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The CHROMacademy Essential Guide to Developing Fast Capillary GC Separations / Developing high throughput separations in capillary GC

The Essential Guide from LCGC’s CHROMacademy presents Developing Fast Capillary GC Separations. 
Dr. John Hinshaw (Senior Staff Engineer at Serveron Corp, CHROMacademy GC Dean) and Tony Taylor (Technical Director, Crawford Scientific), present a concise guide on increasing sample throughput for capillary GC for both existing standard equipment as well as the latest bespoke equipment and approaches for ‘Fast GC’.

Tony Taylor
Technical Director
Crawford Scientific
John Hinshaw
GC Department Dean
CHROMacademy
 
 

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The CHROMacademy Essential Guide to Developing Fast Capillary GC Separations

Going to faster speeds of analysis with GC doesn't always require a new or upgraded instrument, but not all existing instruments are suitable. It all depends upon how fast a separation is required. A modest increase of two to four times shorter retentions using conventional capillary columns with inner diameters of 150 - 200 µm can be achieved quite reasonably on a wide range of conventional laboratory instruments.
Conversely very high analysis speeds can achieve separations that previously needed 10 min or longer in less than 1 min, but such a feat requires significant equipment upgrades or complete replacements. Pushing peaks from a column at high speeds places demands upon autosamplers, inlets, ovens, detectors, and data-handling systems that existing equipment might not be able to handle adequately.

This Essential Guide Tutorial will cover the most important practical issues to consider when increasing analysis speed using conventional equipment as well as taking a look at some of the newer equipment and methodologies required for ultra-high speed analysis.

Developing high throughput or ‘fast’ capillary GC methods involves the consideration of several key aspects relating to the hardware used and the conditions under which the separation is achieved.

Going to faster speeds of analysis with GC doesn't always require a new or upgraded instrument, but not all existing instruments are suitable. It all depends upon how fast a separation is required. A modest increase of two to four times shorter retention using conventional capillary columns with inner diameters of 150 - 200 μm can be achieved quite reasonably on a wide range of conventional laboratory instruments. Figure 1 shows a typical example of how this might be achieved 1

«« Figure 1: Halving analysis time of a spearmint oil sample using simple means Top chromatogram: Polydimethylsiloxane phase 30 m x 0.25 mm x 0.25 μm,
He carrier (25 cm/s) Bottom chromatogram: Polydimethylsiloxane phase 20 m x 0.18 mm x 0.18 μm, H2 carrier (47 cm/s) (from reference 1)

Agilent Technologies
(Santa Clara, California, USA)
.

 
 

Conversely, very high analysis speeds can achieve separations that previously needed 10 min or longer in less than 1 min, but such a feat requires significant equipment upgrades or complete replacements. Pushing peaks from a column at high speeds places demands upon autosamplers, inlets, ovens, detectors, and data-handling systems that existing equipment might not be able to handle adequately.
Newer GC systems incorporate high-speed autosampler injection modes, appropriate inlet designs, and fast data acquisition speeds that encompass the requirements for high-speed operation up to a point. However, specialized GC pneumatics, vacuum systems or dedicated rapid column heating and cooling accessories are required to go even further (see Figure 2).

«« Figure 2: Reconstructed chromatogram of a hydrocarbon mixture of 6 alkanes (recorded at 400 spectra/s).
Column: L = 2 m; 50 μm i.d.; df = 0.1 μm OV-1; linear velocity = 180 cm/s. Inlet pressure = 500 kPa (helium); splitflow = 400 mL/min. Isothermally at
80 °C.
Detector: time-of-flight mass spectrometer (LECO, Michigan, USA); scanned mass range: m/z 30-400; T ion source: 200 ºC.
Compounds: 1. 2,3- dimethylbutane, 2. hexane, 3. heptane, 4. methylcyclohexane, 5. 2,3,4-trimethylpentane, 6. octane. (from reference 2)

Marieke van Deursen, Eindhoven -Technische Universiteit ,Eindhoven, 2002. ISBN 90-386-2873-0.

 
 

Making the decision to go to higher speeds is just the start of what can be an extended method development and validation exercise. A high-speed capable instrument is a platform upon which to deploy a suitable column and method. It might be able to inject and record very narrow peaks while ramping up the column temperature at impressive rates, but without the necessary separation method, it will not deliver the desired results.

We also need to consider other rate limiting factors such as sample preparation or data analysis time which may ultimately be the rate limiting step(s) in any analysis.

This Essential Guide Tutorial will cover the most important practical issues when increasing analysis speed.
Table 1 shows John Hinshaws’ summary of the four proposed ‘speeds’ within capillary GC.

Table 1: Four proposed ‘speeds’ of Capillary GC (reproduced with permission).

 
 

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A useful equation that describes the contributing factors to GC retention time and the ways in which the analysis speed might be increased is shown in Equation 1.

Equation 1: Factors influencing retention in gas chromatography (from reference 3)

 

tR = retention time (as an indicator of analysis speed)
L = column length (m)
ū = carrier linear velocity (cm/sec)
k = retention factor

K. Mastovska and S. J. Lehotay, Journal of Chromatography A, 1000 (2003) 153–180.

Practical ways to reduce retention (analysis) time can be derived from Equation 1 and can be summarized as:

Reduce L (column length) - which reduces the number of theoretical plates (efficiency, N) in direct proportion but affects resolution less (L is proportional to √Rs) and so most approaches to speeding up GC analysis use shorter GC columns (≤ 15m).

Increase ū (carrier linear velocity) – this can be achieved by running at higher carrier gas flow-rate (for a fixed column i.d.), increasing the diffusivity of the carrier by changing to hydrogen gas, using low pressure (vacuum) GC techniques or decreasing the internal diameter of the GC column.

Decrease k (retention factor) – this can be achieved in a number of ways including increasing the oven temperature or rate of temperature increase during gradient temperature programming; increase column internal diameter and decrease film thickness; and changing to an alternative stationary phase.

 
 

The use of hydrogen as carrier gas to increase the speed of analysis is well documented.4-7 For sheer column performance, hydrogen carrier gas offers some strong advantages over helium or nitrogen. Hydrogen yields higher plate numbers at rapid linear velocities and achieves higher velocity at lower pressures. This is illustrated by considering at a simple van Deemter curve showing the plate height against linear velocity for the common carrier gases used for capillary GC.

«« Figure 3: Typical van Deemter curves for common GC carrier gases.

CHROMacademy: GC Channel / Theory and Instrumentation of GC / Gas Supply and Pressure Control.

 
 
Due to the diffusivity of hydrogen, the optimum plate height (minimum value of H on the van Deemter curve) occurs at higher linear velocity. In other words, the maximum efficiency [(highest plate number (N)], occurs at around 20-40 cm/s for helium and around 30-60 cm/s for hydrogen, thus generating efficient GC peaks at higher linear velocity (or column flow). Further, hydrogen shows a relatively flat van Deemter minimum, thus allowing the operator to achieve near-optimal flow at even higher carrier gas linear velocities. The optimum practical gas velocity is a term which describes operation at linear velocities within, around, 25% of the actual minimum plate height value. This operating region is clearly shown in Figure 4.

«« Figure 4: The optimum practical gas velocity working regions of various GC carrier gases. Note that the hydrogen OPGV (solid lines, within 25% of the plate height minimum) can range up to 100 cm/s depending upon application type and column dimension. (from reference 8).

J.V. Hinshaw, Column Connections, LCGC Asia Pacific, 12(2), 1100 (2009).

 
 

So how does one go about finding the optimum setting for improving our GC separations or translating from the use of helium (for example) to hydrogen? The answer is reasonably straightforward, one should attempt to increase the carrier linear velocity until either a) the required separation speed is achieved or b) the efficiency of the peaks reduces to a point where the resolution of analytes within the chromatogram reduces below acceptable levels.

Let’s look at a specific example in which we are translating a method to run with hydrogen carrier. As many readers will know, hydrogen is a bit less than half as viscous as helium or nitrogen at the same temperature, as shown in Figure 5, and so for this reason hydrogen requires a lower pressure drop to achieve the same average carrier gas velocity as for helium or nitrogen.

Column: 50 m x 250 μm
Temperature: 100 oC
Head pressure: 58.6 psig (404 kPa)
Linear Velocity : Helium 60 cm/s / Hydrogen 123 cm/s

The relationship between linear velocity and pressure drop is somewhat nonlinear because of effects stemming from the compressibility of the carrier gas, but in general, the effect of switching from helium to hydrogen on retention time will be to cut retention times roughly in half if the inlet pressure is unchanged.

«« Figure 5: Relative viscosity of common GC carrier gases at various temperatures. (from reference 8).

J.V. Hinshaw, Column Connections, LCGC Europe, Jan 1 (2011).

 
 

We should add the caveat that if the velocity is too high, solutes don’t spend enough time in the column for an efficient separation; if the velocity is too low, solutes broaden excessively by diffusion through the mobile phase. Further, large changes in carrier-gas velocity during temperature programmed analysis can affect relative peak spacing (selectivity) and retention order may swap. If your instrument allows; chose to have the carrier programmed in ‘constant linear velocity mode’, which will avoid selectivity change issues.

The relationships between column length, internal diameter and carrier flow, pressure and linear velocity can be confusing. The mathematical relationships between these parameters are relatively complex but well understood. Most modern GC systems allow the end-user to enter the dimensions of the column and then simply select the required flow, pressure or linear velocity to achieve the desired setting at a particular oven temperature and with a particular carrier gas. It should be noted that flow and linear velocity are ‘calculated’ from the applied pressure and the column dimensions and as such it is important that the column dimensions are accurately entered into the system if the absolute set-points are to be accurate. Figure 6 shows the relative pressures required to achieve a linear velocity of 40 cm/s with various carrier gases, column length and internal diameter.

«« Figure 6: Plots of inlet pressure against column length to achieve an average linear velocity of 40 cm/s
using hydrogen carrier gas at 50 oC
for column internal diameter (mm) of


a) 0.20

b) 0.25

c) 0.32

d) 0.53

(from reference 9).

J.V. Hinshaw, Column Connections, LCGC Europe, Jan 1 (2011).

 
 

For ease of method translation one might use a method translation software tool such as Method Translation Software from Agilent Technologies (Santa Clara, California, USA) which can be found here, or a similar tool. This tool allows the translation of not only the carrier gas settings but can also recommended a temperature programme to preserve elution order/resolution.

A further, very practical, approach can be used to bring about a moderate increase in separation speed, namely operation of the carrier in ‘constant flow’ mode. If the instrument is operated at a constant head pressure, as the temperature increases, then the column flow decreases due to an increase in the viscosity of the carrier (Figure 5). By taking advantage of computerized inlet pneumatics (sometimes called Electronic Pneumatic Control or similar), then as the temperature increases the instrument increases the carrier pressure to maintain constant column flow (increasing linear velocity), which results in earlier elution of the more highly retained sample components (Figure 6). One should note again that this may cause analytes to elute at a lower temperature than in constant pressure mode – which may again require some adaptation of the temperature programme to ensure the required selectivity between analytes, especially the more retained components (Figure 7).

«« Figure 7: Theoretical effects of column temperature on helium carrier flow and linear velocity in constant pressure (top) and constant flow (bottom) modes. Column: 30 m x 0.53 mm, column flow 6.0 mL/min, column outlet pressure 1 atm
(from reference 11).

J.V. Hinshaw, Column Connections, LCGC Europe,
Apr 1 (2008).

 
 
 

Figure 8: Series of chromatographic separations at differing temperature programming rates in constant pressure (P = 4.1 psig) or flow (F = 6.0mL/min.) mode Initial temp 50oC for 2 mins then programmed to 250oC at

a) 3 oC/min.
b) 6 oC/min.
c) 12 oC/min.
d) 24 oC/min

1.0 ml injections at 200:1 split at 200 oC
1 = nC10
2 = nC11
3 = nC12
4 = nC13
5 = 2-octanone
6 = 1-octanol
7 = 2,6-dimethylaniline
8 = 2,4-dimethylphenol

(from reference 11). J.V. Hinshaw, Column Connections, LCGC Europe, Apr 1 (2008).

 

 

 
 

«« Figure 9: Comparison of analysis of commercial paint remover formulation using hydrogen and helium carrier gas maintaining constant resolution Column: 10 m x 0.10 μm HP-Wax, Injection: 0.1 μL, Split ratio: 1000:1 (from reference 12).

Agilent Technologies
(Santa Clara, California, USA).

Figure 9 demonstrates the potential increase in speed of analysis with a simple translation to hydrogen carrier. In this case there was enough resolution between the peak pairs for a simple conversion to the ‘faster’ carrier without changing the temperature programme. For more complex separations this will not be so straightforward and the use of the method translation software mentioned above will be of great utility.
 
 

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Hydrogen is spontaneously explosive when its concentration in air exceeds 4% (at 630oC or above!!). Proper safety precautions should therefore be used in order to prevent explosion within the column oven. The concentration of hydrogen is very unlikely to exceed 4% even in the smallest of laboratories due to the large volume of air against the rate at which hydrogen is produced by a typical generator. Most generator manufacturers will employ several safety features within their instrumentation including:

  • Low volumetric output detection
  • Low reservoir volumes (most <100 mL).
  • Back pressure monitoring (to assess leaks)
  • Internal pressure monitoring to assess production run-away or over production of gas

Most gas chromatographs are manufactured with spring-loaded doors and perforated or corrugated metal column ovens. These help to either dissipate released gas or minimise the explosive force if an explosion were to occur.

Most GC instruments are also equipped with electronic backpressure regulators of pneumatic flow controllers, which monitor carrier and detector gas pressures. If these devices sense a drop in pressure the will automatically shut down the instrument for safety reasons. The only instance in which this check might fail would be a column break at or near the detector, hence maintaining back pressure at the inlet. To guard against this column installation should be carefully and properly carried out and the column should never rest against the internal oven walls as this may reduce its mechanical strength – leading to possible breakage.

Leak testing may also be carried out using an on-board gas detector or portable device.

This built-in hydrogen detector from SIM GmbH (Mulheim, Germany) – includes a probe in the GC-oven with optical and auditory alarms.
If hydrogen is sensed the carrier is switched to an inert source to prevent column damage.

«« Figure 10: Example of GC instrument with a built in hydrogen detector, alarm and fail safe switch over to an inert gas source.

CHROMacademy: GC Channel / Theory and Instrumentation of GC / Gas Supply and Pressure Control.

 
 
Figure 11 shows a portable detector from SRI Instruments (Torrance, California, USA). The device can be connected to a multi-meter for quantitative readings and the flexible probe can be easily positioned within a GC oven or near the detector.

«« Figure 11: Hydrogen detector probe.

CHROMacademy: GC Channel / Theory and Instrumentation of GC / Gas Supply and Pressure Control.

 
 
A hydrogen generator stores only a small amount of hydrogen. The exact amount depends upon the generator capacity but is typically less than 0.20 L. Compared with the 6000–8000 L of gas that is stored in a full-size laboratory gas cylinder when new, it should be obvious that a hydrogen generator presents a considerably lower hazard in terms of the amount of stored flammable gas. Even so, this does not change the rate and total amount of hydrogen that is consumed nor does it remediate the hazards of using flammable gases, it just minimizes the total amount that is present in the laboratory at any one time.
 
 

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Making the assumption that we have selected the appropriate GC stationary phase (see later section), the remaining decisions when specifying a column are its length (in metres), internal diameter (in millimetres) and film thickness (the thickness of the stationary phase layer coated onto the inside of the capillary wall, in microns).

It’s useful to use the resolution equation (below) to visualise the effects which these three parameters have on the separation.

Equation 2: Standard resolution equation.
  Agilent Technologies
(Santa Clara, California, USA).
 
 

Obviously the goal in any chromatographic separation is to obtain the required resolution between all relevant peaks, repeatably, in the minimum timeframe. We can use the resolution equation to highlight the effects of each of the column dimensions on the resolution obtained within our chromatogram. It should be noted that for a ‘fast’ GC separation, we need to generate a high number of theoretical plates at high linear velocity and with the shortest column possible.

N (efficiency) – is a function of both column length and internal diameter. Doubling the column length will double the efficiency (plate number) and result in a 1.4x increase in resolution, which may seem good value, however this will double analyte retention times, significantly increase cost of column purchase and add to the head pressure required to obtained the required carrier gas flow-rates. As a general rule of thumb, column length is dictated by the number of components of interest within your sample, with complex samples requiring longer columns in order to generate enough theoretical plates to achieve the separation. Reducing the column internal diameter is a much better approach to increasing column efficiency and resolution, and most importantly for reducing retention time. Halving the column internal diameter will double the column efficiency, this in turn will allow the column length (and retention time) to be halved without loss of efficiency.

«« Figure 12: Various column geometries which can be used to generate a column effciency of 112,000 plates and the corresponding plates per metre measurement . The red line indicates a column internal diameter below which specialist GC equipment may be required.

Reproduced with permission of Agilent Technologies, Santa Clara, California, USA.

 
 

k (retention factor) – is a function of the stationary phase chemistry, the column internal diameter and the stationary phase film thickness. Adjusting retention to improve resolution is only really effective up to analyte retention factor values around 5, and relatively futile above retention factors of around 10 as the on-column dispersion of the analyte band leads to reduced efficiency that nullifies the advantage. Good choice of stationary phase chemistry combined with a shorter narrow bore column and an optimised temperature programme is required to achieve the correct retention factor range. Remember that retention factor can be increased whilst increasing linear velocity, by making suitable adjustments to the oven temperature programme.

Adjusting the stationary phase film thickness is another valid approach to altering retention time, however this approach is somewhat more complicated. For analytes whose retention factor is less than 5, an increase in film thickness leads to increased resolution, however for analytes with a retention factor considerably greater than 5 the opposite is true and decreased film thickness can lead to increased resolution.

Table 2 shows a brief overview of column data that may be used to inform decisions when translating methods between differing GC column dimensions. Please note that these figures are valid only when considering the same stationary phase material.

 
Column i.d (25 m) Outlet Flow Outlet μ Average μ Inlet Pressure Sample Capacity
mm mL/min cm/s cm/s psi (ng) (0.25 μm film)
50 0.4 500 26 400 1-2
100 0.8 240 36 130 6-13
200 1.5 115 46 37 35-70
320 2.5 75 48 14 110-220
530 4 45 38 3.9 1000 - 2000

Table 2 : Useful data for capillary column translation

 
One of the most useful concepts in method translation is that of Phase Ratio (β) which relates the column internal diameter to the stationary phase film thickness through the following equation:
Where r is the column radius and df the film thickness (both in microns). This parameter can be used to predict how changes in column dimensions might affect analyte retention (or efficiency) and by keeping the phase ratio constant one can select a column which will produce a separation in the same timeframe but with increased efficiency.
 

Consider the situation in Figure 13 below:

30 m x 0.32 mm x 0.5 μm   β = 160 / 2 x 0.5 = 160
10 m x 0.10 mm x 0.2 μm   β = 50 / 2 x 0.2 = 125
Within the constraints of readily available column geometries, the phase ratio is well matched and these two columns are expected to produce a comparable separation in terms of analyte resolution, however the 10 m x 0.10 mm i.d. column will elute analytes around 3 x faster than the 30 m column providing carrier linear velocity and temperature programme are optimized.

«« Figure 13: Comparison of traditional capillary GC and fast GC for the analysis of impurities in styrene monomer.

1. Ethylbenzene
2. p-Xylene
3. m-Xylene
4. Isopropyl benzene
5. o-Xylene
6. n-Propylbenxene
7. p/m-Ethyl toluene
8. Styrene
9. a-Methylstyrene
10. Phenylacetylene
11. b-Methylstyrene
12. Benzaldehyde

(from reference 11).

Agilent Technologies
(Santa Clara, California, USA)
.

Figure 13 demonstrates the power of using smaller internal diameter capillary GC columns which can then be shortened to reduce analysis time.
By keeping the phase ratio approximately constant, resolution is maintained but analysis time is reduced by a factor corresponding to the ratio of the original and final column lengths (in this case 3x).

As we will see in subsequent sections, columns with an internal diameter below around 0.1mm do require some optimization of the system hardware in order to fully realise the benefits of increased speed and efficiency.

 
 

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There are several other important instrument considerations when translating to fast GC methods, especially when using smaller internal diameter columns, and these include:

  • Reducing system dead volume / restricting diffusion
  • Rapid introduction of sample from the inlet
  • Consider sample volume and concentration
  • Rapidity of oven heating and cooling
  • Detector sampling rate

The largest extra column volumes in the typical GC system will be the inlet liner and internal volume of the split/splitless inlet; and the void volume into which the column emerges in the detector.

Inlet Considerations.

Many operators choose to reduce the inlet dead volume by a) reducing the liner internal diameter and b) employing a high split flow to ensure the sample is transferred quickly and efficiently to the GC column – reducing the time available for diffusion of the sample plug.

Figure 14 shows the difference in peak width created using a 2 mm and 4 mm i.d. liner.

«« Figure 14: Peak width resulting from an injection 0.1 μL of acetone in liners of differing i.d. (from reference 11).

Agilent Technologies (Santa Clara, California, USA).

 
 

Whenever one reduces the liner internal diameter, the internal volume available for analyte expansion is reduced accordingly. To mitigate this, it is also typical to increase the split ratio in order to reduce analyte residence time within the inlet. The higher head pressure required to achieve these high split ratio’s will help to restrict the expansion of the analyte plug (see Figure 15).

«« Figure 15: Increasing the split flow or split ratio increases the inlet head pressure, reduces sample transfer time and reduces diffusion of the resulting gaseous sample.

CHROMacademy: GC Channel / Theory and Instrumentation of GC / Sample Introduction.

An additional benefit to increasing the split ratio is a reduction in the actual amount of sample transferred to the column.
As column internal diameter (and/or stationary phase film thickness) is reduced the sample capacity of the column reduces. Increasing the split ratio or decreasing the injection volume will reduce the absolute amount of analyte introduced into the column help to avoid overload characteristics such as peak tailing or fronting.
 
 
Figure 16 can be used to calculate the optimum injection volume for an inlet liner of 2 mm internal diameter, by entering 100 for the liner length and 2 for the liner i.d. It is important that the total volume of gas created by the sample does not exceed the total liner volume - this is represented visually on the liner graphic where we use 0.5 x the total liner volume as the ‘red zone’ indicator.
 

«« Figure 16: Select the liner dimensions, solvent and other operating conditions to calculate gas expansion volume.

CHROMacademy: GC Channel / Theory and Instrumentation of GC / Sample Introduction.

 
 

Detector Considerations

In terms of the detector dead volume, a modern FID detector will be designed to minimize the dead volume created. One should ensure that the capillary column is installed according the manufacturers guidelines as an incorrectly fitted column can give rise to catastrophic dead volume when using narrow internal diameter columns and especially where the internal diameter is below 0.1mm. Similarly, one needs to optimize the detector make-up gas flow-rate (with flame based detectors), as this gas can assist with efficiently transferring the analyte molecules into the flame region of the detector.

 
 

«« Figure 17 Correct column positioning and makeup gas flow-rate are important (especially with FID and flame based detectors) in reducing analyte diffusion (band broadening) when employing fast GC techniques.

CHROMacademy: GC Channel / Theory and Instrumentation of GC / GC Detectors.

 

 
 
Fast chromatography is characterized by narrow peaks with baseline elution profiles in the order of a few seconds to less than one second. In order to properly ‘model’ the profile of the peak, ensure quantitative accuracy and the optimum detector sensitivity, the detector sampling rate should be optimized. Modern GC detectors will typically have sampling rates which are high enough to produce the requisite number of sampling points across the peak (a minimum of 8 data points is recommended for quantitative accuracy), however some MS detectors running in SCAN mode may require some data acquisition parameter re-work in order to derive high enough data collection speed. Older quadrupole MS instruments may not be able to achieve the required sampling rate for fast GC determinations.

«« Figure 18: FID peak height for various fatty acid methyl ester compounds at several detector acquisition rate settings.

Adapted from work by Agilent Technologies
(Santa Clara, California, USA)
.

 
 

Figure 19: Peak shape and retention time effects at various FID acquisition rate settings (reproduced with permission of Agilent Technologies, Santa Clara, California, USA). »»

 

Figures 18 and 19 demonstrate the effects of data acquisition rate on both peak height, retention time and apparent peak efficiency. Note that when data acquisition rates are low, peak height is reduced, the peak apex shifts to later elution time and the peak broadens. The instrument sensitivity will also appear to decrease as the peak height per unit mass injected decreases.

One should attempt to acquire as many data points across each peak as is reasonable; your limiting factors will be the response time of the detector and the size of the data files produced, however in the modern era the latter issue should not cause problems.

In terms of detection speed and the quality/usefulness of data produced, time-of-flight mass spectrometric detectors are increasingly finding a place in the fast and ultra-fast GC application literature.

 
 

Oven Heating and Cooling Considerations

Conventional GC ovens suffer from the inordinate length of time taken to both heat and (especially) cool the GC column using forced air heating / cooling ovens which typically have relatively large thermal mass. If this issue can be overcome, there would be increased scope for decreasing analysis time in combination with the other approaches mentioned above. It should be noted that there are also some significant disadvantages to increasing the temperature programme rate for certain separations. These typically include: separations involving high boiling analytes, convoluted samples with many critical peak pairs and samples which include thermally labile analytes.5

In gradient temperature programmed GC, the analyte rate of migration through the column will double for every 30 oC rise in the oven temperature –this is demonstrated by the smooth curve in Figure 20.

«« Figure 20: Peak migration profile (as both a step function and overlaid with the actual analyte elution profile) for an analyte in temperature programmed GC with an elution temperature of 265 oC

CHROMacademy: GC Channel / Theory and Instrumentation of GC / GC Temperature Programming.

 
 

In simple terms – the analyte moves through half of the column (volume) in the 30 oC prior to elution (265 oC in this instance), three quarters of the column volume in the 60 oC prior to elution etc. etc. This is true for all analytes – and all analytes spend exactly the same length of time moving through the GC column in temperature programmed analysis! Thus all analytes have approximately the same peak width –they are dispersing within the column for the same period of time.

Extrapolating back – all analytes are initially immobilized at the head of the column (focussed), where they remain until the column temperature is suitable for them to begin to vaporize and partition through the column. As each analyte has a different relative vapour pressure, the mechanism of separation in temperature programmed GC relies on this difference in vapour pressure vs. temperature for each analyte. Once the analyte begins to chromatograph, as explained above, it accelerates through the column at the same rate as all other analytes. Therefore, in fast GC temperature programming, where the temperature profile is increased more rapidly, analytes with closely related chemistry / vapour pressure may begin to chromatograph at very similar times, and therefore separation may be reduced.

There are essentially two methods of obtaining very fast temperature programming rates in capillary GC, (1) wrapping the capillary with electrically heated resistive tape (2) using multi-capillary bundles with heating wires inserted between the very small internal diameter capillaries.

To adopt resistive heating of a single capillary the column can be coated with a conductive material, a coil can be wrapped around or run in parallel to the column, or the capillary column can be inserted into a metal tubing. The columns used are short (<10m) and are typically operated at higher carrier gas linear velocity.6-8

«« Figure 21: Column heating via resistive wire
(left, courtesy of Thermo Scientific, West Palm Beach, Florida, USA)
and column jacket
(right, courtesy of Thermedics, Chelmsford, Massachussetts, USA)

 
 

Whilst these systems are capable of very rapid heating (>10 oC/s in many instances) the rate of heating such low thermal mass systems has to be very well controlled in order to prevent retention time irreproducibility. Some systems suffer from the issues of ‘cold spots’ around the injector and detector couplings which can cause pronounced band broadening, especially for higher boiling analytes. High boiling analytes may also broaden as they elute isothermally at the upper temperature of the column, which is typically in the region 375 - 400 oC for most systems.

Wide bore columns with slightly thicker films are required in order to give good working analyte concentration range and it is typical that these systems are used for the separation of samples containing relatively few analytes whose boiling points cover a wide range, primarily due to the use of short columns and the inherent loss of resolution at higher temperature programming rates.

«« Figure 22: Fast analysis of a normal alkane mixture (C10-C42) within 1.5 minutes.

Temperature programme EZ-Flash:
80 oC – 375 oC at 4 oC /s, inlet-pressure = 300 kPa, split-flow = 500 ml/min, ū = 200 cm/s,
temperature-program GC-oven: 80 oC – 140 oC at 30 oC/min, injector temperature program: 350 oC -550 oC at 16 oC /s (from reference 2).

Marieke van Deursen, Eindhoven -Technische Universiteit ,Eindhoven, 2002. ISBN 90-386-2873-0.

 
 

A good rule of thumb for optimum heating rates appears to be 10 oC per void (hold-up) time of the instrument to avoid drastic loss of resolution and increase of upper elution temperatures.

The introduction of the multicapillary bundle column in 1997 9 brought a new approach to rapid temperature programming which offered higher total flow rate, the higher sample capacity and, consequently, the lower minimum detectable concentration of the solutes. These bundles consist of many (circa. 900) short, narrow i.d. (40 μm) columns that are heated by electrically resistive wires placed between the capillaries within the bundle.

«« Figure 23: Typical cross sectional area
of a multicapillary bundle GC column containing
up to 900 separate coated capillaries (from reference 13).

Agilent Technologies
(Santa Clara, California, USA)
.

 
 

The performance of these columns has been demonstrated in the literature and columns of this type are commercially available. To avoid large variations in column repeatability and excessive peak dispersion, the manufacture of each individual capillary has to be very closely controlled and length, internal diameter and film thickness all need to be within very tight tolerances to avoid band broadening issues that render the system unusable.

Whilst a speed increase of around 10 times is possible with this approach, the technique is once again restricted to the analysis of fairly simple mixtures, due to the short columns which are typically used.

«« Figure 24: Fast analysis of hydrocarbon oil index using a deisel fuel / mineral oil mixed calibration standard using conventional and multicapillary columns, conditions as indicated (from reference 13).

 

Agilent Technologies
(Santa Clara, California, USA)
.

The above techniques for decreasing retention factor are very useful for those applications in which use an over-abundance of theoretical plates in order to derive the separation (such as the separation of lower boiling homologues within a gasoline sample), and where plate number may be reduced without adversely affecting the resolution obtained.
 
 
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In gas chromatography, the application of vacuum column-outlet conditions, such as is encountered in GC-MS techniques, is an attractive way to increase the speed of analysis. We have already seen that considerable gains in speed are possible, using short and/or widebore columns where a gain in speed of a factor of 3-5 can easily be obtained. In contrast to other methods for fast GC, the vacuum outlet approach has a significantly increased sample loadability. This mainly arises because larger internal diameter columns can be used.

Vacuum outlet conditions are most readily obtained by using a mass spectrometer as the detection device. Direct coupling of a short and/or wide-bore column to an MS however, will result in operational problems, primarily that the carrier-gas inlet and the injection system have to be operated at sub-ambient pressures.

 

»» Figure 25: Plots of inlet pressure against column length, with hydrogen carrier gas and vacuum compensation on. Column inner diameters (mm):
(a) 0.20, (b) 0.25, (c) 0.32 (d) 0.53; column temperature: 50oC; average linear velocity: 40 cm/s.

The blue shaded area designates negative inlet pressures (from reference 9).

J.V. Hinshaw, Column Connections,
LCGC Europe, Jan 1 (2011)
.

 
 

Secondly, the high carrier flow into the detector might increase the pressure in the ion source of the mass spectrometer to a level exceeding the tolerable limit.

Typical flow for a column of 530 μm, i.d. and a length of 10m are approximately 7-10 mL/min, which exceeds the pumping capacity of most benchtop systems, the best of which are typically limited to 2-4 mL/min.

However, short wide-bore columns can be used in conjunction with MS instruments, if a restriction can be coupled to the column inlet. The flow is now restricted to an acceptable level, the injection system can operate at above-atmospheric pressures but low-pressure conditions exist within the entire column.

Such a column inlet restriction can be readily obtained by coupling a narrow-bore column (e.g. 60 cm x 100 μm) at the inlet position of the analytical column, using a zero dead-volume connector 5. A disadvantage of such a coupling is the possible occurrence of dead volumes, which can cause significant peak diffusion. An alternative solution is the use of an SFC type integral tapered restriction prepared from the column inlet. The restriction is an integrated part of the column which reduces the inherent dead volume within the system.

When the capacity of the pumps is high enough, direct coupling is a possibility and sample introduction is carried out at sub-atmospheric inlet pressures. Fast rotating micro-injection valves (injection times < 100 ms), can be used for fast injections in this regard, which minimizes injection band broadening.

 

«« Figure 26: Schematic drawing of the three restriction types: (A) narrow-bore precolumn (Varian/Chrompack), (B) SFC-restriction (produced in house) and (C) valve restriction.

Restriction column at inlet: 15 m × 250 μm (uncoated) (from reference 2)

Marieke van Deursen, Eindhoven -Technische Universiteit ,Eindhoven, 2002. ISBN 90-386-2873-0.

 
 

«« Figure 27: GC-TOFMS analysis of gasoline (pure) using a wide-bore column with SFC restriction. Inlet-pressure = 4 bar, split-flow = 50 mL/min, Oven = 60 oC then
20 oC/min to 150 oC, injection volume < 0.1 μL (manual).
MS-conditions: scanned mass-range = 41-400, acquisition-rate = 50 spectra/s.


Compounds:
1. 2-methyl-butane
2. 2- methoxy-2-methylpropane (MTBE)
3. benzene
4. toluene
5. ethylbenzene
6. m+p-xylene and
7. o-xylene

(from reference 2).

Marieke van Deursen, Eindhoven -Technische Universiteit ,Eindhoven, 2002. ISBN 90-386-2873-0.

 
 
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Several approaches to increasing sample throughput and reducing have been discussed above.

Experimental approaches and techniques have been contrasted and we have considered practically straightforward as well as more advanced techniques. Below is a concise summary of the discussion, concentrating on the simpler practical approaches to speed up analyses.

 

Parameter

Benefit

Possible downside

Increase speed of injection process

Easy to achieve with high split flow

Possible reduction in analytical sensitivity / issues with ‘backflash’

Decrease column i.d.

Faster optimum carrier gas linear velocity , shorter columns (higher plates/m)

Reduced column capacity, requires high inlet pressure, requires high split ratio or lower injection volume

Increase carrier gas velocity

Easy to achieve with increased head pressure and reduced column internal diameter, impressive speed increases using vacuum outlet approaches

Need to ensure instrument can deliver high carrier head pressure, technology for vacuum outlet applications not yet fully established

Shorten column

Standard i.d. columns (capacity), low pressure drops, standard hardware

Decreased separating power (resolution)

Perform isothermal separation

No cool down cycle, highest resolving power

No thermal or chemical focusing possible to improve peak shape, applies to samples with narrow volatility range, possible column contamination with late eluting compounds

Increase oven programming rate

Elutes full volatility range in a minimum time

Potential changes to resolution and selectivity, may require new hardware

Use alternative carrier gas
(H2 > He > N2)

Same efficiency with shorter columns – time and cost savings with H2

Safety issues with H2, possible initial purchase cost of hydrogen generator

Reduce GC system dead volume

Optimise peak shape and efficiency, easy to achieve with reduced i.d. liner and proper positioning of column in GC inlet and detector

May require new inlet liner purchase, limited injection volume to avoid ‘backflash’

 
 
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References

  1. Agilent Technologies Application Note, ‘Rapid Analysis of Food and Fragrances Using High-Efficiency Capillary GC Columns’ (5989-7509).
  2. Novel concepts for fast capillary gas chromatography, Marieke van Deursen. - Eindhoven : Technische Universiteit Eindhoven, 2002. ISBN 90-386-2873-0.
  3. K. Mastovska and S. J. Lehotay, J. Chrom. A, 1000 153–180 (2003).
  4. R.J. Bartram and P. Froehlich, LCGC N. Am., 28(10), 890 (2010).
  5. J.V. Hinshaw, LCGC N. Am., 28(3), 218 (2010).
  6. J. Heseltine, LCGC N. Am., 28(1), 16 (2010).
  7. J.V. Hinshaw, LCGC N. Am., 26(11), 1100 (2008).
  8. J.V. Hinshaw, LCGC Asia Pacific, 12(2), 1100 (2009). (http://chromatographyonline.findanalytichem.com/lcgc/article/articleDetail.jsp?id=581360&sk=&date=&pageID=3)
  9. J.V. Hinshaw, LCGC Europe, 24(1) (2011).
    (http://chromatographyonline.findanalytichem.com/lcgc/GC/Hydrogen-Carrier-Gas-amp-Vacuum-Compensation/ArticleStandard/Article/detail/709613)
  10. J.V. Hinshaw, LCGC Europe, 21(4) (2008).
    http://chromatographyonline.findanalytichem.com/lcgc/article/articleDetail.jsp?id=504708&pageID=1&sk=&date
  11. Agilent Technologies Application Note, ‘Predictable Translation of Capillary Gas Chromatography Methods for Fast GC’ (5965-7673).
  12. Novel concepts for fast capillary gas chromatography, Marieke van Deursen. - Eindhoven : Technische Universiteit Eindhoven, 2002. ISBN 90-386-2873-0.
  13. A. Hoffmann, B. Tienpont, F. David, P. Sandra, Ultra-Fast Determination of the Hydrocarbon Oil Index by Gas Chromatography using a Modular Accelerated Column Heater (MACH).

 

Essential CHROMacademy Reading and Resources on Developing High Throughput Separations in Capillary GC

Practical Fast Gas Chromatography Using Smaller Diameter Columns and Hydrogen Carrier Gas   
*** CHROMacademy Registered users only *** Lisa Wool,Darren Decke.(2007)

Case Studies: Improved Productivity with Fast GC Accessories    
*** CHROMacademy Registered users only ***Zip.(2009)

Frequently Asked Questions about Hydrogen Carrier Gas     
*** CHROMacademy Registered users only *** John V. Hinshaw 02/01/2009

Hydrogen Carrier Gas and Vacuum Compensation     
*** CHROMacademy Registered users only *** John Hinshaw (2011)

Using Computerized Pneumatics     
*** CHROMacademy Registered users only *** John V. Hinshaw (2008)

Using Computerized Pneumatics - Part 2   
*** CHROMacademy Registered users only *** John V. Hinshaw (2008)

Rapid Quality Control of Flavours and Fragrances using Fast GC–MS and Multi-MS Library Search Procedures   
 *** Registered users only *** Maria Scandinaro,Peter Quinto Tranchida,Rosaria Costa,Paola Dugo,Giovanni Dugo,Luigi Mondello 10/01/2010

Ultra Fast GC Method for the Analysis of Total Hydrocarbons in Water in Compliance with ISO 9377-2 (Mod.)     
*** CHROMacademy Registered users only *** Fausto Munari,Andrea Cadoppi 12/02/2006

John Hinshaw- Fast GC :PITTCON 2010 Theater Interviews-GC     
*** CHROMacademy Registered users only *** John V. Hinshaw (2010)

Technology Forum: Gas Chromatography     
*** CHROMacademy Registered users only *** (2010)

Rapid Analysis of Flavors and Fragrances Using High-Efficiency GC Columns     
*** CHROMacademy Registered users only *** Simon Jones,Mark Sinnott 06/01/2008

 

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The following subjects are covered in CHROMacademy.com

The Theory Of HPLC
Introduction (1.5hrs)
Chromatographic Parameters (3hrs)
Band Broadening (3hrs)
Column chemistry (4hrs)
Reverse phase (partition) chromatography (6hrs)
Ion-Pair Chromatography (3hrs)
Normal phase (absorption) chromatography (3hrs)
Gradient HPLC (3hrs)
Quantitative and Qualitative HPLC (3hrs)
FAST HPLC (4.5hrs)
HILIC (3hrs)
SFC (3hrs)
Ion Chromatography(3hrs)

Theory and Instrumentation of GC
Introduction (1.5hrs)
Chromatographic Parameters (3hrs)
Band Broadening (3hrs)
Gas Supply and Pressure Control (2hrs)
Sampling Techniques (4.5hrs)
Sample Introduction (5hrs)
GC Columns (5.5hrs)
GC Temperature Programming (3hrs)
GC Detectors (2.5hrs)
SFC (3hrs)

Instrumentation of HPLC
Mobile Phase Considerations (3.5hrs)
Solvent Pumping Systems (4hrs)
Autosamplers (4.5hrs)
Detectors (4.5hrs)

Solid Phase Extraction
Molecular Properties (4hrs)
SPE Overview (3.5hrs)
SPE Mechanisms (4.5hrs)
Method Development (5.0hrs)
Primary Sample Preparation Techniques (2hrs)
Liquid / Liquid Extraction Techniques (1.5hrs) Approaches to Automation for SPE (1.5hrs)

Fundamental GC-MS
Introduction (1.5hrs)
GC Considerations (4.5hrs)
GC -MS Interfaces (2.5hrs)

Fundamental LC-MS
Introduction (1.5hrs)
Electrospray Ionisation Theory (6hrs)
Electrospray Ionisation Instrumentation (4hrs)
Mass Analyzers (9.5hrs)
Atmospheric Pressure Chemical Ionisation (3.5hrs)
Atmospheric Pressure Photoionisation (3hrs)
Solvents, Buffers and Additives (3.5hrs)
Vacuum Systems (3hrs)
Flow Rates and Flow Splitting (3hrs)
Orbitrap Mass Analyzers (3hrs)

MS Interpretation
General Interpretation Strategies (11hrs)
Intro to MS Proteomics Research (3.5hrs)

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