Definition and Overview

Chromatography
Chem 221 Instrumental Analysis Spring 2003

Techniques based on the physical and/or chemical separation of 2 or more substances in a mixture Scope and Purview: -Theoretical Basis -Instrumentation -Qualitative and Quantitative Methods
We will focus on Gas Chromatography (GC) but will also make reference to High Pressure Liquid Chromatography

(HPLC)

Theory: Basis for Analytical Separations

Separations are effected due to differences in substances affinities for a mobile phase and a stationary phase. Mobile Phase - typically a liquid (LC) or a gas (GC) Stationary Phase - typically solid or a liquid
Sample Mixture A B (A & B)

The Partition Coefficient (K)

Allows us to quantify the distribution of a compound between the stationary and mobile phases:

K = Cstationary/Cmobile
large K = more time spent in stationary phase = more time spent on the column

Mobile Phase
Stationary Phase Solid Support

increased elution time = larger K

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Assume that K is constant for a compound under chromatographic conditions (thermodynamic constant)

Sample Chromatogram
Retention time

Retention Behavior
tR: not a consistent measure of a compounds
Base Peak Width

(thermodynamics)
Unretained species (K=0)

relative affinity for stationary and mobile phases

-varies with flow rate, temperature, etc.

(kinetics)

Define a new term: Capacity Factor (k)

k = (tR - tM)/tM = tR/tM = k

Adjusted retention
time
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Capacity Factor (k)

Gives a normalized measure of retention Easily calculated from chromatogram Relates directly to the Partition Coefficient:

Bandbroadening
Affects peak width (W) Governed by kinetic processes We need to consider the effects of: -Diffusion
-Eddy Diffusion -Molecular Diffusion -time it takes to partition between the stationary and mobile phases

k = K(VS/VM) = nS/nM
A thermodynamic property.

-Mass Transfer

Eddy Diffusion
Each compound species travels a different path through the particles:
Applies only to a column packed with particles (solid support onto which the stationary phase is adsorbed)

Molecular (Longitudinal) Diffusion

Consider the effect of just a solute in a column filled with just mobile phase (no packing) :
Mobile Phase

AA AA A AA AA
Initial

A AAA A AAA
Final

How affected by:

Particle Size? Mobile Phase Flow Rate? ( as dp ) (rel. independent)

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How affected by:

Diffusion Coeff (DM)?

( DM )

Flow Rate (u)?

( u )

-ALSO: DM varies with mobile phase temp., MW, and viscosity 10

(Stationary Phase)
The partitioning process takes time
-so, the rate at which a compound partitions into and out of the stationary phase will affect bandbroadening

Mass Transfer

More Mass Transfer

Stationary phase mass transfer rate varies with: DS - stationary phase diffusion coefficient K - partition coefficient
-increased K gives increased rate (decr. broadening) -increased DS gives increased rate (decr. broadening)

A A

df - stationary phase thickness - increased df gives decreased rate (incr. broadening) -A faster partitioning process results in decreased bandbroadening
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u - mobile phase flow rate

- increased u gives decreased rate (incr. broadening)

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Still More Mass Transfer

Also must consider Mobile-Phase Mass Transfer:
-varies with only two properties:

Theoretical Plates
Concept derives from distillation theory: The height of a theoretical plate is the length of column in which the equivalent to a single equilibrium separation is achieved.
So, column efficiency can be gauged by the height of a theoretical plate (H) And, the separation power of a column can be assessed by the number of theoretical plates (N), where:

DM - mobile phase diffusion coefficient dp - packing particle size

-increased DM gives increased rate (decr. broadening) -increased dp gives decreased rate (incr. broadening)

How can we quantify each of these band broadening components?

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N = L/H
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Calculating N
The number of theoretical plates on a column is easily calculated from a chromatogram:

The van Deemter Equation

Relates column separation efficiency to bandbroadening components as a function of mobile phase linear flow velocity:

N = 16 (tR/W)2
-assumes that peaks are Gaussian -specific to a particular compound on that column For non-Gaussian peaks (fronted or tailed peaks):

H = A + B/u + (CS + CM)u

Eddy Diffusion Longitudinal (molecular) Diffusion
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N = 41.7(tR/W0.1 1.25 + B/A

)2

Asymmetry Ratio (ratio of base widths on either side of maximum)

Mass Transfer (stationary and mobile phase)

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The van Deemter Plot

HHPLC ~ 10x smaller than HGC

Resolution
BUT: NGC > NHPLC

uopt (gives Hmin)

Overall

separation . . . How do we define resolution?

This is the most critical figure of merit for a

(LGC >> LHPLC)

RS = (2Z)/(WA+WB)
Mass Transfer Eddy Diffusion Longitudinal Diffusion
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Z = (tR)B - (tR)A

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RS Values versus Separation

RS = 0.75 RS = 1.0
(4% overlap)

RS as a Function of Column Properties

If we:
Assume that WA WB Express RS equation in terms of tR Substitute in k and N where appropriate We obtain:

Completely (baseline) Resolved

RS = (N1/2/4) (( - 1)/) (kB/(1 + kB)

= Selectivity Factor = KB/KA = kB/kA = (tR)B/(tR)A

RS = 1.5
(0.3% overlap)
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Relationships with RS
We can solve the RS equation for N:

N and tR:

What Do We Want?
The object is to: Obtain the MAXIMUM RESOLUTION In the MINIMUM TIME

N = 16 (RS)2 (/( - 1))2 ((kB + 1)/kB)2

Even more rearranging and substituting allows calculation of the retention time:

-alas, resolution and time typically work against

each other Lets look at how N, k and affect these two separation qualities
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(tR)B=16(RS)2 (/(-1))2 ((kB+1)3/(kB)2) (H/u)

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Effect of Changing N
Best resolution is obtained with MAXIMUM number of theoretical plates (RS N1/2) How can we increase N?
Optimize mobile phase flow rate (u) Increase the column length (L) -BUT: both methods also increase the retention time

Effect of Changing k
Resolution increases with increasing k
How can we increase k?
Recall that k is related to the thermodynamics of the partitioning process

Change:

DECREASE the height of a theoretical plate (H) -increases column efficiency -doesnt sacrifice time (tR H)
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Temperature (GC) Mobile phase composition (HPLC) Stationary phase composition

BUT: retention time ALSO changes with increasing k

HOW?

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Optimum k
How do RS and tR vary as a function of capacity factor (k) ?
Largest increases in resolution occur with

The Selectivity Factor ()

Optimizing k does not ensure that two compounds will be resolved (they could have identical k values) Need to consider : = 1 k
Keep k >1, but also k <10 GC: vary temperature HPLC: vary mobile phase composition Temperature
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B A

k<5

Largest increases in retention time occur with k>5

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The General Elution Problem

How does one resolve ALL compounds in a mixture in which there is a wide range of k values?

The General Elution Solution!

For GC: dynamically vary temperature as separation progresses 45 oC

145 oC

Temperature Programming

30 - 180 oC

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The HPLC Solution

dynamically vary
mobile phase composition as separation progresses For HPLC:

Instrumentation
Fairly straightforward:

Gradient Elution
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25 - 400 oC
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Mobile Phase Supply & Delivery

Usually use He or H2 -more efficient at high flow rates than N2 20 - 100 mL/min flow rates typical -control with flow controller -measure with soap-bubble meter

Sample Injection
For GC:
Syringe/Septum system At temperature > column temperature Injection volume: 0.2 - 10 L Nanoliter volumes for open

For GC:

tubular columns

For HPLC:

For HPLC:
Sampling loop/Injection valve
Injection volume: 5 - 500 L
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must be degassed and filtered 0.1 - 10 mL/min flow rates typical

PUMP: pulse-free, high-pressure (6 - 10,000 psi)

Nonpolar

Polar

Columns
Size
Packed (GC) 1 - 3 meters long 1 - 5 mm I.D. Open Tubular (GC) 10 - 100 meters long 0.1 - 0. 3 mm I.D. HPLC (packed) 100 - 300 mm long 4 - 10 mm I.D.

More Columns!
Support
Packed (GC)
Glass, Stainless Steel, Copper <150 m diatomaceous earth

Stationary Phase
High B.P. (stable at column temps) Non-reactive Suitable K for analytes Packed (GC)
Coated onto support 1-10% of support mass Nonpolar Polar

Open Tubular (GC)

Glass, fused silica

1 - heptane 4 - propanol

HPLC (packed)

Stainless Steel 3 - 10 m

Open Tubular (GC)

Coated onto wall
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Still More Columns . . .

Plates
Packed (GC)
~2,000 plates/meter 2 - 6,000 plates/column

Detectors
Well consider three types:
Thermal Conductivity Detector (TCD) Flame Ionization Detector (FID) Electron Capture Detector (ECD)

Sample Volume
Packed (GC)
microliters

Open Tubular (GC)

~2,000 plates/meter 20,000 - 100,000 plates/clmn

Open Tubular (GC)

nanoliters

Classify with respect to:

Selectivity Mass Flow versus Concentration based response Linear Dynamic Range Detection limit Destructive?
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HPLC (packed)
~50,000 plates/meter 5,000 - 15,000 plates/column

HPLC (packed)
microliters

Thermal Conductivity Detector (TCD)

Measure change in thermal conductivity due to analyte gases eluting from column (a differential measurement) How? -pass effluent over wire (tungsten or tungsten-rhenium alloy), which is heated by a small current passing through it -temperature of wire (measured as a change in resistance) changes as the thermal conductivity of the effluent changes -signal is based on a change in the temperature (resistance) of the heated wires -so, use a carrier gas with a VERY LARGE thermal conductivity (relative to most compounds): He or H2
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TCD Operating Characteristics

Simple Universal (sensitive to almost ALL compounds) LDR ~ 104 Non-Destructive Detectability: ~10-9 grams (~10 ppm) Concentration-Based Signal

-only useable with packed columns

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Flame Ionization Detector (FID)

Ionize compounds in a flame and measure current due to ions produced
HOW?
H2/Air flame Positive electrode to collect electrons released by ionization of analyte
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FID Operating Characteristics

Simple Selective (specific to organic compounds) LDR ~106 Destructive (sample is burned) Detectability: ~10-11 grams (~50 ppb) Mass-Flow dependent signal

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Electron Capture Detector (ECD)

Measure the decrease in electron flow from a source due to the capture of electrons by eluting compounds How?
63Ni

ECD Operating Characteristics

Complex (needs radioactive source, etc.) Selective (specific towards compounds with electronegative functional groups) LDR ~ 102 - 104 Non-Destructive Detectability: ~10-9 - 10-12 grams Concentration-dependent signal
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e.g., organometallics, halide-containing compounds (pesticides)

- + N2 2e- + N2+
(gives about 10-8 amps) A + e- A- (decreases baseline current)

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Other Detectors
For Gas Chromatography
Mass Spec (well cover later) Infrared Absorption Spec Plasma Emission Etc. Refractive Index (universal) UV/Vis Absorption Spec Fluorescence Spec Conductivity Mass Spec Etc.
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Applications
Qualitative Analysis -retention times (tR) provide some qualitative info regarding species identity:
Match tR values of unknowns with values from standards run under identical conditions BUT: matching tR values do not necessarily indicate identical compounds: tR values should match when run under different experimental conditions (i.e., change temperature)
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For HPLC

Example

More Qualitative Analysis

Biggest limitation: detector selectivity Solution: increase dimensionality of detection
Example: dual detectors

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Kovats Index System

compounds: e.g., homologous series of straightchain hydrocarbons
Retention Index (I):
Define Kovats
Log tR

Quantitative Analysis
Use peak area as the signal that correlates best with analyte concentration DIRECT ANALYSIS
Calibration Curve: plot peak area as a function of amount (volume or concentration) of analyte (from standards)

Provides a quantitative measure of retention Calibrate retention using a series of standard

Pentane x x

I = 100 n
So, I=500 for Pentane Use to quantify retention behavior relative to a set of standards x

Determine amount of analyte in unknown by relating its peak area to the amount of analyte on the calibration curve plot

injection uncertainties
# carbons (n)
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PROBLEM: large signal variations (poor precision) due to

Variable injection rates Volume uncertainty Pre-volatilization
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Solution: Internal Standard

internal standard) to all samples and standards Normalize all standard and sample peak area values to the internal standard peak area values
IF the internal standard behaves like the analyte, then it should experience the same conditions and exhibit the same signal fluctuations Internal standard should have similar retention behavior and volatility as analyte
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Example: Internal Standard

7 6 5 4 3 2 1 0

Add a known/fixed amount of a compound (the

37% RSD Sample Int. Std. Ratio

1 2 3 4 5

37% RSD

0.04% RSD
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Replicate Number

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Area Normalization
If all compounds elute and can be quantified:
Measure peak areas for all compounds Correct peak areas based on relative detector
Due to cmpd specific detector response Calibrate using standards with known concentrations

response factors

Calculate results: % composition = Cmpd Pk Area 100 Cmpd Pk Areas

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