 for analytical procedure validation and development mean for a regulated Good Manufacturing Practice (GMP) laboratory? Are they consistent with the approach taken by 

United States Pharmacopoeia (USP) <1220> on Analytical Procedure Lifecycle

 two documents to describe what USP can do in one?

? Why does it take two documents to describe what USP can do in one?

Good analytical science and ensuring data integrity requires that analytical procedures should be validated for their intended use (1,2). In addition, the applicable US Federal Drug Administration (FDA) Good Manufacturing Practice (GMP) clause is Title 21 of 

 211.194(a), which has been in the regulation since 1978:

The suitability of all testing methods used shall be verified under actual conditions of use (3).

 Chapter 6.15 on quality control (QC) requires validation of test methods:

Testing methods should be validated. A laboratory that is using a testing method and which did not perform the original validation, should verify the appropriateness of the testing method. All testing operations described in the marketing authorisation or technical dossier should be carried out according to the approved methods (4).

Like equipment (analytical instruments and systems) that must be fit for intended use, so must analytical procedures. However, how many readers define what the intended use of their analytical procedure must be?

These regulations apply equally to in-house development as well as pharmacopoeial methods. However, there was no regulatory guidance on how validation or verification was to be achieved. In this column, we take you on a journey covering guidance for the validation of analytical procedures over the past 30 years. The starting point for this journey is to look in the sin bin of non-compliances related to analytical results.

Does Your Analytical Procedure Generate Out of Specification (OOS) Results?

One of the continuing sources of schadenfreude with FDA warning letters and 483 observations is the invalidation of out of specification (OOS) results for a variety of unscientific reasons, such as “human error,” often without a scientific investigation. This results in the invalidation of the result and has resulted in the FDA generating a quality metric for any QC laboratory of percentage of invalidated OOS results (5) as well as a revision of the Guidance on investigating OOS results in May 2022 (6).

Why are there many OOS results in some laboratories? After all, pharmacopoeial procedures always work as written, right? Wrong! Could a laboratory have rushed to validate a hastily developed procedure with the result that a poor procedure most probably is the root cause of OOS results? Therefore, we need a robust method development phase of any analytical procedure to have a sound foundation for validation and ensure that OOS results are rare.

ICH and USP Approaches to Procedure Validation

We focus mainly on publications from two organizations—United States Pharmacopoeia (USP) and International Conference on Harmonization (ICH) for analytical procedure validation. For those who are unaware, 

 was initially called International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use from its inception in 1990. It was a consortium of the regulatory authorities and pharmaceutical industry bodies from the United States, Japan, and Europe. The aim was to develop unified approaches to key regulatory topics so that the pharmaceutical industry only had to perform one activity and it was acceptable in the three regions. Since then, additional regulatory authorities have joined (for example, Brazil and South Korea), and the name of the organization has changed. Conference has been replaced by Council, but it is still known as ICH.

As you can see in Figure 1, the FDA have also published two draft and one final guidance document on validation for analytical procedures and methods, which brings us to an interesting question: procedure or method?

Analytical Method or Analytical Procedure?

It is important to understand what is meant by analytical procedures and if there is a difference with analytical methods:

Analytical procedures refer to the wholeprocess from sampling through to reporting the result.

Analytical methods usually only refer to the instrument portion of the analytical procedure or analytical principle (separation technique).

Therefore, analytical procedure is the preferred term because there may be problems when sampling (for example, light-sensitive analytes), during transport or storage (for example, unstable analyte), or during preparation before analysis, all of which will not be considered if you are focused on the analytical method unless you are the FDA who have bet on both terms.

In The Beginning 1: Initial Validation Guidance

 211 was published in 1978, there was no official guidance available on how to validate an analytical procedure; eventually, the USP issued an informational general chapter <1225> entitled “Validation of Compendial Methods” in 1989 (7).

In addition to general chapter <1225>, USP has also issued two more general chapters:

 <1226> Verification of Compendial Procedures (8), and

 <1224> Transfer of Analytical Procedures (9).

 <1225> served as the foundation for the development of 

 Guidance on Validation of Analytical Procedures, which was published in two parts: 

ICH Q2A Validation of Analytical Methods: Definitions and Terminology

 was published in 1994 and is the parent guideline, and

ICH Q2B Step 4 Validation of Analytical Procedures: Methodology

ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology

 with no changes to the content of the combined document (10) shown in Figure 1.

Method development is ignored, which is problematic because this is vital. Cobbling a procedure together can result in variability contributing to OOS results as mentioned earlier.

 can be followed slavishly (for example, an analytical procedure for measuring assay between 90–110%) and often have LOQ and LOD limits that are determined in the validation. The reason? It is in 

! This is stupid as sound science has not been applied as required by 21 

 211.160(b) (3). We bet you didn’t know that a regulation required you to use sound science.

The focus is mainly on chromatographic analysis with little coverage of spectroscopic analysis.

In The Beginning 3: USP Lifecycle of Analytical Procedures 

 general chapter <1220> on Analytical Procedure Lifecycle (ALM) (11) that became effective on May 1st this year. Bet you didn’t know that either! 

 <1220> has had a long gestation period, starting in 2012 with the first Stimulus to the Revision Process (12) with further Stimuli articles (13–15) and two draft versions of <1220> (16,17) to support the development of the general chapter.

The lifecycle approach is now a regulatory expectation as it features in 

ICH Q12 Technical and Regulatory Considerations for Pharmaceutical Product Lifecycle Management

 is to move from quality by testing to quality by design (QbD), using knowledge, science, and statistical design to achieve more robust pharmaceutical products. QbD is defined by 

A systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management (18).

The key points are: predefined objectives, product and process understanding, sound science, and risk management.

These four points are incorporated in the three-stage lifecycle of <1220> analytical procedures (11) is shown in Figure 2 that comprises:

Stage #1: Procedure Design and Development (PPD). PPD, or method development, is based on the analytical target profile (ATP) for the procedure. Within this phase of the lifecycle, the critical parameters are identified and risk-based ways to control them are implemented. A design space is defined and tested using experimental design software.

Stage #2: Procedure performance qualification (or method validation) of the final design space against the parameters for intended use of the analytical procedure as defined in the ATP and elucidated during the PPD phase of the lifecycle.

Stage #3: Procedure performance verification, which is an ongoing assessment of the procedure’s performance during operational use (for example, trending data and results over time). Any changes made within the design space are considered validated and can be made without formal change control. Changes outside the design space return to either phase 2 for qualification, or if major to revise the ATP with further development. This is the longest portion of the lifecycle and it is imperative that it is controlled and monitored to ensure verification against intended use.

The key to a lifecycle approach for any analytical procedure is to define the intended use as required by the GMP regulations. This is achieved by writing an ATP to provide the foundation for all other work in the lifecycle. Get the analytical target profile (ATP) wrong, and the procedure is not fit for intended use.

 established an Expert Working Group (EWG) to revise 

 on Analytical Procedure Development (20). The aims of the EWG were as follows: 

 required revision as it lacked guidance for near-infrared (NIR) and Raman spectroscopy, especially using multivariate models, resulting in inadequate validation reports in regulatory submissions resulting in multiple questions being asked by regulators.

 needed a new guideline outlining the principles and scientific approaches for analytical procedure development.

Specifically, the feasibility to combine both documents into one for simplification and clarity should be examined.

ICH guidances go through a five-step process:

Step 1: Define the scope and drafting by the EWG.

Step 2: Issue the draft for public comment (the stage that 

Step 3: Review the public comments and update of the document.

Step 4: The final approved version of the document is released.

Step 5: The guidance is incorporated into a member’s national regulatory framework. This can vary from updating regulations or issuing a guidance for industry.

Let us now look at the contents of these two draft guidance documents. Avid readers should also read a review of these two draft documents by Teasdale and others (21), who have been involved in analytical procedure lifecycle development for a number of years.

The first thing to realize is that the expert working group (EWG) have failed to combine both documents to define, simplify and clarify a unified approach to lifecycle management of analytical procedures. This leave analysts in regulated laboratories juggling between two regulatory documents with differing approaches on the same subject. It is not a great start.

 (22) with the continuing failure to mention the most critical part of the lifecycle—procedure development. Given the pharmaceutical industry’s inability to change, this means perpetuating poor science. Furthermore, the failure to consider a lifecycle approach breaks the principles outlined in 

 is a tweak, not a substantive advance—for example, the issues mentioned about lack of validation in regulatory submissions are discussed in 

To be fair to the EWG, an argument is that they wanted to maintain the status quo, but this brings its own problems. Will analysts read 

 and integrate them together? Probably not. In contrast, that has not stopped USP <1220> managing to incorporate all phases in a single general chapter (11).

Let’s start with the major flaws in the draft (22):

define an analytical procedure lifecycle? No.

Does it define an Analytical Target Profile to document intended use of the procedure? No. 

 does mention “performance characteristics” as the nearest to ATP.

Method development is still conspicuous by its absence. This is a critical component of the lifecycle that has been missing throughout the various versions of 

There is no mention of validating the analytical procedures against the intended use as defined by an ATP. This is disappointing because it is the linkage between 

, and it is only shown in the outline in Figure 1. If these documents are to remain separate, they should be linked via the procedure ATP.

 is in the glossary, which is copied from 

The draft guidance jumps straight into a validation study for the procedure, assuming that development is not done, undocumented, or cobbled together. Given the fact that 

 both focus on lifecycle and the proposed 

 does not, it leaves us with the presumption that the authors are happy to accept a suboptimal and unscientific approach.

On the plus side is the expansion of the scope of 

Spectroscopic methods are covered but not in much detail as we shall see later.

Given the large number of biological products and biosimilars that are available and in development, biological assays (for example, polymerase chain reaction (PCR) and cell based and binding assays) are included in an expanded 

 draft. This has resulted in an expansion of calibration methods to accommodate them.

Good results require a good calibration model and the draft guidance includes calibration models that are linear, non-linear, and multivariate. Use any calibration method but keep it appropriate and use simplest model for your data.

Linearity as a validation parameter is only applicable now for linear calibration models.

In Annex 1, there is a good process flow diagram leading from the objective of an analytical procedure that then defines which parameters need to be considered during a validation (not forgetting development as well): specificity, range, accuracy, and precision. For example, a procedure for identification would just focus on specificity in contrast to an assay where all four would be determined. This is better than the table in the current version of 

 in terms of a lifecycle approach to analytical procedures but with a big gap—validation of the procedure which is devolved to 

Minimal versus Enhanced Approaches to Analytical Procedure Development

 discusses minimal versus enhanced approaches. The use of the term minimal is a concern because it is often interpreted as this is all we need to do. Only in the enhanced option is the ATP mentioned and this is defined as:

a prospective summary of the performance characteristics describing the intended purpose and the anticipated performance criteria of an analytical measurement (23).

The ATP defines what is wanted from the procedure but not how it is achieved. However, from the ATP comes the outline of the analytical procedure with knowledge of the analyte and matrix:

Any sampling and sample storage requirements

Instrumental technique used to analyze the sample

Any data interpretation or calculation required

A good idea is to define the ATP for any analytical procedure including the minimal approach. However, without an ATP, is the minimal approach scientifically sound (3)? No. Outlines of the minimal and enhanced approaches are shown in Table I (23). Apart from defining the ATP, the critical component of the enhanced approach is the definition of an analytical control strategy. Here, time is taken to identify the parameters that are critical to ensuring a robust analytical procedure and how they must be controlled. Knowing how a component can influence the outcome is essential. What is not shown in Table I is the iterative nature of the development process between the analytical control strategy and the results from experiments. As more information and knowledge about the procedure are generated, the procedure and control strategy can be refined. Risk management is used to identify critical parameters that may need to be carefully controlled and other that have a lesser or zero impact on the outcome of the procedure. 

Experimental design software can be used either standalone of controlling some analytical instruments to identify and define a design space where the procedure is robust. Having this information before the validation of the procedure as well when it is used operationally is essential as changes within the design space can be made without the need for revalidation. 

 section 10 deals with the submission of analytical procedures. One question that arises is why is this not in 

 as this is where the bulk of the data are generated? This is a good argument for merging the two documents.

One of the remits for the EWG for revising 

 was to include multivariate analysis methods. We want to focus on the development of and validation of spectral libraries used widely for identification of raw materials and active pharmaceutical ingredients (API) by various IR methodologies (NIR, Fourier transform (FT)-IR, and Raman) and how they are covered by the draft 

There is no clear link or explanation of “fit for use” for these methods. A discussion of what fit for use means would help define some of the vague concepts such as “enough” and “small.” This is a general problem with 

. In the revision of these guidances a useful discussion of how to demonstrate fit for use would be very valuable.

Section 8 discusses the development of multivariate analytical procedures, noting that multivariate models are constructed from samples (the bullet points added by us):

...a careful strategy for sample selection is essential for obtaining the relevant in- formation from the analytical data and contributes to the robustness of the resulting model.

Based on the method and measurement principle, the sample population should encompass the sources of variability likely to occur during manufacture and analysis, such as raw material quality, manufacturing process variability, storage conditions, sample preparation, and testing.

Use of risk assessment tools can help to identify sources of variability with the potential to influence the measurements and resulting model outputs.

Obtaining samples with appropriate variability at commercial scale can be challenging. Therefore, development laboratory and pilot scale samples are often utilized to provide enough variability to improve accuracy and robustness of the model.

Inclusion of commercial scale samples is recommended to capture variability related to specific equipment or processing conditions.

Careful consideration should also be given to sample distribution in the calibration and validation sets, as this will influence the model predictive capability.

The key to building an identification library is selecting enough appropriate samples reflecting the expected variation a manufacturer would expect (as noted in the second bullet point above), which can be aided by reducing the number qualified suppliers for each compound. However, finding detailed guidance about how to develop an identification library is lacking, the closest being using Raman to identify incoming glucose samples in section 13.3 Annex C: Example of Multivariate Model Lifecycle Components. However, if you want to identify a material from similar structured compounds, there does not appear to be any advice on building a library. However, there is still the excitement of validating the spectral library in 

Articles

Columns: Focus on Quality

A look at two ICH draft publications reveals that the EWG has much revision work to do for these publications to fully cover the validation lifecycle process.

Quo Vadis Analytical Procedure Development and Validation?

Using Raman spectroscopy and X-ray fluorescence (XRF) to examine the paint on the floor of Jackson Pollock’s studio reveals a list of pigments—published here for the first time—that could be used to verify the authenticity of the abstract expressionist’s paintings.

Jackson Pollock is considered one of the preeminent artists of the 20th century, and perhaps the most important U.S.-born painter. A pioneer in modern art, Pollock used controlled deposition of paint to canvas or masonite to achieve a beautiful effect—one that many have tried to copy. In fact, Pollock has been one of the most plagiarized modern artists, which makes proper authentication important and tricky.

Provenance is key to validating an object as authentic, but scientific analysis is also of great importance. There have been several manuscripts describing the analysis of known Pollock paintings, but few encompassing the most common pigments he used. Recently, we had the opportunity to use an integrated analytical approach to investigate paints on the floor of Pollock’s studio, located at Pollock-Krasner House in East Hampton, New York. As Figure 1 shows, the remnants of paint from Pollock’s many projects remain on the studio floor, and they are ripe for scientific analysis using portable and noninvasive spectroscopic techniques, such as Raman and X-ray fluorescence (XRF). It is almost certain that Pollock created paintings with pigments now present on the studio floor, meaning that they can be used as a reference to help determine the authenticity of any future unknown works. To facilitate future comparisons, a color palette has been provided in Figure 2. The top images in Figure 2 show the isolated colors that have been digitally matched to the actual paint deposited on the workshop floor (shown in the lower portion of Figure 2).

Each area of paint was analyzed by using a Raman spectrometer (Bravo, Bruker) and a handheld XRF instrument (XRF, Tracer 5g, Bruker). The Raman spectrometer used two excitation laser sources (785 and 852 nm) to minimize fluorescence interference, and each laser was actively controlled to shift the emission wavelength to further allow for the removal of any fluorescence interference. Finally, a concave baseline correction was used to remove any residual fluorescence. Acceptable Raman data could be collected on all but the brown pigment.

The handheld XRF spectrometer was equipped with a 4 W Rh X-ray tube, and an energy dispersive spectrometer with a graphene window, allowing a higher transmission of low-energy X-rays. Measurements of each paint target were conducted using three excitation settings—15 kV, 30 kV, and 50 kV—to enhance the elemental resolution in different parts of the energy spectrum. We present only the 30 kV spectra to simplify comparisons. All spectra were collected for 30 s using a collimated spot size of 8 mm.

It is worth noting that Raman is a surface technique in this case; the paint is strongly absorbing, which significantly reduces the depth of penetration. However, the primary X-ray beam penetrates to greater depths, with the detected XRF signal including surface species of interest along with contributions from any underlying paint. Table I gives an overview of the pigments we detected. We identified the pigments using the established literature. The Raman data shown was not manipulated or corrected in any way to facilitate the comparison to data collected from other Pollock paintings.

There were three blue colored paints found on the floor—museum gray-blue, deep blue, and Midas blue-green. The Raman and XRF spectra collected from these paints are shown in Figures 3 and 4, respectively. Raman analysis of the museum gray-blue showed the conclusive presence of phthalocyanine blue, which matched the recorded spectrum of the known reference for this pigment (1). The Raman spectrum from the dark blue paint was interpreted to contain ultramarine (2); the presence of amorphous carbon was also detected. The spectrum from Midas blue-green indicated the presence of Prussian blue (2) and some other highly fluorescing compound. There was a band at 637 cm

 that could not be assigned (there were several possible compounds that have Raman peaks in this area). The XRF results showed prominent peaks at titanium, calcium, and lead, possibly from various underlying white paint layers or mixtures with Prussian blue. The mixed or lime green paint spectra has characteristic Raman bands for carbon black and chrome yellow. The XRF spectrum indicates the presence of chromium, along with lead, barium, and calcium, which may be mixtures with or underlying the lime-green paint. The hunter green paint Raman spectrum also exhibits significant fluorescence, but the two-band signature for Prussian blue was weakly present compared to the Raman spectrum, which was very similar to that collected on Midas blue (not shown). No XRF spectrum was collected from this pigment.

Mid-Gray (Slate) and Dove (Dingy) Gray Pigments

This mid-gray pigment was interpreted to contain carbon black, as shown by the Raman spectrum (2). The XRF spectrum indicates the presence of titanium, barium, and sulfur, suggesting one or more white paints mixed with a carbon-based black paint. The dingy gray paint was found to contain chrome yellow mixed with black (1). The Raman spectrum has bands indicative of carbon black and chrome yellow (Figure 3). Chromium is also present in the XRF spectrum (Figure 4). The presence of titanium and lead further indicated mixing with titanium white and lead white pigments.

The black paint has the signature response of amorphous carbon, as shown in the Raman spectrum (bottom spectrum of Figure 3). Carbon is too light an element to be detectable by portable XRF (2), suggesting that all element peaks in the measured XRF spectrum are from underlying sources.

The dark red paint contains hematite. Hematite bands are present in the Raman spectrum (Figure 5), but are weak shoulders on the titanium oxide peak, which indicates an anatase form. However, the XRF spectrum confirms the assignment (Figure 6). Hematite red was observed by Rosi and associates (3). In comparison, the bright red paint has been determined to be based on a CdS

, as previously reported on authenticated works by Pollock (3). This paint produces substantial fluorescence, with the Raman spectrum shown in Figure 5, and it was the first spectrum successfully collected from this compound that the authors could determine from an extensive literature search. The XRF spectrum was useful in making this identification (Figure 6).

The orange paint was found to contain some carbon and the same BaSO

 complex was observed in the bright red pigment. This identification was also reported by Rosi (3). The XRF spectrum exhibited a strong signal for barium and prominent peaks for sulfur, cadmium, and selenium. The Raman spectrum showed bands attributed to amorphous carbon (not detectable by XRF) and barium white. The mustard-colored paint was found to contain evidence for Mars yellow (FeOOH) and some organic pigment, confirmed by both Raman (Figure 5) and XRF measurements (intense Fe peaks in Figure 6). In the Raman spectrum, the triplet of bands from Mars yellow are evident (2). There were four weak bands present at 1734, 1589, 1286, and 1042 cm

 that were likely because of an unassigned organic pigment. The bands match somewhat the crinacridone red reported by Schulte and associates (5). However, this would be a tentative assignment. The Raman spectrum collected on yellow paint has a strong signature characteristic of chrome yellow (PbCrO

). The XRF spectrum confirmed the interpretation based on prominent Cr and Pb peaks (Figure 6). Chrome yellow has been reported in use by Pollock in other works (3).

The prominent white paint present on the floor of the Pollock studio is titanium white (Figures 7 and 8), as previously observed by Rosi (3). The Raman spectrum contained the well-known doublet for the rutile form of titanium oxide, supported by a strong titanium signal in the XRF spectrum (Figure 8). Minor Zn and Pb peaks in the XRF spectrum likely reflect mixtures of multiple white pig- ments in other paints. The pink paint was interpreted to be azo β-napthol toluidine as identified by the Raman spectrum (Figure 7) with bands at 1620, 1567, 1493, 1440, 1392, and 1326 cm

, as also reported by Rosi (3). The XRF spectrum was weak, also indicating an organic-based pigment. However, the XRF spectrum showed a strong signal for aluminum, confirming the visual inspection that suggested the pink was achieved by mixing the toluidine red pigment with metallic aluminum paint. The Raman spectrum collected from the aluminum-colored paint yielded a spectrum with no bands. Figure 8 shows the XRF spectrum which records a very prominent peak at aluminum, along with other contributions from underlying paints. Bare metals do not have a Raman signature, so no Raman pigment bands would be expected.

Raman analysis of the brown paint yielded only fluorescence, and no XRF was performed on the brown paint. It is commonly difficult to obtain a Raman spectrum from brown paint because there is significant fluorescence interference.

By performing a non-invasive paint analysis of Jackson Pollock’s workshop floor with the integrated use of portable Raman and XRF spectrometers, we came to a few conclusions. We found that most pigments could be readily identified by Raman or XRF analysis. However, the CdS

 complex in the active orange pigment was identified only by XRF analysis; De Faria and Lopes (4) also observed the presence of this uncommon pigment. For bare metals, such as the aluminum pigment, XRF was also essential for making the identification because bare metals do not have a Raman response.

The advantage in this study of portable Raman analysis is that it is primarily a surface technique. As observed from the photographs of the Pollock workshop floor, there were certainly many layers of paint! Raman measurements allowed targeted analysis of only the surface paint, whereas XRF may contain mixed measurements of multiple layers. Additionally, the Raman technique allowed the characterization of the molecular structures of materials, which facilitates highly specific identification for organometallic and organic compounds that do not have unique elemental signatures.

We hope that the pigments identified through our multi-instrumental analysis of a studio floor may provide additional puzzle pieces for future researchers who need to verify whether they really do have a genuine Jackson Pollock—or just a drip painting of unknown origin.

The authors with to express their gratitude to the Pollock-Krasner House curator, Helen Harrison, for allowing us to visit the museum and collect scientific data from paint on the workshop floor.

(2) I.M. Bell, R.J.H. Clark, and P.J. Gibbs, 

 is an Applications Manager, with Bruker Optics. 

 is a Professor of Chemistry at Rutgers University. 

 is Senior Market Applications Scientist, Bruker Nano Analytics. Direct correspondence to: 

Featured Article

Raman and XRF spectroscopy were used to examine paint in artwork, revealing the potential of both techniques to verify the authenticity of famous works of art.

A Different Kind of Art Analysis

In this Part 3 survey article of atomic spectroscopy instrument components, we look at instrument components and subassemblies used for the special components and terms used in atomic spectroscopy instrumentation for inductively coupled plasma–mass spectrometry (ICP-MS), ICP–optical emission spectrometry (ICP-OES), X-ray fluorescence (XRF), energy-dispersive X-ray spectroscopy (EDX), total reflection X-ray fluorescence (TXRF), and laser-induced breakdown spectroscopy (LIBS). Our “under the hood” look continues as we survey the most typical atomic spectroscopy design components. As we continue publishing our four-part component survey series, we note that tutorial articles, and 

The Spectroscopy Instrument Components Terminology Guide

, posted digitally in February 2022, are available to our readers.

In this part 3 of our four-part spectroscopy components survey article, we take a closer look at the special components and terms used in atomic spectroscopy instrumentation for inductively coupled plasma–mass spectrometry (ICP-MS), ICP–optical emission spectrometry (ICP-OES), X-ray fluorescence (XRF), energy-dispersive X-ray spectroscopy (EDX), total reflection X-ray fluorescence (TXRF), and laser-induced breakdown spectroscopy (LIBS). The various instrument acronyms are defined within the table. The atomic spectroscopy techniques and components are described in Table IV, with the table containing four columns of information—the technique or component name, a text description, basic specifications, and references and links (1–4). All topics are in alphabetical order for this table. The list of basic techniques and components used for atomic spectroscopy instrumentation is obviously a very large subject with many details. Here, we are taking a broad look to survey the main techniques or components used for atomic spectroscopy instruments. More topic details are included within 

As with other analytical instruments, atomic spectroscopy instrumentation is advancing rapidly because of discovery research, new materials, improved mechanical tolerances, and continuous developments in electronics, algorithms, and computational power.

The Spectroscopy Instrument Components Terminology Guide 2022

(Springer Science & Business Media, Berlin, Germany, 2000), vol. 56.

 (Royal Society of Chemistry, Burlington House, United Kingdom, 2002), vol. 11.

(4) J.A. Holcombe, G.M. Hieftje, and V. Majidi (eds), 

Focus on Analytical Spectrometry: A Compendium of Applied Spectroscopy Focal Point Articles

 (1994-1997) (Society for Applied Spectroscopy, Frederick, Maryland, 1998).

In this third part of this four-part series on spectroscopy instrument components, we take a closer look at components used in atomic spectroscopy instrumentation—from beryllium and Kapton windows used in X-ray analysis to sampler and skimmer cones and plasma torches for ICP systems.

A Survey of Basic Instrument Components Used in Spectroscopy, Part 3: Atomic Spectroscopy

This year’s molecular spectroscopy award recipient is Lu Wei, an assistant professor of chemistry at the California Institute of Technology in Pasadena, California. From her days as a graduate student at Columbia University, Wei’s work has focused on the development and application of stimulated Raman scattering (SRS) microscopy for bioanalysis, spectroscopy-informed design of vibrational imaging probes, and sample-engineering strategies.

Lu Wei is an assistant professor of chemistry at the California Institute of Technology. Her research focuses on the development and application of stimulated Raman scattering (SRS) microscopy for bioanalysis, on informed design of vibrational Raman imaging probes, and on specialized sample-engineering strategies. Wei has already made several seminal contributions in the field of nonlinear Raman spectromicroscopy, pushing to the next frontier of multiconstituent, high spatial resolution, and biological Raman imaging.

Wei received her PhD in 2015 from Columbia University. During her PhD work, Wei pioneered a bio-orthogonal chemical imaging platform coupling SRS with small bio-orthogonal chemical probes, allowing for the noninvasive imaging of metabolic dynamics in live cells and tissues with high spatial and temporal resolution. Such imaging is used for life science applications (for example, investigations of living cellular metabolism).

She further developed a supermultiplexed optical imaging strategy that exploited a unique pre-resonance SRS excitation region, and achieved remarkable sensitivity with up to 24-plex imaging, which allows the measurement of up to 24 molecular components simultaneously.

Since starting her independent career at the California Institute of Technology (Caltech) in 2018, Wei has been working to develop next-generation Raman microscopy to achieve unprecedented resolution, sensitivity, and multiplexity with improved molecular precision and quantification, especially for complex bioanalysis pathways. One of her key contributions is the development of supermultiplexed and super-resolution vibrational microscopy for subcellular structure—function phenotyping in a complex cellular and biological tissue environment. Toward this end, she devised a label-free super-resolution SRS imaging strategy and obtained the first multiplexed imaging to within a spatial resolution of 100 nm. She also pioneered the development of advanced vibrational chemical probes (such as a photoactivatable and photoswitchable Raman probes), which is a significant component for achieving supermultiplexed and super-resolution vibrational microscopy.

Wei has also pioneered a selective deuterium strategy that has demonstrated the first in situ live-cell quantification and spectral analysis of aggregate composition from native polyQ aggregates, the key hallmark for Huntington’s disease. In addition, she further developed and applied subcellular Raman spectromicroscopy with data-mining cross-correlation to transcriptomics and lipidomics for Raman-guided subcellular pharmaco-metabolomics for metastatic melanoma.

Wei has published 28 papers with 1913 citations and has given 30 oral presentations at scientific conferences. She is a reviewer for multiple journals, including, among others, 

Nature Communications, Proceedings of the National Academy of Sciences of the United States of America (PNAS), Journal of the American Chemical Society, Chemical Science, Angewandte Chromatographie, ChemComm, Journal of Physical Chemistry Letters, Journal of Physical Chemistry B, Small, Langmuir, ACS Omega, Theranostics, Optics Express, Optics Letters, and Biomedical Optics Express

In assessing Lu Wei’s impact on the field, it is useful to look at both her mostly highly cited papers, as well as those considered to be most important by her close mentors and peers. The following publications are noted by the recognition of mentors and peers and by the total number of literature citations to date.

The most recent papers by Wei, published since 2019, represent her current research work in the development of Raman probes, Raman imaging, and SRS, as well as advancing imaging analysis techniques.

In a 2022 paper, Wei and coworkers reported on a general strategy for Raman imaging-based local environment sensing using the hydrogen–deuterium exchange (HDX) of terminal alkynes (termed alkyne-HDX) (1). The paper applied multiple Raman probes to demonstrate that deuterations of the alkynyl hydrogens lead to large shifts of alkyne Raman peaks of up to 130 cm

. These shifted peaks provided signals suitable for high specificity image-based analysis. The work is broadly applicable for using the alkyne-HDX strategy for the quantitative analysis of multiple bioactive molecules in complex biological samples.

A book chapter authored by Wei, Wang, Du, and Lee was published in 2022, containing recent developments in applying SRS for vibrational imaging and mapping of biomolecular analytes in living cells (2). SRS microscopy, when combined with small Raman-active molecular probes, enables detailed biochemical imaging. This chapter reviews a series of vibrational probes reported to date for SRS imaging and their labeling strategies. This imaging technique is useful for studies in cell and tissue metabolism, pharmacokinetics, chemical sensing, cell screening, and phenotyping.

A United States patent application by Wei and collaborators in 2021 described a system using excited fluorescence radiation (3). The patent application introduces stimulated Raman coupled fluorescence (SRCF) and stimulated Raman excited fluorescence (SREF) for imaging fluorescent probes, with improved signal-to-noise (S/N) ratios compared to conventional fluorescence detection. These techniques are especially applicable for multiplexed detection. SRCF is able to achieve high sensitivity down to the single-molecule level by adopting electronic pre-resonance SRS (epr-SRS), which yields a Raman signal enhancement factor of more than 1011 amplification. For both SREF and SRAF, the electron transition induced by the probe beam could optionally be either one-photon or multiphoton excitation. These combined techniques are useful for multianalyte spectral imaging in microscopic biological samples.

In another paper, Wei and Du reported the first general design of multicolor photoactivatable alkyne Raman probes for live-cell imaging and tracking (4). These probes are based on cyclopropenone caging chemistry and demonstrate high sensitivity Raman imaging with customizable or photocontrollable features. Single-cell and organelle-targeting probes were synthesized and tested to achieve images of mitochondria, lipid droplets, endoplasmic reticulum, and lysosomes. This technique should provide new tools for the study of complex biological organelle systems.

In 2021, Wei and coworkers expanded the use of vibrational imaging strategy for Raman microspectroscopy, by introducing a technique they dubbed vibrational imaging of swelled tissues and analysis (VISTA), for scalable and label-free high-resolution images of protein-containing biological samples (5). VISTA exhibits spatial resolution as low as 78 nm for sample images, and renders three-dimensional (3D) images by image isolation of proteins, as well as elimination of scattering lipids. VISTA was able to generate label-free images of the nucleus, blood vessels, neuronal cells, and dendrites in complex mouse brain tissues, making the technique potentially useful for advanced anatomy and physiology analysis of cells and tissues. “I think the VISTA technique, applied to volumetric SRS imaging, will grow into an important tool for label-free tissue imaging (5),” predicts Eric Potma, a professor of chemistry at the University of California in Irvine.

In 2014, Wei and collaborators reported on the use of SRS imaging of alkyne tags as a general strategy for studying small biomolecules in live mouse cells and tissue (6). This paper demonstrated a new key method for tracking alkyne-bearing drugs in mouse tissues and visualizing synthesis of DNA, RNA, proteins, phospholipids, and triglycerides in living tissue by incorporation of the alkyne tags directly into the tissue sample measurement area.

A method for direct imaging for multiple molecular species inside cells was demonstrated by Wei and her colleagues in 2017 (7). They used SRS under electronic pre-resonance conditions to image target molecules inside living cells with high selectivity and sensitivity. As part of this work, the research group synthesized a palette of triple-bond-conjugated near-infrared (NIR) dyes where each dye displays a single distinguishable Raman peak. When combined with available fluorescent probes, a palette of 24 resolvable colors is available for supermultiplex optical imaging in complex biological tissue samples.

Proceedings of the National Academy of Sciences

, Wei and coworkers described an imaging technique to visualize proteins using SRS microscopy coupled with incorporation of deuterium-labeled amino acids (8). The authors noted that incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, and that SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems to generate spatial maps of the new and total proteomes in vivo within cells.

, Wei and her coauthors designed and synthesized a class of polyyne-based molecular probes for optical supermultiplexing (9). By customizing the molecular structure of these probes, the researchers developed 20 distinct Raman frequency molecules. With additional probe modifications, the group created 10-color organelle imaging probes for living cells with high specificity, sensitivity, and photostability. These polyyne-based probes are promising for live-cell imaging and sorting, as well as for use in high-throughput diagnostics and screening.

A bio-orthogonal chemical imaging platform was also introduced by Wei with a team of researchers (10). This work coupled SRS microscopy with Raman- active alkyne and stable isotope probes. This method of bio-orthogonal chemical imaging exhibits excellent sensitivity, specificity, and biocompatibility for imaging small biomolecules present within live cells and tissues. In the paper, Wei and colleagues reviewed other recent research to visualize small biomolecules in live biological samples. This paper also discussed strategies for multicolor bio-orthogonal chemical imaging in this current era of “omics.”

In highlighting Wei’s work for the Emerging Leader in Molecular Spectroscopy Award, the nominators noted that she has made several seminal contributions in the field of nonlinear Raman spectromicroscopy to push the next frontier of biological Raman imaging. As a graduate student at Columbia University, Wei pioneered a bio-orthogonal chemical imaging platform that combined SRS with small-molecule bio-orthogonal probes, enabling noninvasive imaging of metabolic dynamics in live cells and tissues (10). She then further developed a supermultiplex optical imaging strategy that uses a pre-resonance SRS excitation to achieve 24-plex imaging (7).

As she began her independent career at the California Institute of Technology in 2018, Wei began to develop the next generation of Raman microspectroscopy for complex bioanalysis. One of the key contributions she has made during her research leadership at Caltech is to develop what is termed super-multiplex and super-resolution vibrational microscopy. The super-multiplex approach is used for structure-function phenotyping of organisms by taking detailed measurements of cellular and tissue samples. Wei has devised a label-free SRS imaging strategy and was the first to multiplex images to within 100 nm spatial resolution (5). In addition, she also has pioneered the development of photoactivatable (4) and photoswitchable (11) Raman probes, which are key components for implementing supermultiplex and super-resolution vibrational microscopy.

Her other key leadership contributions at Caltech include exploring a selective deuterium strategy that demonstrated the first in situ live-cell quantification and spectral analysis of native polyQ aggregates, the key marker for Huntington’s disease (12), and the development of subcellular Raman spectromicroscopy combined with data mining, using cross-correlation to transcriptomics and lipidomics for Raman-guided subcellular pharmacometabolomics for metastatic melanoma (13). Wei’s work has now attracted broader attention in the field of nonlinear biological Raman imaging.

Wei Min is a Professor of Chemistry at Columbia University and was Wei’s PhD mentor. He has been impressed by Wei’s live-cell imaging research using alkyne-tagged small biomolecules for SRS (6). This paper has attracted much attention from the field, receiving nearly 400 citations. “I believe that her efforts to develop photo-switchable Raman probes are going to have a great impact for molecular spectroscopy (4),” said Potma. The concepts Wei has developed make it possible to optically manipulate the specific performance of Raman probe technology.

Katsumasa Fujita is a Professor at Osaka University in Japan, and has collaborated with Wei since 2015. “I believe Lu’s most significant contribution is development of super-multiplex probes published in 2017 (7),” he said. This work demonstrated advances in the application of Raman probes for supermultiplex imaging, and expanded the number of target analytes observable simultaneously. Fujita believes Wei’s work will become a valuable technology that will attract much attention in the future. “Her metabolic imaging of living organisms using SRS microscopy also presented a new concept in biological and medical research,” he said. “She has also introduced the latest optical microscopy techniques such as super-resolution and sample transparency into SRS microscopy, and is leading the development of new spectroscopic imaging techniques.”

Renee Frontiera is a Northrop Professor of Chemistry at the University of Minnesota. “Lu’s combination of expansion microscopy with stimulated Raman microscopy is really innovative and exciting,” she said. Frontiera explained that Wei’s work allows for imaging of local chemical composition below the optical diffraction limit, and this technique can be applied to a number of different biomedical applications. “Finding expansion conditions suitable for Raman imaging is a huge accomplishment, and the whole field is eager to see where the Wei lab takes this exciting new approach,” she said.

In addition to her Raman research and teaching, Wei has made other impactful contributions to the broader field of molecular spectroscopy. Potma believes that Wei’s most significant contributions involve the development of the now well-known Raman molecular probes, as she pushed the capabilities of Raman tags to probe biological meaningful metabolic processes. “This development has really changed the field of Raman and stimulated Raman scattering (SRS) microscopy,” he said. Wei has further impacted the field by combining SRS with expansion microscopy, thereby allowing label-free imaging with super resolution. “I admire this work a lot, because I tried to do same thing several years ago, but I miserably failed!” he quipped. “Lu has shown that a modified expansion approach is needed to make it work for SRS, and it is this insight that made all the difference. Excellent work!” Min has known Wei for over 12 years, and shares that she was one of his first graduate students when he started his laboratory at Columbia University in 2010. After graduating with her PhD in 2015, Wei stayed in Min’s laboratory as a postdoctoral researcher before starting her own independent career at Caltech in 2018. Min says that Wei has made significant contributions by demonstrating SRS imaging of customized alkyne tags as a generalized method for imaging small metabolites and drugs inside live cells. “Another major contribution would be the development of super-multiplex vibrational imaging technology by developing rainbow-like Raman dyes and ultrasensitive Raman microscopy,” he said.

Fujita further praises Wei’s work. “Lu’s most impactful paper is the one published in 

 in 2017. This paper had a significant impact on the spectroscopy community and the communities in biology and medicine,” he said. Fujita notes that Wei’s approach modifies the vibrational property of dye molecules for multiplex imaging using the sharp Raman emission characteristics. “This approach has provided a new toolkit to chemical biology for the development of probes for biological imaging, which conventional fluorescence probes cannot realize,” he said.

Wei is insightful as a researcher, with a level of determination to match. Min, her PhD mentor at Columbia, saw this firsthand. “Our initial idea of imaging newly synthesized proteins by stimulated Raman scattering (SRS) microscopy was much riskier and more uncertain than what it appears now,” he says, noting that the research was started when the group did not even have an SRS microscope in their laboratory. It was Wei who recognized the potential of the technique, so she took the initiative for this project, and found creative solutions to the challenges. “In order to acquire the key SRS images, Lu flew to our collaborator’s lab in Houston, and worked 15 days straight there,” Min said. “The data she collected there was published in PNAS in 2013, reporting optical imaging of newly synthesized proteome in living cells for the first time (8).” This determination is a quality that Min continues to see in Wei. “Lu is very determined when pursuing research projects,” he said. “Many projects she had worked on at Co- lumbia, as well as several very recent papers from her Caltech lab, were regarded as nearly impossible at the beginning.”

Major research leaders point to a bright future for Wei. “Dr. Wei is a talented young vibrational spectroscopist and is growing into a future leader in the field,” said Min.

Ji-Xin Cheng, a professor at Boston University, agrees. “I consider Lu Wei as a rising star and next generation leader of the chemical microscopy field,” he said. “She is very creative in developing novel probes and new imaging methods for important applications in life science.”

Fujita also foresees Wei having a leading role in life sciences—and beyond. “Lu stands at the crossroads between chemistry, physics, and biology, providing useful tools for cutting-edge science and demonstrating the fascinating biological applications that can be achieved with them,” he said. Fujita anticipates that Wei’s ability to present new concepts and demonstrate their importance may lead her to other useful developments for both basic science and applications. “I am very much looking forward to seeing what she will be interested in, and how she will bring it to the world of vibrational imaging and advance spectroscopy in the future,” he said.

Potma put it more simply. “I think Lu is a game changer,” he said.

(2) H. Wang, J. Du, D. Lee, L. Wei,”Stimulated Raman Scattering Imaging with Small Vibrational Probes,” in Stimulated Raman Scattering Microscopy, J.-X. Cheng, W. Min, Y. Ozeki, and D. Polli, Editors (Elsevier, Amsterdam, The Netherlands, 2022), pp. 289–310. 

https://doi.org/10.1016/B978-0-323-85158-9.00023-3

(3) W. Min, L. Shi, H. Xiong, and L. Wei, 

Stimulated Raman coupled fluorescence spectroscopy and microscopy system and method

Lu Wei of the California Institute of Technology, the 2022 winner of the Emerging Leader in Molecular Spectroscopy Award, is applying stimulated Raman scattering (SRS) microscopy for bioanalysis, spectroscopy-informed design of vibrational imaging probes, and sample-engineering strategies.

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