Practical lesson - Biochemistry 2019
DETERMINATION OF MAGNESIUM
OBJECTIVE: Determine magnesium concentration in unknown sample
THEORETICAL PREPARATION FOR PRACTICAL LECTURE:
Magnesium - significant intracellular cation. Mg2+
as a cofactor in enzymatic systems, its
function in a cell, its importance for the organism. Complex ATP-Mg2+
, examples of where it
works. Hypermagnesemia, hypomagnesemia - causes. Knowledge of the principle of
spectrophotometric measurement (Lambert-Beer's law and its use).
PRINCIPLE OF THE METOD:
Magnesium in the sample reacts with xylidyl blue in an alkaline medium forming a color
complex that can be measured spectrophotometrically. EGTA is included in a reagent to
remove calcium interference.
REAGENTS:
Reagent (A): 0.1 M sodium carbonate, 0.1 M EGTA, 0.1 M sodium triethanolamine, 7.7 mM
potassium cyanide, 0.95 g / l sodium azide.
DANGER: The agent causes severe skin burns and eye damage. Wear protective gloves /
protective clothing. In case of skin or hair contact: Immediately remove / dispose of all
contaminated clothing. Rinse skin with water / shower
Reagent (B): Glycine 25 mmol / L, xylidyl blue 0.5 mmol / l, chloroacetamide 2.6 g / l.
Preparation of the working reagent – PREPARATION IS DONE BY THE LEADER OF
PRACTICAL LECTURE: Pour the reagent B content into Reagent A. Mix gently. The
working agent is stable for 15 days at 2-8 ° C.
Standards: standard 1 - 0,5 mg/dL
standard 2 - 1 mg/dL
standard 3 - 2 mg/dL
standard 4 - 4 mg/dL
MATERIAL (samples)
List of equipment and chemicals Required: unknown sample, different concentration of
magnesium for calibration curve, working reagent, water, ELISA reader, pipettes, tips,
microtubes, tubes, 96-well plate.
PROCEDURE
1. Mark the test tubes 1-12 and prepare the reactions according to the pipetting scheme.
2. Pipette samples (water, standards and unknown sample) – 10 L (small tips) to the
bottom of tubes.
3. Pipette working reagent to each tube – 240 L (large tips) to the tubes containing
samples.
Practical lesson - Biochemistry 2019
4. Pipetting scheme
Mg2+
concentration
(x- axis)
A520
Mean value
A520
A520 sample - A520 BLANK
(y - axis)
Calculated
concentration
1
Blank (water) -
2
3 Standard 1
0,5 mg/dl
-
4
5 Standard 2
1 mg/dl
-
6
7 Standard 3
2 mg/dl
-
8
9 Standard 4
4 mg/dl
-
10
11
Unknown sample
12
5. Mix thoroughly on VORTEX
6. Pipette 200 µL of solution into 96 well microtitrate plate and record the plate number and
well position (eg. plate 1; A1 - B2)
7. Moving to the central lab
8. Measure on an ELISA reader at 520 nm
From the measured absorbance values and known concentrations of magnesium, create a
calibration curve and from the calibration equation calculate the unknown sample
concentration where y is A520 sample - A520 BLANK value
Comparing with the atest
Compare the result of the magnesium concentration in the unknown sample obtained from
your calculated values from the calibration curve with the magnesium concentration specified
in the certificate (calibration package insert). In the case of a different result, analyze the
possible causes (eg. errors in the magnesium determination) and state them at the end of the
report.
Practical lesson - Biochemistry 2019
Unknown sample=
Preparation of calibration standard curve - line
Plot graf in excel – linear regression,
Preparation of calibration standard curve
A standard curve is a type of graph used as a quantitative research technique. Multiple
samples with known properties are measured and graphed, which then allows the same
properties to be determined for unknown samples by interpolation on the graph. The samples
with known properties are the standards, and the graph is the standard curve.
Standard curves are most commonly used to determine the concentration of a substance
such as glucose, protein or DNA. For example, a standard curve for protein concentration is
often created using known concentrations of bovine serum albumin. The property measured
may be absorbance, optical density, luminescence, fluorescence, radioactivity, or other
parameters.
PREPARE PROTOCOL, compare your calculated concentration of unknown sample with
table values.
Practical lesson - Biochemistry 2019
PROTOCOL
Objective :
Principe – summary:
Results - measured values :
Calculations:
Graph – calibration curve
Comparison of results:
Summary:
Answer the test questions:
1. What is blank?
2. Explain the principle of magnesium determination. What will you measure? At which wavelength
will you measure?
3. What is spectrophotometry?
4. Give Beer-Lambert law?
5. Give the principle of determination of unknown concentration with standard calibration curve
Practical lesson - Biochemistry 2019
Magnesium
Magnesium is one of the most abundant cations in the body. It is stored mainly in the bone,
but significant amounts of magnesium are present in gastric and biliary secretions.
Magnesium acts as an essential cofactor for enzymes concerned with cell respiration,
glycolysis, and transmembrane transport of other cations. Plasma magnesium concentration is
normally kept within narrow limits. The kidneys are the main organ of magnesium
homeostasis in maintaining plasma concentrations.
Reference values – human:
Magnesemia – serum, plasma: 0.70 – 0.98 mmol/L
Hypermagnesemia:
Causes - acute and chronic renal failure, decreased magnesium excretion, increased
magnesium intake with diet - dietary supplements, medication, dehydratation
Hypomagnesemia:
Causes - heart disease, ventricular arrhythmia, gastrointestinal malabsorption, fluid loss
Spectrophotometry is the quantitative measurement of the reflection or transmission
properties of a material as a function of wavelength.
Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a
photometer that can measure intensity as a function of the light source wavelength. Important
features of spectrophotometers are spectral bandwidth and linear range of absorption or
reflectance measurement.
The Beer-Lambert law states that the absorbance of a solution is directly proportional to
the concentration of the absorbing species in the solution and the path length. Thus, for a
fixed path length, UV/Vis spectroscopy can be used to determine the concentration of the
absorber in a solution. It is necessary to know how quickly the absorbance changes with
concentration. This can be taken from references (tables of molar extinction coefficients), or
more accurately, determined from a calibration curve.
THE BEER-LAMBERT LAW relates the absorption of light to the properties of the
material through which the light is travelling.
,
where is the absorbance, is the concentration of the solution, is the path length
and the constant - extinction coefficient of the absorber (properties of material).
Practical lesson - Biochemistry 2019
BLANK
Blank (solution), a solution containing no analyte (sample), typically used to zero an
analytical instrument and ensure that any reagents used do not contribute to overall
measurements. Very often it is solvent in case of direct spectroscopy (UV-spectroscopy of
DNA, proteins) or analytical reagent with solvent.
• A spectrophotometer is a device which measures the absorbance of a solution as light
of a specified wavelength is passed through it.
• If we measure the absorbance of a solution containing a known concentration of an
analyte, we can use this value to estimate the concentration of the analyte in an
unknown solution by comparing the two absorbance values
• The range over which absorbance is proportional to concentration varies according to
the analyte and the wavelength of light used. To ensure that there is a direct
relationship between absorbance and concentration, we must prepare a standard curve.
Despite its name, the part of the standard curve that gives a proportional relationship is
a straight line.
• To prepare a standard curve, we prepare a series of dilutions of a standard solution of
our analyte of known concentration. The first tube always contains none of the analyte
(eg. a concentration of 0g/L) and we call this a blank. We use this to calibrate the
Practical lesson - Biochemistry 2019
spectrophotometer to take into account the natural absorbance of the diluents. Each of
the following tubes contain increasing concentrations of the analyte. The absorbances
of each of the standards are read using the spectrophotometer.
• To create the standard curve, we plot a line graph of Absorbance (Y axis) vs
Concentration (X Axis) for each of the standards. A line of best fit is then drawn
through the points.
• To estimate the concentration of an unknown solution of the analyte, we read the
absorbance, and then use the standard curve: