optimize

define alkyl

alkane with one hydrogen removed to make reactive/bondign with something else(alkane)

amine

amide

arene

aldehyde

alkane

alkene

alkynes

ester

carbocylic acid

thiol

ketones

dilution equation - rearrange if want to find volume

molarity equation

moles: divide the mass of your sample in grams by the molar mass of that substance

M to mM

how to find grams when creating molar solution - equation

mobile phase defintion

liquid sample put thro column (water, acetonitrile (ACN), methanol, buffers)

increasing organic solvents like ACN or methanol (ch3-oh) = hydrophobic will interact with m. phase more, come off column faster - better at holding hydrophobic molecules

retention time

time @ which compoound comes out of column, more retention = later elution (if compond interacts strongly with s. phase)

reversed phase HPLC

s. phase: nonpolar/hydrophobic

m. phase: polar

*based on hydrophobic interactions

c18

bonded to silica (silanol groups - Si-OH to Si-C18)

hydrophobic (hydrophobic molecules with react stonger, polar molecule will travel with mobile phase)

18 carbon alkyl chain

long nonpolar hydrocarbon

• (ch2)17-ch3

how pH affects

pH can change charge of molecules whose protons can change which changes interactions with s. phase

charged molecule = more hydrophilic, polar)

related to conc. of hydrogen ions, H+

• more Hions - lower pH - more acidic

• less Hions - higher pH - more basic

common hydrophobic molecules

uncharged, nonpolar (alkanes, hydrocarbons)

fundemental mechanism of seperation

*SAMPIE Continuously MOVES btwn MOBILE and STATIONARY PHASES

liquid chromotagraphy

STATIONARY PHASE IS POLAR SOLVENT

MOBIIE : nOnPolAR

hplc and uplc defintion

high preformance liquid chromatography

ultra = higher pressures, smaller particle

smaller silica particles…

can provide better seperation efficiency but creates greater resistance to flow, require higher pressure

detector

common type uv/vis detector

how much uv light molecules absorb as come out of column

Mass spectrometry

used to measure the mass-to-charge ratio (m/z) of ions to identify, quantify, and structure chemical compound

peak position, peak height/area, peak width, seperation btwn peaks

peak position: retention time

peak height/area: amnt of compound detected

peak width: seperation efficiency

seperation btwn peaks: how well diff compounds seperated

silica

highly porous version of silicon dioxide, SiO2

• network of pores that increase surface area available for int/ (pore size - angstroms)

has silanol grps sticking off (R-Si-OH)

Silanol can participate in hydrogen bonding, acid base interactions, int with polar or charged

bare silica: polar

bonded stationary phase

when silanol gets chemically modified - remove OH and add C18 )from polar to hydrophobic)

endcapping

cover exposed silanol groups to prevent reactions

• chemical treatment to reduce exposed silanol

what molecule will interact most with c18

hydrophobic, nonpolar molecules will interact strongly and come out last

isocratic

mobile phase composition stays consistent

gradient

composition changes during run increasing organix solvent makes easier to elute strongly retained compounds

why would you change the mobile phase or s. phase from typical

some compounds have similar retention times under one set of conditions

changing selectivity of seperation

increasing organic solvent - get retained out of column if hydrophobic molecules wont come off c18

polar defintion

uneven distribution of electrical charge in molecule

electrons = negativley charged

often interact well with water (partial charges can interact and form enegetically favorable interactions)

electronegativy

an atom's tendency to attract shared electrons toward itself when forming a chemical bond

oxygen more electronegative than carbon

commonly polar vs nonpolar

polar functional groups: hydroxyl -oh (alcohols), carbonyl c=o (ketones, aldehyde), amines (nitrogen contribute to polarity, become charged), carboxylic acid -cooh (change state depends on pH)

ACN

dipole dipole int/

hydrogen bonding

ion dipole int/

van der waals int/

acid and bases (r-cooh &r-nh2)

neutral form - more hydrophobic - more retention

charged form - more hydriphilic - less retention

pka

how readily an acid gives up its proton

• relationship btwn ph and pka helps predict whther mostly uncharged or mostly charged

acids ph<pka : mostly protinated/neutral | ph>pka mostly deprontinated/negatice (less retention) - higher pHs, negative charge not lots of free hydrogens to stay attached

buffers

solutions that help resist changes in pH when small amounts of acid or base are added

• phosphate, acetate, amonium acetate/formate

provide resivior of acid/base forms that can absorb change

channeling

large empty space that mobile phase prefers in column, need pack tightly s. phase so the mobile phase has to travel through entire bed

backpressure

more resistance to flow - higher pressure needed

instrument reads this as backpressure

(smaller particles, faster flow rate, longer column, tempature, solvent composition, column diamtere, more viscous liquids = higher pressure needed)

frit

very fine filter

retain s. phase particles (s. phase larger than frit pathways), manage m. phase, support filtration, protect sensitive components, consistent system preform

before column: sample protection

easiest place to precent particulate

in mobile phase

buffer salts, improperly cleaned glassware, microbial contam.

• solvent reservior fitlers

guard column

smaller, replaceable *for protection

• catch irreversibly retained (permanently bind to column) - guard against premature failure

• when m.phase pH is extreme enough = degrade column packing so use g. column with similar phase

• pre column filters

types of stationary phase (in gaurd column)

trap columns

for capture

• concentrate a dilute analyte

• remove salts, buffer components, detergents, or unwanted sample-matrix components

• offline sample prep or on line in flow path

unwanted stuff washed away and anlyte is retained

or

unwanted contamiantnt is retained while desired target passes thro

trap columns - sample conc and desalting

retain target - wash away unwanted matrix components (salt or non-volatile buffer compounds) - elude target (concentrating it)

• peptide conc and desalting

• protien conc and desalting

• small molecule conc and desalting

trap columns - detergent removal

trapping approach depends on detergent type and whats being retained

• sds removal: protien/peptide passes thro for analysis and sds retained

• non-ionic detegernt removal: protien/peptide retained while NID flushed away

trap columns- peptide fractionation and 2d LC trapping

tandem trapping - two diff trapping stages to fractionate peptides, transfer to 2nd trap to conc. abd desalt, then elude

SCX (strong cation exchane) trapping/fractionation - controlled peptide release - peptide concentration and desalting - downstream elution

• SCX trap: peptide fractioantion before controlled release to second trap

• peptide conc and desalting trap: recieves released peptides for conc. and desalting

HILIC

swept volume

dead volume

interstitial volume