CHEM 241 Exam 2

Mass Spectrometry Detector (GC-MS)

Most common for drug testing; determines molecular weight and structural information about the sample to identify the sample; very sensitive

A term

Multiple Paths Term (Eddy Diffusion) -independent of flow velocity -smaller packing beads pack more uniformly and decrease A, minimizing band broadening -not a factor contributing to band broadening in OPEN TUBULAR columns, only packed columns -more gaps/more dissimilar paths you have, greater the A term

Extraction efficiency

Multiple small extractions are more efficient at removing solute A from aqueous phase than one large extraction

What gases are used for GC carrier gases?

N2, He, H2, and Ar -NOT O2 because too reactive -each have own optimum flow rate -carrier gases must be unreactive with analyte

LLC Mobile Phase

Normal Phase LC Reversed Phase LC

Open Tubular v. Packed Columns

OT columns are more efficient than packed columns but have smaller sample capacities -OT = shorter analysis time, high sensitivity, high resolution, more common analytical columns -Packed columns - used for preparative large scale separations

How do we gauge the success of a separation?

Peaks for all components are present and resolved in the chromatogram

What one main chemical parameter is important in determining the retention time of a solute in a typical LC system?

Polarity

What two parameters/characteristics of the solute affect the retention time in GC?

Polarity and Boiling Point -greater dispersions forces, greater boiling point -largest K = longest retention time = larger MW and polarity is the same as SP -ex: butanol --> pentanol --> hexanol in polar SP

Electrophoretic Mobility

Rate of ion migration depends on size of solute (increase size, increase friction, decrease rate) and charge of solute (increase charge, increase rate) -m^2/Vs

Requirement of Gas Chromatography

Solutes must be volatile (gases/get into vapor phase/low boiling point/high vapor pressure) -when VP equals air pressure, liquid boils

Solvent Extractions based on

Some solvents are not miscible; "like dissolves like"; less dense layer is on the top in two layer system (usually organic layer)

How do we gauge the efficiency of a separation?

The "narrowness of peaks is a measure of efficiency; high efficiency peaks are skinnier

How is efficiency in separations measured?

Theoretical Plates (N) -more plates in a column, the better the separation -N= 16tr^2/w^2 -specific for each solute on a given column

Migration time

Time it takes a solute to reach the detector in CE

What electrode to particles move toward in CE?

Toward the cathode; detector is on the cathode side of the capillary; moves in one direction

How does ion exchange chromatography work and what are some common applications?

Water purification/Deionizing water - Ions are detected and replaced with OH- and H+ ions

tr'

adjusted retention time; tr' = tr-tm; also seen as ts

What characteristic of a peak in chromatography is directly proportional to concentration of the solute?

area

Bonded SP

attached to either a packing substrate (bead) or to the inside of surface of the capillary (OTGC)

relative retention (a)

compares adjusted retention times of two solutes -increase a, increase separation between two components, want to be >1 but cannot =1 -a = k'2/k'1 = K2/K1 = tr'2/tr'1

tm

dead or void time, unretained/doesn't interact with the stationary phase (always stays the same)

What is the relationship between N and H?

decrease H, increase N H = L/N

Resolution and Column Diameter (OTGC)

decrease diameter, increase resolution because as make column more narrow, solute is closer to the stationary phase and can get through the column better

Volume flow rate (uv = F)

describes how many mL of solvent flow through column per minute

Linear flow rate (v or ux)

how many cm of column length the solvent travels per minute

What is the relationship between peak width and the diffusion coefficient of a solute?

increase D, decrease N, increase peak width -smaller molecules have larger D and greater peak width -larger molecules have a smaller D and narrower peak width

How to increase N in HPLC

increase flow rate, increase length of column, change polarity of solvent, decrease particle diameter

Eluent Strength trends in LSC

increase polarity, increase eluent strength -more polar MP have stronger eluent strength in LSC (because LSC have polar SP)

How can be speed up the electroosmotic flow in a CE setup?

increase voltage, change pH -increasing voltage however, generates heat and increases longitudinal diffusion (decrease efficiency)

Electrophoresis

migration of ions in solution under the influence of an electric field; separations of ions based on differences in mobilities of ions; typically performed on buffer-soaked paper or gel -ions migrate based on size and charge

C18

most non polar SP in LC

Band Broadening

peaks move down the column and spread out for a variety of reasons -affects the efficiency of the separation by lowering N (decreases efficiency)

What compounds come out first in an LC separation done on a C18 SP column?

polar (because non polar SP, so non polar solutes will be retained the longest)

Partitioning

preferential dissolving based on polarity; partition between two layers

Goals of Separation

recovery of product, purity of product

tr

retention time, longer in column the more interactions with stationary phase

LC sample injection

sample loop rotates to column/waste/etc. -load vs inject position

HPLC basic setup

solvent reservoir --> pump --> injector --> column --> detector --> computer

Resolution

the ability to differentiate between two adjacent peaks on a chromatogram -want resolution to be big so can separate more things -R = 0.589dtr/w1/2av = 2(tr2-tr1)/w1+w2 -IDEAL R = 1.5 (baseline resolved)

What does it mean in a CE setup if a negatively charged solute molecule never makes it to the detector?

the apparent mobility is negative since the electrophoretic mobility is less than the electroosmotic mobility, so the anion will stop migrating

capacity/retention factor (k')

time spent in two phases, number of moles of solute partitioned between two phases, how long solute retained in the column -increase k' = solute retained longer -k'=1, solute spent same amount of time in SP as MP

Why are a heated injector port and column oven necessary in GC?

to volatile sample when injected and to keep solute in the vapor phase

Retention volume (Vr)

volume of mobile phase required to elute (come out) a solute

Rank the solvents in the terms of polarity

water > methanol > ethanol > acetonitrile > THF > chloroform > hexane

LC pump

works similarly to piston pump to insert solvent from the reservoir into the system

Ways to Increase N

(1) Choose optimum flow rate (where H is minimum) (2) Smaller and more uniform packing material or no packing material (OT) - eliminates A term (3) Thinner stationary phase layer in both OT and packed columns decreases H by decreasing C (can equilibrate faster) (4) Longer column gives more theoretical plates

Salting Out in Extractions

-Add NaCl to aqueous layer -Organic compounds are less soluble in salt water -Increases the distribution coefficient (or partitioning coefficient) if the solute is in organic phase

LSC Stationary Phase

-Based on adsorption -solid SP -SP: high porosity with high surfaces for interaction with mobile phase -ex: polar SP - silica gel, alumina

UV-VIS Detector

-Common -linear -UNIVERSAL (solvent adsorption limited) -Works well with gradients -Sensitive but not most sensitive; must choose max wavelength

Flame Ionization Detector (FIT)

-DESTRUCTIVE -NOT UNIVERSAL; only works with species that can be oxidized (burned) = organics, carbon compounds (SELECTIVE) -1000x more sensitive than TC -linear response

What are some advantages of capillaries for electrophoresis?

-Efficient heat dissipation because very small, heat comes off more easily -"wall effect" provides convective stabilization (no convective flow link in gel) -ease of use

Plate Height (H or HETP)

-Height Equivalent of a Theoretical Plate -constant of proportionality between the band variances and the distances it has traveled -uniform across the column -H=L/N -decrease H, increase N, increase resolution -source of band broadening when H increases

LLC Stationary Phase

-LLC most common version of LC -mode of separation is partitioning -liquid SP (can be polar or non polar) -bonded stationary phase - liquid coated onto or chemically bound to exposed silanol groups of silica (solid support)

Thermal Conductivity Detector (TC)

-MOST COMMON -measures a change in voltage as solute band passes through detector -UNIVERSAL -NON-DESTRUCTIVE -400pm/mL detection limit -linear response -doesn't work well at high flow rate, less time to equilibrate -uses a hot filament

LSC Mobile Phase

-MP displaces solvent from SP; make MP stronger by making more similar to SP -Strength of MP = how well it makes things come out of the column/not retained -Eluotropic series - ranks the relative ability of solvent to displace solute based on pentane -ELUENT STRENGTH - a measure of a solvent's adsorption energy; the greater the eluent strength of the solvent (MP), the faster the solutes will be eluted; refers to the strength of the MP = how well it makes things come out of the column

Electron Capture Detector

-NON-DESTRUCTIVE -non-linear -Selective for electron capturing solutes (halides, aromatics, and nitrogen containing compounds); NOT UNIVERSAL -MOST SENSITIVE

What makes CE such a high efficiency separation technique?

-No A term because tube is OPEN (capillary filled with a buffer) -No C term because there is NO SP (no partitioning occurs) -band broadening depends on longitudinal diffusion entirely (B term)

Kovat's Retention Index

-Problem: very difficult to recreate/get replicate results in GC from one lab to the next because of the differences in parameters -KRI is a method to use standards to create a standardized GC separation run -Solute KRI is based on two standards, one of longer and one of shorter retention time

Size Exclusion Chromatography (GPC Gel Permeation Chrom./Molecular Exclusion Chrom.)

-SP consists of small pores that analyses can diffuse into, if they are small enough -used to determine molecular was and to purify systems based on molecular size -larger molecules cannot diffuse into smaller pores so they elute before small molecules -Liquid MP, Porous gel SP -separates based on size, roughly correlates with MW -typical SP is cross-linked, increase cross-lining, decrease pore size

Fluorescence Detector

-SUPER SENSITIVE -Selective - only certain compounds can fluoresce (not universal) -100x more sensitive than UV-VIS -can be used with gradients -can label non-fluorescent compounds with fluorescent tags -linear

General Elution Problem - GC

-Some peaks in typical GC run elute quickly from the column while other care retained for a very long time -Solution: Temperature Programming - increase temperature over the course of the run so that more peaks are present at a shorter amount of time (pressure programming also possible) -increase temp, decrease tr, sharpens peaks

General Elution Problem - LC

-Some samples come out early, some later, and some never come out -Solution: Gradient Elution - change the mobile phase composition with time; slowly make the MP similar to the SP -weak eluent strength to strong eluent strength

Normal Phase LC

-Stationary Phase - Polar; Mobile Phase - Nonpolar -order of elution: non polar to polar -eluent/MP strength: non polar is weak, polar is strong -strongest solvent = water

Refractive Index Detection

-Universal but poor sensitivity -Linear -not used with gradient

SEC Variables/Equations

-Vr = retention volume -Vm = total volume inside and outside of pores -Vo = volume of MP outside of SP pores -Vi = volume inside pores -For large molecules, K=0 and Vr = Vo -For small molecules, K=1 and Vr=Vm -K = Vr-Vo/Vm-Vo

LC columns

-generally packed columns although capillary columns are gaining popularity in certain analyses -decrease particle diameter, increase N, increase R -each particle diameter has an optimal flow rate based on plate height -guard columns important to extend life of an analytical column

Stationary Phase Factors in GC

-like dissolves like, solute interacts with SP longer when of same polarity ex: pentanol retained longer in polar SP -boiling point/volatility - as compound gets bigger in absence of OH, should be retained longer even in polar SP

Reversed Phase LC

-most common -SP - non polar; MP - polar -order of elution: polar to non polar -eluent/MP strength - polar is weak, non polar is strong -strongest solvent = hexane

Ion-Exchange Chromatography (IEC)

-solutes must be ionic/have charge -liquid mobile phase -Resins - amorphous particles of organic materials; combination of polymers -charged sites covalently bound to SP; named after solute ion ATTRACTED to SP -Anion exchangers: positively charged groups on SP (RNR+, RNRH+) -Cation exchangers: negatively charged groups on SP (RSO3-, RCO2-)

What is the difference between split and splitless injections in GC? When might you use each?

-split injections - mechanism by which a portion of injected solution is discarded; only a small portion of sample goes through the column; used for CONCENTRATED samples; small volume introduced into the column -splitless injection - most of sample goes through the column, used for diluted samples; trace-level high-boiling solutes in low-boiling solvents

What is affinity chromatography? How does it work? Where is most commonly used?

-used to isolate a single compound from a complex mixture -use a very specific binding interaction to selectively bind a particular analyte to the SP -antibody-antigen or enzyme-substrate -most common in immunology -Release is accomplished by changing pH, ionic strength, temperature, or other solvent conditions (usually pH); causes antigen to pop off when protein is denatured

Problems with Classical gel electrophoresis

-very labor intensive -many sources of band broadening: multiple paths in gel media, adsorptive interactions, joule heating, simple molecular diffusion - smiling - function of the heating; heat dissipates better on the edges but in the mddiil it has a greater effect

non Gaussian Peak Shapes

1. Fronting - overloaded peaks; too much solute dissolves in the SP and SP starts to look like solute 2. Tailing - when unwanted interactions between solute and SP take place (and SP support)

What are the 3 types of detectors for GC?

1. Thermal Conductivity 2. Flame Ionization Detector 3. Electron Capture Detector

What are the 3 most common detectors in LC?

1. UV-VIS Detection 2. Fluorescence Detection 3. Refractive Index Detection

Detection in CE

1. UV-Vis 2. Fluorescence - most sensitive 3. Mass Spectrometry

Types of Chromatography

Adsorption - SP: solid, MP: gas/liquid Partitioning - SP: liquid, MP: gas/liquid Ion-Exchange - SP: bonded anion/cation, MP: liquid Molecular Exclusion - SP: has pores, MP: liquid Affinity - SP: antigen/antibody interactions, MP: liquid Electrophoretic - no SP

Anode v. Cathode (Electrophoresis)

Anode - positive electrode; anions move towards anode Cathode - negative electrode; cations move toward cathode

Flow Profiles

CE - Plug flow: flat, no band broadening Other LC techniques - Laminar flow: push a liquid through a tube because of friction --> band broadening

Classical v. Capillary Electrophoresis

Classical: gel or paper medium, electrophoretic velocity, poor resolution and heat dissipation Capillary: instrumentation, electroosmotic flow, apparent mobility, separation efficiency

Distribution ratio/coefficient (D)

D = K[H+]/[H+]+Ka (acids) D = KKa/Ka + [H+] (bases) -for organic acids and bases/pH effects

What is the difference between electrophoretic mobility and apparent mobility in CE?

Electrophoretic mobility - constant of proportionality between speed of an ion and electrical field strength; changes based on charge/amount of charge Apparent mobility - sum of electrophoretic mobility and electroosmotic flow for buffer

C term

Equilibration ("resistance to mass transfer") Term -proportional to linar flow velocity; increase ux, increase C -the faster the mobile phase is moving, the less likely solute has the time to equilibrate with SP; solute molecules that interact with SP get left behind by solute molecules in MP --> spread out band -inversely proportional to temperature, increase temp, decrease C because things equilibrate faster at higher temperatures -decreasing SP thickness decreases C

Electroosmotic Flow

Flow of entire solution toward the cathode; this is due to fused silica capillary has exposed silanol groups with have a pKa ~ 2 which means that at pHs about this there is a negative surface charge, so a layer of positive charges to build up near the surface and these are "pulled" toward the cathode -if you decrease pH to 1, electroosmotic flow decreases and almost stops but does not go towards anode

Ideal Flow Rate

Found on Van Deemter Plot -necessary because trade off between B and C term, so need ideal flow rate to maximize N and decrease band broadening as much as possible

GSC v. GLC

GSC - solid SP (adsorption) GLC - liquid SP (partitioning)

Peak Shape

Gaussian Peak Shape -w = width of peak at base -w1/2 = width of peak at half-height

Why is it impractical to have a really large relative retention between two solutes in a separation?

General Elution Problem - have to wait too long for peaks to elute

Van Deemter Equation

H = A + B/ux + Cux -describes sources of band broadening

Gas Chromatography Instrumental Setup

Heated injector port (thru septum) --> injector over --> column oven with capillary column --> detector oven --> detector --> exit

GC Stationary Phases

Ideal Characteristics: must be nonvolatile in the temperature range of the oven, must be unreactive with the solute, mechanically rugged -should buy a column based on polarity application needed

Extreme Basic Conditions

If [H+] << Ka, pH >> pKa, then D=K If [H+] >> Ka, pH << pKa, then D=0 (distribution curve)

Extreme Acidic Conditions

If [H+] >> Ka, pH << pKa, then D=K If [H+] << Ka, pH >> pKa, then D=0 (distribution curve)

How can you increase the number of theoretical plates in CE?

Increase voltage

What is the difference between Isocratic and gradient elution?

Isocratic - composition of the mobile phase stays the same over time Gradient - composition of the mobile phase changes to become more similar to the stationary phase over time

Difference between K and D

K is constant for a solute; D varies with pH

Partition Coefficient (K)

K=[S]org/[S]aq -higher the K, the less polar the solute/compound -K>1: compounds prefer non polar phase/are nonpolar

High Performance Liquid Chromatography (HPLC)

LSC - liquid MP, Solid SP (adsorption) LLC - liquid MP, liquid SP (partitioning) - most common -advantages: high efficiency, great quantitation, automated -disadvantages: expense, complexity -flow rate is a few mL/min or less

B term

Longitudinal Diffusion Term -inversely proportional to flow rate -faster flow rates do NOT give solute band as much time to diffuse (broaden) in the column -increase flow rate, decrease longitudinal diffusion, so decrease B and increase N -B proportional to 2Dt; solute bandwidth proportional to square root of t

What is Chromatography?

Method to separate components in a mixture based on different distribution coefficients between two phases; "like dissolves like" but one phase is "stationary" and one phase is "mobile"

What is the internal standards method?

Method used commonly in gas chromatography; used when sample is not reproducible