketing or Binning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.3 Correcting Baseline Drift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.4 Identifying & Removing Problematic Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.4.1 Removing Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.4.2 Identifying & Removing Regions of No Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.5 Hierarchical Cluster Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.6 Principal Components Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.7 ANOVA-PCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.8 Model-Based Clustering Using mclust . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3 Functions That Are Not Discussed Here 35

4 Colors and Symbol Options 39

5 Technical Background 39

6 Acknowledgements 40

7 Related Packages 41

References 41

1 Introduction
Chemometrics, as defined by Varmuza and Filzmoser[2], is
”. . . the extraction of relevant information from chemical data by mathematical and statistical tools.”
This is an appropriately broad definition, considering the wealth of questions and tasks that can be treated by chemometric
approaches. In our case, the focus is on spectral data sets, which typically have many variables (frequencies) and
relatively few samples. Such multivariate, high p, low n data sets present some algorithmic challenges, but these have
been addressed by knowledgeable folks. In particular, for both the practical and theoretical background to multivariate
chemometric analysis, I strongly recommend the Varmuza/Filzmoser book.[2] Some of the functions described here are
not much more than wrappers for the functions they and others have made available to the R community in their packages.
Another excellent text is the one by Ron Wehrens.[3]
ChemoSpec was developed for the chemometric analysis of spectroscopic data, such as UV-Vis, NMR or IR data (it also
works with chromatographic data, see below).1 The approach is entirely exploratory and unsupervised, in other words,
”top-down”[4]. ChemoSpec is designed to accommodate samples that have different histories, i.e., they fall into different
classes, categories or groups. Examples would be treatment and control groups, or simply different specimens (red flowers
vs. blue flowers). ChemoSpec is designed to be as user friendly as possible, with plenty of error checking, helpful warnings
and a consistent interface. It also produces graphics that are consistent in style and annotation, and are suitable for use
in publications and posters. Careful attention was given to writing the documentation for the functions, but this vignette
serves as the best starting point for learning data analysis with ChemoSpec.
1 ChemoSpec was not developed for and has not been tested with mass spectral data sets (MS), as there are other dedicated packages for

this purpose. See the Chemometrics and Computational Physics Task View for an overview.

2
2 A Sample Workflow

The centerpiece of ChemoSpec is the Spectra object. This is the place where your data is stored and made available
to R. Once your data in stored this way and checked, all analyses are easily carried out. ChemoSpec currently ships with
several built-in data sets; we’ll use one called SrE.IR for our demonstrations. You will see in just a moment how to
access it and inspect it.
I assume you have at least a bare-bones knowledge of R as you begin to learn ChemoSpec, and have a good workflow
set up. For detailed help on any function discussed here, type ?function name at the console. If you type ?ChemoSpec
and click the index link at the bottom, you will see all the available functions, which is also convenient when you can’t
quite remember the name of a particular function.
Finally, some conventions for this document: names of R ”objects” such as packages, functions, function arguments, and
data sets are in typewriter font. File names are in regular font, but blue. The commands you issue at the console
and the output are shown with a light grey background, and are colored according to use and purpose, courtesy of the
excellent knitr package.[5]
By the way, if you try ChemoSpec and find it useful, have questions, have opinions, or have suggestions, please do let me
know. The version you are using already incorporates a great deal of user input, why not add yours? Possible bugs and fea-
ture requests should be documented using the Github issues system at github.com/bryanhanson/ChemoSpec/issues

2 A Sample Workflow
This sample exploration is designed to illustrate a typical ChemoSpec workflow. The point is to illustrate how to carry
out the commands, what options are available and typically used, and the order in which one might do the analysis. You
may wish to put your versions of these commands into a script file that you can source as you go along. This way you can
easily make changes, and it will all be reproducible. To do this, open a blank R document, and type in your commands.
Save it as something like My First ChemoSpec.R. Then you can either cut and paste portions of it to the console for
execution, or you can source the entire thing:
source("My_First_ChemoSpec.R")

A typical workflow is illustrated in Figure 1. Depending upon the nature of your data, some of these steps may be
irrelevant or may be omitted, and the order may need to be changed. Examples are in the following sections.

2.1 Getting Data into ChemoSpec

There are two means of importing raw data sets into ChemoSpec. One is the function files2SpectraObject, which
assumes that your raw data exist as separate files in a single directory, each file containing a frequency column and an
intensity column. A header row may or may not be present, and the data can be separated by any separation mark you
like (typically comma, tab, semi-colon or space). You may also use comma or period as the decimal mark. These options
permit data to be imported from files written by a wide variety of instruments using various conventions.2 It is also
possible to import files in the J-CAMP format.
The second function is matrix2SpectraObject, which assumes you have a single file containing a matrix of the data.
This matrix should have frequencies in the first column, and individual sample intensities in the remaining columns.
There must be a header row in the file, and it must contain the sample names (except the first entry, which marks the
frequencies, is ignored). Other than this requirement, you have all the flexibility describe above.
Please be sure to read the help at ?files2SpectraObject for the details, and be certain to pay special attention to
the . . . argument, as this is how your choice of header, separator, and decimal mark are conveyed to read.table which
does the actual reading.
It’s a very good practice to name your data files using a system that encodes any class membership. For example, if
your data set contains treatment and control groups, or any analogous class/group information, this information should
be available via the file names. The argument gr.crit will be the basis for a grep process on the file/sample names,
and from there, each sample will be assigned to a group and be assigned a color as well. If your samples don’t fall into
2 My experience is that csv files don’t always have comma as the separator, and of course the convention about decimal marks vary a bit

around the world. And an instrument installed in a certain country doesn’t always follow local conventions.

3
2 A Sample Workflow 2.1 Getting Data into ChemoSpec

Raw Data

Inspection & Removal Baseline

Alignment
of Bad Samples Correction

Delete Noise-Only
Binning Normalization
Frequencies

Exploratory Data Analysis Cleaned Data

Figure 1: A typical workflow. For a given data set, some steps may be omitted and the order changed. That is part of
what is meant by exploratory data analysis!

groups, that’s fine too, but you still have to give gr.crit something to go on—just give it one string that is common
to all the file names. Obviously, this approach encourages one to name the files as they come off the instrument with
forethought as to how they will be analyzed, which in turn depends upon your experimental design. Nothing wrong with
having a plan!
The output of files2SpectraObject or matrix2SpectraObject is a Spectra object, which is R-speak for an object
that contains not only your data, but other information about the experiment, as provided by you via the arguments to
the function.
Here’s a typical example (we have to talk hypothetically because I don’t have your data). Let’s say you had a folder
containing 30 NMR files of flower essential oils. Imagine that 18 of these were from one proposed subspecies, and 12
from another. Further, let’s pretend that the question under investigation has something to do with the taxonomy of
these two supposed subspecies, in other words, an investigation into whether or not they should be considered subspecies
at all. If the files were named like this:
sspA1.csv . . . sspA18.csv and sspB1.csv . . . sspB12.csv
Then the following command should process the files and create the desired Spectra object:
ssp <- files2SpectraObject(gr.crit = c("sspA", "sspB"), gr.cols = c("red", "blue"),
freq.unit = "ppm", int.unit = "peak intensity", descrip = "Subspecies Study",
out.file = "subspecies")

This causes files2SpectraObject to inspect the file names for the strings sspA and sspB and use these to assign the
samples into groups. Samples in sspA*.csv files will be assigned the color red and sspB*.csv will be assigned blue (see
Section 4 for some suggestions about planning ahead on color choices, as well as ?colorSymbol). After running this
command, a new file called subspecies.RData will be in your directory, and and Spectra object called ssp will be in your
workspace read for exploration. At a later date, you don’t have to re-import your data, you can use the saved version
and give it whatever name you like as follows (function loadObject is from package R.utils):

4
2 A Sample Workflow 2.2 Preliminary Inspection of Data

SubspeciesNMR <- loadObject("subspecies.RData")

Now it is ready to use.

Working with Chromatograms

While all the language in this vignette and in the package are geared toward analysis of spectra, ChemoSpec can also work
with chromatograms as the raw data. In this case, time replaces frequency of course, but other than that the analysis is
virtually the same. So the only real difference is when you issue the command files2SpectraObject, you will give the
frequency unit along these lines: freq.unit = "time (minutes)".

Built-in Data Sets

ChemoSpec ships with several built-in data sets. SrE.IR is the set used for this vignette. It is composed of a collection
of 14 IR spectra of essential oil extracted from the palm Serenoa repens or Saw Palmetto, which is commonly used to
treat BPH in men. The 14 spectra are of different retail samples, and are divided into two categories based upon the
label description: adSrE, adulterated extract, and pSrE, pure extract. The adulterated samples typically have olive oil
added to them, which has no effect on BPH. There are two additional spectra included as references/outliers: evening
primrose oil, labeled EPO in the data set, and olive oil, labeled OO. These latter two oils are mixtures of triglycerides
for the most part, while the SrE samples are largely fatty acids. As a result, the spectra of these two groups differ: the
glycerides have ester carbonyl stretches and no O–H stretch, while the fatty acids have acid carbonyl stretches and an
O–H stretch consistent with a carboxylic acid OH.
Also included is SrE.NMR which is the corresponding set of NMR spectra, and CuticleIR. The latter is a series of IR
spectra of the cuticle (leaf surface) of the plant Portulaca oleracea. The data were taken by gently pinning the leaf
against an ATR sampling device. The plants were grown at two different temperatures, and two different genotypes
(varieties) were used (a classic G x E, genotype by environment, experiment).
The SrE.IR data set is used as the example in this vignette as the sample spectra are fairly different and give good
separation by most chemometric methods. The CuticleIR spectra differ in much more subtle ways and as as result are
more of a challenge to analyze. For more details about these data sets, type ?data set name at the console.

2.2 Preliminary Inspection of Data

The first thing you should do, and this is very important, is to make sure your data are in good shape. First, you can
summarize the data set you created, and verify that the data ranges and other details look like you expect them to:
data(SrE.IR) # makes the data available
sumSpectra(SrE.IR)
## No gaps were found by check4Gaps
## No plot will be made
##
## Serenoa repens IR quality study
##
## There are 16 spectra in this set.
## The y-axis unit is absorbance.
##
## The frequency scale runs from 399.2123 to 3999.837 wavenumber
## There are 1868 frequency (x-axis) data points.
## The frequency resolution is 1.9286 wavenumber/point.
##
##
## The spectra are divided into 4 groups:
##

5
2 A Sample Workflow 2.2 Preliminary Inspection of Data

## group no. color symbol alt.sym

## 1 adSrE 10 #984EA3 15 d
## 2 EPO 1 #377EB8 2 b
## 3 OO 1 #4DAF4A 3 c
## 4 pSrE 4 #E41A1C 1 a
##
## *** Note: this data is an S3 object of class 'Spectra'

sumSpectra provides several pieces of information, and we’ll discuss some of them as we go along.

2.2.1 Plotting the Spectra

Assuming that everything looks good so far, it’s time to plot the spectra and inspect them. A good practice would
be to check every spectrum for artifacts and other potential problems. There are three functions that can do this for
you:
1. LoopThruSpectra takes the pain out of inspecting quite a few spectra. It shows you one spectrum at a time, and
waits for a return to be typed in the console before proceeding. See the help page for details.
2. plotSpectra is intended for general use and publication-quality graphics. You will generally have to play with the
arguments a bit if plotting more than one spectrum.
3. plotSpectraJS is the interactive version of plotSpectra. It shows your data in a web page with the ability to
offset the spectra and zoom as desired. For really large data sets it may be slow; see the help page for ways to
avoid that.
A basic plot using plotSpectra is shown in Figure 2. In this case we have chosen to plot one spectrum from each
category. Note that the carbonyl and Csp2 − H regions are clearly different in these samples.
# We'll make a fancy title here and re-use in other plots
myt <- expression(bolditalic(Serenoa)~bolditalic(repens)~bold(Extract~IR~Spectra))
plotSpectra(SrE.IR, main = myt,
which = c(1, 2, 14, 16),
yrange = c(0, 1.6), offset = 0.4, lab.pos = 2200)

Depending upon the intensity range of your data set, and the number of spectra to be plotted, you have to manually
adjust the arguments yrange, offset and amplify, but this usually only takes a few iterations. Keep in mind that
offset, and amplify are multiplied in the function, so if you increase one, you may need to decrease the other. Suppose
that you wanted to focus just on the carbonyl region of these spectra; you can add the argument xlim. To demonstrate,
let’s look at fewer spectra, and at higher amplitude, so we can see details, as shown in Figure 3.
plotSpectra(SrE.IR, main = myt,
which = c(1, 2, 14, 16), xlim = c(1650, 1800),
yrange = c(0, 0.6), offset = 0.1, lab.pos = 1775)

These sample plots display the IR spectra in two ways that may be upsetting to some readers: First, the x-axis is
”backwards”, because the underlying spectra were originally saved with an ascending frequency axis (which is not always
the case). This is readily fixed by supplying the xlim argument in the desired order, e.g. xlim = c(1800, 1650) in the
previous example. Second, the vertical scale in these examples is absorbance. When using IR for structural elucidation,
the vertical axis is typically %T, with the peaks pointing downward. You don’t have that choice in ChemoSpec because
the absorbance mode is the appropriate one for chemometrics. Record your original spectra that way and get used to
it.
The argument which in plotSpectra takes a integer vector of the spectra you wish to plot— you can think of this as
the row number if you imagine each spectra to be a row in a matrix, with intensities in the columns (with each column
corresponding to a particular frequency value). You may be wondering how to determine which particular sample is in
each row. This is best accomplished with a grep command. For instance, if you wanted to know what row/sample the
olive oil was in, either of the following methods would locate it for you:

6
2 A Sample Workflow 2.2 Preliminary Inspection of Data

S erenoa repens Extract IR Spectra

1.5

TJ_OO
1.0
absorbance

SV_EPO
0.5

ET_pSrE

CVS_adSrE
0.0

500 1000 1500 2000 2500 3000 3500 4000

wavenumber

Figure 2: Plotting Spectra

7
2 A Sample Workflow 2.2 Preliminary Inspection of Data

S erenoa repens Extract IR Spectra

0.6
0.5
0.4
absorbance

TJ_OO
0.3

SV_EPO
0.2

ET_pSrE
0.1

CVS_adSrE
0.0

1650 1700 1750 1800

wavenumber

Figure 3: Zooming in on a Spectral Region

8
2 A Sample Workflow 2.3 Data Pre-Processing Options

SrE.IR$names # suitable if there are not many spectra

## [1] "CVS_adSrE" "ET_pSrE" "GNC_adSrE"
## [4] "LF_adSrE" "MDB_pSrE" "NA_pSrE"
## [7] "Nat_adSrE" "NP_adSrE" "NR_pSrE"
## [10] "NSI_adSrE" "NW_adSrE" "SN_adSrE"
## [13] "Sol_adSrE" "SV_EPO" "TD_adSrE"
## [16] "TJ_OO"
grep("OO", SrE.IR$names) # use if there are more spectra
## [1] 16

2.3 Data Pre-Processing Options

There are a number of data pre-processing options available for your consideration. The main choices are whether to
normalize the data, whether to bin the data, and whether to scale the data. Data scaling is handled by the PCA routines,
see Section 2.6. Baseline correction is another typical action.

2.3.1 Normalization

Normalization is handled by the normSpectra function. Usually one normalizes data in which the sample preparation
procedure may lead to differences in concentration, such as body fluids that might have been diluted during handling,
or that vary due to the physiological state of the organism studied. The SrE.IR data set is taken by placing the oil
extract directly on an ATR device and no dilution is possible, so normalization isn’t really appropriate. Please see the
help page for the normalization options. The literature contains a number of useful discussions about normalization
issues.[2, 6–9]

2.3.2 Bucketing or Binning

Another type of pre-processing that you may wish to consider is binning or bucketing, in which groups of frequencies
are collapsed into one frequency value, and the corresponding intensities are summed. There are two reasons for doing
this:
1. Compacting large data sets: This is a historical issue, the algorithms in R are quite fast, and large data sets don’t
really slow it down much.
2. Compensating for shifts: This is typically done with aqueous 1 H NMR samples because changes in dilution, ionic
strength, or pH can cause signficant shifts for some types of protons. Spectra with broad, rolling peaks won’t have
this problem (UV-Vis or IR for example).
This example illustrates the process but is not necessary with IR data:
tmp <- binSpectra(SrE.IR, bin.ratio = 4)
## No gaps were found by check4Gaps
## No plot will be made
sumSpectra(tmp)
## No gaps were found by check4Gaps
## No plot will be made
##
## Serenoa repens IR quality study
##
## There are 16 spectra in this set.
## The y-axis unit is absorbance.
##

9
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

## The frequency scale runs from 402.1052 to 3996.945 wavenumber

## There are 467 frequency (x-axis) data points.
## The frequency resolution is 7.71425 wavenumber/point.
##
##
## The spectra are divided into 4 groups:
##
## group no. color symbol alt.sym
## 1 adSrE 10 #984EA3 15 d
## 2 EPO 1 #377EB8 2 b
## 3 OO 1 #4DAF4A 3 c
## 4 pSrE 4 #E41A1C 1 a
##
## *** Note: this data is an S3 object of class 'Spectra'

Compare the results here with the sumSpectra of the full data set (Section 2.2). In particular note that the frequency
resolution has gone down due to the binning process. ChemoSpec uses the simplest of binning algorithms: after perhaps
dropping a few points (with a warning) to make your data set divisible by the specified bin.ratio, data points are
replaced by the average frequency and the sum of the grouped intensities. Depending upon the fine structure in your
data and the bin.ratio this might cause important peaks to be split between different bins. There are more sophisticated
binning algorithms in the literature that try to address this, but none are currently implemented in ChemoSpec.[10, 11]
There are now better ways to approach this problem; see the package speaq.

2.3.3 Correcting Baseline Drift

ChemoSpec uses the functions in the package baseline to correct wandering baselines. The function, baselineSpectra,
can show you the original and corrected baselines if desired, which is useful for choosing a method. Figure 4 shows a
typical usage. Method rfbaseline works well for IR spectra; retC = TRUE puts the corrected spectra into the new
Spectra object so we can use it going forward (and we will).
SrE2.IR <- baselineSpectra(SrE.IR, int = FALSE, method = "rfbaseline", retC = TRUE)

2.4 Identifying & Removing Problematic Samples

In the process of plotting and inspecting your spectra, you may find some spectra/samples that have problems. Perhaps
they have instrumental artifacts. Or maybe you have decided to eliminate one subgroup of samples from your data
set to see how the results differ. To remove a particular sample, or samples meeting a certain criteria, you use the
removeSample function. This function uses a grepping process based on its rem.sam argument, so you must be careful
due to the greediness of grep. Let’s imagine that sample TD adSrE has artifacts and needs to be removed. The command
would be:
noTD <- removeSample(SrE2.IR, rem.sam = c("TD_adSrE"))
sumSpectra(noTD)
## No gaps were found by check4Gaps
## No plot will be made
##
## Serenoa repens IR quality study
##
## There are 15 spectra in this set.
## The y-axis unit is absorbance.
##
## The frequency scale runs from 399.2123 to 3999.837 wavenumber
## There are 1868 frequency (x-axis) data points.
## The frequency resolution is 1.9286 wavenumber/point.
##

10
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

Original spectrum
0.30
0.20
0.10
0.00

0 500 1000 1500

Baseline corrected spectrum

0.30
0.20
0.10
0.00

0 500 1000 1500

Figure 4: Correcting baseline drift

11
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

##
## The spectra are divided into 4 groups:
##
## group no. color symbol alt.sym
## 1 adSrE 9 #984EA3 15 d
## 2 EPO 1 #377EB8 2 b
## 3 OO 1 #4DAF4A 3 c
## 4 pSrE 4 #E41A1C 1 a
##
## *** Note: this data is an S3 object of class 'Spectra'
grep("TD_adSrE", noTD$names)
## integer(0)

The sumSpectra command confirms that there are now one fewer spectra in the set. As shown, you could also re-grep
for the sample name to verify that it is not found. The first argument in grep is the pattern you are searching for; if
that pattern matches more than one name they will all be ”caught.” For example if you used ”SrE” as your pattern you
would remove all the samples except the two reference samples, since ”SrE” occurs in ”adSrE” and ”pSrE”. You can
check this in advance with the grep function itself:
SrE <- grep("SrE", SrE2.IR$names)
SrE2.IR$names[SrE] # gives the name(s) that contain "SrE"
## [1] "CVS_adSrE" "ET_pSrE" "GNC_adSrE"
## [4] "LF_adSrE" "MDB_pSrE" "NA_pSrE"
## [7] "Nat_adSrE" "NP_adSrE" "NR_pSrE"
## [10] "NSI_adSrE" "NW_adSrE" "SN_adSrE"
## [13] "Sol_adSrE" "TD_adSrE"
SrE # gives the corresponding indices
## [1] 1 2 3 4 5 6 7 8 9 10 11 12 13 15

This is what is meant by ”grep is greedy”. In this situation, you have three choices:
1. You could manually remove the problem samples (str(SrE2.IR)) would give you an idea of how to do that; see
also below under Hierarchical Cluster Analysis).
2. removeSample also accepts indices of samples, so you could grep as above, note the index of the sample you
actually want to remove, and use that in rem.sam.
3. If you know a bit about grep and regular expressions, you can pass a more sophisticated search pattern to rem.sam.

2.4.1 Removing Groups

removeSample uses the names of the samples (in Spectra$names) to identify and remove individual samples from the
Spectra object. There is also a function removeGroup which will remove samples belonging to a particular group in
Spectra$groups.

2.4.2 Identifying & Removing Regions of No Interest

Many spectra will have regions that should be removed before analysis. It may be an uninformative, interfering peak like
the water peak in 1 H NMR, or the CO2 peak in IR. Or, there may be regions of the spectra that simply don’t have much
information – they contribute a noisy baseline and not much else. An example would be the region from about 1,800
or 1,900 cm−1 to about 2,500 cm−1 in IR, a region where there are typically no peaks except for the atmospheric CO2
stretch, and rarely (be careful!) alkyne stretches.
Finding these regions might be pretty simple, a matter of inspection coupled with your knowledge of spectroscopy.
Another approach is to use the function surveySpectra to examine the entire set of spectra. This function computes

12
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

S erenoa repens Extract IR Spectra

0.30

0.25
Full Data Set, median +/− iqr

0.20

0.15

0.10

0.05

0.00

1000 2000 3000 4000

wavenumber

Figure 5: Checking for Regions of No Interest

a summary statistic (your choice) of the intensities at a particular frequency across the data set, as well as the mean or
median. In regions with little variation, the mean/median and upper/lower summary lines will be close together. Figure 5
demonstrates the process. There is also an alternative, surveySpectra2 which presents the data in a slightly different
format. See Figure 6.
surveySpectra(SrE2.IR, method = "iqr", main = myt, by.gr = FALSE)

surveySpectra2(SrE2.IR, method = "iqr", main = myt)

In Figure 5 we kept all the groups together by using argument by.gr = FALSE. We also looked at the entire spectral
range. In Figure 7 we can look just at the carbonyl region. The black line is the median value of intensity across the
entire set of spectra. The red lines are the upper and lower interquartile ranges which makes it pretty clear that the
carbonyl region of this data set varies a lot.
surveySpectra(SrE2.IR, method = "iqr", main = "S. repens Detail of Carbonyl Region",
by.gr = FALSE, xlim = c(1650, 1800))

Finally, surveySpectra allows us to view the data set by group, which is really more useful. Let’s look at the carbonyl
region by group (Figure 8). Note that we get warnings because two of the groups have too few members to compute
the interquartile range, and these are not shown. As a result, some panels are empty.
surveySpectra(SrE2.IR, method = "iqr", main = "S. repens Detail of Carbonyl Region",
by.gr = TRUE, xlim = c(1650, 1800))
## Warning in surveySpectra(SrE2.IR, method = "iqr", main = "S. repens Detail of Carbonyl Region",
:
## Group EPO has 3 or fewer members
## so your stats are not very useful...
## This group has been dropped for display purposes!
## Warning in surveySpectra(SrE2.IR, method = "iqr", main = "S. repens Detail of Carbonyl Region",
:
## Group OO has 3 or fewer members
## so your stats are not very useful...

13
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

S erenoa repens Extract IR Spectra

0.1
Centered Spectra

0.0
−0.1
−0.2

iqr
−0.3

500 1000 1500 2000 2500 3000 3500 4000

wavenumber

Figure 6: Checking for Regions of No Interest

## This group has been dropped for display purposes!

For reasons that will become evident in a moment, let’s look at the region between 1800 and 2500 cm−1 (Figure 9).
surveySpectra(SrE2.IR, method = "iqr", main = "S. repens Detail of Empty Region",
by.gr = FALSE, xlim = c(1800, 2500), ylim = c(0.0, 0.05))

From a theoretical perspective, we expect this region to be devoid of interesting peaks. In fact, even when pooling the
groups the signal in this region is very weak, and the only peak present is due to atomospheric CO2 . We can remove
this region, since it is primarily noise and artifact, with the function removeFreq as follows. Note that there are fewer
frequency points now.
SrE3.IR <- removeFreq(SrE2.IR, rem.freq = SrE2.IR$freq > 1800 & SrE2.IR$freq < 2500)
sumSpectra(SrE3.IR)
##
## Serenoa repens IR quality study
##
## There are 16 spectra in this set.
## The y-axis unit is absorbance.
##
## The frequency scale runs from 399.2123 to 3999.837 wavenumber
## There are 1505 frequency (x-axis) data points.
## The frequency resolution is 1.9286 wavenumber/point.
##
## This data set is not continuous along the frequency axis.
## Here are the data chunks:
##
## beg.freq end.freq size beg.indx end.indx
## 1 399.2123 1799.348 1400.136 1 727
## 2 2501.3450 3999.837 1498.492 728 1505
##
## The spectra are divided into 4 groups:

14
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

S. repens Detail of Carbonyl Region

0.30

0.25

0.20
Full Data Set, median +/− iqr

0.15

0.10

0.05

0.00

1700 1750

wavenumber

Figure 7: Detail of Carbonyl Region

15
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

S. repens Detail of Carbonyl Region

0.3

0.2
pSrE

0.1
median +/− iqr

0.0

0.3

0.2
adSrE

0.1

0.0

1700 1750

wavenumber

Figure 8: Detail of Carbonyl Region by Group

16
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

S. repens Detail of Empty Region

0.04
Full Data Set, median +/− iqr

0.03

0.02

0.01

1900 2000 2100 2200 2300 2400

wavenumber

Figure 9: Inspection of an Uninteresting Spectral Region

17
2 A Sample Workflow 2.4 Identifying & Removing Problematic Samples

Gaps in Frequency Data

0.25
0.20
0.15
0.10
0.05
0.00

500 1000 1500 2000 2500 3000 3500 4000

marked regions are skipped in data set

Figure 10: Identifying Gaps in a Data Set

##
## group no. color symbol alt.sym
## 1 adSrE 10 #984EA3 15 d
## 2 EPO 1 #377EB8 2 b
## 3 OO 1 #4DAF4A 3 c
## 4 pSrE 4 #E41A1C 1 a
##
## *** Note: this data is an S3 object of class 'Spectra'

Notice that sumSpectra has identified a gap in the data set. You can see this gap in the data as shown in Figure 10
(sumSpectra checks for gaps, but doesn’t produce the plot); both the numerical results and a figure are provided.
check4Gaps(SrE3.IR$freq, SrE3.IR$data[1,], plot = TRUE)

## beg.freq end.freq size beg.indx end.indx

## 1 399.2123 1799.348 1400.136 1 727
## 2 2501.3450 3999.837 1498.492 728 1505

18
2 A Sample Workflow 2.5 Hierarchical Cluster Analysis

S erenoa repens Extract IR Spectra

1.5
Key
adSrE
EPO
OO
pSrE
1.0
0.5
0.0

SV_EPO

TJ_OO
NP_adSrE
NR_pSrE

NW_adSrE
Nat_adSrE
GNC_adSrE
NA_pSrE
ET_pSrE
MDB_pSrE

CVS_adSrE
LF_adSrE
NSI_adSrE

TD_adSrE
SN_adSrE
Sol_adSrE
clustering method: complete distance method: euclidean

Figure 11: Hierarchical Cluster Analysis

2.5 Hierarchical Cluster Analysis

Hierarchical cluster analysis (HCA from now on) is a clustering method (no surprise!) in which ”distances” between
samples are calculated and displayed in a dendrogram (a tree-like structure; these are also used in evolution and systematics
where they are called cladograms). The details behind HCA can be readily found elsewhere (Chapter 6 of [2] is a good
choice). With ChemoSpec you have access to any of the methods available for computing distances between samples and
any of the methods for identifying clusters. A typical example is shown in Figure 11.
HCA <- hcaSpectra(SrE3.IR, main = myt)

The result is a dendrogram. The vertical scale represents the numerical distance between samples. Not unexpectedly,
the two reference samples which are known to be chemically different cluster together separately from all other sam-
ples. Perhaps surprisingly, the various pure and adulterated oil extracts do not group together precisely. The function
hcaScores does the same kind of analysis using the results of PCA, rather than the raw spectra. It is discussed in the
next section.

19
2 A Sample Workflow 2.6 Principal Components Analysis

Table 1: Principal Components Analysis Options & Functions

PCA options scaling options function

classical PCA no scaling, autoscaling, Pareto scaling c pcaSpectra
robust PCA no scaling, median absolute deviation r pcaSpectra

Diagnostics
OD plots pcaDiag
SD plots pcaDiag

Choosing the correct no. of PCs

scree plot plotScree
alternate style scree plot plotScree2
bootstrap analysis (classical PCA only) cv pcaSpectra

Score plots plotting options

2D plots robust or classical confidence ellipses plotScores
3D plots
—static 3D plots plotScores3D
—interactive 3D plots plotScoresRGL

Loading plots
loadings vs frequencies plotLoadings
loadings vs other loadings plot2Loadings
s-plot (correlation vs covariance) sPlotSpectra

Other
HCA of PCA scores hcaScores
ANOVA-PCA aov pcaSpectra

2.6 Principal Components Analysis

Principal components analysis (PCA from now on) is the real workhorse of exploratory data analysis. It makes no
assumptions about group membership, but clustering (possibly in high dimensions) of the resulting sample scores can be
very helpful in understanding your data. The theory and practice of PCA is covered well elsewhere (Chapter 3 of [2] is an
excellent choice). Here, we’ll concentrate on using the PCA methods in ChemoSpec. Briefly however, you can think of
PCA as determining the minimum number of components necessary to describe a data set, in effect, removing noise and
redundant information. Think of a typical spectrum: some regions are clearly just noise. Further, a typical spectroscopic
peak spans quite a few frequency units as the peak goes up, tops out, and then returns to baseline. Any one of the
points in a particular peak describe much the same thing, namely the intensity of the peak. Plus, each frequency within
a given peak envelope is correlated to every other frequency in the envelope (they rise and fall in unison as the peak
changes size from sample to sample). PCA can look ”past” all the noise and underlying correlation in the data set, and
boil the entire data set down to essentials. Unfortunately, the principal components that are uncovered in the process
don’t correspond to anything concrete, usually. Again, you may wish to consult a more detailed treatment!
Table 1 gives an overview of the options available in ChemoSpec, and the relevant functions.
There’s quite a bit of choice here; let’s work through an example and illustrate, or at least mention, the options as we
go. Keep in mind that it’s up to you to decide how to analyze your data. Most people try various options, and follow
the ones that lead to the most insight. But the decision is yours!
The first step is to carry out the PCA. You have two main options, either classical methods, or robust methods. Classical
methods use all the data you provide to compute the scores and loadings. Robust methods focus on the core or heart of
the data, which means that some samples may be downweighted. This difference is important, and the results from the
two methods may be quite different, depending upon your the nature of your data. The differences arise because PCA
methods (both classical and robust) attempt to find the components that explain as much of the variance in the data set

20
2 A Sample Workflow 2.6 Principal Components Analysis

as possible. If you have a sample that is genuinely corrupted, for instance due to sample handling, its spectral profile may
be very different from all other samples, and it can legitimately be called an outlier. In classical PCA, this one sample
will contribute strongly to the variance of the entire data set, and the PCA scores will reflect that (it is sometimes said
that scores and loadings follow the outliers). With robust PCA, samples with rather different characteristics do not have
as great an influence, because robust measures of variance, such as the median absolute deviation, are used.
Note that neither c pcaSpectra nor r pcaSpectra carry out any normalization by samples. You need to decide if you
want to normalize the samples, and if so, use normSpectra.
Besides choosing to use classical or robust methods, you also need to choose a scaling method. For classical PCA,
your choices are no scaling, autoscaling, or Pareto scaling. In classical analysis, if you don’t scale the data, large peaks
contribute more strongly to the results. If you autoscale, then each peak contributes equally to the results (including noise
”peaks”). Pareto scaling is a compromise between these two. For robust PCA, you can choose not to scale, or you can
scale according to the median absolute deviation. Median absolute deviation is a means of downweighting more extreme
peaks. The literature has plenty of recommendations about scaling options appropriate for the type of measurement
(instrument) as well as the nature of the biological data set.[2, 6–9, 12]
There is not enough space here to illustrate all possible combinations of options; Figure 12 and FIgure 13 show the use
and results of classical and robust PCA without scaling, followed by plotting of the first two PCs (we’ll discuss plotting
options momentarily). You can see from these plots that the robust and classical methods have produced rather different
results, not only in the overall appearance of the plots, but in the amount of variance explained by each PC.
Since we’ve plotted the scores to see the results, let’s mention a few features of plotScores which produces a 2D plot
of the results (we’ll deal with 3D options later). Note that an annotation is provided in the upper left corner of the plot
that describes the history of this analysis, so you don’t lose track of what you are viewing. The tol argument controls
what fraction of points are labeled with the sample name. This is a means of identifying potential outliers. The ellipse
argument determines if and how the ellipses are drawn (the 95% confidence interval is used).
You can choose "none" for no ellipses, "cls" for classically computed confidence ellipses, "rob" for robustly computed
ellipses, or "both" if you want to directly compare the two. Note that the use of classical and robust here has nothing
to do with the PCA algorithm — it’s the same idea however, but applied to the 2D array of scores produced by PCA.
Points outside the ellipses are more likely candidates for outlier status.
class <- c_pcaSpectra(SrE3.IR, choice = "noscale")
plotScores(SrE3.IR, main = myt, class,
pcs = c(1,2), ellipse = "rob", tol = 0.01)
## Warning in plotScores(SrE3.IR, main = myt, class, pcs = c(1, 2), ellipse = "rob", : Group EPO
has only 1 member (no ellipse possible)
## Warning in plotScores(SrE3.IR, main = myt, class, pcs = c(1, 2), ellipse = "rob", : Group OO
has only 1 member (no ellipse possible)

robust <- r_pcaSpectra(SrE3.IR, choice = "noscale")

plotScores(SrE3.IR, main = myt, robust,
pcs = c(1,2), ellipse = "rob", tol = 0.01)
## Warning in plotScores(SrE3.IR, main = myt, robust, pcs = c(1, 2), ellipse = "rob", : Group EPO
has only 1 member (no ellipse possible)
## Warning in plotScores(SrE3.IR, main = myt, robust, pcs = c(1, 2), ellipse = "rob", : Group OO
has only 1 member (no ellipse possible)

Plots such as shown in Figures 12 and 13 can give you an idea of potential outliers, but ChemoSpec includes more
sophisticated approaches. The function pcaDiag can produce two types of plots that can be helpful (Figures 14 and 15).
The meaning and interpretation of these plots is discussed in more detail in Varmuza and Filzmoser, Chapter 3.[2]
diagnostics <- pcaDiag(SrE3.IR, class, pcs = 2, plot = "OD")

diagnostics <- pcaDiag(SrE3.IR, class, pcs = 2, plot = "SD")

Depending upon your data, and your interpretation of the results, you may decide that some samples should be discarded,
in which case you can use removeSample as previously described, then repeat the PCA analysis. The next step for most

21
2 A Sample Workflow 2.6 Principal Components Analysis

S erenoa repens Extract IR Spectra

0.6

centered/noscale/classical Key
robust ellipses by group adSrE
EPO
OO
pSrE
0.4
0.2

● SV_EPO
PC2 score (3.3%)

● NR_pSrE

●
●
0.0

● ●
●
●
● ● ● ●
●●
● ● NW_adSrE
−0.2
−0.4

−2.0 −1.5 −1.0 −0.5 0.0 0.5 1.0

PC1 score (93%)

Figure 12: Classical PCA

22
2 A Sample Workflow 2.6 Principal Components Analysis

S erenoa repens Extract IR Spectra

l1median/noscale/robust Key
robust ellipses by group adSrE
EPO
0.6

OO
pSrE
0.4
PC2 score (19%)

● NR_pSrE
0.2

●
● ●

●
0.0

●●
● ●
●
●

●
●
−0.2

●● SV_EPO
TJ_OO

−1.5 −1.0 −0.5 0.0 0.5 1.0

PC1 score (68%)

Figure 13: Robust PCA

23
2 A Sample Workflow 2.6 Principal Components Analysis

Possible PCA Outliers based on Orthogonal Distance

● NW_adSrE
● NR_pSrE
● ● ● ●
● ● ● ● ● ● ●
● ●
●
1.5
orthogonal distance

1.0
0.5
0.0

5 10 15

Serenoa repens IR quality study

centered/noscale/classical 2 PCs

Figure 14: Diagnostics: Orthogonal Distances

24
2 A Sample Workflow 2.6 Principal Components Analysis

Possible PCA Outliers based on Score Distance

3.5

● SV_EPO
3.0
2.5

●
2.0
score distance

●
1.5

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●
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●
0.5

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●
0.0

5 10 15

Serenoa repens IR quality study

centered/noscale/classical 2 PCs

Figure 15: Diagnostics: Score Distances

25
2 A Sample Workflow 2.6 Principal Components Analysis

S erenoa repens Extract IR Spectra

100
80
60
percent

cumulative percent

individual percent
40
20

centered/noscale/classical
0

1 2 3 4 5 6 7 8 9 10

factor

Figure 16: Scree Plot

people is to determine the number of PCs needed to describe the data. This is usually done with a scree plot as shown in
Figure 16. ChemoSpec has an alternate style scree plot which I actually think is much more informative (Figure 17).
If you are using classical PCA, you can also get a sense of the number of PCs needed via a bootstrap method, as shown
in Figure 18. Note that this method is iterative and takes a bit of time. Comparing these results to the scree plots, you’ll
see that the bootstrap method suggests that 4 or 5 PCs would not always be enough to reach the 95% level, while the
scree plots suggest that 2 PC are sufficient.
plotScree(class, main = myt)

plotScree2(class, main = myt)

out <- cv_pcaSpectra(SrE3.IR, pcs = 5, choice = "noscale")

Now let’s turn to viewing scores in 3D. There are currently two options in ChemoSpec: plotting using lattice graphics,
which produces a static plot that you have to adjust manually, and an interactive plot based on package rgl. Probably
the best place to start is with plotScoresRGL. It it well suited to exploring data, and can be printed out in high quality.
However, the nature of the open GL graphics device means that the title and the legend move with the data, so this may
not give a hardcopy suitable for publications. This interactive plot cannot be invoked in this document, but here are the
necessary commands:
plotScoresRGL(SrE3.IR, class, main = "S. repens IR Spectra",
leg.pos = "A", t.pos = "B") # not run - it's interactive!

For full details, of course take a look at the manual page, ?plotScoresRGL. If you want a similar and probably more
publication-worthy plot, you can use plotScores3D as shown in Figure 19. In this example we’ve set ellipse = FALSE
because the ”adSrE” data points form a very elongated ellipse which forces the plotting limits in such a way that other
points become very hard to see.
plotScores3D(SrE3.IR, class, main = myt, ellipse = FALSE)

In addition to the scores, PCA also produces loadings which tell you how each variable (frequencies in spectral applications)
affect the scores. Examining these loadings can be critical to interpreting your results. Figure 20 gives an example. You
can see that the different carbonyl peaks have a large and opposing effect on PC 1. PC 2 on the other hand is driven

26
2 A Sample Workflow 2.6 Principal Components Analysis

S erenoa repens Extract IR Spectra

●
●
cumulative percent variance shown to right of PC

0.5

●
● ●
●
scores

● ● ●
●
93% ● 96% ● 98% 99% ● 99% 100% 100% 100% 100% 100%
0.0

● ● ●
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●
−0.5

● centered/noscale/classical

1 2 3 4 5 6 7 8 9 10

component

Figure 17: Alternate Style Scree Plot

by a number of peaks, with some interesting opposing peaks in the hydrocarbon region. While the actual analysis of
the data is not our goal here, it would appear that PC 1 is sensitive to the ester vs. acid carbonyl group, and PC 2 is
detecting the saturated vs. unsaturated fatty acid chains (the latter having Csp2 − H peaks).
plotLoadings(SrE3.IR, class, main = myt, loads = c(1, 2), ref = 1)

You can also plot one loading against another, using function plot2Loadings (Figure 21). This is typically not too
useful for spectroscopic data, since many of the variables are correlated (as they are parts of the same peak, hence the
serpentine lines in the figure). The most extreme points on the plot, however, can give you an idea of which peaks
(frequencies) serve to differentiate a pair of PCs, and hence, drive your data clustering.
res <- plot2Loadings(SrE3.IR, class, main = myt, loads = c(1, 2), tol = 0.002)

However, a potentially more useful approach is to use an s-plot to determine which variables have the greatest influence.
A standard loadings plot (plotLoadings) shows you which frequency ranges contribute to which principal components,
but the plot allows the vertical axis to be free. Unless you look at the y axis scale, you get the impression that the
loadings for principal component 1 etc. all contribute equally. The function sPlotSpectra plots the correlation of each
frequency variable with a particular score against the covariance of that frequency variable with the same score. The
result is an s-shaped plot with the most influential frequency variables in the upper right hand and lower left quadrants.
An example is shown in Figure 22 with a detail view in Figure 23. In the latter figure you can clearly see the influence
of the carbonyl peaks. This method was reported in Wiklund et. al.[13]
spt <- sPlotSpectra(SrE3.IR, class, main = myt, pc = 1, tol = 0.001)

spt <- sPlotSpectra(SrE3.IR, class, main = "Detail of S. repens IR Spectra", pc = 1,

tol = 0.05, xlim = c(-0.04, -0.01), ylim = c(-1.05, -0.9))

Finally, you can blend the ideas of PCA and HCA. Since PCA eliminates the noise in a data set (after you have selected
the important PCs), you can carry out HCA on the PCA scores, as now the scores represent the cleaned up data. The
result using the SrE.IR data set are not different than doing HCA on the raw spectra, so we won’t illustrate it, but the
command would be:
hcaScores(SrE3.IR, class, scores = c(1:5), main = myt)

27
2 A Sample Workflow 2.6 Principal Components Analysis

0.95
Explained variance

0.90

●
●
0.85
0.80

Serenoa repens IR quality study centered/noscale/classical

1 2 3 4 5

Number of components

Figure 18: Bootstrap Analysis for No. of PCs

28
2 A Sample Workflow 2.6 Principal Components Analysis

S erenoa repens Extract IR Spectra

PC1 (93%) PC3 (1.8%)

PC2 (3.3%)
● ●

●
●
●

●
●

adSrE EPO OO pSrE

Serenoa repens IR quality study

Figure 19: Plotting Scores in 3D using plotScores3D

29
2 A Sample Workflow 2.6 Principal Components Analysis

S erenoa repens Extract IR Spectra

0.05 0.10

PC 2 Loadings
−0.05
−0.15
0.1

PC 1 Loadings
0.0
−0.2 −0.1

Reference Spectrum
0.20
0.10
0.00

1000 2000 3000 4000

wavenumber
centered/noscale/classical

Figure 20: Loading Plot

30
2 A Sample Workflow 2.6 Principal Components Analysis

S erenoa repens Extract IR Spectra

●
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709.71
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Figure 21: Plotting One Loading vs. Another

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2 A Sample Workflow 2.6 Principal Components Analysis

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2 A Sample Workflow 2.6 Principal Components Analysis

Detail of S. repens IR Spectra

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33
2 A Sample Workflow 2.7 ANOVA-PCA

original data factor matrix

#############################
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Figure 24: aovPCA breaks the data into a series of submatrices

original data factor matrix

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Figure 25: Submatrices are composed of rows which are averages of each factor level

2.7 ANOVA-PCA

Harrington et. al.[14] (and a few others[15]) have demonstrated a method which combines traditional ANOVA with PCA.
Standard PCA is blind to class membership, though one generally colors the points in a score plot using the known class
membership. ANOVA-PCA uses the class membership to divide the original centered data matrix into submatrices. Each
submatrix corresponds to a particular factor, and the rows of the submatrix have been replaced by the average spectrum
of each level of the factor. The original data set is thought of as a sum of these submatrices plus residual error. The
residual error is added back to each submatrix and then PCA is performed. This is conceptually illustrated in Figures 24
and 25.
ANOVA-PCA has been implemented in ChemoSpec via the functions aov pcaSpectra, aovPCAscores and aovPCAloadings.
The idea here is that if a factor is significant, there will be separation along PC1 in a plot of PC1 vs PC2. There
are not enough groups and levels within the SrE.IR data set to carry out ANOVA-PCA. However, the help page for
aov pcaSpectra contains an example using the CuticleIR data set which illustrates how to carry out the analysis. It
also demonstrates another useful function, splitSpectraGroups which allows you to take an existing group designation
and split into new designations. See ?aov pcaSpectra .

2.8 Model-Based Clustering Using mclust

PCA and HCA are techniques which are unsupervised and assume no underlying model. HCA computes distances between
pairs of spectra and groups these in an iterative fashion until the dendrogram is complete. PCA seeks out components

34
3 Functions That Are Not Discussed Here

that maximize the variance. While in PCA one often (and ChemoSpec does) displays the samples coded by their group
membership, this information is not actually used in PCA; any apparent correspondence between the sample group
classification and the clusters found is accidental in terms of the computation, but of course, this is what one hopes to
find!
mclust is a model-based clustering package that takes a different approach.[16–18]. mclust assumes that there are
groups within your data set, and that those groups are multivariate normally distributed. Using an iterative approach,
mclust samples various possible groupings within your data set, and uses a Bayesian Information Criterion (BIC) to
determine which of the various groupings it finds best fits the data distribution. mclust looks for groups that follow
certain constraints, for instance, one constraint is that all the groups found must have a spherical distribution of data
points, while another allows for ellipsoidal distributions. See the paper by Fraley and Raftery[17] for more details. The
basic idea however is that mclust goes looking for groups in your data set, and then you can compare the groupings it
finds with the groupings you know to be true.
ChemoSpec contains several functions that interface with and extend mclust functions. mclust first uses the BIC to
determine which model best fits your data; these results are shown in Figure 26. Next, Figure 27 shows the two groups
that mclust finds in the data. It’s of some interest to visually compare the score plot in Figure 12 with the mclust
results in Figure 27. It looks like mclust groups the two outliers with some of the rest of the data. Next, mclust will
map the true groups onto the groups it has found. Points in error are X-ed out. These results can be seen in Figure 28.
From this plot, you can see that mclust hasn’t done too well with this data set. In general, you have to be very careful
about using mclust’s notion of an error: it is very hard to map the found groups onto the ”truth” in an algorithmic way.
I lean toward not using the ”truth” option in mclust more and more.
model <- mclustSpectra(SrE3.IR, class, plot = "BIC", main = myt)

model <- mclustSpectra(SrE3.IR, class, plot = "proj", main = myt)

model <- mclustSpectra(SrE3.IR, class, plot = "errors",

main = myt, truth = SrE3.IR$groups)
## Warning in coordProjCS(d, dimens = dims, what = "errors", classification = mod$classification,
: classification and truth differ in number of groups

You can also do a similar analysis in 3D, using mclust3dSpectra. This function uses mclust to find the groups, but
then uses non-mclust functions to draw confidence ellipses. This function uses rgl graphics so it cannot demonstrated
here, but the commands would be:
mclust3dSpectra(SrE3.IR, class) # not run - it's interactive!

You have all the options here that you do with plotScoresRGL, namely, classical, robust or no ellipses, control of the
ellipse details, and labeling of extreme points.
I hope you have enjoyed this tour of the features of ChemoSpec!

3 Functions That Are Not Discussed Here

See the help files of course . . .
1. splitSpectraGroups A good example of its use can be found in ?aov pcaSpectra .
2. hypTestScores: Run anova on PCA scores.
3. hmapSpectra: Plot a seriated heat map.
4. evalClusters: Compare various clustering options.
5. clupaSpectra: Align NMR spectra.
6. sgfSpectra Apply Savitzky-Golay filters.
7. plotSpectraDist Plot the distance between each spectrum and a reference spectrum.

35
3 Functions That Are Not Discussed Here

Mclust optimal model: EVE

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Figure 26: mclust Chooses an Optimal Model

36
3 Functions That Are Not Discussed Here

Mclust optimal model: EVE

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Figure 27: mclust’s Thoughts on the Matter

37
3 Functions That Are Not Discussed Here

Mclust optimal model: EVE

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Figure 28: Comparing mclust Results to the TRUTH

38
5 Technical Background

ChemoSpec Primary Scheme

Set1 (qualitative)
ChemoSpec Pastel Scheme

Figure 29: Recommended Color Sets in ChemoSpec

4 Colors and Symbol Options

In ChemoSpec, the user may use any color name/format known to R. For ease of comparison, it would be nice to plan
ahead and use the same color scheme for all your plots. However, if you are just doing preliminary work, ChemoSpec will
choose colors for you automatically.
In addition to colors, "Spectra" objects also contain a list of symbols, and alternative symbols. These are useful for
plotting in black and white, or when color-blind individuals will be viewing the plots. The alternative symbols are simply
lower-case letters, as these are needed for plotScoresRGL, and other rgl-graphics driven functions which cannot plot
traditional symbols.
Two good color schemes are shown in Figure 29; these are generally visually distinct and are part of the RColorBrewer
package. By name, these are:
primary scheme: c(”red3”, ”dodgerblue4”, ”forestgreen”, ”purple4”, ”orangered”, ”yellow”, ”orangered4”,
”violetred2”)
pastel scheme: c(”seagreen”, ”brown2”, ”skyblue2”, ”hotpink3”, ”chartreuse3”, ”darkgoldenrod2”, ”light-
salmon3”, ”gray48”)
These schemes give eight distinct colors, and most people will have trouble distinguishing more than eight. Finally, the
current color scheme of a Spectra object may be determined using sumSpectra or changed using conColScheme.

5 Technical Background
ChemoSpec is written entirely in R, there is no compiled code. Hence, it should be platform independent (please let me
know if you discover otherwise). ChemoSpec uses S3 classes under the hood because frankly they were much faster to
write. For the pros and cons of classes and object-oriented programming in R, see the help archives (search my name for
one thread and some really interesting replies from the big dogs). ChemoSpec employs several different graphics packages
- the choice was one of practicality. In general, I tried to make all the graphics output look similar for consistency.

39
6 Acknowledgements

clupaSpectra plotScree

plotScree2 evalClusters

cv_pcaSpectra

q2rPCA

binData pcaDiag
isWholeNo plotScoresCor

mclust3dSpectra colLeaf mclustSpectra

plotScoresDecoration

shrinkLeaf

labelExtremes coordProjCS
mclust3D

binSpectra
plotHCA plotScores

normVec makeEllipsoid
aovPCAscores sPlotSpectra
sumGroups plot2Loadings seXyIqr
plotScores3D
plotScoresRGL r_pcaSpectra
labelExtremes3d
c_pcaSpectra seXy95
sumSpectra
hcaScores surveySpectra seX
hcaSpectra
seXy
surveySpectra2

rowDist seXyMad
removeGroup
plotSpectraDist chkSpectra

sampleDistSpectra hmapSpectra

normSpectra hypTestScores
files2SpectraObject
plotSpectra
removeFreq matrix2SpectraObject
conColScheme
baselineSpectra
plotSpectraJS
sgfSpectra
loopThruSpectra splitSpectraGroups removeSample groupNcolor
aov_pcaSpectra aovPCAloadings

plotLoadings
avgFacLvls

Figure 30: Map of Functions in ChemoSpec

In understanding the operation of a package, it is useful to know how the functions relate to each other, i.e., which
functions call each other. These relationships are readily visualized with a diagram like Figure 30. This was generated
by first using the foodweb function in package mvbutils[19], then removing the links to function chkSpectra which
generates a lot of clutter since nearly all functions in ChemoSpec call it. Finally, the resulting adjacency matrix was
converted into a graph by gplot in package sna[20].

6 Acknowledgements
The development of ChemoSpec began while I was on sabbatical in 2007-2008, and was aided greatly by an award of
a Fisher Fellowship. These programs are coordinated by the Faculty Development Committee at DePauw, and I am
very grateful to them as well as the individuals who originally created these programs. One of my student researchers,
Kelly Summers, took the data included in CuticleIR in the summer of 2009 as part of a preliminary study. I am also
grateful to Prof. Peter Filzmoser who answered a number of my questions related to the algorithms in his chemometrics
package. Finally, many people have brought bugs to my attention, suggested new features and provided fixes. I am
grateful to all of them. Each is listed in either the code, the NEWW file or the Github issues tracker.

40
References

7 Related Packages
Several other packages exist which do some of the same tasks as ChemoSpec, and do other things as well. The package
closest in functionality to ChemoSpec is hyperSpec written by my friend and collaborator Claudia Belietes (these packages
were developed independently around the same time).[21] There is also a package designed to interconvert Spectra
objects and hyperSpec objects, which allows one to move data between the packages more easily.[22] Package TIMP
is geared toward sophisticated modeling of time-dependent spectral data sets.[23] New packages may appear at any
time (and sometimes they disappear!). A good place to check these things out is the Chemometrics and Computational
Physics Task View.

References
[1] B. A. Hanson, ChemoSpec: Exploratory Chemometrics for Spectroscopy, 2017. R package version 4.4.x.
[2] K. Varmuza and P. Filzmoser, Introduction to Multivariate Statistical Analysis in Chemometrics. CRC Press, 2009.
[3] R. Wehrens, Chemometrics with R: Multivariate Data Analysis in the Natural Sciences and Life Sciences. Springer,
2011.
[4] D. S. Wishart, “Current progress in computational metabolomics,” Briefings in Bioinformatics, vol. 8, no. 5,
pp. 279–293, 2007.
[5] Y. Xie, knitr: A general-purpose package for dynamic report generation in R, 2017. R package version 1.16.
[6] A. Craig, O. Cloareo, E. Holmes, J. K. Nicholson, and J. C. Lindon, “Scaling and normalization effects in NMR
spectroscopic metabonomic data sets,” Analytical Chemistry, vol. 78, no. 7, pp. 2262–2267, 2006.
[7] R. Romano, M. T. Santini, and P. L. Indovina, “A time-domain algorithm for NMR spectral normalization,”
Journal of Magnetic Resonance, vol. 146, no. 1, pp. 89–99, 2000.
[8] R. A. van den Berg, H. C. J. Hoefsloot, J. A. Westerhuis, A. K. Smilde, and M. J. van der Werf, “Centering,
scaling, and transformations: improving the biological information content of metabolomics data,” BMC
Genomics, vol. 7, p. 15, 2006.
[9] S. C. Zhang, C. Zheng, I. R. Lanza, K. S. Nair, D. Raftery, and O. Vitek, “Interdependence of signal processing
and analysis of urine H-1 NMR spectra for metabolic profiling,” Analytical Chemistry, vol. 81, no. 15,
pp. 6080–6088, 2009.
[10] P. E. Anderson, N. V. Reo, N. J. DelRaso, T. E. Doom, and M. L. Raymer, “Gaussian binning: a new kernel-based
method for processing NMR spectroscopic data for metabolomics,” Metabolomics, vol. 4, no. 3, pp. 261–272,
2008.
[11] T. De Meyer, D. Sinnaeve, B. Van Gasse, E. Tsiporkova, E. R. Rietzschel, M. L. De Buyzere, T. C. Gillebert,
S. Bekaert, J. C. Martins, and W. Van Criekinge, “NMR-based characterization of metabolic alterations in
hypertension using an adaptive, intelligent binning algorithm,” Analytical Chemistry, vol. 80, no. 10,
pp. 3783–3790, 2008.
[12] T. K. Karakach, P. D. Wentzell, and J. A. Walter, “Characterization of the measurement error structure in 1D H-1
NMR data for metabolomics studies,” Analytica Chimica Acta, vol. 636, no. 2, pp. 163–174, 2009.
[13] S. Wiklund, E. Johansson, L. Sjostrom, E. J. Mellerowicz, U. Edlund, J. P. Shockcor, J. Gottfries, T. Moritz, and
J. Trygg, “Visualization of gc/tof-ms-based metabolomics data for identification of biochemically interesting
compounds using opls class models,” Analytical Chemistry, vol. 80, no. 1, pp. 115–122, 2008.
[14] P. Harrington, N. Vieira, J. Espinoza, J. Nien, R. Romero, and A. Yergey, “Analysis of variance-principal
component analysis: A soft tool for proteomic discovery,” Analytica Chimica Acta, vol. 544, no. 1-2, pp. 118–127,
2005.
[15] R. C. Pinto, V. Bosc, H. Nocairi, A. S. Barros, and D. N. Rutledge, “Using ANOVA-PCA for discriminant analysis:
Application to the study ofmid-infrared spectra of carraghenan gels as a function of concentration and
temperature,” Analytica Chimica Acta, vol. 629, no. 1-2, pp. 47–55, 2008.

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References References

[16] C. Fraley and A. Raftery, mclust: Model-Based Clustering / Normal Mixture Modeling, 2016. R package version
5.3.
[17] C. Fraley and A. E. Raftery, “Model-based clustering, discriminant analysis, and density estimation,” Journal of
the American Statistical Association, vol. 97, no. 458, pp. 611–631, 2002.
[18] L. Scrucca, M. Fop, T. B. Murphy, and A. E. Raftery, “mclust 5: Clustering, classification and density estimation
using gaussian finite mixture models,” The R Journal, vol. 8, no. 1, pp. 289–317, 2016.
[19] M. V. Bravington, mvbutils: Workspace organization, code and documentation editing, package prep and editing,
etc., 2013. R package version 2.7.4.1.
[20] C. T. Butts, sna: Tools for Social Network Analysis, 2016. R package version 2.4.
[21] C. Beleites and V. Sergo, hyperSpec: a package to handle hyperspectral data sets in R, 2015. R package version
0.98-20161118.
[22] C. McManus, hyperChemoBridge: Bridge between hyperSpec & ChemoSpec, 2016. R package version 1.1.26.
[23] K. M. Mullen and I. H. M. van Stokkum, “TIMP: an R package for modeling multi-way spectroscopic
measurements,” Journal of Statistical Software, vol. 18, no. 3, 2007.

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