Columns

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In This Section, You Will Learn:

• Identifying the Differences between Packed and Capillary columns.

• Calculating Column Efficiency and Resolution.• Introduction to Fast GC.• Introduction to Retention Time Locking.• Understanding Column Selectivity.• Identifying Steps for Column Care.• Performing Isothermal and Temperature Programming Oven

Operation.

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PackedOpen (Capillary)

Regular

BeadColumn

PorousLayerBead

ConventionalPacked andMicro Packed

Porous LayerOpen Tube

Wall CoatedOpen Tube

Column Types

50 ng/peak100 ng/peak100 ng/peakSample Capacity

5-901-401-2010-90Pressure Drop (psi)

0.3-1.01-304-3010-60Flow Rate (ml/min.)

.05-0.30.3-0.750.5302-4I.D. (mm)

5-1005-1005-100.5-10Length(meters)

WCOT (narrow)

WCOT (wide)

Series 530Packed

4

SAMPLEMOBILE

PHASE

STATIONARY

PHASE

Separation is a Partitioning Process

5

10% of ApplicationsAdsorbent

Packed Columns

Adsorbent Packing (GSC)

Capillary - Porous Layer Open Tubular (PLOT)

Carrier Gas

Porous with large surface area

Carrier Gas

Gas Solid Chromatography (GSC)

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Gas Liquid Chromatography (GLC)

Partition PackingPacked Columns

Carrier Gas

Liquid PhaseSolid Support

Very porous with very high surface area

Wall Coated Open Tubular (WCOT)

Liquid Phase

Capillary Columns

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Gas Liquid Chromatography (GLC)

Applies to 90% of applications

• Separation by partitioning or differential solubility in stationary phase.

• Components are separated based upon differences in polarity (interaction of dipole forces).

Generally:“Like disolves like”

orPolar interacts with polar

Example: Alcohols are polar.Carbowax is polar liquid phase.

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A

B

C

D

Flow

Model of the Chromatographic Process

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GOODPOOR

GOODPOOR

Column Separation Characteristics

Efficiency: Ability of the column to produce sharp peaksResolution: Ability of the column to separate two peaks from

each otherSelectivity: Ability of the column to determine chemical and/or

physical difference in two peaks

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Solute

Time

Start

Inert Gas

t mtR

't R

We would like to know the actual time the component spends in the stationary phase.

Let's relate “n” to the length of the column.

orPlates per meter (N) =n

L

Height equivalent to a theoretical plate (HETP) =nL

Thus, the more efficient the column, the bigger the "N" the smaller the "HETP"

n = 5.545t R

'

W h

2t R - t m= t R

't R't R'

Calculating Efficiency

Rt ' = Corrected Retention Time

n = effective theoreticalplates

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column length (cm)

Retention time of inert gas (sec)Linear Velocity =

Measuring Linear Velocity and Flow Rate

Estimate the column length by using the formula Length = Πdkwhere d = the diameter across the cage holding thecolumn k = the number of turns of the column on the cageΠ = 3.14.

The retention time of an inert gas can be estimated by:• Using the retention of the solvent if it is the first component that

elutes.• Injecting cc of butane vapor from a disposable lighter and using the

retention time of the butane peak.

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HET

P

C RESISTANCE TO MASS TRANSFER

A EDDY DIFFUSION

MOLECULAR DIFFUSION

μ ( opt μ)

}

}B{

HETP = A + + C μB

μ

Efficiency & Carrier Gas Linear Velocity

• Efficiency is a function of the carrier gas linear velocity or flow rate.

• The minimum of the curve represents the smallest HETP (or largest plates per meter) and thus the best efficiency. "A" term is not present for capillary columns.

• Plot of HETP vs. linear velocity is know as the Van Deemter plot.• The linear velocity value at the minimum of the curve is the

optimum value for achieving the best efficiency.

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Optimum Flow rate

42-106cm/sec..2-.5 ml/min.0.100 mm (high speed)

21-32 cm/sec..5-1 ml/min.0.200 mm (narrow bore)

20-41 cm/sec.1-3 ml/min.0.320 mm (wide bore)

22-38 cm/sec.3-5 ml/min.HP Series 530 (megabore)Capillary Column

4.2-6.3 cm/sec.20-30 ml/min.1/8”

2.6-3.2 cm/sec.50-60 ml/min.1/4”

Optimum Carrier Linear Velocity

Optimum Carrier Flow RateColumn Diameter

Generally the following guidelines may be used for finding the optimum values for a particular column:

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Where: N = (Plates per meter)nL

HETP = (Height equivalent to a theoretical plate)Ln

Judging Efficiency

>1x10-3<1000>0.03<100Dubious

3-5x10-42000-30000.10-0.15200-300Fair

2-3x10-43000-50000.06-0.10300-500Good

<2x10-4>5000<0.06>500Excellent

HETP, cmNHETP, cmNColumn Efficiency

Capillary0.2 mm (narrow bore)

Packed1/8”

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Effective column efficiency is dependent upon good sample introduction technique. Samples should be introduced in a tight, rapid plug to avoid band broadening.

How To Improve Column Efficiency

1. Use smaller diameter column2. Use a lower % or thinner film of stationary phase3. Use smaller sample size 4. Use longer column.5. Use temperature programming for sharper later eluting

peaks.

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Time ( Min.)

4 5 64.5 5.5

1.0E5

2.0E5

3.0E5

4.0E5

5.0E5

6.0E5

Abu

ndan

ce

RT

= 4.

41

RT

= 4.

59

RT

= 5.

10

1.18 ( RT - RT )2 1

(W + W )1 2

R =

R = 1.5 Results in Baseline Separation

Resolution

• Resolution is a measure of the ability of a column to separate two peaks.

• Resolution is measured in terms of two adjacent peaks which we want to separate. Generally, the most difficult pair is chosen; if these can be pulled apart successfully then all of the others will be resolved as well.

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Decreasing Run Time

Column Parameters:

• Shorter column length• Smaller column i.d.• Thinner liquid phase

Instrument Parameters:

• Different carrier gas• Faster temp program rate• Higher Temp Isothermal analysis• Higher carrier gas flow rate

Use Fast GC!

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Using Fast GC

18.2 min

3.5 min

5 min

1.2 min

toluene

naphthalenes

benzene

C8aromatics

C9 plus aromatics

0.10 mm i.d., hydrogen0.1µl, 800/1

0.32 mm i.d., helium0.5µl, 200/1

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Fast GC Considerations

• Use Small diameter columns – Rapid, high efficiency separations on complex samples

• HP Method Translation Software– Used to predict proper operational parameters– “Free-ware” from HP (http://www.hp.com/go/mts)

• Hardware issues– Fast Temperature program rates used– High inlet pressures required (EPC needed)– Fast Data acquisition rates required– Sample capacity limitations (high split ratios used)– 10-20 meter Column length– Carrier gas change can significantly decrease analysis times

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Efficiency curves for a 25 m x 0.25 mm id WCOT column with 0.4 um of OV-101.

Effect of carrier gas on the resolution of n-heptadecane and pristane.

WCOT Column

25 m x 0.25 mm

Average Linear Velocity (cm/sec)

HETP(mm)

1.2

1.0

.8

.6

.4

.2

10 20 30 40 50 60 70 80 90

C17 at 175º C

k' = 4.95

OV-101 0.4µ

N

He

H2

2

Nitrogen(58 cm/sec)

Helium(58 cm/sec)

Hydrogen(58 cm/sec)

15 m x 0.25 mm

Glass WCOT

SE - 52

Isothermal, 150º C

R = 1.17 R = 1.37

Type of Carrier Gas Effect on Efficiency and Resolution

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Reformate Gasoline

Hydrogen carrier gas speeds up analysis!

min0 5 10 15

min0 2 4

min0 1 2 3 4

1 53

30m x 0.32, 0.5µm HP-Waxhelium

10m x 0.10, 0.2µm HP-Waxhelium

10m x 0.10, 0.2µm HP-WaxhydrogenC

B

A

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Retention Time Locking (RTL)

• Software that allows matching the retention times in any 6890 GCsystem to those in another system, with the same type of column.

• Used by method developers who are responsible for developing, transferring, and maintaining methods for routine analysis.

• Requires the building of a method and pressure-retention time calibration curve for the type of column used.

• Method can be transferred to another instrument with the same type of column and the retention time of a target compound can be locked onto the desired retention time.

• RTL Pesticide Database now available. Retention times for 567 pesticides and suspected endocrine disrupters under locked conditions.

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Liquid Phase Selectivity

1. C112. 4-chlorophenol3. 1-decylamine4. C135. methyl caprate6. C147. acenaphthylene8. 1-dodecanol9. C15

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3

4

5

67

89

1 23

4

6

9

87

5% phenyl methyl silicone

50 % phenyl methyl silicone

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Considerations In Column Selection

Sample to Be Separated:1. Column diameter2. Stationary Phase3. Length4. Film thickness or % loading

a) Capacityb) Retentionc) Inertnessd) Efficiencye) Bleed

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Column Selection

• Try a column that is handy• Ask a colleague for advice• Look for a similar application that has already been published• If in doubt, use a non-polar column such as HP1 or HP5

These approaches are good starting points, but one must optimize performance.

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Type of Column

Carrier Conditions

Oven Conditions

Injection Parameters

Detector Parameters

Sample Information

Example Application Note:

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Gasoline PremiumColumn 15 m/0.3 mm SE-52 film thickness

(um)

0.05

0.15

0.80

8020 programmed 2 /min

1 2 3

4

5

2 3 4

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Analysis of gasoline on columns with varying stationary phase film thickness. (K. Grob and G. Grob, HRC & CC, 2 (1979) 109, reprinted with permission)

Film Thickness vs. Retention Time

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Catalog Information Provides:

• Trade name of phase• Chemical name of phase• Substitute or equivalent phases• Solvent• Temperature minimum and maximum• Example applications

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Column Care: Column Conditioning

• Preconditioning of new columns to remove residual traces of solvent.

• Choose conditioning temperature with these considerations:– High enough to remove non-volatiles.– Low enough to prolong lifetime of column and minimize column

bleed.– Lower conditioning temperature require longer conditioning time.– Verify the maximum temperature limit of the liquid phase from the

column manufacturer.• Conditioning may be done by:

– Repeated oven temperature programming to an elevated temperature until column performance improves.

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PressureFlow

Inlet end of column Detector end of column

Capillary Column Rinsing Reservoir HP part no. 9301-0982

Rinsing

The immobilized phase (cross-linked silicone) permits you to rinse solvents through a column to dissolve or dislodge accumulated deposits that are causing tailing or adsorption.

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Column Storage

• The column should also be safeguarded when not in use.• There are two storage safeguards of greatest importance:

1. Store the column so it will not be scratched. If scratched, thestress to the column upon heating may be great enough to allow the column to break at the weak point.

2. Seal the column ends to protect the liquid phase against diffusing oxygen and contaminants.

• When using fused-silica columns, remember that fused silica is a glass material and eye protection should be used.

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Column Temperature Operation

• Isothermal:– Oven is maintained at a constant temperature throughout the

analysis.– Stop time is set with the initial time.– Rate is set to "zero".– Excessive broadening of later eluting peaks.

• Temperature Programmed:– Used when components have a wide range of boiling points

(>100°).– Reduces analysis time and produces sharper peaks.– Increased column bleed causing baseline drift.– Can have Multiple Ramps.– 6890 can "Fast Ramp" up to 120°/min.

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NormalRun

BlankRun

ColumnCompensatedRun

Column Compensated Chromatograms

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Lab Exercises

The student should perform the following Lab exercises:Lab 1: Calculating Efficiency and ResolutionLab 2: Column SelectivityLab 3: Oven Temperature Operation and Column Compensation

Optional:Lab 4: Retention Time Locking Demo Tool