Download HPLC is a precise tool Lactose fermentation Lactose is disaccharide

HPLC is a precise tool
„
„
„
Use of chromatography for cheese analysis
„
Tiiu-Maie Laht
Tallinn University of Technology
Lactose fermentation
„
„
„
to follow the fermentation
To monitor the starter activity
The organic acids pattern is characteristic
for cheeses with added propionibacteria
Find out the reason of cheese blowing: if
during late blowing of cheeses butyric acid
is formed the Clostridia should be present
Lactose is disaccharide
The major function of lactic starters during
cheese manufacture is to produce lactic acid
from lactose
In some kinds of cheeses like Emmental
lactate is converted into propionate and
acetate
Short-chain fatty acids have strong
characteristic aromas and flavours
1
Formation of organic acids
„
„
„
Lactic acid is major product for
homofermentative starters
Heterofermentative starters produce acetate
and ethanol also
Some lactic starters are able to use also
citrate, aroma compounds acetoin and
diacetyl are formed
Cheese sample preparation
„
„
„
„
„
„
Separation of organic acids using ODS
columns
„
„
„
„
We started analysis of organic acids from
cheese samples using C18 columns
Before cheese we have analysed juices,
fermentation probes and everything worked
For cheese ODS was not a good choice, the
peaks were moving, automatic detection was
complicated and lot of peaks were
overlapping
The shelf-life of the column was short
10g cheese homogenized in water (1:10)
Homogenate is heated for 10 minutes at
75ºC
Centrifugation at 10000rpm/10minutes
Fat is removed, proteins are denaturated
with acetonitrile or i-propanol
Sediment is removed (centrifugation
12000rpm or filtration 0.45µ glass fibre
Injection amount 10-50 µl
Use of HPLC, milk, milk products
„
„
Marsili, R.T., Ostapenko,H., Simmons, R.E and
Green, D.E. (1981). High performance liquid
chromatography determination of organic acids
in dairy products. Journal of Food Science, 46,
52-57.
We modified their method adding RI detector for
detection of sugars during the same run
2
Aminex HPX87- H column
„
„
Has been used for determination of organic
acids in different foods since beginning of
80-s
In a simple isocratic run most important
organic acids and sugars are separated if
you use two detectors in parallel, better
quantification of sugars is achieved with RI,
the ratio UV/RI allows also better
identification of peaks
During ripening of Swiss-type cheese
„
„
„
„
No free sugars are available after some days
Lactic acid is actively formed during vat
process and pressing
Lactate concentration decreased from 120130 mmoles/kg to 30-50mmoles/kg
Propionate (40-60 mmoles/kg) and acetate
(40-50mmoles/kg) are formed, mainly in
warm room
Separation of organic acids
„
„
„
The main organic acids in milk and in
cheese are citric, pyruvic, lactic, uric, formic,
acetic, propionic, butyric and hippuric.
These acids could be separated at ambient
temperature using 0.009n sulphuric acid as
eluent
In parallel you can follow the degradation of
lactose and formation and utilization of
glucose and galactose
HPX-87H column has long shelf-life
„
„
„
Use cation H guard column
If the peaks are not sharp the first step is to
reverse the column and run backwards 0.10.2 ml/min overnight
Cleaning with 5% acetonitrile in 0.009n
sulphuric acid
3
Stability of separation parameters
„
„
„
„
The eluation order of the organic acids is
allways the same
Retention time is very stable
Average recoveries are over 95%
The detection limit depends on substance
and detector
Ion exchanger – the column
„
„
„
Consists of insoluble matrix to which
charged groups have been covalently bound
The charged groups are associated with
mobile counter-ions
These counter-ions can be reversibly
exchanged with other ions with the same
charge without altering the matrix
Ion-exchange chromatography
„
„
„
Separation is achieved on the basis of the
charges carried by soluble molecules
Ion-exchage is capable of separating
molecules differing in charge, the
differencies could be very small
Most biological molecules are polar and can
be charged, so ion-exchange techniques are
widely used
Exchangers could have positive or
negative charges
„
„
Positively charged exchangers have
negatively charged counter-ions available
for exchange and are so termed anion
exchangers
Negatively charged exchangers have
positively charged counter-ions and are
termed cation exchangers
4
The matrix
„
„
„
May be based on anorganic compounds
Synthetic resins, consisting of tightly crosslinked hydrophobic polymer matrices highly
substituted with ionic groups have very
capacities for small ions
The high degree of gross-linking provide
mechanical strength and excellent flow
properties
Charged groups
„
„
„
„
The presence of charged groups is a
fundamental property of an ion-exchanger
The type of group determines the type and
strength of the ion-exchanger
The total number and availability determines
the capacity
Sulphonic and quaternary amino groups are
used to form strong ion exchangers
5