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Bio Lab

Bio

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5 views

Bio Lab

Bio

Uploaded by

ly8182213
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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‫‪OCTOBER / 10‬‬

‫‪Biopharmaceutics‬‬

‫‪Lab.2‬‬

‫حوراء عالوي‬

‫‪CALIBRATION‬‬ ‫زهراء كريم‬

‫‪CURVE‬‬ ‫االء صالح‬

‫نور الزهراء جواد‬

‫اديان حازم‬

‫فاطمة مجيد‬

‫زهراء محمد حنون‬

‫دعاء احمد‬

‫زهراء احمد شاتي‬


Introduction;

◦Calibration curve : Is a reference curve prepared from a series


of standard solution having different concentration plotted
against their absorbance.

Bears -lambert theory


The amount of light absorbed by a sample is directly
proportional to its concentration.

Benefit of calibration curve: to obtain the concentration of


unknown drug sample from its calibration curve by measuring
its absorbance then the concentration could be obtained from
the calibration curve equation.

Aim of the experiment :


Preparing the calibration curve of Diclofenac Sodium in
phosphate buffer (pH6.8 ).
Apparatus and Materials;

Diclofenac Sodium(stock solution)


Spectrophotometer
Beaker
Cylinder
Procedure;

1-Prepare 100 ml of stock solution of Diclofenac Sodium contains 100 mg in


100 ml of Phosphate buffer (6.8). 2-Make several dilutions in the range from
2 mcg/ml to 14 mcg/ml.
3- Set the spectrophotometer to λ max of diclofenac (276 nm)
4-Measure the absorbance of each dilution and the stock solution
5-Use MS Excel to draw the calibration curve.
6-Use excel elements to find calibration curve equation and R2
Discussion;
_ What are the factors affecting absorbance?

The two main factors that affect absorbance are concentration of the substance
and path length.

_ What to do if absorbance is greater than 1?

If the non-linearity occurs at absorbance values higher than one, it is usually


better to dilute the sample into the linear portion of the curve because the
absorbance value has a high relative error.

_ Why is it necessary to include a blank sample in a calibration curve, and how


is it used?

A blank sample, which contains no analyte, is used to establish a baseline


measurement for the instrument response. This allows any background signal
from the solvent, instrument noise, or other non-analyte factors to be
subtracted from the sample readings. Including a blank sample in the calibration
curve helps to improve accuracy by ensuring that the calibration and subsequent
analysis only account for the analyte of interest.

_ What factors can affect the accuracy of a calibration curve?

Several factors can affect the accuracy of a calibration curve, including:

Instrumental limitations, such as detector sensitivity or noise.


Matrix effects from other substances in the sample that might interfere with
the signal.
Preparation errors, like inaccuracies in diluting standard solutions
Environmental conditions (e.g temperature fluctuations
_ What is the aim of calibration curve?
Calibration curves are used to determine the concentration of a sample with an
unknown concentration.

_ Can a calibration curve be negative slope?

If your standard curve has a negative slope, it means that the measured
fluorescence value decreased as the pH increased.

_ How do you use a calibration curve to determine concentration?

Begin with a known chemical, measure its absorbance, and then graph it.
Second, calculate the absorbance of the unknown sample, plot it on a graph,
and apply a calibration curve to get the estimated concentration.

_ Can a calibration curve be a straight line?

Calibration curves allow instrument calibration by predicting the concentration of


an analyte in a sample from the reading of the instrument. This curve is
constructed as the regression straight line that best fits the relationship
between some known concentration standards and their respective instrument
readings.

_ How many points for a calibration curve?

The calibration curve should consist of at least six concentration levels. For
chromatographic assays the calibration standards are prepared and analyzed in
duplicate over the concentration range studied
H.W

1. What action is required for a sample produces absorbance >> 1?

Here are some actions you can take:


1. Dilution .
2. Adjust Path Length.
3. Use Alternative Methods.
4. Instrument Calibration.
By taking one or several of these actions, you can obtain a more accurate
measurement of absorbance for highly concentrated samples.

2. What are the common causes of drug solubility issues in pharmaceutical


formulations?

The solubility of ionizable drugs can be affected by pH. Weak acids and bases
may not dissolve well in certain pH ranges

3. How to solubilize a drug in case of it is not completely dissolved in the


intender solvent for calibration,Explain practical example?

If a drug isn’t fully dissolving during solubilization, you can use several strategies
to improve solubility. Here’s a practical example using a poorly water-soluble
drug to create a solution for analytical calibration.
Example: Improving Solubility of a Drug for Calibration

1. Choose an Appropriate Solvent


2. Use a Co-solvent Approach
3. Heat the Solution
4. Ultrasonication
5. Adjust pH (if applicable)

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