Posted by: tvasailor | May 24, 2012

Overview “Known Unknowns”

New 11/24:  Creating and Using Retention Indices in NIST Software
New 11/15:  Forensic Workshop Using Mass Spec to Identify Unknowns
New:  Training for Wiley KnowItAll New EI Mass Spec Capabilities
New:  FREE Courses for Unknown Identification Using NIST Search

Systematic Process for the Identification of “Known Unknowns” in Commercial Products by GC-MS and LC-MS

Note: Details on other subjects are found in “My Topics” tab under sailboat picture above or on the sidebar links to the right..

Introduction:  In the last 34 years, we have developed a systematic process for the identification of “known unknowns” by GC-MS and LC-MS in commercial products.  We define “known unknowns” as non-targeted species which are known in the chemical literature or mass spectrometry reference databases, but unknown to the investigator.  The process is shown in the following simplified flowchart:


This process is described in detail in the February 2013 copy of LCGC, “MS-The Practical Art.”  The article is entitled:  “Identifying “Known Unknowns” in Commercial Products by Mass Spectrometry.”  A copy with associated ads is shown below:

LCGC PDF with Advertisements

The article originated from work presented at Pittcon in 2012.  The approach uses either NIST libraries or “spectraless” databases such as the CAS Registry or ChemSpider.  The “spectraless” ones are discussed at the bottom of this post.types_of_databases_searchesNIST Search of EI and CID Spectra:  The initial step in our process utilizes computer searches of EI (GC-MS) or CID (LC-MS) spectra against reference databases using the NIST MS search.

I have developed two free “webinar” series courses that detail the use of the NIST search with EI and tandem libraries for the identification of unknowns:

Link to free webinar courses

The computer EI searches normally work better than CID ones, but the latter are still very useful.  We employ both purchased, in-house, and “crowd-sourced” libraries.  The MoNA “crowd-sourced” libraries in NIST EI and Tandem formats are found at the following link:

MoNA EI and Tandem Libraries in NIST format

We use the NIST and Wiley commercial databases, but there are many other specialty databases that others might find useful.

The NIST search interfaces easily with a wide variety of manufacturers’ data processing and structural drawing programs:


“Spectra-Less” Database Searching:  If the NIST search is not successful, then accurate mass data is used to obtain a molecular formula (MF), a monoisotopic mass, or an average molecular weight.  This data is used to search very large databases such as the CAS Registry or ChemSpider  via web interfaces.  We define them as “spectra-less” databases because they contain no computer-searchable mass spectral data.  We had originally used this approach to search the TSCA database or our Eastman Chemical plant material listing.

The candidate structures from the CAS Registry or ChemSpider searches are prioritized by either the number of associated references or key words.  Other ancillary information such as mass spectral fragments in EI or CID spectra; isotopic abundances, UV spectra; types of ion adductsCI data; number of exchangeable protons;  etc. are used to narrow the list to one structure.  This website has many screenshots (SciFinder1, SciFinder2, ChemSpider) that illustrate these approaches with many examples.

Model EI and CID Spectra from NIST Structure Search:  The NIST MS Search program ranks model compounds employing structural searching of both our commercial and in-house databases.  This is particularly valuable for finding model compounds for a proposed structure found in searches of “spectra-less” databases such as the CAS Registry and ChemSpider.

As noted in the table above, there are ~800,000 structures associated with the EI spectra and 100,000 structures with CID mass spectra in our computer-searchable databases.  We use the NIST MS Interpreter  program to automatically correlate fragment ions in the EI and CID spectra with the component’s substructure.

“No Results” from Process:  There will be non-targeted species in the sample which are “unknown unknowns”, those not found in any reference libraries or “spectra-less” databases.  A few thoughts on their identification are discussed in another section.

Future Improvements Needed:  The approach works well for the majority of our samples which are fairly simple and contain components with molecular weights <500 daltons.  On the other hand, improvements are needed for complex samples and components with molecular weights >500 daltons.

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Posted by: tvasailor | December 4, 2022

Part VI: Creating and Using Retention Indices in NIST Software

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This web page highlights a training video demonstrating using AMDIS to determine retention indices (RI’s) with GC-MS data. Also resources are shared to reproduce the techniques demonstrated.

RI’s are used in combination with mass spectrometry to provide nearly unambiguous identification of isomers. This is a very difficult task to accomplish with EI mass data alone.

NIST offers extensive resources including:

-RI’s for >139K compounds
-RI and MS data >114K compounds
-GC methods and RI citations from >400K literature references
-AMDIS to determine RI’s for compounds in GCMS analyses

The results are sent to NIST search for evaluation. The training video illustrates the AMDIS process for determining RI’s for anlytes analyzed by EI GC-MS.

Two Different Formats for Training Video:
YouTube Video
mp4 Format

The following resources are also supplied:

Handout of slides used in video
Resources files for reproducing excercises in video
Definition of Retention Indices from Gary Mallard

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Posted by: tvasailor | November 12, 2022

Unknown Identifications Using NIST EI Hybrid Search

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FORENSIC@NIST 2022 Workshop
Mass Spectral Interpretatin–Tips and Tools for GC-EI-MS and High-Resolution MS Data

A worshop was presented at the Forensics@NIST on a variety of topics. See the link to the Agenda:

Agenda Link

My talk was entitled “Unknown Identifications using NIST Hybrid EI Search” and was an overview of Eastman’s approach to unknown identification as it applied to forensic applications. Links to the talk and other resources are shown:

Link to Video of Talk (YouTube)
Link to Video of Talk (Download directly Goggle Drive)
Handout of Slides (PDF Format)
Handout of Slides (Powerpoint format)
Internet Links to Resources

All the presentations in the workshop were recorded which are excellent resources. I will add links to them on my web page when they are available.

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Posted by: tvasailor | September 1, 2022

Wiley KnowItAll Mass Spec Training

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Wiley released a KnowItAll version with many new powerful electron ionization gas chromatography-mass spectrometry (EI GC-MS) capabilities.  I have developed FREE training videos and associated handouts covering basic and advanced MS topics that will be helpful to the new user.   Also, a Quick Reference Guide is available.

KnowItAll is a vendor neutral data processing solution for spectral analyses which will be very valuable in unknown identifications.  It processes IR, Raman, MS, NMR, and UV data employing a large number of file formats (>170) and a total of 2.5 million reference spectra.

~264K IR, 25K Raman, ~1.25M EI mass spec, 894K NMR, and 30K UV


-Deconvolution/very fast library searching of accurate/nominal mass GC-MS data

-Easy manual processing of nominal and accurate mass GC-MS data
-Fast EI searches of >1.2 million spectra in <0.5 seconds
-Amazing adaptive search for unknown ID by extending usefulness of all libraries
-Transfer structures and spectra between NIST search and KnowItAll
-Easy structure drawing and structure searches
-Conversion of NIST user libraries to Wiley format

-Easy creation of libraries using links to MS interpreter and PubChem

MS Training Videos:
MS Training Video Handouts:


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Always amazed by the power of the NIST hybrid search. See short 10 minute trailer and additional information.


Associated Information

Explanation of EI fragmentation mechanisms for PCP-Related Species
Slides Used for Video
Supporting NMR Data

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Posted by: tvasailor | August 17, 2022

Wiley KnowItAll Software for Unknown Identifications

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Really impressive software suite and associated reference spectra that would be very valuable in unknown identifications. Software processes IR, Raman, MS, NMR, and UV data employing a large number of file formats (>170). The total number of spectra is ~2.5 Million!

~264K IR, 25K Raman, ~1.25M EI mass spec, 894K NMR, and 30K UV

See their website

Example of “Real World” Identification

See the following “Real World” example using their innovative Adaptive Mass Spec Search and IR search of mixtures in solid to identify an “unknown” white powder.

You Tube Video
Hand-Out for Video

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For a chemist, viewing Google search result as structures using “image” approach can be very useful. Much quicker than sorting through text.

As an example, searches of molecular formula can be useful for the identification of unknowns when reference EI and MSMS spectra are not available. The following information compares this approach to our other approaches using ChemSpider and SciFinder. See what you think!

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Solvent Mediated Chemical Ionization (SMCI), as demonstrated by Shimadzu in a poster session, can be a very convenient means for substituting liquids as chemical ionization reagents for GC-MS. Gases in lecture bottles are often inconvenient to purchase, somewhat hazardous to use, and often very difficult to dispose of when spent. NOTE: Ammonia gas if not used properly can be delivered to the MS source as a liquid (see link: Ammonia Gas Connection on 1st page) !

SMCI using propylamine was shown by Eastman Chemical in their recent work to be very useful in the ionization of Surfynol. This compound is very difficult to ionize with many gasses showing the loss of water and no molecular weight information. The propylamine was found to offer similar useful molecular weight information which normally required the use of either ammonia and methylamine as shown in previous work at Eastman.

Other possibilities to try would be solutions of methylamine and ammonia in methanol that can be purchased from Aldrich, methanol-d4, ammonium hydroxide, deuterium oxide, propylamine in methanol-d4, etc. Of course, one could also do negative ion chemical ionization with a variety of solvents to get M-H or chloride adducts. The possibilities are somewhat endless depending on the class of compounds and their relative proton affinities using either pure solvents or mixtures taking into account their relative proton affinities.

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Posted by: tvasailor | April 2, 2022

Helium Conservation in GCMS, GC, and DART Applications

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Currently and in several times in the past, the cost and supply of helium in GCMS and GC applications can be cause for concern. Several manufacturers have taken notice and started to supply products and technical advice to address the problem.

Conserve Helium or Switch to Hydrogen or Even Nitrogen in GCMS and GC Applications? The most straigtforward approach is to conserve helium by minimizing helium useage in GCMS applications. This avoids having to make changes to current well established analytical methods.

A more radical approach is to switch from helium to hydrogen or even nitrogen for GCMS. Switching to other carrier gasses shows promise and is discussed in a separate section below, but there are many challenges.

Thermo has a clever product, iConnectTM Helium Saver, for their Split/Splitless TraceTM GC systems. It uses helium for the injection and nitrogen for all other aspects of the injection process to conserve helium. They state that it can dramatically increase the lifetime of one cylinder of helium:

-3.5 years continuously used 24/7/365 for GC-MS
-14 years when you either shut the helium off of divert to nitrogen overnight and on weekends

Link to their brochure
Link to their website
Development Work Leading to Product
Excerpt Description of Injection Process from Thermo Manual

Agilent has created many useful documents and products. They compare the advantages of using Hydrogen for GC separations compared to helium and even discuss the use of nitrogen as a carrier gas in GC, but not recommended in GCMS. They also discuss a study that dramatically reduced helium useage in their Little Falls Site. For example, a GC-MSD (GC-MS) using their Gas Saver hardware increased the lifetime of a helium cylinder from 109 to 252 days.

Link to Little Falls Site Study

Another Agilent presentation describes in detail the use of their 7890 Helium Conservation Module which uses nitrogen during standby. In one application, it extended the use of a cylinder of helium from 2 to 12 months.

7890 Conservation Module Information

Other Agilent Resources include:

Agilent Cost Saving On-Line Calculator
On-line overview from Agilent On-Line

Chromtech Eco-Saver for All GC Systems

Chromtech Device Link

Markes Mulitigas Thermal Desorber for GCMS

Markes Multigas Desorber

Crawford Scientific, “A Practical Guide”

Link to Guide

*****SWITCH from Helium to Hydrogen or Even Nitrogen for GCMS? *****

Manufacturers discuss the pros and cons of using hydrogen or even nitrogen as a carrier gas instead of helium for GC-MS analyses. Most say limitations, but Agilent, Leco, Bruker, Shimadzu, and Anatune say not so much a problem? By far, Agilent has done the most work in this area.

Topics discussed include chemical noise, detection limits, changes in analyte EI spectra (reduction), tailing, in-situ source cleaning, self-CI, etc. when hydrogen or nitrogen are substituted for helium and how to minimize or avoid such limitations.


Nitrogen was shown successfully used as a carrier in a Shimadzu thermal desorption GC-MS method. In addition, other information found including ideas (e.g. 200 eV vs. 70 eV) for inceasing sensitivity, thoughts on problems in method development, and work in progess on EPA method development. A good article describes maintaining speed in GC analyses using nitrogen, but does not discuss applications in GCMS as a carrier gas.

Leco with hydrogen notes an increase in S/N in all components in a complex tea leave extract as shown in the link to their study above. This is impressive noting a significant decrease in analysis time by a factor of 5 while maintaining good retention indices compared to helium and good EI library search quality. Also, no discussion of any significant delay times in source “acclimation.” Possibly due to their somewhat unique “open-style” EI source?

Bruker with hydrogen says their SCION GC-MS with the Helium Free Analyzer option offers good pumping and injector design. They indicate that all 76 components in EPA Method 8270 including nitro and nitroso compounds gave high quality searches against the NIST library using AMDIS deconvolution. Others noted that nitro compounds can yield spectra showing the in-source reduction of nitro compounds.

Agilent Jet-Clean technology and New Hydroinert Source: Their source actually uses hydrogen in instruments that use helium as a makeup when the instrument is at idle to remove build-up of “dirt” in the source. Thus, using hydrogen as the carrier might also offer similar advantages by minimizing source cleanings. Also, a newly “Hydrointert” source introduced at ASMS 2022 minimizes undesirable chemical reactions

Agilent Jet-Clean info for “in-situ source” cleanings
New Hydroinert Ion Source
Example Applicatons of Hydroinert Source

*****SWITCH from Helium to Nitrogen for DART Applications*****

Direct Analysis in Real Time (DART) mass spectrometry commonly uses helium as the DART gas. With the looming helium shortage, other gases are being evaluated for DART. Nitrogen is inexpensive and readily available, making it a desirable alternative. However, NO+ reagent ions present in positive-ion nitrogen DART result in extensive oxidation for many compounds. Using a narrower aperture on their ceramic insulator cap significantly reduces the problem.

DART ASMS Journal Link



Agilent Webinar (Jim McCurry)
Another Agilent (Shannon Coleman discusses part of Little Falls Study shown above)
Yet Another Agilent (Bryan White)
Axion (switching GC from He to H2)
C&EN Podcast World Supply Outlook

MRI and NMR useage can also be decreased by new technology. Many years ago our Cryrolect GC-IR had a similar approach.

Limiting Amount Needed in Seimins MRI

Party and Weather Balloons account for about 7% of the useage of helium per year. I saw in several places on the internet, but surprised it was such a realtively high number.

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Sold the whole package to an individual at Lake Waccamaw on 10/15/2021. They have a fleet of Holders.

I have decided to sell my two Holder 20’s and all my associated equipment. I once had 4 of these boats and enjoyed them all. Due to travel plans next year, I have decided to start crewing on a J24 when in town, and thus, no longer need the boats.

However, really want to find them a good home where they will be used in one-design racing. I would rather not sell to someone who would paint bottoms and leave them in the water.

The long cockpit Holder 20, Godspeed, is my favorite Holder of the four that I have owned. See the following specification sheet and a link to some pictures. Feel free to make an offer. I need to finish the sailing season on November 13, 2021 before I let this boat go to its new home. After that date, I will pull the boat off the air-lift and store on a trailer in Elizabethon, TN. Price $4,750.

Spec Sheet and Price

Pictures of Boat and Trailer for Godspeed

The short cockpit Holder is a solid boat but just does not have the higher quality hardware added to Godspeed. The fiberglass cleans up nicely and has little or “spidering.” Trailer has new tires within the last 2 years. Price $2,500

-in my opinion, the best type of masts found on Holder 20’s, the black one with a “hinged” base, see pictures
-new windvane “in the box” (remind me to give to you)
-SLO mylar racing main very good shape
-SLO mylar 135 genoa very good shape
-SLO spinnaker very good shape
-grey SLO tent for cockpit
-tapered spinnaker pole requires no harness

Pictures of Boat and Trailer

Other Miscellaneous Items for Sale

-Sailbot Race Horn works with Apple Products Bluetooth (like new $300)
-Holder 20 Waters racing jib very good shape $380
-Holder 20 Waters 135 racing genoa very good shape ($430)
-Holder 20Waters racing main very good shape ($480)
-SLO racing 155 genoa good shape ($430)
-Nissan 4 cycle outboard 2.5 HP, no reverse, just forward and neutral, turn motor to reverse, like new, hardly used long shaft ($450)
-various winch parts/winches with two new plastic tops ordered from Australia (inquire)
-lots of stainless steel hardware including shackles, blocks, cam cleats, screws, line hangers, etc. (inquire to see if you come to buy a boat)
-stanchions, backstay (stainless cable), lifelines (inquire)
-Holder 20 main good practice sail ($100)
-Holder 20 spinnaker good practive sail ($75)
-Holder 20 Bow pulpit, good shape from a Holder 20 converted to Holder 20x ($250)
-Holder 20 boom with reefing hardware, pink, good paint job would help ($75.00)
-7 ft battens, package of 3 ($25.00)
-Holder spinnaker tapered pole, little longer than legal, could be shortened ($75.00)

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Identification of Unknowns by GC-MS and LC-MS Using NIST Search with Commercial and User Libraries

Copy of Talk in PDF

Link to YouTube Video


For the last 20 years, Eastman has routinely employed the NIST search for the identification of unknowns.  Initially, we  primarily utilized EI searches, but in recent years, the increase in the availability and quality of tandem libraries plus associated software has greatly enhanced LC-MS library search results.  More than 50 instruments and associated users are networked at Eastman world-wide.  Our corporate library of >55K entries in NIST format is automatically updated and distributed nightly.

The 2020 edition of the NIST library utilized with the NIST MS 2.4 search software offers many major enhancements.  NIST for the last six years has had an ambitious program to acquire and analyze targeted chemicals (>50,000).  In addition, the novel hybrid search expands the scope of all EI and tandem libraries.  These new improvements and free training resources,  including videos and associated handouts, will be described.  EI/tandem libraries utilized include NIST, Wiley, MoNA, and user-created ones.

ACS 2021 Symposium: Compound Identification with LC/MS and GC/MS: Experimental Method, Data Analysis and Applications

Liquid chromatography and gas chromatography coupled with mass spectrometry (LC/MS, GC/MS) are two major analytical techniques for identifying compounds in complex mixtures. This symposium provides a forum for experts from industry, academia and government agencies to share their novel experimental methods, creative data analysis techniques including mass spectral library searching and software development, and the applications of these techniques, with special emphasis on the use of LC/MS/MS in metabolomics, proteomics, lipidomics, forensics, pharmaceutical and environmental studies.

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