King's College London - Homepage

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.

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.  

What is MALDI?

The 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 types of compounds may be analysed by MALDI?

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.