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B6 B= AC AK AQ AX A_ Af @l @t @{ @ @ @ ? ? ? ? ? ? ? ? > > > > > > > > > > > > > >& >- >4 >; >B >I >P >V >^ >d >k >r >y > > > > > > ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @ @ @% @+ @3 @9 @@ @H @N @U @\ @c @i Aq Aw A~ A A A A A A A A A A A A A A A A A B B A A A A A# A* A1 A8 A> AF AM AS A[ Aa @h @o @v @| @ @ ? ? ? ? > > >@  j   PBCDEF1==#)07>DLRYagnu| !(.6<CJQW_fltz{|@j     `B CLDEFf1LL LLL"L(J0J6J=JDJKJQJYJ`JfJnJtJ{JJJJJJJJJJJJJJJJJJJJJ JJJ J&J.J5J;JCJIJPIWI^IdIlIsIyIIIIIIIIIIIIIIIIIIIII IIII&I-I3I:IAIHINIVI\IcIjIqIxIIIIIIHHHHHHHHHHHHHHHHHHH$H+H1H9H@HFHNHTH[HaHiHoHvH~HHHHHHHHHHHHHHHHHHHHHHHH#H)H0H7H>HEHLHSHYGaHgGnGtG|GGGGGGGGGGGGGGGGGGGGG GGG!G(G.G6G<GCGKGQGXG_GfGlGtGzGGGGGGGGGGGGGGGGGGGGG GGF F&F-F4F;FAFIFPFVF^FdFkFrFyFFFFFFFFFFFFFFFFFFFFF FFFF%F+F3F9F@FGFNFTF\FcFiFqFwF~FFFFFFFFFFFFFFFFFFFFFFFF#F*F1F8F>FFFLFSFZFaFhFoFvF|FFFEEEEEEEEEEEEEEEEE E E E E! E( E0 E6 E= ED EK EQ EY E_ Ef En Et E{ E E E E E E E E E E E E E E E E E E E E E E E E E& E. 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( . 6 = C K Q X _ f l t {                         & - 4 ; B I P V ^ d k r y                         % + 3 9 @ H N U \ c i q w ~                        # * 1 8 > F M S [ a h o v |         @  j  ) 6HP B  xMethylparabene48 Z2C C  * 6M 4  w Ethylparabene48 Z2 C C   + 6@O 5 c  xPropylparabene48 Z2C C  , 6X: > 7  w Butylparabene48 Z2 C C   - PBCDEF1==#)07>DLRYagnu|     ! ( . 6 < C J QW_fltz{|@j  xB . 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F M T Z b h o v }    ' - 5 ; B I P V ^ d k r y       # ) 1 7 > E L R Z ` g n u {    @ K `B CDEF1  "(06=DKQYv_Uf-mtzH:R@\#U  # Eo$,2c9@GM U[*bipIv~ *[XQ~%U k ?    (d.5<mC IQ$W^elrUzODs5L_p$*18?EMSYagnu|        & , 4 : A H O U ] c j q x~ "(06=DKQY_fmtz %,29@GMU[bipv~ !(.6<CIQW]ekry $*28>FLSYagnu|     & - 4 : B H O V ] c k q x ~                        " h) E0 6 > D sK )R Y v_ g m t {  k   k   ' W  ; i   # u  =  8 % d, 2 : @ eG MN FU Q[ mc i o -w }  y   4   E    b 3  c "   .  ^ ! i( . T6 < !B }J P #W l^ e k $s Vy      $ : P b s               # * 1 8 > F L S Z a g o u |    @S L `B CDEF1#)17=DKRX`fmt{ %,39AGNU\bipw} !(/5=CJQX^flsy$+19?FMTZbhov|  '-5;AIOV]djrx")07=EKRY`fnt{ %,3t9[A>GNU\bYjpwo~ $v*z  N    I A )!(g/5{=CJQLX$^ flsz=q tk4;^   =   N  $,+1A9?3EMSZZah@nv|$R{)<M\iu     & - 4 ; A I O V ] d j r x                         " ) 0 7 = E K R Y ` f n t {                         & , 3 9 A G N U \ b j p w ~                        " ( / 6 = C K Q W ^ e l r z                         $ * 2 8 ? F M S [ a h n v |    @Sm  M `B CDEF1")/7=DKRX`fltz %+39?GMT[bhpwvb}I+ y:@cY+ . {   J  '.B5;CIcPW$^dlr=yb5 )T5!P?D#* 1P 7 ? EM L S Z`h.nu|A(h<f*;K Xdo y&-4;AHOV\djpx~")07=EKQX_fltz $,29@GMU[bhpv}  (.5<CIQW^elrz$*18?EMSZahnv|     & - 4 ; A I O U ] c j q x ~                        " ( 0 6 = D K Q Y _ f m t z                         $ , 2 9 @ G M U [ b i p v ~                         ( . 5 < C I Q W ^ e l r z                         $ * 1 8 ? E M S Y a g n u |    @  N PBCDEF1==#)07>DLRY`gmu{ %,3:@HNU\ciqw}{|@ O `B CDEFf1 !(/ 6 < D J Q X _ e m s z                          $ + 2 8 @ F M T [ a i o u }                         ! ' . 4 < B H P V ] d k q y                         # ) 1 7 > E L R Y ` g m u {                          % - 3 : A H N V \ c i q w ~                         ! ) / 6 = D J R X _ f m s z                          % + 2 9 @ F N T Z b h o v }                         ! ' - 5 ; B I P V ^ d k r y                 #) 1 7 > ELRZ`gnu{     % - 3 : A H N V \ c j q w                         ! ) / 6 = D J R X ^ f l s z                         % + 1 9 ? F M T Z b h o v }                         ' - 5 ; B I P V ^ d k r y                         # ) 1 7 > E L R Z ` wg Wn 0u { N @E P `B CDEFf16"(0~6=DNK{QY_ f m t z .Wv_N?~ 9;$,z2-9@GM]U:[ b i p v ~c /[4C2_L 0[ (.5<C#I5QEWT^aemlxrz$*18?EMSYagnu|  &,4:AHOU]cjqx~ "(06=DKQY_fmtz %,29@GMU[bipv~ !(.6<CIQW]ekry $*28>FLSYagnu|     & - 4 : B H O V ] c k q x y~ b F %    X   ]    y  F   ' ]    " ) 0 6 > D "K CR qY _ g Jm t '{  G j g y 3    .  { 0   | L    % , w 2 G : @ G N JU [ c *i o 8w } #   H   4 v    L t       . ? N [ g! r( |. 6 < B J P W ^ e k s y                         # * 1 8 > F L S Z a g o u |    @S Q `B CDEFf1#)17=DKRX`fmt{ %,39AGNU\bipw} !(/5=CJQX^flsy$+19?FMTZbhov|  '-5;AIOV]djrx")07=EKRY`xfnnbtS{A+zEq?2@  , Z  % , 3 90A^GNU\<bjpEw~BJ-^]]J T! 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In addition it is a very simple application that shows the effect of back pressure on retention. Notice the deviations on the system with the pulse damper. Eventually these peaks will fall out of the retention window, and not be detected. TR@RH  0޽h ? X(=^{ 0   (   d  c $   L |  3 rjL )e)e p  L  pThis slide outlines some of the varoius detector capabilities and would be the first step in detector selection.qq@ JH   0޽h ? ̙33H} 0  ,(  ,d ,c $    ,3 r()e)e p   :@ H , 0޽h ? ̙33o 0 /'<(  <d <c $    <3 roL )e)e p   aThis slide show a small sampling of the compound classes that are often analyzed using FL detectors. Remember that some compounds have a native fluorescence while some may require either a pre-column or post column derivitization to provide fluorescent species. Carbamate Pesticide = Carbofuran Aflatoxin = Aflatoxin B1 PAH = Pyrene Vitamin = Vitamin A Amino Acid = Leucine +ACQ (Accutag Chemistry)@#     " H < 0޽h ? ̙33H 0 H(  Hd Hc $   L  H3 r4~L )e)e p  L  :@ H H 0޽h ? ̙33H 0  P(  Pd Pc $   L  P3 rL )e)e p  L  :@ H P 0޽h ? ̙33H 0 @X(  Xd Xc $   L  X3 r\L )e)e p  L  :@ H X 0޽h ? ̙33H 0 ``(  `d `c $   L  `3 rL )e)e p  L  :@ H ` 0޽h ? ̙33 0 xI(  xd xc $   L  xs *L  p  L  3In general, development of a HPLC method often follows the steps outlined here. As you can see, choosing a detector is an important part of the method development process. Correct (or incorrect) detector selection will influence the success of the method. Limits of detection, limits of quantitation are directly influenced by the detector choice. Other factors can be indirectly influenced as well. For example, a very selective detector (MS or FLD) can minimize the need for perfect separation conditions. @H x 0޽h ? ̙33K 0  P(  d c $   L  s *L  p  L  UAll HPLC detectors, regardless of design should provide the functions outlined here. VV@H  0޽h ? ̙33 0 pP(  d c $   L  s *LL  p  L  :@ H  0޽h ? ̙33D 0 (  d c $   L  s *L  p  L  ~NA prefect detector design would provide all of the listed features. Unfortunately, no such detector exists and any detector design is forced to make compromises. As we will see throughout the presentation, knowledge of these compromises and the effects of the obtained results allow users to use detectors for their maximum benefit. OO@H  0޽h ? ̙33 0 !(  d c $   L } s *оL  p  L  Sensitivity is one of the most widely cited detector requirements. Generally speaking, most users demand detector that are highly sensitive to their compounds of interest. Many users describe sensitivity simply as the net response (or signal) of the peak (this may be height count, absorbance units, millivolts, intensity, and others). The true measure of sensitivity is better described as a ratio of the signal intensity divided by the intensity of the baseline noise. @H  0޽h ? ̙33 0 P(  d c $   L  s *ȸL  p  L  :@ H  0޽h ? ̙33 0 @P(  d c $   L  s *L  p  L  :@ H  0޽h ? ̙33{ 0 ;3`(  d c $   L ' s *$L  p  L  The diagram show the generic layout of a photo diode array detector. All of the energy from a broad spectrum lamp (or lamps) usually covering both the ultarviolet and visible wavelength range is passed through a flow cell. The light is then split into its component wavelenghts on a grating. A series of diodes measures the light intensity over the entire wavelenght range. The result is two types of data, a traditional chromatogram (time vs absorbance) at any wavelentgh in the operating range, and a spectrum (wavelength vs absorbace) at any given time point. Data collected from a PDA detector is often refered to as 3 dimensional data. @H  0޽h ? ̙33 0 P(  d c $   L  s *L  p  L  :@ H  0޽h ? ̙33 0 P(  d c $   L  s *p[s p  L  :@ H  0޽h ? ̙33, 0 @|(  ^ S    L  c $L  @  L  r: N2u'lW-VW2u 2ux~ 2u [2u'lW2ux H  0޽h ? ̙33w 0 7/8(  8X 8C    L / 8S 07 @  L  Gradient elution is best suited for reversed-phase HPLC, normal-phase HPLC with bonded polar stationary phases, and ion exchange liquid chromatography. H 8 0޽h ? ̙33  0   HB (  HX HC      HS   @   D 0 Gradient elution is mainly utilized in reversed-phase and ion exchange liquid chromatography. Reversed-phase will here be used as an example. However, all statements made are also valid for ion-exchange chromatography. The gradient is formed by increasing the percentage of organic solvent. Consequently the partition between mobile and stationary phase for a compound will change. The compound will spend a larger part of its time in the mobile phase as the percentage of organic solvent increases. The linear velocity of the compound will thus increase. A compound accelerate through the column! When the compound elutes from the column it has reached a linear velocity often close to the velocity of the mobile phase. It also follows that e.g. a doubling of the column length does not cause a doubling of the migration time. The time it takes for a starting racecar to travel 200 meter is not twice the time it takes to make 100 meter. One can not assign a fixed k value to a compound when gradient elute is applied. k is the partition coefficient and changes during the elution. Calculating k using the formula k =(tr-t0)/t0 is only correct during isocratic elution! H H 0޽h ? ̙33> 0 @X(  XX XC     XS   @   <A scouting run is a convenient way to develop HPLC separations. Unlike using isocratic elution for method development, gradient elution allows the chromatographer to view both early eluting peaks and those strongly retained which might not even be seen with an isocratic elution. Use acetonitrile water for first gradient run if reversed phase. Gradient time can be determine by equation presented on following page. Bands should elute before gradient is complete. If gradient is started with 0% organic, long equilibration times can be expected. The theory is that the brush like features of the bonded phase fold in on themselves. Initial Gradient Run Adjust gradient range to minimize wasted time for a large number of compounds Increase N by increasing t or column length, decrease particle size and flow rate. = = H X 0޽h ? ̙33\ 0  p(  pX pC     pS ;  @   The trade off between flow rate, column length and time is the same for gradient elution. When separation time is a vital issue use a short column (5-15cm) and run at high flow rate. When maximal resolution is crucial it is advisable to use a long column (20-30 cm) at normal flow rate. Compounds with a very steep velocity curve (e.g. proteins) does not benefit from long columns. These compounds reaches quickly (after a few centimeter of acceleration) a k =0 i.e. no stationary phase interaction. When applying steep gradients it is actually counter product to use a long column as the compounds reaches k =0 (no chromatography) before leaving the column. The last part of the column then only add band broadening.H p 0޽h ? ̙33k 0 +#(  X C    # S l%  @   iIsocratic elution is impractical if analytes have widely differing affinities for the stationary phase. j j$>H  0޽h ? ̙33 0 `:(  X C     S   @   <  H  0޽h ? ̙33 0 :(  X C     S PU  @   <  H  0޽h ? ̙33 0 :(  X C     S D]  @   <  H  0޽h ? ̙33  0   p (  X C     S ,d  @   r^Usually gradient slope is expressed as  %/min . Gradient slope for a given column dimension is however more correctly given as  %/ml . It is flow and not time that transport a compound down the column! An increase in flow rate consequently means an decrease in gradient slope, i.e. typically a improvement in resolution and vice versa. The change in plate number with flow rate will counteract this. The net effect is case dependent. However changes in band spacing may happen as with any alteration of gradient slope. The elution times are not very sensitive to flow rate. This is best understood by considering an simplified case: a molecule that does not migrate when the percentage of organic modifier is below 30% but when it is above 30% it move at the same velocity as the mobile phase. The gradient reaches 30% at 20 minutes. At a flow rate of 1 ml/min the compound elute at 22 minutes, i.e. it does not move for 20 minutes and the it goes through the column in two minutes. Increasing the flow rate to 2 ml/min will results in a elution volume of 21 minutes. H  0޽h ? ̙33 0 |t (  ^  S |   w n  c $uw | @  w  The relation between partition coefficient and percentage organic modifier depends on molecular properties, see figure above. This can actually cause a compound to overtake another during a gradient. Analogy: in an 1/4 mile drag race a car passes its competitor half way down the race due to different engine characteristics. This is illustrated in the second diagram above. Larger molecules (e.g. proteins) tend to have a very steep k curves. Band spacing may change as column length is altered. Note elution times with a 10 and a 25 cm column in the diagram above. Observe the small change in migration time with column length. 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