Chem 241 Exam 2: Chromatography

Theoretical Plates

N = tr^2/sigma^2 = 16tr^2/w^2 -measures efficiency in separations -measures how good a column is for a separation -N is specific for each solute on a given column

Relating Resolution to Efficiency

R=[sqrt(N)/4][(a-1)/a][k'2/(1+k'avg)] -R is proportional to sqrt(N) -R is resolution -N is number of plates -k' is retention factor or capacity factor (just k in book) -a = relative retention = t'r2/t'r1 = k'r2/k'r1 = K2/K1 * see pg 126 in notes for summary of most equations

Goals of separation

Recovery of product, purity of product

Q: If two compounds are separated on a 1 m LC column with a R1=2, what would resolution be on the same column if its length was 25 cm?

Resolution is proportional to the sq root of plate # New resolution is 1/2 Rs = 1.0

Adsorption Chromatography

Solute adsorbed on surface of stationary face

Partition chromatography

Solute dissolved in liquid phase bonded to the surface of the open tubular column

"General Elution Problem" (pg 148 in notes)

Some peaks elute quickly from column, while others are retained for very long times

Q: If 2 cmpds of drastically different MW but similar polarity are separated in an LC run, one compound's van Deemter B term contributes more to band broadening than the other. What mcl has the greatest B-term contribution to plate height, H?

The smaller MW compound, because it goes through the column slowly.

Types (modes of chromatography)

Type SP MP 1. Adsorption solid liq or gas 2. Partition liq liq or gas 3. Ion-Exchange bonded ions liq 4. Molecular exclusion pores liq 5. Affinity antigen/antibody liq

Q: What is the most commonly used type of detector in LC applications?

UV-Vis absorbance (not mass spec because it's too expensive)

CH 24: Gas Chromatography

Use: drug testing

Retention volume

Vr = tr * F, where F = uv (mL/min) Vr = Vm +KVs -amount of MP it takes to get solute to elute from column -Vm is "dead V," the amount of liquid that goes through column unretained

Anomalous Peak Shapes (non-Gaussian)

Why? 1. Overloading causes "fronting." Put too much sample in column. Too much solute dissolves in SP; SP begins to look like solute and "like dissolves like." Solubility of solute increases. 2. "Tailing" caused by unwanted interactions between solute and SP (or SP support) commonly seen with proteins. Proteins in solutions tend to adsorb in addition to partitioning of silica support not properly coated

Relative retention

a = tr2'/tr1' = k'2/k'1 = K2/K1 -compares adjusted retention times of two solutes -as a increases, separation between two components increases -don't want a to be = 1, because that means capacity factor is the same -want a >1

Q: Which has greatest eluent strength in normal-phase LC? a) 75% water/25% CH3OH b) 50% CH3OH/50% Hexane c) 50% water/50% CH3OH d) 75% water/25% CH3CN e) 75% CH3OH/ 25% Hexane

a) 75% water/25% CH3OH Most polar combo Look at SP

Some common bonded stationary phases for liquid chromatography

amide (R=spacer-(C=O)NH2) amino (R=(CH2)3NH2) cyano (R=(CH2)3C=-N octadecyl (R=(CH2)17CH3 octyl (R=(CH2)7CH3)

Q: Which solvent has greatest eluent strength in reversed-phase LC? a) 75% water/25% CH3OH b) 50% CH3OH/50% Hexane c) 50% water/50% CH3OH

b) 50% CH3OH/50% hexane Two nonpolar compounds Look at SP

Q:Which method(s) could be used to lower the band broadening contribution of the C-term in van Deemter eqn for an OTGC run? a) faster flow rate b) slower flow rate c) thinner SP layer d) thicker SP layer e) B&C f) A&D g) none of the above

e) Slower flow rate and thinner SP layer -In OTGC, there is no A term (packing). -When a solute dissolves, if SP is thick, it gets "stuck" -slower flow rate is better because -Can also increase temperature

Capacity factor vs partition coefficient

k' vs K k' = CsVs/CmVm K = Cs/Cm -K, partition coefficient, compares the concentration of solute in one phase to the concentration of solute in the other phase k' = K(Vs/Vm) : retention coefficient for solute -as k' increases, solute has a greater retention to stay in the column -k' is the same thing as the "retention factor"

Separation factor

lambda = t2/t1 (lambda >1)

Fraction of time solute spends in MP

q = 1/(1+k')

Important Solvent Extraction Eqn 1

q = V1/(V1+KV2) -q represents fraction of S remaining in phase 1 after 1 extraction -remove phase 2 and add fresh phase 2 to repeat extraction 2 times....n times q^2 = (V1/(V1+KV2)^2 ... etc

Fraction of time solute spends in SP

(1-q) = k'/(1+k') or tr = tm(1+k') adjusted retention time, tr' = tr-tm = ts

Ways to increase N (theoretical plates)

*remember H = L/N or N = L/H 1. Increase length of column. H (plate height) should be constant per distance of a uniformly packed column. Practical limit from pressure considerations. Ex: For a L = 10 cm column which has 1000 plates, the same column would have 2000 plates if it is 20 cm long. (H=0.01 cm) 2. Optimize flow rate to minimize H 3. Smaller packing bead diameter gives more uniform packing and decreases A-term contribution to band broadening. Practical limit is determined by pressure required to pump MP thru column and packing technology. Remember high pressure box shown in Jorgenson lab. 4. Thinner SP layer decreases C term contribution to band broadening. The disadvantage of this approach is that it lowers the capacity of the column also 5. OTLC columns have separation efficiency advantages

Problems with "classical" gel electrophoresis

-(CE is better) -very labor intensive -many sources of zone (band) broadening 1. multiple paths in gel media 2. adsorptive interactions 3. joule heating 4. simple molecular diffusion 5. "smiling": middle of gel gets warmer faster and stuff moves more quickly

Size-Exclusion Chromatography

-AKA gel permeation, gel filtration, molecular exclusion -SP consists of small pores that analytes can diffuse into if small enough -larger mcls elute first -liquid MP

GC: OT columns

-Great for eliminating van Deemter terms to reduce band broadening -high resolution, high sensitivity -less sample

History of chromatography (ch 23)

-Greek word "chromatus" for color -Tswett (1903) first separated plant pigments

LLC

-Liquid-Liquid Chromatography -liquid SP -can be both polar or non-polar -solute dissolves!

LSC

-Liquid-Solid Chromatography -based on **adsorption: polar things are attracted to each other -solid SP -greater polarity elutes faster

Gradient elution

-MP composition changes over time generally from a weak eluent strength to a strong eluent strength

Solvent extraction

-Method to transfer dissolved solute from one phase to another -Typically 2 liquid phases, one aqueous (typically top) and one organic (typically bottom)

Disadvantages of Open Tubular vs Packed columns

-OT techniques have smaller sample capacity -longer retention time

Kovat's Retention Index

-Problem: very difficult to get replicated results in GC from two different labs -KRI (I) is a method to standardize GC separations runs between diff labs by using one analyte with lesser retention and one analyte with more retention I = 100*number of carbons for a linear alkane chain

Electrophoretic Mobility

-Rate of ion migration depends on: 1. Size of solute: greater size increases friction, which decreases rate 2. Charge of solute: greater charge increases rate Uep = q/f*E = muep*E ueq = velocity of ion (m/s) q = charge of ion (C) f = friction coefficient E = electric field (V/m) muep = electrophoretic mobility (m^2/Vs)

Temperature Programming

-Used to solve general elution problem -increasing temperature increases volatility; basically boiling sample out faster -more uniformly spaced peaks

Terms used to describe separations

-Vr : retention volume. Volume of MP required to elute a solute -uv=F : volume flow rate (mL/min) describes how many mL of solvent flow through column per min -v or ux : linear flow rate (cm/min) describes how many cm of column length the solvent travels per minute

Eluent Strength (E^o)

-a measure of a solvent's adsorption energy Ex: methanol (CH3OH)>acetonitrile (CH3CN)> Chloroform (CH3Cl)> Hexane (C6H14)

"Salting Out" in extractions

-adding NaCl to aqueous phase. Organic compounds are even less soluble in salt water, and thus the distribution coefficient (or partition coefficient) increases if the solute is in the organic phase

GC: stationary phases

-aka "bonded SPs" - attached to either a packing substrate or to inside surface of capillary (OTGC) -SP must be non-volatile in the oven temp range (if not, it'll boil away and come out of column) -unreactive with solute -mechanically rugged

Band broadening

-as peaks move down a column, they spread out for a variety of reasons -affects the efficiency of a separation

GC: Chemical Derivatives

-chemical derivatives are used to make a compound more recognizable by the detector -ex: TFA with sugars makes both compounds volatile and very detectable by EDC

GC Detector: Flame Ionization Detector (FID)

-destructive detector -only works with species that can be oxidized (carbon compounds, organics) -not universal (selective) -greater sensitivity than TC (1000 x's more sensitive) -linear response good for making calibration curves

GC Detector: Mass Spectrometry (GC-MS)

-determines MW and structural info about compounds -VERY powerful technique -destructive

Reversed-Phase LC (RPLC)

-evolved bc much cheaper to use water as a MP -less polar SP -more polar MP -order of elution: more polar cmpds to less polar cmpds -eluent/solvent strength (MP): more polar MP is weak, less polar MP is strong -RPLC IS THE MOST COMMON FORM OF CHROMATOGRAPHY

Advantages of Open Tubular vs Packed columns

-greater column length, L -greater linear gas velocity -smaller plate height -smaller retention factor, k -much greater number of theoretical plates, N - greater resolution

The basic HPLC setup

-high performance liquid chromatography ("High Price LC") -advantages: high efficiency, high resolution, great quantitation, automated -disadvantages: expense, complexity, a lot can go wrong

Isocratic elution

-keeping the MP composition constant, just changing the gradient (%)

GC Detector: Thermal Conductivity (TC)

-measures a change in voltage as solute passes through detector -universal detector (any source) -non-destructive detector -400pg/mL detection limits -linear response good for making calibration curves -doesn't work well at high flow rates (F)

Ch 23: Intro to chromatography. What is chromatography?

-method to separate components in a mixture based on different distribution coefficients between the two phases -same principle as solvent extraction (like dissolves like), but one phase is "stationary" and one phase is "mobile" -no longer working with organic and aqueous layers in a separatory funnel -working in a column (which is just a tube)

Capillary Electrophoresis (CE)

-migration of ions in solution under the influence of an E field -separation of ions based on differences in mobilities of ions -typically performed on buffer-soaked paper or gel -Cations go to Cathode -order of elution: positive ions, neutral ions, negative ions. Because everything migrates towards the cathode, because it's negative. -really efficient! Can get 10^5 or 10^6 plates (orders of magnitude higher than HPLC)

Ion-Exchange chromatography

-mobile anions held near cations that are covalently attached to stationary phase -anion-exchange resin; only anions can be attracted to it

Normal-Phase LC

-more polar SP -less polar MP -order of elution: less polar cmpds to more polar cmpds -eluent/solvent strength (MP): less polar is weak, polar is strong

CE: very narrow capillary (open tube)

-no A term because tube is open (no packing) -no C term because NO Stationary Phase H = B/ux

Affinity chromatography

-one kind of molecule in complex mixture becomes attached to molecule that is covalently bound to stationary phase -all other molecules simply wash through

GC Detector: Electron Capture Detector (ECD)

-radioactive nickel component to sample -non-linear response -non-destructive -selective for e- capturing solutes (halides, nitro-containing cmpds (explosives) and aromatics) -sensitivity is super sensitive!

Retention factor

-same thing as "capacity factor" k' (or k) = (tr-tm)/tm tr-tm = total time solute spends in stationary phase tm = time solute spends in mobile phase -if k'=0, solute is unretained (flows right through) -if k'=1, solute spent same amount of time in SP as in MP

Molecular exclusion chromatography

-small molecules penetrate pores of particles -large molecules are excluded and elute faster

Ion-Exchange Chromatography (IEC)

-solutes are ionic -MP is liquid -SP is "resins," combos of polymers. Charged groups are covalently bound to the resins -Cation exchangers means negative SP! -Anion exchangers means positive SP! -This is because it's named after the solute ion attracted to the SP -Useful for deionizing water

Affinity Chromatography

-used in immunology to extract and create antibodies -elution of retained solutes is accomplished by changing pH, ionic strength, temp, or other solvent conditions

Type, SP, MP

1. Adsorption: one polar substance sticks to another polar substance. Solid SP, gas or liquid MP 2. Partition: dissolving of 2 similar liquids. Liquid SP, gas or liquid MP 3. Ion Exchange. Bonded anion or cation SP, liquid MP. 4. Molecular exclusion (AKA size exclusion, gel filtration, gel permeation). SP has pores, liquid MP 5. Affinity: for disease research. Antigen/antibody interactions in SP, liquid MP *Also electrophoresis techniques. These are actually diff from chromatography since no SP, but included in discussion b/c separation equations are the same.

Ways to increase N

1. Choose optimal flow rate (minimum H) from van Deemter (bottom point of curve) 2. Smaller and more uniform packing material minimizes A term 3. OT techniques eliminate A term 4. Thinner SP layers in OT and packed columns (affects C) decreases H by decreasing C contribution 5. Longer column

GC Quantitation: Methods to measure peak area

1. Electronic integration (computer) 2. Triangulation (take area of triangle of peak as precisely as you can, gives you 96% of area) 3. "Cut and weigh" peaks from chart recorder

4 GC Detectors to know

1. Thermal Conductivity 2. Flame Ionization Detector 3. Electron Capture Detector 4. Mass Spectrometry - GC-MS

Detection in High Performance Liquic Chromatography (HPLC)

1. UV-VIS: The most common detector!! Pretty universal, works well with gradients) 2. Fluorescence Detection: 100 x's more sensitive than UV-VIS but more selective. Detects against black background. Can label non-fluorescent cmpds with fluorescent tags.

Split-Injection

1/10 of sample goes into column, 9/10 goes into waste. Good for standardizing amount of sample used, and precise. Need a good syringe.

A term in van Deemter

A = 2*lambda*dp -dp = SP particle diameter -lambda = constant; depends on quality of packing -multiple paths (Eddy diffusion) -independent of flow rate -to reduce A: more uniform SP particle packing, smaller particles -to eliminate A: OT technique

Q: In LC, if the SP beads are reduced in size, it has a positive effect on efficiency of the separation. What term in van Deemter is reduced to account for the bulk of this improvement?

A term - "multiple paths" term

Split-less Injection

All of sample is used.

Resolution and Column Diameter (OTGC)

As column diameter decreases, resolution increases. This is because the narrower the column, the greater chance the solute will go into SP

Internal Standards Methods used in GC

Ax/[x] = F(As/[S]) Ax = area or signal from analyte As = area or signal from IS [X] = Concentration of analyte [S] = Concentration of internal standard (IS) F = response factor

B term in van Deemter

B is proportional to 2Dt -D = diffusion coefficient -longitudinal diffusion term (B/ux) -ux = flow rate -smaller time in column allows for less longitudinal diffusion = less band broadening

GC: branching

Branching of molecules makes them elute faster, because they are more volatile and have a lower boiling point.

Applications

Breast milk, water analysis, urinalysis for drug doping

C term in van Deemter

C*ux -resistance to mass transfer -finite time necessary to equilibrate -trade-off btw B&C term in van Deemter -C gets worse as flow rate increases

Important Solvent Extraction Eqn 2 (pH effects)

D for an acid (HA) D=K[H+]/[H+]+Ka D for a base (B) D=KKa/Ka+[H+] -distribution of acids and bases btw two phases is pH-dependent -to find D, need to know pH of solution, Ka of acid or conjugate acid, K of acid or base -Need D if subjects are protonated or deprotonated -K is constant for a solute, while D varies with pH

Selectivity of GC Detectors

ECD > FID > TCD

Mobile phases in GC

Ex: He, H2, N2, Ar NOT O2 - too reactive! Will oxidize things

Two types of GC

GSC (adsorption) and GLC (partitioning)

GC: packed columns

Generally used for preparative (large) scale separations (large amounts of solute).

van Deemter equation

H = A + B/ux + C*ux -H = plate height = L/N, where L is length of column. Therefore, smaller H's (plate heights) correspond to higher N's -A = multiple paths (of solute thru SP) ("Eddy Diffusion"). Independent of flow velocity. Smaller packing beads pack more uniformly and reduce this contribution to band broadening. This is favorable. We want to minimize band broadening so we get the most theoretical plates (N), and hence the most efficient separation. Not a factor contributing to band broadening in open tubular columns. -B = longitudinal diffusion. Inversely proportional to flow rate. Faster flow rates don't give solute band as much time to diffuse (broaden) in the column. -C = equilibration ("resistance to mass transfer"). Proportional to linear flow velocity. The faster MP is moving, the less likely solute has the time to equilibrate with SP. Remember, solute mcls that interact (partition, etc) with SP get "left behind" by those solute mcls in the MP...spreading out the band. Inversely proportional to temp. Increasing temp decreases C...things equilibrate faster at higher temps. Decreasing SP thickness decreases C. -A, B, and C are contributors to band broadening that we want to minimize. **optimum flow rate (max # of plates, min plate height, skinniest peaks) occurs at bottom of van Deemter curve

plate height

H or HETP (height equivalent of a theoretical plate) -constant of proportionality btw the band variances and the distances it has traveled HETP = L/N = Lw^2/16tr^2 -as HETP decreases, resolution (N) increases -units of length

Special applications with special SP: Separation of Enantiomers

In medicine, want to separate enantiomers because one drug works as a pain reliever, while enantiomer might be a fetus deformer

Resolution and Column Length

Increasing column length increases time in the column, but also increases the resolution

Q: What method is currently used in GC and LC when the quantity of sample analyzed or the instrument response varies slightly from run to run?

Internal standards

Partition coefficient, K

K = [S]2/[S]1 = [S]org/[S]aq -as K increases, substance gets less polar -Variables: [S]1 = q(m/V1) = mol/L = M [S]2 = (1-q)(m/V2) = mol/L = M V1 & V2 = volumes of solvents in phases m = total # of moles of solute present q = fraction of solute remaining in phase 1 at equilibrium -K = (1-q/q)(V1/V2)

GC Detectors from most sensitive to least sensitive

MS > ECD > FID > TCD

Ch 25: Liquid Chromatography

Modes: -preparative LC -semipreparative LC -analytical -high performance LC (Liquid/solid and liquid/liquid)