Exam 3 Lecture 4
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Now in terms of solvents used in HPLC, you have as MP  water + organic solvent
(methanol,acetonitrile,THF)  if you have problems with one solvent, you can try another
First choice in HPLC is acetonitrile  gives you very good separation
Reversed phase chromatography  using C18,C8, or phenyl columns
Acetonitrile  very low UV cutoff?  often times in HPLC you’re using a UV detector  190 
means you can set the UV detector at 190 and above  190 and below you will have UV
absorption and that will affect your analysis  anything above 190 you can use it for detection
Second choice is methanol  has a UV cut off of 205  problem with methanol is its high
viscosity  will have higher backtracker as compared to acetonitrile  but the advantage of
methanol is the expense associated with it  acetonitrile is more expensive (shorter of
acetonitrile so the price has tripled or quadrupled)
THF  the UV cutoff is 12  that’s higher than the other solvents  problem with THF is the
oxidative potential  has epoxides in the solvent  can affect your analysis by oxidizing
compounds  because of the problems with THF  THF can slowly degrade and oxidize by air to
give you peroxides
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So these are solvents often used in HPLC  of course the other components  buffer to
maintain pH at a certain neutral or acidic point TFA to maintain pH  ion-pairing potential of
TFA
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So in terms of detectors for HPLC
The criteria for ideal detector is the sensitivity to analytes you are analyzing and the selectivity to
those compounds
Universability because you don’t want to switch your detectors a lot  UV is pretty universal for
only compounds that contain a chromoform  if they don’t contain chromoform it cant be seen
using UV  should produce a linear response for when you do quantitation you want a standard
curve  so the standard curve needs to be linear  don’t want the peaks broad either
So these are the criteria
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Will compare the UV detector, fluorescence detector, refractive index detector and the mass
spect
For both UV and fluorescence  this applies mostly to the UV  a light source  flow cell
(solvents coming in from here and out)  this is the distance it measures  this is the amount of
material inside this flow cell that can detect it by detector
Since you have compounds coming in here  originally the MP acetonitrile and water at certain
percentage  but if you have compounds coming in and you’re going to have different
absorptivity in this range that will be recorded by detector
So in terms of sensitivity and the usefulness of these detectors which the gradient  because
often times in HPLC we are using gradient elution
So you are looking at UV, refractive index, and then fluorescence, and then masspect
UV is the most commonly used detector in HPLC  compatible with gradient elution  In terms
of sensitivity you’re looking at 0.1 nanogram of material in UV
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Refractive index  refractive index change of the eluent coming off the column  so then its
not compatible with gradient elution  because if you change the gradient (the composition of
MP)  you will change refractive index  and the other problem with refractive index - the
sensitivity  1000 times less than the UV detector  but the advantage is its universal 
anything that comes into the solvent stream is gonna change the refractive index  universal
but less sensitive  like the thermal conductivity detector in GC
This is used for when you have a compound without a UV chromoform (cant be used by UV
detector)
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Fluorescence detector  more sensitive as compared to UV detector because it goes down to 1
pictogram of material  and then its also compatible with the gradient elution because as long
as the solvents you use (methanol, acetonitrile, and THF) they don’t have fluorescence  but
in terms of the detector can only detect compounds that can fluoresce  its more sensitive, but
in terms of selectivity  only compounds that are fluorescent
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Mass spect  for GC its getting more and more commonly used, for HPLC as well  sensitivity is
comparable to UV  compatible with gradient dilution  the advantage of mass spect is that it
gives you the molecular weight of the compound and it gives you fragmentation patterns 
gives you more structural information than the other 3 in his labs they have GC and HPLC MS
But the disadvantage of mass spect  much more expensive than the other detectors  with
the other detectors  30-50,000 dollars with all the pumps  but just the mass spect alone will
be 100,000 dollars  but then the advantage of the mol. Wt and fragmentation pattern is very
worthwhile in terms of the expense.
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Next a few examples of using HPLC in quantitive analysis of drug molecules
1. extraction of APAP tablet  from the textbook  the drug molecule is paracetamol (book was
from England)
a. APAP do extraction  inject into HPLC  UV absorption  determined by UV
b. The solvent front/ peak  coming off at 3.363 minutes  rather exact
c. Standard  if you have just pure compounds, you know the concentration, put it into
HPLC  comes off at exactly the same place but then this 3.36 and 3.34 because you’re
doing 2 separate injections, the difference is only in the second decimal point 
considered to be the same compound  in order to confirm that these 2 are the same
you can mix the 2 together and do a coinjection  if they give you 1 peak you will have 1
compound
d. For this example you are using what is called an external standard  HPLC is a very
precise measurement of peak size, area, with the concentration  so you can use a
standard APAP as the compound  then you have a series of concentrations made and
then do a standard curve like this  this is the concentration of the APAP and the y axis is
the peak area under the curve  what you see is within this concentration range you will
have a linear relationship 
e. inject the sample extract of the tablet  if the compound comes out with a peak area in
this range  what you need to do is draw a line and drop down and then calculate the
concentration of your sample  can calculate how much you have in the tablet  using
external standard 
f. the external standard you are using is the same compound you are analyzing  same
retention time if you have exactly the same concentration you will have the same peak
area  will have a linear relationship
g. often times when you have extraction processes like for tablets  in tablets you have
many other materials in it  can have excipients that can affect recovery  so if your
recovery is very good and its at 90-95% and also reproducible  you can nuse external
standard  the experiment HPLC is very precise in measuring concentration  but if
your recovery for the sample preparation to extract your compounds out of the tablet is
very low 50-60%  the other half cannot be extracted out  then you have to consider
using the next standard called internal standard
h. internal standard mentioned in GC  adding a known compound to the mixture before
you do the extraction  dissolve and break down tablets into little pieces  everything
into solution  add known amount of internal standard  very similar compound to the
one you’re analyzing  but different enough to produce a different peak  so whats the
criteria that you use in terms of selecting internal standard
i. internal standard should closely relate to the structure of the analyte  so the
compound we are analyzing here is hydrocortisone  so whats chosen is
betamethasone (steroid)  using an standard that is a steroid is well 
difference is the methyl group  and then there is a double bond in the standard
but not in hydrocortisone  there is a Fluorine that’s also not in hydrocortisone
 closely related  so the behavior of these 2 compounds ill be similar when you
do the extraction during the sample preparation 1% or 5% of hydrocortisone
and during the extraction  using about 5% of this standard for the extraction
ii. compound should be stable  because the internal standard should not
decompose during the analysis during analysis extraction or storage
iii. photographically resolved  when you do the separation you have 2 peaks 
when you mix the 2 you have to have the 2 peaks separated from each other 
you cannot have overlap and of course the resolution between these 2 you want
to have greater than 2 resolution  so in this case you will have more than
baseline separation
iv. even though they should be separated  they should be close as possible  so
that means you don’t want it to have one here and one close to the solvent front
 bigger separation which means 2 compounds are not very structurally related
v. having the same weight of material  the response should be similar  so if you
have 1 nanomolar concentration  the response of the peak size should be about
the same  the 2 compounds that you look at over here  the 2 peak sizes are
very close together  so they’re close in the terms of these 2, but are separated
in terms of resolution of 2 or higher  and then in terms of peak size, if you have
the same amount of material you’re going to have similar peak size
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several chromatograms over here
one over here that’s just hydrocortisone  so when you have the compound without internal
standard will show 1 peak
when you have the internal standard  so this solution in terms of hydrocortisone is a cream 
so you’re talking about extract of the cream hydrocortisone cream so you can add the internal
standard and it will go through same extraction process  and will have 2 peaks show up 
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need to make sure that these 2 compounds are well-separated from anything else that’s being
extracted  you don’t want the internal standard to overlap with the excipients in the cream
the first one here has known amounts of both the drug molecule and also the internal standard
 which these 3 graphs , and especially with these 2  you can quantitate how much of
hydrocortisone you have in the tablet?  that’s the calculation you can do  shows you what
you can use HPLC for in terms of quantitation
External standard and internal standard
But the other thing we didn’t discuss
In GC  there is derivatization  but in this case we never mentioned derivatization  because
in this case its not necessary in HPLC because you are looking at compounds that don’t really
need to vaporize
But in GC when you have very polar compounds,  ones that contain OH or amines  you need
to derivatize them otherwise the peak shape will be rather tailed  doesn’t give you good
quantitation  very slow/sloped (?) peak and very tailed peak  not good in terms of
integrating compounds for peak area
So we have looked at the different chromatographic separations for both GC and HPLC
So what if you were asked to analyze compounds  in order to select the method of analysis  need to
look at the compounds you are analyzing
1. polarity  in terms of polarity of analytes  compounds at this end will have nonpolar
compounds  will have strongly polar compounds on this end  so this is then looking at the
compounds you are asked to analyze  so for the nonpolar compounds that means they will be
more likely to be volatile  for the strongly polar compounds  less likely to be volatile so then
in terms of determining which method you should peak for this analysis  most likely you will be
using GC  when you have compounds thath are very nonpolar so they are more likely to be
lipid soluble , more likely to be volatile  so you can use GC analysis  if you have compounds
with this range  which compounds would come out first  so the more volatile compounds
will be coming out first, this end will be early, this end will be late  for GC you are going to
increase the temperature of the column oven to elute the compounds off  to have them go
into the MP (helium)  so the more volatile the compounds are, the more likely they will go into
MP and will come out earlier  the ones that are less volatile will spend more time in SP  will
come out later
2. HPLC  discussed in 2 different possibilities  normal and reverse phase
a. Normal  a more polar stationary phase and a less polar mobile phase – so you have
compounds that are strongly polar in this end  these analytes will have more
interactions with SP  will be coming out later  less polar compounds will come out
earlier because they will have less interaction with SP  the organic solvent will elute the
less polar compounds out earlier
b. Reverse  more common  SP are less polar and the MP phase is aqueous solution is
more polar  strongly polar compounds will be coming out first  compounds that are
less polar will have more interactions with the SP like C18,C8 will come out later 
c. Compounds with charges + and -  and want to separate them from opposite charges, or
separate from ones with no charge  you can have ion-exchange chromatography 
running in aqueous solution  charged molecules will be in water
d. This concludes the discussion on the separations