ماهي طريقة وفكرة عمل spectrophotometer

من طرف **mira.kareem** الأحد يناير 16, 2011 2:53 pm

التعامل مع اجهزه المعمل مهم جدا للحصول على نتائج سليمه وواضحه

التحليل الطيفي

تعتمد فكرة التحليل على تسليط حزمة مركزة من شعاع ظوئي على العينة المراد
تحليلها , ثم قياس شدة الشعاع الظوئي الممتص في العينة وبالتالي تقدير
تركيز مادة الاختبار. نبسط اكثر.
ّ
حينما تتعرض الذرات والجزيئات لمصدر طاقة , حراراي, ظوئي ..... الخ تمتص
الالكترونات في الذرات والروابط بين الجزئات هذة الطاقة وتصبح في حالة
مثارة وبالتالي تنتقل الى مستويات طاقة اعلى ثم تعود الالكترونات الى
مستويات الطاقة الاطثر استقرارا وتشع الطاقة الممتصة في صورة جرارة او ظوء
او تفلور.
ينجم عن امتصاص الطاقة , زيادة في الحركة الدورانية او الانتقالية للجزئات.

وحيث ان الظوء المرئي صورة من صور الطاقة( اشعاع كهرومغناطيسي) فعندما
تكون طاقة الروابط الكيمائية للجزئات وللالكترونات في الذرات مساوية لطاقة
الاشعاع الظوئي, يعمل على اثارة الالكترونات وانتقالها الى مستويات طاقة
اعلى وبالتالي امتصاص الظوء

تبنى على هذة الظاهرة العديد من التطبيقات لقياس تراكيز المواد وتحديد هويتها.

في المطياف الظوئي:
هناك مادة يراد تحديد تركيزها ويتم كالاتي:
المادة المراد تحديدها تكون ملونة او تتفاعل مع مركب لتنتج مركب ملون
تسلط حزمة ظوئية لها طول موجي محدد ( اي الطول الموجي للظوء الذي يحدث عندة
اكبر امتصاص للطاقة) حتي تكون حساسية الاختبار دقيقة وبالتالي تقدير
التراكيز المنخفظة للمادة,

اساس الفكرة قانون lambert
يحدد العلاقة بين شدة الشعاع الممتص وتركيز المادة , وهناك علاقتين
النفاذية و
الامتصاصية

النفاذية تعني قدرة الشعاع الظوئي على النفاذ خلال العينة , وفية تقاس
نسبة الشعاع الساقط على العينة على الشعاع النافذ وهي عبارة عن علاقة
لوغارتيمية

الامتصاصية : تحول العلاقة اللوغارتيمية الى علاقة خطية ويتم فيها قياس علاقة شدة الشعاع الممتص بالنسبة لتركيز المادة

طبعا لابد من اجراء معايرة للجهاز , والمحاليل , وقياس العلاقة الخطية لمحاليل قياسية من العينة قبل اجراء القياسات

وباللغه الانجليزيه:
principles of spectrophotometer

A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer
for measuring the intensity of light. The instruments
are arranged so that liquid in a cuvette can be placed
between the spectrometer beam and the photometer. The
amount of light passing through the tube is measured
by the photometer. The photometer delivers a
voltage signal to a display device, normally a
galvanometer. The signal changes as the amount of light
absorbed by the liquid changes.
If development of color is linked to the concentration
of a substance in solution then that concentration
can be measured by determining the extent of
absorption of light at the appropriate wavelength. For
example hemoglobin appears red because the hemoglobin
absorbs blue and green light rays much more effectively
than red. The degree of absorbance of blue or green
light is proportional to the concentration of
hemoglobin.
When monochromatic light (light of a specific
wavelength) passes through a solution there is
usually a quantitative relationship (Beer's law) between
the solute concentration and the intensity of the
transmitted light, that is,
ماهي طريقة وفكرة عمل spectrophotometer Od1

ماهي طريقة وفكرة عمل spectrophotometer Od1

where I sub 0 is the intensity of transmitted light
using the pure solvent, I is the intensity of the
transmitted light when the colored compound is added, c
is concentration of the colored compound, l is the
distance the light passes through the solution, and k is
a constant. If the light path l is a constant, as is
the case with a spectrophotometer, Beer's law may be
written,

ماهي طريقة وفكرة عمل spectrophotometer Od2

ماهي طريقة وفكرة عمل spectrophotometer Od2

where k is a new constant and T is the
transmittance of the solution. There is a logarithmic
relationship between transmittance and the concentration
of the colored compound. Thus,

ماهي طريقة وفكرة عمل spectrophotometer Od3

ماهي طريقة وفكرة عمل spectrophotometer Od3

The O.D. is directly proportional to
the concentration of the colored compound. Most
spectrophotometers have a scale that reads both in O.D.
(absorbance) units, which is a logarithmic scale, and in
% transmittance, which is an arithmetic scale.
As suggested by the above relationships, the
absorbance scale is the most useful for colorimetric
assays.
Using a Spectronic 20 spectrophotometer

The Spectronic 20 spectrometer is widely used
in teaching laboratories. The specific instructions
will differ with other models, but the principles
remain.

The
instrument must have been warm for at least 15 min.
prior to use. The power switch doubles as the zeroing
control.
Use the wavelength knob to set the desired
wavelength. Extreme wavelengths, in the ultraviolet
or infrared ranges, require special filters,
light sources, and/or sample holders (cuvettes).
With the sample cover closed, use the zero
control to adjust the meter needle to "0" on
the % transmittance scale (with no sample in the
instrument the light path is blocked, so the
photometer reads no light at all).
Wipe the tube containing the reference solution
with a lab wipe and place it into the sample
holder. Close the cover and use the light control
knob to set the meter needle to "0" on the absorbance
scale.
Remove the reference tube, wipe off the first
sample or standard tube, insert it and close
the cover. Read and record the absorbance, not the transmittance.
Remove the sample tube, readjust the zero,
and recalibrate if necessary before checking
the next sample.

Why use a reference solution? Can't
you just use a water blank? A proper reference solution
contains color reagent plus sample buffer. The
difference between the reference and a sample is
that the concentration of the assayable substance in the
reference solution is zero. The reference tube
transmits as much light as is possible with the assay
solution you are using. A sample tube with any
concentration of the assayable substance absorbs more
light than the reference, transmitting less light to the
photometer. In order to obtain the best readability and
accuracy, the scale is set to read zero absorbance
(100% transmission) with the reference in place. Now you
can use the full scale of the spectrophotometer. If you
use a water blank as a reference, you might find
that the assay solution alone absorbs so much
light relative to distilled water that the usable scale
is compressed, and the accuracy is very poor.

Protein Assays

Below is a list of assays for the
determination of protein concentration in a solution. This
list includes the sensitivity range, volume/amount of
sample needed, subjective comments on accuracy
and convenience, and major interfering agents. Procedural
details, equipment requirements, and references are
outlined in the individual assay documents.
The criteria for choice of a protein assay are usually
based on convenience, availability of protein for assay,
presence or absence of interfering agents, and need for
accuracy. For example, the Lowry method is very
sensitive but is a two step procedure that requires a
minimum of 40 minutes incubation time. The Bradford
assay is more sensitive and can be read within 5
minutes, however proteins with low arginine content will
be underestimated. Generally, estimates are more
accurate for complex mixtures of proteins. Estimates
of concentration of pure proteins can be very
inaccurate depending on the principle of the assay,
unless the same pure protein is used as a standard.
Criteria will be discussed in the individual documents.

Because different proteins have different amino acid
compositions, the sensitivity of colorimetric assays to
individual proteins may vary widely. The most
reprodicible results are obtained with standards
composed of a mixture of proteins that is as similar as
possible to the unknown. For most purposes, a relative
amount is good enough, but the standard used should be
reported. For example, the Bradford assay is much more
sensitive to bovine serum albumin (BSA)
than to immunoglobulin G (IgG), so that with IgG the
investigator is likely to overestimate the amount of
protein in a sample. With BSA the investigator is likely
to underestimate the amount.

General Reference: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
Absorbance assays

Absorbance at 280 nm
- Range: 20 micrograms to 3 mg
- Volume: Depends on cuvette - volumes range
  from 200 microliters to 3 ml or greater
- Accuracy: Fair
- Convenience: Excellent, if equipment available
- Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets
</li>
Absorbance at 205 nm

- Range: Roughly 1 to 100 micrograms
- Volume: Depends on cuvette - volumes
  range from 200 microliters to 3 ml or greater
- Accuracy: Fair
- Convenience: Very good
- Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets
</li>
Extinction Coefficient
- Range: 20 micrograms to 3 mg
- Volume: Depends on cuvette - volumes
  range from 200 microliters to 3 ml or greater
- Accuracy: ~2% (very good)
- Convenience: Very good
- Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets
</li>

Colorimetric assays

Modified Lowry
- Range: 2 to 100 micrograms
- Volume: 1 ml (scale up for larger cuvettes)
- Accuracy: Good
- Convenience: Fair
- Major interfering agents: Strong acids, ammonium sulfate
</li>
Biuret
- Range: 1 to 10 mg
- Volume: 5 ml (scale down for smaller cuvettes)
- Accuracy: Good
- Convenience: Good
- Major interfering agents: Ammonium salts
</li>
Bradford assay
- Range: 1 to 20 micrograms (micro assay); 20 to 200micrograms (macro assay)
- Volume: 1 ml (micro); 5.5 ml (macro)
- Accuracy: Good
- Convenience: Excellent
- Major interfering agents: None
</li>
Bicinchoninic Acid (Smith)
- Range: 0.2 to 50 micrograms
- Volume: 1 ml (scale up for larger cuvettes)
- Accuracy: Good
- Convenience: Good
- Major interfering agents: Strong acids, ammonium sulfate, lipids
</li>
Amido Black method
- Range: 2 to 24 micrograms
- Volume: 2 ml
- Accuracy: Good
- Convenience: Poor
</li>
Colloidal Gold
- Range: 20 to 640 nanograms
- Volume: 1 ml (scale up for larger cuvettes)
- Accuracy: Fair
- Convenience: Poor
</li>

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