Lecture #10 .. done by: Gada Tuffaha
Review:
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Last time we talked about the U.V detector in HPLC instrument and the main
difference between them was the cuvette; in HPLC the cuvette is a flow cell.
We also know the principle of U.V: In order to have an absorbance you have to have
a chromophore which contains a conjugated part or system.
We also talked about the diode which transforms light into current, which is the
"photodiode cell" that was presented by Einstein and won a Nobel Prize for it.
Now the Multiplier sometimes can't detect the small changes in the current and so you have
to amplify it, like a microphone amplifying the sound.
How to amplify the current? By the multiplier and the principle is as follows: the accelerated
electrons (although an electron has negligible weight it has a substantial kinetic energy,
why? Because it has high velocity and low mass and so high Energy) and these accelerated
electrons will hit static electrons (non-moving) in an Alloy (‫ )سبيكة‬which will cause the
transfer of energy from the accelerated electrons to the static ones, and since the electrons
have a very low mass,this high energy will cause the release of higher number of electrons
just like one billiard ball moving fast hitting a number of balls that are static and be able to
move them!
And so the released electrons from the alloy will be accelerated again, by what?
‫ الطاقة الحركية لشحنة موجودة في مجال كهربائي = الشحنة * فرق الجهد) و بالتالي االلكترونات رح‬:‫(في التوجيهي‬
‫تتسارع طالما في فرق جهد بما انه عندي شحنة في مجال كهربائي رح تكتسب الشحنة (نقصد االلكترونات) طاقة حركية‬
‫) و بالتالي طاقة حركية اعلى و اعلى و بالتالي‬voltage( ‫ هي فرق الجهد‬V ‫ هي الشحنة و‬Z ‫ حيث‬Z*V=‫زائدة تساوي‬
‫سوف تتحرر الكترونات اكثر في المرة القادمة التي ستصطدم فيها بسبيكة أخرى و بالنهاية أحصل على ما يسمى‬
.)amplified current(
And by going through the standard and the test and so a substantial amount of current by
which you can detect the small changes in analyte concentration.
Nowadays we have a modified U.V called the "PHOTODIODE ARRAY":
The idea is like asking your guest: what would you like to eat? And start giving him options
(for example: would you like an apple? Would you like an orange? And so on). This is the
hard principle of U.V = you give a lambda and check for absorbance and so on for each
lambda! & this procedure takes time (for example the U.V scan may take 5 minutes!) and so
if you used this with HPLC (mobile phase is moving and so are the analytes and you have a
very limited time for detecting the conc. of these analytes!) the analytes will be eluted
before you can detect them! And so you must be able to make a U.V scan faster than the
elution of analytes! And this can't be achieved by the normal U.V! that's why the came up
with the photodiode array (the idea is like offering your guest a plate of fruits and he decides
what he wants to eat)this is the principle for the photodiode array: You have more than one
diode approximately each diode is responsible for certain lambda & that’s why they called it
photodiode array, array means: from all angels.
And so you don’t make a selection for certain lambda, but you give the whole light radiation
to the sample and let it choose the lambda it likes, (for example if the sample likes the
240nm it will absorb it, if it dislikes the 220nm it will absorb only a portion from it or even
doesn’t absorb it and so on..) and so the sample will take what it likes and leaves what
doesn't like of course this is according to different absorptivity for each lambda.
Now after the absorption that took place in a short time period we will have (light
separation by monochromator) and since each diode has its own lambda, and from Test
there will be subtraction for density or amount of light absorbed by blank that will give you
the absorbance & this will be done in a limited time, but the problem is the nature of light
(waves) and each wave has a lambda and so upon separation you could have destructive or
constructive combinations of waves and this can be resolved by an (interferogram (by the
principle of feathering or deconvultion)).
Fourier's idea: the wave gives you 2 pieces of information: amplitude and Lambda
(wavelength).
Lambda is qualitative and amplitude is quantitative (if we added another wave from the
same type the amplitude will be increased).Fourier expressed the shape of the wave as an
exponential function and so the wave-function was turned into a signal with x-axis being
qualitative (wavelength) and the y-axis quantitative (amplitude).
And so we ended up with one chromatogram (with Lambda on x-axis and amplitude on yaxis) But we want (retention time and absorbance) and so photodiode array will give you 3d
data: retention time, lambda and absorbance. This will give you a 3d view or map that you
can see everything! (This is the first advantage: it generates 3d map = retention time,
lambda and absorbance)
The second advantage is the following: it gives you an idea whether the compounds
separated are fully resoluted or not (a peak is not hiding behind another or mixed with it) by
viewing the map you can decide about that or you can make a purity test (detects selectivity
or specifity) and by that test is when you are using the (PDA: photodiode array) you analyze
the wave by taking multiple points and gives you a U.V scan for each point and then upon
change in the U.V scan( it must be the same U.V scan for the same compound)it refers to a
mixture of two compounds that gave you a different U.V scan! And so purity drops below
100% and we accept purity to be above 99% (if below 99% it means we don't have selectivity
or specifity) and when this is selective I call this method: Stability indicating method and it
means: Method by which you can quantify the active component apart from its
degredants.it is used widely in medicine manufacture because it is important for us to know
what happens to the drug after time passes (it should separate the degradation product
from API(active pharmaceutical ingredients) and so measures how much degraded from the
drug (shelf life is determined by this, only 10% to be lost or degraded and 90% is active) we
determined shelf life by Arrhenius equation in physical (a form of Vant hoff's equation).
PDA was less sensitive than U.V this problem was solved by increasing the no. of diodes.
Fluorescence detector (we talked about it in Instru) it has an advantage over U.V which is:
Fluorescence detector is more selective and more sensitive, why more selective? (because
compound should be rigid and chromophoric in order to be detected by flurescence), why
more sensitive?( because detection is done on 90 degrees and thus:
cos(90) *vector( ‫محصلة الضوء الساقط= )الضوء الساقط‬
‫ ال يرى الضوء الساقط بل يرى معتم و بالتالي فرصه رؤية بقعة ضوء‬detector‫ و بالتالي ال‬،‫و بالتالي المحصلة=صفر‬
)‫صغيرة في العتمة افضل!(تخيل ضوء مفتوح في النهار مقابل ضوء مفتوح في الليل من نميز بشكل افضل؟‬
Thus we will have negligible background effect and any weak signal can be detected! That’s
why it's more sensitive!
Luminescence (from physical point of view means: to give light after you take it, for
example: you take U.V and give visible light)
Luminescence has 2 forms: fluorescence (detected in Fluor) and phosphorescence (detected
in Phosphorus).
The main difference between them is that fluorescence is in solutions and phosphorescence
is in solids.
Why a compound is fluoroactive? Has chromophore (to absorb light) and rigid! Can't make
twisting or move to spend the energy and thus emit this extra energy as light!
For example takes U.V light and emit visible light.
Fluorescence is seen in nature: ‫قنديل البحر وغيره‬
(Camouflage: ‫خاصية الحرباء‬to be able to give the same color of the environment & is still
unknown)
Other detectors of HPLC other than U.V and Fluorescence:
1-Refractive index:
= speed of light in air / speed of light in media
‫سبب االنكسار هو تغير سرعة الضوء في الوسط‬
Refractive Index is Universal (every compound has refractive index) But not sensitive! (needs
high amounts).
2-Electrochemical detector: detects Redox Rxns, how much electrons going out a compound
to another (standard and test)I can know the conc. ,one of the ways is measuring
conductivity (Unit is Siemens = (ohm^-1) ).
End of Lecture.