chem. 230 – 10/28 lecture. announcements i hw set #3 – due today (short answer problems have...

Chem. 230 – 10/28 Lecture

Announcements I• HW Set #3 – due today (short answer problems

have been posted)• Next Exam: Topics + format (still can bring 3” x

5” notecard)– Gas Chromatography– Supercritical Fluid Chromatography– HPLC (everything except detection)

• Return Application Abstracts– missing a few– most looked good; a few seemed to be focused on

technology rather than application to a particular problem (check to see if you need to improve on your abstract)

Announcements II

• Today’s Lecture– HPLC

• covered so far: classification, packing material geometry and composition, gradient elution, size exclusion chromatography, ion exchange chromatography

• Instrumentation (mobile phase selection and delivery, injection, column dimensions, detection)

• Aerosol-Based Detectors (in more detail)

Liquid ChromatographyInstrumentation – Mobile Phase

Delivery• Mobile Phase Selection

– See slide 18 of lecture for factors influencing selection of mobile phase

– Solvents must meet purity requirements (for column and detector functions)

– Solvent selectivity issue is important because:• Changing solvent affects retention for different

analytes differently• HPLC is less efficient than GC so often more likely to

have overlapping peaks• Changes in pH also are important for acidic/basic

compounds


Delivery

R

CH3CH3 OO

OH

R

CH3O

OH

HPLC-UV Sample 1 (ACN/0.1%TFA)

-500

50100150200250300350

0 5 10 15 20

Time (minutes)

Abs

orba

nce

acetovanilloneacetosyringonecinnamic acidisoeugenolsyringic acid

• Example of solvent changes to affect selectivity:– RP-HPLC Separation of

syringols from guaiacols– Difference is in 2nd MeOH

group– Water/Acetonitrile eluents

produce poor syringol/guaiacol separation factors

– Water/Methanol works better (although greater retention with MeOH of syringol is counter intuitive)

Syringols Guaiacols

HPLC Sample 1 (MeOH/0.1%TFA)

-500

50100150200250300350

0 5 10 15 20

Time (minutes)

Abs

orba

nce

acetosyringoneacetovanillonecinnamic acidisoeugenolsyringic acid

More retention

Less retention


Delivery• Optimization of Mobile

Phase Composition– Separation should be

perfomed on three different water/organic systems

– Then additional separations can be carried out using 3 component mobile phases

– Patterns in retention can be used to optimize mobile phase composition

Acetonitrile (40% in water)

Methanol (50% in water)

THF (30% in water)

20% ACN, 25% MeOH, water


Delivery• Mobile Phase Selection –

pH Buffering– In reversed-phase HPLC,

solute generally must be non-ionized to be retained

– pH is adjusted by adding buffer in water/organic modifier

– pH at pKa means retention factor about half of non-ionized acid retention factor

– In ion-exchange chromatography, pH should be in range needed to produce ions

– In ion-pairing RP-HPLC, an ion-pairing reagent is added

O

OH

O

O-

retained unretained

NH2

Benzyl amine (conj. acid pKa = 9.35)

Non-ionized only at high pH

NH3+

O

O

O-

S

CH3

Ion pair reagent = pentane sulfonic acid (sodium salt)


Delivery• Solvent Flow

– HPLC requires high pressures and thus specific pumps

– The solvent also needs low levels of dissolved gases for pumps to function (through solvent degassing)

– For the simplest “dedicated” HPLC, a single solvent reservoir and pump is needed

– For gradients and/or more method development work, switching between different solvents is needed


Delivery• Pumps

– Most pumps use two piston heads 180º out of phase to reduce pressure fluctuations

– Solvents go into and out of piston heads through one-way “check valves”

– Exit check valve closes on “in” stroke and entrance check valve closes on “out” stroke

pistons

Check valves Out Stroke

open

closed

closed

open

In Stroke


Delivery• Example of pump with

non-functioning check valves

• Fluctuation in pressure and signal can occur

• Changes to retention time also will occur

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

7 7.5 8 8.5 9 9.5 10

Time (min)

Sig

nal

(u

V)

-300

-200

-100

0

100

200

8 8.2 8.4 8.6 8.8 9 9.2

Time (min)

Sig

nal

(u

V)

Bad check valve leaking


Delivery• Solvent Flow (for gradient/greater

flexibility operations)– Dual Pumps (high pressure mixing)– Low Pressure Mixing (stream “open” in proportion to

fraction)

To column

pumps

To column

Mixing chamberpump

Liquid ChromatographyInstrumentation – Injection

• Fixed Loop Injectors (see GC slides for diagram)– Used in almost all cases

– For some injectors, partial filling of loop is possible (V inj < Vloop), but then filling precision must be good

– Special injection valves needed for small injections (< 1 to 5 μL)

– Small injections often needed for microbore columns

• Other Injectors– Traps replace loops (can be used if sample is in weak solvent)– SPME (not as common as for GC but with solvent removing

trapped compounds)– SPME requires special injector

Liquid Chromatography

Instrumentation – Injection• Sample Matrix– Best chromatography solvent – should be weaker than

mobile phase, particularly for larger volume injections– Remember, weaker solvent allows on-column

concentrating– With traps, sample must have weaker solvent, but must

be pulled off with significantly stronger solvent so pulled off in narrow injection plug

– Other concern can be solvent miscibility and solute solubility (example: in reversed phase HPLC, water is a good solvent, but many compounds such as aromatic compounds have limited solubility in weakest solvents)

Liquid ChromatographyInstrumentation – Columns

• Column dimensions– Length: balance between

flow, pressure and efficiency– Diameter:

• Choice depends on separation purpose

• Preparative for isolation of larger quantities

• Microbore usually results in smaller mass detection limits but greater concentration detection limits (good when limited sample)

• Special care is needed using microbore with sample injection, pump stability, and extra-column broadening (tubing diameter and fitting connections)

Type Diameter (mm)

Typical Flow Rate (mL/min)

Preparative

>7.8 > 3

Analytical 4.6 1

Microbore <1 < 0.05

Liquid ChromatographyInstrumentation – Columns

• Column dimensions– Equation for extra-column broadening:

– Extra-Column broadening is more of a problem when using 1) low H columns, 2) early eluting peaks (where Wcol is small)

– Demonstration of Extra-Column Broadening on Narrowest Bore Columns

– note: W can have time or volume dimensions, but hard to get very small volume W for tubing, and some detection

2det

2222ectortubinginjcoltot WWWWW

Liquid ChromatographySome Questions

1. A student is running a RP-HPLC separation using methanol and water. The selectivity ( value) is not good. He decides to switch to ethanol in water. Is this a good decision?

2. A chemist is planning on purchasing an HPLC instrument for developing isocratic analysis methods. Is there an advantage to being able to select multiple solvents?

3. In order to decrease H in a column, which column or packing material dimension should be changed? and in which direction?

4. Why would one want to go to a microbore HPLC system?5. Why is the decrease in H observed often less than predicted

when using smaller diameter packing material or small diameter columns?

6. If injecting large volumes of a sample containing trace levels of benzoic acid in water for a reversed phase separation, will it make any difference what the pH of the sample is?

7. In anion exchange chromatography, what type of sample would allow on-column trapping? What type of samples would give broad peaks if using large injection volumes?

Liquid ChromatographyInstrumentation – Detectors

• Some Generalizations– Relative to GC, HPLC detectors perform poorly and cost

more• Universal Type

– UV absorption (also considered selective)– Refractive Index– Aerosol-based detectors (will cover later)– Conductivity (for ion chromatography)

• Selective Type– Fluorescence– Electrochemical

• Hyphenated Detectors– Photodiode Array Detector (type of UV detector)– Mass Spectrometer


• UV Absorption Detectors– The most common type of detector– Principle: absorption of ultraviolet (or visible) light– Follows Beer’s Law: A = -log(I/Io) = εbC

• I = intensity of light (Io for blank)• ε = molar absorptivity (constant)• b = path length• C = concentration

– Best results for 0.001 < A < 1 – Fast response – sensitivity trade off in path length

(can select cell volumes)

Cell

b

Light beam


• UV Absorption Detectors– Sensitivity to Compounds (ε values)

• Best for compounds with conjugated double bonds, aromatic groups or strongly absorbing functional groups (e.g. R-NO2, R-I, R-Br)

• Poor response for compounds with few or weakly absorbing functional groups (worst for R-CN, R-NH2, R-F; poor for R-OR’, R-OH, R-COOH, R-COOR’)

– Solvents:• Requires use of solvents that absorb poorly in UV


• UV Absorption Detectors– Wavelength Selection:

• Must choose λ > solvent cut-offs

• Most compounds absorb strongly at short wavelengths but many also absorb less moderately at longer wavelengths

• More sensitivity at shorter wavelengths (provided little mobile phase absorption)

• More selectivity at longer wavelengths

Wavelength (nm)

180 220 260

solvent

analyte

Sensitive λ More selective λ


• UV Absorption Detectors– General Properties

• Reasonably good (but variable) sensitivity• Good linearity, reproducibility• Good stability (but baseline drift and warm up time)• Poor as a universal detector

– Types:• Fixed wavelength (absorption at single wavelength)• Variable wavelength (can select one wavelength

using monochromator)• Photodiode array (can measure at multiple

wavelengths simultaneously) – these give some qualitative information and allow more peak overlap


• Application of UV Detection to Weak Absorbers– Use short wavelengths (method must be

selective; not always effective)– Derivatize compounds to add strong

absorber (common for amino acids, carbohydrates)

– Use indirect UV absorption (absorber added to eluent, analytes displace eluent and give negative peak)


• Refractive Index Detectors– Principle:

• liquids with different refractive index will diffract light differently

• Composition will determine refractive index• Any compound with a refractive index different than

the solvent’s is detectable– Advantage:

• Most universal detector (can detect weakly absorbing compounds)

– Disadvantages:• Gradients are not possible• Requires thermal stability• Generally not very sensitive

Liquid ChromatographyInstrumentation – Ion Exchange

Chromatography• Types of Instruments:

– Single column– With analytical plus suppressor columns

• Detection in Single Column Instruments– Other detection methods (fairly common)– Conductivity detection

• Conductivity Detector– Resistance measured (AC circuit)– Conductivity = 1/(resistance)– Ions in solution create conductance– Conductivity depends on ionconcentration and size

Conductivity cell

Electronics

From HPLC column

Liquid ChromatographyInstrumentation – IC

• Difficulties with single column instruments:– Both analytes and ion exchanger

conduct electricity– High concentration of ion exchanger

means high background conductance and difficulties in detecting small concentrations of analytes

– Often large ions used as exchanger (such as potassium hydrogen phthalate for anion exchange)

Liquid ChromatographyInstrumentation – IC

• Suppressor Columns– The purpose of the suppressor is to convert the ion exchanger

to mostly non-ionic compounds– Example below with sodium bicarbonate eluent (Na+HCO3

- is the ion exchanger) in anion exchange

– In the suppressor column, Na+ is replaced with H+

– This converts conductive Na+HCO3- to non-conductive H2CO3

– NaCl is converted to HCl (still conductive)– Na2HPO4 is converted to H+H2PO4

- (less conductive)– This reduces the baseline and increases sensitivity

Separation column

Na+2HPO4

2- Na+Cl-

Suppressor Column

Na+HCO3-

H+ In; Na+ out

To Conductivity detector

H+Cl-


• Electrochemical Detectors– Principle:

• Redox reactions occur at electrodes following column

• Potential cycle used to periodically oxidize/reduce analytes at electrode

• Current depends on concentration of analyte being reduced or oxidized (similar to A in UV detector)

• Electrode potential determines classes of compounds that are detectable (similar to λ in UV detector)

Analyte electrode

Referenceelectrode

Voltage supply/ electrometer

From column


• Electrochemical Detector– Advantages:

• Very sensitive (limits of detection under 1 pg possible)

• Adjustable selectivity• Wide range of compounds can be detected (including

UV inactive compounds)• Advantageous for microbore

– Disadvantages• Electrode fouling• Variable analyte response• Requires ions to “complete circuit”

– Array Detectors:• Can have multiple electrodes in detector (set to

different potentials)


• Fluorescence Detectors– Detection Principle:

• Light promotes molecules to excited electronic state

• Excited molecules transition from lowest excited state back to the ground state and emit light in the process

– Equipment:• High intensity light source• Filters or monochromators to

select wavelengths (before and after cell)

• Sensitive light detector

Light Source

Filter or monochromator

Light detector

M + hν → M*

M* → M*’ (lower vibrational level)

M*’ → M + hν’


• Fluorescence Detectors– Advantages:

• Greater sensitivity possible (for molecules with high fluorescence efficiencies) because easy to detect small signal against zero background (see below)

• Much greater selectivity because few molecules fluoresce, particularly at selected wavelengths

– Disadvantages:• Limited to relatively few molecules (although derivatization is also

possible)

Absorption of light

95% transparent

(equiv. to A = 0.022)

Weak light in black background

Emission of light

Liquid ChromatographyDetector Questions

1. A compound has an absorptivity of 493 M-1 cm-1 at 210 nm and 32 M-1 cm-1 at 280 nm. Why would one even consider setting the wavelength to 280 nm?

2. Describe one way to use a UV detector for detecting weakly absorbing organic compounds.

3. Describe how you could use a photodiode array detector to determine if the odd shaped peak below is from one or multiple compounds.

A (254 nm)

Time

Liquid ChromatographyMore Questions

1. Why is electrochemical detection difficult to use with non-bonded silica HPLC?

2. When weakly absorbing compounds are derivatized, it is more common to use fluorescent derivatizing agents. Why is this?

3. What is the advantage of using suppression in ion chromatography?

4. Why is suppressed ion chromatography not so useful for weak acid anions vs. strong acid anions?

Aerosol-Based Detectors for HPLC

Example Advanced Method Presentation

Aerosol-Based Detectors for HPLC Outline

• Introduction to Technology• Theory Including Three Types of

Detectors• Advantages and Disadvantages of

ABDs• Some Applications• Conclusions• References


Introduction• Limitations of Conventional Detectors

– UV Absorption Detectors:• Not very universal• Poor sensitivity for many classes of compounds

(carbohydrates, fats, amino acids, dicarboxylic acids, etc.)

– Refractive Index Detectors:• Low and somewhat variable sensitivity• Not gradient compatible

– Mass Spectrometer Detectors:• Not all compounds ionize readily• Expensive, large, expensive to operate


Introduction• Processes in Aerosol-

Based Detectors:– Effluent from column is

nebulized producing spray of solvent and solute

– Spray droplets are heated in an oven, evaporating solvent gas and producing aerosol particles from solute

– Aerosol passes to an aerosol detector to produce a signal

HP

LC C

olum

n

N2(g)

Aerosol Detector

Spray Chamber

Nebulizer

Oven

droplet particle


Introduction• Mobile Phase Requirements

– Solvent must be volatile (and cause little column bleed)

• Analyte Requirements– Works best if analyte is non-volatile– Semi-volatile compounds give reduced

response

Aerosol-Based Detectors for HPLCTheory

• Nebulization produces a distribution of drop sizes

• Solvent viscosity and surface tension can affect distribution of droplet sizes

• Evaporation shifts this to distribution of particle sizes based on:

where: dd, dp are drop and particle diameters, C is mass concentration, and ρp is particle density

3/1

pdp

Cdd

1 mg mL-1 solute

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02

diameter (mm)

nu

mb

er (

dn

/dlo

gd

)

DropletsParticles

Size Distributions


• Types of Aerosol-Based Detectors– Depends on method of detecting aerosol particles– Evaporative Light Scattering Detection (ELSD)

(Charlesworth, J. M. Anal. Chem. 1978, 50, 1414)– Condensation Nucleation Light Scattering Detection

(CNLSD) (Allen, L. B.; Koropchak, J. A. Anal. Chem. 1993, 65, 841)

– Charged Aerosol Detector (CAD)/Aerosol Charge Detector (Dixon, R. W.; Peterson, D. S. Anal. Chem., 2002, 74, 2930)


• ELSD principles– Detection by light-

scattering by particles

– Efficient detection when dp ~ λ; less efficient at other sizes

– Non-linear response results

– At low concentrations, dp < λ so sensitivity is poor (detection limits of around 0.1 to 1 μg mL-1)

Detec

tor

concentration

Expanded Region


• Condesation Nucleation Light Scattering Detection– Detection principle also uses particle

light-scattering but overcomes poor detection of small particles by growing small particles to bigger particles by condensation of vapor on to particles

– This technology is very sensitive (a single 3 nm particle can be detected)

– This can translate to very low detection limits (~10 ppb or ~50 pg) under optimal conditions

– Commercialized recently

Particles In

But

anol

cond

enso

r

To light-scattering detector


• Charged Aerosol Detection– Particles charged as aerosol jet collides with ion-rich jet from corona

discharge (commercial version)– Charged particles are collected on a filter with charge passed to

electrometer (current measured)– In another version, particles are charged as they pass near a corona

discharge region– Sensitivity has equalled CNLSD (at least at standard HPLC flows) – Large response range and linearity at lower concentrations

Gamache et al., LCGC North America (2005).

Corona Discharge Wire

Ion Filter (negatively charged rod)

Aerosol Filter

Aerosol In

To Electrometer


Advantages and Disadvantages• Advantages:

– Better performing universal detectors than refractive index detectors

– Universal response for non-volatile analytes– CNLSD and CAD sensitivity is similar to typical UV

sensitivity

• Disadvantages:– Requires analytes of low-volatility, volatile mobile

phases– CNLSD and CAD are often limited by solvent purity and

column bleed– Non-linear calibration often is needed– Cost is higher than UV Detectors


Some Applications• Food

– ELSD has been used extensively to characterize carbohydrates and lipids.

– Methodology requires no derivatizations and allows analysis of whole lipids (as opposed to just fatty acids)

• Polymers (with SEC)– Useful for polymers without chromophores

• Pharmaceutical Industry– ABDs are useful for assessing contaminants in pharmaceutical

products• Biotechnology and Environmental Samples

– Greater potential with CNLSD and CAD for analyzing low concentration samples (some carbohydrate examples)

• Analysis of Cations, Anions and Neutrals– Use in combination with zwitterionic stationary phase allows

simultaneous detection of three categories in single run


Triglyceride Example• By Lísa et al (J. Chromatogr. A,

1176 (2007) 135-142).• Homogenous trigylcerides

shown above without (left) and with “gradient compensation” (right)

• Gradient compensation allows response to remain proportional to area with a gradient

• An alternative is to use a 2 dimensional calibration (Hutchinson et al., J. Chromatogr. A, 1217 (2010) 7418-7427)

• Gradient compensation uses 2 additional pumps pumping eluent after the column to produce a constant eluent composition

• Plant oil samples shown below


Paclitaxel Example• By Sun et al. (J. Chromatogr.

A, 1177 (2008) 87-91).• Looked at impurities in

paclitaxel (a anti-cancer natural product from Pacific yew tree) using UV and CAD

• Shown in upper figure (standards – highest and stressed paclitaxel – lower)

• Paclitaxel impurity response shown to be uniform by CAD but not by UV detection

• Pharmaceutical impurity analysis used for determining acceptable pharmaceuticals

• If no standards available, CAD provides better estimation of impurity levels


Smoke Tracer Example• My work (published in

Dixon and Baltzell and Ward et al. – see my research webpage)

• Detected levoglucosan and related monosaccharide anhydrides

• These are thermal breakdown products from cellulose and hemicellulose

• It was possible to use the levoglucosan concentrations to estimate the total particulate matter (2.5) derived from woodsmoke

O

OH

HH

H

H

OHR

OH

OO

OH

HH

H H

OHO

OH

O

HOH

HH

H

ROH

OH

O

HOH

HH

H

H

OHOH

O

cellulose

levoglucosan

levoglucosan

Chico Winter Air Samplemannosan


Glycan Profiling• My more recent work (with

Thomas Peavy, Biological Sciences) also preliminary work done by Ignaki et al.

• Glycans (glycoprotein oligosaccharides) are difficult to quantify

• Glycans are post-translational modifications and composition can depend on host organism/cells

• Profiles change in cancer cells• Standards are unavailable or

expensive• Currently running surrogate

standards to prepare multi-dimensional calibration (depending on mass concentration and retention time)

• Test standards show errors of ~0 to 25%

Peptide backbone

oligosaccharides

min7.5 10 12.5 15 17.5 20 22.5 25

mV

25

50

75

100

125

150

175

200

ADC1 A, ADC1 CHANNEL A (NOAH\050409000002.D)

6.7

22

7.3

06 7

.617

7.7

91

8.5

31 8

.856

9.1

34 9

.326 9

.604 9

.962

10.

428

10.

980

11.

596

12.

167

12.

585

12.

983 1

4.15

0

14.

736

15.

251

15.

942

16.

565

16.

632

17.

261

17.

813

18.

551

19.

577

19.

607

21.

434

23.

297

23.

846 24.

172

24.

450

24.

776

24.

819

24.

894 25.

328

25.

615

25.

765

26.

242

26.

420

27.

094

27.

451

28.

048

Frog Egg example


Conclusions• ABDs have been replacing RID as a

universal detector (at least for non-volatile compounds)

• ABDs can be used without exact standards for quantification (much as an FID is used in GC)

• Biggest limitations are volatility/non-volatility requirements, cost, and linearity


References• ELSD

– Text (p. 247-248)– Charlesworth, J. M., Evaporative analyzer as a mass detector for liquid

chromatography, Anal. Chem., 50, 1978, 1414-1420. – Review: Koropchak et al., Fundamental Aspects of Aerosol-Based Light-

Scattering Detectors for Separations, Adv. Chromatogr. 40, 2000, 275. • CNLSD

– Allen, L. B. and J. A. Koropchak, Condensation nucleation light scattering: A new approach to development of high-sensitivity, universal detectors for separations, Anal. Chem., 65, 1993, 841-844.

– Same review listed for ELSD• CAD

– Dixon, R. W. and D. S. Peterson, Development and testing of a detection method for liquid chromatography based on aerosol charging, Anal. Chem., 74, 2002, 2930-2937.

– Gamache, P.H., R.S. McCarthy, S.M. Freeto, D.J. Asa, M.J. Woodcock, K. Laws, and R.O. Cole, HPLC analysis of nonvolatile analytes using charged aerosol detection, LCGC North America, 23, 150, 152, 154, 156, 158, 160-161, 2005.


References• For Applications: (See my faculty web page for CAD references)

– Foods:• Asa, D., Carbohydrate and oligosaccharide analysis with a universal HPLC detector, Am.

Laboratory, 38, 16, 18, 2006.• Moreau, R. A.. The analysis of lipids via HPLC with a charged aerosol detector, Lipids, 41,

727-734, 2006.• Lísa, M., F. Lynen, M. Holčapek, and P. Sandra, Quantitation of triacylglycerols from plant

oils using charged aerosol detection with gradient compensation– Pharmaceuticals:

• Loughlin, J., H. Phan, M. Wan, S. Guo, K. May and B. Lin, Evaluation of charged aerosol detection (CAD) as a complementary technique for high-throughput LC-MS-UV-ELSD analysis of drug discovery screening libraries, Am. Laboratory, 39, 24-27, 2007.

• Sun, P., X. Wang, L. Alquier, C. A. Maryanoff, Determination of relative response factors of impurities in paclitaxel with high performance liquid chromatography equipped with ultraviolet and charged aerosol detectors, J. Chromatogr., A, 1177, 87-91, 2008.

– Biotechnology:• Inagaki, S., J.Z. Min, and T. Toyo’oka, Direct detection method of oligosaccharides by

high-performance liquid chromatography with charged aerosol detection, Biomed. Chromatgr., 21, 338-342, 2007.

– Atmospheric Aerosols:• Dixon, R. W. and G. Baltzell, Determination of levoglucosan in atmospheric aerosols using

high performance liquid chromatography with aerosol charge detection, J. Chromatogr. A, 1109, 214-221, 2006.


Questions1. For a complicated sample with several analytes present

at moderate concentrations (around 50 μg mL-1), is it advantageous to use an ELSD (vs. a UV Detector) 1) if the compounds are weak absorbers, 2) if the compounds are strong absorbers?

2. What instrument components will ELSD and CNLSD have in common that are not present in CAD?

3. ABDs can not detect volatile analytes. How should weakly absorbing volatile compounds be determined?

4. With a single calibration standard (over different concentrations), is it possible to estimate concentrations of unknown compounds (e.g. for compounds without any standards)? and under what conditions?

5. Protein concentration can be estimated by looking at absorption from aromatic amino acids? Why might using an ABD be a better way of quantifying unknown proteins?

chem. 230 – 10/28 lecture. announcements i hw set #3 – due today (short answer problems have...

Documents

phase hplc

retention slide

water slide

lecture slide

ionexchange chromatography

abstract slide

size exclusion chromatography

greater retention