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FAQs

Frequently asked questions on Mass Spectrometry

Frequently asked questions specifically concerning techniques offered by the Facility may be found by clicking the drop-down tabs below.

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Gas chromatography- mass spectrometry (GC/MS)

Which compounds are suitable for the GC/MS analysis?

 

gcmsThe analysis of mixtures of volatile and low relative molecular mass compounds (m/z< 800) such as hydrocarbons, fragrances, essential oils and relatively non-polar drugs. Chemical derivatisation, eg trimethylsilylation, can often be employed to increase the volatility of compounds containing polar functional groups (-OH, -COOH, -NH2, etc) thereby extending the range of suitable analytes to such compounds as steroids, polar drugs, prostaglandins, bile acids, organic acids, amino acids and small peptides.

What information does GC/MS provide?

The instruments in the MS Facility can operate in two different modes, full scan and selective ion monitoring. (SIM)

The scanning mode provides a fairly reproducible mass spectral fragmentation pattern ("fingerprint"). Mass spectra are recorded (scanned) at regular intervals (typically 0.5 - 1 per second) during the GC separation and stored in the instrument data system for subsequent qualitative or quantitative evaluation. From such patterns, it is often possible to deduce structural features (mass spectral interpretation) but this requires experience and can be very time-consuming, particularly as a complex mixture might contain hundreds of components. Such "fingerprints" can also be compared with those stored in a standard database (mass spectral library) and several important databases are currently available in the Facility to assist with problem solving. Although library searching is a very useful and timesaving technique, it is important to remember that such searches do not identify compounds - analysts do!

Quantitative work can be performed by integration of selected ion chromatographic peaks. SIM is much more sensitive technique for trace quantitative analysis. Here, instead of scanning a whole spectrum, only a few ions are detected during the GC separation. This can result in as much as a 500-fold increase in sensitivity, at the expense of specificity. Stable isotope-labelled internal standards can be employed.

What sensitivity may be achievable

Depending on the analyte, low picogram to nanogram amounts can be measured using this powerful technique.

How many samples can be run in a day?

The run time per sample is typically 15-60 min depending on the nature of the sample and the GC method used. The column temperature can be kept constant (isothermal) or may be programmed ("ramped") to increase at a predetermined rate, usually linear. In a programmed method, about 5 min has to be allowed for cooling between injections. The autosampler has capacity of 100 samples. When calculating sample numbers, calibration samples, quality control samples, blanks and wash samples have to be included in some cases.

What is sample preparation?

Samples such as water, soil, urine, blood plasma, etc., have to be subjected to a "clean-up" procedure prior to analysis in order to extract and concentrate the more volatile, low molecular mass components. Extraction can be performed by organic solvents or by solid phase extraction (SPE).

Where considered necessary, the extract can be derivatised with a choice of special reagents. For example, thermally labile and polar carboxylic acids groups can be methylated with BF3/Methanol or Trimethylsilated with a variety of commercially available compounds.

How much sample will I need?

GCMS – the autosampler is capable of injecting 0.1-10 μL, so at least 10 μL of sample will be required. A typical injection volume is 1 μL, minimum of 20-50 μL of sample is required. Generally, no more than a microgram of dissolved material should be injected, depending on the capacity of the particular column. Overloading will cause the separation to deteriorate and can lead to serious contamination of the instrument. Concentrated samples can be diluted prior to injection or subjected to a “split” injection where only a specified proportion of the sample enters the column.

Liquid chromatography- mass spectrometry (LC/MS)

Which compounds are suitable for the LC/MS analysis?

lcms01Small Molecular Weight <1kD
drugs, food components, endogenous compounds, vitamins, pesticides, toxins, conjugates (glucuronides, SO4) of compounds with m/z > 50
Middle Molecular weight 1 –10 kDa
synthetic polypeptides and polysaccharides
Large Molecular weight > 10 kDa
polypeptides, proteins, oligonucleotides, polysaccharides 

What is electrospray (ESI) and atmospheric pressure chemical ionisation (APCI)?

In ESI, the sample solution is sprayed into a fine mist of charged droplets containing sample ions by application of a large negative or positive voltage (typically ±4.5 to ±5 kV). A flow of nitrogen drying gas is directed at droplets and individual positive or negative ions are produced. ESI accommodates a liquid flow of 1 μL/min to 1 mL/min, 0.2 μL/min being optimal. This ionisation technique is very suitable for the analysis of polar, thermally labile molecules such as drugs, DNA, RNA, sugars, peptides, and proteins.APCI vaporizes the sample solution at temperature up to 600 °C. Application of a high potential (typically ±3 to ±5 kV) produces a reagent ion plasma, mainly from the solvent vapour. The sample vapour is ionised by ion-molecule reactions with the reagent ions in the plasma. APCI accommodates liquid flows of 100 μL/min to 2 mL/min, 1 mL/min being optimal. This type of ionisation technique is recommended for analysis of less polar, thermally stable molecules such as steroids.  

What type of the LCMS system should I use for quantitative/qualitative work?

Triple quadrupole (TSQ Access, Quattro LC) and ion trap (LCQ DECA XP) instruments available in the MS Facility can be used for both quantitave and qualitative work. However, the ion trap instrument is much more sensitive in full scan mode and has the unique ability to perform MS experiments which may provide additional structural information. Thus, the ion trap is usually preferable for qualitative analysis. The triple quadrupole instruments can analyse samples with great precision and sensitivity in the selective-ion monitoring (SIM) or selective-reaction monitoring (SRM) mode of operation, and therefore are better suited for quantitative work.  

What is the achievable sensitivity?

Detection limits of the LCQ DECA XP are at the low-femtomole levels for full-scan mass spectra and in the low-femtomole to high-attomole levels for the more sensitive but less informative SIM/SRM modes. The triple quadrupole instruments available in the MSF can achieve low pg/ml detection from the complex matrixes. The sensitivity depends greatly on the nature of the compound(s) analysed, sample purity, matrix and the addition of ionisation boosting agents such as formic acid or ammonium acetate.  

Can I skip LC part?

The syringe pump can be used to infuse samples directly into the mass spectrometer or to infuse samples into LC flow by means of a tee piece. The syringe pump is a device that delivers a solution (up to 500 μL) at a specified rate (0.05 to 100 μL/min). This can be very useful in the case of a very clean, concentrated samples. The average analysis time is about 5 minutes for a simple sample, for example a molecular mass determination. An advantage of direct infusion is fast analysis, a disadvantage is the lack of chromatographic separation.  

What should I remember during LC method development?

ESI- optimal flow rate 0.2 mL/min; LC column: internal diameter 2-2.1 mm. APCI- optimal flow rate 1 mL/min; LC column: internal diameter 4.6 mm. Preferable mobile phase solvents: water, acetonitrile, methanol. APCI can also work with a normal phase mobile phase. The mobile phase generally should contain an ionisation boosting agent, such as formic acid, acetic acid, , ammonium acetate, ammonium hydroxide. The concentration of the agent should not be greater than 0.5% or 10mM. Non-volatile salts and buffer, such as phosphate buffer, are totally prohibited .  

How many samples can be run in a day?

Direct infusion - a typical analysis would take about 5 minutes per sample. Sometimes it may take longer to wash the system between samples. LCMS – run time per sample depends totally on the LC method adding about 1.5 minute for the injection cycle. The fastest isocratic run can be only few minutes, the gradient one anything between 10 and 60 minutes. The autosamplers have capacity of 100-160 samples. While calculating sample numbers calibration samples, quality controls samples, blanks and wash samples have to be included

How important is a good sample preparation?

A good sample clean-up, especially from biological samples, is very important. It will greatly affect a sensitivity and robustness of the method. It will prolong a life time of the LC column and time between an ion source cleaning.

How much sample will I need?

Direct infusion - a minimum 100 μL of diluted sample. LCMS – the autosamplers are capable of injectecting 1 μL, so at least 10 μL of sample will be required. A typical injection volume is 5 – 10 μL, minimum of 20-50 μL of sample is required.  

Matrix assisted laser desorption ionization

What is MALDI?

maldiThe acronym MALDI stands for matrix assisted laser desorption ionization and was first used by Franz Hillenkamp & Michael Karas in 1985. They used the technique to ionize the amino acid alanine. MALDI mass spectrometers first became commercially available in the early 1990’s. The technique uses a pulsed laser to ionise a matrix, energy from the matrix is passed to the sample and causes the sample to ionise. 

What is a Matrix?

A matrix consists of crystallised molecules. The most common of which are 3,5-dimethoxy-4-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid, and 2,5-dihydroxybenzoic acid.

They are of a fairly low molecular weight (to allow facile vaporization), but are large enough (with a low enough vapour pressure) not to evaporate during sample preparation or while standing in the spectrometer.  

They are acidic, therefore act as a proton source to encourage ionization of the analyte.  

They have a strong optical absorption in the UV, so that they rapidly and efficiently absorb the laser irradiation.  

They are functionalized with polar groups, allowing their use in aqueous solutions.

How is the Matrix prepared?

The matrix is normally prepared by making a saturate solution of the solid in highly purified water and an organic solvent (normally acetonitrile or ethanol). Trifluoroacetic acid may also be added to encourage ionisation. For best results the matrix should be freshly prepared before use.

How is the sample added to the matrix?

The matrix is normally spotted onto the MAULDI target in solution and the solvent allowed to evaporate to produce a crystalline matrix. Sample may be added to the matrix solution before spotting onto the target, added to the spot of matrix solution on the target in a suitable solvent or added to matrix after it has crystallised on the target in a suitable solvent.

What types of compounds may be analysed by MALDI?

Peptides, nucleotides, oligonucleotides, oligosaccharides, proteins, and lipids, have all been reported as having been analysed by MALDI/MS. The use of the correct matrix for analysis of the samlple is important. A guide as to which matrix to use is given below.

In proteomics, MALDI may be used for the identification of proteins isolated through gel electrophoresis: SDS-PAGE and two-dimensional gel electrophoresis. One method used is to identify proteins by MALDI/MS is by identification from fingerprints; our system is linked to external databases to aid protein identification.

Some synthetic macromolecules, such as catenanes and rotaxanes, dendrimers and hyperbranched polymers, which have molecular weights extending into the thousands or tens of thousands, are amenable to analysis by MALDI mass spectrometry, where most other ionization techniques have difficulty producing molecular ions .

A guide to choice of matrices

2,5-dihydroxy benzoic acid

Common Names :- HB, 2,5-DHB, Gentisis Acid

Solvents :-             water,aetonitrile, methanol, acetone, chloroform

Applications:-         peptides, necleotides, oligononucleotides, oligosaccharidrs

3,5-dimethoxy-4-hydroxycinnamic acid

Common Names :- sinapic acid, snapinic, SA

Solvents :-  acetonitrile, water, acetone, chloroform

Applications:-  peptides, proteins, lipids

4-hydroxy-3-methoxycinnamic acid

Common Name :-   ferulic acid

Solvents :-  acetonitrile, water, propanol

Applications:-         proteins

α-cyano-4-hydroxycinnamic acid

Common Name :-  CHCA

Solvents :-  acetonitrile, water, ethanol, acetone

Applications:-        peptides, lipids, neucleotides

3-hydroxy picolinic acid

Common Name :-  HPA

Solvents :-  ethanol

Applications:  oligononucleotides 

Only matrices usable at the 337nm wave length of the Nitrogen Laser used in the AutoFlex Mass Spectrometer have been Included.

What type of data is obtained from the MALDI Spectrometer?

The data obtained from the MALDI experiment conducted on the AutoFlex is usually nominal mass data and is used for qualitative analysis of the sample. The accuracy of the masses measured will depend on the accuracy of the instrument calibration.

As far as possible samples for MALDI mass spectrometry should not contain inorganic salts. Inorganic salts may be part of protein extracts, for best results it is advisable to remove any such salts by solid phase extraction or washing the final target spots with water. Both methods can also be used to remove salts and other impurities from other types of sample. Samples to be run by MALDI should as far as possible not be prepared in glass apparatus so that they do not pick up alkali and alkaline earth metal ions from the glass which would interfere with MALDI ionisation.

How much sample will I need?

This question is hard to answer. It will depend on the type of sample and matrix being used, the purity of the sample, and the type of MALDI target used. In general an Anchor Chip Target usually produces the best sensitivity for proteins, but unfortunately only one of these is available in the Facility. In general the sample size should be at least 10 fmols.

What is the maximum resolution of the instrument?

With good sample preparation and careful operation of the instrument it is possible to obtain a resolution of about 10,000. The mass range of the instrument extend to about 60,000 Daltons if the reflectron is not used. Working at this level it is not possible to get sufficient resolution to separate the isotope peaks of an ion and it will appear as a hump covering a small range of masses.

How long will it take to run a sample?

MALDI spectra are normally built up by summing the results from a series of scans of the laser. The AutoFlex instrument has the facility to produce a spectrum by summing the results of two or more series of laser scans. The more series of scans that are summed the longer the running of the spectrum is likely to take. If the sample has been well prepared and the analyte is present in reasonable concentration it should be possible to run a MALDI spectrum in ten to fifteen minutes. Time must also be allowed for running of any calibration samples and blanks that may be required.

ICP/MS

I thought ICP-MS suffered too many interferences for most biological work?

 ICP-MS1160-120[1]Historically this was true, restricting ICP-MS usage to heavy metals (Cd, Pb etc) in biological samples, for which interferences are negligible, or very high resolution (and expensive) MS was required. However, the advent of the direct reaction cell (DRC; see below) means that interference-free ICP-MS can now be developed for most sample types and elements.

What elements may be analysed?

All of the naturally occurring elements, except for hydrogen, carbon, nitrogen, oxygen, fluorine, chlorine, helium, neon & argon may be analysed. In general, the most intense isotope will be monitored.

What is the system’s sensitivity?

This depends on the element being considered, the isotope monitored and the matrix in which it is presented. Perkin Elmer quotes detection limits as below:

S 10ppb

B, Si, P, Br, I 1ppb

K, Ca, Se 100ppt

Other metals 10ppt or less  

Typically precision and accuracy are within a few % although this figure increases as you approach the limits of detection for an element (see above). For isotope ratio analysis (see below), this figure falls to nearer 0.1 %.

How many samples can be run in a day?

This depends primarily on the number of elements to be measured, the techniques used and to a lesser extent on their concentration and the nature of the matrix. A typical analysis for 10 elements would take about 6 minutes per sample. The autosampler has room for 149 samples and the instrument can, if needed, be run overnight. There will also be a number of calibrant standards appropriate to your experiment to be run.

What standards and blanks do I need?

Blanks are very important. Ultrapure water should be treated in EXACTLY the same way as the sample and then analysed as an 'analytical blank'. Reagents used for digestion, dilution etc should also be checked for contamination. You may provide your own standards, in which case sample-based (i.e. spiked) standards are best. However, standards will also be prepared by the Centre. You may wish to provide a sample-like certified standard or reference material to be analysed along side your samples

How should the sample be prepared?

Organic solvents should be avoided as they damage the pump tubing. The most usual sample preparation involves an acid digestion, normally with nitric acid and subsequent dilution with water. It is essential that all containers are scrupulously clean and it is good practice to avoid glass vessels where possible. 18.2 M& water should always be used and other reagents should be of an ultra pure grade (e.g. Fluka ‘Trace Select). The sample is normally supplied to the MSF in a 15ml screw top test tube, preferably made of uncoloured plastic (e.g. Elkay 2086-500).

How much sample will I need?

The flow into the instrument is normally 0.4 ml/min, although this can be raised to 1 ml/min (max) if needed. At 0.4ml/min, the typical analysis above would consume 2.4 ml.

Are there any ‘problem’ elements?

Isobaric interferences are dealt with by the instrument’s software, which also warns of potential polyatomic interferences. Sometimes changing the isotope monitored may help (e.g. one normally monitors 44Ca despite its low abundance, as 40Ca is obscured by the massive 40Ar signal from the plasma). Those elements which are found in normal laboratory background (e.g. sodium, potassium, silicon & aluminium) are very difficult to quantitate, especially at low levels. The instrument incorporates a ‘Dynamic Reaction Cell’ which may sometimes be used to separate elements from artefacts (e.g. 56Fe from 40Ar16O) by reaction with ammonia.

Can I measure radioactive species?

Isotope and isotope ratio analysis is common by ICP/MS. but, the MSF is not equipped to handle radioisotopes.

Is HPLC/ICP/MS possible?

Yes, there is a HPLC system associated with the ICP instrument. Solvents should be aqueous; buffers are acceptable, but should be chosen so as not to interfere with the elements of interest and the maximum flow rate is 1ml/min. It is advisable to contact the MSF at an early stage in your method development to ensure compatibility with the instrument. Both samples and standards should be in normal 2 ml vials with septum caps.

Can any technician/student use the instrument?

Generally not. These are expensive and delicate instruments. However, where an individual will have a heavy analytical load and may wish to learn the technique as part of their training, then this can be negotiated with the Centre. However, it will not significantly alter the costs.

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