Lec22.Mobile.phases

7/29/2019 Lec22.Mobile.phases

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INSTRUMENTAL ANALYSIS AND SEPARATIONS

GAS CHROMATOGRAPHY - LECTURE 20Mobile and Stationary Phases

Columns for Gas Chromatography

Capillary columns are commonly made from high purity silica which contains nosignificant metallic oxides that may react with the sample. The surface however does

contain many active silanol groups. Generally prior to the application of the stationary

phase coating, the inner surface is deactivated. One method of deactivation is to react the

free silanol groups with a reagent such as trimethylsilyl chloride. The stationary phase isthen applied, usually by a method known as static coating. The column is filled with the

stationary phase dissolved in a volatile solvent. Then one end of the column is sealed, a

vacuum is applied and the solvent is evaporated, leaving an even coat of the stationary

phase on the column wall. The stationary phase is held in place only by surface tensionforces and it can be disturbed by solvents in the sample or excessive temperature.

Increased stationary phase stability can be achieved either by polymerization of thecoating or by covalently linking it to the column surface. In addition to increasing

durability, immobilization of the stationary phase by these processes allows a much

thicker film to be applied which is important especially for separating highly volatilecompounds. It is also possible to regenerate immobilized stationary phases by washing

the column with solvents such as methylene chloride or tetrahydrofuran to remove

soluble contaminants that accumulate in the column(this can extent column life). The

outer surface of the column is coated with a polymer called polyimide which makes thecolumn strong and flexible. Any flaw in the coating, and the column can break very

easily. There are three major types of gas chromatography columns, packed columns,porous layer open tubular (PLOT) columns, and wall coated open tubular (WCOT)

columns.

The geometry of WCOT capillary columns is fairly simple, consisting of length,internal diameter, and stationary phase thickness. Nevertheless, there are endless

possible combinations of these three factors that could be used for optimizing

chromatography. Doubling the column length effectively doubles the number of

theoretical plates but the resolution between any two compounds is proportional to thesquare root of the plate number so doubling the column length only increases resolution

by about 40%. Doubling the column length also will result in longer analysis times and

small peak heights, so longer columns are not the answer to poor resolution. However,longer columns do spread out the peaks and as a general rule, the more complex the

sample, the longer the column should be.

Column Diameter

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The diameter of a column and the thickness of the stationary phase should always

be considered together because they interact with regards to column performance.

However, the general effect of decreasing column diameter is to increase the speed of

analysis. This is because the optimal carrier gas velocity increases as the diameter

decreases (assuming that the retention factor and plate number are held constant).

Smaller diameter columns usually have lower plate height (higher N)because ofimproved mass transfer. Increasing carrier gas velocity results directly in faster analyses.

On the other hand, larger diameter columns have increased sample capacity which canprovide higher detectability with mass sensitive detectors such as an FID.

Another consideration of column diameter is how it affects the amount of sample

that can be injected. Larger samples require a larger amount of stationary phase to

interact with, otherwise the column will be overloaded, resulting in poor chromatography.

Larger diameter columns have higher capacity and less problems with overloading.Overloading results when a band of a compound in the sample totally saturates the

stationary phase. Once the stationary phase is saturated, no more of the compound can

interact with the stationary phase so it will move through the column as if it were an

unretained compound. Since some of the compound is moving faster than it should, itwill elute from the column a little earlier which will show up on the chromatogram as a

fronting peak. If a compound overloads the mobile phase it will condense on the innersurface of the column (without partitioning into the stationary phase). This effect is most

often seen as later eluting and tailing peaks. It is important to note that overloading is

related to specific compounds in the sample. Generally if complex samples are injected,some compounds will be overloaded, while some compounds may not show up on the

detector because their concentration is below the detection limit. Therefore, the amount

of sample injected is based on a balance between these effects.

Thickness of Coating

The thickness of the stationary phase coating is very important. Thinner coatingsusually provide higher efficiency (higher N or lower HETP) because mass transfer is

quick between the mobile and stationary phase. However, the column capacity is low

and the column is easily overloaded.Very volatile compounds should be analyzed using thick films because there is

not sufficient interaction with thin films and the result is poor separation. Very non-

volatile compounds should be analyzed using thin films, otherwise the retention in the

stationary phase is too long. Polar compounds have a tendency to have excess tailing ofpeaks. This peak tailing can be reduced with thicker stationary phase coatings

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Stationary Phases

The different stationary phases available are described by the term polarity.This term is used in a loose sense as a measure of the interactions between the sample

compounds and the stationary phase. These interactions are mostly based on dipole-

dipole attraction, induced dipole-dipole attraction and van der Waals forces. Becauseseparations are so strongly influenced by temperature, there is no great need for many

types of stationary phases. Common stationary phases are based either on polysiloxanes

or polyglycols. The methylsiloxane group [Si(CH3)2O] can be polymerized to a widerange of thermally stable compounds ranging from low viscosity liquids gums to

silastomer rubbers. These polymers can also be easily modified by substituting polar

groups for methyl groups in the polysiloxane structure thereby producing a wide range of

polarities.

Table. A Few of the Available Stationary Phases For Gas ChromatographyCompound Polarity Max Temp oC Column ID Manufacturer

Poly(methyl siloxane) Low 300-350 HP-1

AT-1DB-1, SE-30

OV-1

ZB-1

RTx-1BP-1

SPB-1

CP-Sil 5 CB

Agilent

AlltechJ&W

Ohio Valley

Phenomenex

RestekSGE

Supelco

Varian

95% Dimethyl, 5%phenyl

Poly(methyl siloxane)

Low 300 HP-5

AT-5, EC-5

DB-5, SE-54

OV-5

ZB-5

RTx-5BP-5

SPB-5,MDN-5

CP-Sil 8 CB

Agilent

Alltech

J&W

Ohio Valley

Phenomenex

RestekSGE

Supelco

Varian

Polyethylene glycol Medium 250 HP-20M

AT-WaxDB-Wax

Carbowax 20M

ZB-Wax

Stabilwax

BP20

Supelcowax 10

CP-Wax 52 CB

Agilent

AlltechJ&W

Ohio Valley

Phenomenex

Restek

SGE

Supelco

Varian

PolyethyleneglycolNitroterephthalic acid ester

(free fatty acid phase)

Medium 250 HP-FFAPAT-1000

DB-FFAP

OV-351

ZB-FFAPStabilwax-DA

BP-21

Nukol, SPB-1000

AgilentAlltech

J&W

Ohio Valley

PhenomenexRestek

SGE

Supelco

Varian

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Mobile Phase

Hydrogen, Helium and Nitrogen are the most common gases used as mobilephases. The lighter the carrier gas, the higher the speed of analysis. Therefore, hydrogen

will give the most plates/sec. However, helium is often used because hydrogen is

potentially explosive. High-speed analysis also results in narrower peaks and betterdetectibility. Longitudinal diffusion is lowest in the heavier gases such as nitrogen so

nitrogen can give the highest N (lowest H) at low mobile phase velocities where

longitudinal diffusion has the highest effect on efficiency.

Figure 1. Velocity vs Theoretical Plate Height for Common Carrier Gasses.

Gases are forced through thecolumn by an applied pressure at thehead of the column. The outlet end of

the column is usually at atmospheric

pressure, or even vacuum (whenattached to a mass spectrometer).

Therefore there is a drop in pressure as

the gas moves through the column.This drop in pressure causes the gas to

expand which can result in peak

broadening. It also causes the gas

velocity to increase as it movesthrough the column (Figure 2).

Therefore it becomes difficult to

optimize gas velocity. Figure 2 showsthe gas velocity profile along the

column. The value pi/po is the ratio of

the inlet velocity(pi) to the outletvelocity (po).

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As the column oven is heated, the viscosity of the mobile phase increases. (the

viscosity of gases generally increases with temperature, which is in contrast to liquids

where the viscosity decreases with increasing temperature). Therefore as the column isheated during the temperature ramp, the flow rate goes down. In order to keep a constant

flow (and reduce peak spreading of later eluting peaks) a process of pressure

programming is used. The constant flow mode increases the pressure at the head end ofthe column, and keeps the mobile phase velocity constant. Pressure programming can

also be used to increase mobile phase velocity as the temperature increases, further

decreasing analysis time and increasing peak height.

Kovats Retention Index

The retention time of an analyte provides at least some information on the

chemistry of the compound. However, retention time is dependant on many operational

factors such as temperature, column length, column diameter, coating thickness, etc.

Therefore it is more satisfactory to use a relative retention value whereby many of thesevariations are compensated for. The Kovats retention index system is based on a scale

defined by the elution of a series of n-alkanes. It is widely used, and information on the

Kovats Index for many compounds can be found in the literature. In this system, normalalkanes (pentane, hexane, heptane, etc.) are given an index value 100 times their carbon

number (ie. Pentane = 500). The elution time of an alkane gets longer with the addition

of each additional CH2 group such that the a plot of the log of retention time vs carbonnumber is linear (there is some deviation, especially at lower carbon numbers). The

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calculation of the Kovats Index for a particular compound is obtained by interpretation of

its position between the two nearest alkanes in the reference series.

Kovats Retention Index = 100Cn + 100 [log tR(A) log tR(n)]

[log tR(n+1) log tR(n)

Where Cn is the carbon number of the alkane eluting just before the analyte

tR(n) is the retention time of the alkane eluting just before the analyte

tR(n+1) is the retention time of the alkane eluting just after the analytetR(A) is the retention time of the analyte

The classical Kovts retention index is measured under isothermal conditions.

However, in the case of temperature-programmed gas chromatography a similar valuecan be calculated utilizing direct numbers instead of their logarithm. In other words, an

equation for the retention index can be developed using a polynomial regression of a

series of alkanes vs. their retention times.

This method of measuring reference retention is dependant only on the analyte,the temperature and the stationary phase composition. A Kovats index for an analyte

should be the same on any column with the same stationary phase irregardless of columnlength, mobile phase, flowrate, etc. However it has been shown that in open tubular

columns with thin films, adsorption effects can contribute to retention, affecting the

accuracy of the Kovats index especially at low temperatures and with polar stationaryphases.

References.

Grant, D. W. Capillary gas chromatography; John Wiley & Sons, Ltd: West Sussex,

England, 1996; pp 295.

Modern Practice of Gas Chromatography; 3rd. ed.; Grob, R. L., Ed.; John Wiley & Sons,Inc.: New York, 1995; pp 888.

Lecture 20 6

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