HPLC and GC Flashcards
Chromatography
- The most important technique for isolating and purifying biomolecules
- Sample interacts with two physically distinct entities: mobile phase andstationary phase.
- The components in the sample will interact differently with the two phases which forms the basis for separation.
- HPLC and GC can be linked to mass spectrometers for mass analysis of the column effluent.
High Performance Liquid Chromatography
•Ideally suited for separation and identification of biologically active molecules.
- egAmino acids, carbohydrates, lipids, nucleic acids, proteins, pigments
•Highly versatile technique: many different chromatographic separations can be effected by using the appropriate column.
- eg. Partition, adsorption, ion exchange, gel exclusion, affinity etc
HPLC - Instrumentation
- Solvent Reservoir
- Capacity at least 500mL
- Must be degassed and filtered
- Typically MeOH, CH3CN, Water
- Isocratic system – only one solvent used orsolvent mixture of one composition
- Gradient system– mixture of 2 solvents that changes during expt
2. Pump
- 500-5000psi
- 1mL/min (analytical) and 100mL/min (preparative)
- Pulse-free solvent flow
- Small holdup volume
3. Injector
- Sample must be introduced onto column in an efficient and reproducible manner
- Usually via microsyringeinjected into a neoprene/Teflon septum
- Typical volume :
Analytical- 1mL-5 mL
Preparative- 100mL-20mL
4. Column
- Prepared from stainless steel or glass-Teflon tubing
- Analytical columns: diameter 2.1, 3.2, 4.5 mm
- Preparative columns: up to 30 mm diameter
- Length typically 10 cm or 20 cm (range 5-100 cm)
- Prepared from stainless steel or glass-Teflon tubing
- Analytical columns: diameter 2.1, 3.2, 4.5 mm
- Preparative columns: up to 30 mm diameter
- Length typically 10 cm or 20 cm (range 5-100 cm)
5. Detector
- The column effluent is continuously monitored
- Must have a high sensitivity as small sample sizes
- Most common detector used is photometric detector: measures UV absorption (sensitivity 1 ng)
- Fluorescence detector is more sensitive (100X) but does not have a linear response
6. Collector
- Column effluent can be collected for further chemical and physical analysis
- Egsent to MS
- Preparative HPLC effluent is collected, solvent removed to isolate compound of interest
HPLC - Output
Each chromatographic peak represents a separated compound
Analysis of Output: capacity factor
Capacity Factor (k’)
k’ = (tR– t0)/t0
The capacity factor is a measure of where the peak of interest is located with respect to the void volume
Analysis of Output: Resolution
Resolution (RS)
RS= 2(tR2– tR1)
(tW1+ tW2)
Rsis a measure of how well two peaks are separated.
The greater the value of Rs, the better the resolution. Large values of Rsindicate a significant time difference between the two peaks and Rsvalues of 1.5 are ideal
Analysis of Output: Tailing factor T
Tailing Factor T
T = Wx/2f
The accuracy of quantitation decreases with increase in peak tailing due to the integrator experiencing problems with where/when the peak starts and ends.
Ideally:
tW= 2f
ieGuassiandistribution
Analysis of Output: Theoretical Plate Number (N)
N = 16(tR/tW)2
The theoretical plate number is a measure of column efficiency and is based on the number of equilibria that may have taken place during the process. The greateseparationr the value of N the more efficient the column.
Quantitative vsQualitative HPLC
Qualitative analysis
Identification of a component in a mixture by retention time tR; can be coupled to MS for m/z
Quantitative analysis
An integrator is used to calculate the area under the peak orpeak height is used.
This is compared to a standard of known concentration which can be run in a separate experiment to give a standard curve (external standardization) oran internal standard is added to the sample.
Preparative vs Analytical HPLC
The purpose of analytical HPLC is to identify components in a mixture and quantify their relative amounts.
Sample size ug
Mass ratio of compound to stationary phase is less than 1:100,000.
Preparative HPLC is used as a purification step and the effluent is collected and solvent removed to isolate the compounds of interest.
Preparative HPLC columns are larger (length and width) and it is possible to separate mg-g.
Stationary Phase
Small diameter, porous materials are used as the adsorbants.
This is where the separation takes place and it is the most important part of the system.
Porous layer beads
- Inert solid core
- Thin porous outer shell of silica, alumina or ion-exchange resin
- 20-45 um
- Used for analytical purposes
- Capacity too low for preparative applications
High Pressure Adsorption Chromatography (Normal Phase)
- Silica or Alumina column (polar stationary phase)
- Analytes adsorbwith different affinities to binding sites in the stationary phase (polar molecules adsorb more strongly than nonpolar molecules)
- Use a non-polar mobile phase (eghexane, EtOAc, MeOH, MeCN, CH2Cl2)
- Steroids, Alcohols, Organic Acids, Vitamins, Pesticides
High Pressure Partition Chromatography (Reverse Phase)
- Non-polar stationary phase and a polar mobile phase
- Separates components according to hydrophobicity
- The active stationary phase is chemically bonded to the inert support (typically silica)
- Most popular column is octadecylsilane(ODS, n-C18H37, C18) – there are ca300 commercial C18columns available.
- Widely used to separate biomolecules including peptides, nucleotides, carbohydrates, amino acids and their derivatives
Normal vsReverse Phase HPLC
Ion-exchange HPLC
- Column is packed with charged functional groups prepared by chemically bonding the ionic groups to the support via silicon atoms.
- Used to separate proteins, peptides and other charged molecules.
Gel-Exclusion HPLC
- Used for proteins and nucleic acids.
- Can separate molecules up to 10, 000, 000 Da.
Must use semi-rigid gels based on cross-linked styrene-divinylbenzene, polyacrylamide and vinyl-acetate copolymer as ‘normal’ gels collapse under the high pressures.
Alternatively, use rigidpackingsprepared from porous glass or silica:
•Compatible with water and organic solvents
Easy to pack and several fractionation ranges
Chiral HPLC
- Separation of enantiomersis very difficult as they have the same physical and chemical properties
- Enantiomersdiffer only in the way they interact with plane-polarised light
- Enantiomersalso differ in how they interact with receptors and one enantiomer may provide the desirable effect while the other may be toxic
- EgThalidomide– the (D, R) form is a sedative whereas the (L, S) form is teratogenic.
- Other eginclude Lipitor, Nexium, Advair
Chiral HPLC
- A chiral stationary phase is used to separate enantiomers.
- Due to the different spatial arrangements around the stereocentresof the enantiomers, they will interact differently with the chiral surface of the stationary phase.
- Most successful chiral columns use proteins (comprised of chiral amino acids)
- Egalpha1-acidglycoproteins (AGP), human serum albumin (HSA) and cellobiohydrolase(CBH)
Chiral HPLC - Ibuprofen
HPLC - Mobile Phase
The column packing material is not the only important consideration for a successful separation. The solvent system is equally important.
Purity
Must be ultra-pure, filtered and degassed. Use HPLC grade or purify lesser grade solvent by microfiltration or distillation
Reactivity
Must not react with the sample or column packing. Many hydrocarbons, alkyl halides and alcohols are suitable.
Detector Compatibility
Most UV detectors monitor the column effluent at 254nm and so the solvent system must not absorb UV in this region.
Pressure Considerations
Viscosity of the solvents will affect the pressure-to-flow rate. Solvents must be chosen that achieve the separation without requiring pressures too high for the system.
Use of Derivatives
Chemical modification before separation is known as derivatization.
Increase sensitivity of detection
Addition of fluorescent group or to increase UV absorption
Eg. Addition of Dansylgroup to amines, peptides and phenols
Gas Chromatography
- A very sensitive technique for separating volatilesubstances
- Stationary phase is a liquid and mobile phase is a gas
- Many substances are not initially appropriate for GC as they are not sufficiently volatile. They can be modified chemically (derivatised)
- Eg: silylationof acids, ROH to give TMS ethers and esters)
- Acylation of amines, ROH to give CF3C=O derivs.
- Akylationof fatty acids, drugs to give Me, Et and Bu esters
GC - Instrumentation
GC Intrumentation: Sample Injection Port
Sample Injection Port
- Located in the oven as sample must be volatilised before loading onto column
- Mixture of vapourisedsolvent and solutes with gas
Sample size 0.1mL – 20mL
GC - Instrumentation: Column
Column
- Several metres in length, usually coiled (save space)
- Fused silica capillary tubes i.d.0.1 mm – 0.53 mm, typical length 20m.
The internal surface of wall coated open tubular (WCOT) columns is coated with the liquid (stationary) phase
GC - Instrumentation: Column Temperature
Column Temperature
- must be controlled to within tenths of a degree.
- optimum column temp. is dependant upon the b.p. of the sample.
Atemperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes.
Column Temperature
Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful
The column temperature is increased (either continuously or in steps) as separation proceeds.
GC - Instrumentation: Detector
Detector
- Flame ionization detector
- Thermal energy of flame causes ionization of the molecules in the flame
- Ions collected by a pair of polarised electrodes
Current produced is amplified and recorded.
Can detect almost all organic compounds; 1 nmoldetection limit
GC - Output
GC trace of the total lipid profile, following phospholipase C digestion and preparation of the TMS ether derivatives, of plasma from a normolipemicsubject. A stainless-steel column (50 cm x 2 mm i.d.), packed with 3 % OV-1TMon Gas ChromQTM(100-120 mesh), was temperature-programmed from 175 to 350ºC at 4ºC/min with nitrogen as the carrier gas at a flow-rate of 40 mL/min. Tridecanoinis the internal standard
HPLC vs GC
Both techniques can be coupled to MS for m/z analysis
Both techniques rely on differences in affinity for mobile and stationary phases.
